The objective of the proposed study is to analyze the cross-talk between the tumor suppressor protein p53 and estrogen signaling pathways in human osteosarcoma cells. The p53 gene is the most frequently altered gene in various human cancers. Inactivation of p53 is considered to be an important step in the development of osteosarcomas. Estrogens are important in the development and functioning of the osteoblast lineage and estrogens stimulate proliferation of osteosarcoma cells. However, consequence of estrogen action in the presence and in the absence of functional wild type p53 is not clear. Although it is known that p300/CBP is a coactivator for both p53-mediated and estrogen receptor-mediated gene regulation, the relationship between p53 and estrogen functions remains unknown. We propose to investigate the functional interrelationship between pathways of p53-mediated and estrogen-mediated gene regulation and its relevance to cell proliferation and perturbation of cell cycle, using PCNA gene expression in osteosarcoma cells as a model. PCNA is a critical component of the cellular DNA replication and DNA repair machineries. PCNA expression in osteosarcoma cells is regulated by p53. Our results show that the PCNA gene is estrogen-inducible. Furthermore, preliminary experiments have revealed a major role for p300/CBP in mediating regulation of PCNA expression by p53. Based on earlier studies and preliminary data, we hypothesize that there is significant cross-talk between p53 and estrogen signaling pathways in regulating PCNA expression. To test this hypothesis, we will analyze the mechanisms by which p300/CBP, a coactivator for both p53 and estrogen receptor, regulates p53-mediated PCNA gene expression. We propose to determine the role of p53-CBP pathway in estrogen-induced PCNA expression and proliferation of osteosarcoma cells. Since the relative amount of PCNA is important in specifying its interaction with other proteins important for the cell cycle, it is also hypothesized that PCNA expression is differentially regulated by coordinated action of p53 and estrogen in different stages of the cell cycle. To test this hypothesis, PCNA transcriptional regulation by p53 and estrogen during the cell cycle will be analyzed. The proposed study will provide important information on the role of estrogen in proliferation and cell cycle checkpoint function in the presence and in the absence of functional p53. It will also provide a paradigm for analyzing cross-talk between steroid hormones and p53 in regulating gene expression in both normal and abnormal cellular processes such as oncogenesis.
