p53-Mediated Regulation of Proliferating Cell Nuclear Antigen Expression in Cells Exposed to Ionizing Radiation

Abstract

The proliferating cell nuclear antigen (PCNA) is a highly conserved cellular protein that functions both in DNA replication and in DNA repair. Exposure of a rat embryo fibroblast cell line (CREF cells) to gamma radiation induced simultaneous expression of PCNA with the p53 tumor suppressor protein and the cyclin-dependent kinase inhibitor p21(WAF1/Cip1). PCNA mRNA levels transiently increased in serum-starved cells exposed to ionizing radiation, an observation suggesting that the radiation-associated increase in PCNA expression could be dissociated from cell cycle progression. Irradiation of CREF cells activated a transiently expressed PCNA promoter chloramphenicol acetyltransferase construct through p53 binding sequences via a mechanism blocked by a dominant negative mutant p53. Electrophoretic mobility shift assays with nuclear extracts prepared from irradiated CREF cells produced four p53-specific DNA-protein complexes with the PCNA p53 binding site. Addition of monoclonal antibody PAb421 (p53-specific) or AC238 (specific to the transcriptional coactivator p300/CREB binding protein) to the mobility shift assay distinguished different forms of p53 that changed in relative abundance with time after irradiation. These findings suggest a complex cellular response to DNA damage in which p53 transiently activates expression of PCNA for the purpose of limited DNA repair. In a population of nongrowing cells with diminished PCNA levels, this pathway may be crucial to survival following DNA damage.

Significance analysis of microarrays applied to the ionizing radiation response

“…Instead, the genes (p48, XPC, gadd45, PCNA) have roles in nucleotide excision repair, a pathway conventionally associated with UVinduced damage (19)(20)(21)(22). We confirmed the induction of these genes by Northern blot (23)(24)(25). Fornace et al reported defective removal of base damage induced by IR in xeroderma pigmentosum cells (26).…”

Repeated exposure of human skin fibroblasts to UVB at subcytotoxic level triggers premature senescence through the TGF-β1 signaling pathway

“…Most of these genes can be sorted according to their potential involvement in the SIPS phenotype. First, genes involved in growth arrest are overexpressed: insulin-like growth factor binding protein 3 (IGF-BP3) (Buckbinder et al, 1995), IGF-BP5 (Caroni and Schneider, 1994), p21 WAF-1 (Sherr and Roberts, 1999), MAX (Ayer et al, 1993) and proliferating cell nuclear antigen (PCNA) (Xu and Morris, 1999). Cyclin D1 is overexpressed as previously found in replicative senescence (Fukami et al, 1995).…”

DNA damage-related gene expression as biomarkers to assess cellular response after gamma irradiation of a human lymphoblastoid cell line

“…The precocity of this induction after irradiation and the parallel with WAF1 mRNA induction indicated that the promoter of PCNA could be transactivated by p53, but probably a threshold of DNA damages should be achieved within the cells. In this respect, Xu and Morris (1999) found that, 1 h after gamma irradiation (12 Gy) of rat embryo ®broblasts, a p53 dependent induction of PCNA mRNA and protein took place, and on human diploid ®broblasts similar induction was seen after UV irradiation (Zeng et al, 1994). Inversely, by Western blot analysing of irradiated human lymphoblastoid cell lines, Wenz et al (1998) found considerable changes in intranuclear distribution and complex formation of PCNA which are not associated with change in PCNA protein level.…”

“…PCNA belongs to a multi-protein complex implicating DNAligase-I (Lig-1), and is involved in joining of Okasaki fragments during replication, and in NER or BER repair process (Tomkinson and Mackey, 1998). The interaction between Waf1 and PCNA blocks replication and/or repair function of PCNA (Li et al, 1994;Waga et al, 1994), but PCNA itself could be directly regulated by p53 (Xu and Morris, 1999).…”