p53-dependent global nucleotide excision repair of cisplatin-induced intrastrand cross links in human cells.

Cisplatin is an extremely effective chemotherapeutic agent used for the treatment of testicular and other solid tumours. It induces a variety of structural modifications in DNA, the most abundant being the GpG- and ApG-1,2-intrastrand cross links formed between adjacent purine bases. These cross links account for approximately 90% of cisplatin-induced DNA damage and are thought to be responsible for the cytotoxic activity of the drug. In human cells, the nucleotide excision repair (NER) process removes the intrastrand cross links from the genome, the efficiency of which is likely to be an important determinant of cisplatin cytotoxicity. We have investigated whether the p53 tumour suppressor status affects global NER of cisplatin-induced intrastrand cross links in human cells. We have used a (32)P-postlabelling method to monitor the removal of GpG- and ApG-intrastrand cross links from two human cell models (the 041TR system, in which p53 is regulated by a tetracycline-inducible promoter, together with WI38 fibroblasts and the SV40-transformed derivative VA13) that each differ in p53 status. We demonstrate that the absence of functional p53 leads to persistence of both cisplatin-induced intrastrand cross links in the genome, suggesting that p53 regulates NER of these DNA lesions. This observation extends the role of p53 in NER beyond enhancing the removal of environmentally induced DNA lesions to include those of clinical origin. Given the frequency of p53 mutations in human tumours, these results may have implications for the use of cisplatin in cancer chemotherapy.

An in vivo hypoxia metagene identifies the novel hypoxia inducible factor target gene SLCO1B3.

A hypoxia-associated gene signature (metagene) was previously derived via in vivo data-mining. In this study, we aimed to investigate whether this approach could identify novel hypoxia regulated genes. From an initial list of nine genes, three were selected for further study (BCAR1, IGF2BP2 and SLCO1B3). Ten cell lines were exposed to hypoxia and interrogated for the expression of the three genes. All three genes were hypoxia inducible in at least one of the 10 cell lines with SLCO1B3 induced in seven. SLCO1B3 was studied further using chromatin immunoprecipitation and luciferase assays to investigate hypoxia inducible factor (HIF) dependent transcription. Two functional HIF response elements were identified within intron 1 of the gene. The functional importance of SLCO1B3 was studied by gene knockdown experiments followed by cell growth assays, flow cytometry and Western blotting. SLCO1B3 knockdown reduced cell size and 3-dimensional spheroid volume, which was associated with decreased activation of the mammalian target of rapamycin (mTOR) pathway. Finally, Oncomine analysis revealed that head and neck and colorectal tumours had higher levels of SLCO1B3 compared to normal tissue. Thus, the knowledge based approach for deriving gene signatures can identify novel biologically relevant genes.

The role of p53 in DNA damage-mediated cytotoxicity overrides its ability to regulate nucleotide excision repair in human fibroblasts.

The p53 tumour suppressor protein plays a pivotal role in the response of mammalian cells to DNA damage. In addition to its regulatory role in cell cycle progression, p53 regulates apoptosis and can therefore influence cellular survival in response to DNA damage. More recent work has revealed that p53 is also involved in the nucleotide excision repair (NER) of structurally diverse types of DNA damage. The relative influence of p53 on NER and cellular sensitivity to DNA damage was investigated in this study using cells that differ in p53 status. Two cell models were selected: 041 TR fibroblasts in which the expression of p53 is regulated by a tetracycline-inducible promoter, and WI38 primary lung fibroblasts together with their isogenic derivative VA13, in which p53 is abrogated post-translationally by SV40 transformation. Cells were exposed to the clinically and environmentally relevant DNA-damaging agents cisplatin (0-5 microM, 2 h), (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (0-0.5 microM, 30 min) and UV-C (0-5 J/m2), each of which induce structurally distinct types of DNA damage known to be subject to p53-dependent NER. Sensitivity of the p53-proficient and p53-deficient cells to this DNA damage was then compared at each dose of DNA-damaging agent using the clonogenic survival assay and the colorimetric MTT assay. p53-proficient cells were more sensitive than p53-deficient cells to cisplatin, (+/-)-anti-benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide and UV-C; these differences in cellular sensitivity were more apparent in the 041 TR cells (up to 3.6-, 5.8- and 1.9-fold, respectively) than the WI38/VA13 cells (up to 2.3-, 1.4- and 1.4-fold, respectively). Thus, despite the well-documented persistence of DNA damage in p53-deficient fibroblasts due to impaired NER, loss of p53 results in reduced DNA damage-mediated cytotoxicity.

Oestrogens induce G(1) arrest in benzo[a]pyrene-treated MCF-7 breast cells whilst enhancing genotoxicity and clonogenic survival.

Carcinogens, such as benzo[a]pyrene (B[a]P), allow cells to evade G(1) arrest (the stealth property), thus increasing the chance that DNA damage will ultimately result in transformation. In this study we have investigated the effects of B[a]P in MCF-7 cells incubated in the presence or absence of oestrogens (beta-oestradiol, oestrone or oestriol). The cytokinesis block micronucleus assay was used to examine cells for chromosomal damage. Micronuclei were scored in 500 binucleate cells per treatment. Increased micronucleus formation (3-fold) occurred following 24 h treatment with 10(-6) M B[a]P alone. Following co-treatment with either 10(-9) M beta-oestradiol, 10(-8) M oestrone or 10(-8) M oestriol, 2- to 3-fold increases in micronuclei were observed with 10(-8) M B[a]P. When MCF-7 cells were pre-incubated for 96 h with 10(-9) M beta-oestradiol, 10(-8) M oestrone or 10(-8) M oestriol prior to the addition of B[a]P for 24 h, up to a 5-fold enhanced sensitivity to micronucleus formation was observed with beta-oestradiol and oestrone, while oestriol appeared to reduce levels of micronucleus formation. B[a]P-induced decreases in cell proliferation (per cent binucleate cells) and plating efficiency were reversed by all three oestrogens. Analysis of cell cycle distributions revealed that treatment with oestrogens or B[a]P alone did not induce marked effects on cell cycle distributions. However, in combination oestrogen and B[a]P induced increases in G(0)/G(1), decreases in S phase and increases in G(2)/M. This work suggests that whilst oestrogens appear to enhance carcinogen-induced DNA damage, they also appear, paradoxically, to trigger mechanisms that facilitate clonogenic survival, which may be relevant to breast cancer initiation.