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 carcinogens 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. We are interested in the role of the AT-Mutated (ATM) gene product in cell cycle checkpoint responses to exposures to environmental carcinogens and in particular the signaling pathways that are generated from broken DNA to the inactivation of cyclin/CDK protein kinase complexes. Normal human fibroblasts 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, pRB, or p21 function. However, lack of p53 results in a progressively increasing proportion of cells losing their G2 checkpoint function that is strongly correlated with the proportion of cells with chromosomal abnormalities. Normal human fibroblasts 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. Furthermore, we have shown that exposure of normal human fibroblasts to reactive oxygen species generated from treatment with t-butyl hydroperoxide results in strong G1 and G2 checkpoint responses. In contrast, ATM-deficient fibroblasts 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 have found that treatment of normal human fibroblasts 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. To better understand the ATM-dependent molecular responses to exposures to environmental toxicants, we are using microarray analysis to investigate the gene expression of normal and AT human lymphoblasts exposed to IR, as well as normal and AT human fibroblasts exposed to IR, UV and t-butyl hydroperoxide. 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.
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