2-Chlorodeoxyadenosine alone and in combination with cyclophosphamide and mitoxantrone induce apoptosis in B chronic lymphocytic leukemia cells in vivo

Cancer Detection and Prevention 28 (2004) 433–442 www.elsevier.com/locate/cdp

2-Chlorodeoxyadenosine alone and in combination with cyclophosphamide and mitoxantrone induce apoptosis in B chronic lymphocytic leukemia cells in vivo Malgorzata Rogalin´ska PhDa, Jerzy Z. Błon´ski MD, PhDb, Margaret Hanausek PhDc, Zbigniew Walaszek PhDc, Tadeusz Robak MD, PhDb, Zofia M. Kilian´ska PhDa,* a

Department of Cytobiochemistry, University of Ło´dz´, S. Banacha 12/16, 90-237 Lo´dz, Poland Department of Hematology, Medical University of Lo´dz, Pabianicka 62, 93-513 Lo´dz, Poland c AMC Cancer Research Center, 1600 Pierce Street, Denver CO 80214, USA

b

Accepted 10 August 2004

Abstract The purpose of the study was to determine some apoptotic events in mononuclear cells obtained from peripheral blood of patients with Bcell chronic lymphocytic leukemia (B-CLL) during and after therapy with 2-chlorodeoxyadenosine (2-CdA; C), and the combination of 2CdA with cyclophosphamide (CC), or 2-CdA with mitoxantrone and cyclophosphamide (CMC). Western blot technique was performed to estimate expression/proteolytic degradation of generally accepted apoptotic markers, i.e., Bcl-2 protein, lamin B, PARP-1, and caspase-3 in leukemic cells isolated from blood samples of patients before treatment and subjected to drug(s) administration. The decrease of antiapoptotic protein Bcl-2 expression and proteolytic cleavage of nuclear proteins—lamin B and PARP-1 were observed in leukemic cells of patients treated according to the above therapy protocols, however, each to a different level among the studied groups. The obtained results indicated also that procaspase-3 was cleaved and activated in leukemic cells of three drug(s) treated groups. However, the cleavage of procaspase-3 and the generation of fragments with mol. mass of 17/20 kDa occurred especially effectively among patients treated according to CMC regimen. The changes in expression/proteolytic degradation of the above selected apoptotic markers, are accompanied by the appearance of apoptotic morphology in leukemic cells originated from blood of patients treated with the above drug(s) in comparison to untreated ones. # 2004 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved. Keywords: B-CLL; Drug-induced apoptosis; Bcl-2; Lamin B; PARP-1; Caspase-3; Western blot; Apoptotic morphology

1. Introduction The balance between cell proliferation, cell differentiation, and apoptosis tightly regulates the normal cell growth and turnover. A disturbance of this balance is thought to be the most important feature of malignant tumors [1–4]. B-cell chronic lymphocytic leukemia (B-CLL) is a hematological neoplasm characterized by the accumulation of mononuclear CD5+ B lymphocytes [5,6]. Most circulating cells appear to be arrested in the G0/G1 phase of the cell cycle and it has been suggested that the clonal excess of B * Corresponding author. Fax: +48 42 635 44 84. E-mail address: [email protected] (Z.M. Kilian´ska).

cells arises from still not well known defect(s) in apoptosis [7,8]. Apoptosis is a highly regulated cellular pathway whereby most cells, including B cells, are removed what leads to homeostasis [9,10]. The central mediator and executioner of apoptotic machinery is a system involving cysteine-aspartate proteases, named caspases [11]. Triggering of the death machinery by different death stimuli (e.g. chemotherapeutic agents, radiation, TNF family members, hormones, viral proteins) culminates in caspase- dependent proteolysis of many cytoplasmic and nuclear proteins, regulatory proteins, a set of proteins involved in cellular signal transduction leading to complete cell disassembly [1,10,11]. The evidence reported by several laboratories recently indicates that many cancer chemotherapy agents

0361-090X/$30.00 # 2004 International Society for Preventive Oncology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cdp.2004.08.001

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induce neoplastic cells apoptosis [4,12]. Lymphoid cells are sensitive to apoptosis induced by glucocorticoid hormones, and other chemotherapeutic agents such as purine analogues, camptothecin, chlorambucil, mitoxantrone, hydroxylated analog of resveratrol-piceatannol, taxol, aspirin and salicylate [13–18]. Our aim was to estimate some apoptotic events in vivo in mononuclear cells isolated from peripheral blood of B-CLL patients treated with purine nucleoside analogue—2chlorodeoxyadenine (2-CdA; cladribine) alone, with its combination with cyclophosphamide or mitoxantrone and cyclophosphamide. In our study apoptosis was assessed using Western blot technique to detect expression of apoptotic regulatory protein—Bcl-2, proteolysis of two nuclear proteins, i.e., poly(ADP-ribose) polymerase-1, lamin B, and processing of apoptosis–associated executioner enzyme—caspase-3, as well as by analysis of morphological signs of programmed cell death. Mechanisms of chemotherapeutic agent-induced apoptosis, including its regulation by cytotoxic drugs may provide information useful for designing more effective therapies by elucidating the main pathways used by different drugs [8,12,19].

2. Materials and methods 2.1. Patients Between December 2000 and March 2002, 17 previously untreated patients with progressive or symptomatic B-CLL were treated with monotherapy with 2-CdA or combined chemotherapy consisting of 2-CdA, cyclophosphamide and mitoxantrone. The characteristics of the patients is shown in Table 1. All of the patients fulfilled the National Cancer Institute-Sponsored Working Group criteria for B-CLL [20]. Pretreatment evaluation included examination of the medical history, a physical examination, a complete blood cell count, a differential count of WBC, a chemical survey, a

bone marrow examination and a serum immuno-globulin level quantitation. The patients had peripheral lymphocytosis greater than 5
109/l and more than 30% lymphocytes in a normal or hypercellular bone marrow. Cell marker studies were performed to confirm B-cell origin and monoclonal proliferation. All patients were CD5, CD19, CD20 and CD23 positive and showed monoclonality for light chain immunoglobulin membrane surface receptors. The clinical stage of disease was determined at the time of initiation of the treatment according to Rai’s classification [21]. Patients in stages 0–II were eligible if they had evidence of active disease, including progressive lymphocytosis, massive splenomegaly or bulky lymphadenopathy, recurrent disease-related infections, weight loss >10% over a 6 month period, temperature of 38 8C related to disease or extreme fatigue. Patients with poor performance status (WHO scale 4), active infection, abnormal liver or renal function and Richter’s syndrome were excluded from the study. The Medical University Ethical Committee, approved the study and the patients had signed the informed consent form. 2.2. Treatment modality The doses and schedule of the treatment agents were based on previous studies [22–24] in cases of B-CLL relapse, refractory B-CLL or low-grade lymphoma. B-CLL patients, previously untreated, were subjected to C (2-CdA), CC (2-CdA + cyclophosphamide) or CMC (2-CdA + mitoxantrone + cyclophosphamide). Eligible patients belong to the group (from central Poland) underwent randomization procedure for the assignment to either C, CC or CMC treatment [22,23]. Six patients received 2-CdA in monotherapy at a dose of 0.12 mg/kg/day by 2 h intravenous infusions for five consecutive days. Four patients received 2-CdA in combination with cyclophosphamide (CC regimen) and seven in combination with cyclophosphamide and mitoxantrone (CMC regimen). The CC regimen consisted of 2-CdA administered at a dose 0.12 mg/kg in 2 h intravenous infusion for 3 days and

Table 1 Clinical characteristics of B-CLL patients before treatment Characteristics

All patients

C

CC

CMC

Number of patients Sex Male Female Median age (years, range)

17

6

4

7

12 5 62.5 (33–78)

5 1 63.5 (36–71)

3 1 61 (28–78)

4 3 61 (33–76)

2 5 2 8

0 2 1 3 103.2 (10.2–606) 12 (4–14) 133 (15–231)

1 1 0 2 110.4 (9–555) 11.4 (5–15) 147 (30–286)

1 2 1 3 82.6 (5,4–376) 11 (5,1–17) 174 (28–314)

Rai stage 0 II III IV WBC (mean number
109/l; range) Hemoglobin (mean concentration g/dl; range) Platelets (mean number
109/l; range)

B-CLL patients were subjected to C(2-CdA), CC(2-CdA + cyclophosphamide) or CMC (2-CdA + mitoxantrone + cyclophosphamide).

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cyclophosphamide 650 mg/m2 i.v. on day 1; in CMC regimen mitoxantrone (10 mg/m2 i.v.) was additionally administered on day 1. 2-chlorodeoxyadenosine (2-CdA; Biodrybin) was obtained from the Institute of Biotechnology and AntibioticsBioton (Poland). Cyclophosphamide (Endoxan-Asta) and mitoxantrone were purchased from Asta Medica (Germany) and Jelfa SA (Poland), respectively. The efficacy of treatment after three courses of chemotherapy was performed in accordance to the NCI Working Group criteria [20]. 2.3. Mononuclear cells isolation Blood samples were obtained from patients 1 day before treatment, during drug(s) administration, i.e. after 1; 5; and 1; 3 days in the case of C and CC, CMC regimen, respectively. Additionally, blood samples were taken from patients after 14 days of drug(s) administration, i.e. on 19 or 17 day, respectively. Blood samples were collected before, during first cycle of each type of treatment, and 14 days later. Mononuclear cells from peripheral blood samples were isolated by centrifugation on Ficoll/Hypaque gradient (400
g, 18 8C, 30 min) according to Bo¨ yum method [25] and the obtained cells were fractionated immediately. At all steps of cell isolation and fractionation, protease inhibitors [26] were added at final concentration of 10 mM and 1 mM in the case of leupeptin, pepstatin A, benzamidine, and phenylmethylsulfonyl fluoride, respectively.

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antisera against caspase-3, anti-rabbit and anti-goat antisera conjugated with alkaline phosphatase were purchased from Sigma Chemical Co (USA). 2.7. SDS-polyacrylamide gel electrophoresis and immunoblotting assay Electrophoretically separated proteins by SDS-polyacrylamide gel electrophoresis [28] on 8.0 or 11.2% slab gels were blotted to immobilon P according to Towbin et al. [29] The membranes were stained with 0.5% Ponceau solution to confirm equal protein loading and transfer [30]. In some cases, actin was used as protein loading control. For immunodetection of antigen(s) immobilized on immobilon P, the alkaline phosphatase technique was used. The membranes were incubated for 1 h at room temperature in 3.0% nonfat dry milk in TBS (10 mM Tris–HCl, pH 7.5, 150 mM NaCl) to saturate non-specific protein binding sites. Then the membranes were incubated overnight with primary antiserum (at appriopriate dilution) in TBS in a cold room. After washing several times in TBS containing 0.05% Tween 20 (TBST), the membranes were incubated with appropriate secondary antiserum conjugated with alkaline phosphatase in TBS for 2 h at room temperature. Then, after washing with TBST, specific antigen-antibody interactions were visualized by incubation with substrate solution (0.33 mg/ml of nitro blue tetrazolium, 0.17 mg/ml of 5-bromo-3-chloro-3-indolyl phosphate in 100 mM Tris–HCl, pH 9.5, 100 mM NaCl and 5 mM MgCl2), prepared according to Leary et al. [31].

2.4. Cellular fractionation 2.8. Analytical procedures The cell pellet was rinsed with cold phosphate buffered saline (PBS) and then suspended (5
107 cells/ml) in isotonic sucrose containing 5 mM MgCl2, 0.5% Triton X-100, 50 mM Tris–HCl (pH 7.5) and protease inhibitors. Cells were then homogenized (10–12 ml) in a Potter homogenizer for 3 min at 80 V and filtered through several layers of gauze. A part of the homogenate was left and kept at 20 8C for further analysis and the rest was spun down at 800
g for 7 min. The crude nuclear pellet was purified by the Blobel and Potter technique [27]. The supernatant was treated as postnuclear fraction. 2.5. Morphological analysis of B-CLL cells Mononuclear cells obtained from peripheral blood of untreated and drug-treated patients were fixed in 4% formaldehyde, stained with 5% Giemsa (solution Sigma Chemical Co) and then the morphology was analysed with light microscopy (Olympus IX 70, Japan). 2.6. Antisera Rabbit polyclonal antisera against Bcl-2, PARP-1 as well as goat polyclonal antiserum against lamin B were from Santa Cruz Biotechnology (USA). Rabbit polyclonal

The protein content was estimated by the method of Lowry et al. [32] and DNA was determined spectrophotometrically.

3. Results The purpose of our study was to investigate potent induction of apoptosis in vivo in mononuclear cells isolated from peripheral blood of B-CLL patients treated with 2-chlorodeoxyadenosine alone and using combinations of 2-CdA with cyclophosphamide or mitoxantrone and cyclophosphamide. The apoptosis induction was estimated by Bcl-2 expression in the homogenate as well as in nuclear and postnuclear fractions obtained from mononuclear cells of B-CLL patients after three different regimen treatments. The activation of proteolysis of two nuclear proteins, i.e., lamin B and PARP-1, after drug(s) administration was also investigated. In addition we included in our study activation of the main executioner enzyme of cell death machinery— caspase-3. Thus, we investigated the expression and processing of procaspase-3 in mononuclear cell homogenates originated from the blood of patients before and during drug(s) treatment. The Western blot analysis using

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Bcl-2, lamin B, PARP-1, and caspase-3 polyclonal antisera was undertaken to compare the expression, and/or proteolytic cleavage of the above proteins, involved in the apoptotic response to 2-CdA or its combinations with other drug(s), in B-CLL cells isolated from the blood of treated versus untreated patients.

after CMC administration. Immuno-reactivity of Bcl-2 in homogenate, nuclear and postnuclear fractions was clearly diminished in the samples obtained 14 days after the last drug(s) administration.

3.1. Bcl-2 expression

3.2.1. Lamin B LaminB, a 67 kDa polypeptide found in all mammalian cells has been identified as DNA-binding protein [34]. Cleavage of this important nuclear lamina component has been reported to occur just prior to or concomitant with some other changes leading to apoptosis [35,36]. The results of our experiments indicate that lamin B is proteolytically degraded in the mononuclear cell homogenate and in the nuclear fraction obtained from blood of B-CLL patients treated with three different regimens. As shown in Fig. 2, a lamin B precursor is degraded to some extent in mononuclear cell homogenates and nuclear fraction isolated from B-CLL patients treated with the chosen regimens. During drug(s) administration and 2 weeks later, we observed some proteolytic fragments of lamin B with molecular mass 26– 32, 36 and 43–46 kDa. The amount of these proteolytic products increased after day 1 and 3 and of the treatment according to CMC and CC regimens, respectively. Even stronger immunostained bands of the proteolytic fragments were detected in the nuclear fraction of mononuclear cells of B-CLL patients after day 3 of CMC administration and 14 days later.

Bcl-2 protein, a major antiapoptotic member of Bcl-2 family is a 26 kDa integral membrane constituent primarily residing in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes [33]. Fig. 1 shows Bcl-2 expression in the mononuclear cell homogenate as well as nuclear and postnuclear fractions of B-CLL patients treated according to C, CC and CMC regimens. A slight decrease of Bcl-2 expression was observed in the homogenate and cellular fractions from B-CLL cells of patients treated with 2-CdA alone. This decrease was seen usually after 5 days of drug administration. Profound analysis of immunoblots of Bcl-2 expression in cells from blood of B-CLL patients treated with CC and CMC regimens revealed that the level of this antiapoptotic protein clearly dropped in cell homogenate preparations. In the case of CC treatment Bcl-2 seems to be preferentially concentrated in the postnuclear fraction. It is interesting to note that the Bcl-2 polypeptide effectively decreased in mononuclear cells obtained from blood of B-CLL patients treated with the combination of three drugs. Bcl-2 expression dropped significantly in homogenate preparations, usually between day 1–3 of CMC treatment. The decrease of this antiapoptotic protein was observed early in nuclear fraction (after day 1). Marked lowering of the Bcl-2 level was noticed in the postnuclear fraction 3 days

3.2. Proteolytic degradation of nuclear proteins

3.2.2. Poly(ADP-ribose) polymerase-1 Poly(ADP-ribose) polymerase-1 is a 113/116 kDa nuclear enzyme activated by DNA strand breaks and is

Fig. 1. Immunoblot analysis of mononuclear cell homogenate, nuclear and postnuclear fractions isolated from peripheral blood of B-CLL patients untreated (0); during drug(s) administration, i.e., after day 1; 5 and day 1; 3 in the case of C and CC, CMC regimens, respectively; and 14 days after last drug(s) administration (19 or 17) by alkaline phosphatase method in the presence of Bcl-2 antiserum. Samples containing 40 mg of proteins were loaded into lanes of 11.2% polyacrylamide gels. The arrows indicate Bcl-2 protein with mol. mass of 26 kDa, tringle-actin (mol. mass of 42 kDa) used as protein loading control (ctrl) of cell homogenate samples.

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Fig. 2. Immunoblot analysis of mononuclear cell homogenate and nuclear fraction isolated from peripheral blood of B-CLL patients untreated (0), during drug(s) administration, i.e., after day 1; 5 and day 1; 3 in the case of C and CC, CMC regimens, respectively; and 14 days after last drug(s) administration (19 or 17) by alkaline phosphatase method in the presence of lamin B antiserum. Samples containing 40 mg of proteins were loaded into lanes of 8.0% polyacrylamide gels. The arrows indicate full length of lamin B with mol. mass of 67 kDa.

involved in DNA repair. It is one of the first described nuclear protein to be cleaved during apoptotic death [37,38]. Fig. 3 shows immunodetection of PARP-1 in the homogenate and in the nuclear fraction of mononuclear cells of B-CLL patients examined in our experiments. The results indicate that among leukemic cells of patients subjected to three regimens, proteolytic degradation of PARP-1 was observed. In the samples obtained from blood patients treated with 2-CdA alone, full length PARP-1 was slightly degradated and its proteolytic products appeared after day 1 of the nucleoside analogue administration. The amounts of immuno-reactive fragments with molecular mass in the range 85–89, 48–65 and 24 kDa increased after 5 days of drug administration. Western blot results revealed

that CC treatment of B-CLL patients caused partial proteolysis of PARP-1 as evidenced by the increase of strongly stained PARP-1 fragments after day 1 of drugs administration. The dominance of the 85–89 kDa proteolytic product was observed during administration period and 14 days later. The treatment of leukemic patients according to CMC regimen resulted in active proteolytic PARP-1 degradation, which was noticed after day 1 of drug administration. Intensive immunostained PARP-1 degradation products of 85–89, 50–65, 44 and 24 kDa were seen after day 1 of treatment. It is interesting that 14 days after CMC administration, a significant decrease of the PARP-1 precursor and increasing amounts of 85–89, 50–65, 44 and 35 kDa fragments in the homogenate and in the nuclear fraction was detected.

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Fig. 3. Immunoblot analysis of mononuclear cell homogenate and nuclear fraction isolated from peripheral blood of B-CLL patients untreated (0); during drug(s) administration, i.e., after day 1; 5 and 1; 3 in the case of C and CC, CMC regimens, respectively; and 14 days after last drug(s) administration (19 or 17) by alkaline phosphatase method in the presence of PARP-1 antiserum. Samples containing 40 mg of proteins were loaded into lanes of 8.0% polyacrylamide gels. The arrows indicate full length of PARP-1 with mol. mass of 113/116 kDa.

3.2.3. Caspase-3 activation The action of enzymes involved in apoptotic degradation of many cellular substrates seems to be carefully regulated [2,11]. We have analyzed the activation of caspase-3 to assess drug(s)-induced apoptosis in mononuclear cell homogenates of B-CLL patients (Fig. 4). The results show the induction of proteolytic cleavage and activation of caspase-3 after the treatment of patients according to all three regimens utilized in the present study. Mononuclear cell homogenates isolated from the peripheral blood of patients before drug(s) administration contained primarily the caspase-3 precursor. In B-CLL cell homogenates of patients treated with C, CC, and

CMC regimens, we observed cleavage of procaspase-3, followed by the appearance of 17/20 kDa fragments, but to a different degree. The amount of 17/20 kDa cleavage products in leukemic cell homogenates isolated from patients treated with 2-CdA alone, only slightly increased during this drug administration. The combined therapy of patients with CC or CMC caused more profound conversion of the caspase-3 precursor into 17/20 kDa fragments. As shown in Fig. 4, during prolonged two- or three-drug therapy, the 32 kDa procaspase-3 was efficiently cleaved generating 17/20 kDa products. In the samples obtained from CMC treated patients, the caspase-3 precursor was almost completely processed to

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B-CLL cells originating from blood of a patient before and after CMC treatment. In drug-treated leukemia cells, Giemsa staining revealed apoptotic morphology, i.e., abnormal shape and volume, blebbing of the plasma membranes, condensation of chromatin or nuclear fragmentation.

4. Discussion

Fig. 4. Immunoblot analysis of mononuclear cell homogenate isolated from peripheral blood of B-CLL patients untreated (0), during drug(s) administration, i.e., after day 1; 5 and day 1; 3 in the case of C and CC, CMC regimens, respectively; and 14 days after last drug(s) administration (19 or 17) by alkaline phosphatase method in the presence of caspase-3 antiserum. Samples containing 40 mg of proteins were loaded into lanes of 11.2% polyacrylamide gels. The arrows indicate caspase-3 precursor with mol. mass of 32 kDa.

the activated form of 17/20 kDa. Its strongly stained band was observed after 3 days of therapy with CMC and persisted 14 days later. 3.2.4. Morphological changes associated with druginduced apoptosis Cytological examination of mononuclear cells isolated from blood of B-CLL patients before and after drug(s) administration revealed signs of apoptosis in treated cells. Fig. 5 shows a representative picture of Giemsa stained

Fig. 5. Photographs of mononuclear cells isolated from peripheral blood of B-CLL patient before treatment (a) and after 3 days of CMC administration (b) stained with Giemsa solution.

Regulation of the cell number is of fundamental importance in multicellular organisms. It is generally accepted that inhibition of apoptosis is a critical event in the development of many cancers, including B-CLL [3,4,8,10,12,19]. Understanding of how to induce apoptosis in cells undergoing neoplastic transformation still represents the principal therapeutic goal of apoptosis research. The introduction of new purine nucleoside analogues (e.g., fludarabine (FAMP); 2-chlorodeoxyadenosine (2CdA, cladribine, leustatin); 20 -deoxycoformycin (DCF)) in the treatment of leukemias and lymphomas was a major improvement in recent years in terms of induction, remission and progression-free survival [39–44]. 2-chlorodeoxyadenosine represents a novel agent that has revolutionized the clinical outlook of hairy cell leukemia patients. This nucleoside analogue has also revealed significant activity against B-CLL [39,42]. It is well documented that molecules of 2-CdA cross the cell membrane, and that their intracellular phosphorylation is necessary for the cytostatic effect to occur. This nucleoside is phosphorylated by deoxycytidine kinase and accumulates as 2-CdATP [43]. The high activity of this kinase in lymphocytes along with their low 50 -deoxynucleotidase activity probably explains its relatively high selectivity for lymphoid cells. Several in vitro investigations have shown synergic interaction between 2-CdA and anthracyclines as well as between other nucleoside analogue-fludarabine and the alkylating agent cyclophosphamide and/or mitoxantrone in the induction of lymphocyte apoptosis [40–42]. However, combinations of 2-CdA with other drug(s) are not always superior to single-agent therapy for lymphoid cells. Hoffman et al. [45] compared the cytotoxicity of two human-derived B-cell lymphoma cell lines incubated with 2CdA alone and in combination with five antineoplastic drugs (cis platin, chlorambucil, daunorubicin, etoposide or paclitaxel). The authors reported that out of the mentioned combinations only the combination of 2-CdA with Cisplatin demonstrated significantly more cytotoxicity than either agent alone. Surprisingly, the combination of 2-CdA and chlorambucil was significantly less cytotoxic that chlorambucil alone, suggesting an antagonistic interaction. In the present study, we have analyzed the induction of apoptosis in vivo by 2-CdA alone or in combinations with other anti-cancer drugs, known to be effective in the treatment of hematological neoplasms. We have investigated such important events of programmed cell death, after

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B-CLL patients’ administration with C, CC, and CMC, as the expression of regulatory protein Bcl-2, expression and proteolysis of lamin B and PARP-1, and the processing of death executionery enzyme—procaspase-3 as well as the appearance of apoptotic characteristics in the morphology of drug-treated leukemic cells. Proteins from the Bcl-2 family which play the key role in apoptosis regulation, include members with opposite functions, i.e., inhibitors and activators [46]. One of the first proteins from Bcl-2 family to be detected was a product of oncogene Bcl-2, found in B cell lymphomas [47]. The polypeptide Bcl-2 represents the strongest inhibitor of apoptosis known to date. Its overexpression was observed in malignant neoplasms of the hematopoietic system as well as in a number of solid tumors, and is responsible for their resistance to chemo- and radiotherapy [48,49]. Robertson et al. [50] observed that CLL patients with low level of Bcl-2 had a higher survival rate relative to those with intermediate or high level of Bcl-2 polypeptide. In the present study, the Bcl-2 expression level in mononuclear cells of B-CLL patients before antileukemic therapy varied among examined individuals. The expression of this antiapoptotic protein slightly decreased in the B-CLL cells of patients treated with 2-CdA alone. Combined therapy consisted of this nucleoside analogue and cyclophosphamide or mitoxantrone and cyclophosphamide caused usually a significant reduction of the Bcl-2 expression, especially, in leukemic cells of patients treated with three drugs. A distinctive feature of apoptotic cell death are the marked changes occurring in the nucleus, which likely reflect proteolysis of several nuclear proteins and fragmentation of DNA [51]. Among nuclear polypeptides, lamins and PARP-1 have received special attention in apoptosis research. Lamins (A, B and C) play a major role in chromatin organization at the nuclear periphery [51,52]. Proteolysis of these proteins appear to be critical for nuclear disassembly during apoptosis [35,36]. It was suggested that cleavage of lamins is required for packaging of condensed chromatin into apoptotic bodies [35]. Lamin B, a DNAbinding protein, can be progressively degraded during apoptosis to major fragments of 46 or 28 kDa [53,54]. This nuclear polypeptide is mainly cleaved by caspase-6. However, it is also documented that it can be proteolysed by serine protease associated with nuclear scaffold [51]. The other nuclear polypeptide useful as apoptotic marker is PARP-1. This important enzyme catalyzes the transfer of ADP-ribose polymers into itself and other nuclear polypeptides in response to the strand breaks and is involved in DNA repair [37,38]. PARP-1 is one of the first nuclear proteins to be cleaved during apoptosis [37,55]. It was reported that during apoptosis this enzyme is degraded mainly to 84/89 and 23/24 kDa fragments [37,56]. Its proteolysis is caused predominantly by caspase-3 [51]. Recently, however, the experiments in vitro have revealed that PARP-1 can be fragmented to polypeptides that range from 40 to 70 kDa. These PARP-1 fragments are produced

by calpain [56,57]. The fragments with similar characteristics were described during necrotic PARP-1 degradation [58]. Results of our experiments indicated that in B-CLL cells obtained from patients treated with 2-CdA alone or using combinations of 2-CdA and cyclophosphamide or mitoxantrone + cyclophosphamide the mentioned above nuclear proteins, i.e., lamin B and PARP-1 are proteolytically degradated. Treatment of patients according to three regimens resulted in diminished immunoreactivity of native lamin B (67 kDa) and full length of PARP-1 (113/116 kDa), and in the appearance of immunoreactive proteolytic products. The extent of lamin B proteolytic cleavage in leukemic cells of patients treated with nucleoside analogue only was lower as compared to those after CC and CMC administration. Proteolytic cleavage of PARP-1 in B-CLL cells from patients treated according to three schedules was observed as early as after 1 day of drug(s) action. However, this effect was especially prominent in samples obtained from mononuclear cells of CMC-treated patients. The full length of PARP-1 was gradually disappearing in the course of the CMC therapy, concomitant with the appearance of proteolytic cleavage products with high immunoreactivity. In our drug(s) induced apoptosis experiments in vivo intensive proteolytic degradation of PARP-1 by additional unknown caspase(s)/protease(s) cannot be excluded. The activation of caspase-3 is one of the key points in signal transmission during the execution phase of apoptosis [2,11]. The active caspase-3 is generated from a 32 kDa precursor. The initial cleavage of proenzyme produced the p20 polypeptide and the small subunit (p12). The p20 polypeptide is further processed, resulting in the appearance of the mature, large subunit—p17 [59]. The active enzyme is heterotetramer composed of p12 and p17 subunits [60]. Our data have revealed that caspase-3 precursor was cleaved and activated in leukemic cells isolated from B-CLL patients treated with 2-CdA and with its combination with cyclophosphamide or mitoxantrone and cyclophosphamide. The treatment of patients according to CC and CMC regiments caused the proteolysis of procaspase-3 what was manifested by higher amounts of cleavage product with mol. mass of 17/20 kDa as compared to 2-CdA monotherapy. In leukemic cells isolated from blood of patients after CMC administration, the caspase precursor was almost completely cleaved leading to the appearance of the highest immunoreactivity in mol. mass of 17/20 kDa. It should be stressed that the changes in Bcl-2 protein expression, expression/proteolytic degradation of lamin B and PARP-1 as well as activation of the caspase-3 precursor occurred concomitantly with the appearance of apoptotic signs (abnormal shape and volume, blebbing of the plasma membranes, chromatin condensation, nuclear fragmentation) in drug(s) treated B-CLL cells. In conclusion, the data presented in this report based on the expression/proteolytic cleavage of the selected proteins involved in apoptosis and analysis of apoptotic morphology,

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provide evidence that 2-CdA, and especially its combinations with cyclophosphamide and mitoxantrone and to a lesser extent its combination with cyclophosphamide, efficiently induce programmed cell death in mononuclear cells of B-CLL patients treated with these chemotherapeutic agent(s). Monitoring of patients’ clinical response to the applied drug(s)-treatment in accordance to the NCI Working Group criteria [20] after three courses was performed. Complete response (CR) required the absence of symptoms and organomegaly, normal complete blood cell count (absolute neutrophil count > 1.5
109/l), hemoglobin concentration > 11.0 g/dl, platelet count 100
l09/l and bone marrow with less than 30% lymphocytes at least 2 months. Partial response (PR) was considered in the case of a 50% or greater decrease in the size of lymph nodes, liver and spleen, and peripheral blood findings either identical to those of CR or improved over pre-therapy value by at least 50%. The patients who had no achieved CR or PR were classified as non-responders. Among studied patients 5 of the 7 treated with CMC obtained response (3CR, 2PR), while in groups who received 2-CdA alone or 2-CdA in combination with cyclophosphamide 4 of the 6 (4PR) and 3 of the 4 (1CR, 2PR) responded to therapy, respectively. Further experiments, extending the number of cured B-CLL patients and using a broader panel of apoptotic machinery proteins, (especially regulatory proteins and the enzymes involved in cell death) are in progress.

Acknowledgements This work was supported partially by Polish State Committee of Science grant No. 6PO5A01821 and by grant 505/430 from University of Lo´ dz.

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