ucn-1028-c and Lymphoma

The present study is focused on the role of oxidative stress in the induction of either necrosis or apoptosis by eicosapentaenoic acid (EPA) in the lymphoma cell lines Raji and Ramos, respectively. To investigate the different death modes induced by EPA, we assessed the importance of some antioxidants and reactive oxygen species in the two cell lines. We observed that different antioxidants counteracted the necrotic effect of EPA on Raji cells to a different extent, and that vitamin E counteracted EPA-induced accumulation of superoxide anion in this cell line. On the contrary, no effects of antioxidants were observed on development of apoptosis induced by EPA in Ramos cells, and vitamin E did not counteract EPA-induced accumulation of superoxide anions in Ramos cells. Moreover, apoptosis was partly inhibited by transcription inhibitors (actinomycin D) and protein synthesis inhibitors (cycloheximide), suggesting dependency upon new protein synthesis prior to apoptosis. Kinase inhibitors (staurosporin and calphostin C) did not alter the EPA-induced apoptosis. The observed cellular accumulation of superoxide anion following EPA incubation may be important for induction of necrosis in Raji cells. In contrast, none of the other investigated parameters indicated a role of oxidative stress promoted by EPA in the induction of apoptosis in Ramos cells.

Topics: Antioxidants; Apoptosis; Eicosapentaenoic Acid; Enzyme Inhibitors; Glutathione Peroxidase; Humans; Lymphoma; Naphthalenes; Necrosis; Protein Kinase C; Protein Synthesis Inhibitors; Superoxides; Transcription, Genetic; Tumor Cells, Cultured; Vitamin E

A newly established human lymphoma cell line (OZ) has the t(14;18)(q32;q21) translocation and expresses large amounts of Bcl-2 compared to CCRF-CEM cells. VP-16 (40 micrograms/mL), a promising agent against lymphoma, caused DNA fragmentation (26.9% of total DNA) typical for apoptosis at 6 h in CCRF-CEM cells, but no significant changes in OZ cells until 24 h after the addition of VP-16. However, coincubation with calphostin C (0.2 microgram/mL), a protein kinase C (PKC) inhibitor, induced DNA fragmentation in VP-16-treated OZ cells (13.5% of total DNA) at 6 h after the treatment. Simultaneous immunoblot analysis revealed that this induction of apoptosis coincided with the downregulation of serine-phosphorylated Bcl-2 (13% of control cells). By contrast, apoptosis induced by VP-16 in CCRF-CEM cells was attenuated by the addition of 0.5 microM phorbol 12-myristate 13-acetate, a potent PKC stimulator. These observations suggest that Bcl-2 function is partly regulated by phosphorylation/ dephosphorylation mechanisms of the PKC system, and that phosphorylated Bcl-2 in lymphoma cells may play a role in the prevention of apoptosis.

Topics: Apoptosis; Chromosomes, Human, Pair 14; Chromosomes, Human, Pair 18; DNA Fragmentation; Drug Synergism; Etoposide; Humans; Lymphoma; Naphthalenes; Phosphoproteins; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Tetradecanoylphorbol Acetate; Translocation, Genetic; Tumor Cells, Cultured; Up-Regulation

Labeling the EBV membrane with octadecylrhodamine-b-chloride (R18) we were able to monitor spectrofluorometrically the early events of EBV fusion, under conditions in which we could affect PKC activity. Binding of EBV to Raji cells induces PKC translocation from the cytosol to the plasma membrane and 32P incorporation into its cellular receptor CR2. CR2 phosphorylation is completely inhibited when cells are preincubated with the PKC inhibitor calphostin c. This treatment also generates a strong inhibition of EBV fusion. Taken together this result suggests a key role of CR2 phosphorylation in the EBV entry into Raji cells.

Topics: Adenosine Triphosphate; Cell Membrane; Cytosol; Fluorescent Dyes; Herpesvirus 4, Human; Humans; Ionomycin; Kinetics; Lymphoma; Membrane Fusion; Naphthalenes; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Polycyclic Compounds; Protein Kinase C; Receptors, Complement 3d; Receptors, Virus; Rhodamines; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured