Human cells deficient in p53 regulated p21(waf1/cip1) expression exhibit normal nucleotide excision repair of UV-induced DNA damage.

Cancer development requires the accumulation of numerous genetic changes, which are believed to initiate through the presence of unrepaired lesions in the genome. In the absence of proficient repair, genotoxic agents can lead to crucial mutations of vital cellular genes via replication of damaged DNA. Many cell cycle regulatory proteins are known to modulate the repair capacity and consequently the fate of cells. We and others have recently shown that p53 tumor suppressor gene product is required for efficient global genomic repair (GGR) but not the transcription coupled repair (TCR) of the nucleotide excision repair (NER) sub-pathways. In order to discern the nature of the p53 modulation to be direct or indirect through a downstream mediator, we have investigated the processing of UV radiation induced lesions in human colon carcinoma, HCT116 cells expressing wild-type p53 but having different p21(waf1cip1) (hereafter p21) genotypes (p21+/+, p21+/-, p21-/-). Following 20 J/m(2) UV, all the three cell lines showed rapid increase in p53 protein but the accompanying increase in the expression of its downstream target protein p21 could only be seen in p21+/+ and p21+/- cells and not in p21-/- cells. Nevertheless, an absence of detectable p21 protein in deficient cells had no demonstrable effect on DNA repair response to UV irradiation, as measured by an immunoassay to detect removal of UV photoproducts from genomic DNA (GGR) and by individual strand specific removal of endonuclease-sensitive CPD from a target gene fragment (TCR). Introduction of cytomegalovirus (CMV)-driven luciferase reporter plasmid, UV damaged in vitro, into the un-irradiated cells of varying p21 background, revealed a relatively small but statistically significant decrease in the reporter expression in the host p21-/- as compared with p21+/+ and p21+/- HCT116 cells. Super-expression of p21 protein upon reintroduction of p21 expression construct, showed an enhanced recovery of UV damaged reporter activity that was not greatly different from a similar enhancement observed with undamaged plasmid reporter DNA. Taken together, the results indicate that (i) the p21 protein does not have a significant role in the repair of genomic DNA at chromosomal level; (ii) the well-established p53 dependent modulation of NER is distinct and independent of its cell cycle checkpoint function; and (iii) the reproducible enhancing effect of p21 expression observed through host cell reactivation (HCR) of extrachromosomal DNA is mainly attributable to an effect exerted on transcription rather than repair.