ucn-1028-c and Lymphoma--B-Cell

Recent studies have demonstrated that the naturally occurring perylenequinone antibiotic calphostin C is a potent inhibitor of protein kinase C and can induce apoptosis in some tumor cell lines by an as yet unknown mechanism. Here we demonstrate that calphostin C induces dose-dependent apoptosis in DT40 chicken lymphoma B-cells, and targeted disruption of lyn, syk, btk, PLCgamma2, or IP3R genes does not prevent or attenuate its cytotoxicity. In our study, calphostin C also induced rapid apoptosis in human acute lymphoblastic leukemia (ALL) cell lines ALL-1 (BCR-ABL+ pre-pre-B ALL), RS4;11 (MLL-AF4+ pro-B ALL), NALM-6 (pre-B ALL), DAUDI (Burkitt's/B-cell ALL), MOLT-3 (T-ALL), and JURKAT (T-ALL), whereas other potent PKC inhibitors did not. In biochemical studies, calphostin C was discovered to induce rapid calcium mobilization from intracellular stores of ALL cell lines, and its cytotoxicity against ALL cell lines was well correlated with the magnitude of this calcium signal. Calphostin C-induced apoptosis was markedly suppressed by BAPTA/AM, a cell-permeable Ca2+ chelator as well as NiCl2, an inhibitor of Ca2+/Mg2+-dependent endonucleases. Inhibition of the Ca2+/calmodulin-dependent phosphatase calcineurin with perfluoreperazine dimadeate (a calmodulin antagonist) or cyclosporin A (a specific inhibitor of calcineurin) also reduced the magnitude of calphostin C-induced apoptosis in ALL cell lines. Calphostin C was capable of inducing calcium mobilization and apoptosis in freshly obtained primary leukemic cells from children with ALL. Taken together, our results provide unprecedented evidence that calphostin C triggers a Ca2+-dependent apoptotic signal in human ALL cells.

Topics: Antibiotics, Antineoplastic; Apoptosis; Biological Transport; Calcium Signaling; Cell Division; Cell Line; Child; Humans; Lymphoma, B-Cell; Naphthalenes; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Tumor Cells, Cultured

The changes of phospholipase D (PLD) activity were investigated during the courses of apoptotic process induced by tumor necrosis factor (TNF)-alpha or anti-Fas/Apo1 antibody in human premyelocyte HL-60 and murine B cell lymphoma A20 cells. The treatment of recombinant TNF-alpha to HL-60 cells resulted in the increased PLD activity as determined by the phosphatidylethanol formation in the presence of 1% ethanol. The enhancement of PLD activity was also observed in the anti-Fas/Apo1 monoclonal antibody-treated A20 cells. However, the activity of PLD was maximized when HL-60 and A20 cells were treated with either TNF-alpha or anti-Fas/Apo1 monoclonal antibody for 6 h. Both TNF-alpha and anti-Fas/Apo1 monoclonal antibody increased PLD activity in a dose-dependent manner up to 200 U/ml and 200 ng/ml, respectively. When the intracellular activity of protein kinase C (PKC) was interrupted by treatment of calphostin-C, both the PLD activation and the apoptosis induced by TNF-alpha and anti-Fas/Apo1 monoclonal antibody appeared to be inhibited. Since PKC is reported to activate PLD, the results indicate that the intracellular signaling cascade via PLD may play a role in the induction of apoptosis induced by TNF-alpha and anti-Fas/Apo1 monoclonal antibody.

Topics: Animals; Antibodies, Monoclonal; Apoptosis; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; fas Receptor; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Lymphoma, B-Cell; Mice; Naphthalenes; Phospholipase D; Protein Kinase C; Receptors, Tumor Necrosis Factor; Signal Transduction; Tumor Necrosis Factor-alpha

Activation of protein kinase C-delta (PKC-delta) by 12-O-tetradecanoylphorbol-13-acetate (TPA) is followed by a gradual decrease in detectable protein 12-24 h later in the mouse B lymphoma cell line A20. Down-regulation is associated with TPA-induced proteolysis and a 50-86% decrease in PKC-delta mRNA 0.5-24 h post-treatment which is due to both a 50% decrease in transcription and accelerated degradation of PKC-delta mRNA as determined using the pulse-chase method. Destabilization of PKC-delta mRNA is also observed when actinomycin D is added to cells pretreated with TPA for 2 h; however, addition of actinomycin D or cycloheximide prior to TPA treatment blocks destabilization. Addition of PKC inhibitors to TPA-treated cells also blocks destabilization of PKC-delta mRNA. Cells treated with TPA for 4 h contain an activity not found in control cells which destabilizes PKC-delta mRNA but not glyceraldehyde-3-phosphate dehydrogenase mRNA in vitro. Addition of TPA to control extracts fails to increase degradation of PKC-delta mRNA in vitro, suggesting that treatment of intact cells is required to induce the synthesis of a factor(s) that destabilizes PKC-delta mRNA. This factor(s) then acts along with transcriptional and post-translational regulatory mechanisms to down-regulate PKC-delta.

Topics: Alkaloids; Animals; Benzophenanthridines; Blotting, Northern; Cell Line; Cell Membrane; Cell Nucleus; Cytosol; Dactinomycin; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Isoenzymes; Kinetics; Lymphoma, B-Cell; Mice; Naphthalenes; Phenanthridines; Protein Kinase C; Protein Kinase C-delta; RNA Processing, Post-Transcriptional; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors; Transcription, Genetic; Tumor Cells, Cultured