The goal of this project is to investigate molecular alterations in cell cycle control during neoplastic transformation. Specifically, the molecular mechanisms involved in cell cycle checkpoint responses to exposures to environmental insults are being investigated in normal human fibroblasts and in fibroblasts that lack normal function of p53, pRB, or the ataxia telangiectasia (AT) cancer susceptibility gene products. Normal human fibroblasts (NHFs) respond to exposure to ionizing radiation (IR) by rapidly delaying entry into mitosis with an associated strong inhibition of p34cdc2/cyclinB protein kinase activity. AT fibroblasts exposed to IR show little delay of entry into mitosis or inhibition of kinase activity. The rapid G2 checkpoint response to IR does not require p53 or p21 function. However, lack of p53 results in cells that lose their G2 checkpoint function with age and this is strongly correlated with the appearance of cells with chromosomal abnormalities. NHFs also respond to exposure to IR by delaying the initiation of DNA synthesis with an arrest in G1 that is accompanied by an increase in levels of p21 protein and an inactivation of G1 cyclin/cyclin-dependent kinase (CDK) activity. AT fibroblasts are defective in this induction of p21 and fail to generate a G1 delay following IR exposure. We are interested in the role of the ATM gene product in cell cycle checkpoint responses to exposures to environmental carcinogens and particularly the signaling pathways that are generated from broken DNA to the inactivation of cyclin/CDK protein kinase complexes. We have shown that exposure of NHFs to reactive oxygen species generated from treatment with t-butyl hydroperoxide results in strong G1 and G2 checkpoint responses. In contrast, ATM- deficient fibroblast are defective in both G1 and G2 checkpoint responses to this oxidative stress and are hypersensitive to the toxic effects of t-butyl hydroperoxide treatment. We found that treatment of NHFs with either IR or reactive oxygen species results in an activation of the pATM-associated in vitro kinase activity, an activity that is lacking in cells lacking pATM. In addition to aiding our understanding of the process of carcinogenesis, these studies hold great potential for providing insight into the mechanism of action of environmental toxins, and particularly those that have been classified as non- genotoxic carcinogens. - cancer, cell cycle control, oncogenes, checkpoints, protein kinases, ataxia telangiectasia, ionizing radiation, DNA damage, oxidative stress
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