Abstract
BackgroundThe multi-step process of carcinogenesis can be more fully understood by characterizing gene expression changes induced in cells by carcinogens. In this study, expression microarrays were used to monitor the activity of 18,224 cDNA clones in MCF-7 and HepG2 cells exposed to the carcinogen benzo(a)pyrene (BaP) or its non-carcinogenic isomer benzo(e)pyrene (BeP). Time and concentration gene expression effects of BaP exposure have been assessed and linked to other measures of cellular stress to aid in the identification of novel genes/pathways involved in the cellular response to genotoxic carcinogens.ResultsBaP (0.25–5.0 μM; 6–48 h exposure) modulated 202 clones in MCF-7 cells and 127 in HepG2 cells, including 27 that were altered in both. In contrast, BeP did not induce consistent gene expression changes at the same concentrations. Significant time- and concentration-dependent responses to BaP were seen in both cell lines. Expression changes observed in both cell lines included genes involved in xenobiotic metabolism (e.g., CYP1B1, NQO1, MGST1, AKR1C1, AKR1C3,CPM), cell cycle regulation (e.g., CDKN1A), apoptosis/anti-apoptosis (e.g., BAX, IER3), chromatin assembly (e.g., histone genes), and oxidative stress response (e.g., TXNRD1). RTqPCR was used to validate microarray data. Phenotypic anchoring of the expression data to DNA adduct levels detected by 32P-postlabelling, cell cycle data and p53 protein expression identified a number of genes that are linked to these biological outcomes, thereby strengthening the identification of target genes. The overall response to BaP consisted of up-regulation of tumour suppressor genes and down-regulation of oncogenes promoting cell cycle arrest and apoptosis. Anti-apoptotic signalling that may increase cell survival and promote tumourigenesis was also evident.ConclusionThis study has further characterised the gene expression response of human cells after genotoxic insult, induced after exposure to concentrations of BaP that result in minimal cytotoxicity. We have demonstrated that investigating the time and concentration effect of a carcinogen on gene expression related to other biological end-points gives greater insight into cellular responses to such compounds and strengthens the identification of target genes.
Highlights
The multi-step process of carcinogenesis can be more fully understood by characterizing gene expression changes induced in cells by carcinogens
DNA adduct analysis DNA adducts were measured in cells exposed to BaP for up to 48 h (Figure 1) in order to establish biologically significant concentrations to be used for the microarray experiments and to enable gene expression changes to be related to DNA damage levels
By linking gene expression data to other phenotypic measures, such as DNA damage levels, cell cycle analysis, and p53 protein expression, we have further elucidated the roles of environment and gene interactions, which may be important in the multi-step process of carcinogenesis
Summary
The multi-step process of carcinogenesis can be more fully understood by characterizing gene expression changes induced in cells by carcinogens. In this study we have analysed the transcriptomes of two distinct human cell lines, MCF-7 derived from a breast carcinoma and HepG2 originating from a hepatocellular carcinoma, after exposure to multiple concentrations of BaP and for different lengths of time to identify the relationship between these variables and gene expression modulation. Both cell lines are known to be metabolically competent in bioactivating carcinogens such as BaP [3,6] and contain wild-type p53 alleles [12,13]. Cells were exposed to the non-carcinogenic isomer of BaP, benzo(e)pyrene (BeP), to try to distinguish between the genotoxic and toxic gene expression responses to BaP
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