(Current Cancer Research) Elena Sotillo, Xavier Graña (Auth.), Greg H. Enders (Eds.) - Cell Cycle Deregulation in Cancer (2010, Springer-Verlag New York)

Contemporary Cancer Research
Jac A. Nickoloff, S ERIES E DITOR
For other titles published in this series, go to

http://www.springer.com/series/7675
Greg H. Enders
Editor
Cell Cycle Deregulation

in Cancer
123
Editor
Greg H. Enders
Fox Chase Cancer Center
Department of Medicine
333 Cottman Ave.
Philadelphia PA 19111-2497
USA
greg.enders@fccc.edu
ISBN 978-1-4419-1769-0 e-ISBN 978-1-4419-1770-6

DOI 10.1007/978-1-4419-1770-6
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Contents
Part I Starting the Cell Division Cycle

1 Escape from Cellular Quiescence . . . . . . . . . . . . . . . . . . 3
Elena Sotillo and Xavier Graña
2 Interplay Between Cyclin-Dependent Kinases
and E2F-Dependent Transcription . . . . . . . . . . . . . . . . . . 23
Jun-Yuan Ji and Nicholas J. Dyson
3 Regulation of Pre-RC Assembly: A Complex Symphony
Orchestrated by CDKs . . . . . . . . . . . . . . . . . . . . . . . . 43
A. Kathleen McClendon, Jeffry L. Dean, and Erik S. Knudsen
Part II Proliferation Under Duress

4 Mitotic Checkpoint and Chromosome Instability in Cancer . . . . 59
Haomin Huang and Timothy J. Yen
5 Mitotic Catastrophe . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Jeremy P.H. Chow and Randy Y.C. Poon
6 p53, ARF, and the Control of Autophagy . . . . . . . . . . . . . . 97
Robert D. Hontz and Maureen E. Murphy
Part III Long-Term Proliferation

7 Regulation of Self-Renewing Divisions in Normal
and Leukaemia Stem Cells . . . . . . . . . . . . . . . . . . . . . . 109
Andrea Viale and Pier Giuseppe Pelicci
8 Maintenance of Telomeres in Cancer . . . . . . . . . . . . . . . . 127
Eros Lazzerini Denchi
9 The Senescence Secretome and Its Impact on Tumor
Suppression and Cancer . . . . . . . . . . . . . . . . . . . . . . . 139
Alyssa Kennedy and Peter D. Adams
v
vi Contents
Part IV Applications in Preventing and Treating Cancer

10 Cell Cycle Deregulation in Pre-neoplasia: Case Study
of Barrett’s Oesophagus . . . . . . . . . . . . . . . . . . . . . . . 157
Pierre Lao-Sirieix and Rebecca C. Fitzgerald
11 Targeting Cyclin-Dependent Kinases for Cancer Therapy . . . . . 167
Neil Johnson and Geoffrey I. Shapiro
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187
Contributors
Peter D. Adams Cancer Research UK Beatson Labs, University of Glasgow,

Glasgow G61 1BD, UK, p.adams@beatson.gla.ac.uk
Jeremy P.H. Chow Department of Biochemistry, Hong Kong University of
Science and Technology, Clear Water Bay, Hong Kong, China
Jeffry L. Dean Department of Cancer Biology, Kimmel Cancer Center, Thomas
Jefferson University, Philadelphia, PA 19107, USA
Eros Lazzerini Denchi Laboratory of Chromosome Biology and Genomic
Stability, Department of Genetics, The Scripps Research Institute, La Jolla, CA
92037, USA, edenchi@scripps.edu
Nicholas J. Dyson Department of Pathology, Harvard Medical School,
Massachusetts General Hospital Cancer Center, Charlestown, MA 02129, USA,
dyson@helix.mgh.harvard.edu
Rebecca C. Fitzgerald Cancer Cell Unit, Hutchison-MRC Research Centre,
Cambridge, CB2 0XZ, UK, rcf@hutchison-mrc.cam.ac.uk
Xavier Graña Fels Institute for Cancer Research and Molecular Biology, Temple
University School of Medicine, Philadelphia, PA 19140, USA; Department of
Biochemistry, Temple University School of Medicine, Philadelphia, PA 19140,
USA, xavier@temple.edu
Robert D. Hontz Program in Molecular and Translational Medicine, Fox Chase
Cancer Center, Philadelphia, PA 19111, USA
Haomin Huang Fox Chase Cancer Center, Philadelphia, PA 19111, USA,
haomin.huang@fccc.edu
Jun-Yuan Ji Department of Pathology, Harvard Medical School, Massachusetts
General Hospital Cancer Center, Charlestown, MA 02129, USA; Department of
Molecular and Cellular Medicine, Texas A&M Health Science Center, College
Station, TX 77843, USA, ji@medicine.tamhsc.edu
Neil Johnson Department of Medical Oncology, Dana-Farber Cancer Institute,
Boston, MA 02115, USA; Department of Medicine, Brigham and Women’s
vii
viii Contributors
Hospital, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115,

USA, neil_johnson@dfci.harvard.edu
Alyssa Kennedy Fox Chase Cancer Center, Philadelphia, PA 19111, USA
Erik S. Knudsen Department of Cancer Biology, Kimmel Cancer Center, Thomas
Jefferson University, Philadelphia, PA 19107, USA,
eknudsen@kimmelcancercenter.org
Pierre Lao-Sirieix Cancer Cell Unit, Hutchison-MRC Research Centre,
Cambridge, CB2 0XZ, UK, pss29@hutchison-mrc.cam.ac.uk
A. Kathleen McClendon Department of Cancer Biology, Kimmel Cancer Center,
Thomas Jefferson University, Philadelphia, PA 19107, USA,
amcclend@kimmelcancercenter.org
Maureen E. Murphy Program in Molecular and Translational Medicine, Fox
Chase Cancer Center, Philadelphia, PA 19111, USA, maureen.murphy@fccc.edu
Pier Giuseppe Pelicci Department of Experimental Oncology, European Institute
of Oncology at IFOM-IEO-Campus, Milan 20139, Italy,
Piergiuseppe.pelicci@ifom-ieo-campus.it
Randy Y.C. Poon Department of Biochemistry, Hong Kong University of Science
and Technology, Clear Water Bay, Hong Kong, China, rycpoon@ust.hk;
http://ihome.ust.hk/∼rycpoon
Geoffrey I. Shapiro Department of Medical Oncology, Dana-Farber Cancer
Institute, Boston, MA 02115, USA; Department of Medicine, Brigham and
Women’s Hospital, Boston, MA 02115, USA; Harvard Medical School, Boston,
MA 02115, USA, geoffrey_shapiro@dfci.harvard.edu
Elena Sotillo Department of Pathology, Children’s Hospital of Philadelphia,
Philadelphia, PA 19104-4399, USA
Andrea Viale Department of Experimental Oncology, European Institute of
Oncology at IFOM-IEO-Campus, Milan 20139, Italy,
andrea.viale@ifom-ieo-campus.it
Timothy J. Yen Fox Chase Cancer Center, Philadelphia, PA 19111, USA,
timothy.yen@fccc.edu
Part I
Starting the Cell Division Cycle
Chapter 1
Escape from Cellular Quiescence
Elena Sotillo and Xavier Graña
Abstract Quiescent: From Latin quies, referring to a state of being at

rest, dormant, inactive, quiet, still (Merriam-Webster, 2009, Online Dictionary:
http://www.merriam-webster.com/dictionary/quiescent). This term refers to a state
of dormancy as opposed to a proliferative state. However, quiescent cells are in
any other regard metabolically active. In many tissues with relative fast cell renewal
rates the primary function of a small group of undifferentiated cells is limited to self-
renewal (stem cells). These cells remain quiescent most of the time dividing only
occasionally. In other tissues, key cell types perform fundamental tissue functions
while remaining quiescent. Both stem cells and cells from tissues that renew via
simple duplication can remain quiescent for long periods of time while retaining the
capacity to re-enter the cell cycle. This chapter will discuss the mechanisms emerg-
ing as responsible for the maintenance of quiescence as well as those pathways that
mediate quiescence entry and exit. We will also review signaling pathways dereg-
ulated during infection by Simian Virus 40 (SV40) and oncogenic transformation,
which result in unscheduled exit from quiescence into the cell cycle, with focus on
SV40 small t antigen.
1.1 Quiescence: The Reversible State

Eukaryotic cells can be in a dividing proliferative state or they can enter non-
dividing states. There are four possible non-dividing states: quiescence (G0),
senescence, differentiation, and apoptosis. Importantly, only quiescent cells can
reversibly re-enter the cell cycle upon appropriate stimuli, whereas terminally dif-
ferentiated (for the most part) and senescent cells, which can also survive for long
periods of time, have permanently withdrawn the cell cycle (Fig. 1.1). In multi-
cellular organisms, commitment to a round of DNA replication and cell division
X. Graña (B)
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine,
Philadelphia, PA 19140, USA
e-mail: xavier@temple.edu
G.H. Enders (ed.), Cell Cycle Deregulation in Cancer, Contemporary Cancer Research, 3
DOI 10.1007/978-1-4419-1770-6_1, C Springer Science+Business Media, LLC 2010
4 E. Sotillo and X. Graña
requires adequate concentration of mitogens in the environment, space, and for

adherent cells, a substrate to attach to. Thus, deprivation of mitogens, lack of adhe-
sion, or growth to high density drive normal cells into quiescence (Fig. 1.1). Recent
studies have uncovered that each of these cell cycle exit-initiating signals elicits a
distinct gene expression signature (Coller et al., 2006). However, to preserve the
reversibility of the quiescent state, a shared “quiescent gene expression program”
that includes genes that suppress differentiation and apoptosis is implemented in all
instances.
It is well established that the quiescent state is associated with an increase in
the expression of the CDK inhibitor p27 (Sherr and Roberts, 1999). Unexpectedly,
the study of the gene expression fingerprints that characterize quiescence has also
revealed that quiescence is not equivalent to growth arrest induced via inhibition
of CDKs. Cells ectopically expressing the p21/p27 CDK inhibitors exhibit a dis-
tinctive program of gene expression that includes a portion of the genes found
Fig. 1.1 Fate of proliferating normal cells upon cell cycle exit. Upon cell cycle exit, cells can enter
three non-dividing stable states: terminal differentiation, senescence, and quiescence. Of these,
only cellular quiescence is reversible. Cellular quiescence can be triggered by mitogenic starvation,
growth to high density, and lack of attachment to substratum. The restriction point (R) is the point
in G1 phase where cells commit to a round of DNA replication and cell division. Cells require
mitogens in the post-mitotic G1 prior to the R. Mitogens activate G1 CDKs, which cooperatively
inactivate pocket proteins and activate the E2F program of gene expression required for cell cycle
progression (see text)
1 Escape from Cellular Quiescence 5
downregulated by all quiescent signals mentioned above, but it does not induce
upregulation of genes that suppress differentiation or inhibit apoptosis (Coller et al.,
2006). In agreement with the observation that CKI inhibitors are upregulated during
differentiation along particular lineages, overexpression of p21 in dermal fibroblasts
induced growth arrest but did not prevent MyoD-induced differentiation. In contrast,
fibroblasts forced into quiescence by contact inhibition or mitogenic withdrawal are
resistant to differentiation signals (Coller et al., 2006). These results show that cellu-
lar quiescence is not a mere consequence of cell cycle exit but rather a unique resting
state that preserves cells in environments that are not suitable for proliferation.
More recently, the mechanisms that control the reversibility of cellular quies-
cence have started to be unveiled. Because the transcriptional repressor Hairy and
Enhancer of Split1 (HES1) is induced by signals that force fibroblasts into qui-
escence but is not regulated when cell cycle exit is induced by overexpression
of CKIs (Coller et al., 2006), Sang et al. tested whether HES1 modulates the
reversibility of cellular quiescence (Sang et al., 2008). Remarkably, it was found that
ectopic expression of HES1 in dermal fibroblasts prevents p21-induced irreversible
senescence, although it cannot reverse this phenotype if senescence is attained
prior to HES1 expression. More importantly, their work also demonstrated that
MyoD-induced differentiation of proliferating fibroblasts is prevented by ectopic
expression of HES1 and that inactivation of HES1 in quiescent fibroblasts is suf-
ficient to induce spontaneous senescence or trigger myogenic differentiation in
response to MyoD activation. Thus, HES1 emerges as a pivotal candidate to control
the reversibility of the quiescent state.
1.2 Overcoming the Restriction Point
1.2.1 The Restriction Point

In unicellularorganisms such as yeast, the availability of nutrients in the envi-
ronment primarily determines their proliferation rate. In contrast, nutrients in the
environment of cells in multicellular organisms are not typically limiting, and thus
proliferation rates are determined by mitogens produced by other cells or by genetic
developmental programs. The cell cycle can be subdivided in two functionally
distinct parts based on their dependency on mitogens for cell cycle progression
(Fig. 1.1). The mitogen-dependent phase spans the period of the cell cycle beginning
with initiation of post-mitotic G1 to the Restriction point (R), which was first defined
by Arthur Pardee (Pardee, 1974). Once cells surpass R, they are committed to a
round of DNA replication and cell division, and the progression and continuity from
one phase to the next depend solely on the cell’s efficiency to faithfully complete
DNA replication, chromosomal segregation, cytokinesis, and other required inter-
mediate steps. On the other hand, normal post-mitotic early G1 cells that encounter
an environment with limiting mitogens, extracellular substrate attachment, or space,
enter a reversible quiescence state.
The main challenge faced by a cell exiting quiescence is to synthesize de novo

all the gene products required for successful cell cycle entry and passage through
R. E2F transcription factors control the expression of many genes whose prod-
ucts are essential, or at least important, for cell cycle progression. In quiescent
cells, repressor E2Fs (E2Fs 4 and 5) form complexes with pocket proteins (typi-
cally p130 and the retinoblastoma protein, pRB) which silence E2F-dependent gene
expression (reviewed in Graña et al., 1998; Mulligan and Jacks, 1998; Blais and
Dynlacht, 2004; Rowland and Bernards, 2006). Mitogens activate intracellular sig-
naling pathways that trigger activation of G1 cyclin/CDK complexes, which in turn
disrupt E2F/pocket protein complexes via phosphorylation of the pocket protein. It
is thought that once the E2F-gene expression program is set in motion to warrant
the expression of sufficient levels of DNA replication enzymes and other cell cycle
proteins and regulators, cell cycle progression becomes insensitive to both positive
and negative external mitogenic stimuli (Fig. 1.1).
1.2.2 G1-Cyclins/CDK, pRB, and E2F Transcription Factors
Since this book contains a chapter devoted to the interplay between CDKs and E2F-
dependent transcription, the focus of this section will be restricted to the events
important for quiescence exit/entry.
G1-cyclins, together with their catalytic partners, the CDKs, are the key effectors
of mitogenic signaling that drive cells out of quiescence in propitious environmental
conditions. There are three mammalian isoforms of cyclin D (D1, D2, and D3) that
exhibit tissue-specific expression. D-type cyclins bind to CDK4 or CDK6 (CDK4-
6) and are activated in mid-G1. E-type cyclins, E1 and E2, bind to CDK2 leading to
its activation later in G1.
Mitogenic stimulation activates RAS, which induces cyclin D1 transcription
(Albanese et al., 1995) and stabilization through RAF/MAPK and PI3K/AKT
mitogenic pathways (Diehl et al., 1998; Henry et al., 2000). Cyclin D/CDK4-6 com-
plexes promote activation of cyclin E/CDK2 complexes through sequestration of
CDK inhibitors (CKIs) from the CIP/KIP family (p21, p27, and p57). The trimeric
complex cyclin D/CDK4-6/CKI shuttles into the nucleus, where it phosphorylates
multiple sites on p130/pRB, relieving repressor E2Fs from pocket protein inhibi-
tion to initiate expression of early E2F-dependent genes, which in turn will generate
more cyclin E (Fig. 1.2). The increase in cyclin E expression and the sequestra-
tion of CKIs by cyclin D/CDK4-6 complexes ensure accumulation of CKI-free
cyclin E/CDK2 complexes that can be phosphorylated on the activating T-loop of
CDK2 by the CDK-activating kinase (CAK) (Kato et al., 1994; Kaldis et al., 1998;
Sherr and Roberts, 2004). As cyclin E/CDK2 active complexes emerge, a positive
feedback loop ensures rapid activation of CDK2 through direct phosphorylation of
CKIs, triggering their degradation, and hyperphosphorylation of pocket proteins
facilitating additional accumulation of cyclin E and CDK2. CDK2 completes the
inactivation of pocket proteins initiated by CDK4-6, which results in forceful elim-
ination of repressor E2F complexes at the promoters and the expression of activator
Fig. 1.2 Mitogens stimulate cell cycle re-entry via activation of the E2F-program of gene expres-
sion. Transition into the G1 phase of the cell cycle from quiescence requires activation of
E2F-dependent gene expression. Expression of E2F-dependent genes is silent in quiescent cells.
Promoters of E2F-dependent genes are occupied by E2F complexes containing repressors E2Fs and
p130, as well as homologs of C. elegans synthetic multivulva class B gene products (MuvB pep).
Mitogenic stimulation results in activation of CDKs by inducing G1 cyclin accumulation and inac-
tivation of CKIs through various mechanisms. G1 CDKs phosphorylate pocket proteins disrupting
their interaction with repressor E2Fs coinciding with the expression of gene products. Among the
upregulated proteins are activator E2Fs (E2F1-3) that are recruited to promoters coinciding with
recruitment of HATs and promoter activity. Cyclin E is an E2F-regulated gene product that helps
inactivate pocket proteins, but also targets other substrates for phosphorylation that are important
for DNA replication and centriole duplication. Antimitogenic signaling negatively regulates CDKs
through upregulation of CKIs
E2Fs (E2F1-3), which are subsequently recruited to multiple E2F-dependent pro-

moters coinciding with expression of E2F-dependent genes. Obviously, there are
other players that participate in the activation of these CDKs and E2F-dependent
gene expression, so readers are directed to more comprehensive reviews (Blais and
Dynlacht, 2004; Rowland and Bernards, 2006; Blais and Dynlacht, 2007). It is
important to note at this time that whereas cyclin D/CDK4-6 primary substrates are
pocket proteins and Smad3 (Liu and Matsuura, 2005), both involved in repression
of cell cycle-dependent gene expression, cyclin E/CDK2 functions are not limited
to pocket protein inactivation in G1. Cyclin E/CDK2 phosphorylates multiple fac-
tors involved in centrosomal duplication, replication origin licensing and firing, and
control of histone synthesis (Moroy and Geisen, 2004) (Fig. 1.2).
Overexpression of G1 cyclins is common in primary tumors and derived tumor
cell lines (Malumbres and Barbacid, 2001). Considering that mitogenic signaling
converges in the activation of G1 cyclin/CDK complexes, deregulation of G1 cyclins
in tumor cells may reduce the threshold of mitogenic stimulation required for pas-
sage through the R or for escaping quiescence. In this regard, early studies showed
that overexpression of either D1 or E shortens G1 phase upon mitogenic stimulation.

However, quiescent primary non-transformed fibroblasts that ectopically express
cyclin D1 and/or E do not exit quiescence if the environment is deprived of mito-
gens or if the cells are arrested by growth to high density (Ohtsubo and Roberts,
1993; Quelle et al., 1993; Resnitzky et al., 1994; Sotillo et al., 2008, 2009). In con-
trast, similar expression of cyclin D1 and E in certain tumor cell lines is sufficient
to trigger exit from quiescence in the absence of any mitogenic stimulation (Calbó
et al., 2002). Experiments performed in our laboratory have shown that in quiescent
tumor-derived T98G cells forced expression of cyclin E leads to formation of active
CDK2 complexes, pocket protein phosphorylation, and activation of the E2F pro-
gram concomitantly with cell cycle entry. Under the same conditions expression of
cyclin E in quiescent normal human fibroblasts (NHF) leads to formation of inactive
complexes failing to trigger cell cycle entry. Concentrations of serum as low as 0.1%
make quiescent NHF responsive to deregulated cyclin E expression, suggesting that
other mitogen-dependent events, besides cyclin E accumulation, are required to
fully activate CDK2 and exit G0. This is consistent with previous work showing
that microinjection of active G1 cyclin/CDK complexes into the nucleus of primary
human WI38 fibroblasts is sufficient to induce DNA synthesis (Connell-Crowley
et al., 1998).
Despite the clear important role of G1 CDKs in mediating passage through R
and triggering E2F-dependent gene expression, ablation of G1 CDKs and cyclins
in mice has evidenced a high level of functional redundancy and compensation
among these G1 cyclin/CDK complexes in triggering inactivation of pocket proteins
and other essential events during the cell cycle (Malumbres and Barbacid, 2009).
Targeted disruption of D-type cyclins, E-type cyclins, CDK4-6, or CDK2 reduces
inactivation of pocket proteins but not below a threshold that could prevent E2F-
dependent gene expression in both proliferating and serum starved and re-stimulated
MEFs (Lee and Sicinski, 2006; Berthet and Kaldis, 2007; Malumbres and Barbacid,
2009). Indeed, even fibroblasts obtained from mouse embryos that simultaneously
lack expression of CDK2, CDK4, and CDK6 proliferate and exit quiescence in
response to serum stimulation (Santamaria et al., 2007). Thus, CDK1 via its bind-
ing with cyclin E appears sufficient to inactivate pocket proteins and induce passage
through R. Of note, serum-starved cyclin E1–/–; E2–/– double knock-out MEFs
are unable to re-enter cell cycle when stimulated with mitogens. However, this is
due to a defect in loading of MCM2 onto chromatin, as pocket proteins are inacti-
vated and E2F-dependent genes expressed (Geng et al., 2003). These results indicate
great plasticity and compensation among cyclins and CDKs in many cell types, with
function of some of them only essential in particular cell types.
1.2.3 Is Inactivation of Pocket Proteins Beyond a Certain

Threshold Sufficient for Passage Through R?
Ablation of the three pocket proteins in MEFs makes these cells bypass cell cycle
exit signals induced by mitogen withdrawal, contact inhibition, and loss of attach-
ment, but cells become apoptotic (Dannenberg et al., 2000; Sage et al., 2000). Pocket
protein mutant MEFs also fail to arrest in response to a variety of signals that cause
G1 arrest. Thus, pocket proteins are important for establishment of a G1 growth
arrest and exit into quiescence, as triple mutant MEFs fail to become quiescent in
response to three independent signals that mediate reversible growth arrest. Ectopic
expression of E2F1 drives quiescent rodent fibroblasts into S phase (Johnson et al.,
1993). Activator E2Fs (E2F1-3) are likely required for cell cycle re-entry, while
E2F-repressor activities are critical for contact inhibition induced cell cycle exit and
other signals that induce G1 arrest (Rowland and Bernards, 2006). Therefore, there
is firm evidence that pocket protein/E2F pathways play critical roles in cell cycle
re-entry/exit, as loss of these pathways makes cells insensitive to extracellular con-
trol in both cycling and quiescent cells. It is important to stress that despite the high
degree of compensation among pocket proteins, it has become clear that distinct
complexes play specialized functions. An evolutionarily conserved complex desig-
nated DREAM has been identified in mammalian cells that contains p130, E2F4,
and mammalian homologs of Caenorhabditis elegans synthetic multivulva class B
(synMuvB) gene products, including LIN-9, LIN-37, LIN-52, LIN-53, and LIN-54
(Litovchick et al., 2007). The DREAM complex binds the promoters of cell cycle-
regulated genes in serum starved quiescent human T98G cells and is required for
their repression. It is yet to be known if other G0 and G1 arrest-inducing signals
result in formation of the same DREAM complex, and whether assembly of this
complex is associated with formation of the common gene expression signature that
defines quiescence in NHF (Coller et al., 2006). Conceivably, E2F-repressor com-
plexes formed in response to G1 arrest signals that do not result in cell cycle exit into
quiescence may be different from the DREAM complex in one or several subunits.
It is also possible that expression of HES1 in cells exiting into G0 is independent of
the formation of DREAM, but it might play a role in defining the type of E2F/pocket
protein complexes that assemble at cell cycle promoters.
1.2.4 What Are Cells Doing as They Exit Quiescence Back

into G1?
The classical video-microscopy experiments of Zetterberg and Larsson defined the

period of time between post-mitotic G1 and R in NIH 3T3 cells (Zetterberg and
Larsson, 1985). In these experiments, removal of mitogens for 1 h affected cell
cycle length of only very young post-mitotic cells progressing through early G1.
Mitogen removal for 1 h in early G1 resulted in elongation of the cell cycle by
as much as 8 h. Others have shown that the time that cells require to exit quies-
cence is proportional to the time that they have spent in this state (Owen et al.,
1989). One logical explanation that was drawn from these early studies is that cells
need time to de novo transcribe and translate the gene products required for pas-
sage to R. More recently, changes in the assembly of DNA pre-replication factors
onto replication origins have also been linked to quiescence. Blow and Hodgson
proposed to define quiescence as a reversible withdrawal from the cell cycle charac-
terized by unlicensed origins and lack of CDK activity (Blow and Hodgson, 2002).
Origins of replication in metazoans are bound by ORC complexes in quiescent and
cycling cells, but other components of the pre-replication complex are not loaded
onto chromatin in quiescent cells (see Chapter 3 by McClendon et al., this volume).
These include CDC6 and CDT1, both required for assembly of the multisubunit
helicase composed of MCM2-7. Expression of MCM-2 in cells of the colonic crypt
corroborates findings of cultured cells, as MCM2 is expressed at high levels in
amplifying cells that are actively proliferating and is not expressed in terminally
differentiated cells. MCM2 expression levels in the stem cells at the base of the
crypts are lower than in the amplifying cells, which are consistent with their infre-
quent division (reviewed in Blow and Hodgson, 2002). A number of the subunits
of the pre-replication complex are not expressed in quiescent cells because their
genes are repressed by E2F-dependent mechanisms and because their protein prod-
ucts are targets of the APCCdh1 ubiquitin ligase, which is active when CDK activity
is low (Diffley, 2004). In this regard, it is important to highlight that CDK-mediated
phosphorylation of CDC6 has been linked to its stabilization and accumulation,
as it prevents APC-mediated ubiquitination (Mailand and Diffley, 2005). CDC6
stabilization, accumulation, and loading onto replication origins may occur con-
comitantly with or downstream of its E2F-dependent transcription. Alternatively,
CDC6 accumulation may be a mitogenically regulated CDK event partially indepen-
dent of the E2F program. It is also notable that CDC6 expression has been linked to
an “attachment checkpoint” that apparently operates at least partially independently
of E2F-dependent transcription in NRK fibroblasts (Jinno et al., 2002). Thus, assem-
bly of pre-RC emerges as another process linked to G1 checkpoints that mediate
quiescence entry and exit.
1.3 Oncogenes That Cooperate to Bypass Quiescence
Cellular oncogenic transformation is associated with unresponsiveness to antipro-

liferative and differentiation signals, bypassing of mitogenic extracellular require-
ments and an increase in proliferative lifespan. As multicellular organisms consist
mostly of quiescent cells, critical oncogenic alterations may primarily deal with
ensuring that cells initiating the transformation process remain in a stable prolifera-
tive state most of the time. In this regard, as the term “Restriction Point” was coined
it was already proposed to be bypassed during malignancy (Pardee, 1974).
A number of studies have focused on identifying activated oncogenes, viral trans-
forming proteins, and/or tumor suppressor genes that, when deregulated alone or
in combination, confer mitogen independency, the ability to bypass contact inhibi-
tion, and insensitivity to lack of substratum attachment. In other words, defining
genetic alterations that bypass signals that ensure entry into quiescence in normal
cells. The most informative studies have been performed testing the effect of com-
binations of oncogenes/inactivation of tumor suppressor genes in primary cells of
different species in culture. That is, testing the ability of these cells to grow in sub-
optimal concentrations of serum, to form foci in cell monolayers and/or to grow in
an anchorage-independent manner. Often, these studies have been followed up by
testing if cells that appear transformed in culture form tumors when injected into
nude mice. If so, these cells are typically designated “malignantly transformed.”
A conclusion of these studies is the fact that the ability of certain oncogenes to
bypass cellular quiescence varies among species, and human cells are more resis-
tant to oncogenic transformation than murine cells (Hahn and Weinberg, 2002). It
is also important to mention here that cells need to become immortal for malig-
nant transformation. This is accomplished in many human cells via stabilization of
chromosomal ends (telomeres), which shorten with each DNA replication cycle, as
somatic cells do not express telomerase. In contrast, the majority of human tumor
cells express telomerase or exhibit an alternative mechanism (ALT) to maintain
chromosomal length (Stewart and Weinberg, 2006; Johnson and Broccoli, 2007)
(see Chapter 8 by Denchi, this volume). In contrast, most studies performed using
murine cells show that these cells become immortal by overcoming stress check-
points, as most cell types used in these studies exhibit telomerase activity. For an
in-depth analysis on immortalization, senescence, cancer, and aging readers are
directed to specific reviews (Sherr and DePinho, 2000; Hahn, 2002; Blasco and
Hahn, 2003; Serrano and Blasco, 2007).
Thus, what oncogenes or inactivated tumor suppressor genes help bypass qui-
escence? Ectopic expression of c-MYC and an activated RAS oncogene in normal
immortal rat fibroblasts (REF-52 cells) induce cyclin E/CDK2 activity and exit from
quiescence in low concentrations of serum (Leone et al., 1997). In quiescent REF-52
cells, cellular RAS is required for activation of CDK activity and E2F-dependent
gene expression in response to mitogenic stimulation, but activated RAS is insuf-
ficient to induce CDK activation in quiescent REF-52 cells placed in low serum.
Coexpression of c-MYC allows cyclin E/CDK2 activation likely via downregula-
tion of CKIs (Leone et al., 1997). Others have shown that ectopic expression of an
oncogenic version of RAS induces premature senescence in human and mouse cells
through activation of the RAF/MAPK pathway leading to activation of p53/ARF.
This arrest is bypassed by depletion of p53 function in mouse cells, but disruption of
both the pRB and p53 pathways is necessary to bypass RAS-induced senescence in
human cells (Serrano, 1997, 1998) (see Chapter 9 by Adams, this volume). Thus, the
c-MYC/RAS pair fails to transform mouse fibroblasts unless the p53/ARF pathway
is mutated, and human cells require alteration of both the p53 and pRB pathways, as
well as activation of telomerase in order to become transformed (Hahn et al., 2002;
Hahn and Weinberg, 2002). This likely explains why normal human fibroblasts as
opposed to immortal REF-52 cells fail to exit quiescence upon coexpression of
c-MYC and an activated RAS oncogene (Sotillo et al., 2008). Thus, because the
same oncogenic signals that induce escape from quiescence also induce senescence,
alteration of the p53/ARF pathway is critical for oncogenesis in mouse cells, while
alteration of both the p53 and pRB pathways is required in human cells. Thorough
transformation assays from the Hahn and Weinberg laboratories have demonstrated
that expression of oncogenic RAS, SV40 large T (LT) and small t (st) antigens, and
the catalytic subunit of telomerase (hTERT) is sufficient to transform NHF (Hahn
et al., 1999), human mammary epithelial cells (Elenbaas et al., 2001), and kidney
epithelial cells (Hahn et al., 2002). Subsequent studies have shown that in this setting
LT and st can be substituted by inactivation of p53, pRB, and PTEN plus constitutive
expression of c-MYC (Boehm et al., 2005). The role of the SV40 tumor antigens
will be discussed in more detail in the next section.
Examination of NHF with various combinations of genetic alterations showed
that NHF-expressing hTERT, a dominant negative version of p53 and deregulated
c-MYC, exit quiescence in low concentrations of serum, but subsequent disruption
of pRB via shRNA made these cells insensitive to serum starvation (Boehm et al.,
2005). Interestingly, as mentioned above disruption of the three pocket proteins in
MEFs appears to be required to make these cells largely insensitive to mitogen with-
drawal, as disruption of either pRB or p130/p107 delays but does not prevent cell
cycle exit (Sage et al., 2000). Interestingly, in addition to its role in irreversible
senescence and apoptosis, p53 has been suggested to participate in cell cycle exit
into quiescence, as p53 expression and activity increase in quiescent NHF, and
p53 inactivation by different means delays reversible cell cycle exit in response to
mitogenic withdrawal (Itahana et al., 2002).
c-MYC has been shown to induce expression of the so-called E2F-activators
(E2F1-3) and to directly interact with their promoters upon mitogenic stimulation
(Leone et al., 2001; Fernandez et al., 2003; Leung et al., 2008). Of note, c-MYC
binding to E2F-promoters seems critical for the loading of E2F1 to these promot-
ers. Besides those genes required for cell cycle entry, E2F1 also regulates a group
of genes involved in apoptosis. It has been shown that activation of the PI3K/AKT
pathway during mitogenic stimulation inhibits E2F1 pro-apoptotic targets, favoring
the role of E2F1 as an inducer of proliferation rather than apoptosis (Hallstrom et al.,
2008; Hallstrom and Nevins, 2009).
The importance that activation of PI3K/AKT pathway has on oncogenic transfor-
mation is underscored by the fact that mutations in PTEN, a key negative regulator
of this pathway, and amplification and abnormal activation of PI3K and AKT are
associated with many types of human cancers (Keniry and Parsons, 2008; Yuan
and Cantley, 2008). It has been shown that ectopic expression of the active sub-
unit of PI3K, p110a, can substitute for st when coexpressed with LT and hTERT in
human epithelial cells, promoting growth in low concentrations of serum as well as
proliferation in soft agar (Zhao et al., 2003). In this scenario, st can also be substi-
tuted by coexpression of activated alleles of AKT1 and RAC, a downstream effector
of the PI3K/AKT pathway. Also, forced coactivation of the RAS/RAF/MEK path-
way with AKT elicited a robust proliferative response leading to activation of
G1 cyclin/CDKs resulting from cyclin D1 accumulation and p27 repression, as
well as removal of p21 from cyclin E/CDK2 complexes (Mirza et al., 2004). In
this regard, the PI3K pathway negatively regulates FOXO transcription factors
via AKT-mediated phosphorylation and exclusion from the nucleus. Activation of
FOXO transcription factors is associated with cell cycle exit into quiescence in non-
hematopoietic cells and has been implicated in the transcriptional activation of p27
as well as downregulation of D-type cyclins (Medema et al., 2000; Schmidt et al.,
2002). Moreover, the PI3K pathway has been shown to cooperate with c-MYC in
the expression of c-MYC-dependent genes by inactivating FOXO, which is impli-
cated in the negative regulation of multiple c-MYC genes (Bouchard et al., 2004).
FOXO transcription factors have been implicated in long-term survival of quiescent
cells (Burgering and Medema, 2003).
The adenoviral oncoprotein E1A has also been long known to have the ability
to stimulate exit from cellular quiescence. Two recent studies suggest how E1A
triggers cell cycling and inhibits the cellular antiviral response and differentiation
(Ferrari et al., 2008; Horwitz et al., 2008). E1A expression in quiescent fibroblasts
results in the global relocation of pocket proteins and the p300/CBP acetyltrans-
ferase on cellular promoters. This process occurs in a sequential manner, leading to
acetylation of lysine 18 on histone H3 and promoter transactivation of a restricted
number of genes involved in proliferation and growth. However, both E1A and
SV40 LT cause a global decrease in the acetylation of histone H3 at this site, which
is apparently due to the restriction of HATs to the subset of proliferation/growth
genes concomitant to the exclusion of these proteins on other gene promoters. Thus,
hypoacetylation at histone H3 lysine 18 may be a general consequence of DNA
tumor oncogenesis, which is linked to quiescence exit (reviewed in Ferrari et al.,
2009).
It is also important to highlight recent findings that suggest that HES1, whose
increased expression in quiescent cells has been associated with conferring the
reversible nature of this state, is found expressed at high levels in rhabdomyosar-
coma tumors and derived cell lines (Sang et al., 2008). Rhabdomyosarcomas
are aggressive tumors that express the muscle differentiation factor MyoD, but
exhibit a block in myogenic differentiation. Forced inactivation of HES1 in a rhab-
domyosarcoma cell line via expression of a dominant negative HES1 mutant or
pharmacological inhibition of Notch, which positively regulates HES1 expression,
promoted MHC expression and differentiation of these cells.
In summary, to endow normal cells with the capability of exiting quiescence
in unfavorable environments, oncogenes/inactivated tumor suppressor genes must
mimic mitogenic signals. Cells with these alterations may produce their own mito-
gens, force surrounding cells to do so, or exhibit constitutively activated downstream
signaling pathways independently of mitogenic stimulation. Some of these alter-
ations as well as others will also help bypass antiproliferative signals from the
environment or will make the cell independent of substrate feeding. Phosphorylation
of pocket proteins and activation of the E2F transcription program are critical,
but exit from quiescence does not always lead to effective proliferation. Thus,
other pathways, such as those that inhibit apoptosis and cell senescence and/or are
involved in monitoring faithful DNA replication, must also be altered.
1.4 SV40 and Exit from Quiescence
1.4.1 SV40 Tumor Antigens and Their Cellular Targets
As described in Section 1.3, transformation assays designed to identify the precise

combination of altered pathways that are required for transformation of a variety
of normal human cells have revealed that expression of SV40 LT and st antigens,
oncogenic RAS, and hTERT suffices to ensure the required alterations (Hahn et al.,
1999; Elenbaas et al., 2001; Hahn et al., 2002). Transformation is also attained with
expression of oncogenic RAS, c-MYC, hTERT, st and inactivation of both p53 and
pRB (Boehm et al., 2005). However, expression of st, hTERT, and pRB inactivation
is not required when comparable transformation assays are performed using rodent
cells. The effects of st on transformation of human cells are thought to be, at least
in part, due to its ability to facilitate proliferation in conditions that promote quies-
cence. This indicates that the ability of st to facilitate a bypass of the quiescent state
may be uniquely critical for transformation of human cells. Thus, in this section we
will discuss the effects of expression of SV40 antigens in human cells with a major
focus on st.
In the early 1960s, Polyomavirus Simian Virus 40 (SV40) was discovered as
a viral contaminant during the production of poliovirus vaccines from cultures
from rhesus monkey kidney cells (Eddy et al., 1962). Soon after its discovery,
SV40 was shown to induce tumors in newborn hamsters (Girardi et al., 1962;
Gerber, 1963). However, it took several years and work from multiple laboratories
to demonstrate that the expression of proteins encoded in the Early Region (ER) of
SV40 was responsible for oncogenic transformation (reviewed in Chen and Hahn,
2003).
SV40 ER encodes three proteins that share 82 amino acids in their amino-
terminal end, which includes a DnaJ chaperone domain. However, their unique
carboxy-terminal extensions are generated through alternative splicing resulting in
the synthesis of three separate protein products designated large T (LT), small t (st),
and 17KT antigens (reviewed in Ali and DeCaprio, 2001; Chen and Hahn, 2003;
Pipas, 2009). The role of both LT and st in cellular transformation and tumorigenesis
has been extensively studied and both proteins have been major tools for identi-
fication and characterization of key signaling pathways commonly altered during
cancer development (Pipas, 2009). The C-terminus of LT targets the tumor suppres-
sor gene product p53 while an LXCXE motif present in the amino-terminal end of
the unique domain targets the three pocket proteins pRB, p130, and p107 (reviewed
in Ali and DeCaprio, 2001) (Fig. 1.3). Thus, LT inactivates two major suppressor
pathways that are found inactivated in most tumor cells. On the other hand, the
unique carboxy-terminal domain of st associates with and inhibits PP2A activities
that are not yet completely defined (Pallas et al., 1990; Yang et al., 1991; Pallas et al.,
1992). PP2A is an heterotrimeric serine/threonine phosphatase that consists of a cat-
alytic subunit (PP2A/C), a structural subunit (PP2A/A), and a variable B subunit
that dictates subcellular localization and substrate specificity (reviewed in Virshup
and Shenolikar, 2009). There are two isoforms each for PP2A/C and PP2A/A sub-
units, and four distinct families of conserved PP2A/B subunits encoded from at
least 15 different genes, many with multiple splice variants. Thus, the combination
of PP2A/A/B/C subunits yields multiple possible heterotrimers to specifically target
a variety of substrates that play key roles in cellular proliferation, DNA damage,
and viability among many other cellular processes. st binds to the PP2A/A/C dimer
through a cystein-rich region interfering with the binding of the PP2A/B subunit,
thus likely precluding specific substrate recognition and/or proper subcellular local-
ization. While in human cells the transforming pathways targeted by LT have been
clearly linked to disruption of pRB/pocket proteins and p53-dependent pathways
Fig. 1.3 SV40 st antigen disrupts PP2A heterotrimeric complexes and upregulates a number of
cell cycle proteins. The early region of SV40 encodes large T antigen (LT) that inactivates p53
and pocket proteins and small t antigen (st) that targets a still not well-defined set of PP2A het-
erotrimeric complexes. st binds to the PP2A dimer composed of a catalytic subunit (PP2A/C) and
a scaffold subunit (PP2A/A), displacing B regulatory subunits. Displacement of B subunits of the
B56 family has been linked to transformation and oncogenic upregulation (c-MYC upregulation).
st expression in quiescent fibroblasts has been shown to upregulate a variety of pathways resulting
in unscheduled expression of key cell cycle regulators and replication factors
by making cells insensitive to checkpoint and antiproliferative signaling, the unique

pathways altered by st are still poorly defined due to the vast number of potentially
critical cellular pathways where distinct trimeric PP2A holoenzymes play critical
but not well understood roles (reviewed in Chen and Hahn, 2003; Skoczylas et al.,
2004). In the next section, we will primarily focus on the analysis of those pathways
targeted by st that promote exit from quiescence.
1.4.2 SV40 Small t Antigen Promotes Exit from Quiescence
The effects of st on transformation of human cells are thought to be, at least in

part, due to its ability to facilitate proliferation in reduced concentrations of growth
factors. This function is dependent on its ability to bind and inhibit PP2A, as st
mutants unable to bind PP2A fail to cooperate with LT driving cell proliferation in
a limiting mitogenic environment (Skoczylas et al., 2004). Expression of st stimu-

lates growth of monkey kidney cells (CV-1) maintained in 0.1% serum to an extent
comparable to serum. In this scenario quiescence exit is accompanied by activation
of the RAF/MAPK pathway (Sontag et al., 1993). It was later shown that activation
of PKCζ through st-mediated inhibition of PP2A would also contribute to further
activate the MAPK pathway, as well as activate NFkB-dependent gene expression
(Sontag et al., 1997) in both CV-1 and NIH3T3 cells in 0.1% serum. In the same
study it was determined that pharmacological inhibition of the PI3K pathway or a
dominant negative mutant of p85 blocks st-mediated activation of PKCζ and NFκB,
as well as st-induced cell proliferation (Fig. 1.3). The importance of the PI3K
pathway for st-mediated transformation has been discussed earlier in this chapter
when referring to combinations of oncogenes able to induce exit from quiescence in
normal cells (Fig. 1.3).
Progress has more recently been made identifying PP2A heterotrimers that
could mediate st transforming activities. In human embryonic kidney (HEK) cells,
PP2A/B56γ has been identified as a potential target of st transforming activity. In
these cells, knockdown of B56γ substitutes for st in transformation assays with
defined combinations of altered genes (Chen et al., 2004). However, it was subse-
quently shown that B56γ cannot substitute for st in cells maintained in suboptimal
mitogen concentrations (Moreno et al., 2004), suggesting that st has other targets
important to bypass serum requirements. A second member of the same family of
B subunits, B56α, has been identified as a negative regulator of c-MYC protein
stability, providing a mechanism to explain how st increases c-MYC expression.
Interestingly, a stable c-MYC mutant that cannot be dephosphorylated substitutes
for st in transformation assays (Yeh et al., 2004; Arnold and Sears, 2006).
Downstream targets of st have also been studied. A number of reports have
described transcriptional activation of cyclin D1 and cyclin A promoters in reporter
assays (Porras et al., 1996; Watanabe et al., 1996; Skoczylas et al., 2005). Cyclin
A protein levels were also shown to be upregulated by st in density-arrested human
fibroblasts stimulated with serum, concomitantly with downregulation of p27. In
this scenario, cyclin A is inactive and cells do not enter the cell cycle unless LT is
coexpressed (Porras et al., 1999).
In search for oncogenes that cooperate to bypass quiescence induced by complete
depletion of mitogens in NHF, our lab found that coexpression of st and cyclin E, but
not their individual expression, was sufficient to bypass quiescence and induce DNA
synthesis (Sotillo et al., 2008). This same combination of oncogenes bypassed quies-
cence induced by growth to high density and lead to continued proliferation and foci
formation in hTERT-NHF. These events are at least partially dependent on PP2A
inhibition. Expression of st alone in quiescent NHF did not lead to accumulation
of cyclins A or D1, but strikingly led to accumulation and loading of the essential
replication factor CDC6 (Sotillo et al., 2009). As MCM2 loading onto chromatin
was also observed in density-arrested cells expressing st, these results suggest that
st induces steps toward licensing of replication origins, a characteristic of cells exit-
ing G0 into G1 (see Section 1.2.4 above). When cyclin E and st were coexpressed
in quiescent NHF, CDC6 further accumulated coinciding with CDK2 activation and
DNA synthesis. In addition, we and others have observed that CDC6 expression, as
well as the expression of other pre-RC components, is linked to phosphorylation of
CDK2 on its activating T-loop (Nevis et al., 2009; Sotillo et al., 2009). Therefore,
deregulation of cyclin E expression in the context of normal cells apparently driven
out of quiescence by st leads to the cooperative and coordinated activation of an
essential pre-replication complex factor (CDC6) and an activity required for origin
firing (CDK2) (Sotillo et al., 2009). Importantly, it was also found that in the context
of this oncogenic-driven exit from G0 and proliferation, CDK2 activity appeared
to be essential (Sotillo et al., 2008). While the direct target of st in this case is
unknown the current data suggest that the selective accumulation of the CDC6 tran-
script is dependent on E2F promoter elements but independent of CDK activation.
This suggests that st controls factors that can selectively regulate the expression of a
gene(s) whose expression is associated with exit from G0. Finally, it is important to
point out that despite the observation that CDC6 expression is required for passage
through an “attachment checkpoint” in NRK fibroblasts (Jinno et al., 2002), expres-
sion of cyclin E and st failed to induce anchorage independent growth of hTERT
NHF, indicating that this oncogene pair does not reverse quiescence induced by
all signals. Altogether these studies show that st changes a fundamental property
of quiescent cells that differentiates them from post-mitotic G1 cells, which is the
status of the cell replication origins. By facilitating passage through the G0/G1 tran-
sition, st may trick certain cells to create an environment proper for viral replication
despite extracellular signaling that would otherwise keep cells in a quiescent state.
Understanding how st disrupts the mechanisms that ensure maintenance of the qui-
escent state will provide insight into the molecular nature of this state, increase our
understanding of how DNA tumor viruses promote their own replication, and may
unveil novel mechanisms for cellular transformation that are exclusive to human
cells.
1.5 Future Directions

The defining characteristic of cells in the quiescent state is maintenance of their
ability to re-enter the cell cycle in propitious conditions. Recent work has shown
that while cells exiting the cell cycle into quiescence initiate programs of gene
expression that are coupled to the quiescent inducing signal, there is a common
gene expression signature that emerges in a time-dependent manner. This signature
appears associated with acquisition of resistance to differentiation, senescence, and
cell death. A transcription factor designated HES1 has been shown to be upregulated
during cell cycle exit into quiescence and appears to be required for maintenance
of the quiescent state, as it blocks both differentiation and senescence in human
fibroblasts. This factor is found deregulated in rhabdomyosarcomas and mediates
a block in myogenic differentiation exhibited by these tumor cells. Work over the
past several decades has led to the identification of oncogenes and tumor suppressor
genes that, when deregulated, facilitate mitogen-independent cell cycle progression
and insensitivity to other signals that induce exit into quiescence. SV40 st antigen
has emerged as a key player in the specific transformation of human cells, as its
unique transforming activity is not required in rodent cells. st appears to facilitate
transformation in certain environments that favor cell cycle exit into quiescence,
such as mitogen starvation and contact inhibition. Recent progress in identification
of the PP2A heterotrimers that are targeted by st, as well as the downstream cell
cycle players that mediate st activities that drive cells out of quiescence are likely to
provide important insights in the near future.
Acknowledgments We thank Manuel Serrano, David G. Johnson, Alison Kurimchak, and Judit
Garriga for critically reading this manuscript and helpful suggestions. Work in this lab has been
supported by a grant project under CA095569 and a Career Development Award (K02 AI01823)
to XG of the National Institutes of Health.
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Chapter 2
Interplay Between Cyclin-Dependent Kinases
and E2F-Dependent Transcription
Jun-Yuan Ji and Nicholas J. Dyson
Abstract Precise control of cell proliferation is essential for normal development

and survival of all multi-cellular organisms. The deregulation of cell proliferation
is a fundamental feature of all types of cancer. One of the key regulators of cell
proliferation is the E2F transcription factor. E2F controls the expression of many
genes that are required for cells to divide and elevated E2F activity is found in most
tumor cells. The activation and inactivation of E2F are tightly linked to the activation
of cyclin-dependent kinases (CDKs). In normal cells, these connections allow the
periodic oscillations in CDK cycle to be coupled with temporal programs of gene
expression. Multiple CDK–cyclin complexes (including CDK1/2–CycA, CDK1/2–
CycB, and CDK7–CycH) have been shown to directly phosphorylate E2F or its
dimerization partner DP. However, in recent genetic studies, one of the strongest
modifiers of E2F-dependent phenotypes was cdk8, a kinase that had not previously
been linked to E2F. In this review, we summarize the effects of CDKs on E2F1
activity and describe a model that may explain the role of CDK8–CycC in E2F
regulation. Since CDKs can both increase and decrease E2F activity, understand-
ing the interplay between E2F and CDK–cyclin complexes may suggest therapeutic
approaches to efficiently block cancer cell proliferation.
2.1 Cell Cycle Progression Is Driven by the Integrated Action

of Cyclin-Dependent Kinases and a Transcriptional Network
The mitotic cell cycle is composed of an S phase (for DNA synthesis) and an
M phase (for mitosis), separated by two gap phases, G1 and G2. Progression through
J.-Y. Ji (B)
Department of Pathology, Harvard Medical School,
Massachusetts General Hospital Cancer Center, Charlestown, MA 02129, USA
e-mail: ji@medicine.tamhsc.edu
24 J.-Y. Ji and N.J. Dyson
these phases is driven by the periodic activation of CDK (cyclin-dependent kinase)–

cyclin complexes. One of the most important discoveries in the field of cell biology
was that these central components of the cell cycle machinery have been con-
served during eukaryote evolution (Nasmyth, 1995; Nurse, 2000). The conservation
of CDK function is so extensive that the human CDK1 (CDC2) gene is able to
substitute for its functional orthologs in yeast (Lee and Nurse, 1987).
Genome sequencing has revealed that mammalian cells contain at least 13 CDKs
and 29 cyclins (Liu and Kipreos, 2000; Malumbres and Barbacid, 2009). CDK1–
CDK6, CDK10, CDK11, and the CDK-activating kinase, CDK7, are all involved in
cell cycle control (Fisher, 2005; Loyer et al., 2005; Malumbres and Barbacid, 2009).
Of these, CDK1 is the most significant: genetic and molecular experiments show
that CDK1 is both necessary and sufficient to drive cell cycle progression in species
as diverse as yeast and humans (Nurse, 1990; Malumbres and Barbacid, 2009). To
some degree, the large number of human CDKs may reflect tissue-specific and fine-
tuned regulation of cell cycle regulation in metazoans. In addition, it has become
clear that some CDKs have activities that are distinct from cell cycle control. For
example, CDK7, CDK8, and CDK9 are all able to phosphorylate the carboxyl-
terminal domain (CTD) of RNA polymerase II and have functions in transcriptional
regulation (Fisher, 2005; Loyer et al., 2005; Phatnani and Greenleaf, 2006). CDK12
and CDK13 bind to L-type cyclins (CycL) and regulate alternative RNA splicing
(Chen et al., 2006; Chen et al., 2007).
Analysis of CDK function in early embryos and embryo extracts showed that
fluctuations in CDK activity are sufficient to regulate cell cycle transitions in the
absence of transcription. However, in most eukaryotic cells, cell cycle progression
is accompanied by dynamic changes in gene expression patterns. The recent analy-
sis of transcriptional profiles in yeast has shown that the transcription of more than
70% of periodically expressed genes continues to oscillate after B-type cyclins have
been removed (Orlando et al., 2008). Although these cells cannot complete DNA
replication or mitosis, some cell cycle events continue to occur in a periodic man-
ner (Haase and Reed, 1999). Computational analysis of the transcriptional network
shows that it has the properties of an oscillator (Orlando et al., 2008). These find-
ings suggest that cell cycle regulation results from the tightly integrated activities
of two oscillators (a CDK oscillator and a transcriptional oscillator) that fluctuate
in tandem. Transcriptional events that respond to changes in CDK activity, and the
periodic expression of CDK regulators, couple these oscillators together and ensure
that changes in CDK activity are coordinated with the appropriate programs of gene
expression.
The interwoven nature of the CDK and transcriptional oscillators is beauti-
fully illustrated by studies of the RB/E2F network. In mammalian cells, RB/E2F
proteins play a critical role in the control of the G1 to S transition. CDKs con-
tribute to both activation and inactivation of E2F-dependent transcription. Moreover,
E2F complexes control the expression of genes encoding CDKs, cyclins, and their
regulators. As described below, genetic studies have revealed a new player in
this network of interactions. Unexpectedly, the levels of CDK8 have a significant
impact on E2F transcriptional activity. Evidence that CDK8 is frequently amplified
2 Interplay Between Cyclin-Dependent Kinases and E2F-Dependent Transcription 25
and overexpressed in colorectal cancer cells suggests that CDK8 levels may be
particularly important in this type of cancer.
2.2 Rb and E2F Proteins Regulate the G1 to S-Phase Transition

in Higher Eukaryotes
The G1 to S-phase transition is a critical event for the control of cell prolifera-
tion. With few exceptions, once cells complete this transition they are committed
to progress through the remainder of the cycle and will divide into two daughter
cells. In higher eukaryotes, the retinoblastoma (RB) tumor suppressor protein and
E2F transcription factors play pivotal roles in the regulation of the G1 to S-phase
transition (reviewed by Dyson, 1998; Lipinski and Jacks, 1999; Müller and Helin,
2000; Zhu, 2005). The E2F transcription factor provides a temporal control over the
expression of hundreds of target genes whose products are necessary for accurate
DNA replication and mitosis (Müller et al., 2001; Ren et al., 2002; Bracken et al.,
2004). In non-dividing cells E2F proteins act together with RB-family proteins to
repress the transcription of these targets, helping to maintain the quiescent state
(G0) (reviewed by Classon and Dyson, 2001; Frolov and Dyson, 2004; Burkhart
and Sage, 2008). This repression must be relieved for cells to proliferate. In response
to the activation of G1 cyclins, E2F switches from a repressor to an activator, and
drives the expression of E2F target genes as cells leave G0/G1 and progress through
the cell cycle (Trimarchi and Lees, 2002; Bracken et al., 2004; Dimova and Dyson,
2005).
E2F was initially identified as a factor required for the activation of the E2
promoter of adenovirus (see Nevins, 1992 for a review) and it is the compos-
ite transcriptional activity of a group of proteins that share similar DNA-binding
domains. The basic unit of E2F is a heterodimer composed of an E2F and a DP
subunit (Dyson, 1998). Eight E2F genes and three DP genes have been identified in
mammals (reviewed in Dimova and Dyson, 2005; DeGregori and Johnson, 2006).
E2F1–E2F6 bind to DNA in association with a DP subunit. Structural studies show
that both DP and E2F subunits contain DNA-binding domains (Zheng et al., 1999)
and E2F/DP dimerization is essential for high-affinity DNA binding and transcrip-
tional activity (reviewed in Dyson, 1998). E2F proteins are often subdivided into
groups that primarily activate (E2F1, E2F2, and E2F3a) or repress transcription
(E2F3b, E2F4, E2F5, E2F6, E2F7, and E2F8) (reviewed in Dimova and Dyson,
2005; DeGregori and Johnson, 2006). Repressor E2Fs are relatively abundant and
broadly expressed, while activator E2Fs are potent and their activities are under
tight control (Trimarchi and Lees, 2002; Attwooll et al., 2004). E2F7 and E2F8
act without a DP subunit and, like E2F6, are thought to repress transcription with-
out directly binding to RB family proteins (Maiti et al., 2005; Milton et al., 2006).
In contrast, the transcriptional properties of E2F1–E2F5 are controlled by a direct
physical interaction with the RB family members. Activator E2Fs have the ability
to potently reverse the effects of repressor E2Fs at target promoters.
Figure 2.1 summarizes the general properties of the RB-E2F regulatory network.
In early G1 phase of the cell cycle, RB family proteins (pRB, p107, and p130) bind
to E2F family members and recruit co-repressor complexes, thereby repressing E2F-
dependent transcription (Fig. 2.1a). Upon growth factor stimulation, CDK–cyclin
(Cyc) complexes, such as CDK4/6–CycD, phosphorylate pRB and partially relieve
its repressive activity. This allows the expression of CycE, which binds to and acti-
vates CDK2. CDK2–CycE further phosphorylates pRB, resulting in its complete
inactivation (Fig. 2.1b). Hyperphosphorylated pRB can no longer bind to E2F, effec-
tively allowing E2F transcription factors to activate transcription (Dyson, 1998;
Nevins, 1998; Müller and Helin, 2000; Fig. 2.1c). Some of the best-known tran-
scriptional targets for E2F are components or regulators of CDKs, such as Cyclin A2
(CCNA2; Schulze et al., 1995; DeGregori et al., 1995; Shan et al., 1996; Ren et al.,
2002), Cyclin B1 (CCNB1; Zhu et al., 2004), Cyclin D1 (CCND1; Lee et al., 2000),
A G1 phase
Co-repressor
complexes target gene
pRB expression
OFF
E2F DP
CDK2-CycE
CDK2-CycA
B
growth factor cyclin A
Stimulations CDK4-CycD pRB E2F-DP cyclin E G1 to S-phase
(eg., EGF) cdc25 transition and
cdk1 mitosis
……
C S phase PCNA
p p p expression of
TK
target genes
pRB ORC DNA replication
target gene MCM
expression ……
ON Apaf1
E2F DP
p73 Apoptosis
……
Fig. 2.1 A summary of the interplay between RB/E2F and CDKs during the G1 to S-phase
transition. (a) During G1 phase, pRB blocks the transcriptional activity of E2Fs by directly
binding to the transactivation domain of activator E2Fs and/or by recruiting transcriptional co-
repressor complexes to target promoters. (b) Growth factor stimulation leads to activation of G1
CDKs, the phosphorylation of pRB (and p107 and p130), the disruption of repressor complexes,
and increased transcription. E2F targets include genes with key functions in cell cycle progres-
sion, DNA replication, and genes that can potentially sensitize cells toward apoptosis. Note that
increased transcription of CycE triggers a feedback loop that promotes the phosphorylation of pRB.
E2F-induction of CycA also increases pRB phosphorylation but CycA-associated kinases can also
phosphorylate E2F1 and down-regulate its activity. (c) In early S-phase, the phosphorylation of
pRB-family members by CDK4–CycD, CDK2–CycE, and CDK2–CycA results in the complete
release of repressor complexes allowing activator E2Fs to drive gene expression. For simplicity,
we have used pRB to refer the three pRB family proteins; the specific and overlapping functions of
pRB family proteins are reviewed by Classon and Dyson (2001) and Burkhart and Sage (2008)
Cyclin D3 (CCND3; Müller et al., 2001), Cyclin E1/2 (CCNE1/2; DeGregori et al.,
1995; Ohtani et al., 1995; Botz et al., 1996; Geng et al., 1996; Shan et al., 1996),
Cyclin G2 (CCNG2; Müller et al., 2001), CDK1 (CDC2; DeGregori et al., 1995;
Shimizu et al., 1995; Tommasi and Pfeifer, 1995; Ren et al., 2002; Zhu et al., 2004),
CDK2 (Shan et al., 1996; Ren et al., 2002), and CDC25A (Vigo et al., 1999; Ren
et al., 2002).
The importance of the RB/E2F network for normal control of cell proliferation
is highlighted by evidence that this regulation is inactivated in most types of tumor
cells (Weinberg, 1995; Dyer and Bremner, 2005; Tsantoulis and Gorgoulis, 2005;
Burkhart and Sage, 2008). Mutation of both copies of the RB gene is rate-limiting
for the development of both familial and sporadic retinoblastoma. The RB tumor
suppressor is also mutated at a high frequency in small cell lung carcinomas and
osteosarcoma (Weinberg, 1995; Dyer and Bremner, 2005). In other cancers, a vari-
ety of alternative events have been shown to functionally inactivate pRB. These
include (a) the loss of the CDK inhibitor p16INK4a by deletion, point mutation, or
promoter methylation, (b) the over-expression of CycD1 by gene rearrangement
or amplification, (c) gain-of-function mutations in genes encoding either CDK4 or
CycD1, (d) the expression of viral oncoproteins, (e) amplification of the E2F3 gene,
and (f) additional changes resulting in increased CDK2–CycE activity (Weinberg,
1995; Sherr, 1996; Nevins, 2001; Johnson and Degregori, 2006). All of these events
have a net effect of deregulating E2F-dependent transcription, generating a cellular
environment that is permissive for cell proliferation.
2.3 CDK Phosphorylation Is One of Several Mechanisms

That Regulate E2F Activity
The levels and activity of E2Fs are regulated at multiple levels. In general, activator
E2Fs are not highly expressed in quiescent cells but are transcribed in response to
growth factor stimulation. The E2F1 promoter contains E2F-binding sites (Johnson
et al., 1993; Hsiao et al., 1994) and this positive-feedback loop helps to amplify
the levels of activator E2Fs. E2F1–E2F3 are constitutively localized in the nucleus
because they contain a nuclear localization signal (NLS). This signal is absent in
E2F4 (Verona et al., 1997) and E2F4-containing complexes have been observed
to translocate from cytoplasm to nucleus during the transition from G0–G1 and
S phases (Lindeman et al., 1997; Verona et al., 1997).
E2F proteins are subject to a variety of post-translational modifications (sum-
marized in Fig. 2.2). E2F1 is activated by acetylation in response to DNA damage,
a change that appears to stabilize the protein (Martínez-Balbás et al., 2000; Ianari
et al., 2004) and this modification may also help to direct E2F1 to specific sub-
set of target genes (Pediconi et al., 2003). E2F1 is also phosphorylated by kinases
that regulate DNA damage responses, such as Chk2 (Checkpoint kinase 2) and
ATM (ataxia-telangiectasia mutated)/ATR (ATM and Rad3 related) (Fig. 2.2). Chk2
modifies E2F1 at Ser364, which stabilizes E2F1 (Stevens et al., 2003). Similarly,
CDK1/2-CycA
CBP/ P/CAF
ATM/ATR Chk2 CDK7-CycH
Ser375
Lys120
Ser31 Lys117 Lys125 Ser364 Ser403 Thr433
1 84 100 120 191 206 283 380 409 426 437
CycA binding DNA binding dimerization pRB

transactivation
(aa84~96) domain with Dp binding
domain (TAD)
HCF binding
(aa97~100) CDK8-CycC
binding
p14ARF binding;
CBP/ P/CAF binding
Fig. 2.2 The structure and regulation of the human E2F1 protein. Binding sites and functional
domains that have been mapped within the 437 amino acid residues of E2F1 are shown. Mapped
sites of lysine acetylation (Lys117, Lys120, and Lys125) and serine/threonine phosphorylation are
indicated. See text for details and references
ATM/ATR was shown to phosphorylate E2F1 at Ser31, a site that is not conserved in
E2F2 or E2F3, during the DNA damage response (Lin et al., 2001). Phosphorylation
of E2F1 at Ser31 also increases its stability (Lin et al., 2001). These DNA damage-
induced modifications are thought to enhance the transcriptional activity of E2F1
and to promote apoptosis through the selective activation of key target genes, such
as Apaf1 (Moroni, et al., 2001) and p73 (Urist et al., 2004).
Other post-translational modifications are thought to suppress E2F activity.
CDK7–CycH, which is the kinase component of the general transcription factor
TFIIH complex, can phosphorylate E2F1 at Ser403 and Thr433. Both of these
sites are located within the TAD (transactivating domain) of E2F1 (Vandel and
Kouzarides, 1999). Phosphorylation of E2F1 on these two sites promotes its degra-
dation, as mutation of these residues to Ala significantly increased the stability of
E2F1-TAD (Vandel and Kouzarides, 1999).
Several mammalian CDK–cyclin complexes have been reported to directly
phosphorylate E2F1 or its dimerization partner DP1, including CDK1/2–CycA
(Dynlacht et al., 1994; Krek et al., 1994; Xu et al., 1994; Krek et al., 1995; Dynlacht
et al., 1997), CDK1/2–CycB (Dynlacht et al., 1997), CDK7–CycH (Vandel and
Kouzarides, 1999), and CDK8–CycC (Morris et al., 2008). These studies paint a
complicated picture because CDK–cyclin complexes target different sites on E2F1
(Fig. 2.2) and these modifications can have different effects on E2F activity. For
example, phosphorylation of Ser375 of E2F1 by CDK1–CycA has been reported to
promote the binding of E2F1 to pRB (Peeper et al., 1995), whereas the more gen-
eral phosphorylation of E2F1 and/or DP1 by CDK2–CycA disrupts the formation of
E2F1–DP1 heterodimers and reduces their DNA-binding properties (Dynlacht et al.,

1994; Krek et al., 1994; Kitagawa et al., 1995). CycA uses a hydrophobic patch to
bind directly to an RXL motif at the N-terminus of the mammalian activator E2Fs in
S-phase (Schulman et al., 1998) and E2F1 has been reported to be phosphorylated
on at least 6 sites in vivo and 7–9 sites in vitro by CDK2–CycA (Xu et al., 1994).
Because different CDKs have similar substrate specificities, it is unclear which
kinases are responsible for phosphorylating E2F1 or DP1 in vivo. Biochemical stud-
ies show that E2F1 and DP1 each contain at least one site that is phosphorylated by
CDK2–CycA but not by CDK2–CycE (Dynlacht et al., 1994) and these results have
suggested a model in which the different substrate specificities of CDK2–CycE and
CDK2–CycA kinases generate a specific period of E2F1 activity: with E2F1 being
activated late in G1 by the phosphorylation of RB-family members by CDK2–CycE
and inactivated in S-phase by CDK2–CycA (Fig. 2.1b).
The levels of activator E2Fs are limited by proteasome-dependent degradation
(Hateboer et al., 1996; Hofmann et al., 1996; Campanero and Flemington, 1997;
Marti et al., 1999). E2Fs have been proposed to be targeted for degradation by
SCFSkp2 complex in mammals (Marti et al., 1999) or by SCFslmb in Drosophila
(Hériché et al., 2003). Degradation of human E2F1 requires the Cul1-E3 ubiquitin
ligase (Marti et al., 1999; Ohta and Xiong, 2001). More recent studies in Drosophila
have shown that the abrupt degradation of dE2F1 that occurs when cells enter S-
phase is mediated by the Cul4Ctd2 E3 ubiquitin ligase and requires PCNA and a PIP
(PCNA-interacting protein) motif located near the N terminus of dE2F1 (Shibutani
et al., 2008). No PIP boxes have been identified in mammalian activator E2Fs,
suggesting that different species may have evolved different mechanisms for the
ubiquitylation of activator E2Fs.
2.4 How Do E2Fs Activate Transcription?

When activator E2Fs accumulate in late G1 and are released from any inhibitory
effects of pRB-family members, they are potent activators of transcription, but pre-
cisely how they exert this effect is unclear. E2F proteins have been reported to
interact with the general transcription machinery, with various transcription factors,
and with proteins that change chromatin structure (Fig. 2.3a). However, at present
it is unclear which of these interactions are the most important for the biological
functions of E2F in vivo.
E2F1 has been shown to physically interact with TATA-box binding protein
(TBP) (Hagemeier et al., 1993; Emili and Ingles, 1995; Pearson and Greenblatt,
1997), a subunit of TFIID complex (Hahn, 2004). TBP can recruit general tran-
scription factors, such as TFIIA and TFIIB, to DNA. pRB has been shown to
repress E2F-dependent transcription in chromatin-free in vitro transcription assays
by blocking recruitment of TFIIA and TFIID, thereby preventing formation of a pre-
initiation complex (PIC) (Ross et al., 1999). Whether E2F1 directly binds to TBP
in vivo is uncertain and many E2F-regulated promoters are TATA-less promoters
A
Co-activators Functions
TBP (TFIID) recruiting and PIC assembly
P300 or P/CAF histone and E2F1 acetylation
Tip60 histone acetylation
HCF recruiting HCF-containing HMTases
Mediator recruiting general transcription factors
B modification of
histone tails
Co-activator
complexes chromatin in
E2F DP open configuration
C
small Mediator
complex target gene
expression
RNA ON
E2F DP GTFs Pol II
D small Mediator
CDK8 complex
sub-module
target gene
p expression
E2F DP RNA OFF
GTFs Pol II
Fig. 2.3 A model for E2F1-mediated transcriptional activation. (a) Transcriptional co-activators
that have been linked to E2F1 are listed. Please see text for references. (b–d) Shows a model
for E2F1 regulation that is suggested by the discovery of an interaction between E2F1 and the
CDK8 module. After phosphorylation and release of pRB, E2F–DP complexes recruit transcrip-
tional co-activators that modify histone tails and render the chromatin in open configuration (b).
P/CAF-mediated acetylation of E2F1 may stabilize E2F1 and enhance DNA binding of E2F1–DP1
complexes. E2F1–DP recruitment of small mediator complex, general transcriptional factors, and
RNA polymerase II activates the expression of E2F target genes (c). Recruitment of the CDK8
sub-module blocks the re-initiation of E2F-dependent transcription, possibly by CDK8-mediated
phosphorylation of E2F1 and disruption of E2F1–DP heterodimers (d)
that do not require TBP (Majello et al., 1998). E2F1 has also been shown to directly
interact with p62 subunit of the TFIIH both in vitro (Pearson and Greenblatt, 1997)
and in vivo (Vandel and Kouzarides, 1999). TFIIH consists of 10 subunits that form
a core complex and a CAK (Cdk-activating kinase) complex, which is composed
of CDK7, CycH, and an assembly subunit MAT1 (reviewed in Hahn, 2004; Fisher,
2005).
A different set of studies have described physical interactions between E2F1 and
histone acetyl transferases (HATs). Examples include p300/CBP (CREB (cAMP-
response element-binding) protein-binding protein) (Fry et al., 1999), PCAF
(p300/CBP-associated factor) (Lang et al., 2001), and the Tip60 complex (Taubert
et al., 2004). These transcriptional cofactor complexes are best known to acety-
late histone tails, presumably allowing E2Fs to promote chromatin with an open
conformation (Fig. 2.3a, b).
E2F1, E2F3a, and E2F4 have also been shown to directly bind to the transcrip-
tion cofactor HCF1 (Host Cell Factor-1) (Luciano and Wilson, 2003; Knez et al.,
2006; Tyagi et al., 2007). HCF1 enhances transcription by recruiting co-activator
complexes, such as MLL (mixed-lineage leukemia) and hSet1 histone H3 lysine 4
(K4) methyltransferases (HMTs) (Tyagi et al., 2007) and H4K16 HAT MOF (Dou
et al., 2005; Smith et al., 2005). Methylation of histone H3K4 at the promoter
regions often positively correlates with active transcription (Barski et al., 2007).
Interestingly, HCF1 can also associate with a transcriptional co-repressor complex
that contains mSin3A/B and HDAC1/2 (Tyagi et al., 2007; Wysocka et al., 2003),
and this property may account for the repressive effect of E2F4 and hypoacety-
lation of E2F responsive promoters during early G1 phase (Tyagi et al., 2007;
Wilson, 2007). A transition from E2F1-bound pRB-mediated or E2F4-mediated
repressive complexes during early G1 to E2F1-bound HCF1-mediated MLL family
HMTs has been proposed to facilitate the activation of E2F target genes required for
proliferation (Tyagi et al., 2007).
Despite these extensive studies, there are still many unanswered questions. It
is unclear, for example, which of these potential mechanisms of E2F regulation
are rate-limiting at key E2F targets. It is possible that the availability of a particu-
lar co-activator may be regulated in vivo to limit E2F-dependent cell proliferation.
Alternatively, given that E2F proteins can interact with several different cofactors, it
is possible that no single co-activator is essential for E2F to drive cell proliferation.
2.5 Drosophila as a Model System to Study E2F Activity In Vivo
The studies described above illustrate a complicated pattern of interactions between

E2F proteins and CDK activity. In different contexts E2F complexes can either
repress or activate the expression of genes encoding cyclins and CDKs, which can
either activate or inactivate the transcriptional activity of E2Fs at various points in
the cell cycle. Understanding the relationship between these groups of proteins is
a difficult problem and one of the limitations of our current models is that they are
largely based on biochemical studies and on experiments in which mutant proteins
have been over-expressed.
Genetic studies in animal models provide a valuable complement to these molec-
ular analyses, helping to identify components of the regulatory network that have
the greatest impact in vivo. Drosophila has proven to be a powerful model sys-
tem for studies of E2F. The Drosophila E2F network is less complex than the
mammalian E2F family, containing just two E2F homologs, dE2F1 (activator), and
dE2F2 (repressor). These two E2Fs provide functions that are analogous to those
provided by the mammalian E2Fs (Stevaux and Dyson, 2002; Trimarchi and Lees,
2002). The streamlined nature of the Drosophila E2F/RBF families has enabled
clear insights into the organization and action of this complex regulatory network
(for a recent review, see van den Heuvel and Dyson, 2008).
One drawback of the Drosophila E2F/RBF network is that dE2f1 homozygous
mutant animals die early in development (Duronio et al., 1995) and this early lethal-
ity represents an obstacle to the analysis of E2F function in the context of animal
development. Although one can produce mutant clones using mitotic recombina-
tion, such clones are small and are generated randomly thus difficult to follow. This
makes the large-scale genetic analysis of mutant clones labor-intensive and unfea-
sible. This hurdle was circumvented in recent studies by the use of transgenic lines
that use RNA interference (RNAi) to selectively target dE2F1 in tissues that are
not needed for animal viability (Morris et al., 2008). Lowering the levels of the
endogenous dE2F1 protein reduces transcription from dE2F1-dependent promoters
and impairs cell proliferation. These changes give visible and reproducible pheno-
types in adult eyes and wings that can be modified by additional genetic changes
(Morris et al., 2008).
dE2f1-RNAi-induced phenotypes have been used to screen mutant collections
and the results provide the first glimpse of the spectrum of genes that are rate-
limiting for E2F1-dependent cell proliferation in vivo. Unexpectedly, one of the
strongest interactions identified in more than 200 dominant modifiers was provided
by an allele of cdk8 (Morris et al., 2008). Mutation of cdk8 strongly suppressed
dE2f1-RNAi phenotypes in both the eye and the wing. Experiments using cultured
Drosophila S2 cells and cdk8 mutant embryos indicate that CDK8 negatively reg-
ulates dE2F1-dependent transcription (Morris et al., 2008). In similar genetic tests,
mutant alleles of cycA, cycB, cycE, and cdk2 all weakly enhanced dE2f1-RNAi
phenotypes, suggesting that they functionally co-operate with dE2F1 (unpublished
observations). Such genetic experiments suggest that, of the CDKs and cyclins
tested, CDK8 is the clearest negative regulator of E2F1 in vivo.
2.6 CDK8–Cyclin C Negatively Regulates E2F1-Dependent

Transcription
CDK8 and its regulatory cyclin partner (cyclin C) are subunits of the CDK8 module
of the Mediator complex, a transcriptional cofactor that acts as an interface between
specific transcription factors and the basal RNA polymerase II (Pol II) transcription
apparatus in yeast and human cells (reviewed by Boube et al., 2002; Conaway et al.,
2005; Kornberg, 2005; Malik and Roeder, 2005; Myers and Kornberg, 2000; Taatjes
et al., 2004; Woychik and Hampsey, 2002).
Biochemical fractionation experiments indicate that the 30 or more mediator sub-

units form two distinct types of complexes: a small mediator complex (also known
as CRSP, or Positive Cofactor 2, PC2) that activates transcription of Pol II-dependent
genes (Malik and Roeder, 2005; Myers and Kornberg, 2000; Näär et al., 2001), and
a large mediator complex that generally represses transcription (Mittler et al., 2001).
The large mediator complex contains all but one (CRSP70/MED26) of the subunits
of the small mediator complex, but also has the CDK8 module, which contains
CDK8, CycC, MED12, and MED13 (Näär et al., 2002; Taatjes et al., 2002; Malik
et al., 2004).
Current models of mediator function suggest that transcription factors bind to
promoter sequences and recruit the small mediator complex. This complex recruits
Pol II and promotes the initiation of transcription (Malik and Roeder, 2005). With
the release of Pol II, many of the general transcription factors are left behind. This
“scaffold complex” contains transcription activator, TFIIA, TFIID, TFIIE, TFIIH,
and the mediator complex (Yudkovsky et al., 2000) and can potentially recruit free
RNA Pol II, TFIIB, and TFIIF, and thereby allowing multiple rounds of transcription
reinitiation to occur. Such reinitiation occurs more efficiently than the first round of
transcription (Yudkovsky et al., 2000).
In general, association of the CDK8 module with the small mediator complex
negatively regulates transcription (Malik and Roeder, 2005). Precisely how the sup-
pression occurs is uncertain, and at least four mechanisms have been proposed:
(a) CDK8 can phosphorylate the RNA Pol II CTD (on Ser5) and Med2 and dis-
rupt the interaction between RNA Pol II and the mediator complex (Hengartner
et al., 1998; Hallberg et al., 2004; Liu et al., 2004; van de Peppel et al., 2005);
(b) CDK8 can phosphorylate the CycH subunit of TFIIH, suppressing the CTD
kinase activity of CDK7–CycH, effectively preventing the initiation of transcrip-
tion (Akoulitchev et al., 2000); (c) only the small mediator complex (lacking the
CDK8 module) interacts with Pol II and forms a holoenzyme complex, suggest-
ing that the CDK8 submodule may serve as a physical barrier between the small
mediator complex and RNA Pol II general transcriptional machinery (Näär et al.,
2002; Samuelsen et al., 2003; Bjorklund and Gustafsson, 2005; van de Peppel et al.,
2005; Elmlund et al., 2006; Knuesel et al., 2009b); and (d) CDK8 has been shown
to directly phosphorylate and inactivate several transcription activators. For exam-
ple, studies in mammalian cells have shown that CDK8 complex, when recruited by
Mastermind, promotes the phosphorylation and ubiquitin-mediated destruction of
Notch intracellular domain (Fryer et al., 2004). Phosphorylation by SRB10 (a bud-
ding yeast homolog of CDK8) promotes the nuclear export of MSN2 (Chi et al.,
2001) or triggers the degradation of GCN4 (Chi et al., 2001). SRB10 has been shown
to directly interact with the activating domains of several different transcriptional
activators, potentially explaining why activator phosphorylation is often concurrent
with transcriptional activation (Ansari et al., 2002; reviewed in Tansey, 2001).
It is worth noting that, in some cases, CDK8/SRB10-mediated phosphorylation
activates, rather than represses, transcription, such as the cases for Gal4 (Hirst et al.,
1999) and Sip4 (Vincent et al., 2001) in yeast. In addition, CDK8 positively reg-
ulates p53-mediated transcription of p21 in mammalian cells treated with Nutlin3
(Donner et al., 2007), a selective small molecule antagonist of MDM2 (Vassilev,

2007). Moreover, CDK8-mediated phosphorylation of Ser10 on histone H3 may
also potentiate transcription (Knuesel et al., 2009a).
These mediator studies provide potential explanations for the genetic interaction
between CDK8 and E2F1 (Fig. 2.3c, d). The fact that CDK8 is a negative regulator
of E2F1 suggests that the small mediator complex functions as a co-activator at some
dE2F1-dependent genes (Fig. 2.3c). As has been observed in other systems, the
CDK8 module may suppress re-initiation and thus limit the burst of transcriptional
activation generated by promoter-bound dE2F1. In keeping with this, the effects of
depleting dE2F1 were partially suppressed by reduction of CDK8, CycC, MED12,
or MED13 (Morris et al., 2008).
There are several lines of evidence to suggest that E2F proteins may also be
substrates for the CDK8 kinase. Experiments using cultured fly and human cells
show that CDK8 associates with dE2F1/E2F1. Moreover, the E2F1 associated with
CDK8 becomes phosphorylated when CDK8 immunoprecipitates are incubated in
a kinase buffer (Morris et al., 2008). Although CDK2–CycA has been shown to
phosphorylate and inhibit the DNA-binding activity of E2F1–DP1 heterodimers, it
is conceivable that this mechanism of regulation is normally carried out by CDK8–
CycC (Fig. 2.3d). Such a model could explain why dE2F1/E2F1 is stable when
bound to RBF1/pRB but is rapidly degraded when it is able to activate transcription.
The fact that mutant alleles of cdk8 suppress dE2F1-RNAi phenotypes, while cycA
and cdk2 alleles do not, is consistent with the idea that CDK8 down-regulates dE2F1
in vivo. However, we note that genetic interactions between cycA and dE2f1 may be
harder to detect if CycA-associated kinases have both positive and negative effects
on dE2F1 activity. Future experiments that map the specific residues of E2F1/DP1
that are phosphorylated by CDK8 may help to clarify this issue. The recruitment of a
CDK to promoters is the salient feature of current models for CDK8 action, and it is
also possible that CDK8–mediated repression of E2F1 may involve other proteins.
2.7 Deregulation of CDK8–CycC in Human Cancers

Consistent with the idea that the levels of CDK8–CycC can have a significant impact
on cell proliferation, the CDK8 and CCNC (encoding CycC) genes are ampli-
fied, mutated, or deleted in various types of human cancers. For example, a point
mutation of CDK8 (D189N) has been found in diverse tumor samples (Greenman
et al., 2007), but the functional consequence of this mutation is still unknown. The
CCNC gene is frequently deleted in acute lymphoblastic leukemia (Li et al., 1996),
osteosarcoma (Ohata et al., 2006), and gastric cancer patients (Yang et al., 2007), but
is highly expressed in colorectal adenocarcinoma, leukemia, and lymphoma cells
(Su et al., 2004).
The variety of changes seen in tumor cells may reflect the fact that the medi-
ator complex regulates a vast number of genes, and the role or importance of
CDK8–CycC will vary depending on the identity of the transcription factors that
drive tumor cell proliferation. A striking example of such context-specific effects

is provided by the recent finding that CDK8 is an oncoprotein in colorectal can-
cers (Firestein et al., 2008). CDK8 was identified in a shRNA screen for genes that
promote β-catenin-driven transcription and are needed for proliferation of colorectal
tumor cell lines. Subsequent experiments demonstrated that the chromosomal region
harboring CDK8 gene shows copy number gains in about 47% of colon cancers.
Over-expression of wild-type CDK8, but not the kinase dead mutant, was found to
promote anchorage-independent growth in untransformed 3T3 cells (Firestein et al.,
2008). In an unexpected convergence of events, E2F1 was found to suppress the
transcriptional activity of β-catenin (Morris et al., 2008). CDK8 and RB1 both reside
on chromosome 13q and copy gains of both genes are found in many colon cancer
patients (Gope et al., 1990; Firestein et al., 2008). The amplification of CDK8 seems
to be particularly advantageous for colorectal cancers. For reasons that are not yet
clear, the components of the CDK8 module enhance β-catenin-driven transcription,
and increased levels of CDK8 helps to promote β-catenin activity in colon cancer
cells. In addition, elevated CDK8 prevents E2F1 from inhibiting β-catenin. In this
cell type, CDK8 appears to co-operate with pRB to sustain β-catenin activity, per-
haps explaining why the RB1 gene is never lost, and is occasionally overexpressed,
in colorectal cancers (Morris et al., 2008).
2.8 Conclusions and Future Directions
The identification of CDK8–CycC as a negative regulator of dE2F1 illustrates the

value of unbiased genetic screens. Because of the relative ease and low cost of
genetic screens and phenotypic analyses, studies in model systems like Drosophila
and Caenorhabditis elegans provide a powerful complement to the molecular and
biochemical analyses that traditionally have been carried out in mammalian cells.
Genetic screens identify components that have a significant functional impact on a
phenotype regardless of the underlying mechanism. Such studies have the advan-
tage that they can discover important and unsuspected connections between genes
and processes. Genetic interactions are, however, only a starting point and need to
be complemented by biochemical studies that characterize the underlying molecular
events.
The genetic interaction between CDK8–CycC and E2F1 raises a new series of
questions for future studies. This interaction suggests that the small mediator com-
plex plays an important role in E2F-mediated activation. It is uncertain, however,
whether the mediator complex acts at all E2F targets or just a subset. Do activa-
tor E2Fs interact with a particular component of the mediator complex, and does
E2F1 recruit mediator, or does mediator recruit E2F1? A second issue is the signifi-
cance of CDK8–CycC phosphorylation of E2F1. The genetic data and the evidence
that CDK8–CycC can phosphorylate E2F1 raise the possibility that CDK8–CycC
may be the key CDK activity that is responsible for the attenuation of activator
E2Fs. Further work is clearly necessary to determine which of the many CDKs that
can phosphorylate E2F1/DP1 dimers are actually responsible for this regulation in
vivo. Answering this question will provide a better understanding of how the E2F
transcriptional program is switched on and off, and how it can be coupled to, or
uncoupled from, the CDK cycle.
In different contexts, deregulated E2F1 can promote cell proliferation or induce
cell death. Knowing how to tip the balance between these outcomes toward cell
death may have many applications in the treatment of cancer cells. The fact
that CDK8 is an oncoprotein in colorectal cancers has raised the possibility that
CDK8 inhibitors might have utility in cancer cells that harbor CDK8 amplifica-
tion (Firestein et al., 2008). Since CDK8 is also an inhibitor of E2F1, CDK8
antagonists may generally enhance E2F1 activity. In combination with appropriate
pro-apoptotic stimuli, CDK8 inhibitors may be useful for stimulating E2F1-induced
apoptosis in many different types of cancer cells.
Acknowledgments We thank Drs. Erick Morris, Gerold Schubiger, and Fajun Yang for critical
comments on this review. J.Y.J. is supported by a post-doctoral fellowship from the MGH Fund for
Medical Discovery. This work was supported by a grant from the NIH (RO1 GM53203). N.J.D. is
the MGH Saltonstall Foundation Scholar.
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Chapter 3
Regulation of Pre-RC Assembly: A Complex
Symphony Orchestrated by CDKs
A. Kathleen McClendon, Jeffry L. Dean, and Erik S. Knudsen
Abstract DNA replication is a tightly regulated process that has critical impli-
cations for human cancers. Pre-replication (pre-RC) assembly is required for
initiation of DNA replication, and virtually all components of the pre-RC are regu-
lated by complex mechanisms that center around the activity of cyclin-dependent
kinases (CDKs). CDK/cyclin complexes both positively and negatively regulate
pre-RC components, including their expression, activity, stabilization, and degra-
dation. Together, these complex mechanisms orchestrate DNA replication and cell
cycle progression in a manner by which genetic material is duplicated only once.
Deregulated pre-RC activity has been shown to result in DNA re-replication and
genomic instability, a hallmark of cancer. Furthermore, deregulation of pre-RC
components and CDK/cyclins is observed in a multitude of human cancers. Thus,
regulation of pre-RC assembly is a critical facet of normal cell biology that has
profound implications related to cancer etiology and diagnosis.
3.1 The Pre-replication Complex
DNA replication is a tightly regulated process that requires coordinated assembly of

protein complexes at distinct periods of the cell cycle. Sites of DNA replication are
determined by binding of origin replication complexes (ORCs) (Bell and Stillman,
1992; Bell and Dutta, 2002; Chesnokov, 2007). The ORC is conserved from yeast to
humans and is believed to remain bound to DNA throughout the cell cycle (Bell and
Dutta, 2002; Chesnokov, 2007). At the end of mitosis, replication control mecha-
nisms are “reset” when Cdc6 and Cdt1 are recruited to ORCs (Bell and Dutta, 2002;
Liang et al., 1995; Coleman et al., 1996; Cocker et al., 1996; Nishitani et al., 2000;
Maiorano et al., 2000). Binding of Cdc6 and Cdt1 to ORCs results in the subsequent
E.S. Knudsen (B)

Department of Cancer Biology, Kimmel Cancer Center, Thomas Jefferson University,
Philadelphia, PA 19107, USA
e-mail: eknudsen@kimmelcancercenter.org
44 A.K. McClendon et al.
recruitment of the mini-chromosome maintenance (Mcm) complex (Mcm2-7) dur-

ing late M-phase and G1 (Bell and Dutta, 2002; Tye, 1999; Maiorano et al., 2006).
Recruitment of Mcm2-7 completes the “pre-replication complex” (pre-RC), the crit-
ical substrate upon which replication initiates. Formation of the pre-RC is essential
for replication initiation and ultimately progression into S phase (Bell and Dutta,
2002; Lau et al., 2007). At the beginning of S phase, the pre-RC is converted to
the initiation complex (IC) through the recruitment of factors such as cdc45 and
Mcm10 (Bell and Dutta, 2002; Maiorano et al., 2006; Merchant et al., 1997; Mimura
and Takisawa, 1998; Sawyer et al., 2004). IC formation results in the subsequent
recruitment of components of the “replisome” (RFC, RPA, and PCNA), the active
complex responsible for DNA replication (Waga and Stillman, 1998; Diffley and
Labib, 2002; Mendez and Stillman, 2003).
While the formation of all protein complexes leading to DNA replication is
highly controlled, regulation of pre-RC assembly for replication licensing is par-
ticularly crucial for maintaining genome integrity. Multiple ORCs form along the
genome to ensure efficient replication of DNA at each cell division (Bell and Dutta,
2002; Chesnokov, 2007; DePamphilis, 2005). Additionally, pre-RC formation is
tightly regulated to ensure that each of these regions is replicated only once (Bell
and Dutta, 2002; Lau et al., 2007). Alterations in the control of replication licensing
can lead to loss or duplication of genetic material, predisposing cells to mutations
and chromosomal instability, factors that have been implicated in promoting tumori-
genesis (Lau et al., 2007; Storchova and Pellman, 2004; Pellman, 2007). A crucial
mechanism by which cells regulate pre-RC formation and insure faithful replication
of the genome is manifested via the complex actions of cyclin-dependent kinases
(CDKs) (Malumbres and Barbacid, 2009; Doonan and Kitsios, 2009).
3.2 Cyclin-Dependent Kinases (CDKs) and General Cell Cycle

Control
Cyclin-dependent kinases (CDKs) are serine/threonine kinases that play critical

roles in cell cycle progression (Malumbres and Barbacid, 2009; Doonan and Kitsios,
2009). As eukaryotic cells progress through the cell cycle, CDK activity is stimu-
lated via association with cyclins. Mammalian cells utilize CDK1, CDK2, CDK4,
and CDK6 for cell cycle progression (Malumbres and Barbacid, 2009; Doonan and
Kitsios, 2009). CDK2, CDK4, and CDK6 are considered to be the interphase CDKs,
and while these proteins are required for proliferation of specific cell types, they are
not essential genes in mice (Malumbres et al., 2004; Kozar et al., 2004; Ortega et al.,
2003; Berthet et al., 2003; Barriere et al., 2007). Furthermore, while CDK2/4/6 have
been shown to act cooperatively to modulate cell cycle progression, some func-
tional redundancy has been indicated in both cells and mice lacking one or more of
the interphase CDKs (Malumbres and Barbacid, 2009; Doonan and Kitsios, 2009;
Malumbres et al., 2004; Kozar et al., 2004; Barriere et al., 2007; Sherr and Roberts,
2004). In contrast, CDK1 is involved in driving mitosis and is essential for cell
3 Regulation of Pre-RC Assembly 45
division (Santamaria et al., 2007). Each of these CDKs is tightly regulated, and
modulation of CDK activation is critical in determining progression versus arrest of
the cell cycle (Malumbres and Barbacid, 2009; Doonan and Kitsios, 2009).
The general model of cell cycle regulation by CDKs begins with mitogenic sig-
naling to stimulate accumulation of D-type cyclins, which form complexes with
and activate CDK4 and CDK6 at the onset of G1 (Malumbres and Barbacid,
2001). Cyclin D–CDK4/6 complexes function to phosphorylate and inactivate the
retinoblastoma tumor suppressor (RB) or related pocket proteins (p107 and p130)
(Mittnacht, 1998; Harbour et al., 1999). Inactivation of RB relieves repression of
the E2F family of transcription factors, allowing for the expression of genes essen-
tial for DNA replication and cell cycle progression (Cobrinik, 2005; Markey et al.,
2002). This initial step in cell cycle progression is antagonized by a family of
CDK inhibitors (CKIs) including the INK4 proteins (p16INK4a , p15INK4b , p18INK4c ,
and p19INK4d ). These CKIs specifically inhibit CDK4 and CDK6 by directly dis-
rupting association to D-type cyclins, ultimately promoting RB-dependent cell
cycle arrest (Sherr and Roberts, 1999; Roussel, 1999; Sherr and McCormick,
2002).
Alleviation of RB-mediated transcriptional repression allows for the expression
of genes such as E-type and A-type cyclins (Cobrinik, 2005; Markey et al., 2002).
E-type cyclins complex with and activate CDK2 to promote further phosphorylation
and complete inactivation of RB, thus allowing for expression of genes required for
driving DNA replication and the G1 to S phase transition (Malumbres and Barbacid,
2009; Doonan and Kitsios, 2009; Harbour et al., 1999; Hochegger et al., 2008).
Much like the negative regulation of cyclin D–CDK4/6 complexes by INK4 pro-
teins, cyclin–CDK2 complexes are inhibited by a second class of CKIs made up
of the Cip/Kip family of proteins (including p21Cip1 and p27Kip1 ). These CKIs
bind both CDKs and cyclins and inhibit the functions of both cyclin A- and cyclin
E-CDK2 complexes (Sherr and Roberts, 1999; Besson et al., 2008). Interestingly,
Cip/Kip proteins also function as assembly factors for cyclin D–CDK complex for-
mation. Inhibition of cyclin D/CDK4 by p16INK4a results in the displacement of
p27Kip1 , which allows for the inhibition of cyclin E/CDK by p27Kip1 and subsequent
G1 arrest (Sherr and Roberts, 1999; Besson et al., 2008).
Upon successful initiation and progression of DNA replication, cyclin B1–CDK1
complexes form to drive mitosis (Malumbres and Barbacid, 2009; Hochegger et al.,
2008; Malumbres and Barbacid, 2005). Final inactivation and degradation of cyclin
B1, as well as various other factors, via the anaphase-promoting complex (APC/C)
results in mitotic exit (Acquaviva and Pines, 2006; Peters, 2006). Furthermore,
degradation of cyclin B1 effectively eliminates CDK activity, ultimately allowing
for new origin licensing and progression of cells into the next cell cycle (Acquaviva
and Pines, 2006; Peters, 2006).
CDKs enforce cell cycle order via a complex array of protein interactions and
mechanisms, all of which dramatically impact pre-RC assembly and replication ini-
tiation. These mechanisms involve both positive regulation of pre-RC formation
in G1 phase of the cell cycle and negative regulation of pre-RCs to prevent re-
replication through S phase and mitosis (Malumbres and Barbacid, 2009; Doonan
A
Anti-Mitogen Mitogen
G0
CyclinD
Ink4 Cdk4/6
P
P RB Cdt1
Cdc6
RB
E2F
MCM
E2F
B
Geminin
APC/C
degradation G1
CyclinE
Licensing
Cdk2
6
3
7
1 2
5 4
CyclinE MCMs
P 6
Cdc6 Cdt1 7 3 2
1
ORC1-6 5 4
C
CyclinA/E
Replisome S
Cdk2
Activation
P
P P P Cdc45
Cdt1 6
Cdc6 3
7 2 MCM10
1
ORC1-6 5 4
D
Cdt1
G2/M
Geminin
ORC1 Cdc6
ORC2-6
Origin
Fig. 3.1 Complex regulation of pre-RC assembly by CDK/cyclins. (a) Mitogenic signaling stim-
ulates CDK4/6–cyclin D activity at the G0–G1 transition of the cell cycle. CDK4/6–cyclin D
complexes inactivate the retinoblastoma tumor suppressor (RB) to relieve repression of the E2F
family of transcription factors, allowing for the expression of genes essential for pre-RC assembly.
CDK4/6–cyclin D function is antagonized by the INK4 family of CDK inhibitors. (b) CDK2–
cyclin E complexes play dual roles in pre-RC regulation in G1. First, CDK2–cyclin E complexes
function to promote further inactivation of RB, thus allowing for expression of genes required for
driving DNA replication and the G1 to S phase transition. Second, CDK2–cyclin E complexes
phosphorylate and stabilize Cdc6. Interestingly, cyclin E also associates with Cdt1 and the Mcm
and Kitsios, 2009). Both aspects of CDK regulation are critical for maintaining
genomic stability and promoting faithful replication of the genome.
3.3 Positive Impact of CDKs on Pre-RC Assembly (G0–G1

Phase)
CDKs are involved in the regulation of pre-RC assembly from the onset of cell
cycle initiation (Fig. 3.1). Activation of the cyclin D–CDK4/6 complex is the first
step in exiting G0 in response to mitogenic signaling (Malumbres and Barbacid,
2001). This kinase complex functions to phosphorylate and partially inactivate the
RB tumor suppressor, allowing for the release of E2F transcription factors and
subsequent transcription of E2F target genes, including Cdc6, Cdt1, and Mcm2-7
(Mittnacht, 1998; Harbour et al., 1999; Cobrinik, 2005; Markey et al., 2002).
Transcription of Cdc6, Cdt1, and Mcm2-7, and the accumulation of these proteins
at ORCs, is required for formation of a functional pre-replication complex (Bell and
Dutta, 2002; Liang et al., 1995; Coleman et al., 1996; Cocker et al., 1996; Nishitani
et al., 2000; Maiorano et al., 2000; Lau et al., 2007). In contrast, activation of the RB
pathway and subsequent G1 arrest is characterized by transcriptional repression of
pre-RC components (Markey et al., 2002; Braden et al., 2006, 2008). Furthermore,
targeted inhibition of CDK4/6 results in the down-regulation of pre-RC components
(Braden et al., 2008). Thus, pre-RC assembly is initially regulated by the ability
of cyclin D–CDK4/6 to release transcriptional repression of pre-RC components
through inactivation of RB, an event that is considered critically important for cells
exiting quiescence.
Once pre-RC components have been transcribed and begin assembling at ORCs,
a second interphase cyclin–CDK complex, cyclin E–CDK2, is responsible for
facilitating and maintaining pre-RC formation for subsequent replication complex
assembly (Malumbres and Barbacid, 2009; Harbour et al., 1999; Lundberg and
Weinberg, 1998). Cyclin E–CDK2 complexes display some functional redundancy
with cyclin D–CDK4/6 complexes in their ability to phosphorylate and inactivate
RB, ultimately promoting transcription of S phase proteins that bind the pre-RC
and initiate DNA replication (Malumbres and Barbacid, 2009; Doonan and Kitsios,
2009; Harbour et al., 1999; Hochegger et al., 2008). Additionally, cyclin E–CDK2

Fig. 3.1 (continued) to promote pre-RC assembly independent of CDKs. Additionally, geminin is
degraded at the onset of G1 to allow Cdt1 association at chromatin. (c) Recruitment of Cdc45 and
Mcm10 in S phase promotes loading of the replisome and initiation of replication. At the onset of
DNA replication, CDK2–cyclin E/A complexes take on a negative regulatory role by phosphorylat-
ing pre-RC components, resulting in their dissociation from chromatin and subsequent degradation.
(d) As cells transition through the G2–M phases of the cell cycle, the chromatin has been stripped
of all pre-RC components. The cell is now “reset” in G0 for another round of cell cycle
has been shown to directly phosphorylate Cdc6, thereby preventing Cdc6 degra-
dation via the anaphase-promoting complex/cyclosome (APC/C), which remains
active until late G1 phase (Mailand and Diffley, 2005). This cyclin E–CDK2-
mediated stabilization is specific to Cdc6, as Cdt1 is not a target of the APC/C
(Mailand and Diffley, 2005; Fujita, 2006). Thus, stabilization of Cdc6 by cyclin
E–CDK2 ensures that the critical components of the pre-RC are not only assembled
for replication licensing, but are also maintained during G1 progression.
In addition to the kinase-dependent function of cyclin E, this interphase cyclin
functions to promote replication complex assembly independent of CDKs. Recent
studies have shown that cyclin E can interact with replication complex components,
Cdt1 and Mcm, to promote loading of Mcm proteins to ORCs (Geng et al., 2007).
Additionally, while CDK2 null fibroblasts were shown to undergo normal cell cycles
(Sherr and Roberts, 2004), cyclin E null cells fail to progress from quiescence to
S phase due to a defect in Mcm protein loading (Geng et al., 2003). A cyclin E
mutant that fails to activate CDKs displayed the same interactions with pre-RCs as
the wild-type cyclin E protein and was able to rescue the G0 to S phase transition
defect in cyclin E null fibroblasts (Geng et al., 2007). Thus, cyclin E plays dual roles
in promoting replication complex assembly through kinase-dependent and kinase-
independent mechanisms, both of which are critical for progression of cells from a
quiescent state.
The positive regulation of pre-RC assembly and subsequent replication initiation
is modulated by interphase cyclins and CDKs via a complex array of mechanisms.
While all of these mechanisms are essential for promoting DNA replication and
cell cycle progression, there must exist counterbalances to ensure that DNA repli-
cation is occurring faithfully and that cell cycle progression is not occurring under
inappropriate conditions. Interestingly, CDKs are critical modulators of the negative
regulation of pre-RC assembly as well.
3.4 Negative Impact of CDKs on Pre-RC Assembly (S–M Phase)
While elevated levels of G1 CDKs function to activate pre-RC assembly and repli-
cation initiation in G1 phase, this same cellular pool of CDKs is also required to
prevent re-initiation during S, G2, and M phases (Malumbres and Barbacid, 2009;
Doonan and Kitsios, 2009). One method of negatively regulating pre-RCs is through
inhibition of Cdc6. As discussed above, phosphorylation on specific residues of
Cdc6 by CDK2 results in stabilization of Cdc6 in G1 phase (Mailand and Diffley,
2005). However, phosphorylation at distinct sites of the protein has been observed to
result in Cdc6 dissociation from ORCs, nuclear export, and proteosomal degradation
(Petersen et al., 1999; Mendez and Stillman, 2000; Petersen et al., 2000; Borlado
and Mendez, 2008). The importance of Cdc6 nuclear export is somewhat contro-
versial, as significant amounts of Cdc6 have also been observed to remain bound to
chromatin throughout S and G2 phases (Mendez and Stillman, 2000; Borlado and
Mendez, 2008; Coverley et al., 2000; Fujita et al., 1999). However, CDK-mediated
phosphorylation of ORC, Cdt1, and Mcm proteins has also been implicated in the
dissociation of pre-RCs, indicating that the phosphorylation of various components
of the pre-RC by CDKs at least partially contributes to the inhibition of pre-RC
assembly and replication initiation (Lau et al., 2007; Malumbres and Barbacid,
2009; Doonan and Kitsios, 2009).
In addition to the CDK-dependent dissociation and degradation of Cdc6, there
are two main modes of negative regulation of Cdt1 in mammalian cells. First, Cdt1
is targeted for ubiquitin-mediated proteolysis via interactions with two distinct E3
ubiquitin ligase complexes, CUL4-DDB1CDT2 and SCFSkp2 . CUL4-DDB1CDT2 tar-
gets Cdt1 for degradation once Cdt1 is bound to PCNA on chromatin (Fujita, 2006;
Zhong et al., 2003; Hu et al., 2004; Senga et al., 2006). This degradation event
occurs in S phase and is stimulated by export/degradation of cyclin D1 (Aggarwal
et al., 2007). Unlike CUL4-DDB1CDT2 , SCFSkp2 -mediated degradation of Cdt1 is
dependent on the phosphorylation of Cdt1 by CDKs and occurs throughout the cell
cycle (Liu et al., 2004; Li et al., 2003). Additionally, both cyclin A–CDK2 and cyclin
A–CDK1 have been implicated in the phosphorylation and subsequent degradation
of Cdt1 (Fujita, 2006; Liu et al., 2004; Sugimoto et al., 2004).
The second mechanism of negative regulation of Cdt1 consists of direct binding
and inhibition of Cdt1 by geminin. Geminin was originally identified as an APC/C
substrate that inhibits pre-RC formation via prevention of Mcm complex loading
(McGarry and Kirschner, 1998). Geminin is degraded by the APC/C complex at
the metaphase/anaphase transition of mitosis, allowing for Cdt1 accumulation and
ORC binding in the next round of the cell cycle (McGarry and Kirschner, 1998;
Wohlschlegel et al., 2000; Tada et al., 2001). More recently, geminin has been identi-
fied as a target of E2F transcription that accumulates as cells enter S phase (Yoshida
and Inoue, 2004). Thus, regulation of Cdt1 by geminin is ultimately controlled by
upstream CDKs and the release of RB-mediated transcriptional repression that is
required for geminin expression.
Negative regulation of pre-RC components is essential for preventing DNA re-
replication. As DNA re-replication has obvious implications in genome instability
and human cancers, it is not surprising that cells have evolved to utilize multiple
mechanisms to control pre-RC assembly and replication initiation.
3.5 Perturbations of Pre-RC Assembly and Cancer
3.5.1 Functional Effects of Deregulated Pre-RC Assembly

Over-expression of pre-RC components (i.e., Cdc6 and Cdt1) and loss of pre-
RC regulatory proteins (i.e., geminin) result in deregulated DNA replication and
cell cycle progression, which can have significant impact on genome stability.
For example, over-expression of pre-RC components has been implicated in DNA
re-replication and chromosomal instability in eukaryotic cells (Lau et al., 2007;
Tatsumi et al., 2006; Vaziri et al., 2003; Karakaidos et al., 2004). Additionally, over-
expression of Cdc6 and Cdt1 has been shown to promote cellular transformation and
tumorigenesis in mouse models either alone or in cooperation with known onco-

genic lesions (Lau et al., 2007; Seo et al., 2005; Gonzalez et al., 2006; Arentson
et al., 2002; Blow and Gillespie, 2008). Furthermore, depletion of geminin has also
been shown to result in DNA re-replication and genomic instability similar to over-
expression of Cdt1 (Zhu et al., 2004; Melixetian et al., 2004; Saxena and Dutta,
2005). Thus, it is not surprising that deregulation of pre-RC components and pre-RC
regulatory factors is frequently observed in various types of human cancer.
3.5.2 Deregulation of Pre-RC Components in Cancer
While mutations of pre-RC components have not been identified in human can-
cers, deregulation of these proteins has been observed in various model systems of
human cancers and tumor specimens. For example, over-expression of Cdt1 and/or
Cdc6 has been observed in a multitude of tumors and tumor-derived cell lines (Lau
et al., 2007; Karakaidos et al., 2004; Williams et al., 1998; Murphy et al., 2005;
Ohta et al., 2001; Bravou et al., 2005). Additionally, deregulation of Mcm2-7 has
also been observed in various types of human malignancies, including breast, cer-
vical, and esophageal cancers (Lau et al., 2007; Williams et al., 1998; Going et al.,
2002; Shetty et al., 2005). Furthermore, since deregulation of pre-RC components
induces characteristics that are considered to be critical for tumorigenesis, expres-
sion signatures of pre-RC proteins have been proposed as potential biomarkers for
cancer diagnosis (Lau et al., 2007; Williams and Stoeber, 2007; Gonzalez et al.,
2005).
Ultimately, deregulation of pre-RC assembly is driven by aberrations in
CDK/cyclin signaling (Malumbres and Barbacid, 2009; Doonan and Kitsios, 2009).
Thus, mutations and deregulated activities of pre-RC regulatory CDK/cyclin pro-
teins are a frequent occurrence in human cancers. For example, deregulation of
CDK4/6, via direct mutation within the genes, loss of p16 or over-expression
of D-type cyclins, has been implicated in a variety of tumors (Malumbres and
Barbacid, 2009; Ortega et al., 2002; Knudsen and Knudsen, 2008). Additionally,
while mutations of CDK2 have not yet been observed in human cancers, dereg-
ulation of cyclin A and cyclin E via various mechanisms has been observed in
a multitude of cancers and is associated with poor prognosis (Malumbres and
Barbacid, 2009; Yasmeen et al., 2003). (Kitahara et al., 1995) Each of these
alterations in cyclin/CDK activity ultimately results in enhanced pre-RC assem-
bly and subsequent DNA replication, processes that have proven to be critical for
tumorigenesis.
3.6 Conclusions
Regulation of pre-RC assembly and replication initiation are critical facets of nor-
mal cell biology that have implications for human cancers. Cyclin/CDK complexes
maintain tight control of over these processes via a complex array of mechanisms.
Perturbations of this regulation can lead to abnormal DNA re-replication, which

can ultimately promote genome instability and cancer. Thus, it is not surprising that
deregulation of either pre-RC components themselves or the cyclin/CDK signal-
ing that controls pre-RC assembly is a common occurrence in many tumors and
has implications as both biomarkers for cancer diagnosis and targets for cancer
therapies.
Since CDKs play such a prominent role in controlling the cell cycle progression,
these proteins have become key therapeutic targets for not only cancer, but numerous
other diseases as well (Lee and Sicinski, 2006). However, like many targeted thera-
pies, not all CDK inhibitors have proven to be effective in clinical trials (Malumbres
and Barbacid, 2009). It is likely that in order to efficiently target CDKs and/or
cyclins for treatment of diseases such as cancer, it will be critical to elucidate the
specific requirements for distinct CDKs and cyclins in different cell types and tis-
sues. Furthermore, tumors are frequently characterized by a spectrum of mutations
that could interfere with the desired outcome of CDK/cyclin inhibition. Thus, under-
standing the dependencies of specific CDKs and cyclins in distinct tissues, as well
as potential cooperating or impeding lesions, will be important for designing treat-
ment regimens involving specific CDK inhibition alone or in combination with other
established therapies.
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Part II
Proliferation Under Duress
Chapter 4
Mitotic Checkpoint and Chromosome
Instability in Cancer
Haomin Huang and Timothy J. Yen
Abstract Chromosome instability (CIN) results in aneuploidy that may promote

tumorigenesis and chemoresistance. Chromosome missegregation allows cells to
rapidly change gene expression patterns on a global scale and provides a mech-
anism that promotes genetic and biochemical diversity that is used by cells to
achieve a transformed state or to survive suboptimal growth conditions. The mitotic
checkpoint is a fail-safe mechanism that monitors the fidelity of the microtubule
attachments to the kinetochore, a macromolecular structure that is situated at cen-
tromeres and essential for chromosome segregation. Over the past two decades
many kinetochore proteins have been identified and characterized in detail. Among
these are the mitotic checkpoint proteins that monitor the integrity of kineto-
chore:microtubule attachments and initiate and propagate the “wait anaphase” signal
that inactivates the anaphase-promoting complex/cyclosome (APC/C). The CIN
phenotype of many cancer cell lines may be attributed to the lack of a robust mitotic
checkpoint as these cells fail to arrest in response to microtubule inhibitors such as
taxol and nocodazole. Interestingly, mutations in the essential mitotic checkpoint
genes are rare. The checkpoint defects exhibited by CIN cells may be due to a com-
bination of factors that affect the mechanical efficiency of the spindle, as well as the
threshold level of “wait anaphase” signal that is required to inhibit the global pool
of the APC/C. In this chapter, we will review our current understanding of CIN and
the mitotic checkpoint and discuss the recent views of the relationships among the
mitotic checkpoint, CIN, tumorigenesis, and chemoresistance.
H. Huang (B)
Fox Chase Cancer Center, Philadelphia, PA 19111, USA
e-mail: haomin.huang@fccc.edu
60 H. Huang and T.J. Yen
4.1 Chromosome Instability (CIN)
4.1.1 Chromosome Missegregation, Aneuploidy, and CIN

Genetic instability is often considered to be the driving force of tumorigenesis.
At the nucleotide level, germline mutations in several different mismatch repair
(MMR) genes were found to result in microsatellite instability (MIN) in hered-
itary nonpolyposis colon cancer (HNPCC) (Fishel et al., 1993; Leach et al.,
1993; Parsons et al., 1993; Shibata et al., 1994; Thibodeau et al., 1993; Umar
et al., 1994). Even though MIN is strongly linked to the development of
HNPCC, it only represents 15% of solid tumors (Tomonaga, 2008; Weaver
and Cleveland, 2006). In contrast to MIN cancer cells that are generally near-
diploid (Eshleman et al., 1998; Jallepalli and Lengauer, 2001), CIN (chro-
mosome instability) cancers exhibit increased rates of chromosome loss and
gains at each division. CIN cells therefore exhibit dramatic differences in kary-
otype within a population (Lengauer et al., 1997, 1998). Interestingly, CIN
tumors generally do not exhibit microsatellite instability (Jallepalli and Lengauer,
2001).
Aneuploidy is clearly an outcome of CIN but the relationship is complex.
Aneuploid tumor cells are not necessarily classified as CIN if they do not gain and
lose chromosomes at each division (Kops et al., 2005b). Aneuploidy may be the
result of a one-time event that provided the cell with a growth advantage. Once the
desired traits are achieved, the ploidy is stably maintained in subsequent genera-
tions (unlike CIN). The CIN phenotype is also not simply caused by aneuploidy.
For example, addition of an extra copy of chromosome 3 into the near-diploid
HCT116 MIN cell line, or its fusion with another MIN cell line, did not result
in a CIN progeny cells (Lengauer et al., 1997). By contrast, a fusion between
a MIN (DLD1) and a CIN (HT29) tumor cells produced cells that retained the
CIN phenotype (Lengauer et al., 1997). These findings suggest that the determi-
nants for CIN may be considered as dominant gain-of-function mutations. A recent
report demonstrated that when diploid non-CIN cell lines, HCT116 and RPE1,
were exposed to a single treatment of anti-mitotic drugs that increased the rate of
chromosome missegregation, the progeny cells exhibited a transient CIN pheno-
type for 20–30 generations (Thompson and Compton, 2008). The number of cells
with deviant chromosome numbers eventually declined, and the ploidy returned
to near basal levels and was stabilized. Not surprisingly, when a CIN cell line
(HT29) was subjected to the identical protocol, it maintained its CIN phenotype.
Collectively, these findings indicate that missegregation of one or multiple chromo-
somes is not sufficient to convert cells to the CIN phenotype and, most importantly,
cannot convert near-diploid cells to aneuploid cells. The latter point is consis-
tent with the general notion that cells do not tolerate chromosome imbalance and
that cells must first overcome this barrier before becoming CIN (Yuen and Desai,
2008).
4 Mitotic Checkpoint and Chromosome Instability in Cancer 61
4.1.2 What Are the Defects That Result in Chromosome

Missegregation in CIN Cells?
Research into mechanisms of chromosome segregation over the past 20 years has
identified many genes that if lost or mutated will result in chromosome missegre-
gation. Defects in the mitotic checkpoint can result in aneuploidy as cells divide
regardless of whether their chromosomes are properly attached to the spindle or
not (Chan et al., 2000, 1999; Gorbsky et al., 1998; Huang et al., 2008a; Jelluma
et al., 2008a, b; Jones et al., 2005; Kops et al., 2005a; Logarinho et al., 2008; Tang
et al., 2004; Taylor and McKeon, 1997). Mutations in proteins that specify proper
attachment of the spindle microtubules to the kinetochore, and those important for
sister chromatid cohesion, will also cause chromosome missegregation (Bakhoum
et al., 2009; Cimini et al., 2001; DeLuca et al., 2006; Feng et al., 2006; Huang
et al., 2007, 2008b; Jallepalli et al., 2001; Kaplan et al., 2001; Pfleghaar et al., 2005;
Vong et al., 2005). Finally, defects during cytokinesis will produce polyploid cells
(Ganem et al., 2007; Storchova and Pellman, 2004). There are literally hundreds
of proteins essential for accurate chromosome segregation, and the loss of any sin-
gle protein would result in aneuploidy. Indeed, mutations in genes that are essential
for mitotic checkpoint signaling, as well as spindle functions have been identified
in some tumor cells. However, no single gene mutation has been found to dominate
and account for the high frequency of aneuploid cancer cells. Efforts to sequence the
genomes of cancer cells should eventually lead to an exhaustive screen for mutations
in genes that are known to be critical for mitosis.
4.2 The Mitotic Checkpoint
The mitotic, or spindle assembly, checkpoint is a fail-safe mechanism that monitors

the fidelity of chromosome segregation in space and time (Chan et al., 2005; Chan
and Yen, 2003; Liu et al., 2003; May and Hardwick, 2006; Musacchio and Salmon,
2007; Yen and Kao, 2005). The molecular details of this checkpoint pathway have
been extensively reviewed so we will cite the features that are most relevant to
this chapter. Until all chromosomes are properly aligned at the spindle equator, the
mitotic checkpoint inhibits the anaphase-promoting complex/cyclosome (APC/C),
an E3 ubiquitin ligase that promotes the degradation of substrates that prevent cells
from entering anaphase (King et al., 1995; Peters et al., 1994; Sudakin et al., 2001,
1995). The core components of the mitotic checkpoint are the Bub1, BubR1, Mps1
kinases, along with Bub3, Mad1, and Mad2 (Cahill et al., 1998; Chan et al., 1999;
Chen et al., 1996; Davenport et al., 1999; Hardwick and Murray, 1995; Hoyt et al.,
1991; Li and Murray, 1991; Li and Benezra, 1996; Roberts et al., 1994; Sudakin
et al., 2001; Taylor et al., 1998; Taylor and McKeon, 1997; Weiss and Winey, 1996;
Winey et al., 1991). All of these proteins seem to act at multiple steps of the signal-
ing pathway that includes monitoring the integrity of the kinetochore–microtubule
attachments, generating, and amplifying the “wait anaphase signal” that then prop-
agates throughout the cell to inhibit the APC/C (Campbell and Gorbsky, 1995; Li
and Nicklas, 1995; McIntosh, 1991; Nicklas et al., 1995; Rieder et al., 1995, 1994).
Loss of any single component will cause cells to exit mitosis regardless of whether
their chromosomes establish proper bipolar attachments and achieve alignment at
the spindle equator.
The mitotic checkpoint appears to monitor two aspects of kinetochore–
microtubule attachments. The Mad1 and Mad2 checkpoint proteins are thought to
monitor the microtubule occupancy at the kinetochore as they are recruited to kine-
tochores that lack microtubule attachments (Chen et al., 1996; Skoufias et al., 2001;
Waters et al., 1998). When the kinetochore is saturated with microtubules (mam-
malian kinetochores can bind approximately 20–25 microtubules), the amounts
of Mad1 and Mad2 on kinetochores are reduced by nearly 100-fold (Hoffman
et al., 2001). The dissociation of Mad1 and Mad2 from kinetochores is not suffi-
cient to silence the checkpoint until the functionality of the microtubule attachment
is satisfied. Productive microtubule attachments occur as a result of their end-on
attachments to kinetochores. This geometry appears to be mediated by a molecu-
lar sleeve that is composed of the Nuf2/Ndc80 complex (Cheeseman et al., 2006;
DeLuca et al., 2005; McCleland et al., 2004). Upon end-on attachment, the intrinsic
dynamic properties of the microtubule in conjunction with various microtubule-
binding proteins at the kinetochore generate opposing poleward-directed forces that
are critical for generating tension between the sister kinetochores. Owing to the ran-
dom nature by which microtubules encounter kinetochores, non-end-on attachments
do occur that are unable to generate proper levels of kinetochore tension. If these
defective attachments go undetected, their persistence can be a source of lagging
chromosomes when cells exit mitosis (Cimini et al., 2001).
In addition to monitoring microtubule occupancy, kinetochores possess an elab-
orate error correction system that will sever non-productive attachments to allow for
new rounds of attachment (Pinsky et al., 2006; Tanaka et al., 2002). Aurora B kinase
is a core component of the error system that is thought to regulate the severing
activity of the microtubule depolymerase, MCAK (mitotic centromere-associated
kinesin) (Andrews et al., 2004; Lampson et al., 2004; Lan et al., 2004). Aurora B
kinase has been reported to specify the differential sensitivity of the mitotic check-
point to the microtubule inhibitors, taxol and nocodazole (Ditchfield et al., 2003).
Microtubules in taxol-treated cells are able to establish kinetochore attachments but
fail to generate tension due to the loss of dynamicity of the microtubule (Waters
et al., 1998). Taxol induces a mitotic delay that is dependent on Aurora B. By con-
trast, the mitotic arrest induced with nocodazole, a drug that blocks microtubule
polymerization that leads to unoccupied kinetochores, was less dependent on Aurora
B (Ditchfield et al., 2003; Famulski and Chan, 2007). Given that Aurora B promotes
the detachment of microtubules from kinetochores, it remains possible that it indi-
rectly activates the checkpoint by transiently creating unoccupied kinetochores. This
may explain why in taxol-arrested cells, Mad2 can still be detected at a few kine-
tochores even though the majority of the kinetochores are attached to microtubules
and lack detectable Mad2 (Waters et al., 1998).
Although all of the checkpoint proteins are localized at kinetochores, their mech-
anistic roles remain unresolved. It is possible that each protein or subsets of proteins
monitor the activities of discrete sets of microtubule-binding proteins and motors
within the kinetochore. Alternatively, the combined activities of all the different
microtubule-binding proteins that act on a single kinetochore are monitored by a
single complex of checkpoint proteins. Regardless of the mechanism, some of the
checkpoint proteins must act as mechanosensors that convert microtubule attach-
ment status to a diffusible signal that is propagated from a defective kinetochore.
The amplification process that allows a localized defect to alter the global biochem-
ical state of the cell may be mediated in part by the rapid turnover of checkpoint
proteins at kinetochores. The proteins that are released from a defective kinetochore
may assume an activated state that is capable of inhibiting the APC/C (May and
Hardwick, 2006; Musacchio and Salmon, 2007). On the other hand, the amplifi-
cation might also be explained by a conventional signal transduction cascade that
is mediated by the various mitotic checkpoint kinases that reside at kinetochores
(Chen, 2004; Ditchfield et al., 2003; Elowe et al., 2007; Huang et al., 2008a; Huang
and Yen, 2009; King et al., 2007; Matsumura et al., 2007; Qi et al., 2006; Rancati
et al., 2005; Wong and Fang, 2007).
Early studies in PtK1 cells showed that a single unattached kinetochore is suf-
ficient to delay mitotic exit (Rieder et al., 1994). More recent studies of a variety
of human cell lines have shown that the length of the delay can vary among differ-
ent cell lines (Gascoigne and Taylor, 2008; Rieder and Maiato, 2004) and perhaps
due to the number of defective kinetochores in a cell (Feng et al., 2006; Huang
et al., 2007, 2008a). These observations suggest that the checkpoint signaling must
achieve a threshold whose magnitude may vary depending on the cumulative bio-
chemical activities of the checkpoint and spindle proteins. Depending on the number
of unattached kinetochores, a cell may vary the length of the delay. As discussed
below, different cell lines or cell types may express different amounts of check-
point and spindle proteins and thus influence the magnitude of the threshold that is
necessary to maintain cells in a checkpoint-inhibited state.
As a complex network, a functional mitotic checkpoint consists of many
upstream regulators and downstream effectors. The hZW10–ROD complex and
CENP-E are examples of proteins that facilitate the actions of the conserved mitotic
checkpoint proteins (Abrieu et al., 2000; Chan et al., 2000; Famulski and Chan,
2007; Famulski et al., 2008; Yao et al., 2000). For instance, hZW10 complex is
essential for recruitment of Mad1 to the unattached kinetochore (Kops et al., 2005a;
Liu et al., 2006), while CENP-E is a binding partner of BubR1 (Chan et al., 1998)
and is a regulator of BubR1 kinase activity (Mao et al., 2003, 2005). More recently,
Chk1 kinase, a component of the DNA damage checkpoint (Zachos et al., 2007;
Zachos and Gillespie, 2007), was found to be required for cells to arrest in mitosis
in the presence of taxol, but not nocodazole. The underlying mechanism of differ-
ential sensitivity of the mitotic checkpoint to these different drugs suggests that the
checkpoint may distinguish between microtubule binding and the functionality of
the attachments. Chk1 may be part of the checkpoint pathway that with Aurora B
and BubR1 monitors the quality of microtubule attachments (tension) as opposed to
whether the kinetochore is occupied by a microtubule. This suggests that there may
be distinct signaling pathways that are activated or more prevalent in response to the
different types of kinetochore attachment defects. This possibility is also supported
by the finding that the phosphorylation patterns of various subunits of the APC/C
differed between taxol and nocodazole treatment (Steen et al., 2008). The identi-
ties of the kinases that are responsible for the different phosphorylation patterns are
currently being pursued.
4.3 Aneuploidy/CIN, Mitotic Checkpoint, and Cancer
While detailed mechanisms of checkpoint protein function remain under intensive

investigation, the identification of these proteins has fueled efforts to study their
contribution to the mechanism of aneuploidy and tumorigenesis. Not surprisingly,
mitotic checkpoint defects are commonly seen in various types of tumors as well
as cancer cell lines (Kops et al., 2005b; Weaver and Cleveland, 2006). Checkpoint
defective cells are classified based on their inability to arrest in mitosis in response
to spindle poisons such as taxol and the vinca alkaloids. Even in the absence of
spindle poisons, these cells often exhibit lagging chromosomes in anaphase or
non-disjunction events that result from premature mitotic exit in the presence of
improperly aligned chromosomes. Unlike some tumor suppressors, oncogenes, and
other cell cycle checkpoint genes that are not essential (Liu and Yen, 2008), all the
mitotic checkpoint genes examined to date in mouse models are essential (Baker
et al., 2004; Dobles et al., 2000; Iwanaga et al., 2007; Kalitsis et al., 2000; Wang
et al., 2004). It appears that the efficiency of the spindle machinery of embryonic
mammalian cells is not high enough to operate without a safeguard. In the absence
of the mitotic checkpoint, the rate of chromosome missegregation may be so high
that it is incompatible with life. Cells that exhibit a defective mitotic checkpoint are
therefore more likely to harbor mutations that compromise rather than eliminate this
activity.
Mutations in the core mitotic checkpoint proteins are uncommon in human can-
cers. For example, SNP (single nucleotide polymorphisms) analysis of the promoter
and coding regions of the six essential spindle checkpoint genes (BUBR1/BUB1B,
BUB3, CENP-E, MAD2/MAD2L1, MAD2L2, and MPS1/TTK) in 441 German famil-
ial breast cancer cases did not reveal a strong correlation with increased risk of
breast cancer (Vaclavicek et al., 2007). By contrast, changes in expression levels
of mitotic checkpoint genes and their proteins are frequently seen (Table 4.1) (Coe
et al., 2006; Grabsch et al., 2003; Lewis et al., 2005; Miura et al., 2002; Ouyang
et al., 2002; Qian et al., 2002; Seike et al., 2002; Shichiri et al., 2002; Shigeishi et al.,
2001a, b; Sze et al., 2004; Wang et al., 2002, 2000; Yuan et al., 2006). While muta-
tions were not identified for Bub1, BubR1, Bub3, Mad1, Mad2, and Mps1/TTK in
a panel of aneuploid breast cancer cell lines, the levels of RNA and protein encoded
by these genes were all elevated as compared to the control breast epithelial cell
line MCF10A (Yuan et al., 2006). Furthermore, the same study showed that 77%
Table 4.1 Status of mitotic checkpoint genes in human cancers that have been examined
Gene Mutation Misregulation
BUB1 5.4% (13/238) 47% (122/261)

BUBR1 11.1% (3/27) 35.7% (70/196)
BUB3 NA 80% (66/82)
MAD1 NA 90% (38/42)
MAD2 32% (25/77) 55% (61/110)
Total 11.9% (41/342) 51.7% (357/691)
Compiled from summary by Weaver and Cleveland (2006).
of 270 primary breast tumor samples exhibited a two-fold to three-fold increase in

BubR1 levels when compared to 18 normal breast ductal tissues. A similar analysis
of a panel of small-cell lung cancer (SCLC) cell lines revealed the presence of extra
copies of 7p22.3, a locus that contains the Mad1 gene (Coe et al., 2006). This raises
a possibility that overexpression of Mad1 may contribute to the development of
this type of cancer. The significance of these observations has been strengthened by
the finding that overexpression of Mad2 in mice promoted aneuploidy and tumori-
genesis (Sotillo et al., 2007). Precisely how overexpression of checkpoint proteins
promotes aneuploidy or tumorigenesis is not known. Increased levels of checkpoint
proteins may delay mitosis and thus increase the chances that kinetochores estab-
lish defective microtubule attachments. Another possibility is that overexpression
may be viewed as a “gain-of-function” mutation where higher levels of checkpoint
proteins promote interactions with other growth pathways that they normally do not
interact with.
Regardless of the defects that promote chromosome missegregation, aneuploidy
might promote tumorigenesis by two ways. One possibility is that chromosome
instability results in large-scale changes in gene expression patterns that provide the
diversity that is necessary for promoting tumorigenesis (Duesberg, 2005; Duesberg
et al., 2005). A second possibility is that chromosome instability might facilitate
loss of heterozygosity (LOH) of genes that are frequently associated with tumori-
genesis. This is illustrated in a study of colorectal cancer where over 50% of early
stage colorectal tumors that were examined exhibited an allelic imbalance (AI) of
chromosome 5q, which is where the adenomatous polyposis coli (APC) tumor sup-
pressor gene is located. Thus, chromosome instability might act at an early step in
the development of colorectal cancer by promoting LOH (Shih et al., 2001).
Whether or not chromosome instability directly drives tumorigenesis remains a
topic for debate (Hahn et al., 1999; Marx, 2002; Zimonjic et al., 2001). Mice with
heterozygous mutations in genes that play critical roles in mitosis, such as BUB1,
BUBR1, BUB3, MAD1, MAD2, CENP-E, RAE1, NUP98, SECURIN, RANBP2, and
CHFR, are haploinsufficient because their cells exhibit increased rates of aneuploidy
(Ricke et al., 2008). In spite of this, the level of spontaneous tumor formation is low.
Of the 13 different CIN mice (including Rae1–Bub3 and Rae1–Nup98 compound
heterozygotes) that were characterized, 10 were found to be prone to cancer only
after exposure to carcinogens (Ricke et al., 2008). The frequency of tumorigenesis

was also not increased after crossing Bub3+/– mice with p53 or Rb heterozygous
mutant mice. The expectation was that chromosome instability resulting from a
reduction in Bub3 would promote LOH of either p53 or Rb and thus increase the
rate of tumorigenesis. The discrepancy between the expected and the observed may
be due to the possibility that the rate of chromosome loss for Bub3+/– mice may
be too low to promote LOH of the tumor suppressor genes (Kalitsis et al., 2005).
Thus, frequency of LOH may increase in mice that are haploinsufficient for other
checkpoint genes.
The ability of aneuploidy to increase cell transformation and tumorigenesis may
depend on other genetic factors. For example, loss of p16ink4a tumor suppressor
gene from hypomorphic BubR1H/H mice (these mutants express < 20% normal
BubR1 levels) increased rates of lung adenocarcinoma by nearly 18-fold over the
BubR1H/H mutant. Interestingly, loss of the p19Arf tumor suppressor did not increase
the incidence of tumor formation relative to parental strain (Baker et al., 2008). The
different outcomes seen with these two tumor suppressors may reflect differences in
their functions. In these studies, loss of p16ink4a decreased the frequency of senes-
cent cells and premature aging in tissues, while p19Arf acted as an anti-aging factor
and prevented cellular senescence in hypomorphic BubR1H/H mice. It is possible
that the loss of p16ink4a may increase the chance that aneuploid hypomorphic BubR1
cells survive and become transformed. The complex relationship between tumor
suppressor and mitotic checkpoint genes is also seen between APC (adenoma-
tous polyposis coli) and BubR1. BubR1+/– mutant mice do not exhibit spontaneous
tumors. When BubR1+/– mice were crossed with heterozygous APCmin/+ mice, the
compound mutant progenies had 10 times more colonic tumors (adenocarcinomas)
than did APCmin/+ mice (Rao et al., 2005). Despite the increase in the frequency of
high-grade colonic tumors, there was a significant decrease in the number of ade-
nomas (polyps) in the small intestine of APCmin/+ /BubR1+/– mice relative to the
APCmin/+ mice (Rao et al., 2005). The reduced number of adenomas might be due
to the fact that chromosome instability resulting from BubR1 deficiency promoted
apoptosis. However, sufficient number of cells with the appropriate chromosome
content survived and became tumorigenic. Striking a delicate balance between ane-
uploidy that can lead to cell death and pathways that allow aneuploid cells to survive
is likely a key factor in the early steps of tumorigenesis.
The complex relationship between chromosome instability and tumorigenesis is
also exemplified in studies of cells and mice that were depleted of the kinetochore
motor protein, CENP-E (Ricke et al., 2008; Weaver et al., 2007). Aneuploid mouse
embryo fibroblasts (MEFs) from CENP-E heterozygous mutant mice increased their
efficiency of transformation with the loss of the p19Arf , or when SV40 large T anti-
gen is overexpressed. Xenograft experiments showed that these transformed MEFs
also produced tumors at higher rates than normal MEFs. Analysis of tumor forma-
tion in CENP-E+/– mice, however, revealed a more complex picture. Ten percent
of CENP-E+/– mice spontaneously developed lymphomas of the spleen and lung
tumors after 19–21 months. This frequency is significantly higher than wild-type
mice. However, the number of spontaneous liver tumors in 19–21-month-old CENP-

E+/– mice was reduced by 50% when compared to wild-type mice. Even after
exposure to the carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA), CENP-E+/–
mice developed 20% less total tumors than wild-type controls. The overall rate of
tumorigenesis was also delayed in CENP-E+/– /p19Arf null mice relative to mice just
lacking p19Arf (Weaver et al., 2007). Based on these complex outcomes, it was sug-
gested that aneuploidy promoted by loss of CENP-E can either promote or suppress
tumor formation.
Aneuploidy is generally not well tolerated by cells and thus may induce apop-
tosis and indirectly reduce the frequency of tumor formation. This is supported by
studies of yeast and mouse cells where it was shown that chromosome gains can
impair proliferation and alter metabolic properties (Torres et al., 2007; Williams
et al., 2008). Interestingly, depending on what chromosome was gained, some cells
became more sensitive to immortalization, while other cells were less sensitive to
immortalization (Hernando, 2008; Torres et al., 2007; Williams et al., 2008). The
combined studies suggest that aneuploidy generates the genetic and biochemical
diversity that is required to achieve the transformed state. However, the intrinsic
and extrinsic factors that influence the cell’s ability to tolerate aneuploidy are also
critical determinants at the early steps of tumorigenesis.
4.4 Mitosis as a Target for Chemotherapy
Research into the mechanisms that specify chromosome segregation has revealed
a plethora of candidate anti-cancer drug targets. As many of the proteins appear
to function specifically in mitosis, such inhibitors should exhibit increased speci-
ficity and reduce undesirable side effects, such as neuropathies, that are associated
with current anti-microtubule agents (Jablonski et al., 2003; Liu and Yen, 2008).
Inhibitors that are being developed for the clinics target the Aurora A, Aurora B,
Plk1 kinases, and the microtubule motors, CENP-E and kinesin-5 (Jackson et al.,
2007; Weaver and Cleveland, 2005). In spite of these new discoveries, the mecha-
nism by which inhibitors of mitosis kill tumor cells remains poorly understood. An
important concern with this class of inhibitors is that they can promote aneuploidy
that drives the selection of drug-resistant cells (Ganem et al., 2007; Rieder and
Maiato, 2004; Storchova and Pellman, 2004). A key toward improving tumor cell
response to anti-mitotic agents is to obtain insights into how the mitotic checkpoint
is linked to cell survival and death pathways.
A prolonged mitotic arrest induced by drugs does not always culminate in death.
Time-lapse studies have revealed that there is considerable variability in how cells
within a population respond to anti-mitotic drugs (Gascoigne and Taylor, 2008; Orth
et al., 2008; Shi et al., 2008). Inhibitors of spindle function will cause the cells to
block in mitosis for variable lengths of time. Some cells will die during mitosis
but the conditions that lead to this outcome are not known. However, there does
not appear to be a strong correlation between death in mitosis and the length of
the arrest. This is consistent with histological studies that suggested the lack of
correlation between the mitotic index and apoptosis in tumors from patients and
mouse xenografts that were treated with paclitaxel and docetaxel (Milross et al.,
1996; Schimming et al., 1999; Symmans et al., 2000).
From a clinical standpoint, an enhanced ability of anti-mitotic drugs to kill cells
before they exit mitosis should significantly reduce tumor recurrance. Although
studies have reported that an active mitotic checkpoint is an important determinant
for activating apoptosis in response to anti-mitotic agents (Masuda et al., 2003; Sudo
et al., 2004; Tao et al., 2005; Taylor and McKeon, 1997). Recent studies that mon-
itored the fates of individual cells that were arrested in mitosis indicated that the
length of the checkpoint arrest did not correlated with death in mitosis (Gascoigne
and Taylor, 2008; Orth et al., 2008; Shi et al., 2008). New data show that death in
mitosis does not depend on the ability of the mitotic checkpoint to prolong mito-
sis. Instead, direct inhibition of factors that promote mitotic exit is more effective
at killing cells in mitosis. This suggests that activation of apoptosis may result from
the cumulative biochemical affects of an extended mitosis (i.e., super-physiological
phosphorylation of cdk1 substrates) (Huang et al., 2009). How cells chose to die
during mitosis as opposed to after they have exited mitosis is not understood. One
model proposes that death in mitosis occurs if apoptosis is activated before cyclin
B1 is degraded and cells exit mitosis (Gascoigne and Taylor, 2008). Consistent with
this idea, cyclin B1/cdk1 has been shown to phosphorylate caspase-9, inhibiting its
ability to induce apoptosis in cells arrested in mitosis by nocodazole (Allan and
Clarke, 2007). It is likely that competing factors dictate whether apoptosis is initi-
ated before the cells exit mitosis. Whether these factors regulate different pathways
that dictate death in mitosis or death after mitotic exit is not known.
Cells with defective spindles that do not die in mitosis will exit as a result of a
crippled checkpoint or “slippage” (Rieder and Maiato, 2004). Slippage is not due
to the silencing of the mitotic checkpoint but is thought to be due to a background
level of APC/C activity that slowly degrades cyclin B1, and thus cdk1 activity, to
levels below that required to maintain the mitotic state (Chibazakura, 2004; Foley
et al., 1999; Foley and Sprenger, 2001; Maiato et al., 2002; Tsuiki et al., 2001).
After cells exit mitosis, they may senesce, die, or proliferate (Andreassen et al.,
2001; Rieder and Maiato, 2004; Weaver and Cleveland, 2005), and these responses
may be influenced by p53 status (Andreassen et al., 2001).
The link between mitotic stress and apoptosis is complex and this connection
has been further complicated by the existence of a caspase-independent mitotic
death (CIMD) pathway (Niikura et al., 2007). CIMD appears to respond to defec-
tive microtubule–kinetochore attachments in the presence of nocodazale, taxol, or
17-AAG, a Hsp90 inhibitor that disrupts localization of several kinetochore proteins.
More importantly, CIMD appears to be dependent on p73 but not p53 and is prefer-
entially activated in cells with an impaired spindle checkpoint. CIN cell lines with
reduced Bub1 levels were found to undergo CIMD in response to spindle defects.
Thus, Bub1 expression may be an indicator of sensitivity of some tumors to mitotic
death mediated by anti-mitotic agents.

Chromosome instability allows cancer cells to rapidly change their gene expression
patterns in a single cell division. These large-scale changes can provide cells with
the genetic and biochemical diversity to undergo transformation and overcome poor
growth conditions. The mechanism by which cells adopt the CIN phenotype appears
to be complex as mutations in genes that encode key components of the mitotic
spindle or its regulatory machinery are rare. The underlying defects for CIN have
not been defined and may ultimately be due to epigenetic changes that alter the
expression levels of multiple proteins so that the spindle functions or activities of
the checkpoint proteins operate suboptimally. In addition, mutations in some tumor
suppressor genes that allow cells to tolerate the aneuploid state are likely to facilitate
CIN. Thus, tolerance of aneuploidy has dual functionality for the neoplastic cell, in
that it can promote tumorigenesis and cellular drug resistance. On the other hand,
aneuploid cells tend to be less fit than wild-type cells, and this weakness may be
exploited to improve response of cancer cells to chemotherapy (Rancati et al., 2008;
Torres et al., 2007).
Of the many genes whose loss has been shown to result in aneuploidy, no
single gene mutation has been found to be responsible for chromosome missegre-
gation in CIN cancer cell lines. The exception may be in colorectal cancer cells
where mutations in the APC gene were found to disrupt spindle functions and
increase rates of chromosome missegregation (Kaplan et al., 2001). In spite of this,
the mechanism that renders the CIN phenotype remains unclear. The defects may
be a combination of reduced spindle checkpoint activities and deficient capacity
to establish stable kinetochore–microtubule attachments. Comprehensive analysis
of mRNA and protein expression along with sequencing of the genomes of CIN
cells would undoubtedly be revealing. Identification of deficiencies in specific net-
works and pathways that provide spindle functions may allow us to exploit them
to enhance the killing of CIN cells. As has been suggested, aneuploid cells with
defects in kinetochore–microtubule capture might be more sensitive to inhibitors of
the mitotic checkpoint given that they may not survive high rates of chromosome
missegregation.
Establishment of the CIN condition requires not only crippling the mitotic appa-
ratus, but additional mechanisms that allow cells to tolerate the aneuploid state. A
comprehensive survey of the activities of various cell death and survival pathways
in CIN cells would likely yield important insights about this complex relationship.
It is possible that mutations that block cell death pathways may play a critical role.
However, it is also possible that aneuploidy itself may select for survivors. Inhibiting
the key survival mechanisms in CIN cells should resensitize them to aneuploidy.
Mitotic checkpoint inhibitors may be used in conjunction with anti-mitotic drugs to
reduce the viability of cells that overcome a checkpoint arrest and thus to improve
treatment outcome.
The mitotic checkpoint guards against aneuploidy and is likely to play a critical
role in determining the sensitivity of tumors to new and existing anti-mitotic agents.
A weakened checkpoint would promote cells treated with anti-mitotic drugs to

exit mitosis and provide an opportunity for the aneuploid cells to survive and pro-
liferate. While it is obvious that targeting cells to die in mitosis would significantly
enhance treatment outcome, the link between mitotic arrest and cell death remains
to be revealed. Regardless, the fact that cells can die in mitosis suggests a connection
between mitotic events and apoptosis. A priori, it seems that cells have evolved a
mechanism to kill themselves in response to an inability to properly segregate chro-
mosomes. Improving the efficiency of anti-mitotic drugs would benefit from efforts
to identify the molecular links between mitosis and apoptosis.
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Chapter 5
Mitotic Catastrophe
Jeremy P.H. Chow and Randy Y.C. Poon
Abstract Mitotic catastrophe is generally defined as a mode of cell death associ-

ated with aberrant mitotic activity. It is characterized by unscheduled activation of
cyclin B1–CDK1, premature chromosome condensation, and cell death. Progress in
the past several years has unraveled a myriad of pathways that can trigger mitotic
catastrophe. In this review, we will discuss several common forms of mitotic catas-
trophe that are highly relevant to cancer. These include mitotic catastrophe triggered
by prolonged mitotic arrest induced by microtubule poisons and the abrogation of
DNA damage and replication checkpoints. Failure of cells to undergo mitotic catas-
trophe is believed to contribute to tumorigenesis. Furthermore, mitotic catastrophe
is exploited as a strategy to induce cell death in cancer treatments.
5.1 Introduction
Proper control of cell number is dictated by a delicate balance between cell cycle
and cell death. Deregulation of these controls is the root of genome instability and
disorders such as cancer. Progress in the past several years has unraveled some of
the underlying principles of a specific form of cell death termed mitotic catastrophe.
Although the biological significance and mechanism of mitotic catastrophe remain
to be fully defined, the prevailing view is that mitotic catastrophe plays a critical
role in maintaining genome stability. Mitotic catastrophe is of particular interest to
cancer research because it integrally links cell death to checkpoints and the cell
cycle. Understanding mitotic catastrophe may reveal principles of tumorigenesis as
well as leads for novel therapeutic designs.
Historically, the term “mitotic catastrophe” was first used (Russell and Nurse,
1987) to describe the lethal phenotype associated with the fission yeast strain that
R.Y.C. Poon (B)

Department of Biochemistry, Hong Kong University of Science and Technology,
Clear Water Bay, Hong Kong, China
e-mail: rycpoon@ust.hk; http://ihome.ust.hk/~rycpoon
80 J.P.H. Chow and R.Y.C. Poon
contain an activated allele of cdc2 (cdc2–3w) and a recessive temperature-sensitive

wee1 mutation (wee1-50) (Russell and Nurse, 1986). These mutations overactivate
the mitotic engine, resulting in premature and aberrant mitosis, particularly with
respect to chromosome segregation and septum formation (Molz et al., 1989).
Mitotic catastrophe is now generally defined as a mode of cell death associated
with premature or inappropriate entry into mitosis. It should be noted that there
is still no consensus definition of mitotic catastrophe. Because of that, there are
disagreements and uncertainties about the precise causes, mechanisms, and out-
comes of mitotic catastrophe. Here, we adopt a relatively simple definition of mitotic
catastrophe to mean a mode of cell death associated with aberrant mitotic activity
(Castedo et al., 2004b). Some scholars have defined mitotic catastrophe with mainly
morphological terms including the formation of large cells with multiple micronu-
clei and decondensed chromatin that is clearly distinctive from apoptosis (Roninson
et al., 2001). Other groups have considered mitotic catastrophe as a state generally,
but not necessary, associated with cell death as an outcome (Erenpreisa and Cragg,
2001; Roninson et al., 2001; Vakifahmetoglu et al., 2008). We are restricting our
definition to a process that ultimately results in cell death, thus excluding situa-
tions in which cells survive aberrant mitosis, such as after mitotic slippage and the
formation of multinucleated giant cells.
A myriad of pathways can trigger mitotic catastrophe, including premature
mitotic entry, prolonged mitotic block, and defective mitosis. Likewise, several
underlying mechanisms appear to be involved in the execution of mitotic catas-
trophe. In this review, we will discuss several common forms of mitotic catastrophe
that are highly relevant to cancer. These include mitotic catastrophe triggered by
prolonged mitotic arrest induced by microtubule poisons and the abrogation of DNA
damage and replication checkpoints. These conditions are characterized by unsched-
uled activation of cyclin B1–CDK1, premature chromosome condensation, and cell
death.
5.2 Normal Control of Mitosis and the Spindle-Assembly

Checkpoint
To appreciate how abnormal mitotic events are brought about during mitotic catas-
trophe, we first briefly review the current paradigm of normal mitotic control in
mammalian cells (Fig. 5.1). We refer the reader in this section to a number of
relevant review articles.
It is well established that CDK1 (also called CDC2) is the key driving force for
mitosis. CDK1 is activated by binding to cyclin B, which then phosphorylates sub-
strates that are critical for entry into mitosis. Destruction of cyclin B provides a
mechanism to rapidly inactivate CDK1 and allow the cell to exit mitosis. In mam-
malian cells, cyclin B1 is believed to be the major mitotic B-type cyclin (Fung and
Poon, 2005). Cyclin B3 is restricted only to developing germ cells and adult testis,
and cyclin B2 does not appear to have an essential function in mice. Different B-type
5 Mitotic Catastrophe 81
Replication DNA Spindle

Block Damage Disruption
β-TrCP
SCF
ATR/ATM MAD2
PLK1
EMI1
CHK1/CHK2
CDC20 CDH1
APC/C APC/C
WEE1
MYT1 Cyclin A2-CDK1/2
Bora CDK1
Cyclin B
Aurora A PLK1 CDC25
Mitosis
Fig. 5.1 Control of cyclin B1–CDK1. Cyclin B1–CDK1 is kept inactive during G2 phase by
Thr14/Tyr15 phosphorylation. Activation of cyclin B1–CDK1 is orchestrated by feedback loops
involving CDC25A/B/C and WEE1/MYT1. How the system is kick-started is uncertain, but may
involve in the Aurora A-PLK1–CDC25 axis. After DNA replication block or DNA damage, the
cyclin B1–CDK1 activation system is suppressed by the ATM/ATR–CHK1/CHK2–CDC25/WEE1
axis. Once the cell is in mitosis, unattached kinetochores activate the spindle-assembly check-
point. This checkpoint is mediated by MAD2’s inhibition of APC/CCDC20 . When the checkpoint
is satisfied, APC/CCDC20 is turned on to destroy cyclin B1. This inactivates CDK1, leading to the
activation of APC/CCDH1 , which in part is responsible in keeping a low level of cyclin B1 expres-
sion during G1 phase. During S phase, APC/CCDH1 is turned off by cyclin A–CDK1/2 and EMI1,
allowing the re-accumulation of cyclin B1 for the next mitosis. See text for details
cyclins are also differentially localized: While cyclin B1 is cytoplasmic during inter-
phase and translocates into the nucleus during mitotic entry, cyclin B2 colocalizes
with the Golgi apparatus and contributes to its fragmentation during mitosis.
CDK1 is present throughout the cell cycle. In contrast, cyclin B1 accumulates
from S phase and forms a complex with CDK1. The complex is kept inactive by
phosphorylation of CDK1Thr14/Tyr15 by MYT1 and WEE1. At the end of G2 phase,
the stockpile of inactive cyclin B1–CDK1 complexes is activated abruptly by mem-
bers of the CDC25 family. Cyclin B1–CDK1 catalyzes its own activation by an
intricate network of feedback loops that simultaneously stimulate CDC25 activa-
tion and WEE1 inactivation (Lindqvist et al., 2009). Thus, cyclin B1–CDK1 is
essentially a bistable system that becomes autocatalytic once a critical portion is
activated (Ferrell, 2002). Despite many years of research, however, how the initial
batch of cyclin B1–CDK1 complexes is activated remains one of the key outstand-
ing questions in the field. Activation of CDC25 by PLK1 may kick-start the system
(van Vugt and Medema, 2005). PLK1 is also involved in the inactivation of WEE1
and MYT1. The activation of PLK1 in G2 phase requires phosphorylation of Thr210

by Aurora A, an event that is assisted by Bora. Binding of Bora to PLK1 is stimu-
lated by cyclin B1–CDK1-dependent phosphorylation, creating yet another positive
feedback loop in the activation of cyclin B1–CDK1 (Lindqvist et al., 2009). Cyclin
A–CDK1/2, another cyclin–CDK pair that is activated slightly earlier, may also par-
ticipate in the activation of cyclin B1–CDK1 (Fung et al., 2007). Phosphorylation
of another residue in CDK1, Thr161, is absolutely required for the activation of the
kinase activity. However, the activity of the enzyme responsible for Thr161 phos-
phorylation (CAK) does not appear to be regulated during the cell cycle (Kaldis,
1999).
At the end of mitosis, cyclin B1 is removed by the ubiquitin–proteasome
system. Specifically, a ubiquitin ligase, designated the anaphase-promoting com-
plex/cyclosome (APC/C) loaded with a targeting subunit termed CDC20, is respon-
sible (Wolf et al., 2007). The timely destruction of cyclin B1 is conferred by a short
sequence at the NH2 -terminal region known as the destruction box (D box). APC/C
is also responsible for degradation of several other substrates including geminin and
securin (Yu, 2007). Degradation of securin releases separase, which in turn cleaves
cohesin to allow sister chromatid separation (Yanagida, 2000). Proteolysis of gem-
inin releases CDT1 from the complex to form the pre-replication complex required
for the next round of DNA replication (Seo and Kroll, 2006).
Activated cyclin B1–CDK1 also negatively regulates itself by stimulating the
activity of APC/CCDC20 through phosphorylation of several subunits of APC/C
including CDC16, CDC23, and CDC27. APC/C is also activated by PLK1 and inac-
tivated by PKA through phosphorylation. Moreover, phosphorylation of CDC20
by cyclin B1–CDK1 is also necessary for APC/CCDC20 activation. Hence, cyclin
B1–CDK1 paves the path for its own destruction by stimulating APC/CCDC20 in a
negative feedback loop (Yu, 2007).
Activation of APC/CCDC20 is initiated only when all the chromosomes have
achieved bipolar attachment to the mitotic spindles. Only one single unattached
kinetochore is sufficient to delay the onset of anaphase (Musacchio and Salmon,
2007). Unattached kinetochores or the absence of tension between the paired kineto-
chores activates a surveillance mechanism termed the spindle-assembly checkpoint.
This checkpoint inhibits APC/CCDC20 , thereby maintaining a high level of active
cyclin B1–CDK1. A growing body of evidence indicates that unattached kine-
tochores attract the components of the checkpoint machinery (including BUB1,
BUB3, MAD1, MAD2, MAD3/BUBR1, MPS1, and CENP-E), catalyzing the for-
mation of diffusible mitotic checkpoint complexes (MCC; components include
MAD2, BUBR1, and BUB3), which in turn inhibit APC/CCDC20 (see Chapter 4).
It is generally accepted that the sequestration of CDC20 by MAD2 is a key step
in the spindle-assembly checkpoint. Binding to CDC20 requires a conformational
change in MAD2 from a less stable open conformation (known as O-MAD2) to the
more stable closed conformation (C-MAD2). Upon conformational change from
O-MAD2 to C-MAD2, the C-terminal CDC20-binding site (which resembles a
“seatbelt” structure) is exposed, thus allowing interaction with CDC20. According
to a currently favored MAD2-template model, the C-MAD2 that binds to MAD1
at the kinetochores serves as a template for the conversion of the cytosolic pool of
O-MAD2 into C-MAD2. The newly activated MAD2 then leaves the kinetochore
to bind and inhibit CDC20. As MAD2 has the same conformation when complexed
with both MAD1 and CDC20, the C-MAD2–CDC20 complexes may autoamplify
the checkpoint signal by recruiting more O-MAD2 to the complex. This model pro-
vides an elegant mechanism for cytosolic propagation of the checkpoint signal away
from kinetochores (Musacchio and Salmon, 2007).
Within minutes after all kinetochores are properly attached, the spindle-assembly
checkpoint is terminated to allow APC/CCDC20 activation and anaphase onset.
How the checkpoint is silenced is not entirely clear, but evidence suggests that
several mechanisms may be involved. These include the stripping of checkpoint
components from the kinetochore by a dynein motility-dependent mechanism, an
energy-dependent MAD2–CDC20 complex dissociation mechanism, an APC/C-
dependent mechanism, and the neutralization of MAD2 by binding to p31comet
(Musacchio and Salmon, 2007).
In marked contrast to CDC20, phosphorylation of CDH1 by cyclin B1–CDK1
alters the conformation of CDH1 and prevents its binding to APC/C, thus keeping
APC/CCDH1 inactive during mitosis. Destruction of cyclin B1 at the end of mitosis
downregulates CDK1 activity and therefore relieves the inhibition of APC/CCDH1
(Baker et al., 2007; Pesin and Orr-Weaver, 2008). The phosphatase CDC14, which
is believed to remove most of the phosphorylation carried out by MPF at the end
of mitosis (Bembenek and Yu, 2003; Stegmeier and Amon, 2004), antagonizes
the inhibitory phosphorylation on CDH1. The activated APC/CCDH1 then degrades
CDC20 and takes over the task of degrading any remaining cyclin B1.
APC/CCDH1 remains active during G1 phase to curb the unscheduled accumu-
lation of mitotic cyclins. Cell cycle-dependent transcription of cyclin B1 provides
an additional level of regulation of cyclin B1 (Fung and Poon, 2005). At the
G1 –S transition, APC/CCDH1 itself is turned off by phosphorylation, allowing the
re-accumulation of cyclin B1. Cyclin A–CDK1/2 phosphorylates CDH1, resulting
in the dissociation of CDH1 from the APC/C core. APC/CCDH1 is also turned off by
EMI1, which begins to accumulate at the G1 /S transition (van Leuken et al., 2008).
During normal prophase, the SCFβ-TrCP targets EMI1 for ubiquitin-mediated degra-
dation. This degradation of EMI1 is promoted by PLK1-dependent phosphorylation
(Eckerdt and Strebhardt, 2006).
5.3 Mitotic Catastrophe Caused by Mitotic Block

and Mitotic Slippage
Agents that suppress microtubule dynamics can artificially activate the spindle-
assembly checkpoint by leaving the kinetochores unoccupied. These include chem-
icals that inhibit microtubules depolymerization (e.g., Taxol) or polymerization
(e.g., vinca alkaloid and nocodazole). Spindle-disrupting drugs are among the most
important chemotherapeutic agents available for a variety of cancers (Weaver and

Cleveland, 2005). Activation of the spindle-assembly checkpoint leads to persis-
tent activation of cyclin B1–CDK1 and stabilization of APC/C targets. While
numerous targets have been proposed, the precise mechanism underlying spindle
toxins-mediated mitotic catastrophe remains controversial (see below).
A direct link between the spindle-assembly checkpoint and mitotic catastrophe is
yet to be fully established. Paradoxical evidence implies that the spindle-assembly
checkpoint is either required or inhibitory to apoptosis. Checkpoint disruption with
dominant-negative BUB1 (Taylor and McKeon, 1997) or BUBR1 (Shin et al.,
2003), siRNA against MAD2 or BUBR1 (Sudo et al., 2004), or overexpression of
Aurora A (Anand et al., 2003) inhibits the cell death induced by nocodazole or
Taxol. Similarly, several human cancer cell lines that contain an impaired check-
point are resistant to nocodazole treatment (Masuda et al., 2003). In disagreement
with these findings, disruption of the spindle-assembly checkpoint by trichostatin A
(Dowling et al., 2005), a mutant CDC20 (Sihn et al., 2003), or siRNA target-
ing BUB1 (Niikura et al., 2007) or BUBR1 (Lee et al., 2004) sensitizes cells to
nocodazole- or Taxol-induced killing.
The role of cyclin B1–CDK1 in spindle poison-induced cell death is also
controversial. Inhibition of CDK1 by chemical inhibitors, a dominant-negative
CDK1, or cyclin B1 antisense oligonucleotides prevents Taxol-induced apoptosis
(Yu et al., 1998; Shen et al., 1998). Likewise, inhibition of CDK1 with the spe-
cific inhibitor RO3306 reduced nocodazole-induced apoptosis (Chan et al., 2008).
However, no reduction of nocodazole-induced apoptosis was found in the pres-
ence of the CDK inhibitor olomoucine (Masuda et al., 2003). Moreover, addition
of CDK inhibitors to Taxol-treated cells enhances apoptosis (O’Connor et al.,
2002; Wall et al., 2003; Pennati et al., 2005). Results from kinase-dead CDK1
expressing or CDK1 conditional knockout cells similarly point to an apoptosis-
protecting role of cyclin B1–CDK1 (O’Connor et al., 2002). Furthermore, contin-
uous spindle-assembly checkpoint function, but not cyclin B1-CDK1 activity, is
required for caffeine-induced apoptosis in nocodazole-arrested cells (Gabrielli et al.,
2007).
Spindle toxin-stimulated cell death is further complicated by mitotic slippage
(also called adaptation) (Weaver and Cleveland, 2005; Rieder and Maiato, 2004).
After a prolonged block in mitosis, some cells can inactivate cyclin B1–CDK1 pre-
cociously and exit mitosis without chromosome segregation and cytokinesis. The
nuclear envelope then randomly reforms around groups of chromosomes. Although
the exact mechanism of mitotic slippage is not known, the central event seems
to be a slow but continuous degradation of cyclin B1. It was found that slippage
is required for the apoptosis induced in response to mitotic kinesin Eg5 inhibi-
tion, which leads to monopolar spindle formation and spindle-assembly checkpoint
activation (Mayer et al., 1999). Slippage-refractory cells are resistant to Eg5
inhibitor-induced apoptosis, but promotion of mitotic slippage with CDK inhibitors
enhances cell death (Tao et al., 2005). However, contradictory evidence suggests that
inhibiting Eg5 activity leads to apoptosis regardless of spindle-assembly checkpoint
function (Chin and Herbst, 2006).
A solution to the problem may be provided by the findings that although cell
death is reduced by mitotic slippage, it is induced in the subsequent multipolar mito-
sis (Chan et al., 2008). Mitotic slippage generates cells that contain tetraploid DNA
contents and two centrosomes, both of which can be duplicated during the subse-
quent S phase. Because centrosomes are microtubule organization centers, cells with
supernumerary centrosomes form multipolar mitotic spindles. The uneven segrega-
tion of genetic material into the daughter cells may result in different fates, including
mitotic catastrophe (Chan et al., 2008), aneuploidy, and transformation. Several
studies have provided evidence that tetraploidization increases chromosome insta-
bility in yeast (Mayer and Aguilera, 1990; Storchova et al., 2006) and mammalian
cells (Cowell, 1980; Fujiwara et al., 2005).
A p53-dependent tetraploidy checkpoint has been proposed to prevent S phase
entry in cells that have undergone adaptation or aborted cytokinesis (Andreassen
et al., 2001). However, the existence of the tetraploidy checkpoint has been disputed
(Fujiwara et al., 2005; Uetake and Sluder, 2004; Wong and Stearns, 2005). One
of the possibilities is that the p53-dependent arrest after tetraploidization is mainly
due to DNA damage or centrosomal stress during the aberrant mitosis (Storchova
and Kuffer, 2008). Mitotic catastrophe per se does not seem to depend on p53 as
it occurs in both p53-positive and p53-negative cells (Lanni and Jacks, 1998; Minn
et al., 1996). Nevertheless, p53 may affect mitotic catastrophe by indirectly regu-
lating cyclin B1–CDK1 activity. The abundance of cyclin B1 mRNA is negatively
regulated by p53 through a transcriptional repression mechanism (Dan and Yamori,
2001).
5.4 Normal Control of the DNA Damage and Replication

Checkpoints
Several checkpoints that monitor DNA integrity prevent precocious entry into
mitosis (Fig. 5.1). Progress in the past several years has unraveled very similar
underlying principles in the DNA replication checkpoint, the intra-S DNA dam-
age checkpoint, and the G2 DNA damage checkpoint in preventing the activation
of CDK1. In essence, DNA damage or replication stress activates sensors that facil-
itate the activation of the PI-3 (phosphoinositide 3-kinase)-related protein kinases
ATM and ATR. ATM/ATR then activates CHK1 or CHK2, which in turn inacti-
vates CDC25s and activates WEE1, culminating in the inhibitory phosphorylation
of CDK1 (Kastan and Bartek, 2004). We refer the reader below to relevant reviews.
Following exposure to ionizing radiation or other genotoxic insults that elicit
DNA double-strand breaks, ATM is autophosphorylated at Ser1981, leading to
dimer dissociation and activation of the kinase. ATR is activated by a broader spec-
trum of stress including ultraviolet irradiation, hypoxia, and replication stress. ATM
and ATR phosphorylate residues in the SQ/TQ domain of CHK1/CHK2, thereby
stimulating the kinase activity of these effector kinases.
The upstream sensors that initiate the activation of ATM/ATR consist of
an intricate network of large protein complexes, of which many components
contain the BRCT domain. These include the RAD9–HUS1–RAD1 (9-1-1) clamp
and the RAD17–RFC clamp loader that facilitate ATR-mediated activation of
CHK1 (Parrilla-Castellar et al., 2004). Another large complex that participates
in ATM/ATR activation is the so-called BRCA1-associated genome surveillance
complex composed of BRCA1, BLM, and MRN (MRE11–RAD50–NBS1) (Wang
et al., 2000; Jhanwar-Uniyal, 2003). Stalled replication forks mainly activate the
ATR–CHK1 pathway. Replication fork progression can be impaired by insufficient
nucleotide supply or lesions and obstacles on the DNA. Several proteins includ-
ing ATRIP (ATR-interacting protein), TopBP1, and Claspin appear to be required
for recruiting ATR to single-stranded DNA present at stalled replication forks to
phosphorylate CHK1 (Cimprich and Cortez, 2008). The ATR–CHK1 pathway is
essential even in the absence of exogenous stresses during unperturbed S phase,
probably for maintaining high rates of replication fork progression (Petermann and
Caldecott, 2006).
Claspin is usually degraded by SCFβ-TrCP -mediated ubiquitination following
the phosphorylation of Claspin by PLK1. This pathway is inhibited after DNA
damage (Freire et al., 2006). In response to genotoxic stress in G2 phase, the phos-
phatase CDC14B translocates from the nucleolus to the nucleoplasm and activates
APC/CCDH1 . This degrades PLK1 and consequently stabilizes Claspin, allowing the
G2 DNA damage checkpoint to be maintained (Bassermann et al., 2008).
CHK1 and CHK2 are believed to be involved in the inactivation of all three
isoforms of the CDC25 family (CDC25A, CDC25B, and CDC25C) (Boutros
et al., 2006). Phosphorylation of CDC25CSer216 by CHK1/CHK2 inactivates its
phosphatase activity either directly or indirectly through the creation of a 14-3-3
binding site. Binding of 14-3-3 masks a proximal nuclear localization sequence
and anchors CDC25C in the cytoplasm, preventing efficient access of CDC25C
to cyclin B1–CDK1. Interestingly, phosphorylation of a proximal site (Ser214) by
cyclin B1–CDK1 inhibits further phosphorylation of CDC25CSer216 . This provides
an elegant mechanistic explanation for the suppression of DNA damage-mediated
CDC25C inactivation during mitosis (Chen and Poon, 2008).
CDC25B is believed to possess a unique role in activating cyclin B1–CDK1
at the centrosome. A growing body of evidence indicates that CHK1 may shield
centrosomal cyclin B1–CDK1 from unscheduled activation by CDC25B during nor-
mal G2 phase and presumably also during the G2 DNA damage checkpoint. The
molecular basis of this activity may be due to CHK1-dependent phosphorylation
of CDC25BSer323 , creating a docking site for 14-3-3 that prevents access of sub-
strates to the catalytic site. Dissociation of CHK1 from the centrosomes at the end
of G2 phase, together with positive regulatory phosphorylation of CDC25BSer353
by Aurora A, enables CDC25B to activate the centrosomal cyclin B1–CDK1 and
initiate mitosis (Boutros et al., 2007).
CDC25A is arguably the most important member of the CDC25 family due to
its nonredundant role in mouse cells. CDC25A is targeted for rapid degradation
by CHK1/CHK2 through a ubiquitin-mediated mechanism. CDC25A stability is
controlled by the APC/CCDH1 complex during mitotic exit and early G1 , but by
the SCFβ-TrCP complexes during interphase. Importantly, the SCFβ-TrCP -dependent
turnover of CDC25A is enhanced in response to DNA damage. Phosphorylation

of CDC25ASer76 by CHK1 is required for the phosphorylation of a phosphode-
gron centered at Ser82 (by an as-yet-unidentified kinase), creating a binding site for
β-TrCP. Interestingly, β-TrCP also binds to a separate nonphosphorylated sequence
in CDC25A (the DDG motif) and plays a role in CHK1-induced ubiquitination and
degradation of CDC25A (Boutros et al., 2007).
There is also evidence that CHK1 can phosphorylate and activate WEE1 by pro-
moting 14-3-3 binding (Rothblum-Oviatt et al., 2001; Lee et al., 2001). Suppression
of CDC25s or activation of WEE1 promotes CDK1Thr14/Tyr15 phosphorylation, thus
preventing damaged cells from entering mitosis. Other mechanisms are also known
to play critical roles in the G2 DNA damage checkpoint. For example, 14-3-3σ
is involved in sequestering cyclin B1–CDK1 in the cytoplasm after DNA damage
(Chan et al., 1999). In addition to its well-known role in the G1 DNA damage check-
point, the p53–p21CIP1/WAF1 axis is also important for the G2 arrest after DNA
damage (Bunz et al., 1998). In p53-deficient cells, a pathway involving the MAP
kinase p38 and MK2 (MAPKAP kinase-2) is important for the G2 DNA damage
checkpoint (Reinhardt et al., 2007). Activation of this pathway by genotoxic stress
is also dependent on ATM/ATR.
5.5 Mitotic Catastrophe Caused by Abrogation of DNA

Integrity Checkpoints
Conceptually, it is vital to prevent the precocious activation of cyclin B1–CDK1

and mitotic entry as long as replication or DNA repair remains incomplete. It is well
known that bypass of the classic checkpoint pathways described above promotes
mitotic catastrophe. For example, cells lacking p53, p21CIP1/WAF1 , or 14-3-3σ fail to
arrest in G2 after DNA damage and undergo mitotic catastrophe (Chan et al., 1999;
Bunz et al., 1998). Depletion of p38 MAP kinase in p53-deficient cells leads to
checkpoint deregulations and mitotic catastrophe during DNA damage (Reinhardt
et al., 2007). Furthermore, the G2 DNA damage checkpoint is partially impaired in
many cancer cells (Fingert et al., 1986). They are unable to maintain G2 arrest and
eventually undergo aberrant mitosis and mitotic catastrophe (Chang et al., 2000).
Uncoupling of the ATM/ATR–CHK1/CHK2 axis can ablate the DNA damage
and replication checkpoints, inducing unscheduled activation of cyclin B1–CDK1
and mitotic catastrophe (Chan et al., 1999; Castedo et al., 2004b; Vogel et al., 2007).
Cells that contain defective ATM, such as those derived from ataxia telangiecta-
sia, often exhibit radio-resistant DNA synthesis and mitotic catastrophe (Lavin and
Khanna, 1999). In support of a role of CHK2 in the G1 DNA damage checkpoint, the
IR-induced G1 arrest is impaired in CHK2–/– mouse embryonic fibroblasts (Takai
et al., 2002). CHK2–/– mice are relatively normal and fertile. In contrast, ATR- and
CHK1-deficient mice die at an early embryonic stage with morphological abnormal-
ities similar to mitotic catastrophe (Brown and Baltimore, 2000; Takai et al., 2000;
Liu et al., 2000). Studies using conditional CHK1 knockout mice also revealed that
CHK1 deficiency causes inappropriate S phase entry, accumulation of DNA dam-

age during replication, and mitotic catastrophe (Lam et al., 2004; Niida et al., 2005).
Since the effect of the ATM/ATR–CHK1/CHK2 pathway is on the inhibitory phos-
phorylation of CDK1, it is not surprising that expression of a nonphosphorylatable
mutant of CDK1 can trigger mitotic catastrophe (Blasina et al., 1997; Chow et al.,
2003; Niida et al., 2005).
Agents that can inhibit the ATM/ATR–CHK1/CHK2 pathway can induce mitotic
catastrophe and many are potential chemotherapeutic agents. Caffeine is a clas-
sic agent that inhibits ATM/ATR (Blasina et al., 1999; Hall-Jackson et al., 1999;
Sarkaria et al., 1999) The checkpoints can also be uncoupled with CHK1 inhibitors
such as UCN-01 (Wang et al., 1996; Graves et al., 2000; Tse and Schwartz,
2004). However, UCN-01 also inhibits MK2 and may induce mitotic catastro-
phe through this pathway (Reinhardt et al., 2007). Likewise, inhibition of CHK2
promotes mitotic catastrophe after DNA damage (Castedo et al., 2004a). The
spindle-assembly checkpoint is required for mitotic catastrophe induced by abro-
gation of the DNA damage checkpoint (Nitta et al., 2004; Vogel et al., 2004),
suggesting a trap in mitosis is required for these types of cell death.
5.6 Mitotic Catastrophe as a Specialized Form of Cell Death

Involving CDK1
Mitotic catastrophe is generally defined as a type of cell death linked to abnor-

mal activation of mitotic kinases. It has been argued whether mitotic catastrophe is
a special case of apoptosis (Castedo et al., 2004b). Mitotic catastrophe frequently
displays phenotypic characteristic of apoptosis and shares several molecular hall-
marks with apoptosis such as caspase activation. Other studies suggest that mitotic
catastrophe represents a form of cell death distinct from apoptosis (Roninson et al.,
2001). The issue whether mitotic catastrophe is a mode of cell death or a process
leading to apoptosis is still controversial (Chu et al., 2004; Skwarska et al., 2007;
Vakifahmetoglu et al., 2008). The many overlapping effects of mitotic catastrophe
and apoptosis add to the uncertainty. For example, CDK1-dependent phospho-
rylations are important in triggering nuclear envelop breakdown and chromatin
condensation during normal mitosis and presumably also in mitotic catastrophe
(Porter and Donoghue, 2003). During apoptosis, these events also occur but are car-
ried out at least in part by caspases. Caspase-6 is essential for cleavage of nuclear
lamins (Orth et al., 1996; Takahashi et al., 1996) and for chromatin condensation
(Ruchaud et al., 2002), especially in cells that express lamin A/C (Slee et al., 2001).
In this connection, caspase-6 also cleaves cyclin B1 itself during mitotic catastrophe,
generating a non-degradable version of cyclin B1 (Chan et al., 2009).
What is the molecular basis of cell death during mitotic catastrophe? Cyclin
B1–CDK1 kinase activity is increased in apoptotic cells induced by different death
signals, suggesting that premature or prolonged activation of CDK1 may cause
apoptosis. Indeed, the fusion of mitotic cells with cells in S or G2 phase results
in mitotic catastrophe, probably due to the unscheduled active cyclin B1–CDK1

complexes (Mackey et al., 1996). Overexpression of cyclin B1 and CDK1 induces
premature chromosome condensation and mitotic catastrophe (Heald et al., 1993;
Jin et al., 1998). Prolonged accumulation of cyclin B1 is also the key requirement
for cell death after spindle disruption (Chan et al., 2008). Knockdown of cyclin
B1 inhibits mitotic catastrophe induced by the bypass of DNA damage checkpoints
(Chan et al., 2009).
While the underlying principles of CDK1-mediated toxicity in general remain
largely unresolved, a few targets that affect survival have been identified. Several
members of the BCL-2 family can be phosphorylated by cyclin B1–CDK1. BAD is
phosphorylated by cyclin B1–CDK1 at Ser128 (Berndtsson et al., 2005; Konishi
et al., 2002). This reduces the interaction between phosphorylated Ser136 and
14-3-3, allowing BAD to translocate to mitochondria and induce cell death. BCL-
2 is also phosphorylated by cyclin B1–CDK1, but the role in mitotic catastrophe
remains to be clarified (Brichese et al., 2002; Furukawa et al., 2000; Ling et al.,
1998; Scatena et al., 1998). Kinases other than CDK1 have also been implicated in
controlling the BCL-2 family during mitotic catastrophe. For example, disruption of
microtubules activates p38 MAP kinase, which induces the translocation of BAX to
mitochondria and enhances mitotic catastrophe (Deacon et al., 2003).
Survivin is another potential target of cyclin B1–CDK1 during mitotic catas-
trophe. Survivin is a member of the inhibitors of apoptosis (IAPs) family and is
believed to inhibit apoptosis. Survivin is also a component of the chromosome
passenger complex, requiring for a sustained spindle-assembly checkpoint arrest
in response to the lack of tension at the kinetochore (Carvalho et al., 2003; Lens
et al., 2003). Survivin accumulates during G2 /M phase and its degradation is con-
trolled by ubiquitination. Phosphorylation of survivinThr34 by CDK1 stabilizes the
protein (O’Connor et al., 2000). Since depletion of survivin by siRNA abolishes
Taxol-induced mitotic arrest and cell death (Carvalho et al., 2003; Lens et al.,
2003), the regulation of survivin by CDK1 probably contributes to the mitotic arrest
rather than apoptosis. In fact, inhibition of CDK1 with chemical inhibitors down-
regulates survivin expression and induces MYC-dependent apoptosis (Goga et al.,
2007). Given that the role of survivin in mitotic catastrophe is ambiguous, it is not
clear whether the cytotoxicity of chemicals that downregulates survivin (such as the
COX-2 inhibitor Celecoxib) involves mitotic catastrophe at all.
5.7 Mitotic Catastrophe and Cancer: Future Directions

Mitotic catastrophe has been studied from two perspectives in cancer research. On
the one hand, failure of some cells to undergo mitotic catastrophe is believed to
contribute to tumorigenesis. On the other hand, mitotic catastrophe is exploited as a
strategy to induce cell death in cancer treatments.
In cells that contain defective checkpoints, mitotic catastrophe represents a final
mechanism to prevent genome instability. Although most cells that bypass the DNA
integrity checkpoints eventually die through mitotic catastrophe, surviving cells

may undergo aberrant mitosis, resulting with mis-segregation of chromosomes and
cytokinesis failure. Likewise, cells that evade mitotic catastrophe after prolonged
activation of the spindle-assembly checkpoint enter a tetraploidy G1 state. If addi-
tional checkpoints are lacking (such as being p53 defective), aneuploid cells can
further undergo DNA replication and centrosome duplication, paving the way to
multipolar mitosis and further genome instability (Fig. 5.2). It has long been real-
ized that many tumors contain a population of polyploid cells (Goga et al., 2007).
It is likely that the ability to execute cytokinesis reduces as the ploidy increases,
giving rise to multinucleated giant cells (King, 2008).
Stresses Checkpoints Outcomes
Checkpoint recovery
Spindle disruption Spindle-assembly checkpoint
Checkpoint bypass
Replication block Replication checkpoint
Mitotic catastrophe
Cell cycle
DNA damage DNA damage checkpoint genome instability
Fig. 5.2 Mitotic catastrophe is an important mechanism to prevent genome instability.

Checkpoints are turned on in response to various stresses to halt the cell cycle. After the stresses
are resolved, the checkpoints are turned off to allow cell cycle progression. Prolonged activation
of the spindle-assembly checkpoints frequently results in mitotic catastrophe. Bypass of the DNA
integrity checkpoints may also induce precocious activation of cyclin B1–CDK1 and mitotic catas-
trophe. Cells that failed to undergo mitotic catastrophe with defective checkpoint execution may
complete mitosis inappropriately, resulting in genome instability
The presence of polyploid giant cells that fail to be killed by mitotic catastro-
phe may also account for resistant to cancer therapy. Following DNA damage (in
particular with relatively low dose of DNA damaging agents), many polyploid cells
appear after an initial phase of mitotic catastrophe and survive for weeks as mono-
or multinucleated giant cells (Blagosklonny, 1999; Puig et al., 2008). Whether these
cells still retain proliferative potential is controversial. Some groups claim that giant
cells have reduced or no proliferative potential (Therman and Kuhn, 1989). Others
showed that giant cells can undergo multipolar mitosis or depolyploidization to
return to near diploid state (Erenpreisa and Cragg, 2001). These studies suggest that
the multistep process of escaping mitotic catastrophe through polyploidization and
then depolyploidization may account for tumor relapse after initial efficient cancer
therapy.
Conversely, strategies have also been developed to sensitize cancer cells to
undergo mitotic catastrophe. Small molecules that inhibit the G2 DNA dam-
age checkpoint should in principle promote mitotic catastrophe when used in
combination with DNA damaging agents. Among these, the CHK1 inhibitor
UCN-01 is in relatively more advanced development (Senderowicz, 2003).
Inhibition of CHK1 with UCN-01 after DNA damage overcomes the DNA damage
checkpoints, inducing premature activation of cyclin B1–CDK1 and mitotic catas-
trophe. However, UCN-01 is also a potent inhibitor of protein kinase C, CDKs,
MK2, AKT (through inhibition of phosphoinositide-dependent kinase 1), and other
kinases. This promiscuous nature of UCN-01 (and protein kinase inhibitors in gen-
eral) makes defining its precise role difficult. For example, the two kinases that can
phosphorylate CDC25CSer216 – cTAK1 and CHK1 – can both be inhibited by UCN-
01 (Busby et al., 2000). Preclinical results using UCN-01 are promising and, thus,
several clinical trials that combine various DNA damaging drugs with UCN-01 are
under way (Blagden and de Bono, 2005; Kortmansky et al., 2005).
CDK1 inhibitors, such as roscovitine, are expected to inhibit mitotic catastrophe.
Indeed, some studies indicate that treatment with roscovitine blocks Adriamycin-
induced mitotic catastrophe (Park et al., 2005). However, since roscovitine itself is
a potent chemotherapeutic agent, there are many more reports showing that roscov-
itine enhances DNA damage-induced cell death (the underlying mechanism is not
obvious, but MYC-overexpressing tumors are particularly sensitive to roscovitine
(Goga et al., 2007)).
As with other potential therapies against cell cycle regulators, it is not immedi-
ately obvious why agents that induce genotoxic stress and mitotic catastrophe should
selectively target cancer cells and spare normal cells (Hunt, 2008). The usual expla-
nation is that the balance of various cell cycle regulators is severely altered in cancer
cells. This sensitizes cancer cells to agents that further disrupt cell cycle and check-
point control. A greater understanding of precisely how checkpoints are controlled
in normal and cancer cells is essential for designing better therapeutic agents.
Acknowledgments We apologize for those whose work that could not be cited due to space
constraints. Related works in our laboratory are supported by Research Grants Council grant
HKUST6439/06 M to R.Y.C.P.
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Yu H (2007) Cdc20: a WD40 activator for a cell cycle degradation machine. Mol Cell 27: 3–16.
Yu D, Jing T, Liu B et al. (1998) Overexpression of ErbB2 blocks Taxol-induced apoptosis by
upregulation of p21Cip1, which inhibits p34Cdc2 kinase. Mol Cell 2: 581–591.
Chapter 6
p53, ARF, and the Control of Autophagy
Robert D. Hontz and Maureen E. Murphy
Abstract The p53 and ARF genes encode well-known tumor suppressor proteins
that respond to oncogenic and genotoxic signals in order to induce growth arrest
or apoptosis. Recently, both of these proteins were found to be intimately tied to
metabolic pathways and to play surprising roles in autophagy. Autophagy (“self-
eating”) is a critical response of eukaryotic cells to stress. During this process,
portions of the cytosol, including cytoplasmic organelles, are sequestered into char-
acteristic double-membrane vesicles called autophagosomes that are delivered to
the lysosome for degradation. This rather non-specific degradation process allows
the cell to adapt to its bioenergetic needs and to prolong survival. The following
sections will outline the evidence for a role of p53 and ARF in autophagy, the role
of this pathway in cancer, and what questions remain to be answered.
6.1 The ARF Tumor Suppressor and Autophagy
The p14ARF tumor suppressor (p19ARF in mouse, and hereafter referred to as ARF)
is transcribed from within the INK4a/ARF locus using an alternate reading frame
from the p16INK4a tumor suppressor. The two overlapping tumor suppressor genes
comprise a locus that is commonly mutated and/or deleted in human cancer (Quelle
et al., 1995; Sherr et al., 2005). While a number of transcription factors coordi-
nately control the expression of the INK4a/ARF locus, the ARF and INK4a genes
have separate promoters and are chiefly regulated in an independent manner (Gil
and Peters, 2006). The ARF gene is tightly suppressed at the transcriptional level in
normal, non-transformed cells, but is upregulated in response to mutational activa-
tion of various oncogenes, including Ha-ras, c-MYC, or β-catenin, or in response
M.E. Murphy (B)

Program in Molecular and Translational Medicine,
Fox Chase Cancer Center, Philadelphia, PA 19111, USA
e-mail: maureen.murphy@fccc.edu
98 R.D. Hontz and M.E. Murphy
to chronic mitogenic stimuli, such as would occur in cultured cells (for review see
Sherr et al., 2005).
ARF is a predominantly nucleolar protein whose best understood function is as
a positive regulator of the p53 tumor suppressor protein. ARF stabilizes p53 by
sequestering and inhibiting the E3 ubiquitin ligase MDM2, which normally ubiq-
uitylates p53 and targets it for proteasome-mediated degradation. As a result, cells
with transcriptionally upregulated ARF undergo cell cycle arrest in a p53-dependent
manner and are likewise sensitized to p53-dependent apoptosis (for review see Sherr
et al., 2005). Interestingly, ARF can also induce cell growth arrest and apoptosis
in a p53-independent manner (Kelly-Spratt et al., 2004; Weber et al., 2000); this
is believed to occur via physical interaction with, and inhibition of, a number of
cell cycle regulators, including nucleophosmin (B23/NPM), DP1, E2F, c-myc (for
review see Sherr et al., 2005). When overexpressed in cells, ARF inhibits the func-
tion(s) of these proteins by sequestering them in the nucleolus. Additionally, ARF
can inhibit nucleophosmin and MDM2 function by enhancing the sumoylation of
these proteins (Tago et al., 2005). ARF has also been shown to suppress cell growth
by inhibiting the processing of precursor rRNA transcripts (Sugimoto et al., 2003).
Each of these diverse activities of ARF is believed to underlie at least part of the
tumor suppressor function of this protein.
6.2 ARF Induces Autophagy

It was first reported in 2006 that both human and mouse ARF can be translated from
an internal methionine (Met48 in human, Met45 in mouse) to generate a short ver-
sion of the protein denoted smARF (short mitochondrial ARF) (Reef et al., 2006).
smARF lacks the N terminus of ARF, which encodes much of ARF’s tumor sup-
pressor functions; specifically, smARF lacks the MDM2-binding domain (which
mediates p53-dependent growth arrest), the nucleolar localization sequence, the
nucleophosmin-binding domain, and the region required to inhibit rRNA process-
ing. Immunolocalization and biochemical fractionation studies determined that, in
contrast to full-length ARF, smARF predominantly localized to mitochondria (Reef
et al., 2006). The mitochondrial localization of smARF is supported by reports that
both human and mouse smARF interact with the protein p32, which is mitochon-
drial (Reef et al., 2007; Itahana and Zhang, 2008). It should be noted that one group
reported that, in addition to the mitochondria, smARF also localizes to the nucleus
and cytoplasm (Ueda et al., 2008).
Full-length ARF and smARF are generated in equal proportions using in vitro
translation reactions; however, smARF has a very short half-life in vivo, as it is
rapidly eliminated by proteasome-mediated degradation. Consequently, smARF is
present at very low steady-state levels (less than 5% of total ARF protein) (Reef
et al., 2006). Kimchi and colleagues found that enforced expression of smARF
resulted in abnormalities in mitochondrial membrane potential, which they found
were caused by increased autophagy. While autophagy is primarily regarded as a cell
survival pathway, chronic induction of autophagy is believed to lead to cell death.
6 p53, ARF, and the Control of Autophagy 99
Kimchi’s group showed that acute overexpression of smARF induces autophagic

cell death, in a p53- and caspase-independent manner.
Conflicting reports exist regarding the potential role of full-length ARF in
autophagy. Abida and Gu engineered a version of ARF in which the internal methio-
nine was mutated and showed that this form could induce autophagy in transfected
cells (Abida and Gu, 2008). In contrast, Reef and Kimchi published findings that
full-length (nucleolar) ARF is incapable of inducing autophagy except when ARF
was expressed at non-physiological levels, at which time ARF was observed in
extra-nuclear compartments (Reef and Kimchi, 2008). This group concluded that, at
physiologically relevant concentrations, only smARF was able to induce autophagy,
and that this occurred predominantly at mitochondria. Because both groups relied
heavily on transient transfection and overexpression of ARF and smARF in cells,
the contribution of full-length ARF to autophagy remains unresolved.
6.3 ARF-Mediated Autophagy Can Enhance Cell Survival

and Promote Tumor Progression
Recently our group reported that ARF has a pro-survival role in autophagy. Rather
than relying on overexpression of this protein in transfected cells, we took advan-
tage of the fact that p53 is a potent repressor of the ARF gene, and that p53-null
cells express high levels of endogenous ARF (for review see Gil and Peters,
2006). Building upon reports that ARF is frequently overexpressed in B-cell tumors
(Eischen et al., 1999), we silenced this gene in B-cell lymphomas using two different
short hairpins for ARF and found that silencing ARF impaired autophagy, as well
as the ability of these cells to establish tumors in vivo (Humbey et al., 2008). These
data support a cytoprotective (rather than cytotoxic) role for ARF in autophagy.
Consistent with this premise, we found that silencing ARF reduced the survival of
lymphoma cells and mouse embryo fibroblasts exposed to nutrient-deprived condi-
tions. These data may explain the large percent of human tumors with mutant p53
that retain high levels of ARF expression and support the premise that ARF and
autophagy promote the survival of some tumors.
The cytoprotective role of ARF and autophagy may be restricted to certain tumor
types. Specifically, we found that silencing ARF in three different primary lym-
phoma cell lines reduced survival and progression in vivo. In contrast, silencing
ARF in a p53-null sarcoma cell line enhanced the progression of this tumor in a
xenograft assay, despite clearly inhibiting autophagy (Pimkina and Murphy, 2009).
These data indicate that autophagy may be cytoprotective only for certain tumors.
Alternatively, ARF’s cytoprotective role may be balanced by its p53-independent
growth suppressive functions, and in the sarcoma cell line tested the latter effect
predominated.
Recently our group used the technique of two-dimensional in-gel electrophoresis
to identify ARF-interacting proteins at the mitochondria. This study revealed that
at the mitochondria, ARF physically interacts with the Bcl2 family member Bcl-xl
(Pimkina et al., 2009). Bcl-xl plays a known role in autophagy; it interacts with
Beclin-1 and negatively regulates the kinase activity of the Vps34/Beclin-1 com-
plex, which is a key mediator of autophagy (Maiuri et al., 2007; Pattingre et al.,
2005). Our group showed that ARF inhibits the ability of Bcl-xl to associate with
Beclin-1 complexes. Interestingly, the Beclin-1/Bcl-xl complex is notoriously dif-
ficult to detect by co-immunoprecipitation; our studies showed that in cells with
high levels of ARF, the Beclin-1/Bcl-xl complex is undetectable. However, in these
same cells, when ARF is silenced, the complex is readily detectable by immuno-
precipitation followed by immunoblotting (Pimkina et al., 2009). While regulation
of Beclin-1/Bcl-xl complex formation is one mechanism whereby ARF induces
autophagy, the fraction of ARF that interacts with Bcl-xl in cells is low, and there
are undoubtedly other mechanisms remaining to be identified.
6.4 The p53 Tumor Suppressor and Autophagy: p53 Induces

Autophagy
The groups of Levine and Jin were the first to demonstrate that the p53 tumor sup-
pressor regulates autophagy. Activation of p53 by DNA damaging agents led to
inhibition of mammalian target of rapamycin (mTOR) and induction of autophagy,
as assessed by autophagosome formation and LC3 lipidation and cleavage (Feng
et al., 2005). There were two mechanisms proposed for the inhibition of mTOR
activity by p53: first, that in a p53-dependent manner, genotoxic stress induced
the activation of the enzyme AMPK, which is a kinase that normally responds to
metabolic stress. This increase in AMPK activity was not as impressive as that
induced by glucose starvation, but it correlated well with the decrease in mTOR
induced by p53. Notably, the authors showed that compound C, which inhibits
AMPK, completely alleviated the inhibition of mTOR by p53, supporting the
premise that p53’s negative regulation of mTOR was via AMPK (Feng et al., 2005).
These authors later showed that in some cell types p53 induction led to the transcrip-
tional activation of PTEN, TSC2, and AMPK β1, which negatively regulate mTOR;
this effect of p53 on mTOR was estimated to occur in a slower time frame than
activation of AMPK by p53, which takes minutes to hours (Feng et al., 2005, 2007).
More recently Budanov and Karin showed that the products of two well-defined
p53 target genes, Sestrin1 and Sestrin2, can bind and activate AMPK, causing it to
phosphorylate TSC2 and thereby inhibit mTOR (Budanov and Karin, 2008). The
combined data indicate that p53 uses multiple avenues in order to inhibit mTOR
and subsequently induce autophagy.
6.5 p53 Transactivates the Autophagy Gene DRAM
Another mechanism whereby p53 induces autophagy emerged with the discovery
by the group of Ryan that p53 directly transactivates the gene encoding DRAM
(damage-regulated autophagy modulator). DRAM encodes a highly conserved lyso-
somal protein with six putative transmembrane domains (Crighton et al., 2006).
p53 binds directly to a consensus p53-binding site in the DRAM promoter fol-
lowing genotoxic stress. Notably, silencing DRAM was shown to markedly inhibit
p53-mediated apoptosis, while overexpression of DRAM alone had limited cyto-
toxic effect on cells. Therefore, DRAM was found to be necessary but not sufficient
for programmed cell death mediated by p53. Interestingly, in support of a role for
autophagy in p53-dependent apoptosis, the authors found that silencing ATG5 like-
wise inhibited apoptosis induced by p53. A role for autophagy in p53-mediated
apoptosis is likewise supported by the findings of Vousden and colleagues, who
showed that the p53 target genes PUMA and Bax induce mitophagy (autophagy
of mitochondria) in response to mitochondrial perturbations, and that inhibition of
PUMA- or Bax-induced mitophagy dampens the apoptotic response (Yee et al.,
2009). These data firmly support a role for autophagy in the successful execution
of apoptosis. Why autophagy might be required for the completion of apoptosis is
not presently clear.
6.6 Nutrient Stress Signals to p53
The groups of Jin and Thompson first showed that nutrient deprivation signals to
p53. As early as 20 min after glucose deprivation, p53 becomes phosphorylated on
serine 15, a hallmark signal of p53 activation (Feng et al., 2005; Jones et al., 2005).
This phosphorylation is mediated by the kinase AMPK, which is thought to serve as
a fuel sensor in the cell by assessing the ratio of AMP to ATP. Phosphorylation
of p53 induces a p53-dependent cell cycle arrest, indicating that like genotoxic
stress, nutrient stress may also use the p53 pathway in order to achieve cessa-
tion of cell proliferation. Whereas Thompson and colleagues showed that p53,
along with phosphorylation on serine 15, was essential for AMPK-mediated growth
arrest in low glucose, it remains unclear whether p53 is a direct or indirect tar-
get of AMPK, and it seems likely that there is an intermediate kinase involved.
Interestingly, Thompson’s group pursued the connection between nutrient stress
and p53 further and showed recently that the anti-diabetes drug metformin and
the AMPK-activating drug AICAR both effectively diminish the growth of p53–/–
tumors, but not matched p53+/+ counterparts (Buzzai et al., 2007).
6.7 p53 Negatively Regulates Autophagy in Unstressed Cells
Seemingly in contrast to the abundant data that p53 induces autophagy in stressed
cells, Kroemer and colleagues reported that p53 inhibits autophagy in unstressed
cells. This group showed that silencing of p53 or pharmacological inhibition of
this protein led to an increase in the steady-state level of basal autophagy, in
both mammalian cells and in Caenorhabditis elegans (Tasdemir et al., 2008a, b).
Consistent with this premise, certain tissues of the p53 knockout mouse, such as
the liver, pancreas, and kidney, have increased basal levels of autophagy compared
to wild-type mice. Similarly, p53–/– MEFs survive more efficiently in response

to metabolic stress compared to wt MEFs (Tasdemir et al., 2008a, b). This group
found that autophagy induced by p53 silencing occurs only in cells in the G1 and
S phases of the cell cycle (Tasdemir et al., 2008c). Further, they showed that silenc-
ing p53 in C. elegans increases the life span of this organism by causing increased
autophagy (Tavernarakis et al., 2008). Somewhat surprisingly, the Kroemer group
obtained data supporting a role for cytoplasmic p53 in the inhibition of autophagy.
Using overexpressed mutant forms of p53 in transfected cells, this group found
that the nuclear export sequence (NES) of p53 is required for its ability to inhibit
autophagy, although whether the NES mutant was otherwise functional or capable
of oligomerizing appropriately was not tested (Tasdemir et al., 2008a). More sur-
prisingly, this group reported that some tumor-derived point mutants of p53 could
likewise inhibit autophagy; again in transfected cells, this group found that tumor-
derived mutant forms of p53 that typically localize to the nucleus (such as R282W)
fail to suppress autophagy, while mutants that consistently localize to the cytosol
(such as R273H) were highly efficient autophagy inhibitors (Morselli et al., 2008).
Unclear from these studies is whether tumor cell lines engineered to stably express
these mutants differ in the basal level of autophagy or whether this was an effect of
acute p53 overexpression. A notable difference between many of the mutant forms
of p53 that varied in autophagy suppression is that several of the mutants inca-
pable of inhibiting autophagy are typically globally denatured (such as R282W),
while those that can inhibit autophagy are largely native in conformation (such as
R273H). These findings may have interesting implications for our understanding of
why different tumor types tend to select for different “hotspot” mutations in p53,
particularly in lieu of the findings that different tumor types may have different
sensitivity to autophagy inhibition.
The findings presented here on the role of p53 and ARF in autophagy are at first
blush somewhat contradictory. For example, stress-activated p53 induces autophagy,
while “unstressed” p53 represses basal levels of autophagy (Fig. 6.1). How these
findings are reconciled is not presently known. One clue may be that p53, particu-
larly the p53-induced genes PUMA and Bax, may selectively induce mitochondrial
autophagy (mitophagy), and this may allow for the release of mitochondrial induc-
ers of apoptosis such as cytochrome c more effectively (Yee et al., 2009). In contrast
Kroemer’s group found that silencing IREα, which controls one arm of the endo-
plasmic reticulum (ER) stress pathway, effectively inhibits the ability of p53 to
suppress autophagy; in other words, p53 may suppress the ER stress pathway, and
silencing of p53 may predominantly signal ER stress and concomitant autophagy.
As such, more careful delineation of the types of autophagy being induced (that
is, mitophagy versus reticulophagy versus non-specific autophagy) along with the
upstream signaling pathway (such as nutrient deprivation, ER stress, or genotoxic
Fig. 6.1 The mechanisms whereby p53 and ARF, in stressed and unstressed cells, regulate
autophagy
stress) may clarify some of these apparent discrepancies. The contribution of ARF
to the autophagy pathway(s) regulated by p53 is unclear: Because the majority of
tumor cell lines inactivate either p53 or ARF, this question can only be addressed in
primary mouse embryo fibroblasts or in tissues from the p53 knockout mouse, com-
pared to the p53/ARF double knockout. Additionally, how ARF induces autophagy
and the rules that govern whether ARF-mediated autophagy is cytoprotective or
cytotoxic need to be delineated. The combined data suggest that the p53 and ARF
tumor suppressors are very intimately tuned into the metabolic state of the cell, per-
haps even more so than to the DNA damage pathway. It is tempting to speculate that
the functions of p53 and ARF originally evolved to monitor the metabolic state of
the cell and to induce growth arrest, autophagy, or apoptosis in response to nutrient
signals; these pathways may then have been adapted to monitor genotoxic stress.
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Part III
Long-Term Proliferation
Chapter 7
Regulation of Self-Renewing Divisions
in Normal and Leukaemia Stem Cells
Andrea Viale and Pier Giuseppe Pelicci
Abstract The identification of a rare population of tumour cells capable of trans-

planting the disease in animal models has radically modified our perception of
the biological organization of tumours. These cells, named tumour-initiating cells
(TICs), or cancer stem cells (CSCs), share phenotypic and functional characteristics
with normal stem cells, most notably their ability to self-renew. Here we discuss reg-
ulation of self-renewal in normal haematopoietic and leukaemic stem cells, focusing
on the role of cell quiescence and inhibitors of the cell cycle.
In the last decade the identification of a rare population of tumour cells capable of
transplanting the disease in animal models has radically modified our perception of
the biological organization of tumours. These cells, named tumour-initiating cells
(TICs), or cancer stem cells (CSCs) because they share phenotypic and functional
characteristics with normal stem cells, have been initially identified in the myeloid
acute leukaemias (Lapidot et al., 1994), and only more recently in solid tumours
(Al-Hajj et al., 2003; Collins et al., 2005; Kim et al., 2005; Li et al., 2007; O’Brien
et al., 2007; Ricci-Vitiani et al., 2007; Schatton et al., 2008; Singh et al., 2004).
Their discovery in tumours of various histotypes has reinforced the hypothesis that
cancer is in reality an anomalous tissue recapitulating the hierarchical organization
found in the normal tissue from which it originates. In this model, a rare population
of stem cells (SCs) would then be a valuable source of progenitor cells in continu-
ous expansion, able to generate more differentiated cells and initiate tumours upon
transplantation into a new host (Bonnet and Dick, 1997).
In the same way as normal haemopoiesis has been the paradigm for studies on
adult stem cells, acute myeloid leukaemias are the paradigm for investigations on
cancer stem cells (Huntly and Gilliland, 2005). Indeed, a huge amount of data is now
available with regard to both the normal and the leukaemic haemopoietic system,
which will be the focus of our review.
A. Viale (B)
Department of Experimental Oncology, European Institute of Oncology at IFOM-IEO-Campus,
Milan, Italy
e-mail: andrea.viale@ifom-ieo-campus.it; andrea_viale@dfci.harvard.edu
110 A. Viale and P.G. Pelicci
7.1 Self-Renewal Potential of Normal Haematopoietic

Stem Cells Is Limited
Haemopoiesis is a tightly regulated process which controls the continuous replace-

ment of differentiated blood cells. It has been estimated that throughout one’s life
the haematopoietic system produces more than 1016 cells. This process relies on the
presence of rare haematopoietic stem cells (HSCs) that divide giving rise to highly
proliferating progenitor cells, sometimes known as transit-amplifying cells, which
after several cycles of division become terminally differentiated. Thus, long-lived
multicellular organisms have developed a strategy which leads to the amplification
of a relatively small number of HSCs into a huge numbers of progeny.
The unique ability of a stem cell to originate two daughter cells with different
replicative and differentiative potentials (one transit-amplifying progenitor and a
new stem cell) is known as self-renewal. Because stem cells maintain tissue home-
ostasis throughout the lifespan, one could assume that they have, in contrast to other
somatic cells, an infinite replication potential. However, since the 1960s–1970s, it
has been clear that during transplantation of bone marrow from a mouse to another,
an assay known as serial transplantation, the ability of HSCs to sustain long-term
multilineage reconstitution of the host is limited to ∼5–6 rounds (Harrison, 1979;
Harrison and Astle, 1982; Harrison et al., 1978), suggesting that HSCs have a
limited replication potential.
Recovering the entire haemopoiesis in irradiated mice is in fact a big challenge
for transplanted HSCs. Not only are they immediately induced to proliferate after
transplantation to quickly regenerate the damaged tissue, but a conspicuous frac-
tion continues to proliferate for longer period, at least 4 months (Allsopp et al.,
2001). Perpetuation of this hyperproliferative state leads ultimately to functional
exhaustion of the HSCs. The number of divisions that one HSC can undergo
during the lifespan of a mammal is relatively constant (∼80–200) and evolution-
arily conserved (Shepherd et al., 2007); as a result of more frequent divisions,
a stem cell reaches its replicative ceiling much sooner, loosing the ability to
self-renew.
7.2 Haematopoietic Stem Cells Are Deeply “Dormant”

Another supposed hallmark of SCs, as compared to their more differentiated pro-
genitors, is that of infrequent division, often referred to as “quiescence” or “G0”. A
variety of evidence supports this idea. For example, several groups described HSCs
as resistant to diverse cell cycle specific cytotoxic agents such as 5-fluorouracil, that
eliminates rapidly dividing cells while sparing non-cycling (long-term reconstitut-
ing) stem cells (Hodgson and Bradley, 1979; Lerner and Harrison, 1990; Randall
and Weissman, 1997).
Due to the growing interest in stem cells, different assays have been developed
to address the issue of quiescent or “resting” cells in vivo. Amongst these, the
7 Regulation of Self-Renewing Divisions 111
method of direct evaluation of total RNA, hypothesizes that quiescent cells have
less RNA than their proliferating counterparts (Darzynkiewicz et al., 1979). Indeed,
detection in a stem cell population of both RNA and DNA content (using RNA-
specific flourochrome Pironin Y and DNA-specific dye Hoechst) demonstrated that
75% of HSCs have low levels of RNA and DNA, compatible with a quiescent
state (Cheshier et al., 1999). Another method most frequently used to analyse in
vivo cell cycle status uses the 5 -bromo-2 -deoxyuridine (BrdU). BrdU, a synthetic
nucleoside analogous to thymidine and incorporated in newly synthesized DNA dur-
ing replication, has been extensively used to study both proliferation (after acute
incorporation) and quiescence. The latter requires exposure of mice to BrdU for long
intervals of time (pulse), followed by several months of no BrdU intake (chase);
the cells that retain BrdU, known as label retaining cell (LRC), are supposed to
be “quiescent” or slowly proliferating cells. Using these approaches authors have
demonstrated that HSCs proliferate slowly: ∼8% enters the cell cycle per day but in
due course all HSCs are recruited into cycle with a frequency of division of around
2 months (Cheshier et al., 1999). These experimental evidences are coherent with
a model of haemopoiesis in which the haemopoietic SC compartment consists of a
homogeneous population of cells that divide asynchronously and infrequently, and
where quiescence is not a functional defined state in which stem cells are “frozen,”
but rather a simple period of time between divisions, however long it may be.
One of the biggest obstacles to studying the cell cycle of HSCs is their extreme
rarity (<0.01% in the bone marrow). Moreover, procedures to isolate HSCs on the
basis of their defined surface marker profile (i.e. multiparametric flow cytometry)
are quite complex, and it is impossible to purify living HSCs based on their cell
cycle properties. For instance, the isolation of quiescent LRC cells using BrdU
includes cell fixation procedures, thus excluding the possibility to evaluate their
long-term reconstituting potential in vivo. These experimental limits have hampered
the understanding of the functional relationship between cell cycle and stemness,
and quiescence remained nothing more than an assumption until few months ago.
Recently, indeed, the use of a new pulse–chase system (Tumbar et al., 2004),
based on transgenic mice that express an inducible histone H2B–green fluorescent
protein (H2B–GFP) in HSC/progenitor cells, has allowed the definitive character-
ization of a HSC subpopulation that spends most of its time outside the cell cycle
in a state of apparent “dormancy” (Wilson et al., 2008). After the induction of the
expression of H2B–GFP (pulse) in HSCs and a chase of several months, this system,
similarly to the BrdU assay, allows the identification of “quiescent” (GFP-positive
LRC) and proliferating (GFP-negative non-LRC) stem cells, but the improvement
is significant. Living GFP-positive HSCs, in fact, can be isolated and their func-
tions characterized. Since GFP intensity will inversely correlate with number of
mitoses, this approach also allows correlations of repopulating activities and pro-
liferative potential. The evaluation of the dynamics of GFP decay in vivo revealed
the existence of two well-defined subpopulations of stem cells: a minor fraction of
“dormant” HSCs (15%) dividing approximately every 150 days and a major fraction
of “activated” and more proliferating HSCs (85%) dividing every 30 days (Wilson
et al., 2008). During homeostasis “dormant” HSCs are in a deep state of quiescence
as suggested by a marked downregulation of almost all genes involved in the assem-

bly and activation of the pre-replicative complex and divide no more than five times
per mouse lifetime (Wilson et al., 2008). Dormancy of HSCs is a re-activable state:
when haematopoietic tissue is lost or damaged by an insult, e.g. chemotherapy,
nearly all “dormant” HSCs are robustly recruited to cell cycle, to later re-enter,
once damage is repaired and homeostasis re-established, into a state of dormancy
(Wilson et al., 2008).
Interestingly, some physiological stimuli, such as G-CSF and IFNα, cause the
activation of HSCs (Essers et al., 2009). This is not surprising if we consider that
both of them are mediators of stressful conditions, like infections and inflammatory
responses. G-CSF, the major cytokine regulator of neutrophilic granulocytes, is well
known for its effects on the stem cell compartment. Indeed, its capability to increase
the number of HSCs in peripheral districts when used in association with cyclophos-
phamide, a process known as “stem cell mobilization” (Duhrsen et al., 1988; Petit
et al., 2002), is probably involved in tissue regeneration (Koda et al., 2007; Srinivas
et al., 2009).
“Dormant” and “activated” HSCs are not independent compartments but are
hierarchically related, with “activated” HSCs derived directly from the prolifera-
tion of “dormant” HSCs. Noteworthy, “dormant” HSCs have a better multilineage
bone marrow reconstitution capacity and their self-renewal potential, as assessed
by serial bone marrow transplantation, is markedly higher than that of proliferat-
ing HSCs, providing a strong demonstration that self-renewal potential inversely
correlates with HSC mitotic division number (Wilson et al., 2008).
Compelling support for the hypothesis that “dormant” HSCs lose some of their
self-renewal capacity at each division comes from the observation that all activat-
ing stimuli, when “chronically” repeated, induce their functional exhaustion (5–6
rounds of serial transplantation, 4–6 sequential 5-FU treatments, 8 consecutive IFNα
treatments) (Viale and Pelicci, 2009).
Thus, under steady-state conditions “dormant” HSCs might be induced to cycle
in order to substitute exhausting “activated” HSCs and maintain tissue homeosta-
sis. This process might happen infrequently at any time, yet continuously during
a lifespan, leading to the progressive exhaustion of the stem cell pool. Under this
assumption, the number of functionally self-renewing stem cells should reflect
the age of an individual and, indeed, in aged mice HSCs are more numerous yet
functionally exhausted (Rossi et al., 2007).
7.3 Genetic Models of Stem Cell Exhaustion
Almost all knockout models that lead to loss of SC quiescence also result in HSC
exhaustion, thus providing genetic evidence that a tight regulation of the HSC cell
cycle might be a key point in the maintenance of self-renewal.
One of the first studies focused on p21cip1/kip1 (hereafter p21), a cyclin-
dependent kinase inhibitor (CDKI). p21-null mice are apparently normal, but the
number of HSCs tends to increase in homeostatic conditions due to a reduction

in quiescence. p21 negatively regulates the cell cycle entry of HSCs and null
animals become more sensitive to repeated 5-FU treatments and serial bone mar-
row transplantation, which in turn lead to exhaustion of stem cells (Cheng et al.,
2000).
A similar phenotype has been described in mice deficient for the growth fac-
tor independent 1 (Gfi-1), a SNAG domain-containing zinc-finger transcriptional
repressor. In its absence, HSCs show enhanced proliferation and are functionally
compromised in competitive and serial bone marrow transplantations. Interestingly,
in the absence of Gfi-1, p21 is markedly downregulated suggesting the possibility
of a common mechanism responsible for stem cell defects (Hock et al., 2004).
Other examples described abnormal signalling through the PI3K–Akt pathway.
One is the forkhead O (FoxO) subfamily of transcription factors, a large family of
proteins involved in several physiological functions such as stress, cell cycle arrest
or metabolism, and playing an important role as downstream mediators of PI3K–
Akt. FoxO members directly regulate proteins that have a significant role in the
regulation of cell cycle checkpoints (G0–G1 transition, G1–S and G2–M); mice
null for FoxO1–3–4 (triple knockout), as well as those null only for FoxO3, show
increased proliferating HSCs. This leads to a marked reduction in number, in turn
correlating with functional impairment of their repopulating potential and premature
exhaustion (Miyamoto et al., 2007; Tothova et al., 2007). Inactivation in the bone
marrow of phosphatase and tensin homologue (PTEN), a negative key regulator
of PI3K kinase, has the same effect, driving HSCs into proliferation and, after a
transitory expansion, to depletion (Zhang et al., 2006). Inactivation of the tuberous
sclerosis complex 1 (TSC1), a tumour suppressor of the complex TSC1–TSC2 that
negatively modulates the function of mTORC1, also markedly induces HSCs to
proliferate with consequent loss in number and repopulating potential (Chen et al.,
2008; Gan et al., 2008). Thus, TSC1 and FoxO, two different branches downstream
of Akt, appear as the key effectors of the regulation of stem cells maintenance by
the PI3K–PTEN–Akt pathway.
In another model is the deletion of Fbw7 to lead to SC exhaustion through their
release from G0. Fbw7 is a F-box protein subunit of the SCF-type ubiquitin ligase
complex (Matsuoka et al., 2008) that ubiquitilates for degradation several mediators
of the G0–G1 transition, such as cyclin E and c-Myc.
Finally, other models involve the “niche” in the regulation of quiescence.
Stemness is not simply a cell autonomous feature, so that each stem cell is intrin-
sically able to self-renew until commitment signals drive it into differentiation, but
rather it appears as a functional state regulated by complex interactions within the
immediate microenvironment, the so-called niche.
A few genetic models correlate occupancy of the niche with maintenance of
quiescence and self-renewal, so that exit from the niche coincides with stem cell
replication and differentiation. One of the best examples is the over-expression
of the transcription factor c-Myc. c-Myc is a proliferative signal and when over-
expressed in HSCs results in their egress from the niche and their exhaustion
(Wilson et al., 2004). Similarly, conditional knockout of CDC42, a Rho GTPase
involved in actin polymerization, or Tie2, the angiopoietin receptor, results in alter-

ations of stem cell–niche interactions that consequently lead to loss of quiescence
and self-renewal (Arai et al., 2004; Yang et al., 2007). Finally, we should note that
genetic models have also been reported, such as those with loss of p18 (cdkn2c)
or HoxB4 over-expression (Antonchuk et al., 2002; Yuan et al., 2004), whose data
are apparently at odds with the assumption that an increase in HSC number corre-
sponds to functional loss. Yet, the anticipated increase in cycling is either not shown
(p18) (Yuan et al., 2004) or even excluded (HoxB4 over-expression) (Schmittwolf
et al., 2005). In these cases, HSC number-increase could depend on other biological
mechanisms, for instance improved survival. Alternatively, HoxB4 over-expression
or loss of p18 could contribute to other biological properties important for HSC
replicative potential.
7.4 Molecular Mechanisms of Stem Cell Quiescence

The G1–S transition of the cell cycle is tightly regulated. As mitogens drive qui-
escent cells to enter the cell cycle, the cyclin D-dependent kinases (cycD-cdk4/6
complexes) phosphorylate Rb that releases E2F from negative constraints leading
to activation of the E2F-responsive genes necessary for S-phase entry (including
cyclin E; see Chapter 2 by Li and Dyson and Chapter 1 by Sotillo and Grana, this
volume). In turn, cyclin E forms complexes with its catalytic partner cdk2 (cycE–
cdk2) that complete phosphorylation of Rb, enabling the full E2F response and entry
into S phase. The actions of cyclin-dependent kinases (cdk) are opposed by two
families of inhibitors: members of the INK4 family (p16, p18, p19) that bind specif-
ically with cdk4/6 and of the Cip/Kip family (p21, p27, p57) that interacts with both
cdk4/6 and cdk2 (Sherr, 2000). Despite the availability of genetic models for each of
the indicated proteins, the molecular machinery that controls cell cycle progression
in SCs remains poorly characterized. Undeniably, the paucity of stem cells effec-
tively prevents most analytical methods, leaving mRNA profiling as the most-easily
accessible approach.
Quiescent stem cells under steady-state condition are mainly characterized by
high expression of cyclin-dependent kinase inhibitors (CDKI). Long-term repopu-
lating (LT) HSCs show high mRNA levels of members of the INK4 (p16, p18, p19)
and Cip/Kip (p21, p27, p57) families of CDKIs and low levels of G1 and S-G2-M
cyclins, especially cyclins E and A (Passegue et al., 2005). This expression pattern
suggests that stem cells are in a functional state characterized by a sustained Rb-
mediated transcriptional repression, probably mediated by p130, the most expressed
Rb family member in HSCs. Once this equilibrium is broken by myelosuppressive
chemotherapy or mobilization (induced by G-CSF and cyclophosphamide), LT-HSC
undergo a dramatic expansion due to a robust recruitment into the cell cycle, but
promptly return to quiescence once the tissue is repaired. Associated with this pro-
cess is early upregulation of cyclin D3 and downregulation of all CDKIs, which
might lead to induction of cyclin E1 and several mitotic cyclins responsible for
the sustained proliferation, as a consequence of the inactivation of E2F repression
(Passegue et al., 2005). Interesting in this dynamic process is the behaviour of p21.
Although with different kinetics (Passegue et al., 2005; Venezia et al., 2004), p21
is highly expressed in stem cells under steady-state condition (Cheng et al., 2000;
Venezia et al., 2004) and further upregulated after the proliferative phase in order to
mediate their return to a condition of quiescence (Venezia et al., 2004).
7.5 Molecular Mechanisms of Stem Cell Exhaustion
Why does stem cell proliferation lead to functional exhaustion? One suggestive
hypothesis is that for a stem cell the early G1 phase of the cell cycle is a crucial step,
the moment when cell fate decisions are made (Calegari et al., 2005; Calegari and
Huttner, 2003). In this view, quiescence would appear as a strategy used by HSCs to
avoid exposure to exhausting stimuli (i.e. differentiation) during G1 transition, and
genes that prevent cell cycle entry would be pivotal in reducing this exposure time
(Orford and Scadden, 2008).
However, an alternative explanation is emerging and recent studies strongly sug-
gest a common paradigm for stem cell exhaustion. In vivo, stem cell functions are
regulated by the interactions with their niches. In the current model, “dormant” stem
cells seem to be localized at the lining edge between bone marrow and endosteum
of the trabecular bone, in contact with specialized cells, known as spindle-shaped
N-cadherin-positive osteoblasts (SNO) (Wilson et al., 2008; Zhang et al., 2003).
Stem cells and SNO form a N-cadherin-based adherent junction that polarizes and
retains stem cells in place, preserving their quiescence and stemness (Wilson et al.,
2007). When injury stimuli activate a “dormant” stem cell, this cell exits from the
osteoblast “dormant” niche (Venezia et al., 2004; Wilson et al., 2004, 2007) and
moves in contact with sinusoids, in a so-called vascular niche, ready to proliferate
(Kiel et al., 2005; Wilson et al., 2007). The two niches have a different O2 level,
functional to the different fate of the hosted stem cell: a hypoxic environment for
dormancy and a normoxic environment for active proliferation (Wilson et al., 2007).
This model seems compatible with the observation that the highest concentration
of HSC activity in the bone marrow is localized in the regions of lower perfu-
sion characterized by a lower O2 tension (Parmar et al., 2007). Thus, hypoxia or
a low-oxygen milieu might protect long-term HSCs from reactive oxygen species
(ROS)-related oxidative stress, preserving stem cell function. Noteworthy, in vivo
the self-renewing potential of HSCs is inversely proportional to their level of ROS
(Jang and Sharkis, 2007); in fact, in serial transplantation assays HSCs with high
level of ROS undergo exhaustion prematurely, whereas those characterized by low
level of ROS maintain their function, appear more quiescent and localize in an
osteoblast niche.
Accordingly, several genetic models of reduced self-renewal involve an increase
of ROS levels in HSCs. For instance, deficiency of the ataxia telangiectasia mutated
gene (ATM), a key molecule in the maintenance of genomic stability, affects the
HSC ability to self-renew through an elevation of ROS levels. High levels of ROS
induce the phosphorylation of p38 MAPK specifically in the stem cell compartment
leading to loss of quiescence (Ito et al., 2004, 2006). Bmi1 is a member of the
Polycomb family of transcriptional repressors essential for the maintenance and
self-renewal of both haematopoietic and leukaemic stem cells. Recently, the defect
in “stemness” of Bmi1 null mice (Lessard and Sauvageau, 2003; Park et al., 2003)
has been also associated to an increase of intracellular ROS (Liu et al., 2009). These
findings are not surprising, as ROS levels go up after activation of mTORC1 (Chen
et al., 2008) or loss of FoxO (Miyamoto et al., 2007; Tothova et al., 2007), both
essential regulators of cell growth.
Since ROS are the major source of endogenous DNA damage (Maynard et al.,
2009) stem cells exhaustion might be due to accumulation of genomic damage.
Mutations arising from such damage would then result in progressive loss of typical
HSC functions, in particular their capacity to replicate. Accordingly, during mouse
aging, HSCs progressively accumulate DNA damage and lose their ability to self-
renew (Rossi et al., 2007).
In this scenario, quiescence, characterized by low levels of ROS and low fre-
quency of DNA replication, could preserve stem cell function by limiting the
accumulation of mutations. Alternatively, the increased DNA damage could be the
mere consequence of hyperproliferation, following the so-called replicative stress.
However, there are no direct data in support of this second hypothesis.
7.6 Existence of Leukaemic Stem Cells
That tumours are morphologically heterogeneous was a concept well defined by

pathologists already in the early nineteenth century, but only several decades later
investigators began to study this heterogeneity from a functional point of view. Ever
since the 1950s–1960s, in vitro and in vivo studies have shown that, within a given
leukaemia sample, very few cells possess clonogenic activity and the ability to trans-
plant the disease in secondary hosts (Bruce and Van Der Gaag, 1963; Griffin and
Lowenberg, 1986; Moore et al., 1973). These observations, whilst not directly prov-
ing the existence of leukaemia stem cells (LSCs), strongly suggested that only a
restricted population of cells have a functional role in the growth and maintenance
of leukaemias.
Evidence of the existence of LSCs arrived only several years later, when the full
development of flow cytometry methods and new xenotransplantation models led to
overcoming several technical issues. Scientists demonstrated that, within different
subtypes of human acute myeloid leukaemias, the rare population of blasts able to
transplant leukaemias in experimental models is characterized by a unique surface
phenotype (CD34+ CD38– ) (Ailles et al., 1999; Lapidot et al., 1994). Amazingly,
this phenotype is the same immunophenotype of normal HSCs. Moreover, these
leukaemic blasts, also called SL-IC (SCID leukaemia-initiating cell), recreate the
entire heterogeneity of the human disease upon transplantation and give rise to
the CD34+ CD38+ cells, as well as to the other subpopulations (Bonnet and Dick,
1997). Although rare, around one per 1×106 leukaemic cells, LSCs themselves are
a functionally heterogeneous cell population, comprising distinct classes of cells

with different self-renewal potential. Indeed LSCs, like their normal counterparts,
can be separated into “long-” and “short-repopulating” cells on the basis of their
capacity to perpetuate the leukaemic clone during serial transplantation assays.
(Hope et al., 2004). It is worth mentioning that the “short-repopulating” cells are
generated by the “long-repopulating” LSCs, thus suggesting a complex hierarchi-
cal organization similar to that of the normal haematopoietic system (Hope et al.,
2004). This organization analogy between normal haemopoiesis and acute myeloid
leukaemias strongly supports the idea that tumours are just atypical tissues and,
as normal tissues, made of subpopulations of cells with different functions and
morphologies.
7.7 Leukaemic Stem Cells Are Quiescent
Several years ago proliferation assays carried out directly in leukaemic patients,
administering 3H-thymidine, demonstrated that tumour cells were almost totally
post-mitotic, with a minority of blasts in active growth (Clarkson, 1969; Dick,
2008).
The dividing population was prevalently represented by fast-cycling blasts, but a
smaller fraction of slow-growing cells, characterized by quite infrequent divisions,
has been described. This “dormant” population, due to its insensitivity to anti-
proliferative drugs, is spared by treatments and supposedly to blame for leukaemic
re-growth and relapse (Clarkson, 1969; Clarkson et al., 1975; Skipper and Perry,
1970). These historical studies suggest that quiescence is another property that LSCs
share with normal HSCs. In different subtypes of human AML only the quiescent
fraction of leukaemic blasts is able to transplant leukaemias, whereas proliferating
cells, even when transplanted in high numbers, always fail to engraft (Guan et al.,
2003). This is not surprising if we consider that ∼80% of human CD34+ CD38–
leukaemic blasts are in G0. On the contrary, the majority of CD34+ CD38+ or CD34–
cells are in G1 or S–G2–M (Ishikawa et al., 2007). Quiescence is a hallmark of LSCs
also in murine models, insomuch as quiescence can be used as an un-biased method
to isolate leukaemic stem cells in vivo (Viale et al., 2009).
7.8 Regulation of Quiescence and Self-Renewal in Leukaemic

Stem Cells
Since self-renewal of LSCs is virtually unlimited, as inferred by their ability to

support continuous expansion of the leukaemic clone and to be propagated inex-
haustibly in mice during serial bone marrow transplantation (Hope et al., 2004;
Warner et al., 2004), one could wonder if quiescence is a relevant feature for
leukaemias or just an un-required “remnant” of a transformed stem cell.
In spite of being the “hot” topic of investigations, unfortunately only few data are
available.
Recently, the cell cycle inhibitor p21 was shown to be very important also
in the maintenance of self-renewal of leukaemic and preleukaemic stem cells
(HSCs expressing leukaemic fusion proteins) (Viale et al., 2009). Noteworthy, acute
myeloid leukaemia-associated oncogenes, when expressed in HSCs, increase their
quiescence through the upregulation of p21. This ability is maintained also in fully
transformed leukaemic blasts and is achieved through a p53 independent mecha-
nism. At first glance, this might seem paradoxical: oncogenes responsible for the
induction of leukaemias promote at the same time stem cells quiescence, instead of
driving them to proliferation. However, in the absence of p21 preleukaemic stem
cells are more sensitive to proliferative stresses than normal stem cells. Indeed,
during myelosuppression or transplantation they immediately undergo dramatic
exhaustion.
By the same token, p21 is highly critical for the initiation and maintenance of
leukaemogenesis (Viale et al., 2009). Although the leukaemia-associated PML-
RAR oncogene induces leukaemia both in the presence or in the absence of p21,
p21-null leukaemias are not transplantable, underlining a defect in LSCs self-
renewal. Interestingly, this behaviour correlates with an active recruitment of LSCs
into the cell cycle and a very drastic reduction in their number. Moreover, AML1–
ETO, another leukaemia-associated oncogene, fails to induce leukaemias in the
same experimental setting. These findings prove that loss of p21, and consequently
loss of quiescence, is critical to the self-renewal of leukaemic stem cells (Viale et al.,
2009).
A possible explanation is that expression of the leukaemia oncogene induces
DNA damage in HSCs, as other oncogenes such as activated Ras usually do in
fibroblasts. In the stem cell the concomitant upregulation of p21 becomes relevant,
because it imposes a cell cycle “pause” (quiescence) that triggers repair of dam-
aged DNA. Indeed, in the absence of p21, preleukaemic or leukaemic stem cells
accumulate very high levels of DNA damage resulting in the loss of their self-
renewal potential (Viale et al., 2009). This ability of p21 to induce a cell cycle
restriction, enabling DNA damage repair, might confer an advantage to HSCs dur-
ing hyperproliferation, as it occurs during stress in the preleukaemic HSCs or after
full transformation in the LSCs, explaining the role of p21 in the maintenance of
leukaemias.
7.9 A Common Paradigm in Stem Cell Exhaustion
In view of what is discussed above, quiescence appears as a common strategy

shared by leukaemic, preleukaemic and normal stem cells to reduce the detrimen-
tal effects of DNA damage on self-renewal (Fig. 7.1). Stem cells, due to their long
lifespan, are an easy target for different agents (metabolic or environmental) that
damage DNA.
Fig. 7.1 Role of p21 and DNA damage in stem cell exhaustion. Quiescence (G0) regulated by
p21 is a common feature of normal, preleukaemic (expressing the initiating oncogene, yet not fully
transformed) or leukaemic (expressing the initiating oncogene and fully transformed) stem cells.
When normal stem cells proliferate they accumulate DNA damage that might lead to a decrease
in their self-renewal. This process, although continuous, is very slow and counteracted by p21 that
maintains quiescence and triggers DNA damage repair. In the absence of p21, HSCs loose their G0
and accumulate DNA damage during hyperproliferation. Preleukaemic and leukaemic stem cells
are challenged by oncogene-induced DNA damage. A concomitant upregulation of p21, increas-
ing their quiescence, limits further accumulation of DNA damage. Losing p21, preleukaemic and
leukaemic stem cells lose quiescence and undergo to a rapid functional exhaustion
Unlike all other cells in the body that respond to a genomic damage with the
elimination of the damaged cell through apoptosis or senescence, stem cells, indis-
pensable to support life, have probably selected a damage repair mechanism which
allows their survival.
Any DNA repair mechanism though inevitably leads to the progressive build up
of mutations that would therefore accumulate in the stem cells. In time, this could
determine two effects: loss of stem cell self-renewal potential and genomic insta-
bility. Notably, both these two effects have been documented in the aging HSC.
Thus, any condition causing loss of quiescence would reduce the stem cell capac-
ity to repair DNA damage; accumulation of DNA damage would in turn lead to
premature exhaustion. In the same way, the transformed stem cell (preleukaemic
or leukaemic), subjected to the constant DNA-damaging effects of the expressed
oncogene, has a continuous need to repair the DNA. In this situation, loss of
quiescence would result even more in a condition incompatible with stem cell
survival.

Cancer continues to be a major global health problem despite our efforts to improve
patient survival. Notably, mortality rates for advanced diseases have remained con-
stant over the last decades (Jemal et al., 2007). This is probably due to the fact that
no specific treatment is available for the metastatic disease, which remains the prin-
cipal cause of death by cancer and for which there is, therefore, an imperative need
of new drugs.
A critical challenge for current drug development is to understand why current
anti-cancer treatments, above all chemotherapy drugs, are frequently not effective in
eradicating the neoplastic disease. Available anti-tumour drugs have been selected
for their anti-proliferation activity, mainly on the basis of in vitro tests on cultured
cancer cells and for their capacity to reduce tumour size in preclinical disease mod-
els and early clinical trials. Consistently their de-bulking activity is usually very
good. However, as discussed, these drugs do not provide disease cure, and as a con-
sequence, they have marginal effect on overall survival. In biological terms, this may
be interpreted as due to an intrinsic heterogeneity of the cancer in response to these
drugs. Tumours would in that case consist of a major cell population that is sensitive
to the available drugs (and therefore responsible for their de-bulking effect) and of
a rare cell population instead inherently resistant (and which would be responsible
of tumour re-growth and clinical relapse).
The cancer stem cell hypothesis may support this interpretation if we assume
the rare dormant cancer stem cells as refractory to anti-proliferative drugs, while
the remaining cells, some of which are actively proliferating, would be sensi-
tive. Built-in in the theory of cancer stem cells is the concept that chemoresistant
stem cells would be capable of bringing back the tumour. In summary, the cancer
stem cell hypothesis suggests that cancer cure can only occur if cancer stem cells
are eradicated, indicating this cellular compartment as the one that should be tar-
geted by anti-cancer drugs. One potential approach could be to render the dormant
cancer stem cells sensitive to chemotherapy by the use, in combination, of anti-
proliferative drugs and agents that boost proliferation of dormant tumour cells. One
such promising agent in leukaemias might be INFα (Essers et al., 2009). An alterna-
tive strategy could take advantage of the unique need of cancer stem cells to repair
DNA damage. In transformed stem cells the downregulation of DNA repair path-
ways might be synthetic lethal with oncogene expression. DNA repair inhibitors
could lead tumour stem cells to accumulate genomic damage to such an extent that
it would result in the loss of self-renewal and consequent tumour exhaustion.
This is an intriguing field and further studies are required before the targeting of
cancer stem cells can become part of new approaches to cancer therapy. Yet, if drugs
specific for cancer stem cells become available it is unlikely that these new drugs
will induce rapid reduction of tumour size, for the same reason that the current drugs
induce rapid de-bulking without providing disease cure. Thus, new modalities may
be necessary for the clinical evaluation of novel anti-cancer drugs based on their
ability to destroy quiescent cancer stem cells.
Acknowledgments We thank Paola Dalton for her scientific editing.
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Chapter 8
Maintenance of Telomeres in Cancer
Eros Lazzerini Denchi
Abstract Tumorigenesis is a complex process that involves several genetic

alterations and many attempts before the “correct” set of mutations occurs to create
a cancer cell of origin. Fortunately, this event is made infrequent by the evolu-
tion of mechanisms able to preserve genomic stability and to limit the proliferation
potential of somatic cells. A crucial role in both of these processes is played by the
nucleoproteins that localize to the tips of our chromosomes: the telomeres. Proper
telomere function is required to maintain genomic stability and in somatic cells
progressive telomere shortening serves as a cellular clock that limits proliferation
potential. In this chapter I will review the mechanisms that allow the proper func-
tion of telomeres in normal cells as well as the mechanisms employed by cancer
cells to bypass their normal regulation.
8.1 Telomere Length in Mammals
Telomeres represent the tips of mammalian chromosomes and are composed of a

long tract of TTAGGG repeats that culminate in a short G-rich single-stranded over-
hang (Fig. 8.1a). Telomeres vary considerably in length between different species
and different cell types. At birth, human telomere measures on average 15 kb while
in laboratory mice telomeres are on average 40–50 kb in length. Two main oppos-
ing forces determine telomere length: telomerase-mediated telomere elongation and
telomere erosion.
Telomerase, a cellular reverse transcriptase, elongates telomeres in germ cells as
well as in several human cancer cells through the addition of TTAGGG repeats
to chromosome ends (Greider and Blackburn, 1985). A less well characterized
mechanism to elongate telomeres is the alternative lengthening of telomeres (ALT)
E.L. Denchi (B)

Laboratory of Chromosome Biology and Genomic Stability, Department of Genetics, The Scripps
Research Institute, La Jolla, CA 92037, USA
e-mail: edenchi@scripps.edu
128 E.L. Denchi
Fig. 8.1 (A) Mammalian telomeres. Chromosome ends in mammalian cells are composed of a
long array of double-stranded TTAGGG repeats and culminate with a short single-stranded G-rich
overhang. (B) The shelterin complex. Six proteins are stably associated with TTAGGG repeats and
constitute the shelterin complex. (C) T-loop. Telomeres can fold into a secondary structure termed
t-loop, where the G-rich overhang invades a portion of the double-stranded telomeric repeat
pathway that is employed frequently in certain type of cancers and will be discussed
in more detail below. Telomere erosion on the other hand occurs at every round
of cellular division and is the result of the intrinsic inability of DNA polymerase
to fully replicate a liner DNA substrate, a problem postulated before the discov-
ery of telomeres by Olovnikov (Olovnikov, 1971). This “end replication problem”
results in a progressive telomere shortening in proliferating cells lacking a telomere-
elongating mechanism. In addition to this progressive telomere shortening, cells
show sporadic telomeric deletions resulting in loss of entire portions of telomeric
DNA possibly through recombination events (reviewed in Baird, 2008).
8.2 Telomere Dysfunction
The essential function of telomeres is to protect chromosome ends from degrada-

tion and ligation. In humans telomerase expression is restricted to adult male germ
line cells, and is undetectable in most somatic cell lines, with the exception of some
stem cell compartments (e.g., hematopoietic stem cells and intestinal crypts) (Aisner
8 Maintenance of Telomeres in Cancer 129
et al., 2002; Forsyth et al., 2002). In proliferating cells, telomeres therefore pro-
gressively shorten and eventually reach a critical length becoming dysfunctional.
When a telomere becomes dysfunctional a canonical DNA damage response is ini-
tiated, with the localization of DNA damage proteins such as γH2AX, 53BP1, and
MDC1 to the ends of the chromosome (di Fagagna et al., 2003; Takai et al., 2003).
Telomere dysfunction activates two main mammalian DNA damage response path-
ways, ATM and ATR, which in turn activate their downstream effector kinases,
CHK2 and CHK1. As a consequence, the cell cycle is inhibited through the repres-
sion of CDK activity (reviewed in Niida and Nakanishi, 2006), p53 is activated, and
ultimately cells enter an irreversible cell cycle arrest mediated by p53-dependent
p21 induction (Brown et al., 1997).
In addition to the induction of a DNA damage checkpoint, dysfunctional telom-
eres are the substrates for DNA repair reactions that create aberrant chromosome
structures. The most frequent repair event observed at dysfunctional telomeres
is non-homologous end joining (NHEJ) that results in end-to-end chromosomal
fusions (van Steensel et al., 1998; Smogorzewska et al., 2002). End-to-end fusions
are deleterious for cell viability because they give rise to dicentric chromosomes,
very unstable structures, that can break upon cellular division thus initiating
cycles of Breakage–Fusion–Bridge (BFB cycles) that can cause rampant genomic
instability (McClintock, 1942).
8.3 Chromosome End Protection: The Shelterin Complex

Telomeric TTAGGG repeats serve as bindings sites for a specific telomere bind-
ing complex, termed shelterin, that protects chromosome ends (de Lange, 2005).
Telomere dysfunction arises when shelterin can no longer be recruited to chromo-
some ends. In mammalian cells six proteins constitute this complex: TRF1(Chong
et al., 1995), TRF2 (Bilaud et al., 1997; Broccoli et al., 1997), RAP1 (Li et al.,
2000), TIN2 (Kim et al., 1999), TPP1 (Houghtaling et al., 2004; Liu et al., 2004; Ye
et al., 2004), and POT1 (Loayza and De Lange, 2003). TRF1 and TRF2 bind directly
to double-stranded telomeric DNA while the rest of the complex is recruited to chro-
mosome ends through protein–protein interactions. RAP1 is recruited to telomeres
exclusively through the interaction with TRF2. TIN2, TPP1, and POT1 form a sub-
complex that can be tethered to telomeres through the TIN2 interaction with either
TRF1 or TRF2. Importantly, POT1 contains two OB fold domains that allow this
protein to bind directly the single-stranded portion of the telomeres. However, at
mammalian telomeres POT1 is primarily recruited through its interaction with TPP1
and not by direct binding to telomere G-overhangs (Hockemeyer et al., 2007).
Localization of shelterin is what allows chromosome ends to be protected from
the DNA damage machinery. Indeed, inhibition of shelterin components is suffi-
cient to induce a DNA damage response, activate the DNA damage checkpoint, and
ultimately trigger apoptosis or senescence (van Steensel et al., 1998; Hockemeyer
et al., 2005, 2007; Palm and de Lange, 2008). The molecular mechanisms by which
130 E.L. Denchi
shelterin is able to hide chromosome ends from the DNA damage response machin-
ery is not fully understood yet, however, major advances have been made in the
recent years. It is now clear that within the shelterin complex certain components
are specialized in suppressing specific branches of the DNA damage response.
Localization of POT1 to telomeres is required to suppress the ATR signaling
pathway (Denchi and de Lange, 2007; Guo et al., 2007), the DNA damage response
pathway involved in the detection of single-stranded lesions in genome. The current
model for POT1 function is that it prevents the activation of the ATR pathway by
competing with RPA – the first essential step in the activation of this pathway – for
binding the single-stranded portion of the telomere.
The mechanism by which telomeres prevent chromosome ends from being recog-
nized as double-stranded breaks (DSBs) appears to be more complex. Localization
of TRF2 at telomeres is required to suppress ATM activation (Karlseder et al., 1999;
Denchi and de Lange, 2007), the key molecule involved in the transduction of these
types of DNA damage. Different molecular mechanisms have been proposed to
explain the ability of TRF2 to suppress this DNA damage response pathway. TRF2
has been proposed to be involved in shaping telomeres in a secondary DNA struc-
ture, the t-loop (Griffith et al., 1999; de Lange, 2004). In the t-loop conformation
the 3 end of the telomere invades a portion of the double-stranded telomere repeat
creating a lasso-like structure (Fig. 8.1c). In this conformation the DNA damage
response pathway would not be activated since the end of the chromosome is hidden
away. TRF2 in vitro has been shown to be able to bend double-stranded telomeric
DNA into t-loop. Whether this still holds in vivo has not been established yet. An
alternative hypothesis is that TRF2 directly inhibits key steps in the ATM-signaling
cascade. Indeed TRF2 overexpression has been shown to inhibit ATM and CHK2,
the key downstream signaling kinase of the ATM pathway (Karlseder et al., 2004;
Buscemi et al., 2009). Whether at physiological levels TRF2 expression is sufficient
to prevent ATM activation remains to be determined.
8.4 Telomere Elongation in Cancer

In humans, telomerase activity is not detected in most cell types (Forsyth et al.,
2002); however, the vast majority of cancers are telomerase positive. The core
telomerase complex is composed of a catalytic component (TERT), an RNA com-
ponent (TERC), and the protein dyskerin (DKC1) (Feng et al., 1995; Cohen et al.,
2007). Telomerase is a reverse transcriptase that is recruited to the tip of the chromo-
some where, using its own RNA as a template, it synthesizes new TTAGGG repeats
(Fig. 8.2a). The limiting factor for telomerase activity is TERT abundance since
TERC is ubiquitously expressed (Feng et al., 1995; Avilion et al., 1996) while TERT
is expressed only in telomerase-positive cells (Nakamura et al., 1997). Moreover,
expression of TERT is sufficient to restore telomerase activity, stabilize telomere
length, and bypass replicative senescence in human cells (Bodnar et al., 1998).
Deregulation of TERT expression in cancer is caused by transcriptional deregulation
Fig. 8.2 Telomere elongation mechanisms. (A) Telomerase. Telomerase is the cellular reverse
transcriptase that adds TTAGGG repeats to chromosome ends. The core components of the telom-
erase enzyme are the catalytic subunit (TERT), the RNA subunit (TERC), and dyskerin. (B) ALT
pathway. Recombination-based model for the alternative lengthening of telomeres (ALT)
and by variation in gene dosage. Positive regulators of TERT expression include the
proto-oncogene Myc (Bahram et al., 1999) and the transcriptional target of the TGF-
beta pathway, SIP1 (Lin and Elledge, 2003). The frequent deregulation of both Myc
and TGF-beta signaling in cancer provides a mechanistic link between cancer pro-
gression and telomerase reactivation. In addition, the tumor suppressor menin is a
direct repressor of TERT (Lin and Elledge, 2003). Finally, gains of the genomic loci
containing TERT and hTR (5p and 3q, respectively) are often observed in human
cancers (reviewed in Cao et al., 2008).
Deregulation of TERT expression and concomitant induction of telomerase activ-
ity are observed in more than 85% of human cancer (Shay and Bacchetti, 1997). The
remaining 10–15% of cancers do not have detectable telomerase but use an alterna-
tive lengthening of telomere (ALT) pathway (Bryan et al., 1995, 1997). The precise
mechanism employed by these cells to maintain their telomeres remains to be fully
elucidated; however, strong evidence links this process with a homologous recom-
bination (HR)-based mechanism (Dunham et al., 2000). In the current model for
the ALT pathway, short telomeres are elongated by a reaction involving strand inva-
sion of a telomere template followed by synthesis of new telomere repeats using the
132 E.L. Denchi
invaded strand as a template (Fig. 8.2b) (reviewed in Henson et al., 2002; Muntoni
and Reddel, 2005; Cesare and Reddel, 2008).
Cancer cells engaged in the ALT pathway have a set of distinctive markers that
allow their identification such as telomeres with an extremely heterogeneous length
and the presence of ALT-associated promyelocytic leukemia (PML) nuclear bod-
ies (APBs). APBs contain telomeric DNA, telomeric proteins, DNA recombination
proteins, and the standard components of PML bodies (PML, SP100). It has been
suggested that APBs are the sites of ALT activity; however, inhibition of ALT does
not result in the loss of APBs. Thus far, no mutations linked to the ALT phenotype
have been identified. In experimental settings it has been shown that the activity of
the recombination complex MRN and SMC5/6 are required for the ALT phenotype
(Jiang et al., 2005; Potts and Yu, 2007; Zhong et al., 2007). Interestingly in certain
settings telomere dysfunction results in high rates of telomere recombination, sug-
gesting that ALT cells might have certain chronic selection mechanisms for telomere
dysfunction. Specifically, inhibition of the critical shelterin components, TRF2 or
POT1, in conjunction with loss of the NHEJ factor, KU, results in extremely high
levels of telomere sister chromatid recombination (T-SCE) (Celli et al., 2006; Palm
et al., 2009). These results show that in normal cells the shelterin complex sup-
presses recombination at telomeres and thus suggest that alteration in the function
of this complex might be a mechanism for cancer cells to maintain telomere length
through the ALT pathway.
8.5 Telomere Instability and Cancer Progression
Telomere length in human cancers is often reduced in cancer cells when compared
to normal surrounding tissues, for instance, in colorectal carcinomas (Hastie et al.,
1990; Engelhardt et al., 1997) and in breast carcinomas (Meeker et al., 2004). These
data suggest that, during the process of cancer development, cells proliferate for
an extended period of time in the absence of a telomere elongation mechanism, and
that reactivation of telomerase (or alternatively activation the ALT pathway) is a late
event in the tumorigenesis process.
This observation supports the notion that telomere shortening acts as a tumor
suppression mechanism and that reactivation of telomere length elongation is an
obligatory step in carcinogenesis. However, telomere dysfunction can also lead to
chromosomal fusions that are a source of genomic instability. Indeed there is strong
evidence linking the presence of high rates of telomere dysfunction with stages
of tumorigenesis characterized by high levels of genomic instability. For exam-
ple, during the transition from adenoma to carcinomas frequent anaphase bridges,
an indicator of end-to-end chromosome fusions, are observed in colorectal carci-
nomas (Rudolph et al., 2001) and oral squamous cell carcinomas (Gordon et al.,
2003).
These opposite effects of telomere dysfunction are confirmed by analysis of
mice with altered telomerase. Mice lacking telomerase activity were generated
by deletion of the TERC gene (Blasco et al., 1997). Terc–/– mice are viable and
do not show any obvious phenotype due the ample reserves of telomere repeats
present in mus musculus. However, when Terc–/– mice are intercrossed for sev-
eral generations, telomeres progressively become critically short and ultimately
become dysfunctional. As expected the tissues mostly affected are those with a
high degree of cellular turnover. In agreement with this, the predominant pheno-
types of these mice include male and female infertility, intestinal atrophy, spleen
atrophy, reduced proliferation of B and T cells, and reduced proliferation of bone
marrow stem cells (Blasco et al., 1998; Herrera et al., 1999; Rudolph et al., 1999a;
Hemann et al., 2001). These mice have confirmed the crucial role of telomerase in
cancer development since they show impaired tumorigenesis when challenged with
skin carcinogenesis (Gonzalez-Suarez et al., 2000), liver carcinogenesis (Rudolph
et al., 1999b), or when crossed with cancer-prone INK4a mice (Greenberg et al.,
1999).
However, these mice have also provided evidence that telomere dysfunction con-
tributes to genomic instability and can promote cancer progression. Indeed in the
absence of p53 loss of telomerase results in increased tumorigenesis and notably the
onset of epithelial cancers with a complex karyotype, indicative of rampant genomic
instability (Chin et al., 1999; Artandi et al., 2000).
Collectively it is now clear that telomere erosion is indeed a strong tumor sup-
pression mechanism. However, when cells accumulate mutations that allow them
to bypass the cell cycle checkpoints initiated at chromosome ends and progress to
mitosis with dysfunctional telomeres, then BFB cycles are initiated and rampant
chromosome instability fuels tumor progression (see model in Fig. 8.3).
Fig. 8.3 Telomere deregulation in cancer. During hyperproliferation telomeres are subjected to
progressive telomere erosion that eventually results in telomere dysfunction. Cells containing dys-
functional telomeres withdraw from cell cycle (in this model blue cells represent senescence cells).
Cells with checkpoint defects enter mitosis in the presence of end-to-end chromosome fusions
resulting in high levels of genomic instability. Finally, reactivation of telomerase activity elongates
critically short telomeres suppressing genomic instability and allowing unlimited proliferation of
cancer cells
134 E.L. Denchi
8.6 Future Perspectives

Despite the major advances seen in the telomere biology field over the last years
there are still fundamental questions that need to be addressed. The most challeng-
ing question is also probably the most fundamental; how do telomeres suppress the
DNA damage response? In this regard the identification of the telomere-associated
complex shelterin, and of proteins within this complex involved in suppression of
specific branches of the DNA damage response is a major step forward. However,
the molecular mechanism employed by these proteins to prevent DNA damage acti-
vation is yet to be discovered and the models that have been thus far proposed still
need to be validated. A major issue here is the fact that our current understanding on
how cells detect DNA damage is limited. Notably, we do not currently know how
cells detect double strand breaks – the structures that most closely resemble telom-
eres. In fact, despite the identification of essential players involved in this DNA
damage response pathway, we still do not fully understand the key initial steps that
initiate a cellular response to these DNA lesions. Hopefully future research will shed
light both on precisely how cells detect these DNA lesions as well as how telom-
eres camouflage chromosome ends in such a way that they remain undetected by the
DNA damage machinery.
Some of the outcomes of telomere dysfunction at the cellular level are also
still poorly understood. In particular, the mechanisms that result in polyploidy
following telomere dysfunction remain unknown. High levels of polyploidy are
detected following telomere dysfunction induced in different experimental sys-
tems such as mutant telomerase expression (Pantic et al., 2006), POT1 inhibition
(Hockemeyer et al., 2006), and TRF2 deletion (Lazzerini Denchi et al., 2006).
Moreover, in preneoplastic lesions critically short telomeres are frequently detected
in polyploid cells (Plentz et al., 2005). Polyploid cells represent a fertile envi-
ronment for the accumulation of gross genomic mutations since extra copies of
chromosomes can allow cells to survive in the presence of mutations that would
be otherwise lethal. Accordingly, polyploid cells have been shown to promote
tumorigenesis in vivo (Fujiwara et al., 2005). For this reason it will be important
to understand the pathways involved in promoting polyploidy following telomere
dysfunction.
A final area of telomere research that remains largely obscure is the alternative
lengthening of telomeres (ALT) pathway. Understanding the process that allows
telomere elongation in the absence of the canonical telomerase pathway is, by
itself, a fascinating problem but it is also of great cancer relevance. A signifi-
cant proportion of cancers utilize this pathway to maintain their telomeres; thus
understanding the underlying genetic alterations that allow this process to occur
will have an important diagnostic value and will, hopefully, provide a therapeutic
opportunity.
Acknowledgments I thank Claire Attwooll, Agnel Sfeir, Keijo Okamoto, and Beatriz Virgen for
comments on the manuscript and helpful discussions.
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Chapter 9
The Senescence Secretome and Its Impact
on Tumor Suppression and Cancer
Alyssa Kennedy and Peter D. Adams
Abstract Cellular senescence is an irreversible proliferation arrest with an

emerging physiological role in tumor suppression. For example, primary cells that
acquire a first oncogenic event often enter cellular senescence and this serves to
block their further neoplastic transformation. Remarkably, the spectrum of factors
secreted by senescent cells differs dramatically from their proliferating counterparts.
For example, the “secretome” of senescent cells includes increased production of
cytokines, matrix metalloproteases, and altered production of many growth factors.
This review discusses these factors, their mechanism of regulation in senescent cells,
and their contribution to the senescent phenotype and its role in tumor suppression.
9.1 Triggers of Cell Senescence

Cellular senescence is an irreversible proliferation arrest instigated by various
triggers. The so-called replicative senescence results, in part, from defective chro-
mosome ends, called telomeres, that progressively accumulate through multiple
rounds of DNA replication (Wright and Shay, 2002). However, in many cell types,
replicative senescence can also result from non-telomeric triggers. We know this
because telomerase, the enzyme that maintains telomeres, on its own is often
insufficient to immortalize some strains of human fibroblast and epithelial cells
(Kiyono et al., 1998). More specifically, most primary cell types enter senescence
in response to specific stresses, such as oxidative stress or reactive oxygen species
(ROS) (Wright and Shay, 2002) and activation of oncogenes or inactivation of tumor
suppressor genes (Chen et al., 2005; Serrano et al., 1997) (Fig. 9.1).
P.D. Adams (B)

Cancer Research UK Beatson Labs, University of Glasgow, Glasgow, G61 1BD, UK
e-mail: p.adams@beatson.gla.ac.uk
140 A. Kennedy and P.D. Adams
Fig. 9.1 Triggers, signals, and effectors of cell senescence. Signaling pathways restricted to mouse
or human are indicated. Dashed lines are inks that are poorly defined mechanistically. See text for
details
9.2 Senescence Signaling Pathways
At least four linked signaling pathways drive the cell senescence program, namely
oncogene signaling, DNA damage signaling, and the pRB and p53 tumor suppres-
sor pathways (Fig. 9.1). The contribution of each of these and some other signals,
such as ROS, to the senescence program has been extensively reviewed elsewhere
(d’Adda di Fagagna, 2008) and will be only briefly described here.
Not surprisingly, oncogene signaling pathways are primarily involved in
oncogene-induced senescence, rather than, for example, replicative senescence. The
signaling pathways involved are best defined for mutated oncogenic Ras (K-Ras,
N-Ras, or H-Ras) and its effectors, although many different oncogenes and growth
regulatory molecules can trigger senescence (d’Adda di Fagagna, 2008). Oncogenic
Ras utilizes much the same signals and effectors as wild-type Ras, including
the Raf–MEK–ERK pathway, the PI3K–Akt pathway, and others (Karnoub and
Weinberg, 2008). Of these, Raf–MEK–ERK signaling is key. Chronic signaling
through this axis drives senescence, in part, through its activation of the p38MAP
kinase p16INK4a stress response pathway (Bulavin et al., 2004; Deng et al., 2004;
Ito et al., 2006; Iwasa et al., 2003), and also through activation of DNA damage
signals.
However, DNA damage signals contribute to senescence in response to both
activated oncogenes and short telomeres. The aberrant or shortened telomeres that
9 The Senescence Secretome and Its Impact on Tumor Suppression and Cancer 141
accumulate during replicative senescence colocalize with activated DNA damage

signaling proteins, such as 53BP1 and γH2AX (d’Adda di Fagagna et al., 2003;
Herbig et al., 2004; Takai et al., 2003), where they are referred to as telomere
dysfunction-induced foci (TIFs). DNA damage foci also form after oncogene-
induced senescence (Bartkova et al., 2006; Di Micco et al., 2006), due to error-prone
rounds of DNA synthesis driven by the activated oncogene. These senescence-
associated DNA damage foci (SDFs) are not necessarily at telomeres. Regardless
of their location, TIFs and SDFs reflect activation of DNA damage signaling path-
ways that are required for initiation and maintenance of the complete senescence
program.
To achieve senescence, these DNA damage signals contribute to activation of
the p53 and pRB tumor suppressor pathways, master regulators of the senescence
program. DNA damage signals are well-known activators of p53, in part by direct-
ing phosphorylation of the protein (Harris and Levine, 2005). Activation of p53
contributes to senescence-associated proliferation arrest by upregulating expression
of the CDKN1a gene, encoding the cyclin/cdk2 inhibitor p21CIP1 (Brown et al.,
1997). In turn, p21CIP1 activates the pRB tumor suppressor pathway, by inhibit-
ing cyclin/cdk2 complexes (Sherr and Roberts, 1999). The pRB protein inhibits cell
proliferation through many effectors, including the E2F family of transcription fac-
tors, required for progression through S phase (see review by Ji and Dyson, Chapter
2, this volume). In addition to being activated downstream of p53, the pRB path-
way can also be activated independent of p53, through upregulation of p16INK4a,
an inhibitor of the cyclin/cdk4 (or cdk6) kinase that inactivates pRB (Kamijo et al.,
1997; Kiyono et al., 1998; Palmero et al., 1997). The centrality of the p53 and pRB
pathways to senescence is underscored by the fact that inactivation of both typi-
cally abolishes senescence, in both human and mouse cells (Harvey et al., 1993;
Shay et al., 1991). That inactivation of both pathways is also frequently observed in
human cancers underscores the role of senescence in tumor suppression (discussed
further below).
9.3 The Altered Secretory Phenotype of Senescent

Cells – The Senescence Secretome
The established view of senescence outlined above sees the mechanisms and impact
of senescence as largely cell autonomous processes. In other words, senescence sig-
naling and its impact are largely confined to the senescent cell itself. However, it
has, in fact, been known for some time that, compared to proliferating cells, senes-
cent cells secrete an altered mix of factors into their environment (Sottile et al.,
1987; West et al., 1989). This altered secretome is now emerging as one of the most
exciting aspects of the senescence program, because of its role in driving senes-
cence and its potential wide-ranging impact on a tissue’s function and response to
damage. Previously, the altered secretome of senescent cells has been monikered
the senescence-associated secretory phenotype (SASP) (Coppe et al., 2008) and the
senescence-messaging secretome (SMS) (Kuilman and Peeper, 2009). Here, it is

referred to simply as the senescence secretome. There are several components to
this altered secretome.
9.3.1 Growth Regulators

Senescent cells show altered expression of many gene products that are empow-
ered to regulate cell proliferation. Specific changes include reduced production of
IGF1 and WNT2 (Ferber et al., 1993; Ye et al., 2007a) and increased IGFBP pro-
teins, TGFβ, HGF, and VEGF (Bavik et al., 2006; Coppe et al., 2006; Linskens
et al., 1995; Shelton et al., 1999; Tremain et al., 2000; Wajapeyee et al., 2008).
Regulation of such growth factors can reinforce the proliferation arrest in the senes-
cent cell. For example, repression of WNT2 contributes to senescence-associated
proliferation arrest, by driving specific chromatin changes in some senescent cells,
formation of so-called senescence-associated heterochromatin foci (SAHF). These
are punctate domains of heterochromatin that derive from large-scale chromo-
some condensation (Funayama et al., 2006; Zhang et al., 2007). Incorporation of
proliferation-promoting genes, such as cyclin A2, into these domains is thought to
repress those genes, thereby contributing to proliferation arrest.
A complex of histone chaperones, including HIRA, ASF1a, and UBN1, has
been reported to drive formation of SAHF in human cells (Banumathy et al.,
2008; Tagami et al., 2004; Zhang et al., 2005). Further supporting this idea, both
HIRA and ASF1a were identified in unbiased whole-genome screens in human
cells for genes required for gene silencing and/or nuclear heterochromatinization
(Gazin et al., 2007; Wajapeyee et al., 2008). Moreover, orthologs of HIRA, ASF1a,
and UBN1 create transcriptionally silent heterochromatin in yeast, flies, and plants
(Goodfellow et al., 2007; Kaufman et al., 1998; Moshkin et al., 2002; Phelps-Durr
et al., 2005; Sharp et al., 2002; Singer et al., 1998). However, it should be noted that
each of these proteins also has other functions in chromatin metabolism (Rocha and
Verreault, 2008).
Formation of SAHF also depends on another specific subnuclear organelle, the
promyelocytic leukemia (PML) nuclear body (Zhang et al., 2005). These bodies
have been implicated in various processes, including tumor suppression and cellular
senescence (Bernardi and Pandolfi, 2007) and assembly of macromolecular regula-
tory complexes (Fogal et al., 2000; Guo et al., 2000; Pearson et al., 2000). Early in
the senescence program, HIRA enters PML bodies and this relocalization is required
for formation of SAHF (Ye et al., 2007b), perhaps through its activation of HIRA.
What is the connection between SAHF, HIRA, and WNT2? Significantly, recruit-
ment of HIRA to PML bodies is triggered by repression of WNT2 expression in
pre-senescent cells. Specifically, repression of WNT2 leads to activation of GSK3
that, in turn, phosphorylates HIRA on serine 697 (Ye et al., 2007a) and drives the
protein into PML bodies, a key event in formation of SAHF. To sum up, through acti-
vation of the HIRA/ASF1a/UBN1 histone chaperone pathway, repression of WNT2
expression is an early trigger for aspects of the senescence program, including

formation of SAHF and proliferation arrest.
9.3.2 Inflammatory Regulators

Senescent cells also often express increased amounts of inflammatory regulators,
specifically cytokines, chemokines, and their receptors (Maier et al., 1990). These
include IL6 and its receptor IL6R/GP80 (Kuilman et al., 2008) and the seven-
spanning transmembrane receptor CXCR2 and its ligands, IL8, CXCL1/GROα,
CXCL5/ENA78, and CXCL7/NAP2 (Acosta et al., 2008; Kuilman et al., 2008;
Shelton et al., 1999; Xue et al., 2007). At least some of these factors contribute to
senescence-associated proliferation arrest. For example, IL6 reinforces senescence-
associated proliferation arrest and formation of SAHF in a cell autonomous
autocrine manner (Kuilman et al., 2008). Similarly, increased CXCR2 and IL8
reinforce the senescence program (Acosta et al., 2008).
One notable class of immune regulatory molecules upregulated by senescent cells
is ligands of the NKG2D transmembrane receptor, which is expressed on NK cells
and T cells. Specifically, senescent cells produce the NKG2D ligands MICA and
ULBP2, as well as IL-15. The latter upregulates expression of NKG2D, thereby
potentiating activity of the pathway (Krizhanovsky et al., 2008; Roberts et al., 2001;
Xue et al., 2007). Activation of NKG2D signaling can trigger interferon (IFN)-γ
production and cytolytic responses (Gonzalez et al., 2008). This is thought to be
important for the role of senescence in tumor suppression and is discussed further
below.
9.3.3 Stromal Regulators

In addition to these growth and inflammatory regulators, senescent cells often
secrete altered amounts of factors predicted to remodel the extracellular stroma
or matrix (ECM). Production of matrix metalloproteases (MMP1 and MMP3),
plasminogen activator inhibitor (PAI), tissue-type plasminogen activator (tPA),
and cathepsin is increased, and expression of fibronectin, collagen, and keratin is
decreased (Kortlever et al., 2006; Krizhanovsky et al., 2008; Millis et al., 1992;
West et al., 1989). In humans, there are 23 MMPs, each with a zinc ion and con-
served methionine residue at the active site (Page-McCaw et al., 2007). MMPs can
degrade the ECM to affect tissue architecture, facilitate cell migration by degra-
dation of the ECM and proteolytically activate or inactivate extracellular signaling
peptides and polypeptides, and cause release of ECM-bound growth factors. As a
result, the effects of MMPs on cell and tissue function are extremely diverse, impact-
ing tissue growth, development, remodeling, and repair. Significantly, one function
of secreted MMPs is to regulate the activity of cytokines, through cleavage of the
cytokines themselves or the ECM by which they are sequestered (Page-McCaw
et al., 2007). This suggests that two components of the senescence secretome, the
cytokines and MMPs, are themselves functionally linked to each other. Consistent
with this, MMP3 has also been linked to the inflammatory response in skin (Wang
et al., 1999).
Notwithstanding some variations between cell types that likely reflect different
functions of the secretome in different senescent cell types (Shelton et al., 1999),
in gene expression array experiments the altered secretome is often one of the
most striking changes observed. Consequently, there is now much interest in the
mechanism of regulation of the secretome in senescent cells.
9.3.4 Regulation of the Secretome
As well as being functionally important in its own right (see below), analysis of the
secretome has brought to the fore other key regulators of senescence, namely the
transcription factors C/EBPβ and NFkB. C/EBPβ is a basic region-leucine zipper
transcription factor with wide-ranging regulatory effects on cell growth, prolif-
eration, and differentiation (Nerlov, 2007). Of relevance here, in mouse embryo
fibroblasts (MEFs), C/EBPβ contributes to senescence through repression of E2F
activity (Sebastian et al., 2005). NFkB is a family of structurally related transcrip-
tion factors, specifically RelA (p65), RelB, c-Rel, p50/p105, and p52/p100, that
regulate immune and inflammatory responses, development, cell growth, and apop-
tosis (Perkins, 2007), by binding to DNA as homo- and heterodimers. Like C/EBPβ,
activation of NFkB has been previously linked to signaling by oncogenic Ras (Finco
et al., 1997; Nakajima et al., 1993).
It has now become apparent that both C/EBPβ and NFkB are key regulators of
the senescence secretome. Gil and coworkers showed that several CXRC2 ligands,
namely IL8, CXCL1/GROα, CXCL5/ENA78, and CXCL7/NAP2, are upregulated
in senescent cells in an NFkB-dependent manner (Acosta et al., 2008). At least two
of these, IL8 and CXCL1/GROα, contain NFkB binding sites in their promoters.
Like NFkB, C/EBPβ also contributes to expression of several cytokines, namely,
IL6, IL8, CXCL1/GROα, and CXCL7/NAP2, in senescent cells (Acosta et al., 2008;
Kuilman et al., 2008). Oncogenic stress enhances binding of C/EBPβ to the IL6
promoter, and C/EBPβ is required for expression of cytokines and execution of the
senescence program. In sum, C/EBPβ and NFkB activate a network of cytokines
that is expressed in senescent cells.
In fact, these transcription factors might cooperate in activation of the secretome,
since both IL6 and IL8 harbor adjacent binding sites for both C/EBPβ and NFkB in
their promoters (Transfac). Older literature indicates that NFkB and C/EBPβ phys-
ically interact with each other in vitro and in vivo (Diehl and Hannink, 1994; Stein
et al., 1993), bind to adjacent sites on gene promoters, and synergistically acti-
vate transcription (Betts et al., 1993; Kunsch et al., 1994; Mukaida et al., 1990).
In another context, C/EBPβ and NFkB function together to cooperatively activate
expression of IL8 (Kunsch et al., 1994; Mukaida et al., 1990; Stein and Baldwin,
1993). In sum, NFkB and C/EBPβ appear to be cooperative transcription activators

of at least some components of the secretome.
What drives activity of NFkB and C/EBPβ in senescent cells? Specific protein
isoforms of C/EBPβ are upregulated in senescent cells, and expression of one NFkB
subunit, RelA, and an upstream activating kinase in the NFkB pathway, IKKβ,
both increase during OIS (Acosta et al., 2008; Kuilman et al., 2008). Recent data
have implicated a p53-independent but ARF-dependent pathway in transcription of
C/EBPβ in mouse cells (Sebastian and Johnson, 2009). However, oncogenic Ras
can also activate NFkB and C/EBPβ through post-translational mechanisms (Finco
et al., 1997; Nakajima et al., 1993). Interestingly, p38MAP kinase is independently
linked to both cell senescence and C/EBPβ (Basak et al., 2005; Bulavin et al., 2004;
Deng et al., 2004; Ito et al., 2006; Iwasa et al., 2003). In sum, C/EBPβ and NFkB
might be activated by a combination of transcriptional and post-transcriptional
mechanisms in senescent cells.
9.4 The Senescence Secreteome in Tumor Suppression

The first evidence that senescent cells exist in vivo came from detection of
senescence-associated-β-galactosidase activity (SA-βgal) (Dimri et al., 1995), likely
lysosomal β-D-galactosidase expressed from the GLB1 gene (Lee et al., 2006).
More recently, it has become the norm to use several readouts to detect senes-
cent cells in vivo, including proliferation arrest (Ki67 or BrdU negative) and one
or more others that reflect different aspects of the senescence program, such as sig-
naling molecules (e.g., p16INK4a and p21CIP1), chromatin changes (e.g., HP1 and
HIRA), DNA damage (e.g., γH2AX and TIFs), secreted factors (e.g., IL8), or mark-
ers of autophagy (e.g., LC3) (Acosta et al., 2008; Braig et al., 2005; Chen et al.,
2005; Collado et al., 2005; Herbig et al., 2006; Kuilman et al., 2008; Michaloglou
et al., 2005; Sedelnikova et al., 2004; Young et al., 2009). Since there does not
appear to be a single unambiguous marker of senescent cells, this combinatorial
approach to their detection in vivo is likely to persist, but become more context
and function specific. As we better understand the physiological functions of senes-
cence, it seems that the specific senescence phenotype is likely to vary depending on
the in vivo context and function, thus rationalizing the use of combinatorial markers
for its detection.
Using such assays, good evidence has emerged for a physiological role for senes-
cence in tumor suppression (Prieur and Peeper, 2008; Shamma et al., 2009). As
discussed below, the senescence secretome likely plays a key contributory role to
this tumor suppression process. Senescent cells are found in benign pre-cancerous
neoplasms associated with oncogene activation and tumor suppressor inactivation,
in both humans and mice (Braig et al., 2005; Chen et al., 2005; Collado et al., 2005;
Courtois-Cox et al., 2006; Dai et al., 2000; Going et al., 2002; Michaloglou et al.,
2005; Shamma et al., 2009). For example, human nevi or moles are benign clonal
neoplasms containing melanocytes made senescent by a mutated Ras or BRAF
oncogene (Michaloglou et al., 2005). In at least some of the mouse models, inactiva-
tion of senescence results in progression to cancerous neoplasms, instead of benign
ones (Dankort et al., 2007; Ha et al., 2007; Sarkisian et al., 2007; Sun et al., 2007).
Underscoring the ability of senescence to block tumor growth, its reactivation in
murine tumors causes tumor regression (Ventura et al., 2007; Xue et al., 2007).
Senescence caused by shortened telomeres also confers tumor suppressor activ-
ity (Cosme-Blanco et al., 2007; Feldser and Greider, 2007; Rudolph et al., 1999).
Importantly from a potential therapeutic perspective, senescence initiated by exoge-
nous genotoxic stress, including chemotherapeutic drugs, also contributes to tumor
regression in vivo (Schmitt et al., 2002; te Poele et al., 2002).
Senescence is likely to serve as a tumor suppression process in at least three
ways (Fig. 9.2). First, since tumor growth depends on cell proliferation, senescence-
associated irreversible proliferation arrest is expected to block tumor growth.
Although this idea makes intuitive sense, it is difficult to show that proliferation
arrest, and not some other component of these senescence program, is primarily
responsible for tumor suppression (Cosme-Blanco et al., 2007; Sarkisian et al.,
2007). Second, by inhibiting inherently mutagenic processes such as DNA repli-
cation and mitosis, proliferation arrest is likely to block acquisition of additional
oncogenic events (McDonald and El-Deiry, 2001). However, long-term genetic sta-
bility of senescent cells in nevi has not been confirmed (Bennett, 2008). Third, the
Fig. 9.2 Senescence as a tumor suppressor mechanism. Acquisition of an activated oncogene or

inactivation of a tumor suppressor initially causes a proliferative burst. Ultimately, senescence
kicks in to arrest proliferation of the cells harboring the oncogenic event. Proliferation arrest is
reinforced through the secretome. Senescence-associated proliferation is likely to arrest tumor
progression by preventing proliferation of neoplastic cells and suppressing accumulation of addi-
tional genetic alterations. In addition, senescence recruits the innate immune system to clear the
genetically altered cells that threaten the host with malignant disease. See text for details
immune regulators, and perhaps other components of the senescence secretome,

produced by senescent cells may trigger clearance of the incipient cancer cells
through recruitment of the innate immune system. Consistent with this idea, var-
ious cells of the immune system, including T cells, NKT cells, and NK cells, are
already known to participate in tumor suppression through removal of cancerous
and/or precancerous cells, a process called immune surveillance (Swann and Smyth,
2007).
Several cell-signaling pathways are involved in immune surveillance of cancer,
including those activated by the NKG2D receptor expressed on NK cells and T
cells. Incipient tumor cells that express appropriate ligands, e.g., MICA, MICB and
ULBPs, are thought to provoke these NKG2D-activated cells to secrete cytokines
and kill the offending cells. Consistent with this idea, mice lacking NKG2D are
deficient in tumor surveillance (Guerra et al., 2008). Recent evidence indicates that
NKG2D-activated NK cells participate in immune surveillance, at least in part,
through clearance of senescent cells. Induction of wild-type p53 expression in a
mouse model of liver cancer caused tumor regression, associated with cell senes-
cence and secretion of inflammatory chemokines, including CXCL1, CCL2, and,
notably, IL15 (Xue et al., 2007). IL15 stimulates proliferation of NK cells and
primes their NKG2D-mediated signaling pathway (Horng et al., 2007). Inactivation
of NK cells with antibodies blocked liver cancer regression after induction of p53
(Xue et al., 2007). In another model, albeit chemical-induced liver damage and not
liver cancer, senescent cells were shown to upregulate expression of the NKG2D lig-
ands, ULBP2, and MICA, thereby making them better targets for clearance by NK
cells (Krizhanovsky et al., 2008). Indeed, a human NK-like cell line, YT, was able to
kill senescent cells in vitro (Krizhanovsky et al., 2008). Together, these results sug-
gest that secretion of chemokines, including NKG2D activators and ligands, directs
the clearance of senescent cells by the innate immune system, specifically NK cells
in this case.
Other components of the immune system, in addition to NK cells, are also likely
to be involved in clearing senescent cells. Other NKG2D-expressing cells, includ-
ing T cells and NKT cells, are also implicated in immune surveillance (Swann
and Smyth, 2007). And, cells made senescent by activated oncogenes produce
other chemokines, such as IL6, IL8, CXCL1, and CCL2, which between them are
chemoattractants and/or activators of B cells, neutrophils, basophils, T cells, mono-
cytes, memory T cells, and dendritic cells (Heinrich et al., 2003; Waugh and Wilson,
2008). In sum, accumulating evidence indicates that a key role of chemokines
secreted by senescent cells is to direct removal of the genetically altered cells by the
innate immune system, thereby eliminating the potentially tumorigenic cells once
and for all.
What might be the role of other components of the senescence secretome in
tumor suppression? Altered expression of growth regulators, such as WNT2, IGF1
and IGFBPs, is likely to reinforce senescence-associated proliferation arrest in vivo
(Wajapeyee et al., 2008; Ye et al., 2007a). So too, in fact, may increased expression
of cytokines and stromal regulators (Acosta et al., 2008; Kuilman et al., 2008). As
discussed above, proliferation arrest is likely to be central to senescence-mediated
tumor suppression. However, the reason for increased production of MMPs by

senescent cells is less obvious. Conceivably, this might also promote prolifera-
tion arrest, perhaps by inactivation of growth factors. Alternatively, MMPs might
facilitate the tumor suppressive role of the immune system, perhaps by facilitating
recruitment and/or access of NK and other immune cells to senescent cells in vivo.
This is obviously an important area for future investigation.
Despite the existence of senescence, and other tumor suppression mechanisms,
cancers obviously do arise. In fact, even clonal benign human nevi can contain hun-
dreds of thousands of cells, a product of many rounds of cell division harboring an
activated oncogene, and these same nevi escape the immune system for decades.
Perhaps related to this, approximately a quarter to one third of human melanomas
are thought to arise from preexisting nevi (Michaloglou et al., 2005). So what deter-
mines the efficiency of execution of the senescence program and its long-term
stability in vivo? In vitro at least, senescent cells can escape the program if they
acquire additional genetic alterations (d’Adda di Fagagna et al., 2003; Di Micco
et al., 2006; Herbig et al., 2004). However, given the apparent heterogeneity of
senescence markers in some benign lesions, including nevi, it is possible that even
in a clonal neoplasm not all the cells ever become terminally or irreversibly senes-
cent (Bartkova et al., 2006; Michaloglou et al., 2005). Conceivably, the speed with
which the senescence program is executed and the stability of the endpoint, and so
its potency as a tumor suppression process, is affected not only by the genetic and
epigenetic status of the cell but also by the microenvironment in which the incipient
senescent or neoplastic cell resides (Adams and Enders, 2008). Understanding the
factors that determine the efficiency of the senescence process may have important
implications for chemoprevention and risk assessment approaches focused on early
neoplasms.
9.5 Summary
Our understanding of senescence has advanced considerably in recent years. A

number of core triggers and signaling pathways are now quite well defined.
At the same time, other signaling mechanisms and phenotypes are emerging,
indicating roles for autophagy, negative feedback signaling, and microRNAs,
for example. One of the most exciting recent developments, and bridging our
understanding of mechanisms, functions, and pathologies, is the senescence secre-
tome. Already, an analysis of the secretome is revealing new insights into
the molecular mechanisms that drive senescence. For example, upregulation of
cytokines in senescent cells is mediated, in part, by transcription factors NFkB
and C/EBPβ, whose roles in senescence were previously underappreciated. The
mode of repression of WNT2 in senescent cells is unknown, but its investiga-
tion has potential to uncover new pathways that regulate senescence independent
of the pRB and p53 tumor suppressors. Moreover, while the senescence secre-
tome directly contributes to the long-standing hallmark of senescence, irreversible
proliferation arrest, it also takes our understanding of the senescence beyond
this proliferation arrest. By influencing the microenvironment and neighboring

cells, the secretome promotes tumor suppression, making senescence-mediated
tumor suppression a tissue level and organismal response. Interestingly, this makes
good sense, given that the incipient cancer cells being cleared have the poten-
tial to harm and kill both the tissue and the organism if they are not properly
countered.
Acknowledgments PDA’s lab is funded by CRUK and the NIH. Thanks to all members of the
Adams lab, past and present, for stimulating discussions.
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Part IV
Applications in Preventing
and Treating Cancer
Chapter 10
Cell Cycle Deregulation in Pre-neoplasia: Case
Study of Barrett’s Oesophagus
Pierre Lao-Sirieix and Rebecca C. Fitzgerald
Abstract Most solid tumours develop through a pre-invasive stage referred to as

intra-epithelial neoplasia (IEN). The lag time between development of IEN and
progression to fully fledged cancer would allow for chemoprevention or therapeu-
tic interventions; however in most cases, IEN remains undetected and the exact
mechanisms for progression remain unknown. Barrett’s oesophagus, the prema-
lignant stage of oesophageal adenocarcinoma, is the perfect model to study IEN
since the oesophagus is readily accessible by endoscopy and allows for temporal
follow-up of patients. Like most IEN, Barrett’s oesophagus and associated dysplasia
are characterized by an increase in proliferation and the expansion of the prolifer-
ative compartment. These features have been associated with an increased risk of
progression to cancer. There is evidence of dysregulation of the G1/S and G2/M
checkpoints which may be caused by an accumulation of abnormally expressed
growth factors and oncogenes. Furthermore, the abnormally high level of duodeno-
gastro-oesophageal acid reflux bathing the distal oesophagus contributes further to
increasing proliferation. The real challenge for future research will be to identify
causal events and to develop better diagnostic methods and therapeutic options.
10.1 Introduction to Pre-neoplasia

Most solid tumours develop through a pre-malignant stage in which the
accumulation of histological abnormalities can be detected as dysplasia. Moderate
to severe (or high-grade) dysplasia is also referred to as intraepithelial neoplasia
(IEN, O’Shaughnessy et al., 2002). Although the progression from IEN may take as
little as a few months, it is believed that most cases will develop over many years.
There are many examples in which IEN has been linked to risk factors such as
tobacco use in lung and head and neck, human papillomavirus in cervical or acid
P. Lao-Sirieix (B)
Cancer Cell Unit, Hutchison-MRC Research Centre, Cambridge, CB2 0XZ, UK
e-mail: pss29@hutchison-mrc.cam.ac.uk
158 P. Lao-Sirieix and R.C. Fitzgerald
reflux in the oesophageal cancers. In some cases, molecular abnormalities such as

loss of APC for colon or BRCA in breast cancers have been identified. However, in
general, little is known about the mechanisms underlying the progression from low-
grade dysplasia to IEN and to cancer because of the difficulty of creating animal
models or in accessing human samples over the course of the disease. A common
phenotype of IEN is an increase in proliferation leading to both an extension of
the proliferative compartment and an increase in the number of proliferating cells
(Freeman et al., 1999). The ease of access to the oesophagus by endoscopy makes
Barrett’s oesophagus (BE), the precursor lesion for oesophageal adenocarcinoma, a
perfect model in which to study IEN and the role of disordered proliferation and cell
cycle dysregulation.
10.2 Introduction to Barrett’s Oesophagus and Oesophageal

Cancer
Barrett’s oesophagus is defined as the metaplastic change of the normal squamous

lining of the oesophagus into a columnar epithelium akin to that of the intes-
tine (Fig. 10.1). Duodeno-gastro-oesophageal reflux is believed to be the primary
Fig. 10.1 Histological sections stained with haematoxylin and eosin representative of BE
with dysplasia (A), BE with low-grade dysplasia (B), BE with high-grade dysplasia (C) and
adenocarcinoma (D)
10 Cell Cycle Deregulation in Pre-neoplasia 159
trigger of this change but it is likely that additional environmental and genetic fac-
tors also predispose to the disease since it is more common in Caucasian males in
Westernised countries. An estimated 10% of BE patients will develop some degree
of dysplasia and around 2.7% will progress to IEN (Inadomi et al., 2009). An
estimated 5–10% of patients with BE will develop oesophageal adenocarcinoma
(AC) with dysplasia as an intermediate stage (Inadomi et al., 2009). The 5-year sur-
vival rate of oesophageal adenocarcinoma is only 13% despite aggressive treatment
modalities (DeLancey et al., 2008). Patients with BE are usually enrolled in surveil-
lance programmes during which they undergo regular endoscopies, the timing of
which depends on the degree of dysplasia (BSG, 2005; Wang and Sampliner, 2008).
These repeated endoscopies allow for cross-sectional mapping of disease stages
as well as longitudinal follow-up. Despite being controversial with regard to cost-
effectiveness, patients with surveillance-detected cancers have a greatly improved
survival (Wang and Sampliner, 2008). The actual 5-year survival is hard to estimate
since most studies are small audits of practice with varying lengths of follow-up of
patients but most experts agree for a survival in excess of 70% (Wang and Sampliner,
2008). The corollary of this is that 90% of BE patients will never develop can-
cer but all are enrolled in an expensive surveillance programme. Therefore, one of
the main areas of research in the context of BE is the identification of biomarkers
that would predict the risk of progression to cancer in order to minimise the num-
ber of patients requiring invasive unnecessary surveillance procedures. Since it was
recognised early on that BE was a pre-invasive disease for which biomarkers were
needed (Naef et al., 1975), proliferation was one of the first hallmarks to be studied
(Fig. 10.2).
Fig. 10.2 Cell cycle abnormalities in Barrett’s oesophagus

10.3 Proliferation in Barrett’s Carcinogenesis

The proliferative status of BE compared to adjacent normal tissue is controversial
with most studies suggesting that BE is more proliferative than normal squamous
oesophagus (NE, Herbst et al., 1978; Fitzgerald and Triadafilopoulos, 1996), while
another study concluded on the contrary (Iftikhar et al., 1992). This comparison is
not particularly meaningful since BE and NE are different tissue types with very
different mechanisms of homeostasis. However, the same controversy was apparent
in early work when the proliferation of BE was compared with other glandular tis-
sues such as normal stomach and duodenum (Fitzgerald and Triadafilopoulos, 1996;
Pellish, 1980; Hong et al., 1995). Recent data from our laboratory suggest that non-
dysplastic BE and normal glandular tissues have similar proliferation levels (Sirieix
et al., 2003). These discrepancies may be due to the different techniques used and
the subjective interpretation of immunostaining. It is likely that the proliferative sta-
tus of non-dysplastic BE reflects the change to a glandular phenotype. However, in
keeping with our data there is general agreement in the literature that the overall pro-
liferative index increases as BE progresses to adenocarcinoma (Hong et al., 1995;
Sirieix et al., 2003; Gillen et al., 1994; Going et al., 2002; Reid et al., 1993), with
an expansion of the proliferative compartment towards the luminal surface (Hong
et al., 1995; Sirieix et al., 2003; Gillen et al., 1994; Going et al., 2002; Sirieix et al.,
2003). Proliferation at the luminal surface of non-dysplastic BE, but not overall
proliferation levels (Chao et al., 2008), was also associated with an increased risk of
progression (Sirieix et al., 2003). These data suggest that it may be the expansion of
the proliferative compartment towards the surface rather than the proliferation rate
per se which is central to BE carcinogenesis.
Over-stimulation of proliferation by growth factors, luminal factors and muta-
tions in oncogenes (Souza et al., 2001) would be expected to increase proliferation.
For example, the wingless and Int pathway (Wnt) controls part of the prolifera-
tion/differentiation switch through regulation of the expression of Eph receptors and
their ligands, the ephrins (Sancho et al., 2003; Batlle et al., 2002). Proliferative and
differentiated intestinal cells in EphB2/EphB3-null mice intermingle (Batlle et al.,
2002) in a pattern reminiscent of non-dysplastic BE with proliferation at the luminal
surface (Sirieix et al., 2003). Furthermore, knockout mice models for members of
the Wnt pathway, Fkh6 (forkhead homologue transcription factor) (Kaestner et al.,
1997) and Nkx2-3 (NK2 homeobox transcription factor) (Pabst et al., 1999), can
result in hyperproliferative, expanded crypt compartments. Components of the Wnt
pathway are frequently dysregulated in BE and AC (Clement et al., 2006) and may
play a role in BE carcinogenesis.
10.4 Factors Influencing Cell Cycle Progression

In the quest for predictive markers of progression to cancer, cell cycle stage and cell
cycle-related proteins have been investigated in BE over the last 20 years. Several
retrospective studies utilising flow cytometry demonstrated that the number of cells
in G1, S, G2 and M phases increased with progression from BE to AC (Reid et al.,

1993; Sciallero et al., 1993). This increase may only reflect the increased prolifer-
ation observed in BE carcinogenesis rather than dysregulation of cell cycle control
(Lao-Sirieix et al., 2004). However, there is some degree of dysregulation in the
expression levels (Sarbia et al., 2001b; Coppola et al., 1999) and heterozygosity
status of Rb (Sarbia et al., 2001b; Boynton et al., 1991), as well as expression of
cyclin D1 (Bani-Hani et al., 2000) and cyclin E (Sarbia et al., 1999), suggesting that
the G1/S transition control system is overridden leading to more sustained prolifer-
ation. Cyclin A and cyclin B1 were also shown to be upregulated in the progression
to AC (Lao-Sirieix et al., 2004; Geddert et al., 2002a). In addition, increased S and
G2/tetraploid phase fractions were associated with an increased risk of progression
from non-dysplastic and dysplastic BE to AC (Chao et al., 2008; Reid et al., 1992;
Rabinovitch et al., 2001; Barrett et al., 1999; Barrett et al., 2003). Hence, all of
these studies suggest that there is a global increase in cell proliferation and over-
expression of cell cycle proteins rather than a disruption in a specific cell cycle
phase as BE progresses towards AC.
10.4.1 Role of Growth Factors and Oncogenes
There are a plethora of reports implicating growth factors and oncogenes in BE

carcinogenesis. In most cases, their expression is increased from BE to AC.
Epidermal growth factor (EGF), its receptor (EGFR), transforming growth factor
alpha (TGFα) and erbB2 have an increased expression with progression to AC
(Filipe and Jankowski, 1993; Brito et al., 1995; Lord et al., 2003; Flejou et al.,
1994; Hardwick et al., 1995; Walch et al., 2000; Walch et al., 2001; Geddert et al.,
2002b; Nakamura et al., 1994) and are also associated with a poor prognosis (Flejou
et al., 1994; Iihara et al., 1993). The products of the ras family (h, k and n) of onco-
genes modulate cell growth by abrogation of cell growth requirements (Forrester
et al., 1987) and increasing frequencies of k-ras mutations but not c-ras have been
identified along the progression to AC (Meltzer et al., 1989; Jankowski et al., 1992;
Trautmann et al., 1996; Lord et al., 2000). Amplification of k- and h-ras was only
seen in established AC, suggesting that this is a late event in carcinogenesis (Galiana
et al., 1995). Amplification of the c-myc oncogene has only been demonstrated in
dysplastic BE and in keeping with the percentage of cases with amplification and
expression of c-myc increased with progression to AC (Sarbia et al., 2001a; Persons
et al., 1998; Tselepis et al., 2003). c-src was detected in 20% of BE and oesophageal
adenocarcinoma (Jankowski et al., 1992) and was upregulated in BE and AC com-
pared to normal columnar tissues (Kumble et al., 1997). Loss of Smad-dependent
TGFβ signalling together with a switch to MAPK signalling in late stages of BE
carcinogenesis allows the bypass of anti-proliferative signals and facilitates invasion
(Onwuegbusi et al., 2006; Onwuegbusi et al., 2007).
The most studied oncogenes in the progression of BE are p16 and p53.
Dysregulation along the progression to cancer may occur via methylation of p16
(Schulmann et al., 2005), as well as mutations (Chao et al., 2008; Paulson et al.,
2008; Neshat et al., 1994), and loss of heterozygosity (Maley et al., 2006) of both
oncogenes. Presence of LOH at p53 and p16 loci (Chao et al., 2008; Maley et al.,
2006; Galipeau et al., 2007) and methylation at the p16 promoter (Jin et al., 2009)
have been demonstrated to be powerful markers of progression to cancer indicating
an initiating role in the carcinogenic process.
Taken together this data suggest the possibility of an autocrine stimulation of
growth, unimpaired by negative regulators which might result in abnormal epithelial
proliferation independent of usual growth requirements.
10.4.2 Role of Luminal Factors
Pulsatile acid and/or bile stimulation of biopsies ex vivo and cell culture in vitro
induces hyperproliferation through involvement of the sodium–hydrogen exchanger
(Na+ /H+ ) (Kaur et al., 2000; Fitzgerald et al., 1996), a major acid extruder in BE
cells, as well as via the activation of the p38 MAPK, p44 ERK1 and Jun-N-terminal
kinase (Souza et al., 2002; Fitzgerald et al., 1998), and was shown to suppress
apoptosis via the p53 pathway (Morgan et al., 2003). Complete normalisation acid
exposure in the oesophagus was shown to decrease the epithelial proliferation of BE
(Lao-Sirieix et al., 2006; Ouatu-Lascar et al., 1999) but incomplete normalisation
led to an increase in proliferation (Peters et al., 1999; Chen et al., 2001). Whether
pharmacological or surgical suppression of acid reflux reduces progression to dys-
plasia and carcinoma is not clear (Peters et al., 1999; Parrilla et al., 2003). Added to
this there has been a suggestion that the use of proton pump inhibitors may induce
proliferation secondary to increases in gastrin levels (Haigh et al., 2003).
Overall these data suggest a combination of dysregulation of cell cycle entry,

autocrine stimulation (growth factors and oncogene activation) and external stim-
ulants (duodeno-gastro-oesophageal reflux) leading to increased proliferation along
the progression from non-dysplastic BE to AC. In some cases, the dysregula-
tion is so profound that it will actually drive carcinogenesis and hence markers
of this dysregulation may be used clinically to identify patients at a high risk of
progression.
Since most epithelial malignancies develop through a stepwise progression from
benign to malignant lesions and most are characterised by uncontrolled proliferation
it is very likely that lessons learned in the case of BE can be applied to other tumour
types. Despite having identified numerous molecular events occurring as an IEN
progress to a full-fledged carcinoma, it remains a challenge to differentiate between
causal and bystander dysregulations. Further progress in this area is required in order
to develop clinically applicable biomarkers to allow for early diagnosis of cancer.
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Chapter 11
Targeting Cyclin-Dependent Kinases
for Cancer Therapy
Neil Johnson and Geoffrey I. Shapiro
Abstract Cyclin-dependent kinase (cdk) activity is universally deregulated in can-

cer. Therefore, cancer cells may be more sensitive to cdk inhibition than normal
cells. Currently, novel classes of inhibitors of cdk4/6 and cdk2/1 are under develop-
ment, capable of inducing G1 or S/G2 cell cycle arrest, respectively. Transcriptional
cdk inhibition affects RNA polymerase II activity and may alter the apoptotic thresh-
old of cancer cells by compromising expression of highly sensitive short half-life
transcripts. Compounds that concomitantly disrupt cell cycle progression and tran-
scriptional control may therefore have potential as single agents. Additionally, cdk
inhibition may modulate cyclin D1 expression, facilitating synergism with signal
transduction inhibitors, as well as p53 and E2F-1 activities, contributing to apop-
totic responses. Cdk activity has also been implicated in DNA damage-induced
checkpoint control and repair, suggesting promise for chemotherapy/cdk inhibitor
combinations. Further preclinical mechanistic experiments, as well as pharmacody-
namic assessments accompanying clinical trials, will be necessary to establish cdk
inhibition as an anti-cancer strategy.
11.1 Introduction
Cyclin-dependent kinases (cdks) comprise a family of Ser/Thr kinases divided
into two groups, including the cell cycle cdks, which orchestrate cell cycle
progression, and the transcriptional cdks, which contribute to transcriptional reg-
ulation (Malumbres and Barbacid, 2009; Meyerson et al., 1992; Sausville, 2002;
Shapiro, 2006). The first group encompasses core components of the cell cycle
machinery, including cyclin D-dependent kinases 4 and 6, as well as cyclin E-cdk2
complexes, which sequentially phosphorylate the retinoblastoma protein, Rb, to
facilitate the G1/S transition (Sherr, 1994). Cyclin A-dependent kinases 2 and 1 are
G.I. Shapiro (B)

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
e-mail: geoffrey_shapiro@dfci.harvard.edu
168 N. Johnson and G.I. Shapiro
required for orderly S phase progression, whereas cyclin B–cdk1 complexes control
the G2/M transition and participate in mitotic progression (Pines, 1994). The func-
tional activation of cell cycle cdks depends in part on the formation of heterodimeric
cyclin–cdk complexes, which may be modulated by association with endogenous
Cip/Kip or INK4 inhibitors (Sherr and Roberts, 1999). Cdks are also regulated by
phosphorylation, including positive events directed by cdk-activating kinase (CAK,
cyclin H/cdk7/MAT1) and negative phosphorylation events (Morgan, 1995). The
transcriptional cdks, including cyclin H-cdk7 and cyclin T-cdk9 (P-TEFb), pro-
mote initiation and elongation of nascent RNA transcripts by phosphorylating the
carboxy-terminal domain (CTD) of RNA polymerase II (Meinhart et al., 2005).
Cancer is characterized by uncontrolled cellular proliferation. Amplification or
mutation of genes encoding cyclins or cdks, or deletion, mutation, or hyperme-
thylation of genes encoding endogenous inhibitors of cdks, ultimately results in
deregulated cdk activity and loss of cell cycle control, a universal feature of malig-
nant cells (Hall and Peters, 1996; Sherr, 1996). In attempts to re-establish cell
cycle control and block cancer cell proliferation, small molecule ATP competitive
inhibitors remain under active study, including agents that selectively target cdk4/6
to induce G1 arrest in cells retaining wild-type Rb, as well as combined cdk2/cdk1
inhibitors, which induce S and G2 phase arrest. Nonetheless, clinical activity has
been modest, especially in solid tumors (Shapiro, 2006). Recently, however, there
has been recognition that cdk activity contributes to the expression of anti-apoptotic
proteins, the regulation of p53 and E2F-1, and multiple aspects of the DNA damage
response, including checkpoint control and repair. These insights will help define the
subset of cdk family members most appropriate for drug targeting, as well as patient
groups most likely to benefit, and will also inform combinations of cdk inhibitors
with other anti-cancer agents.
11.2 Targeting Cdk4 and Cdk6
The frequency of cyclin D–cdk4/6–INK4 pathway alterations in tumor cells and the
growth suppression afforded by reduction of cyclin D-dependent kinase activity in
preclinical models (reviewed in Shapiro and Harper, 1999; Shapiro, 2006) have led
to the development of selective cdk4/6 inhibitors. The first of these, PD0332991,
is an orally active, water-soluble, cell-permeable pyridopyrimidine that potently
inhibits recombinant cdk4 and cdk6 (IC50 = 0.011 and 0.016 μmol/L, respectively)
by competing with the ATP-binding sites (Toogood et al., 2005). Unlike other cdk
inhibitor compounds, there is no appreciable inhibition of other cell cycle or tran-
scriptional cdks. In vitro, PD0332991 treatment causes cells to accumulate in the G1
phase of the cell cycle in a concentration-dependent manner. As expected, the anti-
proliferative effect is dependent on Rb protein expression; the IC50 against a panel
of Rb-positive solid tumor cell lines ranged from 40 to 400 nM, while IC50 values
against two Rb-negative cells lines was >3 μM (Fry et al., 2004). Similarly, in man-
tle cell lymphoma and multiple myeloma primary cells and cell lines, characterized
11 Targeting Cyclin-Dependent Kinases for Cancer Therapy 169
by cyclin D1 overexpression resulting from genomic rearrangement, PD0332991

also induces cytostatic G1 arrest without apoptosis (Baughn et al., 2006; Marzec
et al., 2006).
Additionally, PD0332991 has demonstrated efficacy in a broad spectrum
of human tumor xenografts. In vivo activity is also Rb-dependent, with little
effect against Rb-deficient xenografts (Fry and Garrett, 2000). Among breast,
glioblastoma, non-small cell lung cancer, and prostate carcinoma xenografts, near-
complete stasis was observed. Interestingly, in mice bearing Colo-205 colon cancer
xenografts, PD0332991 produced rapid tumor regression. The mechanism for this
effect remains unclear. It has been postulated that in some cases, the xenograft repre-
sents a balance of proliferating and apoptotic cells, and inhibition of the proliferative
compartment allows naturally dying cells to predominate. Alternatively, inhibition
of cdk4/6 activity via the p16INK4A tumor suppressor protein has been shown to
reduce VEGF expression and compromise angiogenic signaling (Harada et al.,
1999), observations that may account for effects of a cdk4/6 inhibitor compound
observed in vivo that did not occur in vitro.
Additionally, it has also been demonstrated in SW480 colon cancer cells that
selective cdk4 inhibition causes degradation of the NFκB suppressor protein, IκB,
and induces translocation of RelA from cytoplasm to nucleoplasm and eventually to
the nucleolus. The nucleolar translocation of RelA is accompanied by repression of
NFκB-driven transcription as well as apoptosis, providing a mechanism by which a
cdk4 inhibitor may induce apoptosis in certain cell types (Thoms et al., 2007). It is
possible that the association of cdk4 inhibition with repression of NFκB activity also
contributes to the synergism of PD0332991 with bortezomib (Menu et al., 2008) and
dexamethasone (Baughn et al., 2006) recently reported in multiple myeloma cells.
PD0332991 has completed Phase 1 testing in advanced solid tumors with
documented expression of Rb. Dose-limiting toxicities included neutropenia and
thrombocytopenia, so that treatment was administered 21 of every 28 days or 14
of every 21 days. Nineteen of 74 patients had stable disease ≥16 weeks, includ-
ing patients with breast, colon, and ovarian carcinoma who remained on trial >20
months (O’Dwyer et al., 2007). In the context of this trial, three patients with grow-
ing teratoma syndrome were treated. Each of the mature teratomas expressing high
levels of the Rb protein was inoperable and chemotherapy- and radiation-resistant.
These patients achieved either partial response or stable disease lasting at least
18 months (Vaughn et al., 2009). Additionally, in a preliminary report of a phar-
macodynamic study of PD0332991 performed in mantle cell lymphoma, 8 of 17
heavily pre-treated patients achieved partial response or stable disease lasting up to
60 weeks. PD0332991 treatment caused reduced 18 F-fluoro-3 -deoxy-3 -L-fluoro-
thymidine (FLT) uptake on positron emission tomography (PET) scans, consistent
with G1 arrest, as well as reduced phosphorylation of Rb at cdk4 sites, docu-
mented immunohistochemically in lymph node biopsies obtained before and during
treatment (Leonard et al., 2008).
Mechanisms of resistance have also been investigated, illustrated by the effects
of PD0332991 in acute myeloid leukemia (AML) cell lines. In Flt3 internal tandem
duplication (ITD)-expressing cell lines, PD0332991 induces sustained cell cycle
arrest, followed in some cases by apoptosis. In contrast, in Flt3 wild-type AML cells,
PD0332991 induced an initial G1 arrest that was partially overcome after 120 h
through the downregulation of p27Kip1 and the reactivation of cdk2 (Wang et al.,
2007). This is similar to the cell cycle progression observed in cdk4null/null /cdk6–/–
mouse embryonic fibroblasts, in which cdk2 compensates for the loss of the other
G1 cdks in order to facilitate cell cycle progression (Malumbres et al., 2004).
Therefore, in some cases, agents that inhibit both cyclin D-dependent kinases and
cyclin E–cdk2 complexes may produce stronger G1 arrest phenotypes with delayed
emergence of resistance. Alternative mechanisms of cdk2 activation or G1 progres-
sion may well occur in other cellular backgrounds, so that the development of cell
line derivatives resistant to PD0332991 is likely to be instructive.
11.3 Targeting Cdk2 and Cdk1
Cdk2 was initially considered to be a therapeutically relevant candidate for anti-

cancer drug design emanating from experiments that demonstrated growth arrest
or apoptosis when cells were treated with a dominant-negative mutant (Hu et al.,
2001), cdk2 inhibitory peptides (Chen et al., 1999), targeted cyclin A degrada-
tion (Chen et al., 2004), or ectopic expression of p27Kip1 (Wang et al., 1997).
Consequently, many compounds have since been developed with cdk2 inhibitory
activity. However, initial experiments did not take into account the effects of
these treatments on cdk1 activity. Ectopic p27Kip1 expression or loss of cyclin A-
dependent kinase activity was likely to inhibit both cdk2 and cdk1; expression of the
dominant-negative mutant also reduced cdk1 activity, and reported cdk2 inhibitory
peptides were capable of cdk1 inhibition at high concentration. Therefore, it is
not altogether surprising that experiments with embryonic fibroblasts from cdk2–/–
knockout mice (Berthet et al., 2003; Ortega et al., 2003), as well as tumor cells sub-
jected to antisense- or siRNA-mediated cdk2 depletion (Cai et al., 2006; Tetsu and
McCormick, 2003), have demonstrated either absent or minor defects in cell cycle
proliferation, including only slight slowing of the G1/S transition in synchronized
cells. In cdk2-depleted cells, cyclin E–cdk1 complexes (Aleem et al., 2005), as well
as cyclin D-dependent kinase activity, compensate during the G1/S transition, while
cyclin A–cdk1 complexes govern S and G2 progression (Cai et al., 2006; L’Italien
et al., 2006). One exception has been found in melanoma, where there is correlation
in expression between the microphthalmia-associated transcription factor (MITF)
and cdk2. Melanomas with low MITF expression have low levels of cdk2 and are
particularly susceptible to G1 arrest induced by siRNA-mediated cdk2 depletion
(Du et al., 2004).
In contrast to results for other cell cycle cdks, genetic ablation of cdk1 in the
mouse germline results in very early embryonic lethality, indicating cdk1 is required
for cell division during the early stages of embryonic development (Santamaria
et al., 2007). However, no conditional cdk1 knockout strain is available, and the
effects of cdk1 ablation on different tissue types or mature adult cells have not been
assessed. Nonetheless, consistent with data in knockout mice, shRNA-mediated
depletion of cdk1 from non-transformed retinal pigment epithelial cells leads to
potent G2 arrest (Johnson et al., 2009). In contrast, depletion of cdk1 from can-
cer cells results in the formation of novel cyclin B–cdk2 complexes, so that cells
are able to proliferate with only slight slowing of the G2–M transition (Cai et al.,
2006; L’Italien et al., 2006). Therefore, effects of cdk ablation in embryonic mouse
systems may not always predict outcomes in human cancer cells.
Although in most instances, individual depletion of cdk2 or cdk1 cannot com-
pletely block cancer cell proliferation, dual cdk2 and cdk1 depletion or inhibition
produces S phase retardation and G2 cell cycle arrest, and in some instances cell
death (Cai et al., 2006; L’Italien et al., 2006; Payton et al., 2006). The ability of
cdk2 and cdk1 to easily compensate for one another in transformed cells should
guide the prioritization of compounds with equipotency against cdk2 and cdk1 over
those identified in cdk2-selective screens.
11.4 Combined Targeting of Cdks and Anti-apoptotic Proteins
11.4.1 Transcriptional Cdk Inhibition
Several cdk family members play a role in mRNA transcription and processing. In
addition to its contribution to cdk-activating kinase (CAK) activity, cdk7 phospho-
rylates Ser5 of the heptapeptide repeat of the C-terminal domain (CTD) of RNA
polymerase II. Cdk9, in complex with T cyclins (P-TEFb), phosphorylates the CTD
preferentially at Ser2. These events are required for transcriptional initiation and the
elongation of nascent mRNAs, respectively (Meinhart et al., 2005).
Inhibition of cdk9 has largely been studied with potent inhibitory compounds,
including flavopiridol and seliciclib (Shapiro, 2006). The interaction of flavopiri-
dol with the cdk9 ATP-binding site occurs at a Ki of 3 nM, such that it is difficult
to demonstrate competition by ATP (Chao et al., 2000; Chao and Price, 2001; de
Azevedo et al., 2002). Flavopiridol has profound effects on cellular transcription,
causing depletion of labile mRNAs with short half-life, including those encoding
cyclin D1, c-Myc, mitotic regulatory kinases, and Mdm2 (Lam et al., 2001; Lu et al.,
2004). Additionally, transcripts encoding Mcl-1 and XIAP are rapidly depleted fol-
lowing cdk9 inhibition (Lam et al., 2001). Depletion of Mcl-1 may contribute, in
part, to the activity of flavopiridol recently demonstrated in chronic lymphocytic
leukemia (Byrd et al., 2007; Chen et al., 2005) and has prompted the evaluation
of cdk9 inhibitors in other Mcl-1-dependent hematologic malignancies, including
multiple myeloma and mantle cell lymphoma (Gojo et al., 2002; Kouroukis et al.,
2003; Venkataraman et al., 2006).
The depletion of anti-apoptotic proteins such as Mcl-1 and XIAP via cdk9 deple-
tion or inhibition has also induced apoptosis in solid tumor cell lines in which cell
cycle progression has been disrupted by combined inhibition of cdk2 and cdk1 (Cai
et al., 2006). In this scenario, cdk9 inhibition lowers the apoptotic threshold and
facilitates cell death from the S and G2 phases. Therefore, cdk2, cdk1, and cdk9
may together represent a promising subset of the cdk family for drug targeting.
Several such compounds with cell cycle and transcriptional cdk inhibitory activity
have been described, including AZD5438 (Byth et al., 2009), SNS-032 (Conroy
et al., 2009; Heath et al., 2008), and SCH727965, the latter compound a novel
pyrazolo[1,5-α]pyrimidine that has demonstrated promise in early-phase clinical
trials (Nemunaitis et al., 2009; Parry et al., 2007; Shapiro et al., 2008). Seliciclib,
which also targets cell cycle and transcriptional cdks, has recently demonstrated
activity in nasopharyngeal carcinoma, with evidence of reduced cdk2-mediated
phosphorylation of Rb, as well as reduced transcription of proliferation and sur-
vival genes, with decreased expression of cyclin D1 and Mcl-1, consistent with cdk9
inhibition. Seven of 14 evaluable patients had clinical evidence of tumor reduc-
tion; some responses were associated with increased tumor apoptosis, necrosis,
and decreases in plasma EBV DNA post-treatment (Hsieh et al., 2009). Finally,
a selective cdk7 inhibitor has recently been described with preclinical activity in
vitro and in vivo related to both cell cycle and transcriptional cdk inhibitory effects
(Ali et al., 2009).
In some cell types, it is possible that cdk2 and cdk1 may contribute to phospho-
rylation of the CTD of RNA polymerase II. For example, in U2OS osteosarcoma
cells, combined cdk2 and cdk1 depletion alone resulted in reduced RNA polymerase
II phosphorylation, XIAP depletion, and apoptosis (Cai et al., 2006). Cdk2 deple-
tion has also been shown to induce apoptosis in human diffuse large cell lymphoma
cells, with concomitant reduction in Mcl-1 expression (Faber and Chiles, 2007).
11.4.2 Survivin as a Target of Cdk1
Survivin is an inhibitor of apoptosis protein (IAP) phosphorylated by cyclin

B-cdk1 and is rapidly degraded in the absence of phosphorylation by cdk1. MYC-
transformed cells have been shown to be survivin dependent, and the combination
of MYC overexpression and cdk1 inhibition causes synthetic lethality in both in
vitro and in vivo models of MYC-induced lymphomas and hepatoblastomas (Goga
et al., 2007). Similarly, survivin expression is critical for cancer cell survival after
exposure to microtubule-disrupting agents (O’Connor et al., 2002; Wall et al.,
2003). Following mitotic arrest induced by taxanes or vinca alkaloids, cdk1 inhi-
bition results in compromised survivin expression and substantial apoptosis. This
has led to the development of several sequential taxane–cdk inhibitor combinations
(Shapiro, 2004).
11.5 Reduced Cyclin D1 Expression Mediated

by Transcriptional Cdk Inhibition
The cyclin D1 transcript also has short half-life and is depleted by cdk9 inhibi-
tion. Recent experiments have demonstrated in vitro and in vivo synergism of the
anti-HER2 antibody trastuzumab, as well as small molecule EGFR inhibitors with
inhibitors of cdk9, including flavopiridol and seliciclib (Fleming et al., 2008; Nahta
et al., 2002; Wu et al., 2002). The combination of an ErbB targeting agent with
a cdk9 inhibitor results in enhanced loss of expression of cyclin D1 compared to
either drug alone, which in part accounts for synergistic anti-proliferative effects.
Cyclin D1 is essential for HER2-mediated transformation (Yu et al., 2001) and is
also a critical downstream effector of mutant EGFR (Kobayashi et al., 2006). In
the case of non-small cell lung cancer cell lines, cooperativity was also observed in
EGFR wild-type cells, suggesting the combination may expand the range of tumors
that may benefit from EGFR inhibition (Fleming et al., 2008). These results also
raise the possibility that direct cdk4/6 inhibition may synergize with ErbB pathway
inhibitors (Yang et al., 2004; Yu et al., 2006).
11.6 Modulation of the p53 and p21Waf1/Cip1 by Transcriptional

Cdk Inhibition
Mdm2 represents a transcript targeted by cdk9 inhibition, resulting in elevated levels
of p53 (Alonso et al., 2003; Demidenko and Blagosklonny, 2004). In cells par-
ticularly prone to p53-dependent apoptosis, including acute lymphocytic leukemia
(Jackman et al., 2008) and germ cell tumor cells (Mayer et al., 2005), p53 accumula-
tion may directly contribute to cell death. In addition, transcriptional repression can
also prevent the induction of p21Waf1/Cip1 , so that p53 induction can occur without
a concomitant rise in p21Waf1/Cip1 . The same is true after DNA damage in p53 wild-
type cells. For example, in p53 wild-type colon carcinoma cells that arrest after
CPT-11-mediated stimulation of p53 and p21Waf1/Cip1 , flavopiridol-mediated inhi-
bition of the transcriptional induction of p21Waf1/Cip1 can contribute to cell death
(Motwani et al., 2001). Additionally, cdk inhibitor-mediated suppression of p53-
independent induction of p21Waf1/Cip1 following histone deacetylase inhibition can
convert the response from cytostatic to cytotoxic (Almenara et al., 2002).
Interestingly, cdk2 is synthetically lethal to neuroblastoma cells harboring
MYCN amplification. In these cells, selective cdk2 depletion results in apoptosis
that is dependent on the continued expression of MYCN, as well as accumulation of
wild-type p53 (Molenaar et al., 2009). While not yet formally tested, it is tempting
to speculate that increased p53 levels result from reduced RNA polymerase II CTD
phosphorylation that occurs following cdk2 depletion. Therefore, in these cells, as
well as in lymphoma cells, in which cdk2 depletion is accompanied by Mcl-1 deple-
tion (Faber and Chiles, 2007), it is possible that cdk2 contributes substantially to
CTD phosphorylation.
11.7 Cdks and E2F-1 Activity
11.7.1 Cdk Inhibition and the Prevention of Neutralization of

E2F-1 Activity During S Phase
The cell cycle cdks regulate E2F-1 activity in a complex manner (Chapter 2 by
Ji and Dyson). Following cdk-mediated phosphorylation of Rb during G1, E2F-1
activity is derepressed and E2F-1 is released, so that it can direct transcription
of genes required for S phase. However, this transcription is activated only tran-
siently. Orderly S phase progression requires the downregulation of E2F-1 activity,
accomplished in part by phosphorylation mediated by both cdk2 and cdk1 (Dynlacht
et al., 1994; Kitagawa et al., 1995; Krek et al., 1994; Peeper et al., 1995; Xu et al.,
1994). E2F-1 also interacts with the cyclin H-cdk7 as a component of the TFIIH
muti-subunit protein complex. Phosphorylation of E2F-1 by cyclin H-cdk7 is a
prerequisite for ubiquitination and degradation (Vandel and Kouzarides, 1999).
The targeting of E2F-1 phosphorylation via cdk inhibition may lead to the
selective cell death of malignant cells. The disrupted cyclin D–cdk4/6–INK4–Rb
pathway in tumor cells produces high levels of E2F-1 activity. A reduction in cdk
activity during S phase may lead to persistence of E2F-1 activity that is incon-
sequential for normal cells, but may leave transformed cells with inappropriately
persistent high-level E2F-1 activity that is sufficient to induce apoptosis (Chen et al.,
1999; Jiang et al., 2003). E2F-1-dependent apoptosis following cdk inhibition may
occur in a p73-dependent fashion (Chen et al., 2004).
Alternatively, inappropriately persistent E2F-1 activity may suppress transcrip-
tion of Mcl-1, which may also lead to apoptosis (Croxton et al., 2002). Therefore, in
addition to suppression of Mcl-1 transcription by cdk9 inhibition, Mcl-1 expression
may also be reduced by modulation of E2F-1 activity afforded by inhibition of cdks
2, 1, and 7 (Ma et al., 2003).
Interestingly, baseline E2F-1 activity has recently distinguished multiple
myeloma cells that undergo apoptosis in response to cdk inhibitor compounds from
cells that are resistant (Eguchi et al., 2009). Cells lacking p18INK4C have higher
baseline activity of E2F-1 than cells expressing p18INK4C , related to high cdk6 activ-
ity in p18INK4C -deficient cells. These cells, therefore, have lower baseline levels of
Mcl-1 and more readily reach the threshold for mitochondrially-induced apoptosis
after transcriptional cdk inhibition than cells with higher baseline levels of Mcl-1.
Therefore, in this setting, the pretreatment level of p18INK4C serves as a surrogate
to define E2F-1 activity and the predicted response to cdk inhibition.
11.7.2 Role of Cdk8 in Modulation of E2F-1 Activity
E2F-1 has recently been shown to be a potent and specific inhibitor of β-catenin/T-
cell factor-dependent transcription (Morris et al., 2008). Colorectal tumors that
depend on β-catenin for abnormal proliferation thus select conditions that suppress
E2F-1 and enhance the activity of β-catenin. These conditions include retention of
wild-type Rb, as well as amplification of CDK8. Cdk8 is a member of the mediator
complex, which serves to convey information from gene-specific regulatory pro-
teins to the basal RNA polymerase II transcription machinery. Cdk8 represses E2F-1
activity, and elevated levels of cdk8 protect β-catenin-mediated transcription from
inhibition by E2F-1 (Morris et al., 2008). Cdk8 has been shown to be a colorectal
oncoprotein and its kinase activity is necessary for β-catenin-driven transforma-
tion. Suppression of cdk8 expression inhibits proliferation of colon cancer cells
characterized by high levels of cdk8 and β-catenin hyperactivity (Firestein et al.,
2008). Presumably, small molecule-mediated inhibition of cdk8 or inhibition of cdks

2, 1, or 7 may also serve to activate E2F-1 activity and suppress β-catenin-mediated
gene expression in colorectal cancer cells.
11.8 Cdk Inhibition and DNA Damage
11.8.1 Induction of DNA Damage by Cdk Inhibition

Cdk inhibition during S phase, mediated by pharmacologic inhibitors, dn-cdk2, and
siRNA targeting cdk2, elicits an intra-S phase checkpoint that shares components
of the pathway activated by double-strand DNA breaks (Zhu et al., 2004). In A2780
ovarian cancer cells, cdk inhibition has been shown to induce the accumulation
of activated forms of the phosphatidylinositol 3-kinase family member ataxia-
telangiectasia mutated (ATM) and the checkpoint kinase Chk2, as well as nuclear
foci containing phosphorylated substrates of ATM, including p53 and histone H2AX
(γ-H2AX), suggesting that cdk inhibition can induce or predispose to DNA damage.
Cdk2 phosphorylates a number of proteins during S phase, including minichromo-
some maintenance proteins (MCM), which are required for origin replication firing.
Cdk2 inhibition during DNA replication leads to increased MCM complex associa-
tion with DNA and triggers the rereplication of DNA. Overreplication likely results
in the formation of DSB and ssDNA intermediates, which activate ATM and ATR,
and subsequently p53 (Zhu et al., 2004).
An alternative mechanism by which cdk inhibition could mediate DNA dam-
age appears related to the reduced expression of Chk1 (Enders, 2008; Maude and
Enders, 2005). Chk1, along with Chk2, is typically activated by DNA damage in
order to constrain cdk activity. However, following prolonged (i.e., 24 h) cdk inhi-
bition during S phase, the response to cell cycle slowing involves downregulation
of Chk1, perhaps part of a negative feedback loop promoting cell cycle recovery.
Reduced cdk activity may slow or stall DNA replication forks. This block in repli-
cation is detected by ATR (ATM and Rad3-related), which primarily activates Chk1.
Stalled replication forks are dependent on the ATR–Chk1 pathway for stabilization;
when Chk1 activity is compromised double-strand breaks may occur. This mecha-
nism may, in part, explain the increased cytotoxicity and frequency of double-strand
DNA breaks observed when cdk inhibitors are combined with DNA-damaging treat-
ments such as topoisomerase inhibitors (Crescenzi et al., 2005; Maude and Enders,
2005).
11.8.2 Cdk Inhibition in DNA Damage-Induced

Checkpoint Control
In addition to the replicative stress and DNA damage response elicited by cdk inhi-
bition during S phase, cdks have been implicated in checkpoint control following
exposure to standard DNA-damaging agents. Cdk1 and cdk2 are inhibited down-
stream in the DNA damage pathway, affording cell cycle arrest and time for DNA
repair processes to occur. However, emerging evidence indicates that cdks play
critical roles upstream in the initiation of checkpoint control, prior to the termi-
nal events in the checkpoint cascade when their activities are inhibited. Following
double-stranded DNA breaks, ATM is recruited by the MRN (Mre11–Rad50–Nbs1)
complex (Lee and Paull, 2005). MRN, together with the endonuclease CtIP (Sartori
et al., 2007), carries out the process of end resection in order to produce regions
of single-stranded DNA. Single-stranded DNA is coated by RPA proteins, which
serve to recruit ATR (Jazayeri et al., 2006; Zou and Elledge, 2003). In budding
yeast, cdk1 (cdc28) is required for activation of DNA end resection and ultimately
the Mec1 (ATR homolog)-dependent DNA damage checkpoint following a double-
strand break (Ira et al., 2004). In mammalian cells, inhibition or depletion of
cdk1 alone did not affect end resection, likely because of compensation by cdk2.
However, brief exposure to small molecule-mediated combined inhibition of cdk2
and cdk1 in concert with DNA damage did compromise end resection (Johnson
et al., 2009). Activation of endonuclease activity requires cdk-mediated phospho-
rylation of CtIP at Thr 847, analogous to events that occur in yeast (Huertas and
Jackson, 2009). Cdk inhibition therefore compromises ATR recruitment and the
phosphorylation of Chk1, necessary to initiate checkpoint signaling cascades in
response to DNA damage, and ultimately may sensitize cells to DNA-damaging
treatments.
DNA end resection is just one level at which cdk activity regulates the DNA
damage response. When cells are treated with hydroxyurea (HU), which results in
DNA end resection-independent ssDNA break formation and direct ATR activa-
tion, Chk1 phosphorylation is also abrogated with cdk inhibition. Recent studies
have demonstrated that Chk1 and other ATR and ATM targets are not phospho-
rylated as efficiently when cdk1 is depleted or inhibited in non-small cell lung
cancer (NSCLC) cell lines (Johnson et al., 2009). Loss of DNA damage-induced
checkpoint control was caused by a reduction in formation of BRCA1-containing
foci. Furthermore, expression in BRCA1-deficient cells of BRCA1 mutated at
cdk phosphorylation sites S1497 and S1189/S1191 resulted in compromised for-
mation of BRCA1-containing foci compared to those formed in cells engineered
to express wild-type BRCA1. ATR- and ATM-mediated phosphorylation of non-
chromatin-bound proteins is dependent on BRCA1 focus formation (Foray et al.,
2003; Yarden et al., 2002); therefore, when cdk1 is inhibited and BRCA1 foci
formation is reduced, Chk1 and other BRCA1-dependent ATM/ATR substrates
are not phosphorylated (Fig. 11.1). Selective cdk1 depletion or inhibition also
sensitized NSCLC cells to DNA-damaging agents. Importantly, this sensitization
only occurred in transformed cells. In contrast to transformed cells, which con-
tinued to proliferate in the absence of cdk1 because of compensation by cdk2,
non-transformed cells underwent potent G2 arrest in response to cdk1 deple-
tion, which antagonized the response to subsequent DNA damage (Johnson et al.,
2009).
Fig. 11.1 Cdk activity is required to initiate the DNA damage response. Although cdks have
traditionally been considered to be the downstream targets of DNA damage-induced checkpoint
cascades, cdk activity remains elevated initially after exposure to DNA damage. Cdks participate
in several upstream processes. Cdk5 phosphorylates ATM at S794. Cdks also phosphorylate CtIP,
necessary for DNA end resection following a double-strand break (not shown). Cdk2 phospho-
rylates ATRIP at S224. Additionally, cdk1 phosphorylates BRCA1 at S1497 and S1189/S1191,
events required for efficient BRCA1 focus formation. Therefore, cdk activity facilitates the ATM-
and ATR-dependent phosphorylation of non-chromatin bound substrates, including Chk1 and
Chk2. Eventually, activation of checkpoint kinases leads to inhibition of cdk1 and cdk2 activi-
ties, permitting cell cycle arrest. The inhibition of cdk2 permits interaction of BRCA2 with Rad51
(see text), so that homologous recombination repair can occur
It is of interest that while cdk2 can compensate for reduced cdk1 activity
during DNA end resection in NSCLC cells, the same was not true for BRCA1
phosphorylation. With individual cdk depletion, only cdk1 depletion sensitized to
DNA-damaging treatments in these cells. However, cell type-specific differences
may exist. For example, in MCF-7 breast cancer cells, selective cdk2 depletion
resulted in significant abrogation of Chk1 and p53 phosphorylation after treatment
with ionizing radiation (Deans et al., 2006). Cdk2 has also been shown to phos-
phorylate ATRIP and is required to maintain G2 arrest after DNA damage. Cells
reconstituted with mutated ATRIP at cdk phosphorylation sites were not as strongly
G2 arrested after ionizing radiation as cells containing wild-type ATRIP (Myers
et al., 2007). These results suggest that in some cell types, selective cdk2 deple-
tion or inhibition may sensitize to DNA-damaging treatments. Of note, cdk5 has
recently been shown to directly phosphorylate ATM at Ser794, an essential event for
the activation of ATM kinase activity in response to DNA damage in post-mitotic
neurons (Tian et al., 2009). While cdk5 depletion also affects checkpoint control in
non-neuronal cells, the precise mechanism has not yet been defined (Turner et al.,
2008).
11.8.3 Cdk Inhibition and DNA Repair

In addition to phosphorylation of CtIP at Thr847 to stimulate endonuclease activity,
there is also cdk-mediated phosphorylation at Ser327, which is critical for the inter-
action of CtIP with BRCA1 and recruitment of CtIP to sites of DNA damage (Yun
and Hiom, 2009). Following DNA damage, during S and G2, the cdk-dependent
CtIP–BRCA1 interaction directs the cell toward homologous recombination repair,
as opposed to the non-homologous end-joining repair that occurs during G1, when
cdk2 and cdk1 activities are low. Additionally, the role of cdk1 in BRCA1 phos-
phorylation and recruitment to sites of DNA damage is likely also important for
homologous recombination repair. Cdk inhibition, therefore, may sensitize to DNA-
damaging treatments not only by compromising checkpoint signaling but also by
preventing homologous recombination repair. By extension, cdk inhibition may
sensitize cancer cells to PARP inhibition (Ashworth, 2008).
Cdk2 has also been linked to repair of double-strand breaks by non-homologous
end joining (Crescenzi et al., 2005; Deans et al., 2006). In MCF7 breast cancer
cells, combined cdk2 and BRCA1 depletion resulted in a marked reduction in
colony formation compared to individual knockdowns, suggesting that the target-
ing of several repair pathways may be synthetically lethal (Deans et al., 2006), as is
the case with PARP inhibition in a background of BRCA deficiency. Coupled with
high cyclin E and low p27Kip1 expression found in BRCA-deficient cells (Aaltonen
et al., 2008; Chappuis et al., 2005; Deans et al., 2004), it is possible that these cells
are particularly cdk2 dependent. Whether cdk2 inhibition has therapeutic value in
BRCA1-deficient cancers is yet to be clinically tested.
Of note, cdk2 has also been shown to phosphorylate BRCA2, which impairs
its interaction with Rad51, thereby inhibiting homologous recombination (Esashi
et al., 2005). Although this activity of cdk2 appears paradoxical, it is representative
of the interaction of cdks with BRCA proteins designed to insure that checkpoint
control and DNA repair are properly coordinated. Immediately after DNA damage,
cdk activity remains high. Cdk1 and cdk2 activities regulate DNA end resection and
BRCA1 function and ultimately ATR–Chk1 signaling, while cdk2 phosphorylates
BRCA2 and prevents homologous recombination. Later, only after cdk activity is
reduced downstream in the checkpoint cascade to promote cell cycle arrest is the
interaction of BRCA2 and Rad51 facilitated, permitting homologous recombination
repair (Jazayeri et al., 2006).
Finally, cdk9 inhibition may also serve to suppress homologous recombination
DNA repair. This has been shown to occur in a p53-dependent fashion; although
homologous recombination is considered an error-free repair mechanism, its upreg-
ulation can lead to genomic instability, such that p53 interestingly plays a role in
its suppression, in an activity separable from its classic tumor suppressor func-
tions in transcriptionally transactivating target genes implicated in growth control
and apoptosis (Bertrand et al., 2004; Gatz and Wiesmuller, 2006). In p53 wild-
type colon cancer cells, cdk9 inhibition enhances the degree of p53 accumulation
following exposure to camptothecin-mediated DNA damage. Initially, phosphory-
lated p53 binds and inhibits Rad51 and then ultimately mediates transcriptional
repression of Rad51. This inhibition of DNA repair caused by cdk9 inhibition aug-
ments camptothecin-induced apoptosis in p53 wild-type cells (Ambrosini et al.,
2008). Coupled with effects of cdk inhibition on p21Waf1/Cip1 expression, it is of
interest that irinotecan/flavopiridol combinations have produced the greatest benefit
in p53 wild-type gastrointestinal malignancies (Shah et al., 2005).
11.9 Future Perspectives
New generation cdk inhibitor compounds remain under evaluation. The selective
cdk4/6 inhibitor PD0332991 has demonstrated both expected pharmacodynamic
and clinical activity in tumors retaining wild-type Rb, albeit with a primary out-
come of cytostasis when tumor control is achieved (Leonard et al., 2008). Because
of compensatory activity among cdks in transformed cells, equipotent cdk2/cdk1
inhibitors are more likely to arrest cancer cells than drugs more selective for cdk2,
although melanoma, lymphoma, and neuroblastoma represent the first examples
for which selective cdk2 depletion or inhibition may induce cell cycle arrest or
apoptosis (Du et al., 2004; Faber and Chiles, 2007; Molenaar et al., 2009). The
G1 or S/G2 cycle arrest afforded by cdk inhibitors has reduced tumor burden in
carcinogen-induced models of esophageal and colon adenocarcinoma (Boquoi et al.,
2009; Lechpammer et al., 2005), suggesting these compounds could ultimately play
a role in the prevention of premalignant cell progression.
Several cdk2/cdk1 inhibitors also inhibit transcriptional cdk activity, so that the
single agent activity of cdk2/cdk1/cdk9 inhibition will ultimately be tested in both
hematologic malignancies and solid tumors. Mcl-1-dependent cancer cells may be
particularly susceptible, and cells with high baseline E2F-1 activity may be most
prone to apoptosis (Eguchi et al., 2009). Elevation of E2F-1 activity by cdk2/1 inhi-
bition (or cdk8 inhibition) in colon cancer cells may serve to suppress β-catenin
activity, also expected to translate to clinical benefit (Morris et al., 2008).
In addition to modulation of anti-apoptotic protein expression and E2F-1 activ-
ity, the role of cdks in DNA damage-induced checkpoint control and repair suggests
multiple mechanisms by which cdks may be effectively combined with DNA-
damaging agents. While combined cdk2/cdk1 inhibition may be preferable for
inducing cell cycle arrest by a cdk inhibitor alone, selective inhibition of these cdks
may be easier to combine with DNA damage to prevent cell cycle arrest in malig-
nant cells that could inhibit a DNA damage response (Deans et al., 2006; Johnson
et al., 2009). The clinical development of these combinations remains difficult, since
sequence dependence may affect their optimization and a commitment to random-
ized trials is required. Nonetheless, results to date with several chemotherapy/cdk
inhibitor combinations have demonstrated promise (Bible et al., 2005; Goffin et al.,
2003; Shah et al., 2005).
Whether used alone or in combination, pharmacodynamic assessment of cdk
compounds in early-phase trials will be essential for establishing that the expected
targets are modulated so that clinical outcomes can be better interpreted (Haddad
et al., 2004; Hsieh et al., 2009; Tan et al., 2004). Despite the challenges, further
work is justified to determine whether the strategy of cdk inhibition will play a part
in the anti-cancer armamentarium.
Acknowledgments Supported by NIH grants R01 CA090687, P50 CA089393 [Dana-
Farber/Harvard Center (DF/HCC) Specialized Program of Research Excellence (SPORE) in Breast
Cancer], P50 CA090578 (DF/HCC SPORE in Lung Cancer), Susan G. Komen Post-doctoral
Fellowship Award KG080773, and Lymphoma Research Foundation Mantle Cell Lymphoma
Research Initiative and Correlative Grants MCLI-03-006 and MCLC-07-015.
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Index
Note: The letters ‘t’ and ‘f’ following the locators refer to tables and figures respectively
A Andreassen P. R., 68, 85

Aaltonen, K., 178 Andrews P. D., 62
Abrieu, A., 63 Ansari A. Z., 33
Acosta, M., 143–145, 147 Antonchuk, J., 114
Acquaviva, C., 45 APC/C, see Anaphase-promoting com-
AC, see Adenocarcinoma (AC) plex/cyclosome (APC/C)
Acute myeloid leukemia (AML) cell lines, APC, see Adenomatous polyposis coli (APC)
117–118, 169–170 Apoptosis, 3–5, 12–13, 28, 36, 66–68, 80, 84,
Adams P. D., 11, 139–149 88–89, 98, 101–103, 119, 129, 144,
Adenocarcinoma (AC), 159–162, 179 162, 169–174, 178–179
colorectal, 34 Arai, F., 114
lung, 66 Arentson, E., 50
oesophageal, 158–159, 161 Arnold H. K., 16
Adenomatous polyposis coli (APC), 10, 45, Artandi S. E., 133
48–49, 59, 61–66, 68–69, 82–84, Ashworth, A., 178
86, 158 Astle C. M., 110
Aggarwal, P., 49 Ataxia-telangiectasia mutated (ATM), 27–28,
Aguilera, A., 85 85–88, 115, 129–130, 175–177
Ailles L. E., 116 ATM and Rad3 (ATR), 27–28, 85–88,
Aisner D. L., 128–129 129–130, 133, 143, 175–178
Albanese, C., 6 ATM, see Ataxia-telangiectasia mutated
Aleem, E., 170 (ATM)
Al-Hajj, M., 109 ATR, see ATM and Rad3 (ATR)
Ali, S., 180 Attwooll, C., 25
Ali S. H., 14 Avilion A. A., 130
Allan L. A., 68
Allsopp R. C., 110 B
Almenara, J., 173 Bacchetti, S., 131
Alonso, M., 173 Bahram, F., 131
Alternative lengthening of telomeres (ALT), Baird D. M., 128
11, 127–128, 131–132, 134 Baker D. J., 64, 66, 83
Ambrosini, G., 179 Bakhoum S. F., 61
AML, see Acute myeloid leukemia (AML) cell Baldwin A. S Jr, 144–145
lines Baltimore, D., 87
Amon, A., 83 Bani-Hani, K., 161
Anand, S., 84 Banumathy, G., 142
Anaphase-promoting complex/cyclosome Barbacid, M., 7–8, 24, 44–45,
(APC/C), 45, 48–49, 61–64, 68, 47–51, 167
82–84 Barrett M. T., 157–162
DOI 10.1007/978-1-4419-1770-6, C Springer Science+Business Media, LLC 2010
188 Index
Barrett’s oesophagus, case study, 157–162 Broccoli, D., 11, 129

See also Pre-neoplasia, cell cycle Brown E. J., 87
deregulation in Brown J. P., 129, 141
Barriere, C., 44 Bruce W. R., 116
Barski, A., 31 Bryan T. M., 131
Bartek, J., 85 Budanov A. V., 100
Bartkova, J., 141, 148 Bulavin D. V., 140, 145
Basak, C., 145 Bunz, F., 87
Bassermann, F., 86 Burgering B. M., 12
Batlle, E., 160 Burkhart D. L., 25–27
Baughn L. B., 169 Busby E. C., 91
Bavik, C., 142 Buscemi, G., 130
Bell S. P., 43–44, 47 Buzzai, M., 101
Bembenek, J., 83 Byrd J. C., 171
Benezra, R., 61 Byth K. F., 171
Bennett D. C., 146
Bernardi, R., 142 C
Bernards, R., 6–7, 9 Cahill D. P., 61
Berndtsson, M., 89 Cai, D., 170–172
Berthet, C., 8, 44, 170 CAK, see Cdk-activating kinase (CAK)
Bertrand, P., 178 Calbó, J., 8
Besson, A., 45 Caldecott K. W., 86
Betts J. C., 144 Calegari, F., 115
Bible K. C., 179 Campanero M. R., 29
Bilaud, T., 129 Campbell M. S., 62
Bjorklund, S., 33 Cancer stem cells (CSCs), 109,
Blackburn E. H., 127 120–121
Blagden, S., 91 Cantley L. C., 12
Blagosklonny M. V., 90, 173 Cao, Y., 131
Blais, A., 6–7 Carboxy-terminal domain (CTD), 14, 24, 29,
Blasco M. A., 11, 133 33, 168, 171–173
Blasina, A., 88 Carvalho, A., 89
Blow J. J., 9–10, 50 Caspase-independent mitotic death (CIMD)
Bodnar A. G., 130 pathway, 68
Boehm J. S., 12, 14 Castedo, M., 80, 87
Bonnet, D., 109, 116 Cathepsin, 143
Boquoi, A., 179 Cdk-activating kinase (CAK), 6, 24, 30, 82,
Borlado L. R., 48 168, 171
Botz, J., 27 Cdk inhibition
Boube, M., 32 and DNA repair, 178–179
Bouchard, C., 12 ATR–Chk1 signaling, 178
Boutros, R., 86–87 BRCA-deficient cells, 178
Boynton R. F., 161 cdk9 inhibition, 178
Bracken A. P., 25 CtIP–BRCA1 interaction, 178
Braden W. A., 47 DNA damaging treatments, 178
Bradley T. R., 110 p21Waf1/Cip1 expression, 179
Braig, M., 145 p53 wildtype colon cancer
Bravou, V., 50 cells, 178
Breakage–Fusion–Bridge (BFB cycles), transcriptional, 171–172
129, 133 anti-apoptotic proteins (Mcl-1/XIAP)
Bremner, R., 27 depletion, 171
Brichese, L., 89 AZD5438/SNS-032/SCH727965, 172
Brito M. J., 161 flavopiridol, 171
Index 189
mRNA transcription and process, 171 E2F1 with CDK8, association, 34

seliciclib, 172 RNA polymerase II (Pol II)
U2OS osteosarcoma cells, 172 transcription, 32
CDK inhibitors (CKIs) family, 4–7, 11, 45, 51, “scaffold complex”, 32
84, 168, 175, 179 suppression mechanisms, 32
CDKI, see Cyclindependent kinase inhibitor CDK phosphorylation, 27–29
(CDKI) CDK7–CycH, 28
CDKs for cancer therapy, 167–180 CDK–cyclin complexes, 28
Cdk2 and Cdk1, targeting, 170–171 E2F1 or DP1 in vivo, phosphorylation
antisense- or siRNA-mediated cdk2 of, 29
depletion, 170 E2F1, phosphorylation by kinases, 27
cyclin A degradation or ectopic G0–G1 transition and S phases, 27
expression of p27Kip1 , 170 human E2F1 protein, struc-
depletion of cdk2 or cdk1, 171 ture/regulation, 28f
genetic ablation of cdk1, 170 NLS, 27
MITF and cdk2, correlation, 170 proteasome-dependent degradation, 29
Cdk4 and Cdk6, targeting, 168–170 target genes (Apaf1/p73), 28
cyclin D–cdk4/6–INK4 pathway, 168 cell cycle progression, 23–25
PD0332991, inhibitor, see PD0332991 B-type cyclins, 24
treatment CDK oscillator/transcriptional
Cdk inhibition oscillator, 24
in DNA damage-induced checkpoint cell cycle machinery, 24
control, 175–177 genetic and molecular experiments, 24
and DNA repair, 178–179 genome sequencing, 24
induction of DNA damage, 175 L-type cyclins (CycL), 24
Cdks and E2F-1 activity mitotic cell cycle, 23
Cdk8 in modulation, role of, 174–175 RB/E2F proteins, role of, 24
prevention of neutralization during S Drosophila model system to study E2F
phase, 173–174 activity in vivo, 31–32
combined targeting and anti-apoptotic dE2f1-RNAi-induced phenotypes, 32
proteins drawbacks, 32
survivin as target of Cdk1, 172 E2F homologs (dE2F1/dE2F2), 32
transcriptional Cdk inhibition, 171–172 E2Fs activate transcription, 29–31
cyclin D1 expression, reduced, 172–173 cell proliferation, 31
anti-HER2 antibody trastuzumab, 172 E2F1 and HATs, physical interactions,
EGFR wild-type cells, 172 31
future perspectives, 179–180 E2F1 interaction with p62 subunit
p53 and p21Waf1/Cip1 , modulation of, 173 (TFIIH in vitro and in vivo), 30
acute lymphocytic leukemia/germ cell E2F1-mediated transcriptional
tumor cells, 173 activation model, 30f
p21Waf1/Cip1 , 173 TBP, transcriptional factors, 29
p53 wild-type colon carcinoma cells, future directions, 35–36
173 Rb and E2F proteins, 25–27
CDKs/E2F-dependent transcription, 23–36 CDK–cyclin (Cyc) complexes, 26
CDK8–CycC in human cancers, CDKs, components or regulators of,
deregulation, 34–35 26–27
colorectal tumor cell lines, 35 DNA replication and mitosis, 25
point mutation of CDK8 (D189N), 34 E2F proteins, classification, 25
CDK8–CycC negative regulation, 32–34 eight E2F genes/three DP genes,
biochemical fractionation experiments, mammals, 25
33 G1 to S-phase transition, 25
CDK8/SRB10-mediated higher eukaryotes, 25
phosphorylation, 32–33 pRB inactivation, events of, 27
190 Index
CDKs/E2F-dependent transcription (cont.) Chao S. H., 160–162, 171

RB-E2F regulatory network, general Chappuis P. O., 178
properties, 26f Checkpoint kinase 2 (Chk2), 27–28, 85–88,
small cell lung carcinomas and 129–130, 175
osteosarcoma, 27 Cheeseman I. M., 62
Cell cycle machinery, 24, 167 Chen, C., 113, 116
Celli G. B., 132 Cheng, T., 113, 115
Cellular quiescence, 3–18 Chen H. H., 24
future directions, 17–18 Chen L. Q., 162
oncogenes to bypass quiescence, 10–13 Chen, R., 171
adenoviral oncoprotein E1A, 13 Chen R. H., 61–63
ALT, 11 Chen, W., 14–16, 170, 174
c-MYC-dependent genes, 12 Chen, Y., 86
combinations of oncogenes, effects, 10 Chen Y. N., 170, 174
DNA replication cycle, 11 Chen, Z., 139, 145
E2F-activators (E2F1–3), 12 Cheshier S. H., 111
FoxO transcription factors, 12 Chesnokov I. N., 43–44
inactivation of tumor suppressor genes, Chibazakura, T., 68
effects, 11 Chiles T. C., 172–173, 179
“malignantly transformed”, 11 Chin G. M., 84
p53/ARF pathway, 11 Chin, L., 133
PI3K/AKT pathway, 12 Chi, Y., 33
pRB and p53 pathways, 11 Chk2, see Checkpoint kinase 2 (Chk2)
RAF/MAPK pathway, 11 Chong, L., 129
RAS/RAF/MEK pathway, 12 Chow J. P., 88
“Restriction Point”, 10 Chow J. P. H., 79–91
Rhabdomyosarcomas, 13 Chromosome instability (CIN), 60–61
shRNA, serum starvation, 12 chromosome missegregation/
SV40 large T (LT)/small t (st) aneuploidy/CIN, 60
antigens/hTERT, 11 aneuploidy, 60
restriction point, 5–10 anti-mitotic drugs treatment, 60
exit quiescence back into G1, role of CIN/MIN cancer cells, advantage, 60
cells to, 9–10 dominant gain-of-function
G1-cyclins/CDK, pRB, and E2F mutations, 60
transcription factors, 6–8 germline mutations, 60
reversible state, 3–5 missegregation of one or multiple
cell cycle exit, 4f chromosomes, 60
inhibition of CDKs, 4 chromosome missegregation, defects, 61
MyoD-induced differentiation, 5 aneuploidy, 61
non-dividing states, four, 3 genes, lost or mutated, 61
“quiescent gene expression program”, 4 mutations in genes/proteins, 61
spontaneous senescence, 5 sister chromatid cohesion, 61
transcriptional repressor HES1, 5 Chu, K., 88
SV40 and exit from quiescence, 17–18 CIMD, see Caspase-independent mitotic death
small t antigen promotes exit, 15–17 (CIMD) pathway
tumor antigens and cellular targets, Cimini, D., 61–62
13–15 Cimprich K. A., 86
three pocket proteins, ablation of, 8–9 CIN, see Chromosome instability (CIN)
Cesare A. J., 132 CKIs, see CDK inhibitors (CKIs) family
Chang B. D., 87 Clarke P. R., 68
Chan G. K., 61–63 Clarkson B. D., 117
Chan T. A., 87 Classon, M., 25–26
Chan Y. W., 84–85, 88–89 Clement, G., 160
Index 191
Cleveland D. W., 60, 64–65, DePamphilis M. L., 44

67–68, 84 DePinho R. A., 11
Cobrinik, D., 45, 47 Desai, A., 60
Cocker J. H., 43, 47 Dick J. E., 109, 116–117
Coe B. P., 64–65 Diehl J. A., 6, 144
Cohen S. B., 130 Di Fagagna F. D., 129
Coleman T. R., 43, 47 Diffley J. F., 10, 44, 48
Collado, M., 145 7, 12-dimethylbenz[a]anthracene (DMBA), 67
Coller H. A., 4–5, 9 Di Micco, R., 148
Collins A. T., 109 Dimova D. K., 25
Compton D. A., 60 Dimri G. P., 145
Conaway R. C., 32 Ditchfield, C., 62–63
Connell-Crowley, L., 8 DMBA, see 7, 12-dimethylbenz[a]anthracene
Conroy, A., 171–172 (DMBA)
Coppe J. P., 141–142 DNA damage by Cdk inhibition
Coppola, D., 161 induced checkpoint control, 175–177, 177f
Cortez, D., 86 cdk depletion, 177
Cosme-Blanco, W., 146 checkpoint control initiation, 176
Courtois-Cox, S., 145 HU, cells treated with, 176
Coverley, D., 48 MRN (Mre11–Rad50–Nbs1), 176
Cowell J. K., 85 NSCLC cell lines, 176
Cragg M. S., 80, 90 induction of, 175
Crescenzi, E., 175, 178 A2780 ovarian cancer cells, 175
Crighton, D., 100 ATR–Chk1 pathway, 175
Croxton, R., 174 MCM, 175
CSCs, see Cancer stem cells (CSCs) topoisomerase inhibitors, 175
CTD, see Carboxy-terminal domain (CTD) DNA damage signaling, 140
Cyclindependent kinase inhibitor (CDKI), aberrant or shortened telomeres,
112–114 140–141
SDFs and TIFs, 141
D Dobles, M., 64
D’Adda di Fagagna, F., 140–141, 148 Donner A. J., 34
Dai CY., 145 Donoghue D. J., 88
Damage-regulated autophagy modulator Doonan J. H., 44–45, 48–50
(DRAM), 100–101 Double-stranded breaks (DSBs), 130
Dankort, D., 146 Dou, Y., 31
Dannenberg J. H., 8 Dowling, M., 84
Dan, S., 85 DRAM, see Damage-regulated autophagy
Darzynkiewicz, Z., 111 modulator (DRAM)
Davenport J. W., 61 DREAM complex, 9
Deacon, K., 89 DSBs, see Double-stranded breaks (DSBs)
Dean J. L., 43–51 Duesberg, P., 65
Deans A. J., 177–179 Duhrsen, U., 112
De Azevedo W. F., 171 Du, J., 170, 179
De Bono, J., 91 Dunham M. A., 131
DeCaprio J. A., 14 Duronio R. J., 32
Degregori, J., 27 Dutta, A., 43–44, 47, 50
DeLancey J. O., 159 Dyer M. A., 27
De Lange, T., 129–130 Dynein motility-dependent mechanism, 83
DeLuca J. G., 61–62 Dynlacht B. D., 6–7, 28–29, 174
Demidenko Z. N., 173 Dyson, N., 25–26
Denchi E. L., 11, 127–134 Dyson N. J., 23–36, 114,
Deng, Q., 140, 145 141, 173
192 Index
E Fleming I. N., 172–173

E2F-1 phosphorylation, 174 Flemington E. K., 29
E2F transcription program, 13 Fogal, V., 142
Eckerdt, F., 83 Foley, E., 68
ECM, see Extracellular stroma or matrix Foray, N., 176
(ECM) Forkhead homologue transcription factor
Eddy B. E., 14 (Fkh6), 160
EGFR, see Epidermal growth factor receptor Forkhead O (FoxO) subfamily, 113
(EGFR) cell cycle checkpoints, 113
EGF, see Epidermal growth factor (EGF) FoxO1–4 (triple knockout), 113
Eguchi, T., 174, 179 FoxO transcription factors, 12
Eischen C. M., 99 G0–G1 transition, G1–S and
El-Deiry W. S., 146 G2–M, 113
Elenbaas, B., 11, 13 Forrester, K., 161
Elledge S. J., 131, 176 Forsyth N. R., 129–130
Elmlund, H., 33 FoxO, see Forkhead O (FoxO) subfamily
Elowe, S., 63 Freeman, A., 158
Emili, A., 29 Freire, R., 86
Enders G. H., 148, 175 Frolov M. V., 25
Endoplasmic reticulum (ER) stress Fry C. J., 31
pathway, 102 Fry D. W., 168–169
“End replication problem”, 128 Fryer C. J., 33
Engelhardt, M., 132 Fujita, M., 48–49
Epidermal growth factor (EGF), 161 Fujiwara, T., 85, 134
Epidermal growth factor receptor (EGFR), Funayama, R., 142
161, 172–173 Fung T. K., 80, 82–83
Erenpreisa, J., 80, 90 Furukawa, Y., 89
Esashi, F., 178
Eshleman J. R., 60 G
Essers M. A., 112, 121 Gabrielli, B., 84
Extracellular stroma or matrix (ECM), 143 Galiana, C., 161
Galipeau P. C., 162
F Gan, B., 113
Faber A. C., 172–173, 179 Ganem N. J., 61, 67
Famulski J. K., 62–63 Garrett M. D., 169
Fang, G., 63 Gascoigne K. E., 63, 67–68
Feldser D. M., 146 Gatz S. A., 178
Feng, J., 61, 63, 130 Gazin, C., 142
Feng, Z., 100–101 Geddert, H., 161
Ferber, A., 142 Geisen, C., 7
Fernandez P. C., 12 Geng, Y., 8, 27, 48
Ferrari, R., 13 Gerber, P., 14
Ferrell J. E Jr, 81 Gfi–1, see Growth factor independent 1 (Gfi-1)
Filipe M. I., 161 Gil, J., 97, 99, 144
Finco T. S., 144–145 Gillen, P., 160
Fingert H. J., 87 Gillespie D. A., 63
Firestein, R., 35–36, 174–175 Gillespie P. J., 50
Fishel, R., 60 Gilliland D. G., 109
Fisher R. P., 24, 31 Girardi A. J., 14
Fitzgerald R. C., 157–162 Goffin, J., 179
Fkh6, see Forkhead homologue transcription Goga, A., 89–91, 172
factor (Fkh6) Going J. J., 50, 145, 160
Flejou J. F., 161 Gojo, I., 171
Index 193
Gonzalez M. A., 50 Hastie N. D., 132

Gonzalez, S., 50, 143 Hateboer, G., 29
Gonzalez-Suarez, E., 133 HATs, see Histone acetyl transferases (HATs)
Goodfellow, H., 142 HCF1, see Host Cell Factor-1 (HCF1)
Gope, R., 35 Heald, R., 89
Gorbsky G. J., 61–62 Heath E. I., 172
Gordon K. E., 132 Heinrich P. C., 147
Gorgoulis V. G., 27 HEK, see Human embryonic kidney (HEK)
Grabsch, H., 64 cells
Graña, X., 3–18, 114 Helin, K., 25–26
Graves P. R., 88 Hemann M. T., 133
Greenberg R. A., 133 Hengartner C. J., 33
Greenblatt, J., 29–30 Henry D. O., 6
Greenleaf A. L., 24 Henson J. D., 132
Greenman, C., 34 Herbig, U., 141, 145, 148
Greider C. W., 127, 146 Herbst J. J., 160
Griffin J. D., 116 Herbst, R., 84
Griffith J. D., 130 Hereditary nonpolyposis colon cancer
Growth factor independent 1 (Gfi-1), 113 (HNPCC), 60
Guan, Y., 117 Hériché J. K., 29
Guerra, N., 147 Hernando, E., 67
Guo, A., 142 Herrera, E., 133
Guo, X., 130 HES1, see Hairy and Enhancer of Split1
Gustafsson C. M., 33 (HES1)
Gu, W., 99 Hiom, K., 178
Hirst, M., 33
H Histone acetyl transferases (HATs), 13, 31
H2B-GFP, see Histone H2B–green fluorescent Histone H2B–green fluorescent protein
protein (H2B–GFP) (H2B–GFP), 111
Haase S. B., 24 HNPCC, see Hereditary nonpolyposis colon
Haddad R. I., 179–180 cancer (HNPCC)
Haematopoietic stem cells (HSCs), 110–119 Hochegger, H., 45, 47
Hagemeier, C., 29 Hockemeyer, D., 129, 134
Hahn, S., 29, 31 Hock, H., 113
Hahn W. C., 11, 13–15, 65 Hodgson, B., 9–10
Haigh C. R., 162 Hodgson G. S., 110
Hairy and Enhancer of Split1 (HES1), 5, 9, Hoffman D. B., 62
13, 17 Hofmann, F., 29
Ha, L., 146 Homologous recombination (HR)-based
Hallberg, M., 33 mechanism, 131, 178
Hall-Jackson C. A., 88 Hong M. K., 160
Hall, M., 168 Hontz R. D., 97–103
Hallstrom T. C., 12 Hope K. J., 117
Hampsey, M., 32 Horng, T., 147
Hannink, M., 144 Horwitz G. A., 13
Harada, H., 169 Host Cell Factor-1 (HCF1), 31
Harbour J. W., 45, 47 Houghtaling B. R., 129
Hardwick K. G., 61, 63 Hoyt M. A., 61
Hardwick R. H., 161 HR, see Homologous recombination
Harper J. W., 168 (HR)-based mechanism
Harrison D. E., 110 HSCs, see Haematopoietic stem cells (HSCs)
Harris S. L., 141 Hsiao K. M., 27
Harvey, M., 141 Hsieh W. S., 172, 180
194 Index
Huang, H., 59–70 Ji J.-Y., 23–36, 141, 173

Huang H. C., 63, 68 Jinno, S., 10, 17
Huang P. S., 61, 63 Jin, P., 89
Huang, X., 61 Jin, S., 100, 101
Hu, B., 170 Jin, Z., 162
Huertas, P., 176 Johnson D. G., 9, 25, 27
Hu, J., 49 Johnson J. E., 11
Human embryonic kidney (HEK) cells, 16 Johnson, N., 167–180
Humbey, O., 99 Johnson P. F., 145
Huntly B. J., 109 Jones M. H., 61
Hunt, T., 91 Jones R. G., 101
HU, see Hydroxyurea (HU)
Huttner W. B., 115 K
Hydroxyurea (HU), 176 Kaestner K. H., 160
Kaldis, P., 6, 8, 82
I
Kalitsis, P., 64, 66
Ianari, A., 27
Kamijo, T., 141
IAP, see Inhibitor of apoptosis protein (IAP)
Kao G. D., 61
IC, see Initiation complex (IC)
Kaplan K. B., 61, 69
IEN, see Intraepithelial neoplasia (IEN)
Karakaidos, P., 49–50
Iftikhar S. Y., 160
Karin, M., 100
Iihara, K., 161
Karlseder, J., 130
Inadomi J. M., 159
Karnoub A. E., 140
Ingles C. J., 29
Kastan M. B., 85
Inhibitor of apoptosis protein (IAP), 89, 172
Kato, J., 6
Initiation complex (IC), 44
Inoue, I., 49 Kaufman P. D., 142
Internal tandem duplication (ITD)-expressing Kaur, B., 162
cell lines, 169–170 Kelly-Spratt K. S., 98
Intraepithelial neoplasia (IEN), 157–159 Keniry, M., 12
Ira, G., 176 Khanna K. K., 87
Ishikawa, F., 117 Kiel M. J., 115
Itahana, K., 12, 98 Kim C. F., 109
ITD, see Internal tandem duplication Kimchi, A., 98–99
(ITD)-expressing cell lines Kim S. H., 129
Ito, K., 116, 140, 145 King E. M., 63
Iwanaga, Y., 64 King R. W., 61, 90
Iwasa, H., 140, 145 Kipreos E. T., 24
Kirschner M. W., 49
J Kitagawa, M., 29, 174
Jablonski S. A., 67 Kitahara, K., 50
Jackman K. M., 173 Kitsios, G., 44–45, 47–50
Jackson J. R., 67 Kiyono, T., 139, 141
Jackson S. P., 176 Knez, J., 31
Jacks, T., 6, 25, 85 Knudsen E. S., 43–51
Jallepalli P. V., 60–61 Knudsen K. E., 50
Jang Y. Y., 115 Knuesel M. T., 33–34
Jankowski, J., 161 Kobayashi, S., 173
Jazayeri, A., 176, 178 Koda, M., 112
Jelluma, N., 61 Konishi, Y., 89
Jemal, A., 120 Kops G. J., 60–61, 63–64
Jhanwar-Uniyal, M., 86 Kornberg R. D., 32–33
Jiang, J., 174 Kortlever R. M., 143
Jiang W. Q., 132 Kortmansky, J., 91
Index 195
Kouroukis C. T., 171 Ling Y. H., 89

Kouzarides, T., 28, 30, 174 Linskens M. H., 142
Kozar, K., 44 Lin S. Y., 131
Krek, W., 28–29, 174 Lin W. C., 28
Krizhanovsky, V., 143, 147 Lipinski M. M., 25
Kroemer, G., 101–102 Li, R., 61
Kroll K. L., 82 L’Italien, L., 170–171
Kuffer, C., 85 Litovchick, L., 9
Kuhn E. M., 90 Liu, F., 7
Kuilman, T., 142–145, 147 Liu, D., 129
Kumble, S., 161 Liu, E., 49
Kunsch, C., 144–145 Liu, J., 24, 116
Liu, Q., 87
L Liu S. T., 61, 63–64, 67
Label retaining cell (LRC), 111 Liu, Y., 33
Labib, K., 44 Li, X., 49, 62
Lam L. T., 171 Li, Y., 61
Lam M. H., 88 Loayza, D., 129
Lampson M. A., 62 LOH, see Loss of heterozygosity (LOH) of
Lang S. E., 31 genes
Lanni J. S., 85 Lord R. V., 161
Lan, W., 62 Loss of heterozygosity (LOH) of genes,
Lao-Sirieix, P., 157–162 65–66, 162
Lapidot, T., 109, 116 Lowenberg, B., 116
Larsson, O., 9 Loyer, P., 24
Lau, E., 44, 47, 49–50 LSCs, see Leukaemia stem cells (LSCs)
Lavin M. F., 87 Luciano R. L., 31
Leach F. S., 60 Lundberg A. S., 47
Lechpammer, M., 179 Lu, X., 171
Lee B. Y., 145
Lee E. A., 84 M
Lee, J., 87 Mackey M. A., 89
Lee J. H., 176 MAD2 by binding to p31comet, neutralization
Lee M. G., 24 of, 83
Lee R. J., 26 MAD2–CDC20 complex dissociation
Lees J. A., 25, 32 mechanism, 83
Lee Y. M., 8, 51 Maiato, H., 63, 67–68, 84
Lengauer, C., 60 Maier J. A., 143
Lens S. M., 89 Mailand, N., 10, 48
Leonard, J., 169, 179 Maiorano, D., 43–44, 47
Leone, G., 11–12 Maiti, B., 25
Lerner, C., 110 Maiuri M. C., 100
Lessard, J., 116 Majello, B., 30
Leukaemia stem cells (LSCs), 109–121 Maley C. C., 162
Leung J. Y., 12 Malik, S., 32–33
Levine A. J., 100, 141 Malumbres, M., 7–8, 24, 44–45, 47–51,
Lewis T. B., 64 167, 170
Liang, C., 43, 47 Mammalian target of rapamycin (mTOR), 100
Li, B., 129 Mao, Y., 63
Li, C., 109 Markey M. P., 45, 47
Li, H., 34 Marti, A., 29
Lindeman G. J., 27 Martínez-Balbás M. A., 27
Lindqvist, A., 81–82 Marx, J., 65
196 Index
Marzec, M., 169 MITF, see Microphthalmia-associated

Masuda, A., 68, 84 transcription factor (MITF)
Matrix metalloproteases (MMP1 Mitotic catastrophe, 79–91
and MMP3), 143 and cancer: future directions, 89–91
Matsumura, S., 63 depolyploidization, 90
Matsuoka, S., 113 mis-segregation of chromosomes, 90
Matsuura, I., 7 multipolar mitosis and further genome
Maude S. L., 175 instability, 90f
Ma, Y., 174 roscovitine, 91
Mayer, F., 173 UCN-01, 91
Mayer T. U., 84 caused by abrogation of DNA integrity
Mayer V. W., 85 checkpoints, 87–88
May K. M., 61, 63 ATM/ATR–CHK1/CHK2 axis,
Maynard, S., 116 uncoupling method, 87–88
MCAK, see Mitotic centromere-associated replication or DNA repair, 87
kinesin (MCAK) UCN-01 (CHK1 inhibitors), 88
McCleland M. L., 62 caused by mitotic block/slippage, 83–85
McClendon A. K, 10, 43–51 checkpoint disruption, 84
McClintock, B., 129 cyclin B1–CDK1, role of, 84
McCormick, F., 45, 170 microtubules depolymeriza-
MCC, see Mitotic checkpoint complexes tion/polymerization, 83
(MCC) components mitotic slippage or adaptation, 84–85
McDonald E. R., 146 p53-dependent tetraploidy
checkpoint, 85
McGarry T. J., 49
slippage-refractory cells, 84
McIntosh J. R., 62
spindle-assembly checkpoint
McKeon, F., 61, 68, 84
disruption, 84
Mcm, see Mini-chromosome maintenance
spindle-disrupting drugs, 83–84
(Mcm) complex
description/definition, 79–80
Medema R. H., 12, 81
as form of cell death involving CDK1,
Meeker A. K., 132
88–89
MEFs, see Mouse embryo fibroblasts (MEFs) apoptosis, caspase activation, 88
Meinhart, A., 168, 171 BCL-2 family, 89
Melixetian, M., 50 caspase-6, 88
Meltzer S. J., 161 CDK1-dependent phosphorylations, 88
Mendez, J., 44, 48 cyclin B1–CDK1 kinase activity, 88–89
Menu, E., 169 survivin, 89
Merchant A. M., 44 mitotic control/spindle-assembly
Meyerson, M., 167 checkpoint, 80–83
Michaloglou, C., 145–146, 148 APC/CCDC20, activation of, 82
Microphthalmia-associated transcription factor APC/CCDH1 inactive during mitosis, 83
(MITF), 170 CDC25 activation/WEE1
Microsatellite instability (MIN), 60 inactivation, 81
Millis A. J., 143 CDK1 or CDC2, 80
Milross C. G., 68 CDK1Thr14/Tyr15 , phosphorylation, 81
Milton, A., 25 components of checkpoint
Mimura, S., 44 machinery, 82
Mini-chromosome maintenance (Mcm) control of cyclin B1–CDK1, 81f
complex, 44, 46–50 cyclin B1/B3, 80–81
Minn A. J., 85 MCC components, 82
MIN, see Microsatellite instability (MIN) O-MAD2/C-MAD2, “seatbelt”
Mirza A. M., 12 structure, 82–83
Mismatch repair (MMR) genes, 60 PLK1, activation of, 81–82
Index 197
proteolysis of geminin, 82 CIMD pathway, 68

spindle-assembly checkpoint, clinical inhibitors, 67
termination mechanism, 83 crippled checkpoint or “slippage”, 68
subunits of APC/C, 82 mitotic drug arrest, 67
ubiquitin–proteasome system, 82 mitotic index/apoptosis tumors,
normal control of DNA damage/replication correlation between, 68
checkpoints, 85–87 mitotic checkpoint, 61–64
ATM/ATR, activation of, 85–86 Aurora B kinase, 62
ATR–CHK1 pathway, 86 checkpoint proteins (mechanosensors),
BRCA1-associated genome 63
surveillance complex, 86 core components, 61
CDC25 family, isoforms of, 86–87 hZW10–ROD complex and
claspin by PLK1, phosphorylation of, CENP-E, 63
86 inhibits APC/C, 61
DNA damage or replication stress, 85 kinetochores elaborate error correction
G1/G2 DNA damage checkpoint, 87 system, 62
Mitotic cell cycle, 23 Mad1 and Mad2 checkpoint
gap phases (G1 and G2), 23 proteins, 62
M phase (for mitosis), 23 MCAK, 62
S phase (for DNA synthesis), 23 Nuf2/Ndc80 complex, 62
Mitotic centromere-associated kinesin taxol and nocodazole treatment,
(MCAK), 62 differentiation, 63–64
Mitotic checkpoint and CIN in cancer, 59–70 upstream regulators/downstream
aneuploidy/CIN, mitotic checkpoint, and effectors, 63
cancer, 64–67 “wait anaphase signal”, 62
13 different CIN mice, 65 Mitotic checkpoint complexes (MCC)
aneuploidy, genetic factors, 66 components, 82
APC and BubR, relationship Mittler, G., 33
between, 66 Mittnacht, S., 45, 47
core mitotic checkpoint proteins, Miura, K., 64
mutations of, 64 Mixed-lineage leukemia (MLL), 31
DMBA, 67 Miyamoto, K., 113, 116
“gain-of-function” mutation, 65 MLL, see Mixed-lineage leukemia (MLL)
heterozygous mutations in genes, 65 MMP1 and MMP3, see Matrix
high-grade colonic tumors, 66 metalloproteases (MMP1 and
immortalization, cell sensitivity, 67 MMP3)
kinetochore motor protein, CENP-E, 66 MMR, see Mismatch repair (MMR) genes
liver tumors, 67 Molenaar J. J., 173, 179
LOH of genes, 65 Molz, L., 80
mitotic checkpoint defects, occurrences, Moore M. A., 116
64, 65t Moreno C. S., 16
primary breast tumor samples, 65 Morgan, C., 162
spindle checkpoint genes, 64 Morgan D. O., 168
taxol and vinca alkaloids, spindle Moroni M. C., 28
poisons, 64 Moroy, T., 7
tumorigenesis, 65 Morris E. J., 28, 32, 34–35, 174, 179
tumor suppressors/oncogenes/cell cycle Morselli, E., 102
checkpoint genes, 64 Moshkin Y. M., 142
CIN, see Chromosome instability (CIN) Motwani, M., 173
future directions, 69–70 Mouse embryo fibroblasts (MEFs), 8–9, 12,
mitosis for chemotherapy, 67–68 66, 99, 102–104, 144
anti-microtubule agents, side effects, 67 MRN (Mre11–Rad50–Nbs1), 86,
apoptosis, activation of, 68 132, 176
198 Index
MTOR, see Mammalian target of rapamycin O

(mTOR) O’Brien C. A., 109
Mukaida, N., 144–145 O’Connor D. S., 84, 89, 172
Müller, H., 25–27 O’Dwyer, P., 169
Mulligan, G., 6 Ohata, N., 34
Multani Muntoni, A., 132 Ohtani, K., 27
Murphy M. E., 97–103 Ohta, S., 50
Murphy, N., 50 Ohta, T., 29
Murray A. W., 61 Ohtsubo, M., 8
Musacchio, A., 61, 63, Olovnikov A. M., 128
82–83 Oncogene signaling pathways, 140
Myers J. S., 177 chronic signaling, 140
Myers L. C., 32–33 mutated oncogenic Ras (K-Ras/N-Ras/
H-Ras), 140
N Raf–MEK–ERK signaling, 140
Näär A. M., 33 Onwuegbusi BA., 161
Naef A. P., 159 ORCs, see Origin replication complexes
Nahta, R., 172 (ORCs)
Nakajima, T., 144–145 Orford K. W., 115
Nakamura, T., 161 Origin replication complexes (ORCs), 43–44,
Nakamura T. M., 130 47–48
Nakanishi, M., 129 Orlando D. A., 24
Nasmyth, K., 24 Orr-Weaver T. L., 83
Nemunaitis, J., 172 Ortega, S., 44, 50, 170
Nerlov, C., 144 Orth J. D., 67–68
Neshat, K., 162 Orth, K., 88
NES, see Nuclear export sequence (NES) O’Shaughnessy J. A., 157
Nevins J. R., 12, 25–27 Ouatu-Lascar, R., 162
Nevis K. R., 17 Ouyang, B., 64
NHEJ, see Non-homologous end joining Owen T. A., 9
(NHEJ)
NHF, see Normal human fibroblasts (NHF) P
Nicklas R. B., 62 P18INK4C -deficient cells, 174
Niida, H., 88, 129 P300/CBP-associated factor (PCAF), 31
Niikura, Y., 68, 84 P53/ARF/control of autophagy, 97–103
Nishitani, H., 43, 47 ARF induces autophagy, 98–99
Nitta, M., 88 full-length ARF in autophagy, role of,
NK2 homeobox transcription factor (Nkx2–3), 99
160 full-length ARF and smARF, 98
Nkx2–3, see NK2 homeobox transcription smARF, 98
factor (Nkx2–3) ARF tumor suppressor and autophagy,
NLS, see Nuclear localization signal (NLS) 97–98
Non-homologous end joining (NHEJ), 129, ARF, functional aspects, 98
132, 178 oncogenes (Ha-ras/c-MYC/β–catenin),
Non-small cell lung cancer (NSCLC) cell lines, 97, 98
169, 173, 176–177 p14ARF tumor suppressor, 97
Normal human fibroblasts (NHF), 8–9, 11–12, p16INK4a tumor suppressor, 97
16–17 cell survival and promote tumor
NSCLC, see Non-small cell lung cancer progression, enhancement, 99–100
(NSCLC) cell lines Bcl-xl, 99–100
Nuclear export sequence (NES), 102 Beclin-1/Bcl-xl complex, 100
Nuclear localization signal (NLS), 27 cytoprotective role, 99
Nurse, P., 24, 79–80 overexpressed in B-cell tumors, 99
Index 199
p53-null sarcoma cell line, 99 PD0332991 treatment, 168–170

pro-survival role in autophagy, 99 bortezomib and dexamethasone, 169
two-dimensional in-gel electrophoresis dose-limiting toxicities, 169
technique, 99 effects on AML cell lines, 169
future directions IC50 values, 168
autophagy pathway(s), 103 induces cytostatic G1 arrest, 169
ER stress pathway, 102 inhibits recombinant cdk4 and cdk6, 168
stress-activated/“unstressed” p53, 102, in mice bearing Colo-205 colon cancer
103f xenografts, 169
nutrient stress signals to p53, 101 p16INK4A tumor suppressor protein, 169
p53 negative autophagy regulation in pharmacodynamic study, 169
unstressed cells, 101–102 Rb-dependent/Rb-deficient xenografts, 169
“hotspot” mutations in p53, 102 SW480 colon cancer cells, 169
Kroemer group, 102 Pearson, A., 29–30
mammalian cells/Caenorhabditis Pearson, M., 142
elegans, 101 Pediconi, N., 27
tumor-derived point mutants Peeper D. S., 28, 142, 145, 174
(R282W/R273H), 102 Pelicci P. G., 109–121
p53 transactivates autophagy gene DRAM, Pellish L. J., 160
100–101 Pellman, D., 44, 61, 67
p53 target genes, PUMA and Bax Pennati, M., 84
induce mitophagy, 101 Perkins N. D., 144
p53 tumor suppressor and autophagy, 100 Perry, S., 117
mTOR, inhibition mechanisms, 100 Persons D. L., 161
p53 by DNA damaging agents, Petermann, E., 86
activation of, 100 Petersen B. O., 48
p53 target genes, sestrin1 and sestrin2, Peters F. T. M., 162
100 Peters, G., 97, 99, 168
Pabst, O., 160 Peters J. M., 45, 61
Page-McCaw, A., 143–144 Petit, I., 112
PAI, see Plasminogen activator inhibitor (PAI) Pfeifer G. P., 27
Pallas D. C., 14 Pfleghaar, K., 61
Palmero, I., 141 Phatnani H. P., 24
Palm, W., 129, 132 Phelps-Durr T. L., 142
Pandolfi P. P., 142 Phosphatase and tensin homologue (PTEN),
Pantic, M., 134 11–12, 100, 113
Pardee A. B., 5, 10 PI3K–Akt pathway, 113, 140
Park I. K., 116 PI3K–PTEN–Akt pathway, 113
Park S. S., 91 PIC, see Pre initiation complex (PIC)
Parmar, K., 115 Pimkina, J., 99–100
Parrilla-Castellar E. R., 86 Pines, J., 45, 168
Parrilla, P., 162 Pinsky B. A., 62
Parry, D., 172 Pipas J. M., 14
Parsons, R., 12, 60 PIP, see PCNA-interacting protein (PIP)
Passegue, E., 114–115 Plasminogen activator inhibitor (PAI), 143
Pattingre, S., 100 Plentz R. R., 134
Paull T. T., 176 PML, see Promyelocytic leukemia (PML)
Paulson T. G., 161–162 Pocket proteins
Payton, M., 171 p130 and pRB, 6
PC2, see Positive Cofactor 2 (PC2) Polyomavirus Simian Virus 40 (SV40), 11,
PCAF, see P300/CBP-associated factor 13–17, 66
(PCAF) Poon R. Y., 83, 86
PCNA-interacting protein (PIP), 29 Poon R. Y. C., 79–91
200 Index
Porras, A., 16 complex regulation by CDK/cyclins,

Porter L. A., 88 46f
Positive Cofactor 2 (PC2), 33 cyclin D–CDK4/6 complexes, 45
Potts P. R., 132 E-type and A-type cyclins, 45
PP2A mitogenic signaling, 45
catalytic subunit (PP2A/C), 14 serine/threonine kinases, 44
PP2A/B subunits, four families, 14 functional effects of deregulated Pre-RC
PP2A/C and PP2A/A, isoforms, 14 assembly, 49–50
structural subunit (PP2A/A), 14 depletion of geminin, 50
variable B subunit, 14 over-expression of Cdc6 and Cdt1, 49
PRB and p53 tumor suppressor pathways, 140 over-expression of pre-RC components,
PRB, see Retinoblastoma protein (pRB) 49
Pre initiation complex (PIC), 29–30 negative impact of CDKs, 48–49
Pre-neoplasia, cell cycle deregulation in, direct binding/inhibition of Cdt1 by
157–162 geminin, 49
Barrett’s carcinogenesis, proliferation, 160 DNA re-replication, 49
Fkh6 and Nkx2–3, 160 inhibition of Cdc6, 48
luminal surface, proliferation at, 160 S, G2, and M phases, re-initiation of, 48
squamous oesophagus, 160 ubiquitin-mediated proteolysis, 49
Wnt pathway, 160 positive impact of CDKs, 47–48
BE and oesophageal cancer, 158–159 CDK2 null fibroblasts, 48
BE with dysplasia, 159 cyclin–CDK complex/cyclin E–CDK2,
definition of BE, 158 47–48
duodeno-gastro-oesophageal reflux, cyclin D–CDK4/6 complex, activation,
158–159 47
haematoxylin/eosin representative of cyclin E null fibroblasts, 48
BE with dysplasia, 158f RB pathway, activation, 47
identification of biomarkers, 159 Pre-RC components in cancer, 50
oesophageal AC with dysplasia, 159 deregulation of CDK4/6, 50
surveillance-detected cancers, 159 deregulation of cyclin A and cyclin E,
cell cycle progression, factors influencing, 50
160–161 deregulation of Mcm2–7, 50
growth factors and oncogenes, role of, deregulation of pre-RC components, 50
161–162 over-expression of Cdt1 and/or Cdc6,
c-myc oncogene, amplification, 161 50
methylation of p16, 161–162 pre-replication complex, 43–44
ras family (h, k and n), oncogenes, 161 DNA replication, 43
luminal factors, role of, 162 initiation complex (IC), 44
complete/incomplete normalisation “Pre-replication complex” (pre-RC), 10, 17,
acid exposure, 162 43–51, 47, 82
sodium–hydrogen exchanger Price D. H., 171
(Na+ /H+ ), 162 Prieur, A., 145
pre-neoplasia Promyelocytic leukemia (PML), 118, 132, 142
IEN, risk factors, 157–158 PTEN, see Phosphatase and tensin homologue
lowgrade dysplasia to IEN, 158 (PTEN)
molecular abnormalities, 158 Puig P. E., 90
Pre-RC assembly, regulation of, 43–51 Pulse–chase system, 111
CDKs and general cell cycle control, 44–47 PUMA and Bax induce mitophagy, 101
APC/C, 45
cell cycle order, 45 Q
cell cycle progression, 44 Qian, Z., 64
Cip/Kip family of proteins, 45 Qi, W., 63
CKIs family, 45 Quelle D. E., 8, 97
Index 201
R Ricci-Vitiani, L., 109

Rabinovitch P. S., 161 Ricke R. M., 65–66
Rae1–Bub3/Rae1– Nup98 (compound Rieder C. L., 62–63,
heterozygotes), 65 67–68, 84
Rancati, G., 63, 69 RNA interference (RNAi), 32, 34
Randall T. D., 110 RNAi, see RNA interference (RNAi)
Rao C. V., 66 Roberts A. I., 143
Reactive oxygen species (ROS), 115–116, Roberts B. T., 61
139–140 Roberts J. M., 4, 6, 8, 44–45, 48,
Reddel R. R., 132 141, 168
Reed S. I., 24 Rocha, W., 142
Reef, S., 98–99 Roeder R. G., 32–33
Reid B. J., 160–161 Roninson I. B., 80, 88
Reinhardt H. C., 87–88 ROS, see Reactive oxygen species (ROS)
Ren, B., 25–27 Rossi D. J., 112, 116
Resnitzky, D., 8 Ross J. F., 29
Restriction point, 5–10 Rothblum-Oviatt C. J., 87
cell cycle, functional parts, 5 Roussel M. F., 45
E2F transcription factors, 6 Rowland B. D., 6–7, 9
exit quiescence back into G1, role of cells Ruchaud, S., 88
to, 9–10 Rudolph K. L., 132–133, 146
APC-mediated ubiquitination, 10 Russell, P., 79–80
“attachment checkpoint”, 10 Ryan K. M., 100
cell cycle, elongation of, 9
DNA pre-replication factors, 9 S
MCM2 expression levels, 10 Sage, J., 8, 12, 25–27
quiescence, definition (Blow and Salmon E. D., 61, 63, 82–83
Hodgson), 9 Sampliner R. E., 159
video-microscopy experiments Samuelsen C. O., 33
(Zetterberg and Larsson), 9 Sancho, E., 160
G1-cyclins/CDK, pRB, and E2F Sang, L., 5, 13
transcription factors, 6–8 Santamaria, D., 8, 45, 170
activation of E2F-program of gene Sarbia, M., 161
expression, 7f Sarkaria J. N., 88
CAK and CKIs, 6 Sarkisian C. J., 146
D-type cyclins/E-type cyclins, 6 Sartori A. A., 176
histone synthesis, 7 SASP, see Senescence-associated secretory
inactivation of pocket proteins, 8 phenotype (SASP)
mitogenic stimulation, 6 Sausville E. A., 167
primary tumors and derived tumor cell Sauvageau, G., 116
lines, 7 Sawyer S. L., 44
quiescent NHF, 8 Saxena, S., 50
quiescent tumor-derived T98G cells, 8 Scadden D. T., 115
role of G1 CDKs, 8 “Scaffold complex”, 33
phosphorylation of pocket protein, 5 Scatena C. D., 89
pocket proteins inactivation, 8–9 SCFSkp2 complex in mammals, 29
apoptotic cells, 8 Schatton, T., 109
DREAM complex, 9 Schimming, R., 68
E2F1–3, for cell cycle re-entry, 9 Schmidt, M., 12
pocket protein/E2F pathways, roles, 9 Schmitt C. A., 146
proliferation rate, 5 Schmittwolf, C., 114
Retinoblastoma protein (pRB), 6–8, 11–12, Schulman B. A., 29
14–15, 27–31, 35, 140–141, 167 Schulmann, K., 161
202 Index
Schulze, A., 26 replicative stress, 116

Schwartz G. K., 88 ROS/SNO, 115
Sciallero, S., 161 molecular mechanisms of SC quiescence,
SCID leukaemia-initiating cell (SL-IC), 116 114–115
SCs, see Stem cells (SCs) cdk actions opposed by INK4/Cip/Kip
SDFs, see Senescence associated DNA damage family, 114
foci (SDFs) cycD-cdk4/6 complexes, 114
Sears R. C., 16 myelosuppressive chemotherapy or
Sebastian, T., 144–145 mobilization, 114
Sedelnikova O. A., 145 paradigm in SC exhaustion, 118–120
Seike, M., 64 damage repair mechanism, 119
Self-renewing divisions in normal/LSCs, DNA repair mechanism, effects, 120
regulation of, 109–121 role of p21 and DNA damage, 119f
existence of LSCs, 116–117 regulation of quiescence and self-renewal,
evidence, 116 117–118
flow cytometry methods, 116 AML1–ETO oncogene, 118
“long-” and “short-repopulating” cells, bone marrow transplantation, 117
117 cell cycle inhibitor p21, 118
SL-IC, 116 cell cycle “pause”, 118
xenotransplantation models, 116 PML-RAR oncogene, 118
future directions, 120–121 self-renewal potential of HSCs, 110
anti-tumour drugs, 120 daughter cells, SCs originate, 110
cancer stem cell hypothesis, 120 haemopoiesis, 110
genetic models of SC exhaustion, 112–114 self-renewal, 110
abnormal signalling, 113 serial transplantation assay, 110
c-Myc, 113 Senderowicz AM., 91
deletion of Fbw7, 113 Senescence associated DNA damage foci
HoxB4 over-expression, 114 (SDFs), 141
niche, 113 Senescence-associated heterochromatin foci
p21cip1/kip1, 112–113 (SAHF), 142–143
SNAG domain, 113 Senescence-associated secretory phenotype
HSCs are deeply “dormant”, 110–112 (SASP), 141–142
5-fluorouracil, 110 Senescence-associated-β-galactosidase activity
“dormant” and “activated” HSCs, (SA-βgal), 145
comparison, 112 Senescence-messaging secretome (SMS),
“dormant” HSCs, 111–112 141–142
G-CSF and IFNα, 112 Senescence secretome, impact on tumor
GFP-positive LRC/GFP-negative suppression/cancer, 139–149
non-LRC SCs, 111 altered secretory phenotype of senescent
low levels of RNA and DNA, 111 cells
LRC, 111 growth regulators, 142–143
multiparametric flow cytometry, 111 inflammatory regulators, 143
pulse–chase system, 111 regulation of secretome, 144–145
“quiescence” or “G0”, 110 stromal regulators, 143–144
quiescent or “resting” cells in vivo, 110 See also Senescent cells, altered
“stem cell mobilization”, 112 secretory phenotype of
LSCs are quiescent, 117 senescence secreteome in tumor
molecular mechanisms of SC exhaustion, suppression, 145–148, 146f
115–116 benign clonal neoplasms, 145
Bmi1, Polycomb family, 116 cancerous neoplasms, 146
DNA damage/genomic damage, 116 chemokines, 147
“dormant” niche/vascular niche, 115 clonal benign human nevi, 148
G1 phase of cell cycle, 115 components, role of other, 147–148
Index 203
human NK-like cell line, 147 Shay J. W., 131, 139, 141
immune regulators, 147 Shelton D. N., 142–144
immune surveillance, 147 Shenolikar, S., 14
liver cancer, 147 Shen S. C., 84
mutagenic processes, 146 Shepherd B. E., 110
pre-cancerous neoplasms, 145 Sherr C. J., 4, 6, 11, 27, 44–45, 48, 97–98, 114,
proliferation arrest, 146 141, 167–168
SA-βgal, 145 Shetty, A., 50
senescence program, aspects, 145 Shibata, D., 60
tumor suppression process, 148 Shibutani S. T., 29
senescence signaling pathways, 140–141 Shichiri, M., 64
cell senescence program, 140 Shigeishi, H., 64
DNA damage signals, 140 Shih I. M., 65
oncogene signaling pathways, 140 Shi, J., 67–68
p53 and pRB tumor suppressor Shimizu, M., 27
pathways, 141 Shin H. J., 84
triggers of cell senescence, 139–140 Short mitochondrial ARF (smARF), 98–99
oxidative stress or ROS, 139 Sicinski, P., 8, 51
replicative senescence, 139 Sihn C. R., 84
telomeres, 139 Singer M. S., 142
triggers/signals/effectors of cell Singh S. K., 109
senescence, 140f Single nucleotide polymorphisms (SNP)
Senescent cells, altered secretory phenotype of analysis, 64
growth regulators, 142–143 Sirieix P. S., 160
cyclin A2, 142 Skipper H. E., 117
formation of SAHF, 142 Skoczylas, C., 15–16
gene silencing or nuclear heterochro- Skoufias D. A., 62
matinization, 142 Skwarska, A., 88
histone chaperones, 142 Slee E. A., 88
SAHF/HIRA/WNT2, connections and Sluder, G., 85
significance, 142 SmARF, see Short mitochondrial ARF
inflammatory regulators, 143 (smARF)
cytokines/chemokines receptors, 143 Smith E. R., 31
NKG2D signaling, 143 Smogorzewska, A., 129
regulation of secretome, 144–145 SMS, see Senescence-messaging secretome
C/EBPβ and NFκB, 145 (SMS)
CXRC2 ligands, 144 Smyth M. J., 147
MEFs, 144 SNO, see Spindle-shaped N-cadherin-positive
oncogenic stress, 144 osteoblasts (SNO)
regulators of senescence, 144 SNP, see Single nucleotide polymorphisms
stromal regulators, 143–144 (SNP) analysis
ECM, 143 Sontag, E., 16
MMPs, effects and function, 143–144 Sotillo, E., 3–18, 114
Senga, T., 49 Sotillo, R., 65
Seo, J., 50 Sottile, J., 141
Seo, S., 82 Souza R. F., 160, 162
Serrano, M., 11, 139 Spindle-shaped N-cadherin-positive
Shah M. A., 179 osteoblasts (SNO), 115
Shamma, A., 145 Sprenger, F., 68
Shan, B., 26–27 Srinivas, G., 112
Shapiro G. I., 167–180 Stearns, T., 85
Sharkis S. J., 115 Steen J. A., 64
Sharp J. A., 142 Stegmeier, F., 83
204 Index
Stein, B., 144–145 Takisawa, H., 44

Stem cells (SCs), 109–110, 114 Tanaka T. U., 62
Stevaux, O., 32 Tan A. R., 180
Stevens, C., 27 Tang, Z., 61
Stewart S. A., 11 Tansey W. P., 33
Stillman, B., 43–44, 48 Tao, W., 68, 84
Stoeber, K., 50 Tasdemir, E., 101–102
Storchova, Z., 44, 61, 67, 85 TATA-box binding protein (TBP),
Strebhardt, K., 83 29–30
Su A. I., 34 Tatsumi, Y., 49
Sudakin, V., 61 Taubert, S., 31
Sudo, T., 68, 84 Tavernarakis, N., 102
Sugimoto, M., 98 Taylor S. S., 61, 63,
Sugimoto, N., 49 67–68, 84
Sun, P., 146 TBP, see TATA-box binding protein (TBP)
SV40 and exit from quiescence, 17–18 Telomerase or alternative mechanism (ALT),
small t antigen promotes exit, 15–17 11, 127, 131–132, 134
“attachment checkpoint”, 17 Telomere dysfunction-induced foci (TIFs),
cyclin A protein levels, 16 141, 145
effects on transformation of human Telomeres in cancer, maintenance, 127–134
cells, 15 chromosome end protection, shelterin
foci formation in hTERT-NHF, 16 complex, 129–130
HEK cells, 16 DNA damage machinery, 129
monkey kidney cells (CV-1), 16 DSBs, 130
pre-replication complex factor (CDC6), localization of POT1, 130
17 localization of shelterin, 129–130
RAF/MAPK pathway, 16 molecular mechanisms, 130
tumor antigens and cellular targets, 13–15 six proteins in mammalian cells, 129
carboxy-terminal domain, 14 telomeric TTAGGG, 129
cell cycle proteins, upregulation, 15f future perspectives, 134
DnaJ chaperone domain, 14 telomere dysfunction, 128–129
PP2A, see PP2A ATM/ATR, mammalian DNA damage
SV40 antigens in human cells, response pathways, 129
effects/expression, 14 BFB cycles, 129
SV40, discovery of, 14 end-to-end chromosomal fusions, 129
SV40, see Polyomavirus Simian Virus 40 functions, 128
(SV40) NHEJ, 129
Swann J. B., 147 telomere elongation in cancer, 130–132
Symmans W. F., 68 expression of TERT, 130–131
SynMuvB, see Synthetic multivulva class B PML nuclear bodies (APBs), 132
(synMuvB) gene products telomerase complex, composed of, 130
Synthetic multivulva class B (synMuvB) gene telomere elongation mechanisms, 131f
products, 9 T-SCE, 132
Sze K. M., 64 telomere instability and cancer progression,
132–133
T colorectal carcinomas/breast
Taatjes D. J., 32–33 carcinomas, 132
Tada, S., 49 mus musculus (Terc–/– mice), 133
TAD, see Transactivating domain (TAD) oral squamous cell carcinomas, 132
Tagami, H., 142 skin carcinogenesis/liver
Tago, K., 98 carcinogenesis, 133
Takahashi, A., 88 telomere deregulation in cancer, 133f
Takai, H., 87, 129, 141 telomere shortening, 132
Index 205
telomere length in mammals, 127–128 V

“end replication problem”, 128 Vaclavicek, A., 64
mammalian telomeres/shelterin Vakifahmetoglu, H., 80, 88
complex/T-loop, 128f Vandel, L., 28, 30, 174
telomerase, 127–128 Van den Heuvel, S., 32
telomerase-mediated telomere Van de Peppel, J., 33
elongation/erosion, 127 Van Der Gaag, H., 116
Telomere sister chromatid recombination Van Leuken, R., 83
(T-SCE), 132 Van Steensel, B., 129
Te Poele R. H., 146 Van Vugt M. A., 81
Tetsu, O., 170 Vassilev L. T., 34
Therman, E., 90 Vaughn D. J., 169
Thibodeau S. N., 60 Vaziri, C., 49
Thompson C. B., 101 Venezia T. A., 115
Thompson S. L., 60 Venkataraman, G., 171
Thoms H. C., 169 Ventura, A., 146
Tian, B., 177 Verona, R., 27
TICs, see Tumour-initiating cells (TICs) Verreault, A., 142
TIFs, see Telomere dysfunction-induced foci Viale, A., 109–121
(TIFs) Viale, A., 109–121
Tissue-type plasminogen activator (tPA), 143 Vigo, E., 27
Tommasi, S., 27 Vincent, O., 33
Tomonaga, T., 60 Virshup D. M., 14
Toogood P. L., 168 Vogel, C., 87–88
Torres E. M., 67, 69 Vong Q. P., 61
Tothova, Z., 113, 116 Vousden K. H., 101
TPA, see Tissue-type plasminogen activator
(tPA) W
Transactivating domain (TAD), 28 Waga, S., 44
Trautmann, B., 161 Wajapeyee, N., 142, 147
Tremain, R., 142 Walch, A., 161
Triadafilopoulos, G., 160 Wall N. R., 84, 172
Trimarchi J. M., 25, 32 Wang K. K., 159
Tsantoulis P. K., 27 Wang, L., 170
TSC1, see Tuberous sclerosis complex 1 Wang, M., 144
(TSC1) Wang, Q., 64, 88
T-SCE, see Telomere sister chromatid Wang, X., 64, 170
recombination (T-SCE) Wang, Y., 86
Tse A. N., 88 Warner J. K., 117
Tselepis, C., 161 Watanabe, G., 16
Tsuiki, H., 68 Waters J. C., 62
Tuberous sclerosis complex 1 (TSC1), 113 Waugh D. J., 147
Tumbar, T., 111 Weaver B. A., 60, 64–68, 84
Tumour-initiating cells (TICs), 109 Weber J. D., 98
Turner N. C., 177 Weinberg R. A., 11, 27, 47, 140
Tyagi, S., 31 Weiss, E., 61
Tye B. K., 44 Weissman I. L., 110
West M. D., 141, 143
U Wiesmuller, L., 178
Ueda, Y., 98 Williams B. R., 67
Uetake, Y., 85 Williams G. H., 50
Umar, A., 60 Wilson, A., 111–113, 115
Urist, M., 28 Wilson A. C., 31
206 Index
Wilson, C., 147 Yen T. J., 59–70

Winey, M., 61 Ye, X., 142, 147
Wingless and Int pathway (Wnt), 160 Yoshida, K., 49
Wnt, see Wingless and Int pathway (Wnt) Young A. R., 145
Wohlschlegel J. A., 49 Yuan, B., 64
Wolf, F., 82 Yuan T. L., 12
Wong, C., 85 Yuan, Y., 114
Wong O. K., 63 Yu, D., 84
Woychik N. A., 32 Yudkovsky, N., 33
Wright W. E., 139 Yuen K. W., 60
Wu, K., 172 Yu, H., 82–83
Wysocka, J., 31 Yu H. T., 132
Yun M. H., 178
X Yu, Q., 173
Xiong, Y., 29
Xue, W., 143, 146–147
Xu, M., 28–29, 174 Z
Zachos, G., 63
Y Zetterberg, A., 9
Yamori, T., 85 Zhang, J., 113, 115
Yanagida, M., 82 Zhang, R., 142
Yang, C., 173 Zhang, Y., 98
Yang, L., 114 Zhao J. J., 12
Yang, S., 34 Zheng, N., 25
Yang S. I., 14 Zhong, W., 49
Yao, X., 63 Zhong Z. H., 132
Yarden R. I., 176 Zhu, L., 25
Yasmeen, A., 50 Zhu, W., 26–27, 50
Yee K. S., 101–102 Zhu, Y., 175
Yeh, E., 16 Zimonjic, D., 65
Ye J. Z., 129 Zou, L., 176

(Current Cancer Research) Elena Sotillo, Xavier Graña (Auth.), Greg H. Enders (Eds.) - Cell Cycle Deregulation in Cancer (2010, Springer-Verlag New York)

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Contemporary Cancer Research

Jac A. Nickoloff, S ERIES E DITOR

For other titles published in this series, go to

Cell Cycle Deregulation

ISBN 978-1-4419-1769-0 e-ISBN 978-1-4419-1770-6

Library of Congress Control Number: 2010921202

© Springer Science+Business Media, LLC 2010

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Part I Starting the Cell Division Cycle

Part II Proliferation Under Duress

Part III Long-Term Proliferation

Part IV Applications in Preventing and Treating Cancer

Peter D. Adams Cancer Research UK Beatson Labs, University of Glasgow,

Hospital, Boston, MA 02115, USA; Harvard Medical School, Boston, MA 02115,

Elena Sotillo and Xavier Graña

Abstract Quiescent: From Latin quies, referring to a state of being at

1.1 Quiescence: The Reversible State

requires adequate concentration of mitogens in the environment, space, and for

1.2 Overcoming the Restriction Point

1.2.1 The Restriction Point

The main challenge faced by a cell exiting quiescence is to synthesize de novo

1.2.2 G1-Cyclins/CDK, pRB, and E2F Transcription Factors

E2Fs (E2F1-3), which are subsequently recruited to multiple E2F-dependent pro-

that overexpression of either D1 or E shortens G1 phase upon mitogenic stimulation.

1.2.3 Is Inactivation of Pocket Proteins Beyond a Certain

1.2.4 What Are Cells Doing as They Exit Quiescence Back

The classical video-microscopy experiments of Zetterberg and Larsson defined the

1.3 Oncogenes That Cooperate to Bypass Quiescence

Cellular oncogenic transformation is associated with unresponsiveness to antipro-

1.4 SV40 and Exit from Quiescence

1.4.1 SV40 Tumor Antigens and Their Cellular Targets

As described in Section 1.3, transformation assays designed to identify the precise

by making cells insensitive to checkpoint and antiproliferative signaling, the unique

1.4.2 SV40 Small t Antigen Promotes Exit from Quiescence

The effects of st on transformation of human cells are thought to be, at least in

a limiting mitogenic environment (Skoczylas et al., 2004). Expression of st stimu-

1.5 Future Directions

Jun-Yuan Ji and Nicholas J. Dyson

Abstract Precise control of cell proliferation is essential for normal development

2.1 Cell Cycle Progression Is Driven by the Integrated Action

these phases is driven by the periodic activation of CDK (cyclin-dependent kinase)–

2.2 Rb and E2F Proteins Regulate the G1 to S-Phase Transition

2.3 CDK Phosphorylation Is One of Several Mechanisms

ATM/ATR Chk2 CDK7-CycH

1 84 100 120 191 206 283 380 409 426 437

CycA binding DNA binding dimerization pRB

E2F1–DP1 heterodimers and reduces their DNA-binding properties (Dynlacht et al.,

2.4 How Do E2Fs Activate Transcription?

2.5 Drosophila as a Model System to Study E2F Activity In Vivo

The studies described above illustrate a complicated pattern of interactions between

2.6 CDK8–Cyclin C Negatively Regulates E2F1-Dependent

Biochemical fractionation experiments indicate that the 30 or more mediator sub-

(Donner et al., 2007), a selective small molecule antagonist of MDM2 (Vassilev,

2.7 Deregulation of CDK8–CycC in Human Cancers

drive tumor cell proliferation. A striking example of such context-specific effects

2.8 Conclusions and Future Directions

The identification of CDK8–CycC as a negative regulator of dE2F1 illustrates the

A. Kathleen McClendon, Jeffry L. Dean, and Erik S. Knudsen

3.1 The Pre-replication Complex

DNA replication is a tightly regulated process that requires coordinated assembly of

E.S. Knudsen (B)

recruitment of the mini-chromosome maintenance (Mcm) complex (Mcm2-7) dur-