Survival Of B-CLL Cells Research Articles

Potent Antineoplastic Activity of Two Inhibitors of Lymphoid Enhancer Binding Factor-1 (LEF-1) in Chronic Lymphocytic Leukemia (B-CLL).

Abstract 885Recent studies indicate factors governing aberrant activation of WNT signaling in chronic lymphoctic leukemia (CLL) cells. Thus, there is an increased secretion of WNT ligands indicating an autocrine loop leading to the extended survival of CLL cells. Lymphoid enhancer binding factor-1 (LEF-1) is a potent transcription factor regulating the expression of several WNT induced target genes. A comprehensive gene expression profiling from two independent studies revealed that LEF-1 mRNA was ∼3,000 fold overexpressed in B-CLL when compared to its healthy counterpart. Hence LEF-1 is a transcription factor expressed exclusively in CLL cells.The objective of this present study is to demonstrate the therapeutic benefit of inhibiting LEF-1 expression in B-CLL cells using novel small molecule inhibitors CGP049090 and PKF115-584 in vivo and in vitro. JVM-3 cells and primary CLL cells were investigated by siRNA mediated knock down of LEF-1 and viability was assayed after 16h of incubation by flow cytometry. In vitro cytotoxicity and IC50 of the two compounds was enumerated using ATP based cell viability assay. Apoptotic response was investigated in time course experiments. Specificity of the small molecules was demonstrated by co-immunoprecipitation experiments for the LEF-1/βcatenin interaction in primary CLL cells. In vivo efficacy of the small molecules inhibitors were studied using a JVM-3 subcutaneous xenograft model in nu/nu mice.The results indicate there is a high protein expression and nuclear localization of LEF-1 and β-catenin indicating active LEF-1 mediated transcription in CLL cells, whereas LEF-1 remained undetectable in healthy B cells. Preliminary experiments of LEF-1 inhibition using siRNAs resulted in increased apoptosis indicating LEF-1 to be an important player in the survival of B-CLL cells. This observation was extended using CGP049090 and PKF-115584 as they induce both dose and time dependent cytotoxicity in B-CLL, whereas healthy B cells are not significantly affected. The IC50 for CGP049090 and PKF-115584 in CLL cells were 0.7 μM and 0.9 μM, respectively. Healthy B cells were not significantly affected, as ascertained by the fact that IC50 values could not be reached due to lacking total cell death. CGP049090 and PKF-115584 induced apoptotic cell death in primary CLL cells and cell lines by cleavage of caspases 8, 9, 3 and 7 and subsequent cleavage of poly (adenosine diphospate-ribose) polymerase (PARP). Both the inhibitors also altered the expression of several anti apoptotic proteins like XIAP, mcl-1 and bcl-2. Co-immunoprecipitation experiments revealed that both the inhibitors effectively break the β-catenin/LEF-1 interaction, resulting in down regulation of expression of LEF-1 target genes such as c-myc, cyclin D1 and LEF-1. Furthermore, the inhibitors were tested in an in vivo JVM-3 subcutaneous xenograft nude mouse model resulted in >70% inhibition of tumor growth and an increase in the median survival of the treated group without leading to systemic toxicity. Immunohistochemistry analysis of the tumor sections revealed LEF-1 down regulation paralleled by inhibition of proliferation by down regulation of Proliferating Cell Nuclear Antigen (PCNA) and increase in apoptosis (induction of cleaved PARP). In summary, we show that LEF-1 is a potential therapeutic target in the treatment of CLL. Both CGP049090 and PKF115-584 show potent inhibitory effects on the survival of CLL cells in vitro and in vivo without affecting healthy B-cells, suggesting them as potential anti-cancer agents in CLL and other neoplastic malignancies with aberrant LEF-1/TCF transcriptional activity. Further investigations are warranted to determine the feasibility of these small molecules for therapeutic approaches in humans. Disclosures:Schlösser:Novartis: Employment. Schmitt:Novartis: Employment. Hallek:BayerScheringAG: Honoraria, Research Funding.

Role of Platelet-Derived Growth Factor On Vascular Endothelial Growth Factor Synthesis by Mesenchymal Stem Cells in Chronic Lymphocytic Leukemia: Implications for Clinical Progression.

Abstract 804 Background:We have demonstrated that crosstalk between CLL B-cells and marrow-derived mesenchymal stem cells (MSC) can modulate the activation of both. Soluble factors in the CLL-condition media (CM) regulate the migration and proliferation of MSC. We previously found that platelet-derived growth factor (PDGF) receptors were selectively activated in MSC by CLL-CM and are necessary for the downstream Akt activation in MSC. PDGF is capable of promoting vascular endothelial growth factor (VEGF) production in MSC; however, several questions remain unanswered. Can CLL B-cells produce PDGF; is PDGF the specific mediator in CLL-CM responsible for PDGFR activation in MSC; what is the mechanism of PDGF mediated VEGF production in MSC; and is there any clinical significance of PDGF mediated VEGF production by MSC for CLL patients? Methods:MSC were generated from CLL patients and controls as previously described. To determine the specific ligand responsible for PDGFR activation mediated by CLL-CM blocking antibody to PDGF or VEGF or both was used to neutralize the binding activity of PDGF or VEGF in the CLL-CM. Subsequently, the pattern of membrane receptor activation was evaluated using a commercially available receptor tyrosine kinase (RTK) array assay. ELISA was performed to measure PDGF and VEGF levels from plasma and CM of CLL patients and normals. The protein expression of PDGF isoforms in CLL and normal B-cells was assessed using Western blot. To assess the mechanism of PDGF mediated VEGF production by MSC, we used either a PI3K inhibitor, LY294002 (20 mM), or a p38 MAPK inhibitor, SB202190 (100 nM), to block these pathways in MSC. Results:The known ligands for PDGF receptors, PDGF and VEGF, were detected in the CLL-CM (PDGF, 113.7 ± 23.6 pg/ml, VEGF, 186.94 ± 54.57 ng/ml; n = 12). We also found that PDGF is present in CLL cells by Western blot analysis and expression of PDGF in CLL cells was up regulated by hypoxia. By using PDGF- or VEGF-blocking antibody or both to neutralize the binding activity of PDGF or VEGF in CLL-CM, we found only PDGF-blocking antibody was able to neutralize the majority of PDGFRa activation, indicating that PDGF in the CM is the predominant ligand for PDGFR activation in MSC. CLL MSC was exposed to either pooled CLL (n = 5) or pooled normal plasma (n = 5). Interestingly, PDGFR was more potently activated by CLL plasma than the normal plasma, suggesting PDGF levels were elevated in the CLL plasma. Indeed, PDGF in the normal plasma appeared to be much lower compared to the CLL plasma [normal (n = 11): 554.43 ± 88.55 pg/ml; CLL (n=43): 3115.36 ± 428.79 pg/ml. p < 0.0001]. In addition, the plasma PDGF level was found to be weakly correlated with VEGF level in 43 CLL patients assessed using Spearman correlation analysis (r = 0.3, p = 0.001). However, when CLL patients were grouped by clinical prognostic factors, the plasma PDGF level was found to be strongly correlated with plasma VEGF level in CLL patients with high-risk features including more advanced Rai stage 2-4 (r = 0.87, p < 0.0001), ZAP-70 positive (r = 0.8, p =0.04) and CLL patients who had required treatment (r = 0.8, p = 0.0008). To examine the mechanism for PDGF augmentation of VEGF by MSC, we found that PI3K pathway interruption using LY294002 (n = 6, p = 0.04), but not p38 MAPK inhibition (n = 6, p = 0.5), was able to abrogate the PDGF mediated VEGF production in MSC. Importantly, we also found that the secreted VEGF level in the CM of unstimulated CLL MSC appeared to be significantly higher compared to the level from culture media of unstimulated normal MSC [CLL (n = 14) vs. normal (n = 5), 298.1± 61.2 vs. 109.9 ± 52.3, p = 0.03]. Conclusions:These results indicate that PDGF secreted by CLL B-cells is necessary for PDGFR activation in MSC and can lead to increased VEGF production by MSC via a PI3K-Akt dependent mechanism. These findings have clinical implications as plasma PDGF levels are strongly correlated with plasma VEGF levels in high risk CLL. The finding that CLL MSC are more prone to secrete VEGF than normal MSC suggests the stromal microenvironment may function to facilitate CLL disease progression. Finally, as VEGF is known to enhance CLL B-cell survival and drug resistance, interrogation of the PDGF-VEGF regulation in the microenvironment of CLL patients should yield important information that can be used for therapeutic strategies in CLL. Disclosures:Kay:Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.

CCR4: TARC Interaction Provides Supplementary Pro-Survival and Proliferative Signals to Chronic Lymphocytic Leukemia Cells.

Abstract 2327Poster Board II-304A wealth of information is accumulating on a role for antigenic stimulation in the natural history of chronic lymphocytic leukemia (B-CLL). Depending on the specificity of antigen:B-cell receptor (BCR) interactions, mature B cells can recognize and respond to microbial antigens via their BCRs and via Toll-like receptors (TLRs), possibly in a costimulatory manner. Chemokines and their receptors (CXCR3, CXCR4 and CCR7) also play critical roles in leucocyte trafficking as well as cell survival and expansion in B-CLL. Here we report our findings on CC chemokine receptor 4 (CCR4) expression and function in B-CLL. PBMCs from 64 B-CLL cases were screened for the expression of CCR4 on CD5+ B cells. CCR4 expression did not differ significantly when cases were grouped on the basis of IgV gene mutations. However when segregated by expression of CD38, cases with high CD38 positivity (>30%) showed significantly higher percentages of CCR4-expressing cells (52.8 ± 5.2; p< 0.01) than cases with low CD38 positivity (33.7 ± 5.3%). Surprisingly, among CD38 high expressers, those with mutated IgV genes exhibited even more CCR4-expressing cells (78.7 ± 7.8 %; p< 0.01) than those with unmutated IgV genes (44.6 ± 6.4%). Various functional outcomes of CCR4 interaction with its ligand, Thymus and Activation Regulated Chemokine, TARC (CCL17) were assayed. Using Phosphoflow, phosphorylation of Akt, Erk, MAPK, Syk, Btk, NF-kB, STAT-3 and STAT-5 in response to varying concentrations of TARC (0.04-25ng/ml) was assessed. Significant increases in phosphorylation of Btk and STAT-5 were detected in response to varying doses of TARC in different cases. However, TARC did not elicit noticeable changes in phospho- Akt, -Erk, -MAPK, -Syk, -NF-kB and -STAT-3. Based on the percentages of CCR4-expressing cells in B-CLL clones, TARC specifically induced decreases in surface CCR4 expression within 15 minutes, whereas it also exhibited indirect effects on reduction of surface expression CXCR3 and CXCR4 expression in ∼60% of the cases studied. Furthermore a chemotactic response (migration) of CLL cells (studied using transwell cultures) was observed within a 2 hour exposure in the same dose range as above. TARC also exhibited significant downstream effects. It independently rescued CCR4+ B-CLL cells from apoptosis (25 - 32%) as assessed by Annexin V/PI staining after 1 day exposure. Concomitantly in those cases in which B-CLL cells were rescued from apoptosis at day 1, TARC induced proliferation of the leukemic cells in a dose-dependent manner (3H thymidine uptake at day 3). Although there was no appreciable change in percentage of cells expressing CCR4, increases in the density of CCR4 expression were inducible in response to [1] crosslinking BCR with monoclonal anti-IgM antibodies conjugated to dextran (1.47 ± 0.2 fold), [2] TLR agonist, ODN 2006 (1.63 ± 0.26 fold), [3] crosslinking with anti-CD40 mAb + IL-4 (1.9 ± 0.4 fold), and [4] in response to interaction of CD38 with CD31-expressing fibroblasts (1.27 ± 0.09 fold) when B-CLL cells from 8 IgM+ cases were cultured with these stimuli for a 3 day period. These findings suggest a novel role for CCR4:TARC interaction in promoting B-CLL cell survival and promoting cell cycle progression downstream of BCR and TLR triggering in the presence/absence of residual T cell help. This knowledge could specifically aid in understanding the progression of disease in the B-CLL cases with mutated IgV genes and high percentages of CD38+ cells. Disclosures:No relevant conflicts of interest to declare.

Hedgehog-Induced Survival of B-Cell Chronic Lymphocytic Leukemia Cells in a Stromal Cell Microenvironment: A Potential New Therapeutic Target

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by an accumulation of neoplastic B cells due to their resistance to apoptosis and increased survival. Among various factors, the tumor microenvironment is known to play a role in the regulation of cell proliferation and survival of many cancers. However, it remains unclear how the tumor microenvironment contributes to the increased survival of B-CLL cells. Therefore, we studied the influence of bone marrow stromal cell-induced hedgehog (Hh) signaling on the survival of B-CLL cells. Our results show that a Hh signaling inhibitor, cyclopamine, inhibits bone marrow stromal cell-induced survival of B-CLL cells, suggesting a role for Hh signaling in the survival of B-CLL cells. Furthermore, gene expression profiling of primary B-CLL cells (n = 48) indicates that the expression of Hh signaling molecules, such as GLI1, GLI2, SUFU, and BCL2, is significantly increased and correlates with disease progression of B-CLL patients with clinical outcome. In addition, SUFU and GLI1 transcripts, as determined by real-time PCR, are significantly overexpressed and correlate with adverse indicators of clinical outcome in B-CLL patients, such as cytogenetics or CD38 expression. Furthermore, selective down-regulation of GLI1 by antisense oligodeoxynucleotides (GLI1-ASO) results in decreased BCL2 expression and cell survival, suggesting that GLI1 may regulate BCL2 and, thereby, modulate cell survival in B-CLL. In addition, there was significantly increased apoptosis of B-CLL cells when cultured in the presence of GLI1-ASO and fludarabine. Together, these results reveal that Hh signaling is important in the pathogenesis of B-CLL and, hence, may be a potential therapeutic target.

Expression Levels of CD38 in Tumor Cells and T-Cells Are of Prognostic Value in B-CLL.

Purpose: CD38 expression level of tumor cells was identified as predictor for progression, overall survival and response to therapy in B-chronic lymphocytic leukemia. Though CD38 is involved in effector functions of T-cells, the prognostic value of CD38 expression in these cells has not been addressed in B-CLL before.Patients and Methods: CD38 expression levels in B-CLL cells and T-cells from 170 patients were analyzed by flow cytometry and correlated with clinical and molecular risk parameters.Results: CD38 expression levels significantly differed not only in the neoplastic clone but also in T-cells from B-CLL patients with low vs advanced stage and stable vs progressive disease. Combined analysis of CD38 in T-cells and B-CLL cells identified 4 subgroups of patients with distinct clinical course. Multivariate analysis including clinical (Rai stage, anemia) and molecular risk parameters (Zap-70 expression levels, IgVH mutational status) identified CD38 expression levels in B-CLL (RR 6.9; 95% CI: 2.4 to 20.2; p=.0004) and T-cells (RR 2.4; 95% CI 1.2 to 4.8; p=.0106) as independent prognostic factors. Furthermore, CD38 expression in T-cells as a risk factor for increased peripheral tumor load and progressive disease was significantly associated with male gender.Conclusion: Combined analysis of CD38 in tumor cells and T-cells is superior in predicting outcome in B-CLL than either parameter alone. Our data on the prognostic value of CD38+ T-cells in B-CLL in general, and their identification as male risk factor in particular strongly encourage further studies on their role on survival and proliferation of B-CLL cells and the underlying mechanisms.

Involvement of protein kinase C and phosphatidylinositol 3–kinase pathways in the survival of B-cell chronic lymphocytic leukemia cells

AbstractB-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived CD5+ B lymphocytes. TPA (12-O-tetradecanoylphorbol 13- acetate) and interleukin-4 (IL-4) inhibit apoptosis of B-CLL lymphocytes ex vivo. We used specific inhibitors of protein kinase C (PKC), extracellular-regulated kinase (ERK), and phosphatidylinositol 3–kinase (PI3-kinase) to study their involvement in TPA- and IL-4–induced survival of B-CLL lymphocytes. BisI, a specific inhibitor of PKC, induced apoptosis and inhibited the antiapoptotic activity of TPA and IL-4. B-CLL cells have a basal PKC activity that was increased by TPA but not by IL-4. TPA, but not IL-4, induced ERK activation. However, the inhibition of ERK activation did not affect the viability of B-CLL lymphocytes, demonstrating that this pathway is not involved in their survival. Inhibition of PI3-kinase by LY294002 induced apoptosis of B-CLL cells and inhibited the survival effect of IL-4 and TPA. In addition, Akt, a downstream effector of PI3-kinase activity, was phosphorylated by TPA and IL-4 in B-CLL cells, though PI3-kinase had no effect on PKC-dependent phosphorylation of Akt. Furthermore, the inhibition of PKC or PI3-kinase increased dexamethasone- and fludarabine-induced apoptosis ex vivo in the presence of survival factors. These results demonstrate that PKC and PI3-kinase are involved in the survival of B-CLL cells and suggest that inhibitors of these pathways could be combined with the drugs used in the treatment of B-CLL.

Growth and survival of B-chronic lymphocytic leukaemia cells.

Although chronic lymphocytic leukaemia of B cell type (B-CLL) is the most common form of leukaemia in the Western world, several questions about the biology of B-CLL remain to be clarified. To obtain a conceptual model for B-CLL, defined as a relentless accumulation of resting B-CLL cells, it is particularly relevant to ask which cell type is the normal counterpart of B-CLL; what is the site of proliferation; which signals are involved in the recruitment and induction of proliferation and which signals contribute to the survival of the B-CLL cells? The significance of the studies on B-CLL cells in vitro for the interpretation of the in vivo situation may be questioned since they oversimplify the multiple and complex cellular interactions that occur in vivo. However, the in vitro studies have been instrumental in elucidating signals that may regulate growth, differentiation and survival of B-CLL cells. This knowledge, herein reviewed, can be used to put forward a hypothesis on B-CLL cell regulation in vivo.

Prognostic Significance of the Cell Cycle Inhibitor p27Kip1 in Chronic B-Cell Lymphocytic Leukemia

AbstractB-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of resting lymphocytes. The identification of p27kip1, a cyclin-dependent kinase inhibitor that contributes to cell cycle arrest and represents a link between extracellular signals and cell cycle, prompted us to study p27 protein in the lymphocytes from 88 patients with B-CLL and 32 patients with other chronic B-lymphoproliferative disorders. The expression of p27 protein was higher in B-CLL samples with variations among them. In B-CLL, p27 levels were independent of absolute number of circulating lymphocytes, but strongly correlated with both lymphocyte and total tumor mass (TTM) doubling time. High p27 expression was associated with a poorer overall prognosis. In vitro, there was an increased spontaneous survival of B-CLL cells expressing high p27 levels. Interleukin-4 (IL-4) upregulated p27 levels in B-CLL cells, while fludarabine decreased p27 levels. Thus, our results indicate that p27 may be a valuable kinetic marker in B-CLL by providing instantaneous estimation of the disease doubling time. In addition, these results suggest that there is a link between p27 expression and the ability of CLL cells to undergo apoptosis.

Chronic Lymphocytic Leukemic B Cells But Not Normal B Cells Are Rescued From Apoptosis by Contact With Normal Bone Marrow Stromal Cells

The leukemic B lymphocytes from chronic lymphocytic leukemic (CLL) patients have a long survival in vivo, although ex vivo they rapidly die by apoptosis. To further investigate the mechanism of this, we have studied the influence of bone marrow stromal cells from normal subjects on apoptosis of B-CLL cells and normal umbilical cord blood (UCB) B lymphocytes. After 48 hours of incubation in medium alone, leukemic and normal B cells showed, respectively, 22 ± 3% and 31 ± 5% of apoptosis. Cocultures with stromal cells reduced the percentage of leukemic cells undergoing apoptosis (8 ± 2%, P&lt; .0005) and prevented the loss of bcl-2 protein expression. In contrast, stromal cells slightly increased normal B-cell apoptosis (37 ± 6%). Direct contact between leukemic cells and stromal cells was found to be essential for inhibition of leukemic cell apoptosis; indeed, separation of leukemic cells from stromal cells by microporous membrane increased spontaneous apoptosis, and comparable results were obtained with stromal cell conditioned medium. The difference in behavior observed between normal and leukemic B cells plated on stromal cells can be explained by the fact that only a few normal B cells adhere to stromal cells in comparison with B-CLL cells. B-CLL cell adhesion to stromal cells is mediated by β1 and β2 integrins acting simultaneously. Contact between B-CLL cells and bone marrow stromal cells seems to play a major role in the accumulation and survival of B-CLL cells in the bone marrow.

Chronic Lymphocytic Leukemic B Cells But Not Normal B Cells Are Rescued From Apoptosis by Contact With Normal Bone Marrow Stromal Cells

AbstractThe leukemic B lymphocytes from chronic lymphocytic leukemic (CLL) patients have a long survival in vivo, although ex vivo they rapidly die by apoptosis. To further investigate the mechanism of this, we have studied the influence of bone marrow stromal cells from normal subjects on apoptosis of B-CLL cells and normal umbilical cord blood (UCB) B lymphocytes. After 48 hours of incubation in medium alone, leukemic and normal B cells showed, respectively, 22 ± 3% and 31 ± 5% of apoptosis. Cocultures with stromal cells reduced the percentage of leukemic cells undergoing apoptosis (8 ± 2%, P< .0005) and prevented the loss of bcl-2 protein expression. In contrast, stromal cells slightly increased normal B-cell apoptosis (37 ± 6%). Direct contact between leukemic cells and stromal cells was found to be essential for inhibition of leukemic cell apoptosis; indeed, separation of leukemic cells from stromal cells by microporous membrane increased spontaneous apoptosis, and comparable results were obtained with stromal cell conditioned medium. The difference in behavior observed between normal and leukemic B cells plated on stromal cells can be explained by the fact that only a few normal B cells adhere to stromal cells in comparison with B-CLL cells. B-CLL cell adhesion to stromal cells is mediated by β1 and β2 integrins acting simultaneously. Contact between B-CLL cells and bone marrow stromal cells seems to play a major role in the accumulation and survival of B-CLL cells in the bone marrow.

The cellular biology of B-cell chronic lymphocytic leukemia

In conclusion, B-CLL cells through their immunophenotype have the functional potential required to interact with cells in what has been called the immunological synapse, i.e. the cognate interactions between T-cells, antigen-presenting cells and B-cells during immunopoiesis. The data reviewed herein provides substantial evidence to suggest that B-CLL cells in fact can interact, not only with T-cells but also with endothelial cells and stromal cells in the bone marrow. These interactions, in particular signaling through CD40, contribute to extended survival and proliferation of B-CLL cells and, thereby, the risk of complete malignant transformation of the clone. Therefore, this review would suggest that the answers to how B-CLL is initiated may be found in molecules responsible for the normal regulation of immunopoiesis. Transformation to malignancy, by contrast, is likely to be caused by loss of control over the G1 restriction in the cell cycle in B-CLL cells.

Regulation of B-CLL Apoptosis Through Membrane Receptors and Bcl-2 Family Proteins

The accumulation of monoclonal chronic lymphocytic leukemia B (B-CLL) cells may be due to excessive proliferation and longevity. Clinical progression may thus come from a constitutive but altered expression of a number of genes that results in extended B-CLL cells life span, increased proliferative capacity and diminished cell death. B-CLL cells express a number of surface markers that characterise the normal B-cells phenotype. However, B-CLL cells are CD5 positive and most of them also express CD6, surface receptors that are present in just a small subset of normal B-cells. When exploring CD6 function, we found out that cross-linking of CD6 protected B-CLL from anti-IgM-induced apoptosis. CD6 activation blocked anti-IgM- induced Baxa up-regulation and, by doing so, corrected an imbalance in the Bcl-2/Bax ratio that accompanies apoptosis. Here, we review all surface receptors and cytokines that have been described as participating in the induction or protection of B-CLL apoptosis together with data on chemosensitivity and gene modulation, data on the Fas receptor/Fas ligand system, and the implications of all the latter for B-CLL cell survival.

Chronic Lymphocytic Leukemia B Cells Are Resistant to the Apoptotic Effects of Transforming Growth Factor-β

AbstractChronic lymphocytic leukemia (CLL) is the most common leukemia of the western world and is characterized by a slowly progressing accumulation of clonal CD5+ B cells. Our laboratory has investigated the role of transforming growth factor-β (TGF-β) and interleukin-4 (IL-4) in the pathogenesis of B-cell expansion in CLL. In vitro addition of TGF-β did not increase spontaneous apoptosis of B cells from most CLL patients, as determined using the TUNEL method, compared with a twofold increase observed in cultures of normal B cells. There was similar expression of TGF-β type II receptors on both CLL B cells and normal B cells. In contrast to apoptosis, CLL B-cell proliferation was variably inhibited with addition of TGF-β. In vitro addition of IL-4, previously reported to promote CLL B-cell survival, dramatically reduced spontaneous apoptosis of CLL B cells compared with normal B cells. CLL B-cell expression of IL-4 receptors was increased compared to normal B cells. Thus, our results show aberrant apoptotic responses of CLL B cells to TGF-β and IL-4, perhaps contributing to the relative expansion of the neoplastic clone.

Bcl-2, Bax and p53 expression in B-CLL in relation to in vitro survival and clinical progression.

Our previous data have shown that isolated leukemic cells from progressive chronic lymphocytic leukemia (B-CLL) patients respond to growth stimulation in vitro and express high levels of p53, immunoreactive with the configuration-specific antibody PAb 240. We have now analyzed the in vitro survival of B-CLL cells in relation to Bcl-2, Bax alpha and p53 expression and compared this with the clinical progression of the disease. Leukemic cells from patients with progressive disease demonstrated higher in vitro survival, compared with non-progressive B-CLL and normal B cells. All cells were sensitive to treatment with a combination of glucocorticoid and cAMP. Bcl-2 protein levels were not related to clinical progression, as measured by flow cytometry. Competitive PCR showed that Bcl-2 mRNA was over-expressed in most of the B-CLL samples and that p53 mRNA expression was similar between B-CLL groups and normal values and thus not related to clinical progression. However, since Bax alpha expression was lower in progressive than in non-progressive patients, the Bcl-2/Bax alpha ratio at the mRNA level was significantly higher in the progressive group. Our data suggest that the Bcl-2/Bax alpha ratio is important for the regulation of B-CLL cell survival, that p53 over-expression in progressive B-CLL is the result of post-transcriptional modifications and that a directed PKA activation may potentiate the cytolytic effect of glucocorticoids in vivo.

Bcl‐2, Bax and p53 expression in B‐CLL in relation to in vitro survival and clinical progression

Our previous data have shown that isolated leukemic cells from progressive chronic lymphocytic leukemia (B-CLL) patients respond to growth stimulation in vitro and express high levels of p53, immunoreactive with the configuration-specific antibody PAb 240. We have now analyzed the in vitro survival of B-CLL cells in relation to Bcl-2, Baxα and p53 expression and compared this with the clinical progression of the disease. Leukemic cells from patients with progressive disease demonstrated higher in vitro survival, compared with non-progressive B-CLL and normal B cells. All cells were sensitive to treatment with a combination of glucocorticoid and cAMP. Bcl-2 protein levels were not related to clinical progression, as measured by flow cytometry. Competitive PCR showed that Bcl-2 mRNA was over-expressed in most of the B-CLL samples and that p53 mRNA expression was similar between B-CLL groups and normal values and thus not related to clinical progression. However, since Baxα expression was lower in progressive than in non-progressive patients, the Bcl-2/Baxα ratio at the mRNA level was significantly higher in the progressive group. Our data suggest that the Bcl-2/Baxα ratio is important for the regulation of B-CLL cell survival, that p53 over-expression in progressive B-CLL is the result of post-transcriptional modifications and that a directed PKA activation may potentiate the cytolytic effect of glucocorticoids in vivo. © 1996 Wiley-Liss, Inc.

The role of interleukin-10 (IL-10) in chronic B-lymphocytic leukemia: IL-10 prevents leukemic cells from apoptotic cell death.

We examined the possible role of interleukin 10 (IL-10) in the pathogenesis of human chronic B-lymphocytic leukemias (B-CLL). With the use of an in vitro culture technique, we found that IL-10 enhanced the survival of B-CLL cells in a dose-dependent fashion by inhibiting the process of apoptotic cell death. This was demonstrated by transmission electron microscopy and DNA gel electrophoresis. Flow cytometric and immunoblot analyses showed that IL-10 did not significantly upregulate bcl-2 expression as compared with control cultures. B-CLL cells were also found to spontaneously release IL-10 in cultures, and serum IL-10 levels were elevated in five of the eleven B-CLL patients. These findings suggest that IL-10 acts as an autocrine growth factor for B-CLL cells and cytokine-based therapy might be a new approach for the treatment of B-CLL.