This proposal seeks to identify the molecular basis for accumulation of wild type p53 protein in murine breast epithelial cells and its association with tissues susceptible to tumorigenesis. The investigators have documented paradoxical expression of elevated levels of wild-type p53 protein in proliferating cells of the mammary epithelium during adolescence, a period of heightened susceptibility to tumor induction. Accumulation of wold-type p53 protein was also detected in preneoplastic mammary lesions and tumors. These observations suggest a mechanism whereby cellular antagonists of p53 are expressed during critical periods of mammary gland development rendering the epithelium susceptible to somatic mutations. They further demonstrate that the p53-binding protein encoded by mdm2, a potent antagonist of p53 which is estrogen-responsive, is expressed at elevated levels in murine mammary tumors. As p53 and mdm2 form a regulatory circuit, the objective of this proposal is to determine how these factors interact to affect susceptibility of the mammary epithelium to tumorigenesis. Tissues that are susceptible to tumorigenesis (terminal end buds and preneoplastic lesions) are the focus of the experiments because these are the most effective targets for therapy. The approach will be to (1) directly test whether the p53 pathway is compromised in tissues that accumulate elevated levels of wild-type p53 protein; (2) determine the genetic basis for accumulation of p53 protein; (3) quantify changes in the levels of functional p53 protein in the normal developing gland; and (4) evaluate the role of mdm-2 and other candidate genes in regulating p53 expression and function. Induction of cell cycle arrest and apoptosis by gamma- irradiation will be used as end points to determine whether the p53 pathway is functional. Somatic cell hybrids will be prepared to determine whether accumulation of wild type p53 in the cytoplasm and the nucleus are dominant phenotypes over normal levels of p53 protein. These results provide a basis for selecting candidate genes and design of strategies to clone cDNAs of the proteins responsible for accumulation of p53 protein. In addition, dramatic changes in expression of p53 were detected at distinct stages of mammary development. Therefore, a p53 responsive reporter gene will be tested and used to quantify changes in active p53 protein in mammary tissue during development, lactation and involution. The role of mdm2 in these processes will be explored by chanracterizing the expression of each mdm2 transcript throughout development of the mammary gland by in situ hybridization. The function of mdm2 will be tested by ablating its function with antisense inhibitors. Together these experiments will dissect the complex pathways regulating p53 function and tumor susceptibility in mammary tissue.
