DNA methylation analysis using CpG microarrays is impaired in benzopyrene exposed cells
Introduction
Epigenetic mechanisms play an essential role in the cellular processes of transcriptional regulation, chromatin compaction, imprinting and X-chromosome inactivation (reviewed in Bernstein et al., 2007). As well, the disruption of epigenetic mechanisms has emerged as a key mechanism in the process of tumorigenesis with global genomic hypomethylation, as well as gene-specific hypermethylation being observed in nearly all cancers (reviewed by Esteller et al., 2001, Ehrlich, 2002, Esteller, 2007). Various environmental and dietary agents and lifestyles are suspected to be implicated in cancer causation, although the precise targets of environmentally induced epigenetic alterations during cancer development have not been fully identified (Herceg, 2007). While much of our current understanding of epigenetics is based on gene-specific approaches, the development of technologies that allow genome-wide epigenetic profiling (epigenomics; Esteller, 2007) holds great promise in mapping epigenetic patterns susceptible to environmental exposures.
Epigenetic instructions act through precise, intricate patterns of DNA methylation and histone modifications that establish and maintain the chromatin template on which transcription can be initiated or suppressed (Rodenhiser and Mann, 2006). DNA methylation occurs primarily at cytosines within CpG dinucleotides clustered within gene regulatory regions. CpGs are usually unmethylated in actively transcribed genes and methylated in silenced DNA. Together with histone modifications, these reversible epigenetic ‘tags’ can be placed on DNA to ensure proper patterns of gene expression and chromosomal integrity, thereby ensuring that specific genes can be expressed (or repressed) in response to changes in hormone levels, diet or drugs.
Environmental exposures to a wide range of contaminants, including polycyclic aromatic hydrocarbons (PAHs) and heavy metals can have genome-wide effects (Rosenkranz, 1996, Ciganek et al., 2004, Mahadevan et al., 2005). Exposures to these environmental chemicals can result in genetic and epigenetic alterations that can modify endogenous cellular pathways, destabilize the genome, and alter patterns of gene expression in target cells (Nyce, 1989, Nyce, 1997, Lee et al., 1995, Davis et al., 2000, Cooney, 2001). PAHs can affect epigenetic profiles in cells by forming DNA adducts (Denissenko et al., 1996, Hu et al., 2003), inducing cell cycle arrest (Jeffy et al., 2002), activating multiple signal transduction pathways and directly affecting transcription (Wang et al., 1993, Jeffy et al., 1999, Safe and McDougal, 2002). Benzopyrene (BaP) is a common PAH present in cigarette smoke, in certain foods and due to occupational exposures (Lijinsky, 1991, Rosenkranz, 1996, Scherer et al., 2000). DNA adducts involving the BaP metabolite benzo(a)pyrene diolepoxide (BPDE) have been identified in breast epithelial cells and human breast milk, providing evidence that BaP can reach ductal breast epithelial cells from which most breast cancers are thought to arise (Li et al., 1996, Li et al., 2002, Gorlewska-Roberts et al., 2002, Thompson et al., 2002).
Previously, we observed BaP-induced epigenetic events and cell cycle disruptions in breast cancer cell lines (Sadikovic and Rodenhiser, 2006). These effects included robust time- and concentration-dependent loss of proliferation, S/G2M accumulation and apoptosis in p53 positive MCF-7 and T47-D cells. As well, DNA methylation profiling analyses showed dynamic, sequence-specific hypo- and hypermethylation events that reinforce the link between environmental exposures, DNA methylation and breast cancer (Sadikovic and Rodenhiser, 2006). In this report, we follow up our initial studies with microarray experiments to identify BaP-induced epigenetic changes in methylatable CpG regions. We labeled and hybridized DNA from BaP treated and control MCF-7 cells to human CpG-island 12k arrays to identify gene-specific targets and genomic changes in DNA methylation patterns. Depleted and enhanced methylation patterns were identified across the genome; however, subsequent sodium bisulfite analyses revealed no changes in DNA methylation at a number of these loci. Instead, DNA adduct formation appears to be occurring at both methylated and unmethylated CpG dinucleotides and affecting target amplification during sample preparation. These results are relevant to genome screening approaches involving environmental exposures in which DNA adduct formation at specific nucleotide sites may bias target acquisition and compromise the correct identification of epigenetically responsive genes.
Section snippets
Cell treatments
Breast carcinoma MCF-7 cells were grown in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS, 20 mM HEPES Buffer Solution (Gibco), and Penicillin/Streptomycin in humidified tissue culture incubators at 37 °C and 5% CO2. 2 × 106 cells were plated on 15 cm tissue culture plates in 20 ml growth media. After 24 h, triplicate plates of cells (in 30 ml media) were exposed to 0.05% DMSO (as a control) or to 0.5 μM Benzo(a)Pyrene (BaP; Sigma) or [3H] BaP (GE Healthcare) suspended in 0.05% DMSO
DNA methylation microarray analysis in BaP-exposed MCF-7 cells
A DNA methylation-sensitive restriction-based approach was used to analyze the global and gene specific changes in DNA methylation of BaP-treated MCF-7 cells (Fig. 1a). Previous studies from our laboratory showed that MCF-7 cells were the most responsive to chemically induced epigenetic changes in a panel of 4 different breast cancer cell lines (Sadikovic and Rodenhiser, 2006). Differential labeling of the control DMSO-exposed and BaP-treated MCF-7 DNA, and subsequent hybridization on a single
Discussion
Microarray analysis is rapidly becoming a technology of choice for large scale epigenomic studies (Bernstein et al., 2007, Esteller, 2007). Understanding the effects of environmental exposure on epigenomic changes in human tissues and in disease stages will require utilization of these emerging technologies. Benzopyrene is one such environmental pollutant with links to carcinogenesis and epigenetic effects that lead to genomic hypomethylation (Wilson and Jones, 1983), preferential binding to
Acknowledgments
This work was funded by awards from the London Regional Cancer Program and the Lawson Health Research Institute to David Rodenhiser. Bekim Sadikovic is the recipient of studentships from the ‘Hike for Hope’, the Translational Breast Cancer Research Unit and the CIHR Strategic Training Program in Cancer Research.
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