aphidicolin and Ataxia-Telangiectasia

Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block-induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited.

Topics: Alkaloids; Animals; Aphidicolin; Ataxia Telangiectasia; Caffeine; Cell Fractionation; Cell Line; Cell Separation; Checkpoint Kinase 1; Checkpoint Kinase 2; DNA Replication; Enzyme Inhibitors; Flow Cytometry; Humans; Hydroxyurea; Immunoblotting; Microscopy, Fluorescence; Nucleic Acid Synthesis Inhibitors; Phosphodiesterase Inhibitors; Protein Kinases; Protein Serine-Threonine Kinases; Radiation, Ionizing; Replication Origin; S Phase; Staurosporine

The relationship between repair processes and chromosomal aberrations and X-ray-induced cell cycle perturbations were investigated in ataxia telangiectasia (AT) cells with 'intermediate' (AT-INT) and 'classical' radiosensitivity. In the cytogenetic experiments, three AT-INT lymphoblastoid cell lines were X-irradiated in G2-phase and incubated in the presence of inhibitors of DNA polymerases alpha/delta/epsilon (cytosine arabinoside, aphidicolin, 10% v/v DMSO), ribonucleotide reductase (hydroxyurea) and presumed inhibitors of protein kinases (caffeine). Flow cytometric analysis was performed in cells harvested 20 h after irradiation and stained with either propidium iodide or antibody against 5-bromodeoxiuridine in order to investigate cell cycle distribution focusing on G2/Mphase accumulation. From our data it appears that: (i) chromosomal sensitivity to radiation in AT does not always reflect clinical features; (ii) the effects of DNA repair inhibitors are inversely correlated with chromosomal radiosensitivity; and (iii) radiation-induced G2/M phase accumulation is a feature of AT cells and not necessarily correlated with cellular and chromosomal sensitivity to ionizing radiation.

Topics: Aphidicolin; Ataxia Telangiectasia; Cell Cycle; Cells, Cultured; Chromosome Aberrations; Chromosomes, Human; Cytarabine; Dimethyl Sulfoxide; DNA Damage; DNA Repair; Flow Cytometry; G1 Phase; G2 Phase; Humans; Hydroxyurea; Lymphocytes; Nucleic Acid Synthesis Inhibitors; Radiation Tolerance; S Phase; Time Factors; X-Rays

Cell cycle anomalies have been described in ataxia-telangiectasia cells after exposure to ionizing radiation. A recent report demonstrates that cells from these patients lack the ionizing radiation-induced increase in p53 protein seen in controls. We report here that an ionizing radiation-induced p53 response is reduced and/or delayed in cells from four ataxia-telangiectasia complementation groups. On the other hand, p53 induction is normal in all A-T complementation groups after exposure to UV-B light, an agent to which these cells are not hypersensitive. Specific inhibitors of protein kinase C and serine/threonine phosphatases prevented the radiation induction of p53 protein. Agents that produced double-strand breaks in DNA and/or inhibition of transcription caused an induction of p53 in the absence of radiation in control cells but not in ataxia-telangiectasia, but inhibitors of cell cycle progression such as mimosine and aphidicolin led to an increase in p53 in both cell types in the absence of radiation. These results suggest that there is more than one signal transduction pathway responsible for activation of p53, one of which is less efficient in ataxia-telangiectasia cells.

Topics: Aphidicolin; Ataxia Telangiectasia; Cell Cycle; Cell Line, Transformed; DNA Damage; Gene Expression Regulation; Humans; Lymphocytes; Mimosine; Phosphoric Monoester Hydrolases; Precipitin Tests; Protein Kinase C; Radiation, Ionizing; Tumor Suppressor Protein p53; Ultraviolet Rays

The antibiotic, aphidicolin, is a potent inhibitor of DNA polymerase alpha and consequently of de novo DNA synthesis in human cells. We report here that in gamma-irradiated normal human cells, aphidicolin (at 5 micrograms/ml and less) had no significant effect on the rate of the rejoining of DNA single strand breaks or rate of removal of DNA lesions assayed as sites sensitive to incising activities present in crude protein extracts of Micrococcus luteus cells. gamma-irradiated human ataxia telangiectasia cells are known to demonstrate enhanced cell killing and exhibit resistance to the inhibiting effects of radiation on DNA synthesis. Under conditions of minimal aphidicolin cytotoxicity but extensive inhibition of de novo DNA synthesis, the radiation responses of neither normal nor ataxia telangiectasia cells were significantly modified by aphidicolin. Firstly, we conclude that human DNA polymerase alpha is not primarily involved in the repair of the two classes of radiogenic DNA lesions examined. Secondly, the radiation hypersensitivity of ataxia telangiectasia cells cannot be explained on the basis of premature replication of damaged cellular DNA resulting from the resistance of de novo DNA synthesis to inhibition by ionizing radiation.

Topics: Aphidicolin; Ataxia Telangiectasia; Cells, Cultured; Diterpenes; DNA Polymerase II; DNA Repair; DNA Replication; Fibroblasts; Gamma Rays; Humans; Kinetics; Nucleic Acid Synthesis Inhibitors; Skin