benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Leukemia--Lymphocytic--Chronic--B-Cell

The compromised antioxidant defense system in chronic lymphocytic leukemia (CLL) suggested a potential use for reactive oxygen species (ROS) generating arsenic trioxide (ATO) and ascorbic acid. While both ATO and ascorbic acid mediate cytotoxicity in CLL B cells as single agents, the efficacy of ATO is enhanced by ascorbic acid. This effect is dependent on increased ROS accumulation, as pretreatment of B-CLL cells with a glutathione reducing buthionine sulfoximine or catalase inhibiting aminotriazole, enhanced ATO/ascorbic acid-mediated cytotoxicity. Pretreatment with reducing agents such as catalase, or thiol antioxidant, N-acetyl cysteine or GSH also abrogated ATO/ascorbic acid-mediated cytotoxicity. Furthermore, Hu1D10-mediated cell death was enhanced with ATO and ascorbic acid, thus justifying potential combination of ATO/arsenic trioxide therapy with antibodies such as Hu1D10 that also cause accumulation of ROS.

Topics: Amino Acid Chloromethyl Ketones; Amitrole; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Arsenic Trioxide; Arsenicals; Ascorbic Acid; B-Lymphocytes; Buthionine Sulfoximine; Catalase; Cell Line, Tumor; Cysteine Proteases; Cysteine Proteinase Inhibitors; Drug Evaluation, Preclinical; Drug Synergism; Enzyme Activation; Glutathione; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Neoplasm Proteins; Oxidants; Oxidative Stress; Oxides; Reactive Oxygen Species

Chronic lymphocytic leukemia (CLL) is an incurable disease with a natural history of increasing resistance to chemotherapy. A novel approach to overcome chemotherapy resistance may be targeting the endoplasmic reticulum (ER).. The involvement of the unfolded protein response (UPR) in the cell killing effect of xanthohumol (X) was examined in 18 patient samples.. X-induced apoptosis of CLL cells was accompanied by the induction of glucose-regulated protein of 78 kDa (GRP78) and heat-shock protein of 70 kDa (Hsp70) protein levels and by sustained phosphorylation of the eukaryotic translation initiation factor 2 (eIF2alpha), suggesting the involvement of the ER stress transducer, the double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK). The X-box-binding protein 1 (XBP1) mRNA was spliced but no clear activation of activating transcription factor 6 (ATF6) was observed. The proapoptotic outcome was further demonstrated by the up-regulation of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), down-regulation of myeloid cell leukemia 1 (Mcl-1) and B-cell lymphoma 2 (Bcl-2), cleavage of poly-(ADP)-ribose polymerase (PARP) and processing of caspase-3, -4 and -9. Furthermore, X showed proteasome inhibitory activity.. X stimulates the proapoptotic arm of the UPR in ex vivo CLL cells, suggesting that ER stress may play an important role during X-induced apoptosis.

Topics: Activating Transcription Factor 6; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; eIF-2 Kinase; Endoplasmic Reticulum Chaperone BiP; Endoribonucleases; Flavonoids; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Membrane Proteins; NF-kappa B; Propiophenones; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Serine-Threonine Kinases; Unfolded Protein Response

Gossypol, a cottonseed extract derivative, acts as a BH3-mimetic, binding to the BH3 pocket of antiapoptotic proteins and displacing pro-death partners to induce apoptosis. However, knowledge on the molecular underpinnings of its downstream effects is limited. Since chronic lymphocytic leukemia (CLL) cells express high levels of antiapoptotic proteins that act as a survival mechanism for these replicationally quiescent lymphocytes, we investigated whether gossypol induces apoptosis in these cells and what mechanism underlies gossypol-mediated cytotoxicity. Gossypol induced cell death in a concentration- and time-dependent manner; 24-hour incubation with 30 microM gossypol resulted in 50% cell death (median; range, 10%-80%; n = 47) that was not abrogated by pan-specific caspase inhibitor. Starting at 4 hours, the mitochondrial outer membrane was significantly permeabilized (median, 77%; range, 54%-93%; n = 15). Mitochondrial outer membrane permeabiliztaion (MOMP) was concurrent with increased production of reactive oxygen species (ROS); however, antioxidants did not abrogate gossypol-induced cell death. Mitochondrial membrane permeabilization was also associated with loss of intracellular adenosine triphosphate (ATP), activation of BAX, and release of cytochrome c and apoptosis-inducing factor (AIF), which was translocated to the nucleus. Blocking AIF translocation resulted in a decreased apoptosis, suggesting that AIF contributes to gossypol-mediated cytotoxicity in CLL lymphocytes.

Topics: Acetylcysteine; Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Antioxidants; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Biological Transport, Active; Calpain; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Dipeptides; Gossypol; Humans; In Vitro Techniques; Leukemia, Lymphocytic, Chronic, B-Cell; Mitochondrial Membranes; Molecular Mimicry; Reactive Oxygen Species; Superoxides

To investigate the proapoptotic capacities of four arylcoumarin analogues of combretastatins on leukemic cells from B-cell chronic lymphocytic leukemia (CLL), a malignancy characterized by apoptosis deficiency.. The effects of the four compounds on several nuclear, membrane, and mitochondrial events of apoptosis and on expression of proteins controlling the apoptosis were analyzed after treatment of cultured CLL patients' cells.. Treatment with all four compounds resulted in a dose-dependent internucleosomal DNA fragmentation, in stimulation of phosphatidylserine externalization, disruption of the mitochondrial transmembrane potential and caspase-3 activation. DNA fragmentation was prevented in the presence of the pan-caspase inhibitor z-VAD-fmk. Two of the compounds downregulated the expression of Mcl-1, a protein thought to be crucial for the antiapoptotic state in CLL, while Bcl-2 expression was unaffected. No effects were observed on the expression of p27kip1 or the inducible nitric oxide synthase, two proteins, which are constitutively overexpressed by CLL cells and downregulated during the apoptosis induced by other plant-derived molecules (flavopiridol, polyphenols, or hyperforin). This suggests different mechanisms of action for the compounds studied here. Furthermore, normal B lymphocytes from healthy donors appeared less sensitive than CLL cells to the proapoptotic activity of the four compounds.. The four arylcoumarin analogues were able to promote the apoptosis of CLL cells ex vivo through the caspase-dependent mitochondrial pathway. Therefore, these compounds may be of interest to develop new therapies of CLL based on apoptosis restoration.

Topics: Aged; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Bibenzyls; Caspase 3; Caspase Inhibitors; Cyclin-Dependent Kinase Inhibitor p27; Cysteine Proteinase Inhibitors; DNA Fragmentation; Drug Screening Assays, Antitumor; Enzyme Activation; Female; Humans; Intracellular Signaling Peptides and Proteins; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Myeloid Cell Leukemia Sequence 1 Protein; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

We previously reported that flavopiridol-induced apoptosis of B cell chronic lymphocytic leukemia (CLL) patients' cells ex vivo is associated with downregulation of both the inducible nitric oxide (NO) synthase (iNOS) that produces the antiapoptotic molecule NO, and the CDK inhibitor p27kip1 that is thought to block the cell cycle of CLL cells. Here, we show that iNOS downregulation is caspase-dependent and thus can be considered as one of the effector mechanisms of apoptosis, but not a primary triggering event induced by flavopiridol. Furthermore, we also find that this flavone favors the entry into the S and G2 phases of the cell cycle of a subpopulation of the leukemic cells, confirming that flavopiridol might be useful for improving the efficacy of cell cycle-dependent cytostatic agents in the therapy of CLL.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Caspases; Cell Cycle; Cells, Cultured; Down-Regulation; Flavonoids; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Nitric Oxide Synthase Type II; Piperidines

Primary chronic lymphocytic leukemia (CLL) cells are exquisitely sensitive to ABT-737, a small molecule BCL2-antagonist, which induces many of the classical biochemical and ultrastructural features of apoptosis, including BAX/BAK oligomerization, cytochrome c release, caspase activation and chromatin condensation. Surprisingly, ABT-737 also induces mitochondrial inner membrane permeabilization (MIMP) resulting in mitochondrial matrix swelling and rupture of the outer mitochondrial membrane (OMM), so permitting the rapid efflux of cytochrome c from mitochondrial cristae and facilitating rapid caspase activation and apoptosis. BAX and BAK appear to be involved in the OMM discontinuities as they localize to the OMM break points. Notably, ABT-737 induced mitochondrial matrix swelling and OMM discontinuities in other primary B-cell malignancies, including mantle cell, follicular and marginal zone lymphoma cells but not in several cell lines studied. Thus, we describe a new paradigm of apoptosis in primary B-cell malignancies, whereby targeting of BCL2 results in all the classical features of apoptosis together with OMM rupture independent of caspase activation. This mechanism may be far more prevalent than hitherto recognized due to the failure of most methods, used to measure apoptosis, to recognize such a mechanism.

Topics: Adult; Amino Acid Chloromethyl Ketones; Apoptosis; B-Lymphocytes; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Biphenyl Compounds; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Mitochondrial Membranes; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides

We compared antisense phosphorothioate oligonucleotides (PS-ODN) that target BCL-2 such as Genasense (G3139-PS), with other PS-ODN or phosphodiester-ODN (PO-ODN) in their relative capacity to induce apoptosis of chronic lymphocytic leukemia (CLL) B cells in vitro. Surprisingly, we found that thymidine-containing PS-ODN, but not PO-ODN, induced activation and apoptosis of CLL cells independent of BCL-2 antisense sequence or CpG motifs. All tested thimidine-containing PS-ODN, irrespective of their primary sequences, reduced the expression of Bcl-2 protein and increased the levels of the proapoptotic molecules p53, Bid, Bax in CLL cells. Apoptosis induced by thymidine-containing PS-ODN was preceded by cellular activation, could be blocked by the tyrosine-kinase inhibitor imatinib mesylate (Gleevec), and was dependent on ABL kinase. We conclude that thymidine-containing PS-ODN can activate CLL cells and induce apoptosis via a mechanism that is independent of BCL-2 gene interference or CpG motifs.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; B-Lymphocytes; Benzamides; BH3 Interacting Domain Death Agonist Protein; Caspases; Cell Survival; CpG Islands; Drug Screening Assays, Antitumor; Enzyme Activation; Genes, bcl-2; Humans; Imatinib Mesylate; In Vitro Techniques; Leukemia, Lymphocytic, Chronic, B-Cell; Oligodeoxyribonucleotides; Organothiophosphorus Compounds; Phosphorylation; Piperazines; Proto-Oncogene Proteins c-abl; Pyrimidines; Structure-Activity Relationship; Thymidine; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Up-Regulation

B-cell chronic lymphocytic leukemia (B-CLL) is a remarkably heterogeneous disorder. Some patients have an indolent disease whereas others undergo a more agressive presentation needing treatment. New therapeutics approaches are necessary for the treatment of B-CLL. Bortezomib (Btz), is a proteasome inhibitor, currently undergoing clinical trials whose function, at least in part, by stabilizing the IkappaBalpha protein and inhibiting NFkappaB activation.. The objective of this work was to study the effects of Btz on isolated human B-CLL cells, in vitro, and to correlate the differential rates of apoptosis induction with biological variables.. 31 B-CLL samples, from patients in stage A of Binet were used for this study, and the apoptotic effect of Btz on these cells was measured.. Our data show that Btz treatment of B-CLL cells induces apoptosis in a time and dose-dependent manner. The apoptosis induction is mediated in part by inhibition of NFkappaB and is dependent on caspases activation. Interesting, in IgVH mutated cells, Btz have statistically significant differences in their in vitro activity on B-CLL cells according to their BCL-6 mutational status.. Btz is a promising pharmacologic agent for the treatment of B-CLL, but its efficacy seems to be related to IgVH and BCL-6 mutational status, therefore, it could be interesting to further investigate the mechanisms involved in the different behavior of the cells in response to apoptosis induction by this drug.

Topics: ADP-ribosyl Cyclase 1; Adult; Aged; Aged, 80 and over; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Genes, Immunoglobulin; Humans; I-kappa B Kinase; I-kappa B Proteins; Immunoglobulin Heavy Chains; Immunoglobulin Variable Region; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Neoplasm Proteins; NF-kappa B; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-6; Pyrazines; Sulfones; Tumor Cells, Cultured; ZAP-70 Protein-Tyrosine Kinase

Acadesine, 5-aminoimidazole-4-carboxamide (AICA) riboside, induced apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells in all samples tested (n = 70). The half-maximal effective concentration (EC(50)) for B-CLL cells was 380 +/- 60 microM (n = 5). The caspase inhibitor Z-VAD.fmk completely blocked acadesine-induced apoptosis, which involved the activation of caspase-3, -8, and -9 and cytochrome c release. Incubation of B-CLL cells with acadesine induced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), indicating that it is activated by acadesine. Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, 5-iodotubercidin, an inhibitor of adenosine kinase, and adenosine completely inhibited acadesine-induced apoptosis and AMPK phosphorylation, demonstrating that incorporation of acadesine into the cell and its subsequent phosphorylation to AICA ribotide (ZMP) are necessary to induce apoptosis. Inhibitors of protein kinase A and mitogen-activated protein kinases did not protect from acadesine-induced apoptosis in B-CLL cells. Moreover, acadesine had no effect on p53 levels or phosphorylation, suggesting a p53-independent mechanism in apoptosis triggering. Normal B lymphocytes were as sensitive as B-CLL cells to acadesine-induced apoptosis. However, T cells from patients with B-CLL were only slightly affected by acadesine at doses up to 4 mM. AMPK phosphorylation did not occur in T cells treated with acadesine. Intracellular levels of ZMP were higher in B-CLL cells than in T cells when both were treated with 0.5 mM acadesine, suggesting that ZMP accumulation is necessary to activate AMPK and induce apoptosis. These results suggest a new pathway involving AMPK in the control of apoptosis in B-CLL cells and raise the possibility of using acadesine in B-CLL treatment.

Topics: Adenosine; Amino Acid Chloromethyl Ketones; Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Antimetabolites, Antineoplastic; Apoptosis; B-Lymphocytes; Caspases; Cyclic AMP-Dependent Protein Kinases; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Inhibitors; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; MAP Kinase Signaling System; Mitochondria; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Neoplasm Proteins; Neoplastic Stem Cells; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Ribonucleosides; Ribonucleotides; T-Lymphocytes; Tubercidin; Tumor Cells, Cultured

The caspase family of protease is speculated to have a crucial role in apoptosis. The effect of treatment with Idarubicin (IDA) and Medroxyprogesterone acetate (MPA), used alone or in combination, on the activation of Caspase-3 in canine Chronic Lymphatic Leukaemia (CLL) cells was investigated, in order to clarify the mechanism of chemo- and hormone-therapy mediated apoptosis. Caspase activity was determined by a quantitative fluorimetric assay. Apoptosis was monitored by propidium iodide (PI) and nucleosomes assay. Treatment of CLL cells for 24 h with MPA 5 microM did not significantly activate caspase-3 but its activity was increased almost 5-fold more with IDA 1 microM (P < 0.05) than control. Treatment of CLL cells with IDA 1 microM in equimolecular association with MPA was able to increase the activation of caspase-3 induced by IDA of the 61.2% (P < 0.05) in comparison with IDA alone. The activation of caspase-3 was confirmed evaluating apoptosis by PI and nucleosomes assay. Furthermore, both caspase-3 activation and apoptosis triggered by IDA alone or in combination with MPA were significantly inhibited by specific caspase-3 inhibitor AC-DEVD-CMK. These findings provide an explanation for IDA and MPA induced-apoptosis mechanism.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspase Inhibitors; Caspases; Dogs; Drug Synergism; Enzyme Inhibitors; Fluorometry; Idarubicin; Isoenzymes; Leukemia, Lymphocytic, Chronic, B-Cell; Medroxyprogesterone Acetate; Nucleosomes; Propidium; Tumor Cells, Cultured

B-cell chronic lymphocytic leukemia (B-CLL) is an accumulating disease of slowly proliferating cells. CD10 is not normally expressed on the surface of B-CLL cells. The aim of this study was to ascertain whether B-CLL cells, induced into apoptosis, expressed surface CD10, since a correlation between apoptosis and CD10 expression has been demonstrated.. Peripheral blood cells from 31 untreated B-CLL patients were induced into apoptosis by etoposide, fludarabine or Ga(mu)-Ab treatment and tested for CD10 expression by flow cytometry. Normal CD5+ B cells were also induced into apoptosis and tested for CD10 expression.. CD10 positive cells were absent in B-CLL cell suspensions, but were detected following in vitro culture, and their appearance paralleled that of apoptotic cells. Treatment with etoposide, fludarabine or Ga(mu)-Ab enhanced both apoptosis and CD10 expression. Inhibition of apoptosis by VAD-fmk or Ga(delta)-Ab prevented CD10 expression. Cell separation tests following induction of apoptosis demonstrated that CD10+ cells were apoptotic. CD10+ cells were observed in the peripheral blood of two patients within a few hours following fludarabine infusion. In another patient, who failed to respond, no CD10+ cells were seen. Expression of CD10 was observed also in normal CD5+ B cells when these were induced into apoptosis.. This study demonstrates that B-CLL cells, as well as normal CD5+ B cells, become CD10+ following apoptosis induction in vitro. Some of the data obtained also suggest a use for CD10 to monitor apoptosis of B-CLL in a clinical setting.

Topics: ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Amino Acid Chloromethyl Ketones; Antibodies; Antibodies, Monoclonal; Antigens, CD; Apoptosis; B-Lymphocytes; Cell Line, Tumor; Etoposide; Gene Expression Regulation, Neoplastic; Humans; Immunoglobulin A; Immunoglobulin D; Immunoglobulin M; Leukemia, Lymphocytic, Chronic, B-Cell; Membrane Glycoproteins; Neprilysin; Palatine Tonsil; Vidarabine

Monoclonal antibody IDEC-C2B8 (rituximab) has been shown to be highly effective in the treatment of non-Hodgkin's lymphomas (NHL). The present study was designed to investigate relationships between the efficacy of IDEC-C2B8 and expression of CD20, presence of complement, and effects of differently acting chemotherapeutic agents used in lymphoma treatment (doxorubicin, mitoxantrone, cladribine, bendamustine).. DOHH-2, WSU-NHL and Raji lymphoma cell lines and ex vivo cells from patients with chronic lymphocytic leukemia (CLL) (n=17) and leukemic B-cell lymphomas (n=9) were studied. Additionally, the effect of interleukin (IL)-2, IL-4, IL-6, IL-13, granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)alpha on expression of CD20 molecules per cell was determined.. We demonstrate that 10 mg/mL rituximab saturated 80-95% of CD20 molecules per cell in all tested lymphoma samples. Although rituximab induced only a minor increase of apoptosis, combinations of rituximab with different cytotoxic drugs significantly decreased the IC(30)- and IC(50) dosages of the chemotherapeutic agents necessary for induction of apoptosis irrespective of addition of complement, demonstrating a chemosensitizing effect of rituximab in combination with cytotoxic drugs in the neoplastic lymphocytes. This effect seemed to be independent of the percentage of saturated CD20 molecules. After addition of caspase inhibitors to the cell lines incubated with rituximab and cytotoxic agents, caspase-7 and -8 were found, by Western blotting, to be the executioner caspases, possibly explaining the rituximab-sensitized apoptosis. Preincubation of lymphoma cells with cytokines did not alter the expression of CD20; IL-2 and IL-4 even decreased the rate of apoptosis.. We conclude that rituximab sensitizes lymphoma cells to the effect of differently acting cytotoxic drugs used in lymphoma treatment, that this effect does not require complement, and that caspase-7 and -8 may represent the main executioner caspases in chemosensitization by rituximab.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antigens, CD20; Antigens, Neoplasm; Antineoplastic Agents; Apoptosis; Bendamustine Hydrochloride; Burkitt Lymphoma; Caspase 7; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cladribine; Complement Activation; Complement System Proteins; Cysteine Proteinase Inhibitors; Doxorubicin; Drug Synergism; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukins; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Follicular; Mitoxantrone; Neoplasm Proteins; Nitrogen Mustard Compounds; Oligopeptides; Rabbits; Rituximab; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Proteasome inhibitors, including lactacystin and MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), potently induce apoptosis in leukaemic B cells from patients with B cell chronic lymphocytic leukaemia (B-CLL). This pro-apoptotic effect occurs in cells from patients at all stages of the disease, including those resistant to conventional chemotherapy, suggesting that proteasome inhibitors may be useful for treatment of B-CLL. Following initial inhibition of proteasomal activity, these agents induce mitochondrial cytochrome c release and caspase-dependent apoptosis, involving cleavage/activation of caspases -2, -3, -7, -8 and -9. Pre-treatment with the cell permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe)fluoromethyl ketone (Z-VAD.fmk), did not prevent the release of cytochrome c or partial processing of caspase-9 but prevented activation of effector caspases and the induction of apoptosis. These results suggest that the release of cytochrome c is caspase independent and that caspase-9 is the initiator caspase in proteasome inhibitor-induced apoptosis of B-CLL cells. Activation of B-CLL lysates with dATP results in the formation of an approximately 700 kDa caspase-activating apoptosome complex containing Apaf-1. We describe for the first time the formation of a similar approximately 700 kDa caspase-activating apoptosome complex in B-CLL cells induced to undergo apoptosis by proteasome inhibitors.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Apoptosis; Apoptotic Protease-Activating Factor 1; Blotting, Western; Caspase 9; Caspases; Cysteine Endopeptidases; Cytochrome c Group; Cytosol; Enzyme Activation; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Leupeptins; Microscopy, Electron; Molecular Weight; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Biosynthesis; Proteins; Tumor Cells, Cultured

Caspases are known to be involved in the apoptotic killing of chronic lymphocytic leukaemia (CLL) cells by fludarabine. However, it is unclear whether these enzymes are required for the induction of such killing, or whether they simply determine the mode of cell death. To address this question, we examined the effect of the broad-spectrum caspase inhibitor Z-VAD.fmk on six different manifestations of nucleoside cytotoxicity. Our results indicate that while caspase activity is required for nucleoside-induced poly(ADP-ribose) polymerase (PARP) cleavage and DNA fragmentation, other manifestations of cell death (mitochondrial depolarization, exposure of phosphatidyl serine, cell membrane disruption and cell shrinkage) are caspase independent. By showing that caspases influence the mode, but not the extent, of nucleoside cytotoxicity, our results exclude defects in these enzymes as a mechanism of nucleoside resistance in CLL.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Caspase Inhibitors; Caspases; Cell Membrane; Cell Size; Cells, Cultured; Enzyme Inhibitors; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Vidarabine

Glucocorticoids and fludarabine are able to induce typical features of apoptosis in CLL lymphocytes. Cysteinyl aspartate specific proteases (caspases) play a key biochemical role in the apoptotic pathway. Caspase activation following cytotoxic stimuli leads to highly specific proteolytic cleavage of functionally important cellular enzymes. One of them is poly ADP-ribose) polymerase (PARP). To some extent caspase activation seems to be under the control of the Bcl-2 family of interacting proteins. We determined the role of Bcl-2-family proteins Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic), activation of caspase-3 (CPP32/Yama) and activation of PARP in CLL apoptosis. All 21 analyzed CLL samples expressed Bcl-2 and Bax. Four of 13 (31%) samples with a low Bcl-2/Bax ratio exhibited in vitro prednisolone resistance, whereas eight of nine (88%) samples with a high Bcl-2/Bax ratio were in vitro resistant (

Topics: Aged; Aged, 80 and over; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; B-Lymphocytes; bcl-2-Associated X Protein; Benzamides; Blotting, Western; Caspase 3; Caspase Inhibitors; Caspases; DNA Fragmentation; Drug Screening Assays, Antitumor; Enzyme Activation; Female; Gene Expression; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

We analyzed the effect of aspirin, salicylate, and other nonsteroidal antiinflammatory drugs (NSAIDs) on the viability of B-chronic lymphocytic leukemia (B-CLL) cells. Aspirin induced a decrease in cell viability in a dose- and time-dependent manner. The mean IC50 for cells from 5 patients was 5.9 +/- 1.13 mmol/L (range, 4.4 to 7.3 mmol/L). In some cases, 2.5 mmol/L aspirin produced an important cytotoxic effect after 4 days of incubation. No effect was observed with other NSAIDs, at concentrations that inhibit cyclooxygenase, such as ketorolac (10 micromol/mL), NS-398 (100 micromol/mL), or indomethacin (20 micromol/mL), thus suggesting the involvement of cyclooxygenase-independent mechanisms in aspirin-induced cytotoxicity. Salicylate also produced dose-dependent cytotoxic effects on B-CLL cells and the mean IC50 for cells from 5 patients was 6.96 +/- 1.13 mmol/L (range, 5 to 7.8 mmol/L). Both aspirin and salicylate induced DNA fragmentation and the proteolytic cleavage of poly(ADP(adenosine 5'-diphosphate)-ribose) polymerase (PARP), demonstrating that both compounds induce apoptosis of B-CLL cells. Finally, inhibition of caspases by Z-VAD.fmk blocked proteolytic cleavage of PARP, DNA fragmentation, and cytotoxicity induced by aspirin. Mononuclear cells from normal donors showed a lower sensitivity than cells from B-CLL patients to aspirin as determined by analysis of cell viability. B and T lymphocytes from normal donors and T lymphocytes from CLL patients are more resistant to aspirin-induced apoptosis, as determined by analysis of phosphatidylserine exposure. These results indicate that aspirin and salicylate induce apoptosis of B-CLL cells by activation of caspases and that this activation involves cyclooxygenase-independent mechanisms.

Topics: Aged; Amino Acid Chloromethyl Ketones; Annexins; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Aspirin; B-Lymphocytes; Cell Survival; Cyclooxygenase Inhibitors; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Fragmentation; Enzyme Activation; Female; Humans; Indomethacin; Ketorolac; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Neoplasm Proteins; Nitrobenzenes; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteins; Salicylates; Salicylic Acid; Sulfonamides; Tolmetin; Tumor Cells, Cultured

Chlorambucil and prednisolone, two commonly used drugs in the treatment of chronic lymphocytic leukemia (CLL), induce apoptosis in CLL cells. We have investigated the involvement in this apoptotic cell death of caspases, which cleave critical cellular substrates thereby acting as the executioners of the apoptotic process. Induction of spontaneous or chlorambucil/prednisolone-induced apoptosis of freshly isolated B-CLL cells in culture resulted in the activation of the 'effector' caspases, -3 and -7, but generally not of caspase-2. Activation of caspases-3 and -7 was accompanied by the proteolysis of the DNA repair enzyme, poly (ADP-ribose) polymerase. Induction of apoptosis was also accompanied by the processing of caspase-8, the extent of which varied between patients. Induction of apoptosis and processing of all the caspases was inhibited by the cell permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethyl ketone (Z-VAD.fmk). Our results demonstrate a key role for the activation and processing of caspases in the execution phase of apoptosis in CLL cells. Apoptosis of CLL cells resulted in the selective activation of some but not all caspases. Our results suggest that the dysregulation of apoptosis observed in CLL may be due to the signalling leading to the activation of caspases rather than a deletion of pro-caspases. High levels of caspase-8 in CLL cells in conjunction with low levels of CD95 receptor may offer new therapeutic opportunities for the treatment of CLL.

Topics: Aged; Aged, 80 and over; Amino Acid Chloromethyl Ketones; Annexin A5; Apoptosis; Caspase 2; Caspase 3; Caspase 7; Caspase 8; Caspase 9; Caspases; Cysteine Proteinase Inhibitors; DNA Repair; Enzyme Activation; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Leukocyte Count; Male; Middle Aged; Neoplasm Staging; Poly(ADP-ribose) Polymerases; Protein Processing, Post-Translational; Tumor Cells, Cultured