B-cell Chronic Lymphocytic Leukemia Lymphocytes Research Articles

Bcl-2 Family (Bcl-2, Mcl-1, Bim and Bax) and Btk Expression in B-Cell Chronic Lymphocytic Leukemia (B-CLL) Lymphocytes in Different Lymphoid Compartments and Changes during Btk Inhibitor Therapies (ibrutinib or acalabrutinib)

B-cell chronic lymphocytic leukemia (B-CLL) is characterized with variable infiltration by B -CLL lymphocytes of various lymphoid compartments, i.e peripheral blood (PB), bone marrow (BM) and lymph nodes (LN) and lymphoid organs. Interactions with various microenvironments may result in different disease activity and resistance to apoptosis. Novel agents targeting Btk and Bcl-2 show remarkable activity in B-CLL. However, they may have different activity in different lymphoid compartments while Btk inhibitors also may cause significant B-CLL lymphocyte redistribution between compartments. Aim of this study was to evaluate: 1) is there a different expression of Bcl-2 family of anti- and pro-apoptotic proteins (Bcl-2, Bax, Bim, mcl-1) and Btk in B-CLL lymphocytes from different lymphoid compartments; 2) relationship of observed expressions to disease parameters (TTM, TD, stage, B2MG, LDH); 3) changes during treatment with Btk inhibitors. We have included 28 B-CLL patients (18/10 M/F, median age 71 yr, range 48-85) treated with Btk inhibitors (ibrutinib 24, acalabrutinib 4). There were 15 previously treated patients and 13 treatment naïve patients. All evaluated patients had samples from PB and BM before treatment while 19 patients had also LN samples. At least one follow-up sample from PB had 20 patients. Median TTM score was 10.2 (range 3.4-23.9) and TD score was 0.68. Rai stage III/IV had 8 patients. Expression of Bcl-2, Mcl-1, Bim, Bax and Btk was determined by flow cytometry in CD19+CD5+ lymphocytes from fresh samples taken simultaneously by venipuncture (PB) or fine needle aspiration (BM and LN). There is higher expression of Bcl-2, Mcl-1 in LN compared to PB and BM (p<0.05), while there is no change in follow-up PB during ibrutinib/acalbrutinib treatment. There is no significant difference in Bim expression between compartments, while there is trend for higher expression in PB follow-up sample (p=0.054). There is no significant difference in Bax expression between PB, BM and LN compartments, while there is significant drop in Bax expression in PB follow-up sample during treatment (p<0.05). p-Btk has higher expression in LN compared to PB and BM (p<0.05). Follow-up PB p-Btk expression is comparable to pretreatment expression in PB (despite redistribution). We haven't found significant correlation of Bcl-2 family expression and p-Btk expression to the parameters of tumor mass (TTM) and tumor distribution (TD). In conclusion, there is a different pattern of expression of anti-apoptotic and pro-apoptotic Bcl-2 family members and Btk between lymphoid compartments. Our results may indicate higher BCR signaling and higher antiapoptotic activity in B-CLL lymphocytes in lymph node microenvironment. With redistribution during Btk inhibitor treatment B-CLL lymphocytes assume PB phenotype with downregulation of Btk and anti-apoptotic Bcl-2 and Mcl-1 proteins. Downregulation of pro-apoptotic Bax and possible upregulation of Bim protein indicate that there may be more complex interplay between various proteins resulting in different disease course. These results again emphasize the role of LN microenvironment in pathogenesis of B-CLL and may provide insights for treatment strategies combining targeted agents (Btk inhibitors and Bcl-2 antagonists).

Excessive amount and activity of μ-calpain affects apoptotic machinery in chronic B-cell leukemia cells and influences the course of the disease.

B-cell Chronic Lymphocytic Leukemia (B-CLL) is the most common hematological disorder among middle-aged/elderly people in the Western countries. We have shown earlier that B-CLL cells exhibit elevated total amount and available activity of µ-calpain, belonging to a family of ubiquitous, strongly Ca-dependent proteases, involved in the control of proliferation and apoptosis. In this study we attempted to estimate a potential clinical value of μ-calpain in relation to B-CLL clinical staging in patients with extremely high lymphocytosis and studied the molecular mechanisms associating calpain activity with clinical progress of the disease. We observed significant correlations between the amounts of intracellular μ-calpain and clinical staging of the disease, with RAI stage 1 corresponding to the highest calpain amounts in the leukemic cells. There was also a positive, statistically significant correlation between the amount of μ-calpain and phosphorylated (p)ZAP-70 in B-CLL lymphocytes. Calpain activity in the B-CLL cells is associated with decreased activities of pro-apoptotic caspases -3 and -9, and reciprocally with an increased amount of anti-apoptotic Bcl-2. Together, all of these findings make calpain activity in B-CLL cells a promising target modifying the properties of these cells and facilitating therapy. Finally, the proportion of CD19+ B cells with elevated μ-calpain and pZap-70 was markedly reduced in patients after successful therapy.

Synergistic induction of apoptosis in B-cell chronic lymphocytic leukemia cells after treatment with all-trans retinoic acid in combination with interleukin-21 and rituximab.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common adult leukemia in the Western world and characterized by the progressive expansion of malignant B lymphocytes in peripheral blood. In spite of advances in sciences to recognize the number of effective agents for the treatment of chronic lymphocytic leukemia (CLL), this leukemia is thought as incurable one. Introducing a new therapy that has a direct effect on B-CLL lymphocytes and no cytotoxic effects on the other cells is a great wish. Twenty-one patients with B-CLL were enrolled in the study. Peripheral blood mononuclear cells (PBMCs) were isolated from patients' blood samples and were further treated with all-tans retinoic acid (ATRA), interleukin (IL-21), and rituximab at concentrations of 30 ng/ml, 25 ng/ml, and 4 μg/ml, respectively. ATRA, IL21, and rituximab were used alone or in various combinations and their effects on apoptosis were measured using annexin V-fluorescein isothiocyanate apoptosis detection kit. Treatment of the patients' cells with IL21 and rituximab showed a synergistic effect on the induction of apoptosis, in comparison with untreated CLL cells (P < 0.05). The induction of apoptosis by ATRA in combination with IL21 and rituximab were significantly increased compared to untreated CLL cells as a negative control (P < 0.01). Treatment of patients' PBMCs by ATRA in combination with IL21 and rituximab and also IL21 in combination with rituximab showed synergistic induction of apoptosis compared to untreated CLL cells as a negative control (P < 0.01). It seems that ATRA in combination with IL21 and rituximab activate different pathways of apoptosis (extrinsic pathway, intrinsic pathway, and granzyme B pathway).

Aberrant TIRAP and MyD88 expression in B-cell chronic lymphocytic leukemia

TIRAP and Myd88 are adaptor proteins for Toll-like receptors-2 and -4 (TLR2/4) which are engaged in transducing the signal to downstream molecules. Several studies have shown the increased role of infection factors in pathogenesis of B cell chronic lymphocytic leukemia (B-CLL). This prompted us to test whether there is a correlation between MyD88-TIRAP dynamics before and after inflammatory stimuli. We determined the mRNA and protein expression of TIRAP and MyD88 in CD5(+)CD19(+) B-CLL cells and in a subpopulation of normal B CD19(+) lymphocytes. Additionally we determined the influence of lipopolysaccharide Escherichia coli - TLR4-ligand (LPS) and Staphylococcus aureus strain Cowan I - TLR2-ligand (SAC) on TIR-domain-containing adaptor protein, also called MyD88 adaptor-like (TIRAP) and myeloid differentiation primary response protein 88 (MyD88) expression. We have found that the mRNA and protein expression of TIRAP and MyD88 in B-CLL lymphocytes is lower compared with that in normal B lymphocytes. LPS and SAC stimulation in normal lymphocytes significantly altered neither TIRAP nor MyD88 mRNA expression, whereas TIRAP protein level substantially decreased after TLR agonist treatment. We did not observe any changes in MyD88 protein level after B lymphocyte stimulation. There was a significant increase in TIRAP mRNA expression after LPS and SAC stimulation of B-CLL cells. MyD88 mRNA expression levels in B-CLL lymphocytes slightly decreased upon treatment with either stimulator. Stimulation with TLR agonists did not cause changes in TIRAP and MyD88 expression at the protein level in B-CLL lymphocytes. The results of our study suggest that there may exist a, yet unknown, defect of TIRAP and MyD88 proteins in B-CLL lymphocytes.

Different Pattern of Expression of CD52, CD20 and CXCR-4 in Peripheral Blood, Bone Marrow and Lymph Nodes in B-Cell Chronic Lymphocytic Leukemia (B-CLL),

Abstract 3877B-cell chronic lymphocytic leukemia (B-CLL) is characterized by variable clinical presentation with different involvement of various lymphoid compartments i.e. peripheral blood, bone marrow and lymphoid organs such as lymph nodes and spleen. Also there is a well documented intraclonal and interclonal variability of B-CLL cells in different microenvironments regarding a number of surface and intracellular molecules (for example CD38 and ZAP-70). This variable distribution of tumor mass has strong association with prognosis and a well documented influence on response to antibodies like rituximab and alemtuzumab. There is a well documented efficacy of rituximab in cases with with 11q deletion (associated with significant lymphadenopathy), and known resistance to alemtuzumab in pateints with bulky lymphadenopaty (>5cm).Aim of this study was to evaluate level of expression of CD20 and CD52 along with key chemokine receptor CXCR-4 and proliferation marker Ki-67 on B-CLL lymphocytes and intra and interclonal differences dependent on different microenvironment, ie peripheral blood (PB), bone marrow (BM) and lymph nodes (LN).PB, BM and LN samples were taken by conventional techniques (venepuncture and fine needle aspiration) on the same day. The expression levels of CD20, CD52, CXCR-4 and Ki-67 molecules on CD19+CD5+ B-CLL cells were analyzed by flow cytometry. Results were expressed as mean fluorescence intensity (MFI) and analyzed by paired tests.We have analyzed samples taken from 25 typical B-CLL patients with median age of 72 years. There were 14 males and 11 females. Mean beta-2 microglobulin was 4.3mg/l, mean TTM size was 9.8 and mean TD was 0.74. There were 13, 5 and 7 patients in Binet stage A, B and C, respectively. There were 4 previously treated patients (patients were not treated 3 months before sampling) of whom one patient was previously treated with both rituximab and alemtuzumab. Among included patients there were patients with 11q deletion and with 17p deletion. Median expression level (MFI) of CD52 on B-CLL cells was 171, 193, and 352 for PB, BM and LN respectively (p<0.05) and on T cells was 224, 171 and 137 (p<0.05). Median expression level (MFI) of CD20 on B-CLL cells was 7.5, 5.7 and 4.7 for PB, BM and LN respectively (p<0.05). These results were very consistent in this clinically and cytogenetically heterogenous group of B-CLL patients showing the same pattern in almost all patients. Median expression level (MFI) of CXCR-4 was 9.6, 5.9 and 2.6 (p<0.05) and Ki-67 was 1.08, 1.27 and 1.64 (p<0.05) for PB, BM and LN respectively. There was no correlation of CXCR-4 with CD20 and C52 expression in any compartment and there is positive correlation of Ki-67 with both CD20 and CD52 in PB but not in other compartments.Relatively unexpected results demonstrating the lowest level of expression of CD20 on B-CLL cells in lymph nodes compared to PB and BM and the highest expression of CD52 on B-CLL cells in LN compared to PB and BM (but with the opposite pattern on T cells) is inversely related to known efficacy of agents (ie rituximab and alemtuzumab) targeting these molecules in these lymphoid compartments. These results indicate that other factors in selected microenvironment (beside number of molecules on cell surface) regulate sensitivity of B-CLL cells on rituximab and alemtuzumab in vivo. These may include other cells (like T cells) and soluble factors. Also in this study levels of expression were not clearly related to molecules involved in recirculation (CXCR-4) and proliferation (Ki-67) indicating that other factors in microenvironment may be important for the observed expression levels. These results warrant further studies to indentify these factors which may eventually uncover novel therapeutic targets. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

High intracellular content of cyclin‐dependent kinase inhibitor p27Kip1 in early‐ and intermediate stage B‐cell chronic lymphocytic leukemia lymphocytes predicts rapid progression of the disease

Previous studies showed that peripheral blood lymphocytes of B-cell chronic lymphocytic leukemia (B-CLL) displayed a high intracellular level of cell cycle inhibitory protein p27(Kip1). It has been suggested that its' high expression may confer them survival advantage and lead to unfavorable prognosis, but the prognostic significance of p27(Kip1) expression for previously untreated, non-advanced stage B-CLL was not established. We studied a relationship between the intracellular level of p27(Kip1) of lymphocytes of early- and intermediate stage B-CLL patients and their spontaneous apoptosis in vitro, as well as prognostic significance of p27(Kip1) in B-CLL lymphocytes for the risk of disease progression. Intracellular p27(Kip1) content of peripheral blood lymphocytes obtained from 48 previously untreated 0-II Rai stage B-CLL patients was determined by flow cytometry. The viability and apoptosis of those lymphocytes after 72-h culture were also assessed. During the follow-up period (6-71 months, median 59.5), we recorded the time elapsed to the doubling of lymphocyte count, progression to a higher Rai stage and the appearance of indications for cytostatic treatment. The p27(Kip1) expression was neither correlated with initial lymphocyte count, CD38 expression, cell viability nor spontaneous apoptosis ratio after 72-h culture. Higher p27(Kip1) level was related to the probability of earlier occurrence of each of three above-mentioned events. We did not find a prognostic significance of in vitro cell viability nor apoptosis as to the risk of disease progression. Our results indicate that elevated intracellular p27(Kip1) level in leukemic lymphocytes of early- and intermediate stage B-CLL patients contributes to rapid progression of the disease.

Regulation and Function of NOTCH2 in B-CLL Cells.

B-cell chronic lymphocytic leukemia (B-CLL) represents a clonal expansion of self-reactive CD5+ B-lymphocytes which seems to be resistant to apoptosis in vivo. One of the characteristics of B-CLL lymphocytes is the high expression of the B-cell differentiation/activation marker CD23 which we recently identified as a target gene for NOTCH2 signaling. NOTCH2 is implicated in the development/homeostasis of murine CD5+ (B-1a) B-cells, suggesting a function for human NOTCH2 in B-CLL leukemogenesis. Here we show that peripheral B-CLL lymphocytes overexpress a transcriptionally active form of NOTCH2 (N2IC) irrespective of their prognostic marker profile (ie. IgVH mutational status, CD38 expression, and cytogenetics). Although the majority of unstimulated B-CLL samples downregulate their N2IC activity in vitro, DNA-bound N2IC complexes could be maintained by the protein kinase C (PKC) activator TPA (12-O-tetradecanoylphorbol 13-acetate) accompanied by an upregulation of the NOTCH2 target gene CD23 and increased B-CLL cell viability. These effects are sensitive to the PKC-δ selective inhibitor Rottlerin. In 80% of B-CLL cases, NOTCH2 signaling was found to be resistant to the γ-secretase inhibitors (GSI's) Dapt and compound E, indicative for the expression of truncated forms of NOTCH2 which do not require γ-secretase for processing and function. Inhibition of NOTCH2 either by Dapt in GSI sensitive B-CLL cases or, more specifically, by RNA interference downregulates CD23 expression on the mRNA and protein level and sensitizes B-CLL cells for apoptosis. Since self-reactive B-cells are normally eliminated either by chronic (anergy) or apoptotic (negative selection) B-cell receptor (BCR) signaling, we asked whether NOTCH2 modulates B-cell fate decisions triggered by the BCR. For this reason, we stably transduced the human B-cell line BL41 with constitutive active forms of NOTCH2 and found that NOTCH2 inhibits BCR mediated apoptosis induced by surface-IgM cross-linking. In summary, the data demonstrate that NOTCH2 signaling is deregulated in B-CLL cells and might be critically involved in the PKC-dependent maintenance of their malignant phenotype.

Rituximab Consolidation and Maintenance Immunotherapy Improve Outcome in B-Cell Chronic Lymphocytic Leukemia.

Monoclonal antibodies in combination with chemotherapy allowed us to obtain more responses and longer response duration in B-cell chronic lymphocytic leukemia (B-CLL), reducing disease burden to levels detectable only by flow cytometry. Moreover, it has been reported that low-dose rituximab decreases CD20 antigen loss via shaving and promotes enhanced targeting in CLL (Williams, 2006). We performed a phase II study that added rituximab to fludarabine (Flu) as therapy for symptomatic, untreated CLL. Remission status was assessed by a multiparametric flow cytometric method based on the detection of CD19+CD5+CD79b– residual B-CLL lymphocytes. VH mutational status, CD38, ZAP-70 and cytogenetics were obtained in all pts before treatment. We defined as “high risk” pts having at least two of the following markers: unmutated IgVH, CD38>30%, ZAP-70>20%, intermediate/unfavorable cytogenetics (trisomy 12 or del11q or del17p). Eighty-two CLL pts, median age 61 years, received six monthly courses of Flu (25 mg/m2 for 5 days) and four weekly doses of rituximab (375 mg/m2) starting after completion of Flu therapy. According to modified Rai stages, 8 pts had a low stage, 70 an intermediate stage and 4 a high stage. Based on NCI criteria, 66/82 (80%) pts achieved a complete remission (CR), 12/82 (15%) a partial remission (PR) and 4/82 (5%) no response or progression. Hematologic toxicity included mainly neutropenia (grade 3 and/or 4 in 42 pts) and thrombocytopenia (grade 3 and/or 4 in 4 pts). Thirty-five pts in clinical CR or PR, either with CD5+CD19+CD79b– bone marrow (BM) cells >1% (MRD+, n=20 pts) or MRD negative but presenting CD5+CD19+ peripheral blood lymphocytes (PBL) >1000/microl (n=15 pts) within 1 year after completion of the induction treatment, underwent consolidation/maintenance therapy with four monthly cycles of rituximab at 375 mg/m2 followed by twelve monthly doses of rituximab at 150 mg/m2. The median follow-up duration was 46 months. Noteworthy, all B-CLL pts experienced a long progression-free survival (PFS) from the end of induction treatment (68% at 5 years). Nevertheless, CLL pts that underwent consolidation and maintenance therapy (n=35) showed a significant longer response duration (85% at 5 years, Figure). On the other hand, BM and PBL persistently MRD negative (>1 year) pts (n=29) showed a response duration similar to that of the consolidated pts (87% at 5 years). A significant shorter PFS was observed within CD38+ pts (39% vs 78% at 5 years, P=0.002), unmutated pts (45% vs 94% at 2.5 years, P=0.001) and ZAP-70+ pts (36% vs 88% at 6 years; P=0.00002). Notably, within the “high risk” subset (n=30), considering only MRD+ pts in CR or PR (n=20), MRD+ consolidated pts (n=11) showed a significant longer response duration (64% vs 13% at 2 years, P=0.006) in comparison with MRD+ unconsolidated pts (n=9). In conclusion, consolidation/maintenance therapy with rituximab prolongs significantly the response duration in B-CLL, improving also the outcome of the “high risk” subset. [Display omitted]

Laminin-332 (Laminin-5) is the major motility ligand for B cell chronic lymphocytic leukemia

Cell adhesion and motility are central aspects in the pathophysiology of B cell chronic lymphocytic leukemia (B-CLL), but the role of specific extracellular matrix proteins is still to be completely unveiled. Purified peripheral blood neoplastic cells of B-CLL patients migrated poorly on laminins-111,-411,-511, but showed pronounced motility on laminin (LM)-332 in a high percentage of cases. B-CLL cell motility on LM-332 was mediated by the α3β1 integrin and was preferentially observed in cells carrying a mutated IgV H gene profile. Within normal lymph nodes, LM-332 was circumscribed around blood vessels and to areas corresponding to marginal zones, where it was deposited in a pattern reminiscent of reticular fibers. Conversely, in B-CLL involved lymph nodes, a positive LM-332 reticular mesh was diffusely evident, throughout the disrupted nodal architecture. In the present study we identified LM-332 as a crucial motility-promoting factor for B-CLL lymphocytes and as a potential constituent favoring the dissemination of B-CLL lymphocytes through vascular basement membranes and possibly lymph node compartments.

Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B‐cells

Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes as potential disease biomarkers. CD20 expression in B-cell chronic lymphocytic leukemia (B-CLL) is one of the best examples of such a biomarker, but results from the use of different QFC methods vary considerably. We measured CD20 expression on normal and B-CLL B-cells, using FITC and PE conjugates from the same monoclonal antibody (Mab). As a biological control and calibrator, we also measured CD4 expression on T-cells with FITC and PE Mab. Calibration curves were constructed using the CLSI (formerly NCCLS) consensus guidelines for QFC. Calibration with QuantiBRITE PE-labeled microspheres and the use of unimolar PE conjugates provided direct measurement of antibody bound per cell (ABC) for CD4 and CD20. Calibration for FITC conjugates was based on molecules of equivalent soluble fluorochrome (MESF), as determined by NIST RM 8640 microsphere standards. These MESF values were then converted to ABC, using the CD4 T-cell as a biologic calibrator, to normalize FITC and PE results for CD20 expression. On normal B cells, the mean ABC value for unimolar CD20-PE conjugate was 143,500 (CV +/- 19.1%). The mean ABC value for B-CLL B-cells stained with the same conjugate was 21,700 (CV +/- 42.0%). Using the CD4 T-cell as a biologic calibrator for FITC conjugate, the mean ABC value for CD20-FITC on normal B-cells was 199,300. CD20-FITC staining on B-CLL cells was generally too weak for accurate quantification. On normal T-cells, the mean ABC value for CD4 unimolar PE conjugate was (36,800 +/- 10.4)%, and it did not differ significantly in CLL samples. The expression of CD20 on normal and B-CLL lymphocytes can be quantified in ABC units using unimolar CD20-PE conjugates. In addition, CD4 expression on T-cells can be used as a biological calibrator to quantify CD20-FITC ABC, with reasonable agreement between the two conjugates with different fluorochromes. Issues regarding the accuracy of MESF microsphere calibrators and effective F/P ratios for FITC conjugates will require additional laboratory studies.

Induction of apoptosis by proteasome inhibitors in B-CLL cells is associated with downregulation of CD23 and inactivation of Notch2

Recently, proteasome inhibitors (PI) have attracted interest as novel anticancer agents in B-cell chronic lymphocytic leukemia (B-CLL). A prominent feature of B-CLL cells is the high expression of CD23, which is closely related to cell survival and is regulated by Notch2. Since several components of the Notch signaling cascade are tightly regulated by proteasomal degradation, we studied the effect of PI on Notch2 activity and CD23 expression. Exposure of B-CLL cells to PI led to induction of apoptosis, a time- and dose-dependent downregulation of CD23 expression and a decline in DNA binding of transcriptionally active Notch2. In contrast, the transcription factor NF-AT and its putative target gene CD5, which is highly expressed in B-CLL cells, were unaffected. When the late phase of PI-induced apoptosis was arrested by inhibition of caspase 3, the reduction of Notch2 activity was still observed, indicating that reduction of active Notch2 took place already during an earlier phase of apoptosis. Enforced expression of constitutively active Notch2 decreased PI-mediated apoptosis in a human B-cell line. These data indicate that downregulation of CD23 and loss of Notch2 activity are early steps in PI-induced apoptosis of B-CLL lymphocytes and may be part of the full apoptotic response.

Tumor necrosis factor receptor-associated factor 1 gene overexpression in B-cell chronic lymphocytic leukemia: analysis of NF-κB/Rel–regulated inhibitors of apoptosis

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by a resistance toward apoptosis-inducing agents. Nuclear factor-kappaB (NF-kappaB)/Rel has been shown to regulate the expression of antiapoptotic genes, such as members of the inhibitor of apoptosis protein (IAP) and tumor necrosis factor receptor-associated factor (TRAF) gene families. Expression and regulation of NF-kappaB/Rel-dependent inhibitors of apoptosis have not been collectively studied in B-CLL. We examined expression of known NF-kappaB/Rel-regulated antiapoptotic genes by RNAse protection assay, real-time polymerase chain reaction, and immunoblotting in patients with B-CLL. TRAF1 and to a lesser extent TRAF2 were overexpressed in B-CLL lymphocytes as compared with normal CD19(+) B cells. TRAF1 overexpression did not correlate with markers of disease progression or overall survival. Furthermore, we found high constitutive expression of the IAP genes c-IAP-1, c-IAP-2, and XIAP both in normal and B-CLL lymphocytes. Focusing on the regulation of TRAF1, NF-kappaB/Rel activity in B-CLL nuclear extracts was shown to bind to TRAF1 promoter elements. However, IkappaB kinase (IKK) activity was not increased in CLL lymphocytes as compared with normal CD19(+) B cells. The known IKK inhibitor sulfasalazine did not compromise TRAF1 expression. Thus, although our study revealed a common expression pattern of NF-kappaB/Rel-regulated inhibitors of apoptosis, our findings indicate an IKK-independent regulation of TRAF1 in B-CLL.

Homologous recombinational repair vis-à-vis chlorambucil resistance in chronic lymphocytic leukemia

The objective of this study was to further define the role of homologous recombinational repair (HRR) in resistance to the nitrogen mustards in B-cell chronic lymphocytic leukemia (B-CLL). We have demonstrated previously that increased chlorambucil (CLB)-induced HsRad51 nuclear foci formation correlated with a CLB-resistant phenotype in B-CLL lymphocytes. In this report, we measured the protein levels of HsRad51 and Xrcc3 (an HsRad51 paralog) and correlated them with the in vitro CLB cytotoxicity (ld50) in lymphocytes from seventeen B-CLL patients. Both HsRad51 (r=0.75, P=0.0005) and Xrcc3 (r=0.52, P=0.03) protein levels correlated with the in vitro CLB ld50. In addition, multiple linear regression analysis showed a significant correlation between Xrcc3 and Rad51 protein levels versus the CLB ld50 (r=0.78, P=0.0014), suggesting that both proteins influence CLB cytotoxicity. Moreover, since HsRad51 expression varies in cell lines during the cell cycle, we determined proliferating cell nuclear antigen (PCNA) protein levels to assess possible differences in cell cycle progression. There was no correlation between PCNA protein levels and the CLB ld50 (r=0.042, P=0.87) or with HsRad51/Xrcc3 protein levels. Our data suggest that HsRad51 and Xrcc3 protein expression may be predictive of the response in B-CLL patients to treatment with nitrogen mustards.

Deregulation of the ubiquitin system and p53 proteolysis modify the apoptotic response in B-CLL lymphocytes

AbstractWe recently reported increased sensitivity of B-cell chronic lymphocytic leukemia (B-CLL) lymphocytes to apoptotic death activation by the proteasome-specific inhibitor lactacystin. Here, we show that only specific—not nonspecific—proteasomal inhibitors can discriminate between malignant and normal lymphocytes in inducing the apoptotic death response. Indeed, lactacystin and its active metaboliteclasto-lactacystin β-lactone induced apoptotic death in CLL but not in normal lymphocytes. This difference was completely abolished when tripeptide aldehydes such as MG132 or LLnL (which can also inhibit calpains) were used as less specific proteasomal inhibitors. Moreover, B-CLL cells exhibited a constitutive altered ubiquitin-proteasome system, including a threefold higher chymotrypsin-like proteasomal activity and high levels of nuclear ubiquitin-conjugated proteins compared with normal lymphocytes. Interestingly, B-CLL cells also displayed altered proteolytic regulation of wild-type p53, an apoptotic factor reported to be a substrate for the ubiquitin-proteasome system. Nuclear wild-type p53 accumulated after lactacystin treatment used at the discriminating concentration in malignant, but not in normal, lymphocytes. In contrast, p53 was stabilized by MG132 or LLnL in malignant and normal cells undergoing apoptosis, indicating that in normal lymphocytes p53 is regulated mainly by calpains and not by the ubiquitin-proteasome system. This work raises the possibility that two different proteolytic pathways controlling p53 stability may be pathologically imbalanced. This could result in modification of apoptosis control, since in CLL-lymphocytes a highly upregulated ubiquitin-proteasome system, which controls p53 stability among other apoptotic factors, was correlated with an increased propensity of these cells to apoptosis triggered by lactacystin.

Deregulation of the ubiquitin system and p53 proteolysis modify the apoptotic response in B-CLL lymphocytes

We recently reported increased sensitivity of B-cell chronic lymphocytic leukemia (B-CLL) lymphocytes to apoptotic death activation by the proteasome-specific inhibitor lactacystin. Here, we show that only specific—not nonspecific—proteasomal inhibitors can discriminate between malignant and normal lymphocytes in inducing the apoptotic death response. Indeed, lactacystin and its active metaboliteclasto-lactacystin β-lactone induced apoptotic death in CLL but not in normal lymphocytes. This difference was completely abolished when tripeptide aldehydes such as MG132 or LLnL (which can also inhibit calpains) were used as less specific proteasomal inhibitors. Moreover, B-CLL cells exhibited a constitutive altered ubiquitin-proteasome system, including a threefold higher chymotrypsin-like proteasomal activity and high levels of nuclear ubiquitin-conjugated proteins compared with normal lymphocytes. Interestingly, B-CLL cells also displayed altered proteolytic regulation of wild-type p53, an apoptotic factor reported to be a substrate for the ubiquitin-proteasome system. Nuclear wild-type p53 accumulated after lactacystin treatment used at the discriminating concentration in malignant, but not in normal, lymphocytes. In contrast, p53 was stabilized by MG132 or LLnL in malignant and normal cells undergoing apoptosis, indicating that in normal lymphocytes p53 is regulated mainly by calpains and not by the ubiquitin-proteasome system. This work raises the possibility that two different proteolytic pathways controlling p53 stability may be pathologically imbalanced. This could result in modification of apoptosis control, since in CLL-lymphocytes a highly upregulated ubiquitin-proteasome system, which controls p53 stability among other apoptotic factors, was correlated with an increased propensity of these cells to apoptosis triggered by lactacystin.

Metabolism and cytotoxic effects of 2-chloroadenine, the major catabolite of 2-chloro-2′-deoxyadenosine

EHEB cells, a continuous cell line derived from a patient with B cell chronic lymphocytic leukemia (B-CLL), synthesized, when incubated with tritiated 2-chloro-2′-deoxyadenosine (CdA), labeled mono-, di-, and triphosphate ribonucleosides at a much higher rate than CdA deoxyribonucleotides. Further analysis revealed that these ribonucleotides were formed from labeled 2-chloroadenine (CAde), which contaminated commercial tritiated CdA at a proportion of 2–3%. Since CAde is the major catabolite of CdA measured in plasma after oral or intravenous administration of CdA to patients, its metabolism and in particular its potential cytotoxicity were investigated both in EHEB cells and in B-CLL lymphocytes. Phosphorylation of CAde was inhibited by adenine, indicating that its initial metabolism most probably proceeds via adenine phosphoribosyltransferase (EC 2.4.2.7). In both cell types, chloro-ATP was the major metabolite formed from CAde and its concentration increased proportionally at least up to 50 μM CAde. At high concentration, CAde metabolism was accompanied by a decrease in intracellular ATP. Cytotoxicity of CAde, evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, showed an ic 50 of 16 μM in EHEB cells and 5 μM in B-CLL lymphocytes. At cytotoxic concentrations, apopain/caspase-3 activation and high molecular weight DNA fragmentation were observed, indicating that CAde cytotoxicity results from induction of apoptosis. However, since CAde cytotoxicity requires higher concentrations than CdA, it probably does not play a role in the therapeutic effect of CdA in the treatment of hematologic malignancies.

Synthesis of adenine and guanine nucleotides at the ‘inosinic branch point’ in lymphocytes of leukemia patients

The synthesis of purine nucleotides has been studied in human peripheral blood lymphocytes from healthy subjects and patients affected by B-cell chronic lymphocytic leukemia (B-CLL). The rate of the synthesis was measured by following the incorporation of 14C-formate into the nucleotides of lymphocyte suspensions. The whole sequence AMP→ADP→ATP was found reduced in B-CLL lymphocytes; in the case of guanylates only the last step of the sequence GMP→GDP→GTP was significantly lower in the same cells. From the analysis of these results, combined with previous data, we conclude that purine metabolism undergoes an imbalancement during CLL, which is partially compensated, and point out the importance of studying concomitantly purine metabolism and nucleic acid synthesis in leukemia cells.

Differential Induction of Apoptosis by Fludarabine Monophosphate in Leukemic B and Normal T Cells in Chronic Lymphocytic Leukemia

AbstractFludarabine (F-ara-A), an adenine nucleoside analog with efficacy in B-cell chronic lymphocytic leukemia (B-CLL), has also been shown to have a long-lasting suppressive effect on T lymphocytes. In heterogeneous clinical samples, apoptosis cannot be detected by standard methods in small cellular subsets. We developed, therefore, a combined assay of in situ end-labeling of nicked DNA by terminal deoxynucleotide transferase, with measurements of cellular DNA content and surface antigens (CD3, CD4, CD8, and CD19) by multiparametric flow cytometry. This assay was used to determine F-ara-A–induced apoptosis in different lymphocyte subsets from CLL patients and normal controls treated with F-ara-A in vitro. Apoptosis was also correlated to bcl-2 protein levels. We observed a direct effect of F-ara-A on both B-CLL and T lymphocytes. The response to F-ara-A in B-CLL lymphocytes in vitro was Rai stage–dependent, the early-stages being more responsive (P = .01). Higher levels of spontaneous apoptosis were observed in B-CLL lymphocytes from early stage patients (P = .02). No difference was observed in spontaneous apoptosis of normal T cells in B-CLL, although T lymphocytes in late-stage disease were more sensitive to F-ara-A–induced apoptosis. Incubation with cyclosporin A did not affect B-CLL and T-lymphocyte survival compared with control cultures. Results suggested a direct apoptotic effect of F-ara-A on B-CLL lymphocytes that decreases with increasing clinical stage. No correlation was found between bcl-2 and spontaneous or F-ara-A–induced apoptosis. Apoptosis occurred at all cell-cycle stages and was not restricted to cells in S phase. The mechanisms of this stage-dependent apoptosis in CLL remain to be elucidated.

Differential Induction of Apoptosis by Fludarabine Monophosphate in Leukemic B and Normal T Cells in Chronic Lymphocytic Leukemia

Fludarabine (F-ara-A), an adenine nucleoside analog with efficacy in B-cell chronic lymphocytic leukemia (B-CLL), has also been shown to have a long-lasting suppressive effect on T lymphocytes. In heterogeneous clinical samples, apoptosis cannot be detected by standard methods in small cellular subsets. We developed, therefore, a combined assay of in situ end-labeling of nicked DNA by terminal deoxynucleotide transferase, with measurements of cellular DNA content and surface antigens (CD3, CD4, CD8, and CD19) by multiparametric flow cytometry. This assay was used to determine F-ara-A–induced apoptosis in different lymphocyte subsets from CLL patients and normal controls treated with F-ara-A in vitro. Apoptosis was also correlated to bcl-2 protein levels. We observed a direct effect of F-ara-A on both B-CLL and T lymphocytes. The response to F-ara-A in B-CLL lymphocytes in vitro was Rai stage–dependent, the early-stages being more responsive (P = .01). Higher levels of spontaneous apoptosis were observed in B-CLL lymphocytes from early stage patients (P = .02). No difference was observed in spontaneous apoptosis of normal T cells in B-CLL, although T lymphocytes in late-stage disease were more sensitive to F-ara-A–induced apoptosis. Incubation with cyclosporin A did not affect B-CLL and T-lymphocyte survival compared with control cultures. Results suggested a direct apoptotic effect of F-ara-A on B-CLL lymphocytes that decreases with increasing clinical stage. No correlation was found between bcl-2 and spontaneous or F-ara-A–induced apoptosis. Apoptosis occurred at all cell-cycle stages and was not restricted to cells in S phase. The mechanisms of this stage-dependent apoptosis in CLL remain to be elucidated.

Expression of adhesion molecules in 113 patients with B-cell chronic lymphocytic leukemia: Relationship with clinico-prognostic features

The B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease, its clinical and biological behavior possibly being influenced by surface molecules expressed in B-lymphocytes. These molecules mediate cell adhesion, mobility and homing. Expression of surface adhesion molecules of the integrin family (CD11a/CD18 or LFA-1, CD11c/CD18), of the immunoglobulin-related family (CD54), of the selectin family (CD62L or LAM-1) and the lymphocyte homing receptor (CD44) were analyzed in peripheral cells from 113 B-CLL patients. The association with three prognosis-related parameters (Rai stage, bone marrow pattern and doubling time) was determined. The study included only patients with B-CLL lymphocytes of typical morphology, which always expressed CD5 and CD23. Low expression of integrins, particularly CD18, was associated with advanced disease (Rai stages III–IV) and diffuse bone marrow pattern, even after adjusting for other prognosis-related variables. Expression of CD54 was associated independently with rapid doubling time (less than 12 months). The association persisted after adjusting for stage and bone marrow pattern; CD44 was expressed in all patients. No correlations were found between expression of CD62L and the prognostic variables analyzed. In conclusion, CD54 expression and low CD18 expression are both significantly associated with poor prognostic features.