British Journal of Haematology, 2002, 116, 528537
Cytotoxicity of 2-chlorodeoxyadenosine and arabinosylcytosine
in leukaemic lymphoblasts from paediatric patients:
significance of cellular nucleoside transporter content
Adrienne M. P. Wright, 1 Alan R. P. Paterson, 1,2 Bernard Sowa, 1 John J. Akabutu, 2,3
Paul E. Grundy 2,3 and Wendy P. Gati 1,2 1Department of Pharmacology, 2Department of Oncology and
3
Department of Pediatrics, University of Alberta and Cross Cancer Institute, Edmonton, Alberta, Canada
Received 16 May 2001; accepted for publication 27 September 2001
Summary. 2-chlorodeoxyadenosine (2-CdA) and arabino-
sylcytosine (araC) are nucleoside drugs that are used to
treat various leukaemias, although 2-CdA has not been
tested extensively in children with acute lymphoblastic
leukaemia (ALL). Nucleoside cytotoxicity depends on the
conversion of these agents to 5-phosphate derivatives, fol-
lowing drug entry into cells via nucleoside transport (NT)
processes. This study compared es nucleoside transporter
content, determined using a flow cytometric assay with
SAENTA [5-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5-thio-
adenosine] fluorescein, and cytotoxicities of 2-CdA and araC
in fresh lymphoblasts from previously untreated paediatric
ALL patients and the human T-lymphoblast cell line, CCRF-
CEM. Lymphoblast samples from individual patients ranged
widely in sensitivity to both 2-CdA (IC50, 6 nmol/l to
> 5 lmol/l; mean 418 nmol/l; n 8) and araC (IC50,
59 nmol/l to > 5 lmol/l; mean 1050 nmol/l; n 7),
although IC50 values for the two drugs were correlated
In the current treatment of acute lymphoblastic leukaemia
(ALL), the most common form of childhood cancer,
approximately 25% of patients fail to achieve long-term,
disease-free survival despite intensive combination chemo-
therapy (Rivera et al, 1991), an outcome attributed to the
emergence of drug-resistant disease. 2-chlorodeoxyadeno-
sine (2-CdA), a purine nucleoside analogue with important
antileukaemic activity in both resting and proliferating
lymphocytes (Piro, 1992), is currently used in the treatment
of a number of indolent lymphoid malignancies (Piro et al,
1988; Kay et al, 1989; Kurzrock et al, 1991). 2-CdA has
also shown notable antileukaemic activity in previously
Correspondence: Dr Wendy P. Gati, Department of Pharmacology,
University of Alberta, 970 Medical Sciences Building, Edmonton,
Alberta, T6G 2H7 Canada. E-mail: wendy.gati@ualberta.ca
528
(r 078, P 0032, n 7). Cellular es nucleoside trans-
porter content varied more than 35-fold among samples
from 10 patients. The correlation between es nucleoside
transporter content and drug sensitivity was statistically
significant for araC (r )093, P 0023, n 5), but not
for 2-CdA (r )057, P 023, n 6). Exposure of CCRF-
CEM cells to araC resulted in a substantial araC concen-
tration-dependent increase in the relative survival of es
transporter-deficient cells, whereas the increase was slight
following exposure to 2-CdA. We conclude that, in ALL
lymphoblasts, es nucleoside transporter content is a deter-
minant of araC sensitivity and that a deficiency in NT may
impart resistance to araC.
Keywords: acute lymphoblastic leukaemia, nucleoside
transport, SAENTA fluorescein, 2-chlorodeoxyadenosine
(2-CdA), arabinosylcytosine (araC).
untreated paediatric acute myeloid leukaemia (AML)
patients (Santana et al, 1991, 1992; Kearns et al, 1994).
The success of 2-CdA in treating these malignancies, along
with its low clinical toxicity (Carson et al, 1984; Santana
et al, 1991, 1992; Kearns et al, 1994), has made 2-CdA a
candidate for cytotoxic evaluation in the treatment of
paediatric ALL.
Phase I and Phase II studies by Santana et al (1991,
1992) reported some antileukaemic activity of 2-CdA
treatment in a small number of paediatric ALL patients,
although those studies included patients in their second or
third relapse, poor candidates for reinduction with any
single agent. In vitro studies of freshly isolated ALL cells by
Kumagai et al (1994) showed that sensitivity to 2-CdA
appeared to be independent of either a high-risk karyotype
(Philadelphia chromosome or 11q23 abnormalities) or a
previous relapse history, factors that are usually predictive
2002 Blackwell Science Ltd
 Nucleoside Transporters and Drug Toxicity in Leukaemic Blasts
of an unfavourable response to chemotherapy. Further-
more, potentially cytotoxic concentrations of 2-CdA have
been attained in cerebrospinal fluid (CSF) with a well-
tolerated, conventional treatment regimen, suggesting a
potential role for systemic 2-CdA in the treatment of
meningeal leukaemia, a major cause of relapse in this
disease (Kearns et al, 1994).
An aim of the present study was to compare the cytotoxic
potential of 2-CdA and arabinosylcytosine (araC; cytara-
bine) towards leukaemic lymphoblasts obtained from the
bone marrow of newly diagnosed paediatric patients with
ALL, using an in vitro tetrazolium dye reduction assay
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay] of cell viability. AraC was chosen for
this comparison because of its current use in remission
induction and consolidation protocols in ALL therapy; araC
has also been used in combinations prophylactically, to
reduce the possibility of central nervous system (CNS)
involvement in childhood ALL (Morra et al, 1993). Pieters
and colleagues (Pieters et al, 1994; Klumper et al, 1995)
showed that the MTT chemosensitivity assay may be a good
predictor of clinical outcome in acute leukaemia of child-
hood, and a retrospective study in adult AML also demon-
strated the prognostic value of the MTT assay (Sargent &
Taylor, 1989).
The cytotoxicity of 2-CdA and araC depends upon the
formation, inside cells, of 2-CdATP and araCTP respectively
(Griffig et al, 1989; Rustum & Raymakers, 1992). Thus, a
prerequisite for the antileukaemic activity of the nucleoside
drugs is entry into target cells, which is mediated by
nucleoside transporters (Belt et al, 1993; Cass, 1995). In
mammalian cells, several nucleoside transporter subtypes
may be expressed, including equilibrative (facilitated diffu-
sion) and concentrative (Na-dependent) transporters. Both
2-CdA and araC are substrates for the equilibrative nucle-
oside transport (NT) processes, es and ei (Crawford et al,
1988), which are distinguishable by their differential
sensitivity to nitrobenzylthioinosine (NBMPR), a potent
inhibitor of the es nucleoside transporter (Paterson & Oliver,
1971). 2-CdA has also been shown to be a substrate for two
of five Na-linked, concentrative transporters, namely cif
(N1) (King & Cass, 1994) and cs (N5) (Paterson et al, 1993).
Of the five concentrative nucleoside transporters expressed
in mammalian cells, only cs is sensitive to NBMPR (Paterson
et al, 1993).
Although several NT processes have been identified in
human leukaemia cells, the es transporter appears to
account largely for inward fluxes of nucleosides (Belt et al,
1993; Alessi-Severini et al, 1995). Nevertheless, the cellular
content of es nucleoside transporters in leukaemic blasts
varies widely among samples from adult patients with acute
leukaemia (White et al, 1987; Jamieson et al, 1993; Gati
et al, 1997), suggesting that the cellular es transporter
content may account in part for differences in clinical
response to nucleoside drugs. It has been reported that es
nucleoside transporter abundance in human leukaemia
cells correlated with both araC influx and levels of araCTP
formed (Wiley et al, 1983; Young et al, 1985). This
laboratory has reported a correlation between es nucleoside
2002 Blackwell Science Ltd, British Journal of Haematology 116: 528537
529
transporter abundance and sensitivity to araC in fresh
leukaemia cells from adult patients with AML and ALL (Gati
et al, 1996, 1997, 1998). Thus, we hypothesized that the
es-mediated, inward flux of 2-CdA and araC in lymphoblasts
from paediatric ALL patients may be a determinant of
cytotoxicity and that es nucleoside transporter deficiency
may be a factor in the emergence of resistance to these
agents.
The present study examined fresh lymphoblasts from
paediatric patients with ALL and a human T-lymphoblast
cell line to test relationships between the cellular content of
es nucleoside transporters and cell sensitivity to 2-CdA and
araC. The study used SAENTA [5-S-(2-aminoethyl)-
N6-(4-nitrobenzyl)-5-thioadenosine] fluorescein, a fluores-
cent ligand for the es nucleoside transporter, in the flow
cytometric measurement of transporter abundance in cells
(Jamieson et al, 1993; Buolamwini et al, 1994; Gati et al,
1996). This study also reports, for the first time, the es
nucleoside transporter content of fresh lymphoblasts from
children with ALL.
MATERIALS AND METHODS
Cell preparation. Fresh leukaemic cells were obtained,
with informed consent, from 11 bone marrow aspirates and
one peripheral blood sample from paediatric ALL patients.
All patients were newly diagnosed and immunophenotyp-
ing was performed at the Department of Laboratory
Medicine, University of Alberta Hospital, Edmonton, AB,
Canada. Leukaemic blasts were isolated using a conven-
tional, density-gradient procedure in which the heparinized
bone marrow or blood sample was diluted 1:1 with Roswell
Park Memorial Institute (RPMI)-1640 medium (Sigma,
St. Louis, MO, USA) containing 2 mmol/l Hepes buffer and
10% fetal bovine serum (RH10F). The cell suspension was
layered over Ficoll-Paque and centrifuged (30 min, 400 g).
Cells from the Ficoll-Paque interface were washed twice
(10 min, 150 g) with RH10F medium and resuspended (a)
in RH10F medium at a concentration of 1 107 cells/ml for
the MTT cytotoxicity assay, and (b) in phosphate-buffered
saline (PBS) of the following composition: 137 mmol/l NaCl,
27 mmol/l KCl, 81 mmol/l Na2HPO4 and 11 mmol/l
KH2PO4 (pH 74) at a concentration of 6 106 cells/ml
for the flow cytometric SAENTA fluorescein-binding assay.
CCRF-CEM human T-lymphoblasts (hereafter, CEM cells)
were maintained in logarithmic growth in RPMI-1640
medium containing 10% fetal bovine serum in a humidified
atmosphere of 5% CO2 in air at 37C.
Chemicals. 2-CdA was prepared (Kazimierczuk et al,
1984) by Dr J. S. Wilson, Department of Medical Microbi-
ology and Immunology, University of Alberta, Canada.
AraC and MTT were obtained from Sigma Chemical Co.,
and NBMPR (Paul et al, 1975) and 5-(SAENTA-8)-fluo-
rescein (Buolamwini et al, 1994) were provided by Alberta
Nucleoside Therapeutics, Department of Pharmacology,
University of Alberta. Phycoerythrin-labelled annexin V
(annexin V-PE) was from Pharmingen Canada (Mississauga,
ON, Canada) and 7-aminoactinomycin D (7-AAD) was from
Molecular Probes, (Eugene, OR, USA). Ficoll-Paque was
530 A. M. P. Wright et al
purchased from Pharmacia Biotech (Baie dUrfe, PQ,
Canada). Cell culture materials were from Gibco BRL
(Mississauga, ON, Canada).
MTT assay of drug sensitivity. The in vitro sensitivity of
fresh leukaemic lymphoblasts to 2-CdA and araC was
determined using the MTT dye reduction assay (Alley et al,
1988). Briefly, 100-ll portions of cell suspensions (1 106
cells) were added to 100 ll RH10F medium in 96-well
microculture plates containing graded concentrations of
either 2-CdA or araC (15000 nmol/l); between three and
six replicate microcultures were prepared for each drug
concentration. Control assays lacked only drugs. Plates
were incubated in a humidified atmosphere of 5% CO2 in air
at 37C for 72 h. After incubation, 30 ll of MTT solution
prepared in RH10F (1 mg/ml) was added to each well and
plates were incubated for a further 2 h. The plates were
then centrifuged (7 min, 50 g) and the supernatant fluid
aspirated from each well. Following this, the crystalline
formazan product was dissolved by the addition to each well
of 150 ll dimethylsulphoxide (spectrophotometric grade,
Aldrich, Milwaukee, WI, USA) and the absorbance of the
well solutions was measured in a microplate reader (Titertek
Multiskan Plus, ICN Biomedicals, Mississauga, ON, Canada)
at 540 nm (A540). The A540 of each microwell solution was
proportional to cell concentration. Cell viabilities in the test
wells were expressed as percentages of those in control
wells, which lacked the cytotoxic test agent.
The cytotoxic effects of the nucleoside drugs in fresh
lymphoblasts were quantified by determining the concen-
tration of antileukaemic agent producing a half-maximal
response in the MTT assay. A computer program (PRISM,
Version 10, GraphPad Software Inc., San Diego, CA, USA)
was used to fit the following logistic equation (1) to plots of
cell viability (% of control) versus the log of the drug
concentration:
b fluorescein, X is the total concentration of SAENTA
Y A2 A1 A2 =1 X=IC50 1 fluorescein, Bmax is the maximum specifically bound
in which Y is the response (cell viability,% of control), A1
and A2 are the upper and lower limits for Y, X is the drug
concentration, IC50 is the concentration of drug producing a
half-maximal response, and b is a constant related to the
steepness of the plot.
Flow cytometric enumeration of es nucleoside transporter sites
on primary leukaemic blasts. Freshly isolated leukaemic
lymphoblasts from patients were washed twice (5 min,
150 g) and resuspended at 3 106 cells/ml in PBS for
determination of the total binding of SAENTA fluorescein, or
in PBS containing 5 lmol/l NBMPR for measuring non-
specific binding of SAENTA fluorescein. Prior treatment of
cells with NBMPR under these conditions (30 min, 22C)
prevented the site-specific binding of SAENTA fluorescein to
the es transporter sites. To determine total binding of
SAENTA fluorescein, cells suspended in PBS were incubated
with graded concentrations (0120 nmol/l) of SAENTA
fluorescein for at least 15 min at 22C before flow
cytometric analysis of cell-associated fluorescence. Non-
specific binding was determined by incubating NBMPR-
2002 Blackwell Science Ltd, British Journal of Haematology 116: 528537
treated cells with the graded concentrations of SAENTA
fluorescein in medium containing 5 lmol/l NBMPR. Cell
suspensions were protected from light during these incuba-
tions. The autofluorescence of leukaemic cells was measured
in PBS in the presence and absence of 5 lmol/l NBMPR.
Cell-associated fluorescence and light scatter signals were
analysed using a FACSCAN flow cytometer [Becton Dickinson
Immunocytometry Systems (BDIS), San Jose, CA, USA].
SAENTA fluorescein molecules that were cell associated
were excited with 488 nm light from an argon laser and the
emitted fluorescence was collected at 530 nm (FL1). Fluo-
rescence signals from 10 000 cells for each condition were
acquired and analysed using BDIS LYSIS II software. Analysis
of SAENTA fluorescein fluorescence was restricted to signals
that fell within an electronic gate encompassing the blast/
lymphocyte region of the light scatter dot plot (Sutherland
et al, 1994). The mean relative fluorescence intensity (RFI)
obtained from histograms of SAENTA fluorescein fluores-
cence was used as a measure of cell-associated SAENTA
fluorescein, and the es transporter site-bound content of
SAENTA fluorescein was calculated for each concentration
of the fluorescent ligand as the difference between the RFI
values for total SAENTA fluorescein binding and non-
specific SAENTA fluorescein binding. The latter (non-
specific binding) values were determined from plots of
SAENTA fluorescein fluorescence versus concentration of
SAENTA fluorescein measured in the presence of 5 lmol/l
NBMPR.
The equilibrium binding constants, KD and Bmax for
SAENTA fluorescein were determined by non-linear regres-
sion analysis (Microcal ORIGIN, Version 41) of the equilib-
rium binding data by using the following equation (2):
Y Bmax  X=KD X 2
in which Y is the specific binding (RFI) of SAENTA
SAENTA fluorescein (RFI) per cell, and KD is the equilibrium
dissociation constant for SAENTA fluorescein at es nucle-
oside transporter sites.
Flow cytometric assay of drug sensitivity. In some experi-
ments, nucleoside cytotoxicity was determined by flow
cytometric analysis of CEM T-lymphoblast populations. Cell
suspensions (75 104 cells in 15 ml RH10F) were incu-
bated in 24-well microculture plates for 96 h in the absence
(controls) or presence of graded concentrations of 2-CdA or
araC. The microcultures were centrifuged, washed in HSC
buffer (140 mmol/l NaCl, 25 mmol/l CaCl2 and 10 mmol/l
Hepes, pH 74) and stained with 20 nmol/l SAENTA
fluorescein (or 20 nmol/l SAENTA fluorescein in the pres-
ence of 5 lmol/l NBMPR for non-specific binding), a ligand
concentration that yields an estimate of the Bmax value and,
thus, a measure of the cellular content of es nucleoside
transporters. Cells were counterstained with annexin
V-PE and 7-AAD, to identify apoptotic and necrotic cells
respectively (Schmid et al, 1992; Koopman et al, 1994).
Cell-associated fluorescence signals were recorded using a
 Nucleoside Transporters and Drug Toxicity in Leukaemic Blasts 531
FACSCAN flow cytometer, with excitation at 488 nm and
simultaneous collection of fluorescence signals emitted
from SAENTA fluorescein, annexin V-PE and 7-AAD.
CELLQUEST software was used to enumerate viable cells in
populations that survived each concentration of 2-CdA or
araC, by setting a gate that excluded apoptotic (annexin
V-PE-positive) and necrotic (7-AAD-positive) cells, and to
simultaneously determine the cellular content of es nucleo-
side transporters (specific binding of SAENTA fluorescein) in
the electronically isolated, viable cell subsets.
Statistical analysis. Wilcoxons signed rank test was used
to determine levels of significance, using P < 005 to
indicate a significant difference between groups.

RESULTS
Cytotoxicity of 2-CdA and araC in fresh leukaemic
lymphoblasts
The MTT dye reduction assay was used to determine the
sensitivity of freshly isolated ALL cells to 2-CdA and araC.
A linear relationship was demonstrated between cell
number per well and the reduced dye product (formazan)
formed, measured colorimetrically as A540 (data not shown).
That linearity occurred over a range of cell concentrations
(104)106 cells per well) in four patient samples.
Figure 1 and Table I illustrate the variability in response
to both agents among patient samples. The IC50 values for
araC toxicity towards the preB-cell ALL lymphoblasts from
Patients 4 and 8 were 372 nmol/l and 374 nmol/l,
respectively, and, although several-fold greater than those
for 2-CdA in the same experiment, these values are within a
concentration range (60800 nmol/l) that is achievable in
plasma with conventional araC chemotherapy (Rustum &
Raymakers, 1992). However, cells from preB-cell ALL
Patient 6 were less sensitive to both 2-CdA and araC (IC50
values of 1660 nmol/l and 2650 nmol/l respectively, Fig 1).
Similarly, resistance to both 2-CdA and araC (Patient 9) was
shown in lymphoblasts from T-cell ALL (Table I).
Table I shows that interpatient variation in sensitivity to
the nucleoside analogues was considerable, with IC50 values
for 2-CdA ranging from 6 nmol/l to > 5 lmol/l, and for
araC from 59 nmol/l to > 5 lmol/l. The leukaemic cells
showed significantly (P < 005) greater sensitivity to 2-CdA
(n 8) than to araC (n 7). In the present study, IC50
values for 2-CdA were < 100 nmol/l in one-third of the
patient samples tested; concentrations of 2-CdA achievable
in the plasma were in the 2385 nmol/l range (Kearns et al,
1994).
Figure 2 tested the correlation between IC50 values for
2-CdA and araC cytotoxicity in leukaemic lymphoblasts
samples from seven paediatric patients with ALL in which
both parameters were determined (Table I), yielding a
statistically significant relationship (r 078, P 0032).
Cellular content of es nucleoside transporters in fresh
leukaemic lymphoblasts
The data of Fig 3 illustrate the equilibrium binding of
graded concentrations of SAENTA fluorescein to cells from
preB-cell ALL Patient 8 and T-cell ALL Patient 9, as
Fig 1. Sensitivity of leukaemic lymphoblasts from acute lym-
phoblastic leukaemia (ALL) Patient 5 (T-cell, top) and Patient 6
(pre-B-cell, bottom) to 2-chlorodeoxyadenosine (2-CdA) (,) and
arabinosylcytosine (araC) (d). Cells were exposed to graded con-
centrations of each drug for 72 h. Cell viability was measured using
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay at the end of the incubation period. Logistic curves
were fitted to the data to obtain IC50 values from the concentration-
effect relationships, as described in Materials and methods. Each data
point represents the mean SD of six replicate measurements in a
single experiment.
determined using flow cytometry. The RFI value obtained
from each fluorescence histogram (RFI versus frequency of
events) was a quantitative measure of bound SAENTA
fluorescein. Specific binding was calculated as the difference
between SAENTA fluorescein binding in the absence (total
binding) and presence (non-specific binding) of an excess of
NBMPR. These experiments showed that SAENTA fluores-
cein binding was saturable and es transporter-selective in
that bound SAENTA fluorescein was reduced by prior
treatment of cells with NBMPR. Non-specific binding of
20 nmol/l SAENTA fluorescein was 81% and 22% of the
total binding in cells from Patients 8 and 9, respectively,
after correction for autofluorescence. Mass law analysis
of the binding data for lymphoblasts from Patient 8 yielded
the constants Bmax 578 16 RFI units/cell and
KD 206 024 nmol/l. The binding constants for
Patient 9 were Bmax 114 04 RFI units/cell and
KD 285 036 nmol/l. Conversion of RFI values to es
nucleoside transporter sites (NBMPR binding sites) using a
factor (200) determined in human cell lines (Gati et al,

2002 Blackwell Science Ltd, British Journal of Haematology 116: 528537

532 A. M. P. Wright et al
Table I. Immunophenotype and in vitro sensitivity to 2-CdA and araC* of leukaemic lymphoblasts from children with ALL.

Patient number Immunophenotype Bone marrow blasts (%) IC50, 2-CdA (nmol/l) IC50, araC (nmol/l)

1 preB-cell 93 799 2,290

2 preB-cell 99 nd nd
3 T-cell nd 408 nd
4 preB-cell 95 79 372
5 T-cell 77 6 65
6 preB-cell 95 1660 2,650
7 preB-cell nd nd nd
8 preB-cell 95 84 374
9 T-cell 97 > 5000 > 5000
10 preB-cell nd nd nd
11 preB-cell 77 171 1,540
12 T-cell nd 136 59

*IC50 values were determined from concentration-effect plots following 72-h exposure of lymphoblasts to the nucleoside
analogues. The MTT dye reduction assay was used to measure cell viability in the drug-treated cultures.
Not determined. All samples used in the present study are listed in the table. Although some samples were not tested for drug
sensitivity, SAENTA fluorescein binding data for those samples are shown in Fig 4.
Peripheral blood sample.

shown), indicating that SAENTA fluorescein was bound

with high affinity to the es transporter in leukaemic
lymphoblasts.

Correlation of chemosensitivity and cellular content

Fig 2. Correlation of sensitivity to 2-chlorodeoxyadenosine (2-CdA)
and to arabinosylcytosine (araC) in leukaemic lymphoblasts from
acute lymphoblastic leukaemia (ALL) patients. The coordinates of
each data point are logarithms of the IC50 values for 2-CdA and
araC in individual patient samples, determined according to the
methods of Fig 1. A statistically significant (P 0032) correlation
of sensitivity to the two nucleoside drugs was found in the lym-
phoblast samples tested.
1997) would yield Bmax 11 600 320 sites/cell and
Bmax 2280 80 sites/cell for lymphoblasts from Patients
8 and 9 respectively.
Marked patient to patient differences were apparent in
the maximum binding of SAENTA fluorescein in leukaemic
lymphoblasts from 10 paediatric patients with ALL, as
shown in Fig 4. The Bmax values ranged from 400 to 147
RFI units/cell (80029 400 es transporter sites/cell). The
KD values for SAENTA fluorescein binding to the es
transporter ranged from 0663 to 111 nmol/l (not
of es nucleoside transporters
The present study investigated whether the abundance of es
nucleoside transporters in fresh leukaemic lymphoblasts
was a determinant of 2-CdA or araC cytotoxicity. Figure 5
shows tests of the correlation of 2-CdA and araC sensitivity
with es nucleoside transporter content of the blasts. The
correlation between 2-CdA sensitivity and es transporter
abundance was weak (Fig 5, top), and did not reach
statistical significance (r )057, P 023). In contrast,
a strong, statistically significant correlation between araC
sensitivity and es transporter abundance was found (Fig 5,
bottom), that is, the IC50 value for araC was inversely
correlated with the Bmax value for SAENTA fluorescein
binding (r )093, P 0023).
Proportional increases in es nucleoside transporter-deficient
CEM cell subsets in cultures exposed to araC,
but not in those exposed to 2-CdA
Replicate samples of CEM cells were exposed to graded
concentrations of 2-CdA or araC for 96 h, then stained with
SAENTA fluorescein, annexin V-PE and 7-AAD for the
measurement of es nucleoside transporter content, and the
recognition of apoptotic and necrotic cells, respectively, as
described in Materials and methods. IC50 values for 2-CdA
and araC under these conditions were 28 nmol/l and
43 nmol/l respectively (not shown).
Figure 6 shows examples of flow cytometry histograms of
SAENTA fluorescein-stained control and drug-treated CEM
cell populations that contained only viable cells (non-
apoptotic and non-necrotic according to annexin V-PE and
7-AAD staining). In these experiments, cells that were

2002 Blackwell Science Ltd, British Journal of Haematology 116: 528537

 Nucleoside Transporters and Drug Toxicity in Leukaemic Blasts
Fig 3. Equilibrium binding of SAENTA [5-S-(2-aminoethyl)-
N6-(4-nitrobenzyl)-5-thioadenosine] fluorescein to leukaemic lympho-
blasts from preB-cell acute lymphoblastic leukaemia (ALL) Patient 8
(top) and T-cell ALL Patient 9 (bottom). Cells were incubated with
graded concentrations of SAENTA fluorescein for 30 min at 22C in
the absence (j) or presence (n) of 5 lmol/l nitrobenzylthioinosine
(NBMPR). Cell-associated fluorescence was determined by measur-
ing the mean fluorescence intensity of 10 000 cells for each data
point. Specific binding (s) was determined as the difference
between total (j) and non-specific (n) fluorescence. Hyperbolas
were fitted to the specific binding data to yield Bmax and KD values,
as described in Materials and methods. The constants so determined
were: Patient 8, Bmax 578 16 RFI, KD 206 024 nmol/l;
Patient 9, Bmax 114 04 RFI, KD 285 036 nmol/l;
The inset figures show Scatchard analyses of the specific binding
data.
stained with SAENTA fluorescein, or SAENTA fluorescein in
the presence of 5 lmol/l NBMPR, yielded histograms that
specified the fluorescence intensity values of total binding
(solid line) or non-specific binding (overlay, dotted line,
marker M1) respectively (Fig 6). Fluorescence intensity
2002 Blackwell Science Ltd, British Journal of Haematology 116: 528537
533
Fig 4. Interpatient variation in the cellular content of es nucleoside
transporters in leukaemic lymphoblasts from children with acute
lymphoblastic leukaemia (ALL). Bmax values [in relative fluores-
cence intensity (RFI) units] were determined by measuring
the equilibrium binding of SAENTA [5-S-(2-aminoethyl)-
N6-(4-nitrobenzyl)-5-thioadenosine] fluorescein, as described for
the experiments of Fig 3. Open bars, preB-cell ALL; hatched bars,
T-cell ALL.
values for specific binding were recognized (marker M2) as
those of the total binding histogram that did not fall within
the range of non-specific binding fluorescence signals
(marker M1). In control samples (no drug, Fig 6A), a small
number of cells (58%) were dimly fluorescent after staining
for total SAENTA fluorescein binding (solid line), emitting
signals that fell within the range of marker M1 (non-specific
binding). As these cells lacked measurable specific binding of
SAENTA fluorescein, they were regarded as es transporter-
deficient cells. Similarly, Fig 6B shows a SAENTA fluores-
cein histogram of CEM cells that survived 40 nmol/l 2-CdA,
a concentration that killed 64% of the original cell
population. Under these conditions, the transporter-defi-
cient subset (78%) was only slightly greater than that of the
control sample in Fig 6A. In contrast, cells that survived
10 nmol/l araC (Fig 6C), a concentration that killed 97% of
the cells, included a large (40%) proportion of transporter-
deficient cells. These results suggested that cells with a high
es nucleoside transporter content were selectively elimina-
ted by araC.
CEM cells that were exposed to graded concentrations of
2-CdA or araC, and analysed according to the procedure of
Fig 6, yielded the data shown in Fig 7. The increase in es
transporter-deficient CEM cell subsets in araC-surviving cell
populations was dependent on araC concentration (Fig 7A)
and on araC cytotoxicity (Fig 7B). In contrast, the propor-
tion of transporter-deficient cells in CEM cultures exposed to
2-CdA changed only slightly with graded increases in 2-CdA
concentration or the extent of cell kill (Fig 7). In the
experiments with 2-CdA, a 100-nmol/l concentration of the
nucleoside was also tested, but the number of surviving cells
was insufficient to allow a precise analysis of flow cytometry
data (not shown).
534 A. M. P. Wright et al

Fig 5. Correlation of 2-chlorodeoxyadenosine (2-CdA) (top) and

arabinosylcytosine (araC) (bottom) sensitivity with es nucleoside
transporter content of lymphoblasts from paediatric acute lym-
phoblastic leukaemia (ALL) patients. Es transporter content is
expressed as the Bmax (in RFI units) for SAENTA [5-S-(2-amino-
ethyl)-N6-(4-nitrobenzyl)-5-thioadenosine] fluorescein binding,
determined as in the experiments of Fig 3. IC50 values are the
concentrations of nucleoside analogues that reduced cell viability
by 50%, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay as in the experiments of
Fig 1.

DISCUSSION
The present study examined the role of nucleoside transport
in the cytotoxicity of 2-CdA and araC in leukaemic
lymphoblasts from previously untreated paediatric patients
with ALL, by comparing the cellular es nucleoside trans-
porter content and nucleoside drug sensitivity of freshly ALL, an immunophenotype often associated with an unfa-
isolated lymphoblasts. vourable outcome (Mahony, 1994), two T-cell ALL samples
A wide interpatient variation in sensitivity of the leukae- in this study were also among those that were most sensitive
mic blast samples to 2-CdA and araC was reflected in IC50 to the nucleosides. Pieters et al (1994) have shown a wide
values that ranged more than 800-fold and 80-fold respec- variation in sensitivity to a number of antitumour agents in
tively. Although the leukaemic cell sample that was most leukaemic blasts from patients with ALL. Campana et al
resistant to 2-CdA and araC was from a patient with T-cell (1993) also observed considerable interpatient variation in

2002 Blackwell Science Ltd, British Journal of Haematology 116: 528537

 Nucleoside Transporters and Drug Toxicity in Leukaemic Blasts
Fig 6. Increases in es nucleoside transporter-deficient cell subsets in
arabinosylcytosine (araC)-treated CEM cultures, but not in those
treated with 2-chlorodeoxyadenosine (2-CdA). CEM cells were
exposed to graded concentrations of the nucleoside drugs for 96 h,
then stained with SAENTA [5-S-(2-aminoethyl)-N6-(4-nitroben-
zyl)-5-thioadenosine] fluorescein in the absence (total binding) or
presence of nitrobenzylthioinosine (NBMPR) (non-specific binding),
and with phycoerythrin-labelled annexin V (annexin V-PE) and
7-aminoactinomycin D (7-AAD). The samples were analysed by
flow cytometry as described in Materials and methods. Representative
samples from four replicate data sets are shown. (A) SAENTA flu-
orescein binding in control cells (no drug), showing fluorescence
intensity ranges for non-specific binding (overlay: dotted line,
marker M1) and total binding (solid line). Marker M2 (specific
binding) designates fluorescence signals emitted from the total
binding sample that exceeded values identified as non-specific
binding. Signals from the total binding sample that fell within the
range of marker M1 were calculated as a percentage of total events
(M1 + M2) to determine the proportion of cells that did not bind
SAENTA fluorescein specifically and were therefore regarded as es
transporter-deficient (control value shown, 58%). (B) Histogram of
total SAENTA fluorescein binding in cells surviving exposure to
40 nmol/l 2-CdA, indicating that 78% of cells were transporter-
deficient. (C) Histogram of total SAENTA fluorescein binding in cells
surviving exposure to 10 nmol/l araC, indicating that 40% of cells
were transporter-deficient.
lymphoblast sensitivity to vincristine, 6-thioguanine, araC
and teniposide, using a novel stroma-supported immuno-
cytometric assay (SIA) to evaluate blasts from patients with
ALL. In contrast to results reported here, in vitro studies by
Kumagai et al (1994) showed little interpatient variation in
cytotoxicity of 100 nmol/l 2-CdA to the 20 samples of
paediatric ALL lymphoblasts examined. A possible explana-
tion for the difference may be found in the design of the
535
study by Kumagai et al (1994), in which the SIA assay was
used to assess cell viability after exposure of cells to 2-CdA
for periods of 96120 h. Those results also showed that
2-CdA was effective in cells from patients who had relapsed
after initial treatment with multiagent chemotherapy and in
cells from patients who had an unfavourable karyotype.
In the present study, IC50 values for 2-CdA were lower
than those for araC in most lymphoblast samples, and a
statistically significant linear correlation between IC50
values for 2-CdA and araC was observed. The latter finding
may indicate a common cytotoxic mechanism or resistance
mechanism for these nucleoside drugs in ALL blasts.
Although the leukaemic lymphoblasts were generally more
sensitive to 2-CdA than to araC, 2-CdA may have little
advantage over araC in the treatment of ALL of childhood, as
higher plasma concentrations of araC are obtained clinically.
Previous studies have shown that the cellular content of
es nucleoside transporters was a determinant of araC influx
(Wiley et al, 1982), araCTP formation (Wiley et al, 1985)
and araC cytotoxicity (Gati et al, 1996, 1997, 1998) in
freshly isolated blast samples from adult patients with acute
leukaemia. Furthermore, es nucleoside transporters have
been implicated in the cellular efflux of 2-CdA in cultured
lines of leukaemic lymphoblasts, enabling manipulation of
2-CdA cytotoxicity in cells exposed sequentially to 2-CdA
and NBMPR (Wright et al, 2000). The present study
compared the cell sensitivity to 2-CdA and araC with the
es transporter content of lymphoblasts from paediatric
patients with ALL. It was hypothesized that es transporter-
mediated inward flux of 2-CdA and araC may be a requisite
of nucleoside cytotoxicity in the leukaemic lymphoblasts,
and that a low es transporter abundance might be a
predictor of resistance to these agents. A rapid and sensitive
flow cytometric assay, using the es transporter-specific

Fig 7. The proportion of es transporter-deficient cells in CEM cell populations after exposure to 2-chlorodeoxyadenosine (2-CdA) (h)
or arabinosylcytosine (araC) (d). CEM cells were exposed to graded concentrations of the nucleoside drugs for 96 h, stained with SAENTA
[5-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5-thioadenosine] fluorescein, phycoerythrin-labelled annexin V (annexin V-PE) and 7-aminoactino-
mycin D (7-AAD), and analysed by flow cytometry as described in Materials and methods. The proportions of es transporter-deficient cells were
determined as described in the examples of Fig 6. (A) Nucleoside concentration-dependence of changes in the proportion of es transporter-
deficient cells. (B) Changes in the proportion of es transporter-deficient cells as a function of nucleoside cytotoxicity. The data are means SEM
(n 4).

2002 Blackwell Science Ltd, British Journal of Haematology 116: 528537

536 A. M. P. Wright et al
ligand, SAENTA fluorescein, was used to determine the
cellular content of es transporters in the ALL blasts. Bmax
values for SAENTA fluorescein binding ranged > 35-fold
among samples from 10 ALL patients, reflecting a wide
variation in cellular es transporter abundance.
A statistically significant correlation between sensitivity
to araC and es nucleoside transporter abundance was found
in this study, suggesting that the es transporter is a
determinant of araC cytotoxicity in ALL blasts. Accordingly,
lymphoblast samples that were relatively deficient in es
transporters were resistant to araC, and araC-sensitive
samples with low es transporter abundance were not found.
Thus, the data (Fig 5, bottom) suggest that the IC50 value
for araC would be > 1 lmol/l in ALL lymphoblasts in which
the Bmax value for SAENTA fluorescein binding (es nucleo-
side transporter content) is less than about 10 RFI, and that
such cells are likely to be resistant to araC at concentrations
of the drug achieved in plasma during therapy at conven-
tional doses. In contrast, the corresponding correlation with
2-CdA was not statistically significant, suggesting that, in
fresh leukaemic lymphoblasts, nucleoside transport is a less
important factor in the cytotoxicity of 2-CdA than in the
cytotoxicity of araC. These ideas are consistent with our
findings in the CEM lymphoblast cell line, in which
nucleoside transporter expression is limited to the es
transporter (Belt et al, 1993). The present experiments
showed a substantial, araC concentration-dependent in-
crease in the proportion of es transporter-deficient cells
within CEM cell populations that survived exposure to araC.
Those results suggest that nucleoside transport-proficient
CEM cells were selectively killed by araC, and that trans-
porter-deficient cells were araC-resistant. In contrast, only a
slight increase in the proportion of transporter-deficient cells
was found in cell populations surviving exposure to 2-CdA,
indicating that es nucleoside transporter abundance was
apparently a less important determinant of sensitivity to
2-CdA in CEM cells.
Although the present experiments support the hypothesis
that es nucleoside transporter deficiency may impart resis-
tance to araC in fresh ALL blasts, these studies suggest that
mechanisms unrelated to the es nucleoside transporter
probably account for 2-CdA resistance in lymphoblast
samples from some patients. Correlations of es transporter
content and sensitivity to nucleoside drugs may be con-
founded by the expression, in some cells, of other nucleoside
transporters, such as the cs, cif or ei nucleoside transporters
that mediate the uptake of 2-CdA, but are not recognized by
SAENTA fluorescein. Because of the limited size of lympho-
blast populations obtained from bone marrow samples in
the present study, no attempt was made to detect other
nucleoside transporters in these cells, or to measure other
mechanisms of 2-CdA resistance, such as a deficiency in
deoxycytidine kinase (Kearns et al, 1994). A comparison of
the es nucleoside transporter content of leukaemic lympho-
blasts and clinical response to therapy with nucleoside
drugs in the controlled setting of a clinical trial will be
needed to determine the clinical significance of nucleoside
transport deficiency in resistance to 2-CdA and araC in ALL
of childhood.
2002 Blackwell Science Ltd, British Journal of Haematology 116: 528537
ACKNOWLEDGMENTS
We are indebted to the paediatric healthcare staff at the
University of Alberta Hospital and to John Chan for help in
the provision of bone marrow samples. We thank Carmen
Harris for technical assistance. This study was supported by
the Alberta Cancer Board, the Alberta Heritage Foundation
for Medical Research and the Peter Jang Memorial Fund.
A.M.P.W. was the recipient of a Studentship Award from
the Alberta Heritage Foundation for Medical Research
during the conduct of this study.
REFERENCES
Alessi-Severini, S., Gati, W.P., Belch, A.R. & Paterson, A.R.P.
(1995) Intracellular pharmacokinetics of 2-chlorodeoxyadeno-
sine in leukemia cells from patients with chronic lymphocytic
leukemia. Leukemia, 9, 16741679.
Alley, M.C., Scudiero, D.A., Monks, A., Hursey, M.L., Czerwinski,
M.J., Abbott, B.J., Mayo, J.G., Shoemaker, R.H. & Boyd, M.R.
(1988) Feasibility of drug screening with panels of human tumor
cell lines using a microculture tetrazolium assay. Cancer Research,
48, 589601.
Belt, J.A., Marina, N.M., Phelps, D.A. & Crawford, C.R. (1993)
Nucleoside transport in normal and neoplastic cells. Advances in
Enzyme Regulation, 33, 235252.
Buolamwini, J.K., Craik, J.D., Wiley, J.S., Robins, M.J., Gati, W.P.,
Cass, C.E. & Paterson, A.R.P. (1994) Conjugates of fluorescein
and SAENTA (5-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5-thioad-
enosine): Flow cytometric probes for the es nucleoside transporter
elements of the plasma membrane. Nucleosides and Nucleotides,
13, 737751.
Campana, D., Manabe, A. & Evans, W.E. (1993) Stroma-supported
immunocytometric assay (SIA): a novel method for testing the
sensitivity of acute lymphoblastic leukemia cells to cytotoxic
drugs. Leukemia, 7, 482488.
Carson, D.A., Wasson, D.B. & Beutler, E. (1984) Antileukemic and
immunosuppressive activity of 2-chloro-2-deoxyadenosine. Pro-
ceedings of the National Academy of Sciences of the United States of
America, 81, 22322236.
Cass, C.E. (1995) Nucleoside transport. In: Drug Transport in
Antimicrobial Therapy and Anticancer Therapy (ed. by N.H.
Georgopapadakou), pp. 403451. Marcel Dekker, New York.
Crawford, C.R., Ng, C.Y.C., Noel, D. & Belt, J.A. (1988) Nucleoside
transport in L1210 murine leukemia cells. Journal of Biological
Chemistry, 265, 97329736.
Gati, W.P., Paterson, A.R.P. & Belch, A.R. (1996) Use of SAENTA-
fluorescein in a flow cytometric assay of cellular es nucleoside
transporter site abundance, a correlate of chemosensitivity to
cytarabine in acute myeloid leukemia. Proceedings of the American
Association for Cancer Research, 37, 405.
Gati, W.P., Paterson, A.R.P., Larratt, L.M., Turner, A.R. & Belch,
A.R. (1997) Sensitivity of acute leukemia cells to cytarabine is a
correlate of cellular es nucleoside transporter site content mea-
sured by flow cytometry with SAENTA-fluorescein. Blood, 90,
346353.
Gati, W.P., Paterson, A.R.P., Belch, A.R., Chlumecky, V., Larratt,
L.M., Mant, M.J. & Turner, A.R. (1998) Es nucleoside transporter
content of acute leukemia cells: Role in cell sensitivity to
cytarabine (araC). Leukemia and Lymphoma, 32, 3554.
Griffig, J., Koob, R. & Blakley, R.L. (1989) Mechanism of inhibition
of DNA synthesis by 2-chlorodeoxyadenosine in human lympho-
blastic cells. Cancer Research, 49, 69236928.
 Nucleoside Transporters and Drug Toxicity in Leukaemic Blasts
Jamieson, G.P., Brocklebank, A.M., Snook, M.B., Sawyer, W.H.,
Buolamwini, J.K., Paterson, A.R.P. & Wiley, J.S. (1993) Flow
cytometric quantitation of nucleoside transporter sites on human
leukemic cells. Cytometry, 14, 3238.
Kay, A.C., Carrera, C.J., Carson, D.A., Beutler, E. & Piro, L.D. (1989)
Phase II trial of 2-chlorodeoxyadenosine in low grade lymphoma.
Blood, 74, 121A.
Kazimierczuk, Z., Cottam, H.B. & Robins, R.K. (1984) Synthesis of
2-deoxytubercidin, 2-deoxyadenosine and related 2-deoxy-
nucleosides via a novel direct stereospecific sodium salt glycosy-
lation procedure. Journal of the American Chemical Society, 106,
63796382.
Kearns, C.M., Blakley, R.L., Santana, V.M. & Crom, W.R. (1994)
Pharmacokinetics of cladribine (2-chlorodeoxyadenosine) in
children with acute leukemia. Cancer Research, 54, 12351239.
King, K.E. & Cass, C.E. (1994) Membrane transport of 2-chloro-
2-deoxyadenosine is required for cytotoxicity. Proceedings of the
American Association for Cancer Research, 35, 577.
Klumper, E., Pieters, R., Kaspers, G.J., Huismans, D.R., Loonen,
A.H., Rottier, M.M., van Wering, E.R., van der Does-van den
Berg, A., Hahlen, K., Creutzia, U. & Veerman, A.J.P. (1995)
In vitro chemosensitivity assessed with the MTT assay in
childhood acute non-lymphoblastic leukemia. Leukemia, 9,
18641869.
Koopman, G., Reutelingsperger, C.P.M., Kuijten, G.A.M., Keehnen,
R.M.J., Pals, S.T. & van Oers, M.H.J. (1994) Annexin V for flow
cytometric detection of phosphatidylserine expression on B cells
undergoing apoptosis. Blood, 84, 14151420.
Kumagai, M., Manabe, A., Coustan-Smith, E., Blakley, R.L., Beck,
W.T., Santana, V.M., Behm, F.G., Raimondi, S.C. & Campana, D.
(1994) Use of stroma-supported cultures of leukemic cells to
assess antileukemic drugs. II. Potent cytotoxicity of 2-chloro-
deoxyadenosine in acute lymphoblastic leukemia. Leukemia, 8,
11161123.
Kurzrock, R., Talpaz, M. & Gutterman, J.U. (1991) Hairy cell
leukemia: review of treatment. British Journal of Hematology, 79,
1720.
Mahony, D.H. (1994) Acute lymphoblastic leukemia in childhood.
In: Principals and Practice of Pediatrics (ed. by F.A. Oski, C.D. De
Angelis, R.D. Feigin, J.A. McMillan & J.B. Warshaw), pp. 1701
1710. J.B. Lippincott Co, Philadelphia.
Morra, E., Lazzarino, M., Brusamolino, E., Pagnucco, G., Casta-
gostino, C., Bernasconi, P., Orlandi, E., Corso, A., Santagostino,
A. & Bernasconi, C. (1993) The role of systemic high-dose
cytarabine in the treatment of central nervous system leukemia.
Clinical results in 46 patients. Cancer, 72, 439445.
Paterson, A.R.P. & Oliver, J.M. (1971) Nucleoside transport. II.
Inhibition by p-nitrobenzylthioguanosine and related com-
pounds. Canadian Journal of Biochemistry, 49, 271274.
Paterson, A.R.P., Gati, W.P., Vijayalakshmi, D., Cass, C.E., Mant,
M.J., Young, J.D. & Belch, A.R. (1993) Inhibitor-sensitive, Na+-
linked transport of nucleoside analogs in leukemia cells from
patients. Proceedings of the American Association for Cancer
Research, 34, 14.
Paul, B., Chen, M.F. & Paterson, A.R.P. (1975) Inhibitors of
nucleoside transport. Structure-activity study using human ery-
throcytes. Journal of Medicinal Chemistry, 18, 968973.
Pieters, R., Kaspers, G.J., Klumper, E. & Veerman, A.J. (1994)
Clinical relevance of in vitro drug resistance testing in childhood
2002 Blackwell Science Ltd, British Journal of Haematology 116: 528537
537
acute lymphoblastic leukemia; the state of the art. Medical Pedi-
atric Oncology, 22, 299308.
Piro, L.D. (1992) 2-Chlorodeoxyadenosine treatment of lymphoid
malignancies. Blood, 79, 843845.
Piro, L.D., Carrera, C.J., Beutler, E. & Carson, D.A. (1988) 2-Chloro-
deoxyadenosine: an effective new agent for the treatment of
chronic lymphocytic leukemia. Blood, 72, 10691073.
Rivera, G.K., Raimondi, S.C., Hancock, M.L., Behm, F.G., Pui, C.H.,
Abromowitch, M., Mirro, Jr, J., Ochs, J.S., Look, A.T., Williams, D.L.,
Murphy, S.B., Dalh, G.V., Kalwinsky, D.K., Evans, W.E., Kun, L.E.,
Simone, J.V. & Crist, W.M. (1991) Improved outcome in child-
hood acute lymphoblastic leukemia with reinforced early treat-
ment and rotational combination chemotherapy. Lancet, 337, 6166.
Rustum, Y.M. & Raymakers, R.A.P. (1992) 1-b-Arabinofuranosyl-
cytosine in therapy of leukemia: preclinical and clinical overview.
Pharmacology and Therapeutics, 56, 307321.
Santana, V.M., Mirro, J., Harwood, F.C., Cherrie, J., Schell, M.,
Kalwinsky, D. & Blakley, R.L. (1991) A Phase I clinical trial of
2-chlorodeoxyadenosine in pediatric patients with acute leuke-
mia. Journal of Clinical Oncology, 9, 416422.
Santana, V.M., Mirro, J., Kearns, C., Schell, J.M., Crom, W. &
Blakley, R.L. (1992) 2-Chlorodeoxyadenosine produces a high
rate of complete remission in relapsed acute myeloid leukemia.
Journal of Clinical Oncology, 10, 364370.
Sargent, J.M. & Taylor, C.G. (1989) Appraisal of the MTT assay as a
rapid test of chemosensitivity in acute myeloid leukemia. British
Journal of Cancer, 60, 206210.
Schmid, I., Krall, W.J., Uittenbogaart, C.H., Braun, J. & Giorgi, J.V.
(1992) Dead cell discrimination with 7-amino-actinomycin D in
combination with dual color immunofluorescence in single laser
flow cytometry. Cytometry, 13, 204208.
Sutherland, D.R., Keating, A., Nayar, R., Anania, S. & Stewart, A.K.
(1994) Sensitive detection and enumeration of CD34+ cells in
peripheral and cord blood by flow cytometry. Experimental
Hematology, 22, 10031010.
White, J.C., Rathmell, J.P. & Capizzi, R.L. (1987) Membrane
transport influences the rate of accumulation of cytosine arabi-
noside in human leukemia cells. Journal of Clinical Investigation,
79, 380387.
Wiley, J.S., Jones, S.P., Sawyer, W.H. & Paterson, A.R.P. (1982)
Cytosine arabinoside influx and nucleoside transport sites in
acute leukemia. Journal of Clinical Investigation, 69, 479489.
Wiley, J.S., Jones, P.E. & Sawyer, W.H. (1983) Cytosine arabinoside
transport by human leukemia cells. European Journal of Cancer and
Clinical Oncology, 19, 10671074.
Wiley, J.S., Taupin, J., Jamieson, G.P., Snook, M., Sawyer, W.H. &
Finch, L.R. (1985) Cytosine arabinoside transport and metabo-
lism in acute leukemias and T cell lymphoblastic lymphoma.
Journal of Clinical Investigation, 75, 632642.
Wright, A.M.P., Gati, W.P. & Paterson, A.R.P. (2000) Enhance-
ment of retention and cytotoxicity of 2-chlorodeoxyadenosine in
cultured human leukemic lymphoblasts by nitrobenzylthioino-
sine, an inhibitor of equilibrative nucleoside transport. Leukemia,
14, 5260.
Young, I., Young, G.J., Wiley, J.S. & van der Weyden, M.B. (1985)
Nucleoside transport and cytosine arabinoside (araC) metabolism
in human T lymphoblasts resistant to araC, thymidine and
6-methylmercaptopurine riboside. European Journal of Cancer and
Clinical Oncology, 21, 10771082.