title: Suppression of cell growth by wild-type p53 in tumor-derived rat esophageal epithelial cells.
abstract: Our laboratory previously identified a frameshift mutation in codon 176 of the p53 gene in the cell line RE-149DHD derived from transformation of rat esophageal epithelial cells by benzo(a)pyrene dihydrodiol. This mutation correlated with tumorigenic potential of the cells. To investigate the effect of rat wt-p53 expression on cell growth we constructed a recombinant expression vector, pcDNA3-RP53, in which wt-p53 cDNA 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 vector. Exogenous p53 DNA fragments were successfully amplified from three of the four cell lines with the sense primer complementary to T7 promoter sequences downstream of the CMV promoter. Restriction digestion analysis of these amplified PCR fragments showed fragments of the expected size for exogenous p53 DNA, indicating successful introduction of 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 of primers, spanning codon 176 of the p53 gene, was used to amplify a 183-bp fragment of p53 mRNA from all transfected cell lines, the parental RE-149DHD cell line and normal esophageal tissue. Subsequent SSCP analysis of the RT-PCR products from all transfected cell lines and the parental cell line RE-149DHD showed the same mobility shift, indicating expression of endogenous mutant p53 alone, and no exogenous expression of p53 mRNA in all four stable clonal cell transfected with the pcDNA3-RP53 vector. This data suggests that wt-p53 may play a negative role in growth regulation of tumor-derived rat esophageal epithelial cells.
authors.affiliation: OHIO STATE UNIV,SCH PUBL HLTH,COLUMBUS,OH 43210. OHIO STATE UNIV,SCH MED,DEPT PATHOL,COLUMBUS,OH 43210. OHIO STATE UNIV,SCH MED,DEPT OTORHINOLARYNGOL,CANC ETIOL & CHEMOPREVENT LAB,COLUMBUS,OH 43210.
authors.forename: D
authors.initials: D
authors.lastname: Wang
authors.forename: J
authors.initials: J
authors.lastname: Lang
authors.forename: C
authors.initials: C
authors.lastname: Weghorst
authors.forename: C
authors.initials: C
authors.lastname: Sabourin
authors.forename: G
authors.initials: G
authors.lastname: Stoner