Fludarabine Induces Differential Expression of Proteins in Human Leukemia and Lymphoma Cells

The purine analog fludarabine (FdAMP) is widely used for chemotherapy of B-lymphoid malignancies, and multiple mechanisms of action leading to apoptosis have been proposed. We examined changes at the protein level induced in the Raji cell line (Burkitt's lymphoma) by fludarabine nucleoside (FdA). Raji cells are sensitive to FdA. Raji cells treated with FdA (3 μ M, 24 hours), accumulate multiple phosphorylated forms of p53 in the nucleus that in turn degrade to phosphorylated forms of p40. Using CD antibody microarrays to determine surface expression profiles for Raji cells treated with FdA, we found up-regulation of the following CD antigens: CD20, CD54, CD80, CD86, and CD95. FdA thus induces changes in the genetic program of the cells that might be exploited to obtain synergy with therapeutic antibodies.     

Fludarabine induces differential expression of proteins in human leukemia and lymphoma cells

The purine analog fludarabine (FdAMP) is widely used for chemotherapy of B-lymphoid malignancies, and multiple mechanisms of action leading to apoptosis have been proposed. We examined changes at the protein level induced in the Raji cell line (Burkitt's lymphoma) by fludarabine nucleoside (FdA). Raji cells are sensitive to FdA. Raji cells treated with FdA (3 micro M, 24 hours), accumulate multiple phosphorylated forms of p53 in the nucleus that in turn degrade to phosphorylated forms of p40. Using CD antibody microarrays to determine surface expression profiles for Raji cells treated with FdA, we found up-regulation of the following CD antigens: CD20, CD54, CD80, CD86, and CD95. FdA thus induces changes in the genetic program of the cells that might be exploited to obtain synergy with therapeutic antibodies.     

Fludarabine and cladribine induce changes in surface proteins on human B-lymphoid cell lines involved with apoptosis, cell survival, and antitumor immunity

Fludarabine and cladribine are purine analogues used to treat hematological malignancies. Alone or in combination with therapeutic antibodies, they are effective in treating patients with chronic lymphocytic leukemia and non-Hodgkin's lymphoma. However, the mechanisms of action of these drugs are not well understood. Plasma membrane proteins perform a variety of essential functions that can be affected by malignancy and perturbed by chemotherapy. Analysis of surface proteins may contribute to an understanding of the mechanisms of action of purine analogues and identify biomarkers for targeted therapy. The surface of human cells is rich in N-linked glycoproteins, enabling use of a hydrazide-coupling technique to enrich for glycoproteins, with iTRAQ labeling for quantitative comparison. A number of plasma membrane proteins on human leukemia and lymphoma cells were affected by treatment with a purine analogue, including decreases in CD22 (an adhesion and signaling molecule) and increases in CD205 (a "damaged cell marker") and CD80 and CD50 (T-cell interaction molecules). Purine analogues may affect B-cell receptor (BCR) signaling and costimulatory molecules, leading to multiple signals for apoptosis and cell clearance. Fludarabine and cladribine induce differential effects, with some cell survival proteins (ECE-1 and CD100) more abundant after fludarabine treatment. Cell surface proteins induced by fludarabine and cladribine may be targets for therapeutic antibodies.     

Comprehensive proteomic analysis of the effects of purine analogs on human Raji B-cell lymphoma

Cladribine (CdA) and fludarabine (FdAMP) are purine analogs that induce apoptosis in chronic lymphocytic leukemia and non-Hodgkin's lymphoma, but the mechanisms are undefined. The effects of CdA and fludarabine nucleoside (FdA) on the cytosolic, mitochondrial, and nuclear proteomes in human Raji lymphoma cells have been determined using two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry. Differentially abundant proteins have provided new insights into CdA- and FdA-induced apoptosis. Treatment with these purine analogs induced changes in proteins involved with intermediary metabolism, cell growth, signal transduction, protein metabolism, and regulation of nucleic acids. Differentially abundant mitochondrial 39S ribosomal protein L50, mTERF domain-containing protein 1, Chitinase-3 like 2 protein, and ubiquinone biosynthesis protein COQ9 have been identified in cells undergoing apoptosis. Up-regulation of several stress-associated proteins found in the endoplasmic reticulum (ER) including GRP78, ERp57, and ORP150 suggests that purine analog-induced apoptosis may result from ER stress and unfolded protein response. While mitochondria-dependent apoptosis has been associated with purine analog cytotoxicity, the likely involvement of the ER stress pathway in CdA- and FdA-induced apoptosis has been shown here for the first time.     

On CK2 regulation of chronic lymphocytic leukemia cell viability

Specific inhibition of signaling elements essential for the viability of B-cell chronic lymphocytic leukemia (CLL) cells offers great promise for the design of more efficient therapies. The protein serine/threonine kinase CK2 is frequently upregulated in cancer, and it is overexpressed and hyperactivated in primary CLL cells from untreated patients. We have shown that inhibition of CK2 induces apoptosis of CLL cells, whereas it does not significantly impact normal lymphocytes, demonstrating the selectivity of the CK2 inhibitors toward leukemia cells. Notably, although co-culture with OP9 stromal cells and BCR stimulation both promote leukemia cell survival in vitro, they do not prevent apoptosis of CLL cells treated with CK2 inhibitors. PI3K signaling pathway was previously shown to be essential for CLL cell viability, an observation we confirmed in all patient samples analyzed. Further, we observed that CK2 blockade decreases PTEN phosphorylation, leading to PTEN activation, and that apoptosis of CLL cells upon CK2 inhibition is mediated by PKC inactivation. This suggests that activation of PI3K/PKC signaling pathway is involved in the pro-survival effects of CK2 in CLL cells. Sensitivity to CK2 inhibition does not correlate with expression of ZAP-70 or CD38, or with IGVH mutation status. However, it positively correlates with the percentage of CLL cells in the peripheral blood, β2 microglobulin levels, and Binet clinical stage. CK2 appears to play an important role in the biology of CLL and constitutes a promising target for the development of leukemia-specific therapies.     

Analysis of apoptosis regulatory genes altered by histone deacetylase inhibitors in chronic lymphocytic leukemia cells

Histone deacetylases (HDACs) play a key role in the regulation of acetylation status not only of histones but also of many other non-histone proteins involved in cell cycle regulation, differentiation or apoptosis. Therefore, histone deacetylase inhibitors (HDACi) have emerged as promising anticancer agents. Herein, we report the characterization of apoptosis in B-cell chronic lymphocytic leukemia (CLL) induced by two HDACi, Kendine 92 and SAHA. Both inhibitors induce dose-, time- and caspase-dependent apoptosis through the mitochondrial pathway. Interestingly, Kendine 92 and SAHA show a selective cytotoxicity for B lymphocytes and induce apoptosis in CLL cells with mutated or deleted TP53 as effectively as in tumor cells harboring wild-type TP53. The pattern of apoptosis-related gene and protein expression profile has been characterized. It has shown to be irrespective of TP53 status and highly similar between SAHA and Kendine 92 exposure. The balance between the increased BAD, BNIP3L, BNIP3, BIM, PUMA and AIF mRNA expression levels, and decreased expression of BCL-W, BCL-2, BFL-1, XIAP and FLIP indicates global changes in the apoptosis mRNA expression profile consistent with the apoptotic outcome. Protein expression analysis shows increased levels of NOXA, BIM and PUMA proteins upon Kendine 92 and SAHA treatment. Our results highlight the capability of these molecules to induce apoptosis not only in a selective manner but also in those cells frequently resistant to standard treatments. Thus, Kendine 92 is a novel HDACi with anticancer efficacy for non-proliferating CLL cells.     

Lysophosphatidic acid (LPA) protects primary chronic lymphocytic leukemia cells from apoptosis through LPA receptor activation of the anti-apoptotic protein AKT/PKB

Lysophosphatidic acid (LPA) protects epithelial and fibroblast cell lines from apoptosis. In B-cells, LPA acts as a growth factor promoting cell proliferation. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+/CD5+ B-lymphocytes primarily through a block in apoptosis. The mechanisms underlying this defect are not fully understood. We investigated whether LPA could be a survival factor in CLL cells. Herein, we demonstrate that LPA protects B-cell lines BJAB and I-83 and primary CLL cells but not normal B-cells from fludarabine- and etoposide-induced apoptosis. Furthermore, LPA prevented spontaneous apoptosis in primary CLL cells. The LPA1 expression was found to be increased in primary CLL cells compared with normal B-cells correlating with LPA prevention of apoptosis. Treatment of primary CLL cells with the LPA receptor antagonist, diacylglycerol pyrophosphate, reverses the protective effect of LPA against apoptosis, and down-regulation of the LPA1 by siRNA blocked LPA-mediated protection against spontaneous apoptosis in primary CLL cells. The protective effect of LPA was inhibited by blocking activation of the phosphatidylinositol 3-kinase/AKT signaling pathway. These results indicate that LPA is a survival factor in B-cell lines and primary CLL cells but not normal B-cells. Thus, drugs targeting the LPA receptors might be an effective therapy against B-cell-derived malignancies such as CLL.     

Critical role of p53 upregulated modulator of apoptosis in benzyl isothiocyanate-induced apoptotic cell death

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, decreases viability of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of Bcl-2 family proteins in BITC-induced apoptosis using MDA-MB-231 (breast), MCF-7 (breast), and HCT-116 (colon) human cancer cells. The B-cell lymphoma 2 interacting mediator of cell death (Bim) protein was dispensable for proapoptotic response to BITC in MCF-7 and MDA-MB-231 cells as judged by RNA interference studies. Instead, the BITC-treated MCF-7 and MDA-MB-231 cells exhibited upregulation of p53 upregulated modulator of apoptosis (PUMA) protein. The BITC-mediated induction of PUMA was relatively more pronounced in MCF-7 cells due to the presence of wild-type p53 compared with MDA-MB-231 with mutant p53. The BITC-induced apoptosis was partially but significantly attenuated by RNA interference of PUMA in MCF-7 cells. The PUMA knockout variant of HCT-116 cells exhibited significant resistance towards BITC-induced apoptosis compared with wild-type HCT-116 cells. Attenuation of BITC-induced apoptosis in PUMA knockout HCT-116 cells was accompanied by enhanced G2/M phase cell cycle arrest due to induction of p21 and down regulation of cyclin-dependent kinase 1 protein. The BITC treatment caused a decrease in protein levels of Bcl-xL (MCF-7 and MDA-MB-231 cells) and Bcl-2 (MCF-7 cells). Ectopic expression of Bcl-xL in MCF-7 and MDA-MB-231 cells and that of Bcl-2 in MCF-7 cells conferred protection against proapoptotic response to BITC. Interestingly, the BITC-treated MDA-MB-231 cells exhibited induction of Bcl-2 protein expression, and RNA interference of Bcl-2 in this cell line resulted in augmentation of BITC-induced apoptosis. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with the induction of PUMA protein in the tumor. In conclusion, the results of the present study indicate that Bim-independent apoptosis by BITC in cancer cells is mediated by PUMA.     

Morphological and immunophenotypic features of chronic lymphocytic leukemia

In this review, we summarize the morphological features and immunophenotypic profile of chronic lymphocytic leukemia (CLL) cells, discuss the value of these investigations as front line diagnostic tests, and emphasize their correlation with the clinical features, disease progression, molecular genetics and pathogenesis of CLL. In CLL, the morphology of the circulating cells is characteristic and typical in the majority of cases. However, 15% of patients, either at diagnosis or during the course of the disease, show atypical morphology reflected by either (1) an increased (> 10%) number of circulating prolymphocytes, designated CLL/PL, or (2) an increased (> 15%) number of circulating lymphoplasmacytic and cleaved cells, designated 'atypical' CLL. There is strong evidence of a close association between atypical morphology (CLL/PL) and atypical (CLL) and clinical features, e.g. disease progression, advanced stage and survival, molecular genetics, particularly trisomy 12, but also the rare cases with t(11;14) or t(14;19), p53 abnormalities, unmutated immunoglobulin (Ig) VH genes and origin of the cell (naive, pregerminal center cell). CLL cells have a distinct immunological repertoire different from that of other lymphoproliferative disorders. The typical CLL phenotype is CD5+, CD23+, FMC7-, weak expression of surface Ig (sIg) and weak or absent expression of membrane CD22 and CD79b. The latter marker identifies an extracellular epitope of the B-cell receptor (BCR) beta chain and its weak or absent expression in CLL may derive from the expression of a truncated form. This, together with the low expression of CD22, might explain the abnormal signal transduction of CLL cells similar to that of anergic B lymphocytes. Because no single marker is specific for CLL, a composite phenotype considering this set of 5 or 6 markers compounded into a scoring system helps to distinguish CLL from the other B-cell malignancies. Immunophenotypic analysis has also been shown to be useful for minimal residual disease detection and adds valuable prognostic information because the expression of certain markers, such as FMC7 or CD38, seems to be associated with a poor outcome. In addition, CLL cells express a variety of Bcl-2 family proteins with a profile that favors inhibition of apoptosis which, together with the interaction with microenvironmental (e.g. stromal) cells and the release of cytokines, explains the long life span and subsequent accumulation of CLL cells in various organs. Despite controversies relating to the expression of adhesion molecules (selectins and integrins) in CLL cells, it appears that some of these molecules do play a role in the pathogenesis, biology and clinical patterns of the disease. In conclusion, morphology and immunophenotype are the two essential investigations, which must be carried out in all cases of CLL. Both provide relevant information in terms of diagnosis, course of the disease, prognosis and pathogenesis.     

Quantification of the leukocyte common antigen (CD45) in mature B-cell malignancies.

CD45 is a glycoprotein expressed on all lymphohematopoietic cells. Its expression increases during normal B-cell differentiation and remains stable on mature cells. Although it is widely known that CD45 antigen expression is decreased in B-acute lymphocytic leukemia (ALL), only scarce and contradictory information is available on CD45 expression on mature B-cell malignancies. In healthy adults (n = 15), CD45 expression on B lymphocytes was lower than that on T cells. In patients with chronic lymphocytic leukemia (CLL; n = 22), CD45 expression on malignant cells was lower than that on the whole lymphocyte population of healthy adults (n = 28) and on normal B lymphocytes (n = 15). In 6 of the 22 CLL patients, the malignant cell population could be separated from the normal lymphocyte population on the CD45-side scatter (SSC) plot. In 16 CLL patients, there was some degree of overlap between the malignant and normal cells with respect to CD45 expression. For these patients, there was an inverse correlation between CD45 expression on the whole lymphocyte population and the percentage of malignant cells in this population. In two patients with mantle cell lymphoma (MCL), CD45 expression on the malignant cells appeared lower than that on normal B cells and on the whole lymphocyte population. In six patients with hairy cell leukemia (HCL), CD45 expression on hairy cells was comparable to that on the whole lymphocyte population of healthy adults, but slightly higher than that of normal B cells. Evaluation of CD45 expression may help to characterize mature B-cell malignancies.     

AICAR induces Bax/Bak-dependent apoptosis through upregulation of the BH3-only proteins Bim and Noxa in mouse embryonic fibroblasts

5-Aminoimidazole-4-carboxamide (AICA) riboside (AICAR) is a nucleoside analogue that is phosphorylated to 5-amino-4-imidazolecarboxamide ribotide (ZMP), which acts as an AMP mimetic and activates AMP-activated protein kinase (AMPK). It has been recently described that AICAR triggers apoptosis in chronic lymphocytic leukemia (CLL) cells, and its mechanism of action is independent of AMPK as well as p53. AICAR-mediated upregulation of the BH3-only proteins BIM and NOXA correlates with apoptosis induction in CLL cells. Here we propose mouse embryonic fibroblasts (MEFs) as a useful model to analyze the mechanism of AICAR-induced apoptosis. ZMP formation was required for AICAR-induced apoptosis, though direct Ampk activation with A-769662 failed to induce apoptosis in MEFs. AICAR potently induced apoptosis in Ampkα1 ^?/? /α2 ^?/? MEFs, demonstrating an Ampk-independent mechanism of cell death activation. In addition, AICAR acts independently of p53, as MEFs lacking p53 also underwent apoptosis normally. Notably, MEFs lacking Bax and Bak were completely resistant to AICAR-induced apoptosis, confirming the involvement of the mitochondrial pathway in its mechanism of action. Apoptosis was preceded by ZMP-dependent but Ampk-independent modulation of the mRNA levels of different Bcl-2 family members, including Noxa, Bim and Bcl-2. Bim protein levels were accumulated upon AICAR treatment of MEFs, suggesting its role in the apoptotic process. Strikingly, MEFs lacking both Bim and Noxa displayed high resistance to AICAR. These findings support the notion that MEFs are a useful system to further dissect the mechanism of AICAR-induced apoptosis.     

Differential cell killing by lymphomagenic murine leukemia viruses occurs independently of p53 activation and mitochondrial damage

Upon inoculation into AKR mice, mink cell focus-forming murine leukemia virus (MCF MLV) accelerates thymic lymphoma formation. During the preleukemic phase of disease, we observed the induction of apoptosis in thymic lymphocytes. A similar induction of apoptosis was observed for cultured mink epithelial cells after MCF13 MLV infection. In this study, the relevance of viral pathogenicity to cell killing was determined by testing the susceptibility of various cell types from different species to lymphomagenic MLVs. We observed that the cytopathic effect of lymphomagenic MLVs was restricted to mink cells. Southern blot analysis of MLV-infected cells revealed an accumulation of the linear form of unintegrated viral DNA, particularly in mink cells after MCF13 MLV infection. Thus, a strong correlation was observed between viral superinfection, which results in the accumulation of high levels of unintegrated viral DNA, and cell killing. Immunoblot analysis for MCF13 MLV-infected mink epithelial cells did not show a significant change in total p53 levels or its phosphorylated form at Ser-15 compared with that in mock-treated cells. Moreover, a time course analysis for mink epithelial cells infected with MCF13 MLV did not reveal mitochondrial depolarization or a significant change in Bax levels. These results demonstrate that MCF13 MLV induces apoptosis preferentially in cells in which superinfection occurs, and the mechanism involved is independent of p53 activation and mitochondrial damage.     

Analysis of apoptosis regulatory genes altered by histone deacetylase inhibitors in chronic lymphocytic leukemia cells

Histone deacetylases (HDACs) play a key role in the regulation of acetylation status not only of histones but also of many other non-histone proteins involved in cell cycle regulation, differentiation or apoptosis. Therefore, histone deacetylase inhibitors (HDACi) have emerged as promising anticancer agents. Herein, we report the characterization of apoptosis in B-cell chronic lymphocytic leukemia (CLL) induced by two HDACi, Kendine 92 and SAHA. Both inhibitors induce dose-, time- and caspase-dependent apoptosis through the mitochondrial pathway. Interestingly, Kendine 92 and SAHA show a selective cytotoxicity for B lymphocytes and induce apoptosis in CLL cells with mutated or deleted TP53 as effectively as in tumor cells harboring wild-type TP53. The pattern of apoptosis-related gene and protein expression profile has been characterized. It has shown to be irrespective of TP53 status and highly similar between SAHA and Kendine 92 exposure. The balance between the increased BAD, BNIP3L, BNIP3, BIM, PUMA and AIF mRNA expression levels, and decreased expression of BCL-W, BCL-2, BFL-1, XIAP and FLIP indicates global changes in the apoptosis mRNA expression profile consistent with the apoptotic outcome. Protein expression analysis shows increased levels of NOXA, BIM and PUMA proteins upon Kendine 92 and SAHA treatment. Our results highlight the capability of these molecules to induce apoptosis not only in a selective manner but also in those cells frequently resistant to standard treatments. Thus, Kendine 92 is a novel HDACi with anticancer efficacy for non-proliferating CLL cells.     

CD79a detected by ZL7.4 separates chronic lymphocytic leukemia from mantle cell lymphoma in the leukemic phase.

Both B-cell chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are characterized by a lymphoproliferation of neoplastic CD5+ B-cells, but an accurate differential diagnosis between these two malignancies is vitally important for guiding treatment options. Because CD79a has been identified as a pan-B marker, we intended to use it in place of CD19 to identify B-cells and to use CD23 to distinguish between CLL and MCL in the leukemic phase. Anti-CD79a (clone ZL7.4) was used to detect the Igalpha/mb1 protein in fresh CD5+ B-lymphocytes by dual-channel flow cytometry. Expression of CD19 and CD23 were similarly assessed. As expected, CD19 was expressed in all specimens, whereas CD23 expression was zero in 3/4 MCLs, weak in 1/4 MCLs, and 2/8 CLLs (10-19%) and stronger in 6/8 CLLs (> or =45%). However, although all the CD19+/CD5+ cells of MCL expressed high CD79a levels, CD79a expression was negligible or absent in 8/8 CLL specimens (mean positivity for CD79a = 2.41 +/- 2.71%). CD79a (ZL7.4) levels may provide a more reliable distinction than CD23 levels between CLL and MCL. If these results hold up in a larger series, we recommend that the ZL7.4 antibody should be considered in routine marker panels for CLL and low-grade lymphoma.     

Role of CTLA4 in the Proliferation and Survival of Chronic Lymphocytic Leukemia

Earlier, we reported that CTLA4 expression is inversely correlated with CD38 expression in chronic lymphocytic leukemia (CLL) cells. However, the specific role of CTLA4 in CLL pathogenesis remains unknown. Therefore, to elucidate the possible role of CTLA4 in CLL pathogenesis, CTLA4 was down-regulated in primary CLL cells. We then evaluated proliferation/survival in these cells using MTT, ³H-thymidine uptake and Annexin-V apoptosis assays. We also measured expression levels of downstream molecules involved in B-cell proliferation/survival signaling including STAT1, NFATC2, c-Fos, c-Myc, and Bcl-2 using microarray, PCR, western blotting analyses, and a stromal cell culture system. CLL cells with CTLA4 down-regulation demonstrated a significant increase in proliferation and survival along with an increased expression of STAT1, STAT1 phosphorylation, NFATC2, c-Fos phosphorylation, c-Myc, Ki-67 and Bcl-2 molecules. In addition, compared to controls, the CTLA4-downregulated CLL cells showed a decreased frequency of apoptosis, which also correlated with increased expression of Bcl-2. Interestingly, CLL cells from lymph node and CLL cells co-cultured on stroma expressed lower levels of CTLA4 and higher levels of c-Fos, c-Myc, and Bcl-2 compared to CLL control cells. These results indicate that microenvironment-controlled-CTLA4 expression mediates proliferation/survival of CLL cells by regulating the expression/activation of STAT1, NFATC2, c-Fos, c-Myc, and/or Bcl-2.     

Arsenic trioxide augments Chk2/p53-mediated apoptosis by inhibiting oncogenic Wip1 phosphatase

The oncogenic Wip1 phosphatase (PPM1D) is induced upon DNA damage in a p53-dependent manner and is required for inactivation or suppression of DNA damage-induced cell cycle checkpoint arrest and of apoptosis by dephosphorylating and inactivating phosphorylated Chk2, Chk1, and ATM kinases. It has been reported that arsenic trioxide (ATO), a potent cancer chemotherapeutic agent, in particular for acute promyelocytic leukemia, activates the Chk2/p53 pathway, leading to apoptosis. ATO is also known to activate the p38 MAPK/p53 pathway. Here we show that phosphatase activities of purified Wip1 toward phosphorylated Chk2 and p38 in vitro are inhibited by ATO in a dose-dependent manner. Furthermore, DNA damage-induced phosphorylation of Chk2 and p38 in cultured cells is suppressed by ectopic expression of Wip1, and this Wip1-mediated suppression can be restored by the presence of ATO. We also show that treatment of acute promyelocytic leukemia cells with ATO resulted in induction of phosphorylation and activation of Chk2 and p38 MAPK, which are required for ATO-induced apoptosis. Importantly, this ATO-induced activation of Chk2/p53 and p38 MAPK/p53 apoptotic pathways can be enhanced by siRNA-mediated suppression of Wip1 expression, further indicating that ATO inhibits Wip1 phosphatase in vivo. These results exemplify that Wip1 is a direct molecular target of ATO.     

VDAC1-based peptides: novel pro-apoptotic agents and potential therapeutics for B-cell chronic lymphocytic leukemia

The voltage-dependent anion channel 1 (VDAC1), localized in the outer mitochondrial membrane, mediates metabolic cross-talk between the mitochondrion and the cytoplasm and thus serves a fundamental role in cell energy metabolism. VDAC1 also plays a key role in mitochondria-mediated apoptosis, interacting with anti-apoptotic proteins. Resistance of cancer cells to apoptosis involves quenching the mitochondrial apoptotic pathway by over-expression of anti-apoptotic/pro-survival hexokinase (HK) and Bcl-2 family proteins, proteins that mediate their anti-apoptotic activities via interaction with VDAC1. Using specifically designed VDAC1-based cell-penetrating peptides, we targeted these anti-apoptotic proteins to prevent their pro-survival/anti-apoptotic activities. Anti-apoptotic proteins are expressed at high levels in B-cell chronic lymphocytic leukemia (CLL), an incurable disease requiring innovative new approaches to improve therapeutic outcome. CLL is characterized by a clonal accumulation of mature neoplastic B cells that are resistant to apoptosis. Specifically, we demonstrate that the VDAC1-based peptides (Antp-LP4 and N-Terminal-Antp) selectively kill peripheral blood mononuclear cells (PBMCs) obtained from CLL patients, yet spare those obtained from healthy donors. The cell death induction competence of the peptides was well correlated with the amount of double positive CD19/CD5 cancerous CLL PBMCs, further illustrating their selectivity toward cancer cells. Moreover, these VDAC1-based peptides induced apoptosis by activating the mitochondria-mediated pathway, reflected in membrane blebbing, condensation of nuclei, DNA fragmentation, release of mitochondrial cytochrome c, loss of mitochondrial membrane potential, decreased cellular ATP levels and detachment of HK, all leading to apoptotic cell death. Thus, the mode of action of the peptides involves decreasing energy production and inducing apoptosis. Over 27 versions of cell-penetrating VDAC1-based peptides were designed and screened to identify the most stable, short and apoptosis-inducing peptides toward CLL-derived lymphocytes. In this manner, three optimized peptides suitable for in vivo studies were identified. This study thus reveals the potential of VDAC1-based peptides as an innovative and effective anti-CLL therapy.     

Sensitization of IL-2 signaling through TLR-7 enhances B lymphoma cell immunogenicity

The innate ability of B lymphoma cells to escape control by tumor-reactive T cells must be overcome to develop effective immunotherapies for these diseases. Because signals from both the innate and adaptive immune systems direct the acquisition of strong immunogenicity by professional APCs, the effects of IL-2 and the TLR-7 agonist, S28690, on the immunogenic properties of chronic lymphocytic leukemia (CLL) B cells were studied. IL-2 with S28690 caused CLL cells to proliferate and increased their expression of B7-family members, production of TNF-alpha and IL-10, and levels of tyrosine-phosphorylated STAT-1 and STAT-3 proteins. S28690 increased CD25 expression on CLL cells and sensitized them to IL-2 signaling. However, IL-2 did not change TLR-7 expression or signaling in CLL cells. The ability to stimulate T cell proliferation required additional activation of protein kinase C, which inhibited tumor cell proliferation, "switched off" IL-10 production, and caused essentially all CLL cells (regardless of clinical stage) to acquire a CD83(high)CD80(high)CD86(high)CD54(high) surface phenotype marked by the activation of STAT-1 without STAT-3. These findings suggest that TLR-7 "licenses" human B cells to respond to cytokines of the adaptive immune system (such as IL-2) and provide a strategy to increase the immunogenicity of lymphoma cells for therapeutic purposes.     

Modulation of NF-kappa B activity and apoptosis in chronic lymphocytic leukemia B cells

Chronic lymphocytic leukemia (CLL) is an indolent malignancy of CD5+ B lymphocytes. CLL cells express CD40, a key regulator of B cell proliferation, differentiation, and survival. In nonmalignant B cells, CD40 ligation results in nuclear translocation and activation of NF-kappaB proteins. Based on observations that in some CLL cases, the tumor cells express both CD40 and its ligand, CD154 (CD40 ligand), we proposed a model for CLL pathogenesis due to CD40 ligation within the tumor. To evaluate this issue, we used freshly isolated CLL B cells to examine constitutive and inducible NF-kappaB activity by electrophoretic mobility shift assay. We consistently observed high levels of nuclear NF-kappaB-binding activity in unstimulated CLL B cells relative to that detected in nonmalignant human B cells. In each case examined, CD40 ligation further augmented NF-kappaB activity and prolonged CLL cell survival in vitro. The principle NF-kappaB proteins in stimulated CLL cells appear to be quite similar to those in nonmalignant human B cells and include p50, p65, and c-Rel. In a CD154-positive case, blocking CD154 engagement by mAb to CD154 resulted in inhibition of NF-kappaB activity in the CLL cells. The addition of anti-CD154 mAb resulted in accelerated CLL cell death to a similar degree as was observed in cells exposed to dexamethasone. These data indicate that CD40 engagement has a profound influence on NF-kappaB activity and survival in CLL B cells, and are consistent with a role for CD154-expressing T and B cells in CLL pathogenesis. The data support the development of novel therapies based on blocking the CD154-CD40 interaction in CLL.