Differential effects of hypoxia and acidosis on p53 expression,repair of UVC-damaged DNA and viability after UVC in normal and tumor-derived human cells

Hypoxia and low pH are commonly associated with the tumor microenvironment. We have examined the effects of hypoxia alone (HA) and hypoxia coupled to low pH (HApH) on p53 expression, nucleotide excision repair (NER) and cellular sensitivity to UVC in normal human fibroblasts and human tumor cells. p53 expression was measured using Western blotting, NER using host cell reactivation (HCR) of a UV-damaged reporter gene and cell sensitivity using the MTT assay. HApH resulted in a transient increase in p53 expression in normal fibroblasts at 6 h and in tumor cells at 6–18 h. In normal fibroblasts HApH resulted in a transient increase in HCR at early times (12–24 h) and a concomitant decrease in UVC sensitivity. In contrast, for the tumor-derived cells, the increased HCR of the UVC-treated reporter gene was delayed (36–40 h) and UVC sensitivity increased or remained the same after HApH treatment. These results suggest that early upregulation of p53 and increased repair of UV-damaged DNA after HApH treatment is required for increased cell viability after UVC. HA treatment alone also resulted in a transient increase in HCR of the UVC-damaged reporter gene at early times (12–24 h) in normal fibroblasts and a delayed increase (36–40 h) in the tumor-derived cells. However, the enhanced p53 expression was less or even absent for treatment with HA alone, and HA had no significant affect on cell viability after UVC for any of the cell lines. These results indicate a different cellular response following HApH compared to HA alone.     

Tumor suppressor p53 dependent recruitment of nucleotide excision repair factors XPC and TFIIH to DNA damage

Functional tumor suppressor p53 is mainly required for efficient global genomic repair (GGR), a subpathway of nucleotide excisions repair (NER). In this study, the regulatory effect of p53, on the spaciotemporal recruitment of XPC and TFIIH to DNA damage sites, was investigated in repair-proficient and -deficient human cells in situ. Photoproducts were induced through micropore UV irradiation of discrete subnuclear areas of intact cells and the specific lesions, as well as recruited repair factors, were detected by immunofluorescent intensity and density of the damaged DNA subnuclear spots (SNS). Both cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) were visualized in situ at SNS within irradiated nuclear foci. The in situ repair kinetics revealed that p53-WT normal fibroblasts are proficient for the repair of both CPD and 6-4PP, whereas, p53-Null Li-Fraumeni syndrome (LFS) fibroblasts fail to efficiently repair CPD but not 6-4PP. Colocalization experiments of the NER factors showed that in normal human cells, XPC and TFIIH are rapidly and efficiently recruited to DNA damage within SNS. By contrast, recruitment of both XPC and TFIIH to DNA damage in SNS occurred much less efficiently in p53-Null or p53-compromised cells. The total cellular levels of XPC and XPB were similar in both p53-WT and -Null cells and remained unchanged up to 24h following UV irradiation. The results also showed that dispersal of recruited XPC and TFIIH from DNA damage SNS occurs within a short period after DNA damage. Such dispersal requires functional XPA, XPF and XPG proteins. Taken together, the results demonstrated that p53 plays a pronounced role in the damage recognition and subsequent assembly of repair machinery during GGR and the recruitment of XPC and TFIIH to CPD is p53-dependent. Most likely mechanism of this p53 action is through its downstream effector protein, DDB2.     

Efficient repair of bulky anti-BPDE DNA adducts from non-transcribed DNA strand requires functional p53 but not p21(waf1/cip1) and pRb

Wild-type p53 protein is known to regulate the global genomic repair (GGR), removing bulky chemical DNA adducts as well as cyclobutane pyrimidine dimers from the genome overall and from non-transcribed strands (NTS) in DNA. To investigate the role of cellular factor(s) relevant to p53 regulated DNA repair processes, we examined the repair kinetics of chemical carcinogen, anti-benzo[a]pyrene-diol epoxide (anti-BPDE), induced bulky DNA adducts in normal human mammary epithelial cells (HMECs) and HMEC transformed by human papillomavirus (HPV)-16E6 or -16E7 oncoproteins, which, respectively targets p53 or pRb proteins for degradation. The results show that the removal of anti-BPDE DNA adducts from the genome overall and NTS by GGR was significantly reduced in HPV-16E6 protein expressing cells as compared to that in normal and HPV-16E7 protein expressing cells, indicating the role of p53 and not pRb in nucleotide excision repair (NER). We further determined the potential effects of the p53-regulated p21(waf1/cip1) gene product in NER in human colon carcinoma, HCT116 cells expressing wild-type p53 but different p21(waf1/cip1) genotypes (p21+/+, p21+/-, p21-/-). The results donot show a discernible difference in the removal of anti-BPDE DNA adducts from the genome overall and the transcribed strand (TS) and NTS irrespective of the presence or absence of p21(waf1/cip1) expression. Based on these results, we suggest that: (i) the wild-type p53 function but not p21(waf1/cip1) expression is necessary for GGR of chemical induced bulky DNA adducts; (ii) the Rb gene product does not play a significant role in NER; and (iii) the modulation of NER by p53 may be independent of its function in the regulation of cell cycle arrest upon chemically induced DNA damage.