SUPPRESSION OF CELL-GROWTH BY WILD-TYPE P53 IN TUMOR-DERIVED RAT ESOPHAGEAL EPITHELIAL-CELLS

Our laboratory previously identified a frameshift mutation in codon 17 6 of the p53 gene in the cell line RE-149DHD derived from transformati on of rat esophageal epithelial cells by benzo(a)pyrene dihydrodiol. T his mutation correlated with tumorigenic potential of the cells. To in vestigate the effect of rat wt-p53 expression on cell growth we constr ucted a recombinant expression vector, pcDNA3-RP53, in which wt-p53 cD NA is under transcriptional control of a cytomegalovirus promoter, and introduced the vector into RE-149DHD cells. A stable clonal cell line was isolated from each of four colonies transfected by pcDNA3-RP53 ve ctor. Exogenous p53 DNA fragments were successfully amplified from thr ee of the four cell lines with the sense primer complementary to T7 pr omoter sequences downstream of the CMV promoter. Restriction digestion analysis of these amplified PCR fragments showed fragments of the exp ected size for exogenous p53 DNA, indicating successful introduction o f the pcDNA3-RP53 vector into RE-149DHD cells. However, expression of exogenous p53 mRNA was not detected by RT-PCR in these cell lines. To further confirm absence of exogenous p53 expression, a different set o f primers, spanning codon 176 of the p53 gene, was used to amplify a 1 83-bp fragment of p53 mRNA from all transfected cell lines, the parent al RE-149DHD cell line and normal esophageal tissue. Subsequent SSCP a nalysis of the RT-PCR products from all transfected cell lines and the parental cell line RE-149DHD showed the same mobility shift, indicati ng expression of endogenous mutant p53 alone, and no exogenous express ion of p53 mRNA in all four stable clonal cell transfected with the pc DNA3-RP53 vector. This data suggests that wt-p53 may play a negative r ole in growth regulation of tumor-derived rat esophageal epithelial ce lls.