Abstract
The p53 tumor suppressor gene is a transcriptional activator involved in cell cycle regulation, apoptosis, and DNA repair. We have shown that p53 is required for efficient nucleotide excision repair of UV-induced DNA photoproducts from global genomic DNA but has no effect on transcription-coupled repair. In order to evaluate whether p53 influences repair indirectly through cell cycle arrest following DNA damage or plays a direct role, we examined repair in vivo in human cells genetically altered to disrupt or regulate the function of p53 and p21. Both primary human fibroblasts and HCT116 colon carcinoma cells wild type for p53 but in which the p21 gene was inactivated through targeted homologous recombination showed no decrease in global repair of UV photoproducts. Human bladder carcinoma cells mutant for p53 and containing a tetracycline-regulated p21 cDNA showed no significant enhancement of repair upon induction of p21 expression. All of the cell lines, including the mismatch repair-deficient, MLH1 mutant HCT116 cells, were proficient for transcription-coupled repair. Clonogenic survival of HCT116 cells following UV irradiation showed no dependence on p21. Therefore, our results indicate that p53-dependent nucleotide excision repair does not require the function of the p21 gene product and is independent of p53-regulated cell cycle checkpoints.
Highlights
The p53 tumor suppressor gene product is an integral and critical component of the mammalian cellular response to DNA damage, loss of which contributes to genomic instability and carcinogenesis [1]
The G1-S phase cell cycle arrest occurs by the binding of p21 to cyclin-dependent kinases responsible for phosphorylation of cell cycle proteins such as the retinoblastoma protein that provide for entry into S phase [23]. p21 binds to proliferating cell nuclear antigen (PCNA) and inhibits DNA replication following damage by photoproducts; XP, xeroderma pigmentosum; global genomic repair (GGR), global genome repair; TCR, transcription-coupled repair; PE, plating efficiency; PCNA, proliferating cell nuclear antigen
Effects of p21 Knockout on nucleotide excision repair (NER) Activity in Normal Diploid Fibroblast Cell Lines—Initial studies on the effect of heterozygous and homozygous disruption of the p21 gene on GGR and TCR were performed in normal human fibroblasts
Summary
LF1 LF1p21ϩ/Ϫ LF1p21Ϫ/Ϫ HCT116 HCT116p21Ϫ/Ϫ HCT116p53Ϫ/Ϫ EJ EJp21 (ϩTet) EJp21 (ϪTet) (Time of UV) EJp21 (ϪTet) (12 hours prior to UV). It has been suggested that p53 indirectly regulates DNA repair following DNA damage through a checkpoint function governed by p21, thereby “allowing time” for repair [21]. If this hypothesis is correct, induced expression of p21 in the absence of wild type p53 or inhibition of p21 expression in the presence of wild type p53 should reproduce the effect of p53 function on NER. To ascertain whether the role of p53 in NER is through cell cycle arrest following DNA damage, we have evaluated the effect of p21 on NER activity in vivo systematically using a panel of both primary and transformed human tumor cell lines. The role of p53 in regulating NER appears to be independent of its cell cycle checkpoint activity and is potentially mediated by directly regulating the expression and/or activity of target NER gene products
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