title: Analysis of p53 transactivation through high-affinity binding sites.
abstract: Alterations or elimination of the p53 protein is frequently occurring during human carcinogenesis. Overexpression of wild-type p53 has a profound growth-inhibitory effect on many cell lines, including strong and apparently non-sequence specific repression of a number of promoters. Consistent with the hypothesis that it acts as transcriptional regulator, wild-type p53 protein binds DNA and activates transcription of several promoters. We have studied DNA binding and transactivation (TA) properties of human wild-type and mutant p53 proteins representing four major mutational hotspots. DNA-gel retardation was used to detect specific p53-DNA complexes in nuclear extracts, with radiolabelled oligonucleotides representing high affinity p53-binding sites (HBS) as a probe. p53-specific complexes were identified by competition with unlabelled 'self' oligos and by double band-shifts in the presence of anti-p53 antibodies. To show transactivation by p53, TK promoter-driven CAT reporter gene was placed 3' of the p53-binding site. CAT activity was assayed after co-transfection of reporters with either wild-type (WT) or mutant p53 expression constructs into human cells that do not express p53 (SKOV3). We found that wild-type p53 has strong transactivating effect on the reporter. All mutants, with the exception of His273, were inactive in TA-assay. p53 is a target of several oncogenes found in DNA tumor viruses. We examined the effect of either SV40 T-ag or 55 kDa EIB protein of Ad5 on DNA binding and transactivation by p53 in transformed COS-1 and 293 cell lines, respectively. COS-1 extracts produced strong p53-dependent band-shift of the HBS oligos, that was doubleshifted by anti-p53 but not anti-T-ag antibodies, indicating that T-ag is not part of the complex. COS-1 cells had a high level of WT p53-dependent expression of transfected CAT reporter, indicating the presence of transactivation-competent p53, acting through the HBS element. In human Ad-transformed 293 cells, endogenous p53 was also transactivation competent and capable of DNA binding. In summary, we found efficient transactivation of HBS motif by WT and His273-p53. Studies of COS-1 and 293 cells suggest that a proportion of p53 in transformed cells display wild-type DNA binding and TA properties and that expression of transcriptionally inactive mutant p53 proteins in these cells does not interfere with WT-dependent transactivation.
authors.affiliation: Cedars-Sinai Medical Center, UCLA School of Medicine 90048.
authors.forename: A M
authors.initials: AM
authors.lastname: Chumakov
authors.forename: C W
authors.initials: CW
authors.lastname: Miller
authors.forename: D L
authors.initials: DL
authors.lastname: Chen
authors.forename: H P
authors.initials: HP
authors.lastname: Koeffler
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis; D004247:DNA / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000502:physiology*
chemical_list: D015534:Trans-Activators; D016159:Tumor Suppressor Protein p53; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase