Inactivation of the p53 tumor suppressor gene is the most common genetic abnormality in human cancers. It is highly involved in regulation of the cell cycle and apoptosis after cell injury. While the rise and fall of total p53 levels are often correlated with transactivation, it does not reflect the dynamic changes in phosphorylation state which influence DNA binding. Since p53 exists as many phosphorylated species, it is likely that only a limited number are bioactive. The purpose of this project is to study how changes in p53 phosphorylation relate to molecular function. The project utiliizes several approaches. First, 2D PAGE is being used to separate p53 phosphoisoforms and note proportional changes and appearance of new species in response to DNA damaging agents such as CAPE, hydrogen peroxide or irradiation. Individual isoforms will be related to p53-mediated cellular responses to identify bioactive isoforms. Second, r-p53 from the baculovirus expression system treated with okadaic acid is being used to identify normal phosphorylated and hyperphosphorylated p53 digestion fragments by mass spectral (MS) analysis in collaboration with other NIEHS researchers. MS analysis will also be similarly applied to purified p53 from normal cells in response to DNA damaging agents. Third, tumor cell lines expressing wild type p53 will be compared to normal human diploid cells for differences in response to cell cycle arrest and apoptosis and which p53 phosphoisoform expression pattern is correlated with each outcome. Apoptosis induced by phosphatase inhibitors in wt p53 expressing tumor lines will be examined for effects on p53, p21 and bax. Fourth, phosphorylation of the p53-dependent gene, p21-waf1, is also be studied as a potential regulatory phenomenon in cell proliferation and apoptosis. Thus, these studies will attempt to link specific biological activities to individual p53 isoforms and stimulation of p53-dependent genes.
