The leukemogenic property of BCR-ABL in chronic myeloid leukemia (CML) is critically dependent on its protein tyrosine kinase activity. STI571 inhibits the BCR-ABL kinase activity, the growth and the viability of BCR-ABL expressing cells. In this study, we report the apoptotic effect of STI571 in combination with daunorubicin (DNR) on peripheral blood mononuclear cells from 11 CML patients and four BCR-ABL-positive cell lines: AR230, LAMA84, K562 and KCL22. Primary blast cells were identified by flow cytometry on the basis of their low CD45 expression. Nucleus fragmentation, exposure of phosphatidylserines and decrease in mitochondrial membrane potential were measured using acridine orange, FITC-annexin V and DiOC6(3), respectively, to evaluate apoptosis. On cell lines, the effect of DNR was negligible, whereas STI571 induced 10 to 35% of apoptosis in 18 h. STI571 sensitized AR230, LAMA84 and K562 cells to DNR when apoptosis was measured at the mitochondrial and membrane but not the nuclear levels. On CML blast cells, phosphatidyl serine exposure was significantly induced by both DNR and STI571 and was higher when these drugs were used in combination (P < 0.0003). However, the effects of this drug combination were only additive and no sensitization of blast cells to DNR by STI571 was observed. Interestingly, sensitization was evidenced in CML but not normal lymphocytes. These results suggest that other mechanisms additional to Bcr-Abl tyrosine kinase activity could be responsible for DNR resistance, and further investigations are needed to understand its origin.
Chronic myeloid leukemia (CML) is a malignant disease of pluripotent hematopoietic stem cells and is characterized by excessive accumulation of the myeloid lineage. It involves myeloid, monocytic, erythroid, megakaryocytic, B lymphoid, and occasionally T lymphoid lineages.1 The hallmark of CML is the Philadelphia (Ph) chromosome, derived from a t(9;22) translocation which is detected in virtually all cases of the disease. The molecular consequence of this translocation is the formation of a hybrid BCR-ABL gene, encoding a fusion protein with enhanced tyrosine kinase activity.2,3,4
The Bcr-Abl protein can transform hematopoietic cells so that their growth and survival in vitro become independent of cytokines.5,6 It can also protect hematopoietic cells from programmed cell death (apoptosis) in response to cytokine withdrawal, and to DNA damage induced by chemotherapy or irradiation.7,8 Since the main transforming property of the Bcr-Abl protein is exerted through its constitutive tyrosine kinase activity, direct inhibition of such activity seems to be the best therapeutic target.
The structure of several protein kinases and their inhibitors has been determined with the aid of crystallography. It is now possible to rationally design compounds aimed at blocking the ATP binding site or the active site of the enzyme with high specificity. One of these compounds, STI571 (formerly CGP57148, Novartis Pharmaceuticals), a 2-phenylaminopyrimidine derivative, is a potent inhibitor of the Abl protein tyrosine kinase.9 STI571 specifically inhibits CML10 and BCR-ABL-positive acute lymphoblastic leukemia11 progenitor cell proliferation with a maximal differential effect at 1 μM and induces apoptosis selectively in CML cell lines.12 In clinical trials, significant hematologic responses to STI571 have been observed in interferon-α refractory CML patients.13 However, in advanced stages of the disease and in acute leukemia, relapses have been a major problem.14
Bcr-Abl is described to inhibit apoptosis induced by antitumoral drugs.7 It was thus important to investigate whether tyrosine kinase inhibition by STI571 was able to restore sensitivity to anthracyclines in BCR-ABL expressing cells. In the present work we studied the ability of STI571, daunorubicin (DNR) and their combination to induce in vitro apoptosis of CML primary cells and cell lines.
Materials and methods
STI571 was kindly provided by Novartis Pharma (Basel, Switzerland). A 10 mM stock solution was prepared by dissolving the compound in sterile phosphate-buffered saline (PBS) or dimethylsulfoxide (DMSO). DNR was obtained from RPR-Bellon (Neuilly, France).
Cells and cell culture
Mononuclear cells (MNC) were obtained from EDTA-anticoagulated peripheral blood from 11 adults with CML by Ficoll centrifugation (Lymphoprep, Nycomed Pharma, Oslo, Norway). Five patients were in accelerated phase (AP), and seven in blast crisis (BC).
K562, KCL22, AR230, LAMA84 cells and peripheral blood MNC were grown in RPMI 1640 medium (Gibco-BRL, Eragny, France) supplemented with 10% fetal serum (FCS) and 1 mM L-glutamine, 10 mM Hepes (Gibco-BRL), 100 U/ml penicillin, and 50 μg/ml streptomycin in a humidified 95% O2 and 5% CO2 atmosphere at 37°C. All the experiments with cell lines were carried out using cells in exponential growth phase.
Apoptosis was induced on leukemic cell lines at 3 × 105/ml or on MNC at 106/ml by incubation for 18 h in culture medium without drug (control), with STI571 (1 μM), DNR (200 nM) or STI571 plus DNR in combination.
Analysis of apoptosis by flow cytometry
Blast cells and lymphocytes were identified by flow cytometry (FCM) on the basis of their CD45 expression and scatter properties.15,16 FITC-annexin V (Coulter-Immunotech, Roissy, France) was used as specified by the manufacturer to measure the exposure of phosphotidylserine on the external membrane. Cells (3 × 105) were centrifuged and resuspended in 100 μl of culture medium containing 5 μl of PC5-anti-CD45 antibody (Beckman-Coulter, Margency, France) and incubated for 10 min at room temperature. Afterwards, 500 μl 1 × binding buffer containing 5 μl FITC-annexin V solution were added to the cell suspension. The samples were incubated on ice for 10 min before analysis at 525 nm with an XL cytometer (Coulter-Immunotech). The FITC-annexin V stained cells were considered as apoptotic and the negative cells as viable after analysis of 104 blast cells. The cell lines did not require CD45 staining but were otherwise analyzed in the same way for annexin-V binding. The mitochondrial membrane potential was measured by flow cytometry using 100 ng/ml DiOC6(3) as a probe,17 with cells exhibiting low DiOC6 fluorescence being considered as apoptotic. Nuclear apoptosis was evaluated by fluorescence microscopy after staining with 1.5 μg/ml acridine orange as previously described.18 Cells with condensed chromatin and/or fragmented nuclei were considered as apoptotic. At least 100 cells were enumerated.
Cell sorting and FISH analysis
Frozen MNC from five patients were thawed and incubated overnight in culture medium. The next day, the viability was checked and the cells were centrifuged, resuspended in 100 μl of culture medium containing 5 μl of PC5-anti-CD45 antibody and incubated for 10 min at room temperature. They were then diluted in 500 μl of PBS and analyzed by FCM using an ELITE cell sorter (Beckman-Coulter). Lymphocytes (high CD45 expression and low side scatter properties) and blast cells (low CD45 and low to medium side scatter) were sorted. The purity of sorted populations was estimated by re-analysis and was greater than 95%. At least 3 × 104 cells were layered on microscope slides by cytospin. The slides were fixed in methanol/acetic acid (3/1) for 20 min at 4°C and stored desiccated at −20°C until FISH analysis.
Hybridization of interphase nuclei with fluorescently labelled probes for ABL and BCR (Vysis, Downers Grove, IL, USA) was done as previously described.19
The paired Student's t-test was used to analyzed the data. Due to the large range in spontaneous apoptosis during the culture of patient samples, the drug-induced apoptosis was considered after subtraction of spontaneous apoptosis for statistical analysis.
Apoptosis can occur through multiple pathways which ultimately lead to chromatin condensation and nuclear fragmentation. These nuclear events are usually preceded by alterations in mitochondrial membrane potential (ΔΨm) and loss of membrane asymmetry resulting in phosphatidylserine (PS) exposure.
In this study, we exposed four Bcr-Abl expressing cell lines to 0.2 μM DNR, 1 μM STI571 or both for 18 h. DNR alone was unable to induce significant apoptotic morphology in nuclei of these cell lines under conditions that induced 30% apoptotic death in U937 cells.20 In contrast, STI571 induced apoptosis as detected at the nuclear level in the four cell lines (Figure 1). The combination of DNR and STI571 did not increase the rate of apoptosis induced by STI571 alone.
When apoptosis was measured at the mitochondrial level, no effect was observed with DNR in BCR-ABL expressing cell lines, whereas STI571 induced from 10 to 40% apoptotic cells (Figure 2). The addition of DNR to STI571 increased the effect of STI571 alone on the ΔΨm in LAMA84, AR230 and K562 (P < 0.05) but was without effect on KCL22. However, the induction of apoptosis by DNR after STI571 treatment remained relatively modest and the sensitization of Bcr-Abl positive cells to DNR was low.
When apoptosis was measured at the membrane level using FITC-annexin V, DNR alone was again ineffective on the four cell lines. STI571 induced apoptosis on LAMA84, AR230 and KCL22 (Figure 3). In K562, STI571 failed to induce PS exposure, although apoptotic cells were detected with the nuclear and mitochondrial probes (Figures 1 and 2). The combination STI571+DNR significantly increased the rate of PS exposure as compared to STI571 alone in LAMA84 (P < 0.05) and in AR230 (P < 0.01), but not in KCL22. The effect of the combination remained very low on K562, which seems to be poorly able to expose its PS on the outside leaflet of the membrane.
Taken together, these results confirm that BCR-ABL expressing cells are resistant to DNR and suggest that STI571 consistently induces apoptosis which can be detected at the nuclear, mitochondrial or membrane levels in these cells. However, STI571 inhibition of Bcr-Abl tyrosine kinase does not seem to sensitize the cells to DNR induction of nuclear apoptotic morphology (Figure 1). A faint sensitization to DNR by STI571 was observed in three of the four cell lines when using membrane or mitochondrial probes (Figures 2 and 3).
In view of the heterogeneous response of cell lines to STI571+DNR, similar experiments were performed on peripheral blood MNC from 11 CML patients with circulating blast cells. After incubation with the drugs, the cells were stained with PC5-anti-CD45 and with FITC-annexin V. Blast cells and lymphocytes were identified on the basis of CD45 expression, and annexin V binding was analyzed on these gated populations (Figure 4).
DNR, STI571 and STI571+DNR all induced significant apoptosis on CML blast cells (P < 0.01, 0.003 and 0.0003, respectively, as compared to control, untreated cells; Figure 5a). STI571+DNR induced more apoptosis than DNR or STI571 alone in all samples (P < 0.002 and 0.003, Figure 5b and c). Moreover, the mean increase in cell killing efficiency of the combination (20.6% apoptosis) did not differ significantly from the sum effect of both drugs separately (23.7%, P > 0.2). If a sensitization of blast cells to DNR had occurred due to kinase inhibition, the increase in cell killing would had been expected to be significantly greater than the sum of both effects.
When the same measurements were performed on the lymphocyte population of CML samples a significant apoptosis was found when using STI571+DNR in combination (P < 0.006, Figure 6). STI571 alone was without effect above spontaneous apoptosis, and did not show a differential effect either between CML and normal lymphocytes (Figure 6). The combination STI571+DNR was significantly more efficient in inducing apoptosis in CML lymphocytes than in normal lymphocytes. These results suggest than STI571 was able to sensitize CML lymphocytes to DNR in contrast to its effect on CML blast cells or on normal lymphocytes.
The distribution of the results on CML lymphocytes was larger (range 0 to 58% apoptosis, mean 22.4%, median 14.6%) than in normal lymphocytes (Figure 6). This could be due to varying amount of Bcr-Abl expressing lymphocytes in the blood of CML patients. Thus, FISH experiments were performed on sorted lymphocytes and blast cells of five advanced phase CML patients. While the blast cells from all samples were BCR-ABL positive (100%), the lymphocytes exhibited the BCR-ABL rearrangement with a mean value of 14% (median = 14%, range 0–50%, n = 5).
The high tyrosine kinase activity of the Bcr-Abl protein is thought to play a major role in the pathogenesis of CML. STI571, an inhibitor of the Abl tyrosine kinase,21 selectively inhibits the growth10 and induces apoptosis in BCR-ABL-positive cells.22 In this study, we investigated the induction of apoptosis by STI571, DNR and their combination on Bcr-Abl-positive cell lines and fresh blood cells from CML patients.
Previous reports had indicated that BCR-ABL expression renders cells resistant to chemotherapeutic agents.7 Overexpression of the MDR-1 gene, leading to P-glycoprotein activity and resistance in blast crisis has been previously described.23 Multiple mechanisms such as BCR-ABL and Pgp overexpression were responsible for the in vitro resistance to apoptosis induction19 and to STI57124 in BCR-ABL-positive cell lines. Relapse is frequently observed in patients treated with STI571 in advanced stage of CML,13 probably due to one or more of the above mechanisms. It was thus of interest to investigate if the combination of STI571 and an anthracycline was able to increase the apoptotic response of CML cell lines and primary blast cells during accelerated phase and blast crisis.
In the present study, DNR alone was found to induce low rates (<10%) of apoptosis in the four BCR-ABL expressing cell lines at a concentration that led to apoptosis in 50% of U937 cells20 and of blast cells from 31 acute leukemias.16 STI571 alone was able to induce 10 to 40% apoptosis in four BCR-ABL-positive cell lines and to sensitize three of them to the pro-apoptotic effect of DNR. However, variations in the magnitude of this response could be observed when apoptosis was measured in different sub-cellular compartments. It is intriguing why STI571 treatment favored apoptotic responses of the BCR-ABL cell lines to DNR at the membrane and mitochondrial levels while no response was detected in the nucleus in these conditions. Nuclear morphologic alterations are late events of apoptosis and it is possible that DNR activates apoptotic pathways that do not lead to nuclear fragmentation during the experiment time lapse. Longer incubations may be required to verify this hypothesis. It is clear from our results that STI571 does not completely reverse the resistance of BCR-ABL expressing cells to DNR as the response to the combination of both drugs does not reach the amplitude of the response which was previously observed in acute leukemia cells in response to DNR alone.16,20
Combinations of DNR and other drugs with STI571 have been previously tested in vitro for their anti-proliferative effects on BCR-ABL-positive cell lines and CML cells, producing varying results pending of drugs25 and cell lines.26 Another report has shown that a combination of STI571 with cytarabine and doxorubicin induced a rate of apoptosis equivalent to the sum of the effects of each drug separately on BCR-ABL expressing cell lines.27
In the present study, we were able to discriminate between CML blast cells and lymphocytes in blood MNC using CD45 labeling,15 and thus to measure apoptosis in either population separately. We confirmed that STI571+DNR induced a rate of apoptosis close to the sum of the individual effects of the two drugs on CML blast cells. It seems paradoxical that inhibition of the Bcr-Abl tyrosine kinase was unable to reverse the apoptotic resistance of CML blast cells to DNR. Nevertheless, this can be attributed at least in part to the additional molecular abnormalities acquired by these cells during the accelerated phase of the disease. In fact, several cytogenetic changes have been reported during the transition to blast crisis, such as trisomy 8, isochromosome 17,28,29 abnormalities on p53 (chromosome 17p13)30 and c-MYC (8q24). These molecular alterations may also be responsible for resistance of CML blast cells to cytotoxic agents.
Interestingly, in the present study, a significant effect of the combination DNR+STI571 was found in CML lymphocytes while no effect was detected in normal lymphocytes. There was, however, a large distribution of apoptotic response (ranging from 0 to 58%) which could be explained by the rate of Bcr-Abl expressing lymphocytes (0 to 50%) as determined in five patients.
Altogether, these results suggest that combination therapy with STI571 and DNR could increase the pro-apoptotic effect of each drug on CML blast cells while no synergy between the two drugs was evidenced. Other mechanisms additional to Bcr-Abl tyrosine kinase activity could be responsible for DNR resistance, and further investigations are needed to understand its origin and to allow selection of the most efficient combination therapy.
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This work was supported by grants from Fondation de France, France (RT, FXM, FB) and the Leukaemia Research Fund, UK (JVM).
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Tabrizi, R., Mahon, F., Cony Makhoul, P. et al. Resistance to daunorubicin-induced apoptosis is not completely reversed in CML blast cells by STI571. Leukemia 16, 1154–1159 (2002). https://doi.org/10.1038/sj.leu.2402498
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