aphidicolin and Mast-Cell-Sarcoma

Cytotoxic T lymphocytes secrete a pore-forming cytolysin, perforin, that damages membranes of target cells. They also ligate Fas receptors on target cells and provoke apoptotic death. A20 (B lymphoma) and P815 (mastocytoma) cell lines were examined for their susceptibility to perforin-mediated lysis and to Fas-induced apoptosis after blockade of the cell cycle at the G1/S interface. Cells were arrested at the G1/S interface by inhibition of DNA synthesis with thymidine or aphidicolin. Subsequently, the treated cells were incubated either with CTL cytotoxic granules or the Fas-specific monoclonal antibody Jo-2. We show that arrest of the cell cycle at the G1/S interface markedly reduced the susceptibility of target cells to perforin-mediated lysis. In contrast, growth arrest with thymidine or aphidicolin increased susceptibility of A20 and P815 cells to Fas-mediated apoptosis. Susceptibility to lysis by intact CTLs was not affected significantly by blockade of target cells with aphidicolin or thymidine. When cells surviving exposure to perforin-containing granules were isolated on Ficoll density gradients and cell-cycle profiles were examined by flow cytometry, the ratio of G1 to G2 cells increased among the survivors exposed to granules in contrast to controls incubated with buffer alone. The data suggest that cells in G1 phase of the cell cycle are less susceptible to the perforin pathway than cells in G2 and S phases but are more susceptible to the Fas pathway.

Topics: Animals; Aphidicolin; Apoptosis; Cell Cycle; Cell Death; Cell Line; Cytoplasmic Granules; Cytotoxicity, Immunologic; fas Receptor; Lymphoma, B-Cell; Mast-Cell Sarcoma; Membrane Glycoproteins; Mice; Perforin; Pore Forming Cytotoxic Proteins; T-Lymphocytes, Cytotoxic; Thymidine; Tumor Cells, Cultured

Two heat-sensitive (arrested in G1 at 39.5 degrees C) and two cold-sensitive (arrested in G1 at 33 degrees C) clonal cell-cycle mutants of the murine P-815-X2 mastocytoma line were tested for DNA polymerase alpha, beta and gamma activities. After transfer of mutant cells to the respective nonpermissive temperature, DNA polymerase alpha activities decreased more slowly than relative numbers of cells in S phase. Furthermore, numbers of DNA-synthesizing cells decreased to near-zero levels, whereas polymerase alpha activities in arrested cells were as high as 15-40% of control values. After return of arrested cells to the permissive temperature, polymerase alpha activities increased essentially in parallel with relative numbers of cells in S phase. In contrast to the changes in thymidine kinase (Schneider, E., Müller, B. and Schindler, R. (1983) Biochim. Biophys. Acta 741, 77-85), the decrease of polymerase alpha during entry of cells into proliferative quiescence thus appears to be under rather relaxed control, while after return of arrested cells to the permissive temperature the increase in polymerase alpha is tightly coupled with reentry of cells into S phase. For DNA polymerase beta and gamma activities, no obvious correlation with changes in the proliferative state of cells was detected.

Topics: Animals; Aphidicolin; Cell Cycle; Cell Line; Cold Temperature; Diterpenes; DNA Replication; DNA-Directed DNA Polymerase; Hot Temperature; Kinetics; Mast-Cell Sarcoma; Mice; Mutation; Nucleic Acid Synthesis Inhibitors

The influence of aphidicolin on cell multiplication and DNA synthesis was examined using synchronous mouse mastocytoma P-815 cells. Aphidicolin was cytotoxic specifically to the cells of the S phase of the cell cycle. This cytotoxicity was reversed by appropriately washing the drug-treated cells, but not by the addition of deoxyribonucleosides. Aphidicolin, a potent inhibitor of DNA synthesis, selectively inhibited the activity of partially purified DNA polymerase alpha from the nucleus and the cytosol of mastocytoma cells, but did not affect the activity of DNA polymerase beta. Furthermore, aphidicolin had no effect on the synthesis of RNA and protein, and produced no changes in cell size at least for one generation.

Topics: Animals; Antibiotics, Antineoplastic; Aphidicolin; Cell Division; Cell Survival; Cells, Cultured; Diterpenes; DNA-Directed DNA Polymerase; DNA, Neoplasm; Mast-Cell Sarcoma; Mice; Sarcoma, Experimental