Fludarabine-induced apoptosis of B chronic lymphocytic leukemia cells includes early cleavage of p27kip1 by caspases.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of growth arrested clonal B lymphocytes that undergo apoptosis when treated with fludarabine. To further explore the mechanism for the cell cycle arrest, we examined the expression and activity of cyclin-dependent kinases and inhibitors in primary B-CLL cells. We observed high levels of p27kip1, cyclin D2, cyclin E, cdk2, and cdk4 expression in freshly isolated B-CLL cells. Despite high levels of cyclins and cdks, little cdk2 or cdk4 activity was observed with p27kip1 in complex with cyclinD2/cdk4 and cyclin E/cdk2. Remarkably, when B-CLL cells were treated in vitro with fludarabine, p27kip1 underwent caspase-specific degradation accompanied by an increase in cdk4 activity. We conclude that the G0/G1 arrest of B-CLL cells may protect against apoptosis and that the decrease in p27kip1 expression by caspase cleavage may be a key step in chemotherapy-induced apoptosis in B-CLL.     

Drug-induced apoptosis in chronic lymphocytic leukemia.

Glucocorticoids and fludarabine are able to induce typical features of apoptosis in CLL lymphocytes.Cysteinyl aspartate specific proteases (caspases) play a key biochemical role in the apoptotic pathway. Caspase activation following cytotoxic stimuli leads to highly specific proteolytic cleavage of functionally important cellular enzymes. One of them is poly ADP-ribose) polymerase (PARP). To some extent caspase activation seems to be under the control of the Bcl-2 family of interacting proteins. We determined the role of Bcl-2-family proteins Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic), activation of caspase-3 (CPP32/Yama) and activation of PARP in CLL apoptosis. All 21 analyzed CLL samples expressed Bcl-2 and Bax. Four of 13 (31%) samples with a low Bcl-2/Bax ratio exhibited in vitro prednisolone resistance, whereas eight of nine (88%) samples with a high Bcl-2/Bax ratio were in vitro resistant (相似文献   

Role of myeloid cell factor-1 (Mcl-1) in chronic lymphocytic leukemia

The primary abnormality in chronic lymphocytic leukemia (CLL) is a defect in apoptosis, probably related to alterations in the expressions of Bcl-2 family members. In transgenic mice over expressing the anti-apoptotic Bcl-2 family member, myeloid cell factor-1 (Mcl-1), B cell lymphomas occur. Moreover, mice conditional for the loss of Mcl-1 display a profound reduction in B and T lymphocytes. This suggests that Mcl-1 is an essential survival factor in lymphocytes. In the present study, we have evaluated the role of Mcl-1 in CLL. Mcl-1 protein expression was measured by Western blot analysis in the CLL cells of 45 patients and correlated with clinical variables and survival. Mcl-1 levels were similar in 29 patients to normal B and T lymphocytes, were decreased in 8 patients and increased in 12 patients. An inverse correlation was found between Mcl-1 expression and Rai stage (P = 0.001). When assessed by flow cytometry, Mcl-1 expressions were normally distributed among CLL cells in individual patients and the mean levels correlated with those obtained by Western blotting. To evaluate the role of Mcl-1 in drug resistance, Mcl-1 levels were sequentially measured in the leukemic cells of 4 CLL patients during therapy with fludarabine (Flu). The Mcl-1 levels were found to increase in 2 patients while the peripheral blood lymphocyte counts dropped, suggesting that the residual drug-resistant cells had the highest Mcl-1 levels. Primary CLL cells were also treated with chlorambucil (CLB) or Flu in vitro and the Mcl-1 levels decreased correlating with the sensitivity of these cells to undergo apoptosis. Drug sensitivities of the CLL cells to CLB and Flu were also measured by MTT assay and the concentrations of drug required to decrease cell viability by 50% (IC50) varied from 1.9 to 9.27 microM for Flu (median, 9.4 microM) and 10 to 32.5 microM (median, 5.5 microM) for CLB. The sensitivities of the leukemic cells to CLB correlated inversely with Mcl-1 levels (P < 0.05). These results suggest that Mcl-1 may contribute to cell survival in CLL.     

Role of beta2 integrins in the prevention of apoptosis induction in chronic lymphocytic leukemia B cells.

Immunologically committed lymphocytes, especially mature, leukemic B cells, proliferate then accumulate without further cell division in chronic lymphocytic leukemia patients (CLL). These mature, leukemic B cells often produce autoantibodies. Under normal circumstances, immunologically committed lymphocytes that are autoreactive are deleted by a programmed cell death mechanism. In CLL cells, these mechanisms appear to be inhibited; therefore, cells accumulate rather than be destroyed. To understand the mechanism by which cell survival is selected over death in CLL cells, we studied the role of beta2 integrins and their ligands in the regulation of apoptosis. CLL cells were treated with monoclonal antibodies directed against beta2 integrins. Antibodies directed against the I-domain of the alpha chain of CD11b/CD18 inhibited apoptosis. The identity of the physiological ligand or counter-receptor for beta2 integrins that was required for the inhibition of apoptosis induction was sought. The ligand iC3b, but not ICAM-1 or fibrinogen, was identified as a ligand that could prevent apoptosis of CLL B cells. Free iC3b levels were elevated in CLL patients indicating that this ligand is available in vivowhere it may interact with beta2 integrins on CLL B cells and sustain their viability by preventing activation of the programmed cell death pathway. Leukemia (2000) 14, 34-39.     

Increased bone marrow angiogenesis in B cell chronic lymphocytic leukemia.

Recent studies have shown that angiogenesis may be involved in the pathogenesis of hematopoietic malignancies, apart from its well-characterized role in the growth and metastasis of solid tumors. In this study, we quantified the degree of angiogenesis in B cell chronic lymphocytic leukemia (B-CLL) by measuring the microvessel density and hotspot density in bone marrow trephine biopsy sections with B-CLL involvement (n = 12) and compared it to normal bone marrow sections (n = 11). The B-CLL samples had a mean microvessel count/high power field (hpf) of 7.64 while the control samples had a mean microvessel count/hpf of 2.11 (P = 0.0001). The mean hotspot density in the B-CLL sections (14.83/hotspot) was also significantly higher (P = 0.0008) than the mean hotspot density in control bone marrow sections (7.09/hotspot). Both the microvessel density and hotspot density correlated positively with the clinical stage of the B-CLL patients. In a separate cohort of B-CLL patients, the median urine level of the angiogenic peptide, basic fibroblast growth factor (2216.5 pg/g, n = 14), was significantly higher (P = 0.0001) than the bFGF level in normal controls (1,084 pg/g, n = 58). These results indicate that angiogenesis may be involved in the pathogenesis of B-CLL.     

Protease activation and glucocorticoid-induced apoptosis in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is at present an incurable disease. All of the drugs used in the treatment of CLL induce apoptosis in the cells, and in vitro responses to glucocorticoid or analogs correlate with in vivo sensitivity to these agents. Since CLL lymphocytes accumulate rather than proliferate, the idea that CLL is a disease involving defective apoptosis is particularly attractive. Recent studies have identified many of the central components of the apoptotic pathway that appear to be conserved from one cell type to another. Thus, investigation into the functionality of these molecules should reveal where the defect(s) in apoptosis may lie in CLL cells. Protease activation is a central event during apoptosis. and leads to many of the familiar characteristics of apoptosis. Here we will examine the role of apoptotic proteases in CLL and speculate on their contribution to disease emergence and drug resistance.     

Prodigiosin induces apoptosis of B and T cells from B-cell chronic lymphocytic leukemia.

We have previously reported that prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosene) induces apoptosis in human hematopoietic cancer cell lines with no marked toxicity in nonmalignant cell lines. In this study, we demonstrate that prodigiosin induces apoptosis of B-cell chronic lymphocytic leukemia (B-CLL) cells (n=32 patients). The dose-response for the cytotoxic effect of prodigiosin was analyzed in cells from 12 patients showing an IC(50) of 116+/-25 nM. Prodigiosin induced apoptosis of B-CLL cells through caspase activation. We also analyzed the cytotoxic effect of prodigiosin in T cells from B-CLL samples and no differences were observed with respect to leukemia cells. This is the first report showing that prodigiosin induces apoptosis in human primary cancer cells.     

Chemosensitivity of B cell chronic lymphocytic leukemia and correlated expression of proteins regulating apoptosis, cell cycle and DNA repair.

B cell chronic lymphocytic leukemia (B-CLL) cannot be cured with conventional chemotherapy. This clinical enigma appears to be at least partially due to the fact that B-CLL cells are resistant to programmed cell death (apoptosis) and that they are arrested in G0/G1 phase of the cell cycle. The reasons for the dysregulation of these two key cellular events in B-CLL are unclear. The present study aimed at determining correlations between the expression levels of proteins regulating apoptosis, cell cycle and DNA repair in B-CLL cells and normal B cells. In addition, the differential sensitivity of B-CLL cells to drug-induced apoptosis was quantified. We show that in B-CLL cells levels of the death-suppressor Bcl-2 correlated positively with those of the pro-apoptotic protein Bax and of the cyclin-dependent kinase (cdk) inhibitor p27Kip1. In B-CLL cells levels of the anti-apoptotic Bcl-xL showed a positive correlation with levels of the 80 kDa regulatory component (Ku80) of the DNA-dependent protein kinase that is involved in DNA double-stranded break repair. These correlations were not detected in normal B cells. The sensitivity of leukemic cells to FLUD but not to ADM, CPM or to DEX was reduced in pre-treated patients. These data support the hypothesis that in B-CLL cells death-modulators and molecules modulating cell cycle and DNA repair are regulated in a coordinated manner. Leukemia (2000) 14, 40-46.     

Bcl-2 and apoptosis in chronic lymphocytic leukemia

Opinion statement The Bcl-2 family of pro- and antiapoptotic proteins are key regulators of the apoptosis cascade and the mitochondrial-mediated pathway of caspase activation. Of this family, Bcl-2 was the first identified and remains the best characterized. Aberrant expression of Bcl-2 is common in chronic lymphocytic leukemia (CLL) and is associated with poor response to chemotherapy and decreased overall survival. Bcl-2 is an attractive target for novel therapeutic agents. Antisense oligonucleotides directed against Bcl-2 are effective in vitro and are being evaluated in clinical trials in CLL. Small molecule Bcl-2 inhibitors are in preclinical development and should be ready for clinical evaluation in the next few years. Strategies that induce apoptosis and bypass Bcl-2 may also be therapeutically useful in CLL. Thus, over the next decade, one can envision incorporating measurements of apoptotic proteins such as Bcl-2 into the risk assessment and treatment algorithms for individual patients. In addition, we anticipate that in the next decade, rationally designed therapies targeting specific molecular defects in the malignant CLL lymphocytes will be introduced into the clinic.     

Merkel cell polyomavirus (MCPyV) in chronic lymphocytic leukemia/small lymphocytic lymphoma

Merkel cell polyomavirus (MCPyV) is a novel polyomavirus that shows a strong association with Merkel cell carcinoma (MCC). Recent studies have demonstrated MCPyV in some cases of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), a malignancy with a similar demographic as MCC. We tested for the presence of MCPyV by PCR and immunohistochemistry in 18 cases of CLL/SLL. Very low-level MCPyV DNA was detected in 33% of CLL/SLL cases by real-time PCR, but only one case demonstrated immunohistochemical positivity for MCPyV. MCPyV was not identified in 17 cases of follicular lymphoma, suggesting either that MCPyV is involved in CLL/SLL pathogenesis or that the immunodeficiency state of CLL/SLL induces low-level MCPyV reactivation.     

The configuration of the immunoglobulin genes in B cell chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (CLL) lacks a consistent genetic abnormality. However, immunoglobulin V(H) gene segment mutation analysis has provided insights into the pathogenesis of these diseases and allowed the development of powerful prognostic markers. Immunoglobulin gene chromosomal translocations are rare in CLL and involve a distinct subset of genes including BCL3, BCL11A and CCND2. BCL2 translocations in CLL appear to arise via a different mechanism from comparable translocations seen in B cell non-Hodgkin lymphoma.     

Role of angiogenesis in chronic lymphocytic leukemia

Angiogenesis is a physiologic process of new blood vessels formation mediated by various cytokines called angiogenic and angiostatic factors. Although its potential pathophysiologic role in solid tumors has been extensively studied for more than 3 decades, enhancement of angiogenesis in chronic lymphocytic leukemia (CLL) and other malignant hematological disorders has been recognized more recently. An increased level of angiogenesis has been documented by various experimental methods both in bone marrow and lymph nodes of patients with CLL. Although the role of angiogenesis in the pathophysiology of this disease remains to be fully elucidated, experimental data suggest that several angiogenic factors play a role in the disease progression. Biologic markers of angiogenesis were also shown to be of prognostic relevance in CLL. The current findings provide the rationale for investigating antiangiogenic agents in CLL. In the current review angiogenesis in CLL is discussed and its potential diagnostic and therapeutic applications.     

CD5-induced apoptosis of B cells in some patients with chronic lymphocytic leukemia.

Although B chronic lymphocytic leukemia (B-CLL) is characterized by prolonged survival of CD5(+) B cells in vivo, these cells apoptose spontaneously in vitro. The effect of CD5 ligation on apoptosis was studied in 27 newly diagnosed patients with B-CLL, in relation to the expression of surface IgM (sIgM), CD79b, CD38, CD72 and CD19. B cells from 15 patients (group I) were resistant to anti-CD5-induced apoptosis, whereas apoptosis above spontaneous levels was seen in the remaining 12 studied (group II). Group II was then subdivided on the basis of differences in the time required to reach maximum apoptosis: whilst B cells from seven patients underwent apoptosis by 18 h, those from the remaining five needed 36 h to apoptose. The expression of sIgM, CD5, CD79b and CD38 was higher in group II than group I, suggesting that signaling for apoptosis might operate via CD79, and that CD38 expression was required. As shown by flow cytometry and confirmed by Western blotting, apoptosis was associated with a decrease in the ratios of Bcl-2/Bax and Bcl(XL)/Bax, due to an increase in the level of Bax, but no change in that of Bcl-2. This heterogeneous apoptotic response to CD5 ligation offers an explanation for the incomplete success of anti-CD5 monoclonal therapy, and might help identify patients who would respond to such treatment.     

Discontinuous deletions at 11q23 in B cell chronic lymphocytic leukemia.

Loss of genomic material in 11q is one of the most common structural chromosome aberrations in B cell chronic lymphocytic leukemia (B-CLL). In order to characterize the deletions of 11q23 in B-CLL, we performed fluorescence in situ hybridization (FISH) with eight YAC (yeast artificial chromosome) probes on peripheral leukocytes of 30 patients. These YACs form a contig spanning 7.8 Mb at 11q23.1-q23.3. We found deletions in nine out of 30 cases (30%) and five of them had discontinuous deletions in this region. The region represented by YAC 755b11 (1.6 Mb in size) was involved in all cases with deletions, supporting the hypothesis that this region might contain a novel gene of pathogenic importance to B-CLL. A more distal region represented by YAC 785e12 (760 kb in size) was deleted frequently and specifically. Whether there is another novel gene of pathogenic importance to B-CLL and what is its potential relationship to the deletions in the region represented by YAC 755b11, are issues that require further studies.     

Alkylating agents and nucleoside analogues in the treatment of B cell chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in the Western world. The natural clinical course is highly variable and chemotherapy is usually not indicated in early and stable disease. Treatment is needed in the progressive form of this leukemia. Chlorambucil, with or without steroids, has been for many years the drug of choice in the treatment of CLL. More recently, treatment approaches have included nucleoside analogues, (NA) fludarabine (FAMP) and cladribine (2-CdA, 2-chlorodeoxyadenosine), which seem to be the treatment of choice for patients failing standard therapies. Their role as first line therapy is being investigated in randomized trials and the results have recently been published. These studies have shown a higher overall response and complete remission (CR) rate and longer response duration in patients treated initially with NA than with chlorambucil or cyclophosphamide-based combination regimens. In contrast, overall survival is similar in patients treated with NA and alkylating agents. However, the randomized trials were designed as crossover studies which may influence survival. Combined use of NA with other cytotoxic drugs, cytokines, monoclonal antibodies and other agents may increase the CR and prolong survival time. However, the results of randomized trials comparing combination treatment with NA alone are not yet available. In conclusion, alkylating agents still have an important place in the routine management of the majority of CLL patients. NA should be routinely used as second line treatment and possibly as first line therapy in younger patients, who are candidates for potentially curative treatment such as stem cell transplantation and/or monoclonal antibodies.     

Chromosome abnormalities in B cell chronic lymphocytic leukemia and their clinical correlations

Cytogenetic analysis was successfully performed on 31 of 40 patients with chronic B cell leukemia. Clonal abnormalities were seen in 16 patients using various culture methods. Fourteen of these had unstimulated cultures established of which 13 had the clonal abnormality. Trisomy 12 was observed in seven patients while a 14q32 translocation was present in four. Race, age, hemoglobin, WBC, percentage of lymphocytes and prolymphocytes in BM and PB, platelets, Smig, lymph node, spleen, liver, pattern of bone marrow infiltration, therapy free interval, and overall survival were all compared. Significant correlations between the presence of clonal abnormalities and prior therapy (p less than 0.005) and an increase in prolymphocytes in bone marrow (p = 0.05) and/or peripheral blood (p = 0.0014) were observed.     

Immune complexes and apoptosis in B-cell chronic lymphocytic leukemia

The progressive accumulation of B-cell chronic lymphocytic leukemia (B-CLL) cells in vivo is attributed to resistance to apoptosis, although this can be modulated in vitro by a variety of cellular and humoral factors (cell-cell, cell-matrix interactions, cytokines). We have previously reported that IgG immune complexes (IC) delay B-CLL cell apoptosis through a paracrine mechanism, which depends on monocytes and NK cells. On the other hand, despite the fact that IC effectively bind to type II Fc gammaRs expressed on B-CLL cells, they are unable to deliver transmembrane signals. We speculate that this lack of responsiveness of resting B-CLL cells to IC could be overcome by activation. The analysis of this possibility would be relevant since the presence of circulating IC is a common feature in B-CLL patients.