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Blood, Vol. 91 No. 3 (February 1), 1998:
pp. 1001-1007
Cyclosporin A Induces Apoptosis in Childhood Acute
Lymphoblastic Leukemia Cells
By
Chikako Ito,
Raul C. Ribeiro,
Frederick G. Behm,
Susana C. Raimondi,
Ching-Hon Pui, and
Dario Campana
From the Departments of Hematology-Oncology and Pathology and
Laboratory Medicine, St Jude Children's Research Hospital, Memphis;
and University of Tennessee College of Medicine, Memphis, TN.
 |
ABSTRACT |
In an effort to identify novel antileukemic agents that can bypass
the mechanisms of multidrug resistance, we found that cyclosporin A
([CyA] 5 µmol/L) produced a median cell kill of 69% (range, 47%
to 85%) in seven B-lineage acute lymphoblastic leukemia (ALL) cell
lines (OP-1, SUP-B15, KOPN-55bi, RS4;11, NALM6, REH, and 380) and three
T-lineage ALL cell lines (MOLT4, CCRF-CEM, and CEM-C7) after 4 days of
culture. At 10 µmol/L, median CyA toxicity was 99% (range, 88% to
>99%). CyA was equally toxic to both a multidrug-resistant cell
line, CEM-VLB100, which overexpresses gp-170
P-glycoprotein, and one resistant to topoisomerase II inhibitors, CEM-VM1-5, which has a mutation in the topoisomerase II gene. CyA was
also toxic to primary leukemic cells maintained in stroma-based culture, a system that substantially prolongs in vitro cell survival. Against lymphoblasts from 21 patients with B-lineage ALL, the compound
(at 5 µmol/L) reduced the leukemic cell number by a median of 87%
(range, 27% to >99%) compared with results for parallel control
cultures lacking CyA. Seven of these samples were from cases with
unfavorable genetic features (eg, Philadelphia-chromosome or
MLL gene rearrangements); three were obtained at relapse.
Against T lymphoblasts (from six patients), the median reduction in
cell number was 79% (range, 30% to >99%). At 10 µmol/L, the cell
kill exceeded 97% in all cases studied. The mechanism of CyA
cytotoxicity was found to be the activation of apoptosis, which was
suppressed by phorbol myristate acetate but not by inhibitors of
ceramide-mediated apoptosis, phosphatidyl inositol-3 kinase activity,
or tyrosine kinase activity. These findings demonstrate high levels of
CyA-induced toxicity against ALL cells at concentrations achievable in
vivo, thus providing a strong rationale for clinical testing of this agent in patients with ALL.
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INTRODUCTION |
THE FUNGAL undecapeptide cyclosporin A
(CyA) is widely used in the treatment of allograft rejection,
graft-versus-host disease, and a variety of autoimmune
disorders.1,2 Its cellular effects depend on its binding to
a ubiquitous cytosolic protein (cyclophilin). The complex formed by CyA
and cyclophilin targets the Ca2+- and calmodulin-dependent
protein phosphatase calcineurin.1,2 The resuting
interference with Ca2+-dependent signaling is accompanied
by a block in transcriptional activity mediated by key transcription
factors, such as NF-AT, Oct-1, and NF-kB, and suppression of a variety
of other regulatory molecules, including interleukin-2, MYC, and
tyrosine kinases of the SRC family.1,2 Disruption of the
pathways controlled by these molecules leads, in turn, to impairment of
normal lymphoid cell activation.
Reports of CyA toxicity against leukemic T-cell lines3-6
and, more recently, a complete remission induced by CyA alone in a
patient with a lymphoproliferative disorder,7 suggest that this agent can inhibit critical cellular functions not only in normal
lymphoid cells but also in their malignant counterparts. In this study,
we tested the activity of CyA against childhood acute lymphoblastic
leukemia (ALL) cells growing in vitro. To directly assess the effects
of this compound on leukemic lymphoblasts from patients, we took
advantage of a stromal-layer culture system that promotes long-term
survival of ALL cells.8-15 The results reported here
demonstrate that CyA triggers apoptosis of both T and B leukemic
lymphoblasts, including those with molecular and genetic features of
drug resistance, suggesting its potential value as an antileukemic
agent.
| |
SUBJECTS AND METHODS |
Patients and cell lines.
Bone marrow cells were collected at diagnosis or relapse from 27 patients with ALL (21 B-lineage and six T-lineage cases) aged 7 months
to 16 years (median, 8 years; Table 1).Bone marrow cells from three healthy donors were also studied. Approval
for these studies was obtained from the St. Jude Institutional Review Board, with informed consent given by the parents or guardians of each
child. In all B-lineage cases, more than 80% of the lymphoblasts were
positive for CD19, class II antigens, and terminal deoxynucleotidyl transferase (TdT) and lacked surface Igs. Six of 21 cases were classified as pre-B ALL, with lymphoblasts expressing cytoplasmic µ heavy chains. In T-lineage cases, more than 80% of the cells were
positive for CD7, cytoplasmic or surface CD3, and TdT. In all T-lineage
ALL cases, drug testing was performed immediately after cell
collection, whereas B-lineage ALL samples were used either fresh or
after cryopreservation; previous experiments have shown that the latter
does not affect the ability of these cells to grow in stroma-supported
cultures.13 In all experiments, cell viability exceeded
80% by trypan blue dye exclusion.
Mononuclear cells were collected by density-gradient centrifugation
(Lymphoprep; Nycomed, Oslo, Norway) and washed three times in phosphate-buffered saline (PBS) and once in AIM-V (GIBCO, Grand Island, NY), a chemically defined, serum-free, cytokine-free tissue culture medium. The ALL cell lines selected for this study (OP-1, SUP-B15, KOPN-55bi, RS4;11, NALM6, REH, 380, CCRF-CEM, CEM-C7, and
MOLT4) are maintained in our laboratory, and the cell lines CEM-VLB100 and CEM-VM1-5 were provided by Dr W.M. Beck,
University of Illinois Cancer Center (Chicago, IL).16,17
All cell lines were maintained in RPMI 1640 tissue culture medium
(Whittaker Bioproducts, Walkersville, MD) with 10% fetal calf serum
(Whittaker).
Cell culture and treatment with CyA.
Bone marrow stromal cells depleted of T cells by CD6/CD8-mediated
rabbit complement lysis were derived from seven normal donors (5 to 26 years old) and prepared in 96-well flat-bottomed plates (Costar,
Cambridge, MA) as previously described.12-15 To prepare test cultures, we removed the media from the bone marrow stroma and
washed the adherent cells seven times with AIM-V tissue culture medium.
Leukemic lymphoblasts or normal bone marrow cells were resuspended in
AIM-V, and 1 to 3 × 105 cells were placed on the stromal
layer in each well. Continuously growing cell lines were resuspended in
RPMI 1640 with additives, and 1 to 3 × 104 cells were
plated in each well in the absence of stroma. CyA (Calbiochem Inc, San
Diego, CA) was diluted in absolute ethanol (stock
solution, 5 mmol/L) and further diluted in AIM-V or RPMI 1640 immediately before it was added to the cultures at the final concentrations indicated in the results. In some experiments, a CyA
preparation for intravenous administration (Sandimmune; Sandoz, Basel,
Switzerland) was also used. Teniposide was obtained from Bristol Myers
Squibb (Wallingford, CT) and daunorubicin from Sigma (St Louis, MO).
Phorbol myristate acetate, fumonisin B1, sphingosine 1-phosphate,
staurosporine, and genistein were purchased from Sigma. Cultures were
incubated at 37°C with 5% CO2 and 90% humidity for the
times indicated in the results.
Assessment of drug effects.
At the beginning and end of the cultures, the cell number and
immunophenotype were determined by flow
cytometry.9,11,13,14 Briefly, cultures were transferred to
Falcon tubes (Becton Dickinson, Lincoln Park, NJ). Cells from B-lineage
cases were incubated with CD19 (Leu 12) monoclonal antibody conjugated
to fluorescein isothiocyanate (FITC), and those from T-lineage cases
with CD7-FITC. Normal bone marrow cells were labeled with CD34
conjugated to peridin chlorophyll protein, CD19 PE, as well as with
CD13 and CD33 conjugated to FITC. All monoclonal antibodies and
isotype-matched unreactive controls were purchased from Becton
Dickinson (San Jose, CA) or Dako (Carpinteria, CA). Staining with
antibodies was omitted in experiments with cell lines. After two washes
in PBS with 0.2% bovine serum albumin and 0.2% sodium azide, the
cells were resuspended in 0.5% paraformaldehyde and analyzed with a
FACScan flow cytometer and Cell Quest software (Becton Dickinson). In
each case, we outlined "gates" surrounding the area of the
light-scatter dot plot that comprised all viable leukemic cells, and
used them to enumerate cells that were present before and after culture
with or without CyA. For primary leukemias and normal bone marrow
cells, the results were corrected for the percentage of cells in each
sample expressing different antigens. The formula, (no. of cells
recovered with CyA/no. of cells recovered without CyA) × 100, was
used to calculate relative cell recovery after CyA treatment. All
results are reported as the mean of duplicate experiments. Drug
concentrations that produced 50% cell killing (LC50)
were calculated with the AllFit software from the National Institutes
of Health.18
DNA gel electrophoresis to detect apoptotic DNA fragmentation was
performed after separating fragmented and intact DNA by centrifugation
as previously described.8 We also studied cell labeling by
Annexin-V conjugated to FITC (Trevigen, Gaithersburg, MD), which binds
to phosphatidylserine exposed on the surface membrane of cells
undergoing apoptosis.19 Cell membrane permeabilization was
detected by standard propidium iodide labeling.
| |
RESULTS |
Toxicity against leukemic cell lines.
CyA at 5 µmol/L was markedly toxic to all of the leukemic cell lines
studied (Table 2). After 4 days of culture,
the median percentage of cell kill among the seven B-lineage lines
(OP-1, SUP-B15, KOPN-55bi, RS4;11, NALM6, REH, and 380) and the three T-lineage lines (MOLT4, CCRF-CEM, and CEM-C7) was 69% (range, 47% to
85%). The extent of CyA cytotoxicity was dose-dependent in each of
these cell lines (Table 2). At 10 µmol/L, CyA was highly cytotoxic
against all lines, with a median cell kill of 99% (range, 88% to
>99%) after 4 days of incubation. With most of the cell lines,
CyA-induced toxicity was still appreciable when the drug was added at a
concentration of 2 µmol/L (median, 36%; range, 10% to 73%). In
cultures containing vehicle only (0.1% ethanol), the number of cells
recovered was identical to that of control cultures without additives
(data not shown). In comparative experiments, the antileukemic potency
of CyA from Calbiochem diluted in ethanol and the CyA preparation for
clinical use (Sandimmune) was identical (data not shown).
To determine whether CyA cytotoxicity would bypass the common
mechanisms of drug resistance, we tested different concentrations of
the compound against the CEM-VLB100 cell line, which
overexpresses gp-170 P-glycoprotein and is resistant to several
anticancer drugs,16 as well as the CEM-VM1-5 cell line,
which carries a mutation in the topoisomerase II gene and is resistant
to topoisomerase II inhibitors,17 and compared the results
with those obtained with the parent cell line CCRF-CEM. As expected,
CEM-VLB100 was relatively resistant to daunorubicin
(LC50, 40.1 v 2.2 ng/mL in CCRF-CEM cells), while
CEM-VM1-5 showed resistance to teniposide (LC50, 16.8 v 0.04 µmol/L in CCRF-CEM cells). By contrast, the
concentrations of CyA that were cytotoxic in all three lines were
similar (LC50, 2.9 µmol/L in CCRF-CEM, 3.7 in
CEM-VLB100, and 5.6 in CEM-VM1-5).
Toxicity against clinical samples of leukemic cells.
To assess the toxic effects of CyA against fresh samples of leukemic
lymphoblasts from patients with ALL, we took advantage of a
stroma-based culture system that allows prolonged cell
culture.8-15 After 7 days of such culture, the median cell
recovery among 27 cases studied was 103% (range, 50% to 550%). CyA
at 5 µmol/L produced variable levels of cytotoxicity in these cases
(Fig 1). Against B-lineage ALL (21 cases),
the reduction in leukemic cell number was 27% to more than 99%
(median, 87%) of that in parallel 7-day cultures not exposed to the
compound but containing 0.1% ethanol (the CyA vehicle). Likewise, CyA
at 5 µmol/L was cytotoxic in all six cases of T-cell ALL (range of
cell kill, 30% to >99%; median, 79%; Fig 1). CyA cytotoxicity was
dose-dependent, and in some cases remained substantial at
concentrations as low as 1 µmol/L. At 2 µmol/L CyA, the percentage
of cell kill for 20 cases of B-lineage ALL was 12% to 61% (median,
32%). Cytotoxicity was not detectable at this concentration in only
one case. Similar levels of cell kill were obtained at this drug
concentration in T-cell samples (median, 58%; range, 16% to 83%).
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| Fig 1.
CyA toxicity against ALL cells from patients after 7 days
of culture on bone marrow-derived stromal layers. Data are the mean of
duplicate experiments. Horizontal bars indicate the median % cell kill
for each concentration of CyA. Intraassay variability was <10% in
all experiments.
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Cells from the seven B-lineage ALL cases with adverse genetic features
(no. 1 to 7), including t(9;22), t(4;11), or t(11;19),20 were susceptible to CyA (at 5 µmol/L) to the extent that the levels of cell kill did not differ significantly from those determined in
other B-lineage cases: median, 95% (range, 67% to >99%) versus 85% (27% to >99%) at 5 µmol/L, and 29% (12% to 59%) versus
33% (<1% to 61%) at 2 µmol/L. Notably, three of the samples with
high-risk genetic features (no. 1, 3, and 4) were obtained at relapse.
Likewise, the four cell lines with t(9;22) (OP-1, SUP-B15, and
KOPN-55bi) or t(4;11) (RS4;11) were as susceptible to CyA cytotoxicity
as cell lines without these high-risk features (Table 2).
To determine whether CyA cytotoxicity represented a direct effect on
leukemic lymphoblasts or an indirect effect mediated by damage to
stromal layers, we incubated the stromal layers with CyA at various
concentrations (1 to 10 µmol/L) for 2 days, washed them, and then
seeded them with leukemic lymphoblasts. In two independent experiments
with samples from patients no. 4 and 8, the percentage of cell recovery
after 7 days of culture on CyA-pretreated stroma was identical to that
achieved with unmanipulated stromal layers. For example, the percentage
of cell recovery after culture on unmanipulated stroma was 111% and
103% in the two cases; after culture on stroma pretreated with CyA 5 µmol/L, it was 111% and 102%, respectively. Thus, any indirect
toxicity to stromal layers does not appear to influence the recovery of
ALL cells exposed to CyA.
Toxicity against normal immature hematopoietic cells.
Previous studies with colony-forming assays have indicated that CyA has
no toxicity against normal hematopoietic cells.21-24 To
further address this issue, we prepared stroma-supported cultures of
normal bone marrow mononucleated cells from three individuals with and
without CyA, and assessed the effects of the compound on the expansion
of phenotypically immature cells characterized by low side-scatter,
expression of CD34, and absence of markers associated with B-lineage
(CD19) or myeloid-lineage (CD13 and CD33)
differentiation.25,26 At the beginning of the cultures, cells with these characteristics represented less than 0.1% of bone
marrow mononucleated cells in all three samples. After 7 days of
culture without CyA, the mean ±SD percentage of CD34+,
CD19 , CD13, CD33 cell
recovery was 879% ± 684% of those originally seeded. CyA at 2 µmol/L did not significantly change the expansion of these cells in
culture (mean cell recovery, 849% ± 620%). At 5 µmol/L, the
number of CD34+, CD19, CD13,
CD33 cells was lower than in control cultures (percent
cell recovery, 233% ± 288%), indicating an inhibitory effect of
the compound on the growth of normal hematopoietic cells. Nevertheless,
the mean number of CD34+, CD19,
CD13, CD33 cells in these cultures had
doubled despite CyA-mediated inhibition of cell growth.
Molecular mechanism of CyA-induced cell death.
CyA appeared to trigger apoptosis in all cell lines and leukemic cases
studied as early as 24 hours after its addition to cultures, as
indicated by typical changes in cell morphology observed by inverted
microscopy (ie, nuclear fragmentation and cell shrinkage; not shown).
CyA-induced morphologic deterioration was reflected by changes in
light-scatter properties of the cells determined by flow cytometry,
resulting in a reduction in forward light-scatter and an increase in
90° light-scatter (Fig 2A).
27 Exposure of phosphatidyl serine residues on the cell membrane (revealed by binding to Annexin-V19; Fig 2B) and massive
DNA fragmentation in multiples of 180 base pairs28 (Fig 2C)
determined in experiments with cell lines confirmed that CyA induced
apoptosis in ALL cells.
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| Fig 2.
CyA induces apoptosis in ALL cells. The T-lineage ALL
cell line CEM-C7 and the B-lineage ALL cell lines OP-1 (carrying the Philadelphia chromosome) and RS4;11 (with MLL gene
rearrangements) were incubated with CyA (10 µmol/L) or the vehicle
(ethanol 0.1%) for 48 hours. (A) Flow-cytometric dot plots illustrate
changes in cell light-scatter properties typical of apoptosis caused by exposure to CyA. These consisted of decreased cell size (forward scatter, FSC; x-axis) and increased cell granularity (side scatter, SSC; y-axis). (B) Isometric contour plots illustrate binding of Annexin-V to phosphatidyl serine residues on the cell membrane (a
feature of apoptosis) and propidium iodide labeling due to cell
membrane permeabilization caused by CyA. (C) In all 3 cell lines, CyA
induced a DNA ladder of a multiple of 180 base pairs, characteristic of
apoptosis. Molecular mass markers are indicated.
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To investigate the signaling pathways leading to apoptosis in leukemic
cells exposed to CyA, we used a series of compounds that have been
reported to inhibit apoptosis induced by a variety of stimuli. Phorbol
myristate acetate (1 to 10 ng/mL), which stimulates protein kinase C
activity and inhibits radiation- and glucocorticoid-induced apoptosis,29,30 suppressed the toxic effects of CyA on the pre-B ALL cell line NALM6 (Fig 3) and in the T-cell line MOLT4 (not
shown). All other compounds tested at nontoxic concentrations, including the ceramide-mediated apoptosis inhibitors fumonisin B1 (1 to
50 µmol/L) and sphingosine-1-phosphate (0.1 to 10 µmol/L),31-33 the phosphatidyl inositol-3 kinase
inhibitor wortmannin (100 nmol/L),34-36 and the tyrosine
kinase inhibitors staurosporine (3 to 50 nmol/L) and genistein (1 to 50 nmol/L),37 lacked any discernible effect on CyA-induced
cytotoxicity in both cell lines (Fig 3, and
not shown).
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| Fig 3.
CyA-induced apoptosis is suppressed by phorbol myristate
acetate (PMA). B-lineage ALL cell line NALM6 was incubated for 1 hour
with the compounds indicated before addition of CyA (5 µmol/L). After
48 hours of culture, the number of viable cells in cultures with and
without CyA was compared. Only preincubation with PMA (1 ng/mL)
inhibited CyA-induced apoptosis, while fumonisine B1 (FMN B1, 50 µmol/L), sphingosine 1-phosphate (SPP, 10 µmol/L), staurosporine (3 µmol/L), genistein (10 µmol/L), and wortmannin (WM, 100 nmol/L) had
no effect. At higher concentrations, these compounds were markedly
toxic. Bars represent the mean ± SD of 4 measurements per test.
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DISCUSSION |
In this study, we found that CyA is toxic to leukemic lymphoblasts via
induction of apoptosis, which was detectable as early as 24 hours after
exposure of the cells to the drug. Although previous studies had
indicated that CyA selectively produces toxicity in T
cells,3-6 we did not find its effects to be restricted to cells of this lineage. To the contrary, the compound was equally active
against B and T lymphoblasts; moreover, both continuously growing cell
lines and fresh samples of leukemic cells from patients were
susceptible to the toxic effects of CyA. Although the toxicity of CyA
observed on primary leukemic cells could be due to an effect of the
compound on the stromal layers used to support ALL cell survival in
vitro, we think this indirect mechanism unlikely, as our experiments
using stroma pretreated with CyA failed to reveal a decrease in its
capacity to promote ALL cell survival. In addition, the cytotoxic
effects of CyA were also observed in the ALL cell lines growing in
vitro without stromal cells.
The suppressive effects of CyA on normal lymphocyte function include
inhibition of T-cell activation in vitro in response to mitogenic
lectins, CD3 ligation, phorbol ester, and Ca2+
ionophore,1,2 as well as suppression of B-cell activation in response to B-cell receptor cross-linking.38 Notably,
treatment of murine splenic B cells with CyA induces cell
death.39 Our findings indicate that CyA also suppresses the
growth of malignant human lymphoid cells. The precise biochemical
mechanisms underlying CyA-induced apoptosis in leukemic lymphoblasts
remain to be clarified, but appear to be, at least in part, distinct
from those operating in cells undergoing apoptosis after exposure to
tumor necrosis factor,31,32 daunorubicin,33 and
ligation of Fas,32,37 surface Ig,35 or
CD38,36 as inhibitors that rescue cells from these agents
failed to diminish CyA toxicity. Nevertheless, CyA toxicity was
markedly suppressed by exposure to phorbol ester, reminiscent of
observations with radiation- and steroid-induced apoptosis in
thymocytes.29,30 Interestingly, other compounds known to
interfere with Ca2+-mediated signaling and T-cell
activation,1,2 such as FK-506 and rapamycin, did not induce
apoptosis in ALL cells (our unpublished observation, September 1996).
FK-506, which is approximately 100-fold more active than CyA in
inhibiting T-cell activation, had no effect on leukemic cell growth or
survival,6 while rapamycin had a cytostatic but not
cytotoxic activity against ALL cell lines.
Our findings have clear clinical implications. Currently, about 30% of
children and 65% of adults with ALL can be expected to relapse one or
more times.40 Patients with the Philadelphia-chromosome or
11q23 abnormalities involving the MLL gene, representing
approximately 6% of children and 30% of adults with ALL, have an
especially dire prognosis.40 Thus, new antileukemic agents
capable of overcoming the different mechanisms of drug resistance are
urgently needed. It is noteworthy in this regard that all of our cases,
including those studied at relapse and/or displaying adverse
cytogenetic features, as well as cell lines with molecular features
directly linked to multidrug resistance, were susceptible to
CyA-induced apoptosis.
The findings of CyA antileukemic activity in this study complement
previous observations that CyA increases the sensitivity of
multidrug-resistant cells to anticancer agents through a specific interaction of CyA with the 170-kD drug-efflux pump
P-glycoprotein.41,42 For example, in experiments with mice
engrafted with L1210 lymphoid leukemia and treated with etoposide, the
addition of CyA significantly prolonged survival.43 Of
note, in our study, the cell line CEM-VLB100, which
overexpresses P-glycoprotein, was nevertheless susceptible to
CyA-induced apoptosis. Clinical trials of CyA administered intravenously as a modulator of drug resistance have shown that steady-state serum levels of 3 to 5 µmol/L,44,45 which
are cytotoxic in vitro, are readily achieved at tolerable administered doses. The unique antileukemic activity of CyA, which may bypass the
common mechanisms of drug resistance, and its relatively lower toxicity
against immature normal hematopoietic cells21-24 provide a
strong rationale for clinical testing of this agent in patients with
high-risk ALL.
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FOOTNOTES |
Submitted June 17, 1997;
accepted September 25, 1997.
Supported by Grants No. RO1-CA58297, P30-CA21765, and CA20180 from the
National Cancer Institute, and by the American Lebanese Syrian
Associated Charities.
Address reprint requests to Dario Campana, MD, PhD, Department of
Hematology-Oncology, St Jude Children's Research Hospital, 332 N
Lauderdale, Memphis, TN 38105.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Elaine Coustan-Smith for flow-cytometric analysis of apoptosis
and John Gilbert for editorial assistance.
| |
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Abstract
Introduction
Abstract