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 ionizing radiation (IR) are being investigated in normal human fibroblasts and in cells that lack normal function of p53, pRB, or the ataxia telangiectasia (AT) cancer susceptibility gene product. Normal human fibroblasts respond to exposure to 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. In at least one case, loss of G2 checkpoint function and the appearance of chromosomal abnormalities was accompanied by a reduction in ATM protein levels. We are particularly interested in the role of the ATM gene product in cell cycle checkpoint responses to exposures to environmental carcinogens and particularly how signals are generated from broken DNA to inactivation of cyclin/ CDK protein kinase complexes. We have developed and characterized several antisera to the ATM protein and are investigating the expression and function of this cancer susceptibility gene product. 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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