UV-induced DNA incision and proliferating cell nuclear antigen recruitment to repair sites occur independently of p53-replication protein A interaction in p53 wild type and mutant ovarian carcinoma cells.
The tumour suppressor gene TP53 plays an important role in the regulation of DNA repair, and particularly of nucleotide excision repair. The influence of p53 status on the efficiency of the principal steps of this repair pathway was investigated after UV-C irradiation in the human ovarian carcinoma cell line IGROV-1 (expressing wild-type p53) and in the derived clone IGROV-1/Pt1 (with p53 mutations at codons 270 and 282). Clonogenic survival after UV-C irradiation showed that IGROV-1/Pt1 cells were approximately 2-fold more resistant to DNA damage than parental cells. Modulation of p53 protein levels, cell cycle arrest and apoptosis were induced in UV-irradiated IGROV-1 cells, but not in the p53-mutant cell line. Exposure to UV or cisplatin induced down-regulation of p53-replication protein A (RPA) interaction in parental, but not in IGROV-1/Pt1 cells. However, persistent binding of p53 to RPA did not affect the early steps of DNA repair. In fact, both UV-induced DNA incision and the recruitment of proliferating cell nuclear antigen (PCNA) to DNA repair sites occurred to a comparable extent in p53-wild type and -mutant cell lines, although PCNA remained associated with chromatin for a longer period of time in IGROV-1/Pt1 cells. Global genome repair, as detected by immunoblot analysis of cyclobutane pyrimidine dimers, was not significantly different in the two cell lines at 3 h after UV irradiation. In contrast, lesion removal at 24 h was markedly reduced in IGROV-1/Pt1 cells, being approximately 25% of the initial amount of damage, as compared with approximately 50% repair in parental cells. These results indicate that the presence of mutant p53 protein and its persistent interaction with RPA do not affect the early steps of nucleotide excision repair in IGROV-1/Pt1 cells. Thus, repair defects in p53-mutant ovarian carcinoma cells may be attributed to late events, possibly related to a reduced removal/recycling of PCNA at repair sites.
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D015153:Blotting, Western; D002470:Cell Survival / Q000528:radiation effects; D004249:DNA Damage / Q000528:radiation effects*; D004260:DNA Repair / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005260:Female; D006801:Humans; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D017354:Point Mutation / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D051716:Replication Protein A; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C497167:RPA1 protein, human; D051716:Replication Protein A; D016159:Tumor Suppressor Protein p53