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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1742-1748
Differential Induction of Apoptosis by Fludarabine Monophosphate in
Leukemic B and Normal T Cells in Chronic Lymphocytic Leukemia
By
Ugo Consoli,
Iman El-Tounsi,
Alex Sandoval,
Virginia Snell,
Hans-Dieter Kleine,
Wendy Brown,
Johnnie R. Robinson,
Francesco DiRaimondo,
William Plunkett, and
Michael Andreeff
From the Section of Molecular Hematology and Therapy, Department of
Hematology, and Department of Clinical Investigations, The University
of Texas M.D. Anderson Cancer Center, Houston; and the Institute of
Hematology, University of Catania, Italy.
 |
ABSTRACT |
Fludarabine (F-ara-A), an adenine nucleoside analog with efficacy in
B-cell chronic lymphocytic leukemia (B-CLL), has also been shown to
have a long-lasting suppressive effect on T lymphocytes. In
heterogeneous clinical samples, apoptosis cannot be detected by
standard methods in small cellular subsets. We developed, therefore, a
combined assay of in situ end-labeling of nicked DNA by terminal deoxynucleotide transferase, with measurements of cellular DNA content
and surface antigens (CD3, CD4, CD8, and CD19) by multiparametric flow
cytometry. This assay was used to determine F-ara-A-induced apoptosis
in different lymphocyte subsets from CLL patients and normal controls
treated with F-ara-A in vitro. Apoptosis was also correlated to bcl-2
protein levels. We observed a direct effect of F-ara-A on both B-CLL
and T lymphocytes. The response to F-ara-A in B-CLL lymphocytes in
vitro was Rai stage-dependent, the early-stages being more responsive
(P = .01). Higher levels of spontaneous apoptosis were
observed in B-CLL lymphocytes from early stage patients
(P = .02). No difference was observed in spontaneous apoptosis of normal T cells in B-CLL, although T lymphocytes in late-stage disease were more sensitive to F-ara-A-induced apoptosis. Incubation with cyclosporin A did not affect B-CLL and T-lymphocyte survival compared with control cultures. Results suggested a direct apoptotic effect of F-ara-A on B-CLL lymphocytes that decreases with
increasing clinical stage. No correlation was found between bcl-2 and
spontaneous or F-ara-A-induced apoptosis. Apoptosis occurred at all
cell-cycle stages and was not restricted to cells in S phase. The
mechanisms of this stage-dependent apoptosis in CLL remain to be
elucidated.
| |
INTRODUCTION |
FLUDARABINE
(9- -D-arabinofuranosyl-2-fluoroadenine; F-ara-A) is an adenine
nucleoside analog resistant to adenosine deaminase that has been
extensively used to successfully treat various hematological malignancies.1,2 Although F-ara-A's major action is
inhibition of DNA synthesis, clinical investigations have shown strong
therapeutic activity in indolent lymphocytic malignancies with very low
growth fraction.3-5 Other mechanisms of action have been
shown for F-ara-A, such as incorporation into RNA, effect on
methylation, DNA repair, and nicotinamide adenine dinucleotide
metabolism.2 The mechanism by which F-ara-A induces
apoptosis in both proliferating and quiescent cells has not yet been
completely established, however, and is an active focus of
investigation. Evaluation of the effects of F-ara-A in clinical samples
in vivo and in vitro is difficult because of the complex interactions
among different cell populations that influence growth or maintenance
of the leukemic clone as well as its response to chemotherapy. In
chronic lymphocytic leukemia (CLL), an often indolent disease, F-ara-A
proved to be a markedly cytoreductive agent,6 but it also
induced profound T lymphopenia.7 Subsequent studies showed
that CD4 and CD8 T-lymphocyte subsets are rapidly depressed when
F-ara-A therapy is initiated, revealing that this lymphocytotoxic agent
may exert preferential cytotoxicity toward T lymphocytes.8
This observation raises the possibility that the F-ara-A effect on
B-CLL cells could be exerted, at least in part, by a reduction of T
lymphocytes and their cytokine production or other functions. Indeed,
the role of T lymphocytes in B-cell activation, proliferation,
differentiation, and isotype switching is well recognized.9
Little is known, however, about the interaction of T lymphocytes and
leukemic B cells in CLL. Although T lymphocytes represent a minority of
the circulating lymphocytes, their absolute number is generally
increased in CLL.10,11 A role for T lymphocytes in B-CLL
may also be postulated from published observations of a reduction in
B-CLL cells after treatment with cyclosporin A (CyA).12,13
Although CyA's direct effect on B-CLL cannot be excluded,14 it may have affected B-CLL through inhibition
of T-lymphocyte cytokine production.15,16 In CLL cells, CyA
has also been shown to inhibits cytokine-induced
proliferation.17
We investigated the in vitro effect of F-ara-A in B and T peripheral
blood lymphocytes from 25 CLL patients and 4 normal controls. We also
evaluated the effect of CyA in 5 CLL samples. This approach was
intended to address the question of whether F-ara-A directly affects
the B-CLL lymphocyte compartment or whether its effect could be
modulated indirectly through T lymphocytes. We explored the correlation
between bcl-2 protein expression and F-ara-A-induced and spontaneous
apoptosis. Finally, to test whether recruitment of cells in the S phase
was required for F-ara-A-inducted apoptosis, we investigated the
possible correlation of apoptosis with cell-cycle stage. For this
purpose we developed an in situ end labeling (ISEL) technique to
simultaneously detect DNA strand breaks associated with
apoptosis,18 cell surface antigens, and cellular DNA
content.
| |
MATERIALS AND METHODS |
Reagents.
F-ara-A was provided by Dr V.L. Narayanan, Drug Synthesis and Chemistry
Branch Division of Cancer Treatment, National Cancer Institute,
Bethesda, MD. CyA was purchased from Sandoz (East Hanover, NJ). Phycoerythrin (PE)- and peridinin chlorophyl (PerCP)-conjugated monoclonal antibodies (MoAb) specific for CD3, CD4, CD8, CD19, CD5, and
control antibodies with irrelevant specificities were purchased from
Becton Dickinson (San Jose, CA); fluorescein isothiocyanate (FITC)
bcl-2 MoAb and isotype IgG1 control were from Dako Corporation (Carpinteria, CA). Terminal deoxynucleotide transferase (Tdt), biotin-16-2 -deoxyuridine-5-triphosphate (b-dUTP), and Tdt reaction buffer were obtained from Boehringer-Mannheim Co (Indianapolis, IN).
Hoechst 33342 was purchased from Polysciences, Inc (Warrington, PA).
Patients, cell isolation, and incubation conditions.
Patient samples were obtained with informed consent according to
institutional guidelines. All CLL patients fulfilled the National
Cancer Institute (NCI) criteria for the diagnosis of CLL19
and had been without treatment for at least 2 months before the analysis. Immunophenotyping by dual-parameter
flow cytometry showed coexpression of CD5 and B-cell antigens and
isotypic light-chain expression. Rai clinical staging system was
applied.20 CLL patients were grouped in two categories
according to the Rai stage at the time of the analysis: group 1, patients at stages 0 and I (18 patients) and group 2, patients at
stages II, III, and IV (7 patients).
Freshly obtained peripheral blood from CLL patients and 4 healthy
subjects was fractionated by Ficoll-Histopaque-1077 (Sigma, St Louis,
MO) sedimentation. Mononuclear cells were resuspended in RPMI 1640 medium (GIBCO-BRL, Gaithersburg, MD) supplemented with
streptomycin, penicillin, and 10% fetal calf serum (GIBCO) at a
concentration of 0.5 to 1 × 107 cells per milliliter.
Cells were incubated at 37°C in an atmosphere of 5% CO2
with various drugs for the times indicated.
Immunofluorescence detection and flow cytometry.
Cells obtained from the cultures were washed twice in cold
phosphate-buffered saline (PBS). One million cells were resuspended in
100 µL of PBS and 1% bovine serum albumin (BSA) and incubated for 30 minutes at 4°C with PE- or PerCP-conjugated MoAbs at the concentration of 10 µg/mL. PE- and PerCP-conjugated MoAb with irrelevant specificities were used as controls. Cells were then washed
twice in cold PBS and processed for bcl-2 or apoptosis measurement as
described following.
Bcl-2 immunofluorescence detection and quantitation.
Bcl-2 proto-oncogene protein staining was performed in combination with
detection of CD19 and CD5. Briefly, after staining with CD5-PE and
CD19-PerCP, cells were washed twice and fixed in 1% paraformaldehyde
for 15 minutes on ice, followed by permeabilization with 70% ethanol
for 15 minutes on ice; cells were then washed in cold PBS before the
addition of 10 µg/mL of FITC-conjugated anti-bcl-2 or isotype IgG1
MoAb. Intensity of bcl-2 expression was measured on a logarithmic
scale, and bcl-2 quantitation was assessed by Quantum Simply Cellular
microbeads with QuickCal Software (Flow Cytometry Standard Corporation,
Triangle Park, NC) as previously described21,22 and
expressed as antibody-binding capacity (ABC) as an estimate of the
number of antibody molecules bound per cell. A FACScan flow cytometer
(Becton Dickinson) equipped with an argon laser (488 nm) was used to
measure fluorescence. Data were analyzed using Lysys II software
(Becton Dickinson).
Measurement of apoptosis: ISEL of DNA strand breaks associated with
apoptosis.
For multiparametric analysis of surface antigens, ISEL for apoptosis
and DNA content, cells stained with MoAbs were fixed in 2 mL of 1%
freshly prepared paraformaldehyde in PBS (pH 7.4) for 15 minutes at
4°C on a horizontal shaker. Samples were washed once and stored at
 20°C in 70% ethanol for up to 2 weeks. After being rehydrated in
PBS, cells were resuspended in 50 µL of cacodylate buffer containing
0.2 mmol/L potassium cacodylate, 2.5 mmol/L Tris-HCl (pH 6.6), 2.5 mmol/L CoCl2, 0.25 mg/mL BSA, 7 U Tdt, and 0.5 µmol/L
biotin-16-dUTP. Cells were incubated in this solution at 37°C for 1 hour, then rinsed twice in PBS and resuspended in 100 µL avidin FITC
at 2.5 µg/mL in 4× SSC with 0.1% Triton X-100 and 1% BSA. After
30 minutes of incubation at room temperature in the dark, cells were
rinsed in PBS with 0.1% Triton X-100 and resuspended in 500 µL of
PBS for flow cytometric analysis. Control reactions lacked Tdt. We
measured a total of at least 3 × 104 cells per sample,
setting a "live gate" on CD3+ cells to ensure
acquisition of at least 1 × 104 cells for those samples
in which T-lymphocyte subpopulations were a small fraction of the
entire number of cells.
DNA staining and analysis.
For DNA staining we used a 20-fold concentrated Hoechst 33342 stock
solution dissolved in distilled water to a concentration of 10 µg/mL
with 10% Tween 20. One hundred microliters of this solution were mixed
into the cell suspension, resulting in a final concentration of 0.5 µg/mL of Hoechst 33342 and incubated at 4°C for 8 hours. Samples
were measured with a FACSVantage flow cytometer equipped
with a Coherent Enterprise laser (Becton Dickinson, Mountain View, CA).
The Enterprise laser was tuned to emit at 325 nm and 488 nm
simultaneously. The laser power was adjusted to 100 mW for 488 nm for
ISEL and MoAb excitation and to 40 mW for UV (Hoechst 33342 DNA-fluorescence). The filters used for the fluorochromes excited at
488 nm were 530 nm, 585 nm, and 650 nm for FITC, PE, and PerCP,
respectively; a blue filter (470 nm) was used for Hoechst fluorescence.
Data acquisition was done with a Hewlett Packard 3000 System combined
with Lysys II software. Compensation and amplification were set with
specific isotype controls. A pulse processor was used for
doublet-discrimination by calculating width, height, and area of the
analog fluorescence signal of the DNA parameters.
Statistics.
Statistical analysis was performed using Student's t-test and
the Spearman rank correlation coefficient.
| |
RESULTS |
F-ara-A-induced apoptosis in B and T lymphocytes from CLL patients and
healthy individuals.
Cultured lymphocytes from healthy individuals and CLL patients were
treated with F-ara-A (3 µmol/L),23-25 and the degree of apoptosis was evaluated in the different lymphocyte compartments at 24, 48, and 72 hours. As shown in Fig 1,apoptotic ISEL+ cells could be determined in
CD19+, CD3+, CD4+, and
CD8+ antigen-defined cell populations. In the
example shown in Fig 1 (patient No. 24), after 72 hours spontaneous
apoptosis was 6.5%, 16.2%, 16%, and 10.6% in CD19+,
CD3+, CD4+, and CD8+ cells,
respectively. In the same antigen-defined populations F-ara-A-induced
apoptosis was 11.9%, 62.2%, 64.6%, and 67.4%, which reflect the
relative resistance of F-ara-A-induced apoptosis in CD19+
cells but not in CD3+ T lymphocytes in these patients
(stage IV). An assay that detects apoptosis in the entire population
only would have missed the disproportionate apoptosis between B-CLL and
T lymphocytes. In fact, although CD3+/ISEL+
cells are only 1.73% (Fig 1B, number in parenthesis) of the entire population, they represent 62.2% of the CD3+ lymphocytes.
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| Fig 1.
Flow cytometric determination of apoptosis by ISEL in
antigen defined subpopulations (patient No. 24, Table 1). Peripheral lymphocytes were incubated in vitro in the presence or absence of
F-ara-A (3 µmol/L) or CyA (100 ng/mL). Apoptosis was evaluated after
72 hours in CD19+, CD3+,
CD4+, and CD8+ lymphocytes. The number in
the top right quadrant represents the percentage of apoptotic cells in
the antigen-defined population. The numbers in parentheses represent
the percentage of antigen-defined apoptotic cells within the total
number of peripheral lymphocytes.
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In CLL samples we observed a time-dependent increase in the percentage
of F-ara-A-induced apoptotic cells (Fig
2). Within the CD19+ cell
compartment 27.1 ± 16.5% of apoptotic cells were already detectable
after 24 h in group 1 patients vs. 5.6% ± 7.1% in group 2 (Fig 2A).
The number of CD19+ apoptotic cells in the two groups
constantly increased during the second day of incubation, reaching 58.3 ± 23.7% for group 1 versus 26.5 ± 23.3% for group 2 (P = .01) at 72 hours. Time-dependent induction of apoptosis
was also observed in the CD3+ compartment of the two
patient groups (Fig 2A). Interestingly, F-ara-A treatment induced less
apoptosis in T cells in group 1 (43.2 ± 15%) compared with 66.9 ± 23.4% in group 2 patients (P = .04). We then
investigated whether F-ara-A-induced apoptosis in CLL T cells had
differential effects on CD4+ and CD8+ T-cell
subpopulations. In five samples analyzed CD4+ and
CD8+ T-lymphocyte subcompartments were equally affected by
F-ara-A (a representative experiment is shown in Fig 1).
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| Fig 2.
Kinetics of F-ara-Ainduced (A) and spontaneous (B)
apoptosis in T-normal and B-CLL peripheral lymphocytes from 25 patients. Suspensions of peripheral lymphocytes were incubated alone or in the presence of 3 µmol/L F-ara-A for 72 hours. Patients were grouped according to Rai stages 0 to I (solid bars) and stages II to IV
(open bars). Apoptosis was measured by using ISEL in antigen-defined
lymphocyte subpopulations and expressed as percentages. Bars represent
the mean values in each group plus standard deviation. Statistical
significance was evaluated at 72 hours, using the Student
t-test.
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We also tested F-ara-A effects on normal B and T cells from four
healthy subjects. After 24 hours of incubation with F-ara-A, we
detected 52.5% ± 17.7 and 7.9% ± 4.2 apoptotic lymphocytes within
CD19+ and CD3+ cells, respectively (Fig
3). Data suggest that, under these in vitro
conditions, normal B cells from healthy subjects are more susceptible
to F-ara-A-induced apoptosis than B-CLL lymphocytes (52.5 ± 17.7
for healthy subjects v 27.1 ± 16.5% and 5.6% ± 7.1% for group 1 and group 2 CLL patients, respectively), whereas the susceptibility of normal T cells is comparable to group 1 CLL patients
but lower than group 2 (7.9% ± 4.2 for healthy subjects v
10.5 ± 10 and 24.8 ± 21.1 for group 1 and group 2 CLL patients, respectively).
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| Fig 3.
Spontaneous and F-ara-A-induced and apoptosis
(% ± SD) in CD19+ and CD3+ peripheral
lymphocytes from four healthy subjects after 24-hour incubation in
vitro. Solid bars, F-ara-A; open bars, control.
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Spontaneous apoptosis in B and T lymphocytes from healthy individuals
and CLL patients.
The extent of spontaneous apoptosis over a 72-hour period was measured
in lymphocyte subsets of peripheral blood cells from 25 patients with
CLL and 4 normal individuals. Figure 2B shows spontaneous apoptosis in
CD19+ and CD3+ lymphocytes after 24, 48, and 72 hours in group 1 and group 2 CLL patients. Few apoptotic cells were
detected after 24 hours in CD19+ and CD3+
compartments of group 1 and 2 patients. A slight increase in the
percentage of spontaneously apoptotic cells was observed in both B- and
T-cell compartments with time in the two groups of patients. After 72 hours, CLL lymphocytes showed 16.5 ± 13.5% spontaneous apoptosis in
the CD19+ compartment of group 1 patients, compared with
8.1 ± 3.9 in group 2 (P = .02). Group 1 and group 2 did not
differ significantly in spontaneous apoptosis of CD3+
lymphocytes (7.3 ± 4.2% v 12.0 ± 6.0%;
P = .09).
Normal B lymphocytes (Fig 3) showed a higher percentage of apoptotic
cells after 24 hours than did T lymphocytes (24.2% ± 14.3 v 2.9% ± 1.5), suggesting that under these in vitro
conditions B cells were more susceptible to spontaneous apoptosis than
T cells.
Effect of CyA on lymphocytes.
In 5 CLL patients (3 patients group 1 and 2 patients group 2),
lymphocytes were cultured in the presence of CyA (100 ng/mL). The
percentage of apoptotic cells was assessed in T and B-CLL lymphocytes
at 72 (Fig 4) and 120 hours (not shown). No
significative differences in the number of apoptotic cells were
observed in either the T- or B-lymphocyte compartments in the presence
of CyA as compared with controls. The same results were found in the
CD4+ and CD8+ T-cell compartments at 72 hours
(example shown in Fig 1).
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| Fig 4.
Comparison of spontaneous, CyA, and F-ara-A-induced
apoptosis (% ± SD) in CD19+ and CD3+
lymphocytes from 5 CLL patients after 72 hours incubation in vitro.
Solid bars, T cells; open bars, B cells.
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Bcl-2 measurements.
Twenty patients with CLL were tested for bcl-2 expression, and the
results are shown in Table 1. The
quantitation of bcl-2 antibody-binding sites was achieved by direct
immunofluorescence of Quantum Simply Cellular microbeads as described
in Material and Methods. Results were expressed as ABC (bcl-2
molecules/cell).25 In all 20 cases, leukemic cells showed
detectable levels of bcl-2. Mean bcl-2 antigen density in all tested
CLL cases was 43,150 ± 20,850 ABC, levels higher than those found in
normal CD3+ and CD19+ lymphocytes (29,000 and
37,000 ABC, respectively).26 Although higher levels of
bcl-2 were observed in group 2 (51,915 ± 26,627 ABC) than in
group 1 patients (39,394 ± 17,669 ABC), the difference was not
statistically significant (P = .3). Figure
5 shows bcl-2 ABC in CLL patients divided
by Rai stage. No further correlations were found between bcl-2 levels
and the extent of F-ara-A-induced (r2 = .002;
P = ns), spontaneous apoptosis (r2 = .03;
P = ns), and B2 microglobulin levels
(r2 = .28; P = ns).
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Table 1.
Percentage of Spontaneous and F-ara-A-Induced Apoptosis
in Antigen-Defined Peripheral Lymphocytes in CLL Patients and Bcl-2 Protein Expression
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| Fig 5.
Bcl-2 levels in CD5+/CD19+
lymphocytes of 20 CLL patients according to Rai stage. Quantitation of
bcl-2 was expressed as ABC (bcl-2 molecules/cell) as described in
Materials and Methods.
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Apoptosis and cell cycle.
Peripheral lymphocytes from two CLL patients were simultaneously
analyzed for apoptosis, surface markers, and DNA content. Figure
6 shows correlated DNA histogram and ISEL measurements in CD19+ cells. Cells with a low degree of DNA
fragmentation (region 1) had G0/1 DNA content, whereas
increased intensity for ISEL was accompanied by a shift into the
hypodiploid area (region 2). The few cells with S/G2M DNA
content were predominantly ISEL+.
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| Fig 6.
Simultaneous determination of apoptosis (ISEL) and DNA
content (Hoechst 33324) in B-CLL CD19+ lymphocytes after
24-hour treatment in vitro with 3 µmol/L fludarabine (patient No. 5, group 1). B-CLL cells are mainly in the G0/1 cell cycle phases when
they become positive for ISEL. Among the
CD19+/ISEL+ lymphocytes, cells with diploid
DNA content (early apoptotic cells, region [reg] 1) can be
distinguished from cells with subdiploid DNA content (late apoptotic
cells, reg 2).
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| |
DISCUSSION |
The primary aim of this study was to determine the induction of
apoptosis in different subpopulations during F-ara-A treatment. We
observed that induction of apoptosis appeared at the same time in both
populations of B and T lymphocytes, being more likely a direct effect
of F-ara-A than a consequence of interactions between these two cell
populations. When a high rate of spontaneous apoptosis was observed, it
involved predominantly B-CLL cells rather than T lymphocytes. In
several patients, an effective induction of apoptosis by F-ara-A of the
T lymphocytes did not affect B-CLL lymphocytes. Finally, incubation of
T lymphocytes in the presence of CyA did not enhance the rate of
spontaneous apoptosis of B-CLL cells.
Taken together, our data strongly suggest a direct effect of F-ara-A on
B-CLL lymphocytes. Although lymphocyte subset analysis in vivo revealed
that T lymphocytes are more sensitive to the cytotoxic effect of
F-ara-A than B lymphocytes,6 T lymphocytes from four normal
subjects proved to be more resistant than B lymphocytes in vitro in
both F-ara-A-induced and spontaneous apoptosis. In vivo data indicate
that F-ara-A-related lymphophenia is related to the decreased number
of CD4+ cells.27 Our in vitro results showed no
difference, however, in susceptibility to F-ara-A treatment and
spontaneous apoptosis between CD4+ and CD8+
cells, suggesting that prolonged in vivo CD4 lymphophenia is related to
slow recovery rather than to selective F-ara-A cytotoxicity. In our
study, although spontaneous apoptosis was significantly higher in B
lymphocytes obtained from patients with low-risk disease, pronounced
interpatient variation was evident. As mentioned in the results, no
significant differences were observed for spontaneous apoptosis of T
lymphocytes in the two groups of patients.
The role of CyA in CLL is still controversial. Originally, CyA was used
to treat autoimmune disorders associated with B-CLL,28 and
occasional antileukemic effects were described
thereafter.12,29 CyA was also used successfully in two
patients with angioimmunoblastic lymphadenopathy with dysproteinemia
(AILD), a disease in which abnormal T cells induce B-cell
hyperreactivity.30 Whether CyA acts directly on malignant B
cells14,31 or indirectly through different pathways is not
clear. In our in vitro model, we did not observe any direct or indirect
induction of apoptosis by CyA on CD19+, CD3+,
CD4+, and CD8+ lymphocyte compartments over a
120-hour period. A recent clinical report concerning five CLL patients
treated with CyA showed no encouraging results.13
Despite the low incidence of bcl-2 rearrangements in CLL, estimated at
4%,32 bcl-2 protein levels in B-CLL cells are equivalent to or higher than those found in cell lines containing t(14:18). Hypomethylation in the 5 end of the bcl-2 gene has been associated with transcriptional activation of this proto-oncogene. Hanada et
al31 showed in three cases that CLL cells with increased levels of bcl-2 protein survived longer in culture and underwent delayed spontaneous apoptosis. Bcl-2 overexpression was also shown to
increase relative resistance to  -irradiation and
chemotherapy33,34 and to prolong in vitro survival of
normal35 and acute lymphoblastic leukemia B
lymphocytes.36 In our study, however, we found no correlation when bcl-2 levels were related to the percentage of F-ara-A-induced or spontaneous apoptosis, which suggested that F-ara-A-mediated cytotoxicity may be bcl-2 independent. Similar data
recently presented by Kitada et al37 and Robertson et
al38 suggested that other factors such as p53, CD40L, or
Mcl-1 could be key regulators of apoptotic or antiapoptotic pathways
for B-CLL lymphocytes.39-41
DNA strand breaks are characteristic of apoptosis and may be exploited
in the ISEL assay, which seems to be more effective than conventional
assays in being more sensitive.42 Unlike extracted DNA
analysis, ISEL is capable of identifying individual cells, thus
allowing apoptotic cells to be quantified. The combination of this
technique with simultaneous multicolor staining of membrane antigens
and DNA content provides the opportunity to study induction of
apoptosis in subpopulations of cells in heterogeneous samples and to
establish correlations with other biological
parameters.43,44 The technique may be useful in selection
of cytotoxic drugs that target a specific population. As in the
representative patient shown in Fig 1, we observed that in 6 of 7 patients with stage III-IV CLL (patient Nos. 19, 20, 21, 22, 23, 24;
Table 1), F-ara-A was relatively ineffective on CD19+
lymphocytes but showed higher cytotoxicity in CD3+ cells.
The overall use of F-ara-A in those refractory patients may, therefore,
not be beneficial.
The mechanism of action of F-ara-A in proliferating cells is mainly
cell cycle-specific, and incorporation of F-ara-A into DNA during S
phase is required for the induction of apoptosis in a T lymphoblastoid
cell line.45 The clinical efficacy of F-ara-A is higher,
however, in indolent lymphoid malignancies than in intermediate- and
high-grade lymphoid neoplasms. In direct measurements of apoptosis and
cell cycle, as already shown in normal lymphocytes,46 we
showed an S phase-independent F-ara-A induction of apoptosis in B-CLL
lymphocytes.
F-ara-A had a direct effect on malignant and normal B and T
lymphocytes. Its cytotoxicity was lower at Rai stages II to IV than at
stages 0 to I, and normal T cells of CLL patients at stage II, III, and
IV were more sensitive to F-ara-A than at lower stages. The
F-ara-A-induced apoptosis effect was not S phase-dependent and was
observed in G0/1 cells.
| |
FOOTNOTES |
Submitted September 2, 1997;
accepted October 24, 1997.
Supported in part by National Institutes of Health Grant Nos. CA 55164 and CA 16672. U.C. is supported in part by a scholarship from the
Fondazione Catanese per lo Studio e la Cura delle Malattie Neoplastiche
del Sangue, Catania, Italy.
Address reprint requests to Ugo Consoli, MD, Institute of Hematology,
University of Catania. Ospedale Ferrarotto Alessi, via Citelli 6, 95124 Catania, Italy.
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.
| |
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Abstract
Introduction
Abstract