Effect of oxidative stress on wild-type and mutant p53 in cancer cells

p53 is a major tumor suppressor that is commonly compromised in cancer cells. The present research focus was to analyze the effect of oxidative stress on both wild-type and mutant p53 from cancer cell lines with respect to their sequence specific DNA binding properties as well as to their levels and cellular localization. We have found that stress affects sequence specific DNA binding of wild-type p53 to several of the gene regulatory sequences studied here, although the specificity and affinity varied depending on the cell line. On the contrary, this stress failed to alter DNA binding of mutant p53 from breast cancer cell lines significantly. This was contrasted with DNA binding observations made with mutant p53 from thyroid cancer cell lines ARO (Arg273His) and WRO (Pro223Leu) where both the mutants bound to the survivin and p21 gene regulatory sequences. In order to explain the differences in sequence specific DNA binding and levels of wild-type and mutant p53 protein in the cancer cell lines, the serine 15 (Ser15) residue on p53 was tested for phosphorylation. A basal level of Ser15 phosphorylation of mutant p53 was correlated with the high level of p53 protein in particular cell lines. Another focus of the project was to develop a method to incorporate an unnatural amino acid into a protein of interest using an amber stop codon suppression system. We were able to incorporate 3-nitro-tyrosine (3-nTyr) using a modified tyrosyl tRNA synthetase from M.jannaschii into a full length model protein, histidinol dehydrogenase encoded by a gene carrying an amber stop codon at the 225th position. Overall this research advances the knowledge of p53 in cancer cell lines and also sets the stage for designing a novel protein labeling method.