Induction of multiple PCR-SSCPE mobility shifts in exons in cultures of normal human urothelium exposed to low-dose gamma radiation

Abstract

We have previously shown that primary explant cultures of human urothelium exposed to low doses of gamma -radiation subsequently accumulate a high level of stable p53 but it was not clear from those studies whether this protein stabilization occurred through an event in another gene involved in p53 protein control or possibly an epigenetic event. In these experiments, primary urothelial cultures from five different patients were exposed to either 0.5 or 5 Gy gamma -radiation from a 60Cobalt source and allowed to grow for 7-10 division cycles to allow development of any radiation-induced, non-lethal changes in the cells. C-myc, Bcl-2 and stable p53 proteins were found to be elevated in cultures following both radiation doses. PCR-SSCPE analysis of the p53 gene was performed on cultures in order to determine whether genetic mutations could be the underlying basis for persistent increased stable p53 expression. Following 0.5 Gy exposure, the cultures also developed multiple distinct 'foci' of rapidly dividing cells which strongly overexpressed p53. These grew on a background of morphologically normal cells. When such foci were selectively analysed for their p53 mutation status by PCR-SSCPE, there was evidence that they contained cells which had developed changes to the p53 gene postirradiation. These changes appeared to occur more frequently in focal cells than in cells of normal morphological appearance in the same culture. These results may have mechanistic importance given the controversy regarding low-dose radiation effects and p53 -related genomic instability.