Mechanisms involved in p53 regulation

Abstract

Inactivation of the tumor suppressor protein p53 is a very important and common step in the process of carcinogenesis. The overall purpose of this project was to gain a better understanding of the mechanisms involved in the regulation of p53 function. To gain insight into these mechanisms, we chemically mutagenized A1-5 cells expressing high levels of temperature sensitive p53 val135 (tsp53) and selected for clones that were capable of growth at the permissive temperature for p53 activation. The clones generated, called ALTR (for A&barbelow;1-5 L&barbelow;ow T&barbelow;emperature R&barbelow;esistant), could grow at the permissive temperature. Using the ALTR cell system and the parent A1-5 cells, we determined that nuclear translocation of p53 could result in a change in the conformation from mutant to wild-type but that these may be two separable events. We also investigated, in depth, the mechanism by which p53 was inactivated in one ALTR cell line, ALTR 9. We identified calpain mediated degradation of p53 as a partial mechanism of p53 inactivation in these cells. Our results suggest that degradation of p53 by calpain can lead to the functional inactivation of p53 and that this degradation can be regulated by genomic stress. To gain insight into the significance of cytoplasmically sequestered p53 protein in tumors, we chose a neuroblastoma derived cell line, SK-N-SH, that expresses a wild-type but cytoplasmically sequestered p53 protein. We report here, that down regulation of p53 by HPV-16 E6 resulted in the morphological conversion of SK-N-SH cells to substrate-adherent fibroblast-like S-type cells. The morphologic conversion was accompanied by a loss of neurofilament expression, a marker for the neuronal N-type cells, an increase in the expression of vimentin, a lack of responsiveness to RA induced neuronal differentiation, and loss of anchorage independent growth. These results suggest that p53 is required for the maintenance of the neuroblastic tumorigenic phenotype. Both the ALTR cell system and the SK-N-SH cells provided us with insight into the mechanisms involved in p53 inactivation resulting in tumor formation.