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.     

Bcl-2/Bax ratios in chronic lymphocytic leukaemia and their correlation with in vitro apoptosis and clinical resistance.

The bcl-2 gene is overexpressed in the absence of gene rearrangements in most cases of B-cell chronic lymphocytic leukaemia (B-CLL) and the proto-oncogene product Bcl-2 has been shown to be a regulator of apoptosis. The activity of this protein is opposed by Bax, a homologous protein that accelerates the rate of cell death. B-lymphocyte Bcl-2 and Bax protein levels were found to be significantly altered in B-CLL and increased Bcl-2/Bax ratios were observed in both the treated and untreated patients compared with those of normal controls. These alterations were particularly pronounced in those treated patients found to be clinically unresponsive to chemotherapy. In order to determine whether Bcl-2/Bax ratios affected cell survival via an anti-apoptotic mechanism, cell death was induced in B-CLL cells in vitro using chlorambucil, and apoptosis was monitored by Annexin V and propidium iodide staining. Confirmation that the labelled cells were apoptotic was achieved by morphological assessment of cytospin preparations of cell-sorted populations. Drug-induced apoptosis in B-CLL cells was inversely related to Bcl-2/Bax ratios.     

Active alpha 2 and beta adrenoceptors in lymphocytes from patients with chronic lymphocytic leukemia.

A single population of alpha 2 adrenoceptors was characterized in intact B lymphocytes from patients with chronic lymphocytic leukemia (B-CLL) by binding and saturation studies with 3H labelled yohimbine and by competition studies with alpha adrenergic antagonists. While the affinity of B-CLL alpha 2 adrenoceptors was low (KD: 9.81 nM-20.98 nM), each cell expressed a large number of receptors. No binding of the alpha 1 adrenoceptor antagonist prazosin was observed and the binding properties of beta adrenergic receptors (assayed with 3H-labelled dihydroalprenolol), were similar to those described for normal lymphocytes. Reaction of B-CLL with the beta adrenergic agonist isoproterenol raised the levels of cAMP in 11/13 patients tested, and in 8 of these, incubation with the alpha 2 adrenergic agonist clonidine prevented the effect and reduced the basal cAMP levels. The presence of active alpha 2 adrenoceptors on B-CLL lymphocytes may be involved in the regulation of metabolic pathways that affect cell functions and favor neoplastic growth.     

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.     

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   

Activation of the alpha2beta1 integrin prevents c-erbB2-induced scattering and apoptosis of human mammary epithelial cells in collagen

Constitutive overexpression of c-erbB2 in the mammary epithelial cell line MTSV1-7 has been shown to result in epithelial-mesenchymal conversion, anchorage-independent growth and loss of organized morphogenesis in collagen. To elucidate the events leading to this drastic change, MTSV1-7 cells and its subclone HB2 (which shows a more strictly epithelial phenotype) were transfected with the hybrid trk-neu receptor consisting of the extracellular domain of the trkA nerve growth factor (NGF) receptor and the transmembrane and cytoplasmic domains of c-erbB2 (neu). In cells expressing this construct, c-erbB2 homodimerization can be mimicked by addition of NGF. In trk-neu transfectants of HB2 cells, modest expression led to increased cell proliferation upon NGF treatment. When clones with higher expression levels were grown in collagen, NGF instead induced cell scattering, diminished viability and dramatically increased apoptosis. Interestingly, both the dissociation of colonies and loss of cell viability could be completely reversed by treatment of the cells with antibodies that activate the adhesive capacity of the alpha2beta1 integrin. Long-term NGF treatment of high-expressing transfectants generated fibroblastic clones displaying a reduced expression of integrin alpha2 and E-cadherin, and extensive apoptosis in collagen. These results, which indicate that strong c-erbB2 signalling may lead to downregulation and/or inactivation of the alpha2beta1 integrin, promoting apoptosis in collagen, provide one possible explanation to the increased apoptosis frequently seen in early tumour development.     

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.     

T cell receptor alpha expression in B-type chronic lymphocytic leukemia

Normal B lymphocytes are characterized by rearrangement and expression of immunoglobulin genes, but not of T cell receptor genes. These properties might assist in lineage assignment, but there are examples of fresh leukemic cells and of cell lines where exceptions to this rule have been noted. We have studied cell samples of patients with B-CLL for expression of TCR alpha and beta chain genes. Using in situ hybridization with fluorescein-labeled probes, TCR alpha mRNA was found to be expressed in 14 of 18 samples and TCR beta mRNA in 7 of 16 samples. Specificity of hybridization was demonstrated by near complete blockade of TCR alpha hybridization with unlabeled TCR alpha, but not with unlabeled TCR beta probe. Furthermore, in Northern blot analysis a truncated 1,4 kb message for TCR alpha was readily detectable. No significant cell surface staining with the anti-TCR alpha/beta monoclonal antibody WT31 was observed. A contribution of T cells within the leukemic sample could be excluded since only samples with leukemic cell counts of greater than 50,000 cells/mm3 and only samples with 5% or less CD2+ T lymphocytes were studied. Our data show that a large proportion of B-CLL samples may express a truncated version of the TCR alpha message, indicating that this gene can be activated in leukemic B cells frozen at a late stage of differentiation.     

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.     

Interleukins, TNF-alpha and beta-2M in patients with B cell chronic lymphocytic leukemia

In order to investigate the possible existence of a prognostic factor for B cell chronic lymphocytic leukemia (B-CLL), we determined the serum levels of TNF-alpha, IL-1a, IL-1b, IL-2, sIL-2R, IL-6, IL-10 and beta-2M in 20 patients. We observed significant changes in sIL-2R and beta-2M levels, whereas in all stages of disease, TNF-alpha and other interleukins exhibited only mild changes. An excellent correlation between sIL-2R and beta-2M levels and disease activity wes reported. Patients with aggressive disease (Rai stages III and IV and Richter's syndrome) had increased levels. Patients who responded to therapy and with improved clinical status had decreased sIL-2R and beta-2M levels. However, patients with progressive disease and no response to therapy were associated with increased levels of sIL-2R and beta-2M. In conclusions, as serum levels of sIL-2R and beta-2M are increased in the aggressive stages of B-CLL, they may be used as reliable markers for monitoring B-CLL activity, showing response to treatment and early relapse and/or disease progression.     

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.     

Inhibition of PDE3B augments PDE4 inhibitor-induced apoptosis in a subset of patients with chronic lymphocytic leukemia.

PURPOSE: cAMP phosphodiesterase (PDE) 4 is a family of enzymes the inhibition of which induces chronic lymphocytic leukemia (CLL) apoptosis. However, leukemic cells from a subset of CLL patients are relatively resistant to treatment with the PDE4 inhibitor rolipram, particularly when this drug is used in the absence of an adenylate cyclase stimulus such as forskolin. Elevated cAMP levels induce compensatory up-regulation of several cyclic nucleotide PDE families in other model systems. We here examine the hypothesis that CLL cells that survive treatment with rolipram do so as a result of residual PDE activity that is not inhibited by this drug. EXPERIMENTAL DESIGN: We examined by Western analysis the effect of rolipram treatment on CLL expression of PDE3B, PDE4A, PDE4B, PDE4D, and PDE7A. We also examined the ability of rolipram (PDE4 inhibitor) or cilostamide (PDE3 inhibitor), alone or together, to induce apoptosis or elevate cyclic AMP in leukemic cells from patients with CLL. RESULTS: Rolipram increased levels of PDE4B and, to a variable extent, PDE4D. When combined with forskolin, rolipram also increased levels of a second family of PDEs, PDE3B. Addition of the specific PDE3 inhibitor, cilostamide, modestly augmented rolipram-induced apoptosis in five of seven "rolipram-resistant" CLL samples. CONCLUSIONS: Although this work confirms that PDE4 appears to be the most important PDE target for induction of apoptosis in CLL, combination therapy with PDE3 and PDE4 inhibitors or use of dual-selective drugs may be of benefit in a subset of relatively PDE4-inhibitor resistant CLL patients.     

Role of caspases and apoptosis-inducing factor (AIF) in cladribine-induced apoptosis of B cell chronic lymphocytic leukemia.

We have evaluated the role of caspases and the mitochondrial apoptosis inducing-factor (AIF) in apoptosis induced by cladribine (2CdA), in vitro, in cells from patients of B-CLL and in peripheral blood lymphocytes from normal donors. In sensitive B-CLL cells, apoptosis was characterized by cell shrinking, loss of mitochondrial membrane potential (DeltaPsi(m)), phosphatidylserine exposure, activation of caspases 3, 7, 8 and 9, reduction of Mcl-1 levels, translocation of AIF from mitochondria to nucleus and chromatin condensation. No significant variations in the levels of Bcl-2, Bax and Bak proteins were noticed upon treatment with 2CdA. Co-treatment of cells with the pan-caspase inhibitor Z-VAD-fmk attenuated some morphological and biochemical characteristics of apoptosis and delayed 2CdA-induced DeltaPsi(m) loss, but did not prevent cell death. Z-VAD-fmk did not prevent 2CdA-induced AIF translocation but in this case apoptotic cells displayed only peripheral chromatin condensation, characteristic of AIF action. Reduced or negligible caspase 3 expression did not prevent 2CdA toxicity in cells from four patients. Cells from three patients that responded poorly to 2CdA lacked expression of caspases 9 or 3. Cells from another patient resistant to 2CdA expressed caspases 3, 7, 8 and 9 but they were not activated by treatment. These results indicate that execution of apoptosis is carried out independently by AIF and caspases, which are responsible for the development of apoptotic phenotype in response to 2CdA. Although caspases can also collaborate in DeltaPsi(m) loss, proapoptotic proteins from the Bcl-2 superfamily may be the key inducers of DeltaPsi(m) loss and apoptosis in B-CLL cells sensitive to 2CdA.     

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.     

The small organic compound HA14-1 prevents Bcl-2 interaction with Bax to sensitize malignant glioma cells to induction of cell death

A functional imbalance between proapoptotic Bax and antiapoptotic Bcl-2 is likely to participate in the resistance of cancer cells to therapy. We show here that ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1), a small organic compound recently proposed to function as an inhibitor of Bcl-2, increases the sensitivity of human glioblastoma cells to radiotherapy and chemotherapy. This sensitizing effect is lost if Bcl-2 expression, but not Bcl-xL expression, is knocked down or if cells only express a mutant of Bax that does not interact with Bcl-2. This points to a specific Bcl-2 inhibitory function of HA14-1 and implies that it selectively involves hindrance of Bcl-2 binding to Bax, which HA14-1 inhibits in cell-free assays and in cells in receipt of an apoptotic stimulation. Moreover, HA14-1, in combination with a cytotoxic treatment, slows down the growth of glioblastoma in vivo. Thus, the inhibition of Bcl-2 achieved by HA14-1 might improve treatment outcome.     

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.