Impact of p53 function on metabolic activation of environmental carcinogens
Supervisor(s): Professor David Phillips
Section of Molecular Carcinogenesis
Team: Human Biomonitoring and Carcinogen Activation
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Summary
The p53 protein is an important tumour suppressor that is inactivated in many cancers; more than 50% of all human tumours contain a mutation in the TP53 gene. Exposure to environmental carcinogens has been linked to tumour development and various carcinogens have been found to be associated with different characteristic mutations in TP53. Recent findings in our team indicate that p53 expression can influence the metabolism of different environmental carcinogens, suggesting a role for p53 in cytochrome P450 (CYP)-mediated bioactivation.
In order to investigate the role of p53 in xenobiotic metabolism we will use a panel of isogenic cell lines (e.g. colorectal HCT116) developed through targeted homologous recombination that differ only with respect to their endogenous TP53 status. HCT116 cells having wild-type TP53 [TP53(+/+)], heterozygous TP53 [TP53(+/-)], complete knock-out of TP53 [TP53(-/-)], or mutant TP53 [TP53(R248W/-) or TP53(R248W/+)] will be treated with environmental carcinogens from different chemical classes (e.g. benzo[a]pyrene, 3-nitrobenzanthrone, 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP), 4-amino-biphenyl, 7,12-dimethylbenz[a]anthrancene, tamoxifen and aristolochic acid). These cells will be characterised for (i) their capability to metabolically activate the carcinogens, (ii) their expression of xenobiotic-metabolising enzymes (both at gene and protein level), and (iii) their expression of transcription factors involved in metabolism regulation such as the aryl hydrocarbon receptor (AHR).
In addition, expression of AHR, TP53 and related transcripts will be silenced with siRNA and the AHR gene will be knocked-out by gene targeting technology in the isogenic HCT116 cells to study the influence on carcinogen metabolism. The role of specific phase I enzymes (e.g. CYPs) will be investigated by treatment of cells with activated reactive intermediates (e.g. benzo[a]pyrene-diol-epoxide, N-hydroxy-PhIP, N-hydroxy-4-aminobiphenyl, alpha-hydroxy-tamoxifen) and results will be compared to those obtained with their parent compounds. Finally, the influence of TP53 status on other cellular processes such as apoptosis and cell cycle will be explored.
These data will provide the basis to examine hypotheses on a novel role of p53 in carcinogen metabolism. It is anticipated that these results will provide significant insights into the mechanism of p53 function in chemical carcinogenesis, information that may ultimately assist in developing strategies for cancer prevention.
References:
- Hockley, S.L., et al. (2008) Identification through microarray gene expression analysis of cellular responses to benzo(a)pyrene and its diol-epoxide that are dependent or independent of p53. Carcinogenesis Vol 29, No 1, p202-10
- Arlt, V.M., et al. (2008) Metabolic activation of benzo[a]pyrene in vitro by hepatic cytochrome P450 contrasts with detoxification in vivo: experiments with hepatic cytochrome P450 reductase null mice. Carcinogenesis Vol 29, No 3, p656-65
- Hockley, S.L., et al. (2007) AHR- and DNA-Damage-Mediated Gene Expression Responses Induced by Benzo(a)pyrene in Human Cell Lines. Chem Res Toxicol, Vol 20, No 12, p1797-810
