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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2836-2843
In Vitro Evaluation of Fludarabine in Combination With Cyclophosphamide
and/or Mitoxantrone in B-Cell Chronic Lymphocytic Leukemia
By
Beatriz Bellosillo,
Neus Villamor,
Dolors Colomer,
Gabriel Pons,
Emili Montserrat, and
Joan Gil
From the Departament de Ciències Fisiològiques II,
Universitat de Barcelona, Campus de Bellvitge, L'Hospitalet, Spain;
and the Unitat d'Hematopatologia, Servei d'Hematologia, Institut
d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS),
Hospital Clínic, Barcelona, Spain.
 |
ABSTRACT |
B-chronic lymphocytic leukemia (B-CLL) is characterized by the
accumulation of long-lived CD5+ B lymphocytes. We have
analyzed the effect in vitro of the combination of fludarabine with
cyclophosphamide and/or mitoxantrone on cells from 20 B-CLL patients.
Mafosfamide, the active form of cyclophosphamide in vitro, increased
the cytotoxicity of fludarabine in all of the patients studied and
produced a significant synergistic effect (P < .01) after 48 hours of incubation. The addition of mitoxantrone to this combination
increased the cytotoxic effect in cells from 8 patients, but in the
remaining 12 patients no significant increase was observed. The effect
of fludarabine and mafosfamide was dose-dependent. Mafosfamide and
fludarabine had a synergistic effect in inducing apoptosis of B-CLL
cells as determined by DNA staining with propidium iodide and analysis
of phosphatidylserine exposure. Mafosfamide significantly increased the
apoptosis induced by fludarabine on CD19+ cells
(P = .007), but not on CD3+ cells (P
= .314). Cell viability was correlated with a decrease in Mcl-1
levels and an increase in p53 levels. These results support that
fludarabine in combination with cyclophosphamide and/or mitoxantrone can be highly effective in the treatment of B-CLL.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
B-CELL CHRONIC lymphocytic leukemia
(B-CLL) is characterized by the accumulation of long-lived,
functionally inactive, mature appearing neoplastic B
lymphocytes.1 Most circulating cells appear to be arrested
at the G0 phase of the cell cycle and the clonal excess of
B cells is mainly caused by defects that prevent programmed cell death
rather than by alterations in cell cycle regulation.2
Patients with B-CLL in early clinical stages (Binet A, Rai 0) and
stable disease should not be treated unless the disease progresses. In
contrast, most patients with poor prognostic features, such as an
advanced clinical stage, diffuse bone marrow infiltration, or rapidly
increasing blood lymphocyte levels, require therapy. For many years,
chlorambucil, either alone or in combination with prednisone, has been
the treatment of choice for B-CLL.3 In the last few years,
purine analogs, particularly fludarabine, have changed the treatment
possibilities of B-CLL. Fludarabine has demonstrated high efficacy in
the treatment of this form of leukemia.4-8 However,
although fludarabine produces the highest response rate ever reported
for a single agent in B-CLL, responses are not sustained and all
patients eventually relapse. In addition, fludarabine does not prolong
survival of individuals with B-CLL in comparison with chlorambucil and
CAP (cyclophosphamide, doxorubicin, and prednisone).9,10
For all these reasons, there is increasing interest in assessing
whether the results obtained with fludarabine alone could be improved
by combining it with other drugs.11,12 Recently, pilot
studies have analyzed the use of fludarabine in combination with
prednisone, doxorubicin, cyclophosphamide, epirubicin, chlorambucil,
and mitoxantrone in the treatment of B-CLL.6,11,13-19 Among
these regimens, those who have given the most promising results are the
combination of fludarabine and cyclophosphamide11,14-16 and
these 2 with mitoxantrone.19
The aim of this study was to analyze the chemosensitivity of B-CLL
cells in vitro to the combination of fludarabine, mafosfamide (the
active form of cyclophosphamide in vitro), and mitoxantrone, alone and
in combination.
| |
MATERIALS AND METHODS |
Patients.
Twenty patients (9 men and 11 women) with B-CLL who had not received
treatment for the previous 6 months, with a median age of 72 years
(range, 42 to 86 years), were studied. B-CLL was diagnosed according to
standard clinical and laboratory criteria. The median peripheral blood
leukocytosis was 102 × 109 leukocytes/L (range, 19 to
510 × 109/L). Leukemic cells phenotyped for cell
surface markers by flow cytometry were positive in all cases for CD5
and CD19. According to Binet's classification,20 at the
time of inclusion, 12 patients were at stage A, 3 patients were
at stage B, and 5 patients were at stage C
(Table 1).
Isolation of B-CLL cells.
Mononuclear cells were isolated from peripheral blood samples by
centrifugation on a Ficoll/Hypaque (Seromed, Berlin, Germany) gradient and cryopreserved in liquid nitrogen in the presence of 10%
dimethyl sulfoxide (DMSO).
Reagents.
Fludarabine monophosphate was obtained from Schering AG (Berlin,
Germany). Mafosfamide was obtained from ASTAMedica AG (Frankfurt, Germany). Mitoxantrone was obtained from Lederle Laboratories (Gosport,
Hampshire, UK). 3,(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) and propidium iodide (PI) were obtained from Sigma Chemicals Co (St Louis, MO).
Cell culture.
B-CLL lymphocytes were cultured immediately after thawing at a
concentration of 2 to 5 × 106 cells/mL in RPMI 1640 culture medium (GIBCO BRL, Paisley, Scotland) supplemented with 10%
heat-inactivated fetal calf serum (Bio Whittaker, Verviers, Belgium), 2 mmol/L glutamine, and 0.04 mg/mL gentamicin at 37°C in a humidified
atmosphere containing 5% carbon dioxide.21 Factors were
added at the beginning of the culture.
Cell viability assay.
Cell viability was determined by the MTT assay.22 B-CLL
lymphocytes (5 × 105 cells/well) were incubated in
96-well plates in the absence or in the presence of factors in a final
volume of 100 µL. After 48 hours, 10 µL of MTT (5 mg/mL in
phosphate-buffered saline [PBS]) was added to each well for a further
6 hours. The blue MTT formazan precipitated was dissolved in 100 µL
of isopropanol:1 mol/L HCl (24:1) and the absorbance values at 550 nm
were determined on a multiwell plate reader.
PI DNA staining.
Quantification of apoptosis by PI staining and fluorescence-activated
cell sorting (FACS) analysis was performed as described previously.23 Briefly, cells were harvested and fixed in
70% ethanol. Cells were centrifuged, washed in PBS, and resuspended in
0.5 mL PBS containing PI (5 µg/mL) and RNase (100 µg/mL). Tubes were incubated for 30 minutes at 37°C and placed at 4°C in the dark overnight before flow cytometry analysis to identify the sub-G0 peak corresponding to apoptosis.
Analysis of apoptosis by annexin binding.
Exposure of phosphatidylserine was quantified by surface annexin V
staining as described previously.24 One million cells were
incubated for 24 hours with the indicated factors. Cells were then
washed in PBS and incubated with phycoerythrin (PE)-conjugated anti-CD19 (DAKO, Glostrup, Denmark) or PE-conjugated anti-CD3 (Caltag
Laboratories, Burlingame, CA) for 15 minutes in the dark. Cells were
then washed, resuspended in 200 µL of binding buffer (10 mmol/L
HEPES, pH 7.4, 2.5 mmol/L CaCl2, 140 mmol/L NaCl), and
incubated with 0.5 µg/mL of Annexin V-fluorescein isothiocyanate (FITC; Bender MedSystems, Vienna, Austria) for 5 to 15 minutes in the
dark. Cells were washed again and resuspended in binding buffer. PI (5 µg/mL) was added to each sample before flow cytometric analysis
(FACScan; Becton Dickinson, Mountain View, CA). Samples were acquired
using Lysis-II software and data were analyzed with the Paint-a-gate
Pro software (Becton Dickinson). To analyze a sufficient number of
cells, a live-gate in side scatter (SSC) versus CD19 or SSC versus CD3
was drawn and at least 5,000 CD19+ cells or
CD3+ cells were acquired.
Western blot.
Cells were lysed in 80 mmol/L Tris HCl, pH 6.8, 2% sodium dodecyl
sulfate (SDS), 10% glycerol, and 0.1 mol/L dithiothreitol (DTT), and equal amounts of protein were separated by
electrophoresis on 12% polyacrylamide gel and transferred to
Immobilon-P (Millipore, Bedford, MA) membranes. The membranes were
incubated with monoclonal antibodies against p53 (Oncogene Science Inc,
Uniondale, NY), Bcl-2 (DAKO), or polyclonal antibodies against Mcl-1
(Santa Cruz Biotechnology Inc, Santa Cruz, CA) or Bax (Pharmingen, San
Diego, CA). Antibody binding was detected using a secondary antibody (swine antimouse or mouse antirabbit Ig; DAKO) conjugated to
horseradish peroxidase and an enhanced chemiluminescence (ECL)
detection kit (Amersham, Buckinghamshire, UK).
Statistical analysis.
The interactions and synergisms between drugs were analyzed by a
Multilevel regression model.25 Levels of significance
between samples were determined using the t-test for nonpaired
samples and the analysis of variance (ANOVA), Fisher's Protected Least Significant Difference (PLSD). Synergy between drugs for individual patients was determined by comparing the viability obtained when B-CLL
cells were incubated with the combination of the drugs and the
viability expected that was calculated by multiplying the viability of
the cells when incubated with each factor alone. Correlations between
Mcl-1 levels, p53 levels, and cell viability were determined by the
Pearson method.
| |
RESULTS |
Cytotoxic effect of the combination of fludarabine with mitoxantrone
and mafosfamide.
B-CLL cells from 20 patients were incubated for 48 hours with
pharmacological concentrations of fludarabine (F; 1 µg/mL), mitoxantrone (M; 0.5 µg/mL), and mafosfamide (Mf; 1 µg/mL)
alone or in combination (Table 2).
The statistical analysis of the values obtained for these 20 patients
shows that the viability of control cells was 82% ± 16% (95%
confidence interval [CI], 75 to 89). Incubation of B-CLL cells with
fludarabine, mafosfamide, or mitoxantrone alone produced a significant
decrease in cell viability (P < .01). The strongest effect
was produced by fludarabine, which reduced viability by 25 units (U)
(95% CI, 22 to 28.9), followed by mitoxantrone (20 U; 95% CI, 16.7 to
23.7) and mafosfamide (9 U; 95% CI, 5.6 to 12.5). All of the
combinations of 2 drugs were more cytotoxic than any drug alone. The
combination of mitoxantrone with either fludarabine or mafosfamide
produced a significant additive effect (P < .01) and the mean
value viability was 36% ± 17% and 53% ± 21%, respectively.
Interestingly, the combination of mafosfamide with fludarabine produced
a significant synergistic effect (P < .01) and induced an
additional loss of 17 U (95% CI, 12.47 to 22.33) to the expected
viability that would have been obtained if the combination of these 2 drugs had produced only an additive effect.
Although the comparison of fludarabine + mitoxantrone versus
fludarabine + mafosfamide showed no statistical significance (P = .172), in cells from 8 patients (patients no. 2, 3, 4, 7, 11, 16, 17, and 18) a higher effect was observed when the cells were incubated with
the combination of fludarabine + mafosfamide (P < .05 in all cases).
When considering the mean values of the 20 patients studied, the
addition of mitoxantrone to the combination of fludarabine and
mafosfamide did not induce a significant increase in the cytotoxic effect obtained by these 2 drugs (P = .08). However, the
analysis of individual patients showed a significant increase in
cytotoxicity in cells from 8 patients (patients no. 1, 3, 6, 7, 8, 9, 10, and 17) when the cells were incubated with the 3 drugs
(P < .05 in all cases). We analyzed whether there
was a correlation between this greater sensitivity and any biological
or clinical characteristics of the patients (Table 1). No correlation
was found with clinical stage, peripheral blood leukocyte count,
trisomy of chromosome 12, or immunophenotype. Interestingly, a
significant correlation was found with previous treatment. Thus,
whereas in 5 of 6 (83.3%) previously treated patients (3 of whom had
received purine analogues) the combination of fludarabine, mafosfamide,
and mitoxantrone was necessary to achieve the highest cytotoxic effect.
This was the case in only 3 of 14 patients (21.4%) with no prior
therapy (P = .036;  2 with continuity correction).
Correlation with in vivo response.
Five of the 20 patients included in this study received treatment with
the combination of fludarabine, cyclophosphamide, and mitoxantrone
(FCM). Two patients achieved partial response (patients no. 8 and 9)
and 2 patients achieved complete response (patients no. 7 and 14). The
remaining patient (patient no. 17) could not be evaluated, because the
treatment had to be discontinued due to toxicity. However, a decrease
in the peripheral blood leukocyte count was observed (from 40 to 6.4 × 109/L).
An additional patient who was eventually excluded from the analysis
because she was receiving chlorambucil at the time of the study did not
respond to FCM. Interestingly, the cells from this patient had not
responded in vitro to any of the combinations assayed.
Although the number of patients is too small to make statistical
studies, it appears that there is a tendency for better clinical responses in patients who showed higher rates of apoptosis in vitro
when treated with the combination of all 3 drugs.
Dose-dependence of the cytotoxic effect of fludarabine and
mafosfamide.
To study the synergism between fludarabine and mafosfamide in more
detail, B-CLL cells from 6 patients were incubated for 48 hours with
various concentrations of fludarabine, ranging from 0.25 to 5 µg/mL,
in the presence or absence of 1 µg/mL mafosfamide. As seen in
Fig 1, fludarabine produced a
dose-dependent cytotoxic effect in all of the cases studied, although
the sensitivity differed from one patient to another. The addition of
mafosfamide increased fludarabine-induced cytotoxicity and, in patients
no. 1, 11, 12, and 13, the combination produced a significant
(P < .05) synergistic effect. In all patients studied, the
effect of the combination of 0.25 µg/mL fludarabine and 1 µg/mL
mafosfamide was greater than the effect of 1 µg/mL fludarabine alone.
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| Fig 1.
Potentiation of the cytotoxic effect of fludarabine by
mafosfamide. Cells from 6 patients were incubated for 48 hours with
various concentrations of fludarabine ranging from 0.25 to 5 µg/mL,
without ( ) or with 1 µg/mL mafosfamide ( ). Cell viability was
determined by the MTT assay as described in Materials and Methods and
is expressed as the percentage with respect to control cells at the
beginning of the culture. Data are shown as the mean value ± SD of
triplicate cultures. Statistical significance of the synergism between
fludarabine and mafosfamide was assayed by ANOVA (Fisher's PLSD).
*P < .05.
|
|
Dose-dependence was also found when B-CLL cells were incubated with
increasing doses of mafosfamide (0.25 to 2.5 µg/mL) in the presence
of a constant dose of fludarabine (1 µg/mL;
Fig 2). These results indicate that 0.25 µg/mL mafosfamide is sufficient to increase the cytotoxic effect of
fludarabine in most patients. Furthermore, synergy was produced by the
addition of the various concentrations of mafosfamide to the cells
incubated with fludarabine in 4 of the 6 patients studied (patients no.
1, 11, 12, and 13).
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| Fig 2.
Potentiation of the cytotoxic effect of mafosfamide by
fludarabine. B-CLL lymphocytes from 6 patients were incubated for 48 hours with various concentrations of mafosfamide ranging from 0.25 to
2.5 µg/mL, without () or with 1 µg/mL fludarabine (). Cell
viability was determined by the MTT assay as described in Materials and
Methods and is expressed as the percentage with respect to control
cells at the beginning of the culture. Data are shown as the mean value ± SD of triplicate cultures. Statistical significance of the
synergism between fludarabine and mafosfamide was assayed by ANOVA
(Fisher's PLSD). *P < .05.
|
|
Induction of apoptosis by fludarabine, mafosfamide, and mitoxantrone
in B-CLL cells.
To analyze whether the cytotoxic effect produced by the different
combinations of these 3 drugs was due to potentiation of apoptosis, we
studied the effect of these combinations on the percentage of apoptotic
cells by flow cytometry. Figure 3 shows the
histograms obtained by FACS analysis of the hypodiploid peak obtained
by PI staining of cells from patient no. 17 incubated with fludarabine,
mitoxantrone, and mafosfamide alone and in combination. When the cells
were incubated with just 1 drug, the greatest effect was produced by
fludarabine. The use of 2 drugs produced a higher number of apoptotic
cells, especially with the combinations of fludarabine + mafosfamide
and fludarabine + mitoxantrone. Finally, the combination of all 3 drugs
produced the highest number of apoptotic cells. Similar results were
obtained with cells from 5 additional patients (results not shown).
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| Fig 3.
Induction of apoptosis by fludarabine,
mafosfamide, and mitoxantrone on B-CLL cells. Cells from
patient no. 17 were incubated for 48 hours with fludarabine (F; 1 µg/mL), mitoxantrone (M; 0.5 µg/mL), and/or mafosfamide (Mf; 1 µg/mL). DNA content was quantified by PI staining and flow cytometry
analysis as described in Materials and Methods.
|
|
The apoptotic effect induced by the combination of fludarabine + mafosfamide was analyzed in cells from 13 patients by the same
method. Fludarabine and mafosfamide alone increased the number of
apoptotic cells (control, 34% ± 18%; fludarabine, 53% ± 22%; mafosfamide, 49% ± 23%), and the combination of the 2 drugs
significantly increased the number of apoptotic cells (77% ± 21%)
when compared with fludarabine (P = .009) or mafosfamide
(P = .003) alone.
Furthermore, apoptosis was also quantified by flow cytometry analysis
of phosphatidylserine exposure. Annexin V-FITC binding was studied in
CD19+ and CD3+ cells from 11 B-CLL patients
after 24 hours of incubation. At this time, incubation of B-CLL
lymphocytes with fludarabine induced apoptosis of CD3+
(P = .017) cells, but not of CD19+ cells (P = .67; Fig 4). No significant effect was
produced by incubation of cells with mafosfamide alone on either type
of cell. Interestingly, mafosfamide significantly increased the
apoptosis induced by fludarabine on CD19+ cells (P = .007), but not on CD3+ cells (P = .314).
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| Fig 4.
Comparison between the induction of apoptosis in B cells
and T cells from 11 B-CLL patients. Cells were incubated with
fludarabine (F) and/or mafosfamide (Mf) for 24 hours and
phosphatidylserine exposure was measured by binding of annexin V-FITC
to CD19+ or CD3+ cells as described in
Materials and Methods. Statistical significance was determined using
the t-test for nonpaired samples: *versus control; §versus
fludarabine.
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Effect on the expression of apoptosis-regulatory proteins.
Using antibodies specific for Bcl-2, Bax, Bcl-X, and Mcl-1, we studied
whether the combinations of fludarabine with mafosfamide and/or
mitoxantrone produced changes in the levels of these
apoptosis-regulatory proteins. No modification of Bcl-2 nor Bax levels
was observed. Bcl-X was not detected in control cells, and its presence
was not induced by any of the drug combinations (data not shown). Analysis of Mcl-1 in 10 patients showed a decrease in the levels of
this protein when cells were incubated with the 3 drugs in combination
(data not shown). The effect of the drugs alone and of all the
combinations was studied in cells from 3 patients
(Fig 5). Fludarabine alone produced a
decrease in patients no. 2 and 3, but mitoxantrone or mafosfamide did
not affect Mcl-1. Lower levels of protein were detected after
incubation with fludarabine + mitoxantrone or fludarabine + mafosfamide, whereas incubation with mitoxantrone + mafosfamide only
produced a decrease in cells from patient no. 3. Finally, the
combination with the 3 drugs produced the lowest levels of Mcl-1. The
timing of the decrease in Mcl-1 protein was parallel to the decrease in
cell viability (data not shown). A correlation was found between Mcl-1
levels and cell viability in response to the different treatments for 48 hours (r = .8; P < .001).
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| Fig 5.
Effect of the combination of fludarabine with
mitoxantrone and/or mafosfamide on p53 and Mcl-1 levels. Cells from
patients no. 2, 3, and 17 were incubated for 48 hours with fludarabine
(F; 1 µg/mL), mitoxantrone (M; 0.5 µg/mL), and/or mafosfamide (Mf;
1 µg/mL). Cell viability was determined by the MTT assay as described
in Materials and Methods and is expressed as the percentage with
respect to control cells. Western blots of p53 and Mcl-1 were
quantified and the values are expressed as the percentage with respect
to control cells (Mcl-1) or cells incubated with the 3 drugs (p53).
Similar p53 levels were obtained for the 3 patients.
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|
DNA-damaging agents increase p53 levels26,27 and B-CLL
cells with p53 gene mutations are more resistant to chlorambucil and
fludarabine.28,29 Therefore, we analyzed the effect of the
combinations of these drugs on p53 protein. First, p53 levels were
determined in cells from 10 patients. In all cases, the levels of p53
in untreated cells were very low or undetectable, and the incubation of
the cells with the combination of the 3 drugs induced the accumulation
of p53 (data not shown). The effect of the drugs alone and combined was
studied in cells from 3 patients. As shown in Fig 5, after 48 hours of
incubation with fludarabine, mitoxantrone, or mafosfamide alone, an
increase in p53 levels was observed, except for 1 patient for whom no
modification was detected in response to mafosfamide. A higher increase
was observed when cells were incubated with fludarabine + mafosfamide
or fludarabine + mitoxantrone, and the combination of the 3 drugs
produced the highest p53 levels of all conditions. This increase in p53
levels correlated with the decrease in cell viability in the patients analyzed (r =  .82; P < .001) and
with the decrease in Mcl-1 (r = .65; P = .001).
Furthermore, the increase in p53 protein was detectable after 12 hours
of incubation, preceding the loss of viability (data not shown).
| |
DISCUSSION |
The results of this study demonstrate the synergism of the combination
of fludarabine and cyclophosphamide on neoplastic cells from B-CLL and
that the cytotoxic effect of this combination can be increased in some
cases by mitoxantrone. Several studies have found a correlation between
the response to different treatments in vitro and the effect of therapy
on the patient's clinical response.29-32 In this regard,
our results are consistent with the promising clinical results obtained
with the combination of fludarabine and
cyclophosphamide11,14-16 and the combination of fludarabine with cyclophosphamide and mitoxantrone.19
The basis for the treatment of cancer with combinations of drugs is
their additive or synergistic effects. A number of studies have
analyzed the effect of the combination of fludarabine with other drugs
in vitro on B-CLL cells.33-36 However, neither the effect
of the combination of fludarabine with cyclophosphamide nor the effect
of these 2 combined with mitoxantrone has been reported.
The dose-response assays show that doses of fludarabine less than 1 µg/mL (0.25 to 0.5 µg/mL) in combination with 1 µg/mL mafosfamide
produce a higher effect than that obtained with 1 µg/mL fludarabine
alone. Furthermore, our results also show that lower doses of
mafosfamide (0.25 to 0.5 µg/mL) when combined with fludarabine could
produce a similar effect to that obtained with 1 µg/mL mafosfamide.
These results support the use of these drugs together.
The mechanism of the synergism between fludarabine and mafosfamide in
B-CLL cells is not known. Cyclophosphamide is an alkylating agent that
induces DNA damage and fludarabine inhibits DNA and RNA synthesis as
well as DNA repair.13,37 The effectiveness of these drugs
in B-CLL cells may be attributed to the apparent requirement for
continual DNA housekeeping, even in nonproliferating cells.38 Inhibition of DNA interstrand cross-link removal
by fludarabine may account for the synergistic cytotoxicity of this combination.39
DNA-damaging agents increase p53 levels by posttranslational
stabilization and induce p53-dependent cell death.26,27 The importance of the p53 pathway in B-CLL was demonstrated by the finding
of p53 mutations.40 Importantly, B-CLL cells from patients with p53 gene mutations were more resistant to chlorambucil or fludarabine,28,29 and p53 mutations or deletions are
associated with drug resistance and short survival.28,41,42
We found a high correlation between the cytotoxicity of the different
combinations of drugs and the increase in p53 protein. Furthermore,
this increase precedes loss of viability. These results suggest that 1 of the mechanisms by which the combination of these drugs increases its cytotoxicity is the potentiation of DNA damage and stabilization and
activation of p53. One of the mechanisms involved in p53 stabilization in response to DNA damage is its phosphorylation by ataxia
telangiectasia mutated (ATM).43 Very interestingly, ATM has
been found inactivated in B-CLL cells.44,45
The levels of Mcl-1 protein were decreased by all the combinations that
induced apoptosis and a high correlation between levels of Mcl-1 and
viability was found. However, the levels of Bcl-2 and Bax were not
modified. These results are consistent with those reported by Kitada et
al.46 Our results indicate that higher cytotoxic effects
correlate with concurrent low levels of Mcl-1 and high levels of p53.
The timing of the decrease in Mcl-1 protein is parallel to the decrease
in viability, and so it is very difficult to demonstrate a causal
effect, but the decrease in Mcl-1 protein may contribute to the
potentiation of apoptosis.
The results obtained by PI staining of cells with a subdiploid content
of DNA and by analysis of phosphatidylserine exposure show that the
synergistic effect of fludarabine and mafosfamide is due to
potentiation of apoptosis. Consistent with Consoli et al,47
fludarabine was cytotoxic to CD3+ cells of B-CLL patients.
However, the addition of mafosfamide to this drug produces a higher
increase in the apoptosis of B lymphocytes than of T lymphocytes.
In conclusion, this in vitro study shows that cyclophosphamide
synergizes with fludarabine in inducing cytotoxicity and apoptosis in
B-CLL cells. In addition, the combination of mitoxantrone with these 2 drugs significantly increases the cytotoxic effect in some cases,
especially in previously treated patients. These results provide
experimental support to clinical trials assessing the effect of
fludarabine combined with cyclophosphamide11,14-16 and these 2 with mitoxantrone.19
| |
ACKNOWLEDGMENT |
The authors thank L. Quintó from the "Unidad de
Epidemiología y Bioestadística" for his help in
statistical analysis. We thank Dr F. Bosch, Dr J. Esteve, Dr A. Carrió, M. Perales, O. Casanovas, and C. Pastor for their help
and suggestions and thank R. Rycroft for language assistance.
| |
FOOTNOTES |
Submitted October 16, 1998; accepted May 30, 1999.
B.B. is a recipient of a research fellowship from "Fundación
de la Asociación Española Contra el Cáncer."
Supported by "Fondo de Investigaciones Sanitarias de la Seguridad
Social" (FIS 95/0873, FIS 94/0665), "Comisión
Interministerial de Ciencia y Tecnologia" (SAF 98-0100),
"Generalitat de Catalunya" (97 SGR/74), and Schering AG (Berlin, Germany).
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.
Address reprint requests to Joan Gil, PhD, Departament de
Ciències Fisiològiques II, Campus de Bellvitge, Universitat
de Barcelona, 08907 L'Hospitalet, Spain; e-mail:
joangil{at}bellvitge.bvg.ub.es.
| |
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ABSTRACT
INTRODUCTION