Isolation and characterization of sixteen novel p53 response genes

The EB-1 cell line is a stable transfectant of EB, a p53 null colon carcino ma cell line, with an inducible promoter controlling expression of a wild t ype p53 cDNA. The induced p53 is transcriptionally active and gives rise to apoptosis in these cells. Using this cellular model for presence or absenc e of the transcription factor p53 and transactivated genes, the Suppression Subtractive Hybridization (SSH) technique permitted the isolation of 17 mR NA candidates (GIPs-(G) under bar enes (i) under bar nduced by (p) under ba r 53), whose expression appears to be p53-dependent. Identity has been esta blished for nine of the 17 isolated candidates. These are HGFL/MSP, Zap-70, APOBEC2, Ponsin/ SH3P12/CAP/FLAF2, CDCreI2b/H5/PrutI2, Igc, lats 2, cytoke ratin 15 and PIG-3 (quinone oxidoreductase). The latter gene is the only GI P previously demonstrated to be p53 regulated. Of the eight remaining GIPs, sis correspond to Unigene clusters. One candidate, GIP #1, is significantl y homologous (72% identity) to a chicken zinc finger protein, CTCF, which b inds to insulator elements and thus attenuates enhancer cross-talk between physically adjacent promoters. The p53-dependent expression of GIPs was con firmed by dependence of expression upon induction of wt p53 expression in t he EB-1 cellular model and by up-regulation following activation of an endo genous wt p53 by treatment with adriamycin.