p53 activation following DNA damage results in the initiation of either cell cycle arrest or apoptosis. Prime factors determining cell fate include cell type, context and the extent and type of DNA damage sustained and the presence of a normal p53 genotype. It is well recognized that p53's tumor suppressor properties in apoptosis and growth control are inactivated by single base mutation producing a mutated, inactive protein. Up to 50 percent of cancers contain a mutated p53 gene. However, the remaining half of cancers contain a normal p53 genotype but continue to be malignant. We have focused some of our attention on selected tumor lines that contain a normal (wild type) p53 gene to determine if epigenetic factors are involved in some cancers or if other pathways counteract the wt p53 gene product. Activation of p53 may have very different outcomes in normal and tumor cell types bearing a wt p53 genetype and should reveal important aspects about p53 activity in normal versus tumor cell phenotype. Activation of p53 may include an increase p53 protein over basal levels, nuclear translocation and posttranslational modification by phosphorylation and acetylation. Of these factors, we have hypothesized that site-specific phosphorylation is intimately involved in cellular decisions to produce arrest or apoptosis, that may produce conformational changes in p53 or interaction with specific transcriptional cofactors. We believe specific phosphorylation-induced alterations in conformational structure of p53 constitute the primary factor that imparts selectivity for particular promoter regions responsible for p53 regulated gene activation. There are p53-independent mechanisms of accomplishing growth arrest or cell death. However, we have found that exposure to ionizing radiation and reactive oxygen species like hydrogen peroxide, H2O2, produce a well-defined p53-dependent arrest initially but at higher concentrations elicit apoptosis. Exactly how p53 is altered to transcriptionally activate specific target genes but not others is has not yet been elucidated. Current projects involve site-specific phosphorylation changes in specific cell types or between propagating cells and differentiated cells. Several amino and carboxy-terminal sites on p53 have been found with a constitutive level of phosphorylation by our group; first by using mass spectrometry in collaboration with the MS Group at NIEHS and also by use of phosphospecific antibodies in cell and nuclear lysates from wild type (wt) p53 expressing cell types. In one set of studies focusing on cancer biology, the wt p53 cell types we have been using are either diploid immortalized cells or tumor cell lines. The level of phosphorylation at different amino acid sites of p53 and the relative levels of p53 were both increased in both diploid and tumor wt p53 cells after exposure to different types of genotoxic stress with H2O2 or irradiation treatments. However, cell cycle studies showed differences in growth arrest characteristics after genotoxic stress. For example, it turned out that pRb tumor suppressor defects were responsible for lack of G1 checkpoint arrest in the tumor cell line instead of altered p53 phosphorylation. However, oxidative stress experiments in the same tumor cell line caused apoptosis. Further experiments suggested the presence of a kinase that was specific for an amino-terminal phosphorylation site on the wt p53 that preceded apoptosis in the tumor line. In another set of experiments, another hypothesis is being tested that involves cell death and neuronal cells. We hypothesize that neuronal cells will experience a unique pattern of p53 phosphorylation during apoptosis that is different from epithelial cell responses during either genotoxic stress or toxicant exposures. A very relevant potential cytotoxicant for neuronal cells is nitric oxide. The hypothesis that neuronal cells will show unique p53-dependent cell death responses has been evaluated under conditions of active cell division and also in a differentiated state with no cell turnover. We have been studying peripheral and central nervous system derived cell lines that serve as in vitro models of neuronal response. Our group has been collaborating with Dr. Freed at NIDA/NIH in Baltimore in using some of their recently formed neuronal cell lines to test this hypothesis. The intent of such studies is to determine differences in wt p53 gene expression in various neuronal cells in response to apoptotic reagents to determine possible roles in neurodegeneration and cell death. Results of these studies will be published and presented at the American Association for Cancer Research 2004 and the Society of Neurobiology 2004 annual meetings.
