The Circadian Clock - Pacemaker and Tumour Suppressor PDF

2003 Nature Publishing Group
The word circadian is derived from a Latin phrase

meaning about a day. Daily oscillation of physiological
and behavioural processes in plants and animals have
been reported since the time of Alexander Great, in the
fourth century BC. However, it wasnt until the middle of
thelast century that such oscillating rhythmswerefound
to be driven by an internal timing machine the circa-
dian clock which can maintain biological rhythmsof
about 24 hoursin theabsenceof external cues
1
.
Mutagenic studiesin fruitfly were the first to indicate
that the circadian clock could be regulated genetically
2
.
Thefirst circadian gene, Period,wascloned from fruitflies
in themid-1980s(REFS3,4). Sincethen, therapid advances
in the field of circadian biology research have revealed
that these clocks are operated by numerous gene prod-
ucts that function in interacting feedback loops in all
speciesstudied. Circadian geneshave been discovered in
all species studied, although they might have developed
following independent evolutionary pathwaysin different
kingdoms
5
. Clocks provide organisms with a survival
advantage, by organizing their behaviour and physiology
around cyclic changesin theenvironment.
Circadian rhythmsregulate hundredsof functionsin
the human body, including sleep and wakefulness, body
temperature, blood pressure, hormone production,
digestive secretion and immune activity. Disruption of
these rhythms can have a profound influence on our
health. For example, disruption of circadian rhythmshas
been linked to insomnia, jet lag, stomach ailments, coro-
nary heart attacks and depression
6
, and is commonly
observed among cancer patients
7,8
.
There has been a long history of research seeking a
link between circadian clocks and tumour suppres-
sion. Studies of animal models and human tumour
samples have revealed that the disruption of circadian
rhythms is an important endogenous factor that con-
tributes to mammalian cancer development
912
. CANCER
CHRONOTHERAPY is based on the asynchrony that exists
i n cell proli ferati on and drug metaboli sm between
normal and malignant tissues. Administration of can-
cer therapy based on ci rcadi an ti mi ng has shown
encouraging results, but still lacks a strong mechanis-
tic foundation
13
. Further investigations into the mole-
cular li nk between the ci rcadi an clock and growth
regulation will generate novel therapeutic strategies
for improving the efficacy of cancer treatment. What
is our current understanding of the role of the circa-
dian clock in growth control, and how does it affect
tumour suppression and cancer treatment?
Circadian clockwork
Eight core circadian genes have been identified so
far. They are Clock
14
, casein kinase I (CKI)
15,16
,
cryptochrome1(Cry1) and chryptochrome2(Cry2)
1719
,
THE CIRCADIAN CLOCK:
PACEMAKER AND TUMOUR
SUPPRESSOR
LoningFu and ChengChi Lee
The circadian rhythm s are daily oscillations in various biological processes that are regulated by
an endogenous clock. D isruption of these rhythm s has been associated w ith cancer in hum ans.
O ne of the cellular processes that is regulated by circadian rhythm is cell proliferation, w hich often
show s asynchrony betw een norm al and m alignant tissues. This asynchrony highlights the
im portance of the circadian clock in tum our suppression in vivoand is one of the theoretical
foundations for cancer chronotherapy. Investigation of the m echanism s by w hich the circadian
clock controls cell proliferation and other cellular functions m ight lead to new therapeutic targets.
Department of Molecular
and Human Genetics,
Baylor Collegeof Medicine,
OneBaylor Plaza,Houston,
Texas77030,USA.
Correspondenceto C.C.L.
e-mail: ching@bcm.tmc.edu
doi:10.1038/nrc1072
3 5 0 | MAY 2003 |VOLUME 3 www.nature.com/reviews/cancer
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NATURE REVI EWS | CANCER VOLUME 3 |MAY 2003 | 3 5 1
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in the nucleus but apparently do not directly bind to
DNA. The PASdomains are functionally important, as
they provide surfaces that allow heterodimerization
among different clock proteins. The Clockand Bmal1
genes encode basichelixloophelix (bHLH)-PAS
transcription factors. The levelsof mRNAsand proteins
of thesecircadian genes, except thoseof Clockand CK1,
oscillatethroughout the24-hour period
27
(TABLE1).
The molecular clockwork of the mammalian central
pacemaker has been the subject of several recent
reviews
5,2729
. Briefly, it can be explained by a model of
transcription-translation feedback loops of circadian
genes(FIG. 1). The anatomy of the mammalian circadian
clock contains three components: input pathways, the
central pacemaker and output pathways. The input
pathways transmit information from environmental
cues to the central pacemaker. The central pacemaker
synchronizeswith the environment to generate endoge-
nousrhythms. Theoutput pathwaysconvert theinstruc-
tions from the central pacemaker into daily oscillations
in various physiological and behavioural processes
27,28
(FIG. 2). Thecentral pacemaker in mammalsresidesin the
SUPRACHIASMATIC NUCLEI (SCN) of the anterior hypothala-
mus
30,31
(BOX 1). The SCN is composed of multiple, sin-
gle-cell circadian oscillators that, when synchronized,
generate coordinated circadian outputs
32,33
. Ablation of
SCN leadsto lossof circadian rhythm in rodents
30,31
.
Among all environmental cues, light is the most
powerful circadian synchronizer
34,35
. In mammals, the
circadian photoreception pathways are distinct from
those of visual perception. Mice lacking rods and cones
respond normally to light-induced melatonin
suppression and PHASESHIFTSin behaviour rhythms
36,37
.
Period1 (Per1), Period2(Per2) and Period3 (Per3)
2024
,
and Bmal1 (REFS25,26). The three Per genes encode
PERARNTSIM (PAS)-domain proteinsthat function
Summary
The circadian clock is the internal timing machine that can sustain rhythms of about
24 hours in the absence of external cues. The circadian clock is operated by the
feedback loops of the circadian genes in the mammalian central pacemaker, as well as
in most peripheral tissues.
The mammalian central pacemaker is located in the suprachiasmatic nuclei (SCN) of
the brain and controls the activity of peripheral clocks through the neuroendocrine
and autonomic nervous systems. The circadian clock regulates hundreds of functions
in the human body.
Disruption of circadian rhythms has been linked to mammalian tumorigenesis and
tumour progression, and has been used as an independent prognostic factor of
survival time for patients with certain metastatic cancers.
Normal and malignant tissues often show asynchronies in cell proliferation and
metabolic rhythms. Based on these observations, cancer chronotherapy has been
developed to improve the efficacy in cancer treatment and the quality of patients life.
The circadian clock functions in vivoas a tumour suppressor at the systemic, cellular
and molecular levels. The central clock is capable of generating 24-hour cell-
proliferation rhythms in peripheral tissues through the activity of the neuroendocrine
and autonomic nervous systems.
Molecular clocks in peripheral tissues control cell-proliferation rhythms by regulating
the expression of cell-cycle genes. The core circadian genes are also involved in
regulating cell proliferation. The circadian clock in peripheral tissues responds directly
to DNA damage and could be important in the control of the cell cycle and apoptosis.
The molecular clockworks and cell-cycle clocks in peripheral tissues can be regulated
simultaneously by the central clock, through interacting signalling pathways.
Further study of the mechanism of the circadian clock in tumour suppression and
the DNA-damage response has important implications for cancer therapy.
Table 1 |Mammalian circadian regulators
Circadian regulators Role in mammalian circadian clock Direct and indirect targets References
in growth regulation
Period 1 (Per1) Involved in circadian phase resetting in SC N 2022,57,123,
and in peripheral tissues; m utations shorten 131133,136,141
circadian period in rodents
Period 2 (Per2) M utation alters behaviour rhythm icity, c-M yc, cyclin D 1, p53, 11,22,24,60,197
results in neoplastic grow th phase and M dm 2, cyclin A, G add45
deficient D N A-dam age response in rodents,
and causes advanced sleep disorder in
hum ans; stim ulates Bmal1expression
Period 3 (Per3) M utations do not affect behaviour rhythm icity 23
in rodents
C asein kinase I (C KI) Phosphorylates Per to control Per stability -C atenin 15,16,173175
and nuclear localization; m utations alter
behaviour rhythm icity in rodents
C lock Physically associates w ith B m al1; binds to c-M yc? 14,31,140
E-box sequences to stim ulate Per and Cry
transcription; m utations alter behaviour
rhythm icity in rodents
B m al1 Physically associates w ith C lock; binds to c-M yc 11,25,26,30,31
E-box sequences to stim ulate Per and Cry 123,140
transcription; m utations alter behaviour
rhythm icity in rodents
C ry1 and C ry2 Physically associate w ith and stabilize c-M yc 11,1719,31
Per; m utations alter behaviour rhythm icity
in rodents; m ight also be im portant in
photoreception; suppress B m al1/C lock
transcription activity
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However, the phases of circadian gene expression in
peripheral tissues are delayed by a few hours, relative
to those of SCN, and ablation of SCN abolishes circa-
dian gene oscillation in peripheral tissues
49,50
. So, the
peripheral clocks are either driven or synchronized by
the SCN pacemaker. Under certain conditions, how-
ever, such as when food supply is restricted at resting
phase, the peri pheral clocks can be completely
uncoupled from the SCN central clock, and take their
phasing cues from the feeding time rather than from
the SCN
51,52
.
Clock control. Recently, i t was shown that the tran-
scri pti on feedback loops of ci rcadi an genes can be
regulated by intracellular redox pathways
53
. This indi-
cates that peripheral clock timing can be affected by
the intracellular metabolic rate, which is independent
of the SCN pacemaker
54
. The phase resetti ng i n
peripheral tissues by restricted feeding is a relatively
slow process that can be quickly reversed when food
i s provi ded wi th a normal feedi ng schedule. The
phase reversal of the peri pheral clock seems to be
under the control of hormones of the neuro-
endocrine system. Hormones such as glucocorticoids
inhibit the uncoupling of peripheral clocks from the
central pacemaker
55
.
The SCN central clock and peripheral tissue clocks
both regulate cell functions by controlling the expres-
sion of clock-controlled genes. These genes are not,
however, required for clock function. Whereas some
clock-controlled genes are regulated indirectly by the
molecular clock, some are regulated directly, such as
by the Bmal1Clock heterodi mer, whi ch bi nds to
E-box sequences in gene promoters
11,27
. Recent studies
i ndi cate that 210% of all mammali an genes are
clock-controlled genes
5561
. Most of these show ti s-
sue/organ-speci fi c expressi on patterns and are
involved in organ function. Only a small set of clock-
controlled genes are expressed i n multi ple organs.
Among them are genes that encode key regulators of
cell-cycle progression
11,5659
.
Loss of circadian rhythm and tumorigenesis
The finding that disruption of circadian rhythms led to
increased mammary tumour development was first
reported in the 1960s(REFS62,63). These studiesindicated
that disruption of circadian endocrinerhythms either
through constant light exposure or by PINEALECTOMY
accelerates breast epithelial stem-cell proliferation,
inducesmammary-gland development and increasesthe
formation of spontaneous mammary tumours in
rodents
64,65
. In addition, light-induced circadian-clock
suppression also increasescarcinogenesisin rodents
66,67
.
Several epidemiological studies have revealed a role
for the circadian clock in human breast cancer devel-
opment
9,10,68,69
. They showed that disruption of circa-
dian cycles, such as in people that work predominantly
at night, is a risk factor for breast cancer development.
The breast cancer risk increased with the number of
years, or number of hours per week, that individuals
spent working at night. These studies were carefully
Certain human blind subjects with no significant
perception of light still retain the circadian response
to light
38
. Light signals are received by a subset of
MELANOPSIN-EXPRESSING RETINAL GANGLION CELLS, and are
transmitted directly to the SCN through the retinohy-
pothalamic tract (RHT)
3942
(BOX 1). In addition, the
mammalian circadian genes Cry1and Cry2are also
involved in photoreception by certain retinal neurons
43
.
The RHT produces neurotransmitters that activate a
cascade of events in SCN neurons, leading to circadian
phase resetting
27,28
.
Peripheral clocks. Circadian rhythms are regulated in
peripheral tissues by similar interacting loops of core
circadian gene products. These peripheral clocks are
regulated by the SCN pacemaker, through both the
autonomic nervoussystem (ANS) and neuroendocrine
systems
4446
(BOX 1). The rhythmic expression of core
ci rcadi an genes i s observed i n most peri pheral ti s-
sues
47,48
, and can be induced in cultured fibroblasts
49
.
SUPRACHIASMATIC NUCLEI
(SCN). Themammalian master
circadian clock. TheSCN are
small bilateral structureslocated
next to thethird ventricleand
just abovetheoptic chiasm in
mammalian brain. Each SCN
nucleuscontainsabout 10,000
neuronsthat aresynchronized to
generatecoordinated circadian
outputsin vivo.
PHASESHIFT
Thedisplacement of waveform
in time. When awaveform is
displaced by acomplete
wavelength, it isdescribed as
having aphaseshift of 360
degrees. When awaveform is
displaced by ahalf awavelength,
it isdescribed ashaving aphase
shift of 180 degrees.
P
C KI
C KI
B m al1
B m al1
D egradation
C KI
Per
Per
Per
Per
C ytoplasm
N ucleus
Per
R ev-Erb
R ev-Erb
R ev-Erb
C lock
C lock
B m al1
B m al1
C ry
C ry
C ry
C ry
Per
Figure 1 | Mammalian core circadian gene feedback loops. C ircadian rhythm s are
generated by the feedback loops of the core circadian genes. In the SC N neurons, the
intracellular levels of C lock rem ain steady throughout the 24-hour period, w hereas B m al1
expression levels are high at the beginning of a subjective day and low at the beginning of a
subjective night. The high level of B m al1 prom otes the form ation of B m al1C lock
heterodim ers. These bind to E-box sequences in the prom oters of the Cry, Per and RevErb
genes to activate transcription at the beginning of a circadian day. The B m al1C lock
heterodim er can also inhibit Bmal1transcription. After transcription and translation, the
R evErb protein enters the nucleus to suppress the transcription of Bmal1and Crygenes.
As the Per proteins, such as Per2, accum ulate in the cytoplasm , they becom e
phosphorylated (P) by C KI. The phosphorylated form s of Per are unstable and are degraded
by ubiquitylation. Late in the subjective day, how ever, C ry accum ulates in the cytoplasm ,
prom oting the form ation of stable C KI/Per/C ry com plexes, w hich enter the nucleus at the
beginning of a subjective night. O nce in the nucleus, C ry1 disrupts the C lock/B m al1-
associated transcriptional com plex, resulting in the inhibition of Cry, Per and RevErb
transcription, and derepression of B m al1 transcription
193,194
. It is not clear w hether Per and
C ry m ust dissociate from the C KI/Per/C ry com plex to inhibit the activity of C lock/B m al1
heterodim er and to stim ulate Bmal1transcription in the nucleus. The interacting positive and
negative feedback loops of circadian genes ensure low levels of Per and C ry, and a high level
of B m al1 at the beginning of a new circadian day
5,2729
. Solid lines indicate direct regulation,
and dashed lines indicate indirect regulation.
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Similar observations have been made in cancer
patients. When circadian rhythms determined by
levels of salivary cortisol were measured in patients
with metastatic breast cancer, early mortality was
observed more frequently in patientswho had lost nor-
mal diurnal salivary cortisol variation. After adjusting
for other factors that might affect survival time, the cir-
cadian rhythm of salivary cortisol remained asa statisti-
cally significant predictor of survival time for patients
with breast cancer
72
. The circadian rhythm of rest/activ-
ity wasalso monitored in patientswith metastatic colo-
rectal cancer. The 2-year study showed that patients
with clearly defined rest and activity rhythmshad a five-
fold higher survival rate and experienced significantly
less fatigue than patients who had lost rest/activity
rhythm. So, the rest/activity rhythms provide a novel
independent prognostic factor for survival and tumour
responseof patientswith metastatic colorectal cancer
73
.
Chronotherapy
Most conventional cancer therapeutic strategies are
aimed primarily at maximizing cytotoxicity and avoid-
ing acquired drug resistance, whereas the ability of the
host biological system to cooperate with the therapy
has not been taken into full consideration. Anticancer
drugsusually act selectively on proliferating cellsor at a
specific phase of the cell cycle. These drugsnot only tar-
get cancer cells, but also normal host tissues that are
engaged in active cell proliferation. So, the balance
between the level of damage to normal tissues and the
targeting efficiency to tumours, or the therapeutic
index, is not always favourable. Chronotherapy has
been developed in an attempt to improve the efficacy of
cancer treatment and the quality of patients life.
The principle of chronotherapy in oncology is to
take advantage of the asynchronies in cell prolifera-
ti on and drug metaboli c rhythms between normal
and malignant tissues by administering therapy at a
specific time of the day. This could potentially mini-
mize the damage to host tissues and maximize drug
toxicity to tumours
13,74
. The efficiency of anticancer
drugs in vivois determined by their absorption, distri-
bution, intracellular metabolism and elimination. All
these processes show ci rcadi an vari ati on in vivo
75
.
Some of them, such as the rhythm of tumour blood
flow, can be di sti nct from that of normal ti ssue
76
.
Once inside the cell, the effect of a cytotoxic drug is
mainly determined by the circadian phase of that cells
proliferation cycle
74,77,78
.
Circadian variation of mitotic activity in normal
human tissues was first described in 1938 (REF. 79). It is
now well known that cell proliferation in rapidly renew-
ing mammalian tissuesfollowsdaily oscillation patterns
in vivo
77,80,81
. Themitotic rhythmsof mammalian cancers
havebeen studied since1940 (REF. 82). So far, accumulated
evidence indicates that mammalian tumours, even at
advanced stages, arenot temporally disorganized masses.
Theproliferation of tumour cellscan bestably entrained
to thehost circadian rhythm or follow a tumour-specific
rhythm, in vivo. The coupling of proliferating rhythm
between host and tumour cells is usually observed in
designed, and included a large number of participants,
as well as long follow-up peri ods. The study by
Schernhammer et al.
9
included 78,562 women from
the Nurses Health Study and involved a 10-year fol-
low-up period. So, circadian rhythms could be more
important than family history in determining breast
cancer risk
70
.
Disruption of circadian rhythm not only increases
the risk of tumour development, but also accelerates
cancer progression in tumour-bearing animals and in
cancer patients. Carcinoma- or sarcoma-bearing rats
showed increased tumour growth and reduced sur-
vival time when kept in alternative light/dark (L/D)
cycles such as L/D for 14:10 hours followed by D/L
for 10:14 hours every 3 days than those kept i n
constant L/D cycles of 12:12 hours
71
. A recent study
showed that ablation of SCN in mice resulted in loss
of circadian rhythm in wheel-running activity, body
temperature, plasma corticosterone and lymphocyte
numbers. Osteosarcomaor pancreati c adenocarci -
noma both grew 23 ti mes faster i n mi ce beari ng
SCN lesi ons than i n controls, leadi ng to si gni fi cant
reductions in survival time
12
.
MELANOPSIN-EXPRESSING
RETINAL GANGLION CELLS
A small subset of retinal
ganglion cellsthat are
intrinsically photosensitiveand
expresstheopsin-likeprotein
melanopsin. Theseneurons
project directly to the
suprachiasmatic nucleusof the
mammalian central circadian
clock, aswell asto the
intergeniculateleaflet and the
olivary pretectal nucleusin the
brain. Micethat aredeficient in
melanopsin show attenuated
responsesto light stimuli.
PINEALECTOMY
Ablation of thepineal gland.
Thepineal gland isacone-shape
gland that islocated at the
posterior end of thethird
ventriclein thebrain. Thepineal
gland producesmelatonin, a
hormonethat isimportant for
regulating circadian rhythmicity
in humans. Thelevel of
melatonin risesat night and falls
during theday.
Aldosterone
C ortisol
U rine output
Lym phocytes
M onocytes
Platelets
Eosinophils
Aw ake Sleep Aw ake Aw ake Sleep
Figure 2 | Circadian regulation of hormones, urine and the
immune system.The circadian oscillation of hundreds of
biological processes enables a hum an body to adapt to 24-
hour light/dark cycles. a| The serum levels of cortisol and
aldosterone, and urine volum e
195
, oscillate in circadian cycles in
hum ans. The cortisol level peaks in healthy individuals at early
m orning and reach their low est levels before bedtim e.
D isruption of cortisol circadian rhythm can result in fatigue and
restlessness, w eight loss, insom nia and coronary heart
disease. Aldosterone is a steroid horm one that is secreted by
the cortex of the adrenal gland and regulates the bodys
electrolyte balance. The level of aldosterone is norm ally low
during the day and high during sleep in hum ans. D isruption of
aldosterone rhythm s result in sodium and w ater retention,
increased blood pressure and coronary heart disease. U rine
volum es have inverted circadian oscillating patterns, com pared
to that of aldosterone, in healthy hum ans. b| The circadian
oscillation of lym phocyte, m onocyte, platelet and eosinophil
levels in healthy young adults. The activity of the im m une
system , usually represented by the num ber of lym phocytes,
peaks in the late evening and is low est in the early m orning.
D isruption in this circadian rhythm could lead to im m une
suppression. Adapted from REF. 113.
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B ox 1 | The mammalian central clock and how it controls peripheral tissues
Circadian rhythms are generated by a small subset of neurons that are located in the suprachiasmatic nuclei (SCN),
which is located in the anterior hypothalamus in the brain
30,31
. The longitudinal viewof mouse brain (a) illustrates the
direct light input pathway from the eye to the SCN, and the direct targets of SCN fibres. Light signals are received by a
small group of melanopsin-expressing retinal ganglion cells in the eye and transmitted to the SCN neurons directly
through the retinothypothalamic tract (RHT)
3942
. The retinal ganglion cells also project to the intergeniculate leaflet
(IGL) and the oliveray pretectal nucleus in the brain, which transmit light signals indirectly to the SCN
40
(not shown).
The SCN central pacemaker controls physiological and behavioural rhythms through diffusible molecules that are
produced by the SCN neurons
188190
, as well as by targeting other regions of the brain directly. Four different targets of
SCN fibres have been identified in rodent brain. Three of them are confined within the medial hypothalamus. They are
the endocrine neurons that produce corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH)
and gonadotropin-releasing hormone (GnRH); the autonomic paraventricular neurons (aPVN); and the intermediate
neurons. Intermediate neurons include the neurons in the subparaventricular nucleus of the hypothalamus (sPVN), the
neurons of the dorsomedial nucleus of the hypothalamus (DMH), and the neurons of the medial preoptic nucleus
(MPN, not shown). Outside of the hypothalamus, the SCN neurons project to the paraventricular nucleus of the
thalamus (PVT) and the IGL
191
. A cross-section of mouse brain (b) shows the location of the SCN nuclei. The SCN are
small bilateral structures located aside the third ventricle (3V) and just above the optic chiasm. Each SCN nucleus
contains about 10,000 neurons that are synchronized to generate coordinated circadian outputs in vivo
32,33
. The SCN
controls the circadian rhythmicity of plasma corticosterone production through the neuroendocrine and autonomic
nervous systems in mice (c). The SCN controls the activity of the autonomic nervous system through the autonomic
neurons of the paraventricular nucleus (aPVN). These neurons project from the hypothalamus to preganglionic
parasympathatic and sympathetic neurons in brainstem nuclei, such as the dorsal motor nucleus of the vagus (DMV)
and in the intermediolateral cell columns (IML) of the spinal cord
44
. The SCN indirectly controls the secretion of
adrenocorticotropic hormone (ACTH) from the pituitary gland, through the CRH-producing endocrine neurons in the
hypothalamus
192
. Circadian variations in the activity of the autonomic nervous system and in ACTH production result
in rhythmic release of corticosterone from the adrenal gland into the blood. So, plasma levels of corticosterone undergo
a 24-hour circadian variation in vivo. See REFS44 and99for indepth reviews on the neuroanatomy output pathways of
SCN to peripheral tissues. LV, lateral ventricle. Part ais modified from REF. 99.
O ptic nerve
aPVN
SC N
C R H
R H T Eye
Light
SC N O ptic
chiasm
LV
a
c
b
Light
G nR H
sPVN
D M H
SC N
C R H
TR H
IG L
PVT
Forebrain
C erebellum
aPVN
H ypothalam us
Pituitary gland
3v
Pituitary gland
Spinal
cord
D M V
IM L
Adrenal
gland
AC TH
C orticosterone
R E V I E WS
treatment with theserhythms
9094
. Theresultsof theani-
mal model studies have been extrapolated to random-
ized clinic trials of patients undergoing treatment for
advanced-stage cancers
74,9597
. Some of these clinical tri-
als involve more than 2,000 patients, mostly with
metastatic colorectal cancer
97
. The resultsof these stud-
ies indicate that chronotherapy reduced drug toxicity
and improved patients performance. Chronotherapy
was two- to eightfold more effective than conventional
therapy patients showed improvement in tumour
response rate and the duration of the response, and
decreased frequency of tumour metastasis
74
, although it
did not increase the long-term survival of patients with
metastatic colorectal or breast cancer. Chronotherapy
has, however, been shown to significantly increase the
survival time of children with acute lymphoblastic
leukaemia (ALL) this approach increased survival
time by 4.2-fold in an 8-year study, and by 2.6-fold in a
15-year disease-freesurvival analysis
98
.
Circadian clocks as tumour suppressors
So what i s the mechani sm by whi ch the ci rcadi an
clock affects tumour growth?The circadian clock has
been shown to function as a tumour suppressor at the
systemic, cellular and molecular levels in vivo.
At thesystemic level. The SCN central clock regulates
cell proliferation and apoptosis in peripheral tissues
through the ANSand neuroendocrine systems, such as
the hypothalamicpituitaryadrenal (HPA) and hypo-
thalamicpituitarygonadal (HPG) axes
4446,99
(FIG. 3).
In vivo, theANSinnervatesall peripheral tissues, except
skeletal muscle. It also controls cell proliferation and
death in innervated tissues and organs in a tissue- or
cell-type-specific manner through G-protein-coupled
transmembrane-receptor-mediated pathways
100106
.
Hormones produced by the HPA and HPG axes, such
as oestrogen and glucocorticoids, are widely known to
control cell proliferation and apoptosis in peripheral
tissues
107111
. The activity of the ANSand neuroen-
docrine systems is regulated by the SCN clock, and
shows a 24-hour rhythmic activity in vivo
45
, providing
an explanation for the circadian-coordinated cell-pro-
liferation rhythm in peripheral tissues. Disruption of
ANS and neuroendocri ne rhythms could lead to
deregulation of cell-proliferation rhythm in peripheral
tissuesand promote oncogenesis
112
.
The ANSand neuroendocrine system also func-
tion at the systemic level to suppress tumour devel-
opment through i mmunomodulati on (FI G. 3). Both
ANSand hormones of the HPA axis, such as gluco-
corticoids, have been shown to control cytokine pro-
ducti on, leukocyte di stri buti on, proli ferati on and
apoptosis. This means that the immune response is
also regulated by the central ci rcadi an clock
113
.
Disruption of circadian rhythms could therefore lead
to immune suppression, which could disrupt cancer
immunosurveillance and promote tumour develop-
ment
12,71,102104,111115
. Immune products such as
cytokines can also act to modulate the activity of the
SCN clock and the HPA and HPG axes, providing an
slow-growing tumours, although, in these cases, DNA
synthesis and mitotic indices are often considerably
hi gher i n tumour cells throughout the 24-hour
period
8385
. The altered circadian rhythms, or ultradian
rhythms (less than 24-hour oscillation), in cell prolif-
erati on are often observed i n fast-growi ng or
advanced-stage tumours
8589
. So, cancer treatment can
be optimized by exploring the cytokinetic asynchrony
between tumour and host tissues, and applying anti-
cancer drugs at a time of the day that is associated with
maximal tumour susceptibility and host tolerability
74
.
The efficacy of chronotherapy with various anti-
cancer drugs was first tested in animal models. These
studies indicated that both in vivohost tolerability and
drug efficacy were affected by circadian rhythms, and
the best therapeutic index was achieved by coupling
Light
SC N
H ypothalam us
G onads Adrenal gland
Interferons
Stress, social environm ent
O ther regions of the brain
Autonom ic
nervous
system
Pituitary gland
Pineal gland
M elatonin
G lucocorticoid
Interleukin-1
Peripheral tissues
C ell proliferation
and apoptosis
Figure 3 | The circadian clock controls cell proliferation
and apoptosis at the systemic level. Light and other
environm ental cues reach the suprachiasm atic nuclei (SC N )
through various input pathw ays. The SC N clock
synchronizes w ith the environm ent to generate endogenous
rhythm s, w hich are transm itted through output pathw ays to
peripheral tissues. R epresentative output pathw ays, such
as the autonom ic nervous system (AN S), the
hypothalam icpituitarygonadal (H PG ) and the
hypothalam icpituitaryadrenal (H PA) axes, are show n. The
pineal gland and peripheral tissues can also feed back to
SC N or H PA axes, through production of m elatonin, to
regulate hom eostasis. M elatonin binds to receptors on SC N
neurons to induce phase shifts
196
. The adrenal glands
produce glucocorticoids, w hich have negative feedback on
the hypothalam us to term inate the release of corticotropin-
releasing horm one
117
. The products of im m une activity, such
as interferon- and -,and interleukin-1, can also m odulate
the activity of SC N , as w ell as the H PA axis
116,118
. Feedback
pathw ays are indicated by dashed lines.
R E V I E WS
of multiple molecular pathways contribute to the can-
cer-prone phenotype of the Per2-mutant mice.
Deregulation of the Myc-mediated growth-regulatory
pathway is one possible mechanism by which disrup-
tion of the circadian clock could promote tumour for-
mation. The expression pattern of c-MycmRNA shows
a low-amplitude circadian oscillation in all mouse tis-
sues studied, but expression is significantly increased
throughout the 24-hour period in Per2mutants. In
addition, the c-MycP1 promoter is suppressed directly
by the core circadian regulators, indicating that c-Mycis
a clock-controlled gene. The expression of Myc-target
genesCcns1and Ccna1/2also show circadian oscillation
patterns in vivo, and this oscillation is significantly
altered following Per2mutation
11
.
Per2-mutant mice are more susceptible to lym-
phoma after exposure to -radiation. This observation
might be explained by the fact that lossof Per2activates
the Myc signalling pathways(FIG. 4) that induce cell pro-
liferation and apoptosis. Induction of cell-cycle entry by
c-Myc also sensitizes cells to apoptosis. Suppression of
c-Myc-induced apoptosis, such as by co-expression of
Bcl-x
L
, is sufficient to promote tumour progression
without additional oncogenic mutations
132
. One of the
key mediators in c-Myc-induced apoptosis is the
tumour suppressor p53 (REF. 133). Loss of p53 activity is
necessary and sufficient for c-Myc-accelerated lym-
phomagenesis in mice
134,135
. The Per2-mutant thymo-
cytes are also deficient in p53-mediated apoptosis in
response to -radiation. So, deregulation of c-Myc and
deficiency in p53-mediated apoptosis are likely to
underlie the high incidence of radiation-induced lym-
phoma in Per2-mutant mice
11
(FIG. 4). As the core circa-
dian genes show coordinated expression in vivo, the
model in which a c-Myc signalling pathway mediates
circadian control of cell proliferation needs to be tested
in additional animal models, such as in mice that are
deficient in other corecircadian genes.
There has been a long debate about whether the
circadian clock and the cell-cycle clock are connected
in vivo. In our experiments, -radiation-induced apop-
tosisiscircadian time-dependent in both wild-type and
Per2-mutant thymocytes. When irradiated at the early
stage of active phase or at the early stage of resting
phase, Per2-mutant thymocytes show a G2/M-specific
resistance to radiation-induced apoptosis
11
. So, the cir-
cadian clock not only regulates the expression of cell-
cycle genes it could also be involved in controlling
cell-cycle checkpoint function.
In DNA-damage response. The core circadian genes
respond directly to -radiation. Disruption of Per2
abolishes the response of all core circadian genes to
-radiation. So, the molecular clock itself can be modu-
lated by genotoxic stress in peripheral tissues. The abil-
ity of circadian genes to mediate the DNA-damage
response seems to be cell autonomic, as Per2-mutant
thymocytes have attenuated p53 induction in response
to -radiation in vitro
11
. It has been shown recently that
the clock genes also respond to low levels of ultraviolet
(UV) irradiation in cultured human keratinocytes
136
.
immune-regulatory, circadian-paced feedback loop
116118
.
So, by anticipating and adapting to external and internal
cues, the SCN clock controls overt rhythmicity in cell
proliferation in peripheral tissuesin vivo.
At thecellular and molecular level. In cells of periph-
eral tissues, the SCNs clock controls cell proliferation
and apoptosis by regulating the expression of circa-
dian-controlled genes. Recent studies have shown that
about 7% of all clock-controlled genes that have been
identified in rodents regulate either cell proliferation
or apoptosis
5659
. These clock-controlled genes include
c-Mycand Mdm2, the tumour-suppressor genes Trp53
and Gadd45, as well as genes that encode the cas-
pases, cycli ns, transcri pti on factors, and ubi qui ti n-
associated factors that are involved in regulating the
cell cycle and apoptosis
11,5659
. The rhythmic expression
of several cyclins, as well as that of the tumour sup-
pressor p53, is also observed in human oral mucosa.
These expression patterns are synchronized with the
ci rcadi an osci llati on patterns of Per1 and Bmal1
expression in the same tissue
119,120
.
Apart from controlling the expression of cell-cycle
genes and tumour-suppressor genes at the transcrip-
tional and post-transcriptional levels, the core circa-
di an genes are also i nvolved di rectly i n modulati ng
the intracellular signalling pathways that regulate cell
proli ferati on. Recently, i t has been shown that the
core circadian regulator CKI also functions in pro-
moti ng cell proli ferati on by stabi li zi ng -cateni n.
Overexpressi on of CKI mi mi cs the effect of WNT
signalling, resulting in cytoplasmic accumulation of
-cateni n and i ts subsequent nuclear locali za-
ti on
121123
. -Cateni n i nteracts wi th transcri pti on
factors of the T-cell-specific transcription factor/lym-
phoid enhancer factor-1 (TCF/LEF) family to regu-
late transcri pti on
124
and promote tumori genesis
125
.
Genes activated by -catenin/TCF/LEF include mem-
bers of the AP1 transcription family, c-Junand Fra1
(REFS126,127), and c-Mycand Ccnd1(whi ch encodes
cyclin D1) (REFS128130). It is not known whether the
role of CKI in cell proliferation is independent of its
role in circadian-clock function.
Interestingly, -catenin is destabilized by glycogen
synthase kinase-3 (Gsk3) in the absence of WNT
signalling
124
. Gsk3 is a functional homologue of the
core ci rcadi an gene Shaggyi n frui tfly
131
. Although
di rect molecular evi dence for the role of Gsk3 i n
mammali an ci rcadi an-clock functi on has not been
shown, by antagoni zi ng CKIs abi li ty to promote
-catenin stability, Gsk3 could also be involved in cir-
cadian control of cell-cycle progression. Therefore, as
the molecular clockworks regulate the cell cycle and
apoptosisin cellsof peripheral tissues, mutationsin cir-
cadian genescould conceivably result in deregulation of
these processesand tumour development.
Per2 regulation ofp53 and c-Myc.Micewith disruptions
in the core circadian gene Per2have recently been
shown to display salivary-gland hyperplasia and develop
spontaneous lymphoma
11
. It is likely that deregulation
R E V I E WS
at the begi nni ng of a subjective ni ght results i n the
release of intracellular Ca
2+
in SCN neurons, which, in
turn, activates signalling pathways, such as the mito-
gen-activated protein kinase (MAPK)/extracellular-
signal-regulated kinase (ERK) pathway, and the cal-
cium/calmodulin and c-AMP-protein kinase A (PKA)
pathways (FIG. 5). Light exposure at the end of a subjec-
tive ni ght activates ni tri c oxi de (NO) and c-GMP
pathways
148150
. The activation of these pathways leads
to phosphorylation of c-AMP/calcium-responsive ele-
ment-binding protein and subsequent activation of
Per1via a c-AMP-responsive element in the promoter
of the Per1gene. Activation of Per1is involved in the
light-induced phase resetting in the SCN clock
47,148152
.
Mammalian peripheral clocksdo not respond directly
to light stimuli,but areinstead regulated by cyclic changes
in the levels of neurotransmitters, growth factors, and
hormones such as glucocorticoids and retinoids
55,153,154
.
Thesecontrol expression of thecorecircadian genesPer1,
Per2 and Cry1 throughout the 24-hour period
153
.
Retinoidsinteract with nuclear receptors,such asretinoid
X receptor- (RXR) and retinoic-acid receptor-
(RAR), to regulate the transcriptional activity of
Clock/Bmal1(REF. 154). Glucocorticoidsinteract with glu-
cocorticoid receptors(GRs),and localizeto thenucleusto
regulate the expression of genesthat contain glucocorti-
coid-responsive elements(GREs)
155
. The Per1gene con-
tainstwo almost-perfect GREs one in its5 promoter
and another in itsfirst intron
156
.
A phaseshift in theexpression patternsof thecorecir-
cadian genes Per1, Per2and Cry1can be induced by
restricting nutrient supply
55,153
. Oscillationsin circadian
gene expression can also be induced in cultured rat
fibroblasts and NIH3T3 cells by serum shock, or by
treatment with dexamethasoneor tetradecanoylphorbol
13-acetate (TPA). This induction does not require
denovo protein synthesis, but is mediated directly
through varioussignalling pathways, such asthe c-AMP,
protein kinase C (PKC), Ca
2+
and MAPK pathways
157,158
(FIG. 5). The RasMAPK signalling pathway has been
shown to control circadian output pathways in fly and
chick
159,160
. It islikely that similar pathwaysareinvolved in
maintaining peripheral clock control in mammals.
The c-AMPPKA, PKC and MAPK pathways are
well known for their roles in regulating cell prolifera-
tion. The MAPK family contains three well-character-
ized subfamilies of kinases. They are the ERKs, the
c-JUN amino-terminal kinases (JNKs) and the p38
MAPKs
161
. Among these three subfamilies, the ERK sig-
nalling pathway controlscell proliferation and apoptosis
in response to diverse stimuli, such as growth factors,
cytokinesand carcinogens. It also respondsto polypep-
tide hormones and neurotransmitters through interac-
tionswith thec-AMP signalling pathway
106,161164
(FIG. 5).
Deregulati on of ERK pathways i s commonly
observed i n human cancer cells, and i nhi bi tors of
these pathways have been tested as anticancer agents
in clinical trials
165168
. ERK signalling leads to the acti-
vation of AP1 transcription factors, which are dimeric
basi c leuci ne zi pper protei ns from the Junand Fos
families
169
. The AP1 transcription factors regulate a
The molecular pathways by which the circadian
clock controlsthe DNA-damage response in peripheral
tissues remain unclear. It has recently been shown
that casein kinase II (CKII) is involved in controlling
circadian rhythms in Arabidopsis, Neurospora and
Drosophila
137140
. In fruitflies, CKIIdirectly phosphory-
lates PER, which regulates its ability to enter the
nucleus
140
(FIG. 1). In mammals, CKII regulatescell-cycle
progression by phosphorylating and activating c-Myc.
The interaction of CKII and c-Myc has been associated
with variousmammalian cancers
141143
. CKII also phos-
phorylates p53 in response to UV irradiation
144
. It is
important to determine whether CKII functions in the
mammalian circadian clock, and whether it is involved
in the circadian-clock-controlled DNA-damage
responsein vivo.
Peripheral clock control
A bri ef li ght pulse at the begi nni ng of a subjective
night activates a cascade of events in the SCN neurons
, such as activation of expression of the immediate-
early genes c-Fosand JunB, as well as the ci rcadi an
genes Per1and Per2(REFS145147). Light exposure at the
end of a subjective night, however, only inducesPer1
(REF. 47). So, the response of the SCN central clock to
light stimuli is circadian time-dependent. Light stimuli
Per2
-irradiation
p53
or
c-M yc
C ancer
Apoptosis
G enom ic
instability
B m al1/C lock B m al1/N pas2
G enom ic instability,
cell proliferation
Figure 4 | A model for the role of Per2 in tumour
suppression. C ore circadian gene products regulate a
num ber of oncogenes, such as c-Myc. O verexpression of
c-Myc has been show n to lead to D N A dam age, hyperplasia
and tum origenesis. H eterodim eric circadian regulators such
as B m al1C lock and B m al1N pas2 negatively regulate c-Myc
expression at the transcriptional level. Loss of Per2 function
reduces Bmal1expression throughout 24-hour light/dark
cycles, leading to decreased intracellular levels of
B m al1N pas2 or B m al1C lock, and derepression of c-M yc.
Follow ing -irradiation, the loss of Per2 function partially
im pairs p53-m ediated apoptosis, leading to genom ic
instability and accum ulation of dam aged cells. These cells can
still progress through the cell cycle in the presence of genom ic
D N A dam age, due to the high level of c-Myc expression,
resulting in tum our form ation after -radiation. Solid lines
indicate the pathw ays that have been dem onstrated by recent
studies. The dashed line indicates a regulatory pathw ay(s) that
is still not fully understood
11
.
R E V I E WS
wide range of cellular processes, including cell prolif-
eration, apoptosis and differentiation. Genes that are
di rectly or i ndi rectly controlled by AP1 i nclude
Ccnd1, Trp53, Cdkn1a and Cdkn2a (REFS170173).
Persi stent activati on of AP1 leads to abnormal
cell-cycle progression and transformation
169
.
It isunlikely that AP1 controlscircadian gene expres-
sion in peripheral tissues, however, as the induction of
c-Fosand Per1occur at about the same time in SCN,
and mice deficient in c-Fosshow normal Per1induction
by light
146,174
. It ismost likely that induction of the circa-
dian genes Per1and Per2, along with the immediate
early-response genesc-Fosand c-Jun,isregulated simul-
taneously through interacting signalling pathways in
peripheral tissues.
In addi ti on to controlli ng ci rcadi an rhythm i n
peripheral tissues, retinoids and glucocorticoids also
regulate cell proliferation. These factors are therefore
used as cancer therapeutics. The retinoid-acid recep-
tors RAR, RAR and RXR i nduce G1 cell-cycle
arrest by suppressing Ccnd1expression
175177
, and dis-
ruptions in RAR are associated with acute promye-
locytic leukaemia
178
. GR signalling activates genes that
inhibit components of the ERK pathway
179
. The GR
also i nteracts di rectly wi th AP-1, whi ch blocks
the abi li ty of AP-1 to activate transcri pti on
180
.
Glucocorti coi ds can also modulate c-AMP and
MAPK si gnalli ng through non-genomi c mecha-
nisms
181184
. Glucocorticoids inhibit cell proliferation
i n many types of cells by promoti ng apoptosi s and
cell-cycle arrest, which are often correlated with the
downregulation of c-Myc and cyclin D3(REFS185187).
Together, this evidence indicates that molecular
clockworksand cell-cycle clocksin peripheral tissue are
regulated by a complex interaction of pathways that
include glucocorticoids, retinoids, c-AMP, PKC, WNT,
Ca
2+
and MAPK signalling
153,154,157,158,161164,175177,179,181
(FIG. 5). In vivo, production of extracellular signals
such as growth factors, cytokines, neurotrasmitters and
hormones are controlled by the SCN central
clock
45,99,102,104,113
(FIG. 3). The SCN therefore controls the
24-hour rhythmic activities in peripheral tissues, by
controlling intracellular signalling (FIG. 5). Theperipheral
clocks, synchronized by the central clock and operat-
ing in their own local environments, respond to these
signals to regulate the genes that control cell-cycle pro-
gressi on, such as c-Myc, Ccnd1 and Trp53 (REFS
11,119,120). However, under certain conditions, such as
i n the case of DNA damage, the peri pheral clocks
respond immediately to the damage, and could then
operate i ndependently of the central pacemaker, to
control local cell-cycle checkpoints, activate apoptosis
and suppressmalignant growth
11
.
Future directions
A large amount of in vivoevi dence has shown that
the ci rcadi an clock i s i nvolved i n tumour suppres-
sion, and that cancer should no longer be treated as a
local disorder. However, the ability of the biological
clock to suppress malignant growth and to cooperate
wi th cancer treatment has not been fully explored.
cAM P
R as G R s
G sk3
C KI C R EB
M EK
ER K
JN K
AP-1
+
N eurotransm itters,
peptide horm ones
G row th
factors
G lucocorticoids
Akt PKA R ap1
B raf
R af1
-C atenin
TC F/LEF
C ore circadian
genes
M APK1, 2
c-M yc, cyclin D 1,
p53
C ell-cycle progression
or apoptosis
Figure 5 | Signalling pathways linking the circadian
clock to cell-cycle regulation in peripheral tissues.
Production of extracellular signals is regulated by the central
clock, and can result in cell-cycle progression or apoptosis
in peripheral tissues. For exam ple, circadian-regulated
production of grow th factors activates the m itogen-
activated protein kinase (M EK , M APK 1,2)/extracellular-
signal-regulated kinase (ER K ) pathw ays, w hereas
neurotransm itters and peptide horm ones regulate the
c-AM P/protein kinase A (PK A) signalling pathw ays
106,161164
.
These pathw ays interact through R ap1, w hich inhibits R as
signalling, and B raf, w hich activates R af1 signalling in a cell-
type-dependent m anner. Signalling through the c-AM P/PK A
and M EK pathw ays, how ever, leads to activation of the
c-AM P response-elem ent binding protein (C R EB ). C R EB
activates the transcription of core circadian genes in
peripheral tissues
157,158
. Additionally, ER K signalling
activates the transcription factor AP-1, w hich regulates
production of c-M yc, cyclin D 1 and p53, and thereby
m ediates cell proliferation and apoptosis
169
. The c-AM P
pathw ay also indirectly prom otes -catenin stability, through
AK T and glycogen synthase kinase-3 (G sk3) signalling.
The core circadian gene product C K I also stabilizes
-catenin, and thereby activates TC F/LEF (T-cell-specific
transcription factor/lym phoid enhancer factor-1)
121123,162
.
This leads to activation of c-M yc and cyclin D 1.
G lucocorticoids can inhibit AP-1 activity directly through
ligand-bound glucocorticoid receptors (G R s), or indirectly
by inhibiting the c-JU N am ino-term inal kinase (JN K )
signalling pathw ay
180184
. So, peripheral clocks and cell-
cycle clocks are intim ately linked to each other by
interacting signal-transduction pathw ays.
R E V I E WS
mani pulati ng pati ents ci rcadi an clocks such as
through light therapy, restriction of meal time, mela-
tonin administration or glucocorticoid administra-
ti on can i mprove cancer treatment and slow
tumour progression.
The challenge and the excitement of biomedical
research in the new millennium are to integrate knowl-
edge from different fields of biological research and
apply them to solve important problems related to
human health at the systemic level. The circadian clock
provides a unique system for studying the mechanisms
of cancer in vivoand for developing novel therapeutic
strategiesfor cancer.
Future research should be focused on studyi ng the
mechanisms by which the circadian clock controls cell
proliferation, apoptosis and responses to genotoxic
stress. Advanced cancers are often autonomic in cell-
proliferation rhythm. It will be important, therefore, to
study how tumour-cell proliferation escapes the con-
trol of the central pacemaker, and whether somati c
mutations in clock genes or deregulation of entraining
pathways for peripheral clocks are involved. This infor-
mation is particularly needed to improve the efficacy
of current chronotherapy. In addition, as slow-grow-
i ng tumours can fall under the control of the host
circadian clock, it will be important to study whether
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Online links
DATABASES
The following terms in this article are linked online to:
C a n c e r.g o v: http://w w w .cancer.gov/cancer_inform ation/
acute lym phoblastic leukaem ia | breast cancer | colorectalcancer |
osteosarcom a | pancreatic cancer
L o c u sL in k : http://w w w .ncbi.nih.gov/LocusLink/
-catenin | B cl-x
L
| Bmal1| Ccna1| Ccna2| Ccnd1| CK | Cry1|
Cry2| cyclin D 3 | Fos | Fra1| Gadd45 | G sk3 | J un | J unB|
M APK | Mdm2| Myc | Per1| Per2| Per3| PKC | R as | Trp53
FURTHER INFORMATION
A tim e to h e a l c h ro n o th e ra p y tu n e s in to b o d y s rh yth m s:
H ttp://w w w .fda.gov/fdac/features/1997/397_chrono.htm l
B io lo g ic a l c lo c k s: http://w w w .seoulin.co.kr/U p/clock/clock.htm l
B io tim in g tu to ria l:
http://w w w .cbt.virginia.edu/tutorial/H ISTB AC K.htm l
F ro m c irc a d ia n rh yth m to c a n c e r th e ra p y:
http://w w w .eortc.be/hom e/chrono/From circadianrhythm s.htm l
Access to this interactive links box is free online.

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The word circadian is derived from a Latin phrase

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