The general goal of this project is to better understand the multistage process of cancer development in differentiating epithelia. Because established carcinomas are generally refractory to conventional treatments, knowledge of the genetic defects and cellular markers of carcinoma stages is important for early cancer detection and for the rational design of chemopreventive and therapeutic strategies.
The aims are based on the following hypotheses: 1) p53 protein forms control G1/S and G2/M decision points in epidermal cells, selecting among pathways of terminal differentiation, growth arrest or apoptosis after DNA damage. Normal and malignant cells will be tested for activation of each pathway and dependence on p53 expression by immunoblotting, flow cytometry and indirect immunofluorescence. 2) Kinetically and functionally distinct p53 protein forms respond to DNA damage: a transient response of p53 latent for DNA binding carries out transcriptional repression of growth- associated genes and DNA damage-sensing functions while a sustains response of a p53 form(s) active for sequence-specific DNA binding specializes in transcriptional activation. These functions may be differentially lost during malignant conversion or progression. The principal investigator has found that p53 (regular splice) and p53 as (endogenous alternative splice in mouse cells and (tissues) are induced with different kinetics after treatment of cells with DNA damaging agents. The two proteins also have different DNA binding efficiencies and cell cycle associations, suggesting different functions. These forms and functions will be measured by assays of sequence specific and non-specific DNA binding, and transcriptional modulation of reporter plasmids with or without p53 binding sites, immunoblotting of p53 and p53as and of protein products of downstream target genes. 3) Expression of wt p53 protein in active forms or in active complexes occurs due to cellular factors which form complexes with p53 and regulate its activity. Experiments will include the characterization of two cellular factors, a p80 (which appears to be wild type conformation-specific and is not mdm2) and a p40 (which appears to be p53 conformation independent) which have been coimmunoprecipitated with p53 proteins from cells and which bind to GST/p53 fusion proteins; the characterization will include microsequencing, obtaining or constructing expression vectors in order to verify their association with p53 nd to characterize their role, if any, in p53 activity and contribute to benign tumor formation or malignant conversion. Three transcripts selected by differential display in cells at these transformation stages will be verified by northern blotting, their genes identified and expression vectors used to examine roles in differentiation, cell growth control and cotransformation of cells with a defective p53 gene. Knowledge of different functional p53 protein forms in cells and cooperating factors may improve the resolution, prognosis or treatment plan for human tumors with selective loss of distinct p53 functions.
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