aphidicolin and Lymphoma--B-Cell

The conserved protein kinase Chk1 is believed to play an important role in checkpoint responses to aberrant DNA structures; however, genetic analysis of Chk1 functions in metazoans is complicated by lethality of Chk1-deficient embryonic cells. We have used gene targeting to eliminate Chk1 function in somatic DT40 B-lymphoma cells. We find that Chk1-deficient DT40 cells are viable, but fail to arrest in G(2)/M in response to and are hypersensitive to killing by ionizing radiation. Chk1-deficient cells also fail to maintain viable replication forks or suppress futile origin firing when DNA polymerase is inhibited, leading to incomplete genome duplication and diminished cell survival after release from replication arrest. In contrast to embryonic cells, however, Chk1 is not required to delay mitosis when DNA synthesis is inhibited. Thus, Chk1 is dispensable for normal cell division in somatic DT40 cells but is essential for DNA damage-induced G(2)/M arrest and a subset of replication checkpoint responses. Furthermore, Chk1-dependent processes promote tumour cell survival after perturbations of DNA structure or metabolism.

Topics: Amino Acid Sequence; Animals; Aphidicolin; Cell Division; Cell Survival; Checkpoint Kinase 1; Chickens; DNA Damage; DNA Replication; Enzyme Inhibitors; Gene Targeting; Genes, cdc; Humans; Lymphoma, B-Cell; Molecular Sequence Data; Protein Kinases; Radiation, Ionizing; Sequence Alignment; Tumor Cells, Cultured

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