Apoptosis Of B-chronic Lymphocytic Leukemia Cells Research Articles

CPG Oligodeoxynucleotides Enhance Immunogenicity In Vitro in All Cytogenetic Subgroups of B-Cell Chronic Lymphocytic Leukemia (B-CLL), but Preferentially Augment Apoptosis in B-CLL with Good Prognosis Cytogenetics

CPG Oligodeoxynucleotides Enhance Immunogenicity In Vitro in All Cytogenetic Subgroups of B-Cell Chronic Lymphocytic Leukemia (B-CLL), but Preferentially Augment Apoptosis in B-CLL with Good Prognosis Cytogenetics

Tetrocarcin-A—induced ER stress mediates apoptosis in B-CLL cells via a Bcl-2—independent pathway

AbstractTetrocarcin-A (TC-A), an antibiotic agent isolated from actinomycetes, has recently been described to antagonize Bcl-2 functions, thereby sensitizing tumor cells to cell death signals under control of Bcl-2. In this study, we analyzed the direct proapoptotic effect of TC-A in the B-chronic lymphocytic leukemia (B-CLL) model. We focused on the signal cascade triggered by TC-A in B-CLL cells and identified activated mitochondrial as well as endoplasmatic reticulum (ER) stress signals. The expression levels of known effector molecules mediating mitochondrial signaling, such as Bax and Bid, and the antagonistic molecule Bcl-2 did not influence sensitivity of B-CLL cells to TC-A. Furthermore, the molecular chaperone and sensor of ER stress, HSP70, though significantly up-regulated in B-CLL cells undergoing TC-A—triggered apoptosis, was ineffective to exert its anti-apoptotic function described in multiple cell death pathways. Autologous T cells of B-CLL patients were significantly less sensitive to TC-A as were also T cells from healthy donors when compared with their normal B-cell fraction. Furthermore, sensitivity of B-CLL cells to TC-A treatment in vitro was dependent neither on the expression levels of CD38—a prognostic factor for survival of B-CLL patients as well as for their response to therapy—nor on the clinical stage or pretreatment status of patients. From our data showing that TC-A induced a cell death pathway via ER stress preferentially in B cells and that it acted independently of important markers of drug sensitivity and of clinical markers, we conclude that TC-A might represent an attractive candidate drug for further evaluation in preclinical trials.

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.

Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the defect of apoptosis in B-CLL: association with protein kinase Cδ

In the present study we analyzed the role of phophatidylinositol-3 kinase (PI-3K) in B chronic lymphocytic leukemia (B-CLL) cells. PI-3K is activated by many stimuli and is linked to several different signaling pathways. We demonstrated that inhibition of PI-3K by a specific inhibitor, LY294002, induced apoptosis in B-CLL cells in vitro. This effect was specific for the inhibition of PI-3K because inhibition of other signaling pathways such as extracellular signaling-regulated kinase (ERK), p38, or p70S6 kinase did not affect spontaneous apoptosis. Furthermore, PI-3K was constitutively activated in freshly isolated B-CLL cells. Corresponding to enhanced apoptosis, LY294002 down-regulated expression of the antiapoptotic proteins X-linked inhibitor of apoptosis protein (XIAP) and Mcl-1. Next, we investigated which factors downstream of PI-3K were activated in B-CLL cells. We demonstrated that protein kinase B/Akt is expressed in all tested CLL samples but no activation of Akt was detected. In contrast, we observed a constitutive activation of protein kinase Cdelta (PKCdelta) in freshly isolated B-CLL cells. PKCdelta is linked to PI-3K and is phosphorylated at Thr505 in response to PI-3K activation. We further demonstrated that tyrosine phosphorylation and activity of PKCdelta were dependent on PI-3K activity in B-CLL cells. Inhibition of PKCdelta by the specific inhibitor Rottlerin strikingly enhanced apoptosis. In contrast, peripheral blood B cells of healthy donors were resistant to inhibition of PI-3K or PKCdelta. We conclude that activated PI-3K might be important in the pathogenesis of B-CLL, and survival signals might be mediated via PKCdelta. Therefore, inhibition of PI-3K or PKCdelta may be an innovative approach to treat B-CLL.

Interleukin-6 dimers produced by endothelial cells inhibit apoptosis of B-chronic lymphocytic leukemia cells

AbstractTumoral lymphocytes from patients with B-chronic lymphocytic leukemia (B-CLL) are long-lived cells in vivo, but they die rapidly by apoptosis in vitro. Here, it is reported that endothelial cells (ECs) inhibit the apoptosis of B-CLL cells, as determined by 4 different flow cytometric methods, and that this antiapoptotic effect is mediated mainly by soluble factor(s), as can be deduced from the following findings. First, EC-conditioned medium (ECCM) inhibited the apoptotic rate in B-CLL to approximately 50% of control. Second, the antiapoptotic effect mediated by EC/B-CLL cell contact was more apparent than real; using a fluorescence-based phagocytosis assay, it was demonstrated that this effect was due to the phagocytic capacity of ECs, which internalized apoptotic cells. Third, the protective effect of ECCM was associated neither with proliferation nor differentiation signals. Fourth, the survival factor was a dimeric form of IL-6 because anti–IL-6 antibodies completely neutralized the antiapoptotic effect mediated not only by the crude ECCM but also by the 45- to 55-kd active fractions obtained after gel filtration, which contained high levels of IL-6. These IL-6 dimers (IL-6D) were noncovalently associated. Sixth, human recombinant IL-6D(hrIL-6D) inhibited B-CLL apoptosis, whereas hrIL-6 monomers (hrIL-6M) did not. Binding and functional competition experiments showed not only that monomers and dimers had similar affinity for the IL-6R, but also that hrIL-6Minhibited the antiapoptotic activity of hrIL-6D. These data suggest that IL-6D derived from ECs promote the survival of B-CLL cells.

Apoptosis or plasma cell differentiation of CD38-positive B-chronic lymphocytic leukemia cells induced by cross-linking of surface IgM or IgD

Previously, we demonstrated that B-chronic lymphocytic leukemia (B-CLL) cells could be divided into 2 groups depending on the expression of CD38 by the malignant cells. The 2 groups differed in their signal-transducing capacities initiated by cross-linking of surface IgM; only in CD38-positive cells was an efficient signal delivered, invariably resulting in cell apoptosis. In this study, we investigated the effect of surface IgD cross-linking in 10 patients with CD38-positive B-CLL. Exposure of the malignant cells to goat antihuman delta-chain antibodies (Gadelta-ab) caused [Ca(++)]i mobilization and tyrosine kinase phosphorylation in a manner not different from that observed after goat antihuman mu-chain antibody (Gamu-ab) treatment in vitro. However, Gadelta-ab-treated cells failed to undergo apoptosis and instead displayed prolonged survival in culture and differentiated into plasma cells when rIL2 was concomitantly present. Cross-linking of surface IgD failed to induce proliferation of the malignant cells in vitro. Moreover, treatment with Gadelta-ab did not prevent apoptosis of B-CLL cells induced by Gamu-ab. Collectively, these experiments demonstrated that IgM and IgD expressed by the same cell may deliver opposite signals under particular circumstances and provide some clues for the understanding of the pathophysiology of B-CLL. (Blood. 2000;95:1199-1206)

Antisense-mediated suppression of Bcl-2 highlights its pivotal role in failed apoptosis in B-cell chronic lymphocytic leukaemia.

Although advances have been made in the development of more effective treatment modalities, B-cell chronic lymphocytic leukaemia (B-CLL) remains incurable due to the development of drug resistance. Defective programmed cell death mechanisms rather than dysregulation of cell cycle appears to predominate in B-CLL and it is likely that a failure to initiate apoptosis contributes to chemoresistance. Most B-CLL cells contain high levels of the anti-apoptotic protein Bcl-2 and high Bcl-2/Bax ratios have been associated with in vitro resistance to cytotoxic agents. In this study we evaluated the cellular responses to a Bcl-2 antisense oligonucleotide in terms of Bcl-2 mRNA and protein expression and the induction of apoptosis. The antisense molecule induced a specific reduction in Bcl-2 mRNA and protein expression over the 48 h culture period and was associated with increased apoptosis. The study indicates that Bcl-2 protein is central to the mediation of resistance to apoptosis in B-CLL. Therefore Bcl-2 antisense oligonucleotides might be useful in the treatment of B-CLL.

In Vitro Evaluation of Fludarabine in Combination With Cyclophosphamide and/or Mitoxantrone in B-Cell Chronic Lymphocytic Leukemia

B-chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5(+) B lymphocytes. We have analyzed the effect in vitro of the combination of fludarabine with cyclophosphamide and/or mitoxantrone on cells from 20 B-CLL patients. Mafosfamide, the active form of cyclophosphamide in vitro, increased the cytotoxicity of fludarabine in all of the patients studied and produced a significant synergistic effect (P <.01) after 48 hours of incubation. The addition of mitoxantrone to this combination increased the cytotoxic effect in cells from 8 patients, but in the remaining 12 patients no significant increase was observed. The effect of fludarabine and mafosfamide was dose-dependent. Mafosfamide and fludarabine had a synergistic effect in inducing apoptosis of B-CLL cells as determined by DNA staining with propidium iodide and analysis of phosphatidylserine exposure. Mafosfamide significantly increased the apoptosis induced by fludarabine on CD19(+) cells (P =.007), but not on CD3(+) cells (P =. 314). Cell viability was correlated with a decrease in Mcl-1 levels and an increase in p53 levels. These results support that fludarabine in combination with cyclophosphamide and/or mitoxantrone can be highly effective in the treatment of B-CLL.

Ceramide-induced killing of normal and malignant human lymphocytes is by a non-apoptotic mechanism.

Synthetic ceramides induce apoptotic death of Jurkat and HL60 leukaemia cell lines. By contrast we show here that ceramide induces non-apoptotic killing of malignant cells from patients with B-chronic lymphocytic leukaemia (B-CLL) and of normal B lymphocytes. The protein phosphatase inhibitor okadaic acid readily induces apoptosis of B-CLL cells, indicating that this death pathway is fully functional in these cells. The ability of ceramide to activate the apoptotic protease caspase 3 in HL60 cells but not in B-CLL cells, as well as the lack of correlation of ceramide-mediated killing of different B-CLL isolates with expression of the apoptosis-regulating proteins bcl-2 and bax reinforce the conclusion that ceramide killing of B-CLL cells is by a non-apoptotic mechanism. Fludarabine treatment or gamma-irradiation of B-CLL cells resulted in ceramide elevation and in killing by both apoptotic and non-apoptotic mechanisms, suggesting that a ceramide-triggered non-apoptotic mechanism may play a role in the killing of these cells. Therefore, the results here show that ceramide can induce either apoptotic or non-apoptotic death, depending on the cellular context. The inability of synthetic dihydroceramide to kill B-CLL cells or normal B lymphocytes suggests that non-apoptotic killing by ceramide is via interaction with a specific, but unidentified, cellular target.

Aspirin and Salicylate Induce Apoptosis and Activation of Caspases in B-Cell Chronic Lymphocytic Leukemia Cells

AbstractWe analyzed the effect of aspirin, salicylate, and other nonsteroidal antiinflammatory drugs (NSAIDs) on the viability of B-chronic lymphocytic leukemia (B-CLL) cells. Aspirin induced a decrease in cell viability in a dose- and time-dependent manner. The mean IC50 for cells from 5 patients was 5.9 ± 1.13 mmol/L (range, 4.4 to 7.3 mmol/L). In some cases, 2.5 mmol/L aspirin produced an important cytotoxic effect after 4 days of incubation. No effect was observed with other NSAIDs, at concentrations that inhibit cyclooxygenase, such as ketorolac (10 μmol/mL), NS-398 (100 μmol/mL), or indomethacin (20 μmol/mL), thus suggesting the involvement of cyclooxygenase-independent mechanisms in aspirin-induced cytotoxicity. Salicylate also produced dose-dependent cytotoxic effects on B-CLL cells and the mean IC50 for cells from 5 patients was 6.96 ± 1.13 mmol/L (range, 5 to 7.8 mmol/L). Both aspirin and salicylate induced DNA fragmentation and the proteolytic cleavage of poly(ADP(adenosine 5′-diphosphate)-ribose) polymerase (PARP), demonstrating that both compounds induce apoptosis of B-CLL cells. Finally, inhibition of caspases by Z-VAD.fmk blocked proteolytic cleavage of PARP, DNA fragmentation, and cytotoxicity induced by aspirin. Mononuclear cells from normal donors showed a lower sensitivity than cells from B-CLL patients to aspirin as determined by analysis of cell viability. B and T lymphocytes from normal donors and T lymphocytes from CLL patients are more resistant to aspirin-induced apoptosis, as determined by analysis of phosphatidylserine exposure. These results indicate that aspirin and salicylate induce apoptosis of B-CLL cells by activation of caspases and that this activation involves cyclooxygenase-independent mechanisms.© 1998 by The American Society of Hematology.

Aspirin and Salicylate Induce Apoptosis and Activation of Caspases in B-Cell Chronic Lymphocytic Leukemia Cells

We analyzed the effect of aspirin, salicylate, and other nonsteroidal antiinflammatory drugs (NSAIDs) on the viability of B-chronic lymphocytic leukemia (B-CLL) cells. Aspirin induced a decrease in cell viability in a dose- and time-dependent manner. The mean IC50 for cells from 5 patients was 5.9 ± 1.13 mmol/L (range, 4.4 to 7.3 mmol/L). In some cases, 2.5 mmol/L aspirin produced an important cytotoxic effect after 4 days of incubation. No effect was observed with other NSAIDs, at concentrations that inhibit cyclooxygenase, such as ketorolac (10 μmol/mL), NS-398 (100 μmol/mL), or indomethacin (20 μmol/mL), thus suggesting the involvement of cyclooxygenase-independent mechanisms in aspirin-induced cytotoxicity. Salicylate also produced dose-dependent cytotoxic effects on B-CLL cells and the mean IC50 for cells from 5 patients was 6.96 ± 1.13 mmol/L (range, 5 to 7.8 mmol/L). Both aspirin and salicylate induced DNA fragmentation and the proteolytic cleavage of poly(ADP(adenosine 5′-diphosphate)-ribose) polymerase (PARP), demonstrating that both compounds induce apoptosis of B-CLL cells. Finally, inhibition of caspases by Z-VAD.fmk blocked proteolytic cleavage of PARP, DNA fragmentation, and cytotoxicity induced by aspirin. Mononuclear cells from normal donors showed a lower sensitivity than cells from B-CLL patients to aspirin as determined by analysis of cell viability. B and T lymphocytes from normal donors and T lymphocytes from CLL patients are more resistant to aspirin-induced apoptosis, as determined by analysis of phosphatidylserine exposure. These results indicate that aspirin and salicylate induce apoptosis of B-CLL cells by activation of caspases and that this activation involves cyclooxygenase-independent mechanisms.© 1998 by The American Society of Hematology.

Interleukin 10 induces apoptotic cell death of B-chronic lymphocytic leukemia cells.

Recent studies have established that interleukin (IL)-10 induces growth and most notably differentiation of normal human B lymphocytes. We studied here the effects of IL-10 on the proliferation and survival of B-chronic lymphocytic leukemia (B-CLL) cells. IL-10 was found to inhibit 54-96% of the spontaneous tritiated thymidine incorporation observed in 3 of 12 B-CLL samples. Furthermore, IL-10 decreased the viable cell recovery of all five B-CLL samples tested, irrespective of whether cells were spontaneously synthesizing DNA or not. After 1 wk, B-CLL populations cultured with IL-10 were lost while those cultured without IL-10 survived. Flow cytometric analysis, DNA gel electrophoresis, and Giemsa staining all revealed that IL-10 induced B-CLL cells to die from apoptosis. This IL-10-mediated apoptosis was dose dependent and specific as it could be inhibited by a neutralizing anti-IL-10 antibody. B-CLL cells undergoing apoptosis in response to IL-10 showed decreased Bcl-2 protein levels. Addition of IL-2, IL-4, interferon gamma, and anti-CD40 monoclonal antibody prevented the IL-10-mediated apoptosis of B-CLL cells. None of the malignant B cell populations obtained from eight non-Hodgkin's lymphomas and three hairy cell leukemias underwent apoptosis after IL-10 treatment, thus suggesting that the apoptotic effect of IL-10 is specific for B-CLL cells. Thus, IL-10 inhibits the DNA synthesis and most notably the survival of B-CLL cells, findings that call for considering IL-10 in the immunotherapy of chemoresistant B-CLL.

Interleukin 4 protects chronic lymphocytic leukemic B cells from death by apoptosis and upregulates Bcl-2 expression.

B chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of slow-dividing and long-lived monoclonal B cells arrested at the intermediate stage of their differentiation. We previously showed that interleukin 4 (IL-4) not only inhibits but also prevents the proliferation of B-CLL cells. We report here that IL-4 protects the B-CLL cells from death by apoptosis (programmed cell death [PCD]). IL-4 inhibits spontaneous and hydrocortisone (HC)-induced PCD of highly purified B cells from 12 unselected CLL patients, as shown by sustained cell viability and lack of DNA fragmentation. IL-1, -2, -3, -5, -6, -7, tumor necrosis factor alpha, and transforming growth factor beta have no protective effect. The in vitro rescue from apoptosis by IL-4 is reflected by an increased expression of Bcl-2 protein, a proto-oncogene directly involved in the prolongation of cell survival in vivo and in vitro. Hence, IL-4-treated B-CLL cells express significantly more Bcl-2 than unstimulated, HC-treated, or fresh B-CLL cells. Furthermore, subcutaneous injection of IL-4 into one CLL patient enhances Bcl-2 protein expression in the leukemic B cells. These data may suggest that IL-4 prevents apoptosis of B-CLL cells using a Bcl-2-dependent pathway. Given our recent observations that fresh T cells from B-CLL patients express IL-4 mRNA, we propose that IL-4 has an essential role in the pathogenesis of CLL disease, by preventing both the death and the proliferation of the malignant B cells.