benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Lymphoma--B-Cell

Apoptin, a chicken anemia virus-derived protein, selectively induces apoptosis in transformed but not in normal cells, thus making it a promising candidate as a novel anticancer therapeutic. The mechanism of apoptin-induced apoptosis is largely unknown. Here, we report that contrary to previous assumptions, Bcl-2 and Bcl-xL inhibit apoptin-induced cell death in several tumor cell lines. In contrast, deficiency of Bax conferred resistance, whereas Bax expression sensitized cells to apoptin-induced death. Cell death induction by apoptin was associated with cytochrome c release from mitochondria as well as with caspase-3 and -7 activation. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad spectrum caspase inhibitor, was highly protective against apoptin-induced cell death. Apoptosis induced by apoptin required Apaf-1, as immortalized Apaf-1-deficient fibroblasts as well as tumor cells devoid of Apaf-1 were strongly protected. Thus, our data indicate that apoptin-induced apoptosis is not only Bcl-2- and caspase dependent, but also engages an Apaf-1 apoptosome-mediated mitochondrial death pathway.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-X Protein; Breast Neoplasms; Capsid Proteins; Caspase 3; Caspase 7; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Cytochromes c; Enzyme Activation; Female; Fibroblasts; Humans; Intracellular Signaling Peptides and Proteins; Lymphoma, B-Cell; Male; Mitochondria; Prostatic Neoplasms; Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Tumor Cells, Cultured

Sphingolipid metabolites are important regulators of cell growth and apoptosis. To clarify the biological roles of cell surface sialylation in the effects of sphingomyelinase (SM) treatment on cell viability, the human diffuse large B cell lymphoma cell line, HBL-2 with or without treatment with Vibrio cholerae neuraminidase, was incubated with exogenous bacterial SM which is a key enzyme of ceramide production from sphingolipids in cell membranes. SM treatment enhanced viability of HBL-2 cells compared to non-treatment after 6 h of incubation. On the other hand, viability of HBL-2 cells was decreased by SM treatment with neuraminidase pre-treatment after 6 and 24 h of incubation, and ceramide production on cell surfaces of SM treated cells was enhanced by neuraminidase treatment as shown by flow cytometric analysis. Furthermore, treatment with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor which specifically reduces the activity of UDP-glucose:ceramide glucosyltransferase in combination with SM treatment, causes the viability of HBL-2 cells to be decreased more with neuraminidase pre-treatment than without it. Exogenous C6-ceramide induced HBL-2 cell death, and there was no difference in the effects of C6-ceramide after 6 h of incubation between treatment and non-treatment with neuraminidase. Together these data suggest that alteration in susceptibility of HBL-2 cells to SM by neuraminidase treatment may precede the process of ceramide production, and that cell death through the activation of SM, which induces ceramide production, is regulated by cell surface sialylation in DLBCL.

Topics: Amino Acid Chloromethyl Ketones; Biotinylation; Cell Death; Cell Line, Tumor; Cell Membrane; Cell Survival; Ceramides; Enzyme Inhibitors; Flow Cytometry; Glucose; Glucosyltransferases; Humans; Lectins; Lymphoma; Lymphoma, B-Cell; Lymphoma, Non-Hodgkin; Models, Biological; Neuraminidase; Sialic Acids; Sphingomyelin Phosphodiesterase; Temperature; Time Factors; Uridine Diphosphate

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

CH31 cells have been used for analysis of B cell tolerance, since engagement of membrane immunoglobulin (mIg) results in loss in mitochondrial membrane potential (DeltaPsim), followed by cell death. We have reported that the dominant-negative (dn) form of c-Jun N-terminal kinase (JNK) substantially prevented a loss of DeltaPsim at 24 h, with partial protection around 48 h after anti-IgM stimulation. In this study, we demonstrate that anti-IgM induced a sustained activation of p38 mitogen-activated protein (MAP) kinase. The p38MAP kinase inhibitor SB203580 substantially prevented loss of DeltaPsim at 14 h, with partial prevention (18-24 h) after anti-IgM stimulation. The dnJNK1-mediated prevention of anti-IgM-induced mitochondrial dissipation was enhanced by SB203580 at 42 h, but not 24 h, after stimulation, suggesting that activation of either p38 MAP kinase or JNK may be sufficient for the initiation of early phase of anti-IgM-induced loss of DeltaPsim while both may be necessary in the late phase.

Topics: Amino Acid Chloromethyl Ketones; B-Lymphocytes; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Imidazoles; Immunoglobulin M; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Lymphoma, B-Cell; Membrane Potentials; Mitochondria; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Pyridines; Tumor Cells, Cultured

Cross-linking of the Ag receptors on the immature B cell lymphoma, WEHI-231, leads to growth arrest and apoptosis. We now show that although commitment to such B cell receptor (BCR)-mediated apoptosis correlates with mitochondrial phospholipase A(2) activation, disruption of mitochondrial function, and ATP depletion, it is executed independently of caspase activation. First, we demonstrate a pivotal role for mitochondrial function in determining B cell fate by showing up-regulation of cytosolic phospholipase A(2) expression, induction of mitochondrial phospholipase A(2) activity, arachidonic acid-mediated collapse of mitochondrial transmembrane inner potential (Delta psi(m)), and depletion of cellular ATP under conditions of apoptotic, but not proliferative, signaling via the BCR. Importantly, disruption of Delta psi(m), ATP depletion, and apoptosis can be prevented by rescue signals via CD40 or by Delta psi(m) stabilizers such as antimycin or oligomycin. Second, we show that commitment and postmitochondrial execution of BCR-mediated apoptosis are not dependent on caspase activation by demonstrating that such apoptotic signaling does not induce release of cytochrome c from the mitochondria or activation of effector caspases, as evidenced by poly(ADP-ribose) polymerase or Bcl-x(L) cleavage. Indeed, apoptotic signaling via the BCR in WEHI-231 B cells does not stimulate the activation of caspase-3 and, consistent with this, BCR-mediated disruption of Delta psi(m) and commitment to apoptosis take place in the presence of caspase inhibitors. In contrast, BCR signaling induces the postmitochondrial activation of cathepsin B, and resultant apoptosis is blocked by the cathepsin B inhibitor, (23,35)trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester (EST) suggesting a key role for this executioner protease in Ag receptor-driven apoptosis of WEHI-231 immature B cells.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; B-Lymphocytes; Caspase 3; Caspase Inhibitors; Caspases; Cell Differentiation; Cysteine Proteinase Inhibitors; Cytochrome c Group; Enzyme Activation; Growth Inhibitors; Humans; Intracellular Membranes; Jurkat Cells; Ligands; Lymphoma, B-Cell; Membrane Potentials; Mice; Mitochondria; Phospholipases A; Phospholipases A2; Receptors, Antigen, B-Cell; Signal Transduction; Tumor Cells, Cultured

A family of mitogen-activated protein (MAP) kinases comprising the extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 MAP kinases are involved in proliferation and apoptosis. However, there are some arguments concerning the role of these kinases in Ag-induced B cell apoptosis. Two of the B lymphoma cell lines (CH31 and WEHI-231) susceptible to anti-IgM-induced apoptosis were used as a model. To address these issues, we examined the kinetics of anti-IgM-induced activation of MAP kinases and established cell lines overexpressing a dominant-negative (dn) mutant form of JNK1 (dnJNK1). Anti-IgM induced a sustained JNK1 activation with a peak at 8 h, with a marginal activation of ERK1/ERK2 in CH31 cells. The sustained JNK1 activation was not a secondary event through a caspase activation. The peak point of the JNK1 activation was just before the onset of a decline in mitochondrial membrane potential, which preceded anti-IgM-induced cell death. Following anti-IgM stimulation, dnJNK1 prevented a decline in mitochondrial membrane potential at 24 h, with a prolonged inhibition up to 72 h in WEHI-231, although it did so only partially during a later time period in CH31. The dnJNK1 cells also demonstrated diminished procaspase-3 activation and a decreased rate of apoptosis upon anti-IgM stimulation, with a concomitant increased arrest in G(1) phase, which could be explained by enhanced levels of cyclin-dependent kinase inhibitor p27(Kip1) protein. Thus, anti-IgM-induced JNK activation might be implicated in cell cycle progression as well as in apoptosis regulation, probably involving p27(Kip1) protein.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antibodies, Anti-Idiotypic; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p27; Enzyme Activation; G1 Phase; Gene Expression Regulation, Neoplastic; Immunoglobulin M; Intracellular Membranes; JNK Mitogen-Activated Protein Kinases; Lymphoma, B-Cell; Membrane Potentials; Mice; Microtubule-Associated Proteins; Mitochondria; Mitogen-Activated Protein Kinases; Mutation; Receptors, Antigen, B-Cell; Tumor Cells, Cultured; Tumor Suppressor Proteins

Concentrations of rotenone (ROT) that block electron flow through mitochondrial complex I (100 nM) did not significantly alter either cell viability or the growth of PW cells. However, 10- to 50-fold higher concentrations (1-5 microM) were found to induce a dose-dependent cell cycle arrest predominantly at the G2/M stage of the cycle and apoptosis. Apoptosis was dependent on the cell cycle arrest, since apoptosis but not the G2/M arrest was prevented with the broad spectrum caspase inhibitor zVADfmk. Biochemical features of apoptosis included mitochondrial cytochrome c release, reactive oxygen species generation, and the activation of procaspase 3. Thus, ROT inhibition of mitochondrial electron transport may be insufficient to induce apoptosis in PW cells. Instead, apoptosis in these cells occurs as a consequence of disruption of the cell cycle and is only indirectly dependent upon mitochondrial electron transport.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; B-Lymphocytes; Caspase Inhibitors; Cell Cycle; Cell Division; Cell Survival; Cysteine Proteinase Inhibitors; Cytochrome c Group; Electron Transport; G2 Phase; Humans; Lymphoma, B-Cell; Mitochondria; Mitosis; Reactive Oxygen Species; Rotenone; Tumor Cells, Cultured; Uncoupling Agents

Anti-CD20 monoclonal antibodies have been successfully employed in the clinical treatment of non-Hodgkin's lymphomas in both unmodified and radio-labeled forms. Previous publications have demonstrated that the antitumor effects of unmodified anti-CD20 mAb are mediated by several mechanisms including antibody-dependent cellular cytotoxicity, complement-mediated cell lysis, and induction of apoptosis by CD20 cross-linking. In this report, we demonstrate induction of apoptosis by three anti-CD20 monoclonal antibodies [1F5, anti-B1, and C2B8 (Rituximab)]. The magnitude of apoptosis induction was greater with the chimeric Rituximab antibody than with the murine 1F5 and anti-B1 antibodies. Apoptosis could be enhanced with any of the antibodies by cross-linking with secondary antibodies (or Fc-receptor-bearing accessory cells). The signaling events involved in anti-CD20-induced apoptosis were investigated, including activation of protein tyrosine kinases, increases in intracellular Ca2+ concentrations, caspase activation, and cleavage of caspase substrates. Our results indicate that anti-CD20-induced apoptosis can be attenuated by PP1, an inhibitor of protein tyrosine kinases Lck and Fyn, chelators of extracellular or intracellular Ca2+, and inhibitors of caspases, suggesting that anti-CD20-induced apoptosis may involve modulation of these signaling molecules. We also demonstrated that varying the expression of Bc1-2 did not affect the magnitude of anti-B1-induced apoptosis, possibly because of the sequestering effects of other Bc1-2 family members, such as Bad. These studies identify several of the signal-transduction events involved in the apoptosis of malignant B cells that transpire following ligation of CD20 by anti-CD20 antibodies in the presence of Fc-receptor-expressing cells or secondary goat anti-(mouse Ig) antibodies and which may contribute to the tumor regressions observed in mouse models and clinical trials.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antigens, CD20; Apoptosis; B-Lymphocytes; bcl-Associated Death Protein; Burkitt Lymphoma; Calcium; Calcium Signaling; Carrier Proteins; Caspases; Chelating Agents; Cysteine Proteinase Inhibitors; Enzyme Activation; fas Receptor; Humans; Immunoglobulin Fc Fragments; Ionomycin; Lymphoma, B-Cell; Lymphoma, T-Cell; Mice; Phosphoprotein Phosphatases; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-bcl-2; Receptor Aggregation; Recombinant Fusion Proteins; Rituximab; Signal Transduction; Tumor Cells, Cultured

IgM cross-linking induces G1 arrest and apoptosis in murine B-lymphoma cells. It prevents pRb phosphorylation by decreasing cyclin-dependent kinase 2 activity via the up-regulation of cyclin kinase inhibitor p27Kip1. Anti-IgM also causes an increase in cytosolic free calcium and a loss of c-myc mRNA and protein. This down-regulation of c-Myc is prevented by CD40L, which rescues cells from anti-IgM-induced apoptosis. In this study, we addressed the mechanism(s) of anti-IgM-induced p27Kip1 accumulation. We examined effects of early events in B-cell receptor-mediated signaling, c-Myc down-regulation, and an increase in free calcium on p27Kip1. Down-regulation of c-myc alone had no effect on p27Kip1; neither did an increase in free calcium alone. Together, these two events led to p27Kip1 induction, growth arrest, and apoptosis. CD40L, the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, and cyclosporin A all prevented anti-IgM-induced p27Kip1 accumulation, suggesting that both the decrease in c-Myc expression and an increase in free calcium are necessary for p27Kip1 up-regulation.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Calcium; Caspase Inhibitors; CD40 Ligand; Cell Cycle Proteins; Cell Line; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclosporine; Cytosol; Down-Regulation; Enzyme Inhibitors; G1 Phase; Genes, Tumor Suppressor; Immunoglobulin M; Lymphoma, B-Cell; Membrane Glycoproteins; Mice; Microtubule-Associated Proteins; Proto-Oncogene Proteins c-myc; Tumor Suppressor Proteins

We have investigated whether the increases in ceramide levels that occur during apoptosis in SKW 6.4 cells induced by anti-Fas antibody depend on the activation of caspases. Using cells prelabelled to equilibrium with [14C]acetate, it was shown that the amount of ceramide approximately doubled after 24 h incubation with anti-Fas, but the time course of ceramide changes was slower than that of anti-Fas-induced cell death. Complete inhibition of the effects of anti-Fas on cell death and on ceramide production was observed when the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-(O-methyl)fluoromethane (zVAD.fmk) was added together with anti-Fas, but N-benzyloxycarbonyl-Phe-Ala-fluoromethane (a structurally similar cathepsin B inhibitor) had no effect. Treatment of cells with the Ca2+-ionophore A23187 also doubled ceramide levels, but in this case the effect was complete within 2 h, was not blocked by zVAD.fmk and was not associated with increases in nuclear fragmentation. These results suggest that ceramide is not an upstream messenger in Fas-mediated apoptosis and may instead be produced as a consequence of processes downstream of the activation of caspases and increases in cytosolic calcium concentration.

Topics: Acetates; Amino Acid Chloromethyl Ketones; Antibodies; Apoptosis; Calcimycin; Calcium; Ceramides; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; fas Receptor; Humans; Kinetics; Leukemia, Promyelocytic, Acute; Lymphoma, B-Cell; Signal Transduction; Time Factors; Tumor Cells, Cultured