p53-mediated transcription induces resistance of DNA to UV inactivation

Abstract

A possible role of p53-dependent transcription in the induction of DNA repair was explored by transfecting a UV-irradiated chloramphenicol acetyl transferase (CAT) reporter plasmid (pRGC.FOS.CAT), containing a minimal FOS promoter driven by a consensus p53 binding site, into a p53 negative-mouse cell line [(10)1]. When a p53-expressing plasmid (pSV.p53) was cotransfected into these cells, CAT expression levels persisted even after prolonged UV irradiation. In comparison, CAT expression from pSV2.CAT, which lacks a p53-responsive element in its SV40 promoter, dropped o much more precipitously after UV irradiation in the absence or presence of WT p53 expression. A similar sharp drop was observed with three other constructs when the reporter gene was under the control of the ras, b-actin or fos promoter. Mouse cells (A1-5) that constitutively express a temperature-sensitive mutant (135 AV) of mouse p53 also generated, at 328C, higher levels of enzyme expressed from UV-irradiated pRGC.FOS.CAT than from UV-irradiated pSV2.CAT. The frequency of cyclobutane pyrimidine dimers in UV-irradiated pRGC.FOS.CAT was determined with T4 endo V, and the probability of having an undamaged CAT coding strand was calculated by the Poisson distribution for various times of UV-irradiation. The observed relative CAT expression levels from irradiated pSV2.CAT and pRGC.FOS.CAT in the absence of p53 were consistent with those numbers. These results show that WT p53-mediated transcription directs a resistance of the transcribed DNA to UV inactivation and reactivates the reporter gene. Furthermore, some single point substitution mutants of p53 that maintain a near normal ability to activate transcription had lost their ability to extend CAT gene expression after UV irradiation. Conversely, other mutants with reduced transcriptional activity retained this ability. This indicates that although resistance to UV inactivation is transcriptionally-dependent, these two activities are genetically distinct. These data, taken together, suggest that the transcription of UV-damaged DNA by a p53-dependent process promotes its repair.

γ-Irradiation enhances transgene expression in leukemic cells

“…p53 expression peaked after UV exposure and maintained transgene expression when the transgene promoter included a fixation site to p53 but not when SV40 promoter was used. 6 Enhancement of transgene expression observed with CMV promoters could be explained by over-expression of NF-kB after irradiation, which seems to mediate various gene-induced overexpressions.…”

New p53 target, phosphatase of regenerating liver 1 (PRL-1) downregulates p53

“…For the luciferase reporter assay, pRGC-Luc (Huang et al, 1998), Wt p21-Luc (Resnick-Silverman et al, 1998 or Mt p21-luc reporter with or without PRL-1 (Flag-PRL-1; kindly provided from Dr Sung Hyun Kang and Dr Kwang Hee Bae) was transfected into cells using LipofectAMINE Plus (Gibco-BRL). For assays using PRL1-p53-luc and PRL1 1.1 kb-luc, the reporters were co-transfected with or without the p53 plasmid (Kim et al, 2007).…”

p53, Oxidative Stress, and Aging

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