Abstract
Global loss of DNA methylation and locus/gene-specific gain of DNA methylation are two distinct hallmarks of carcinogenesis. Aberrant DNA methylation is implicated in smoking-related lung cancer. In this study, we have comprehensively investigated the modulation of DNA methylation consequent to chronic exposure to a prototype smoke-derived carcinogen, benzo[a]pyrene diol epoxide (B[a]PDE), in genomic regions of significance in lung cancer, in normal human cells. We have used a pulldown assay for enrichment of the CpG methylated fraction of cellular DNA combined with microarray platforms, followed by extensive validation through conventional bisulfite-based analysis. Here, we demonstrate strikingly similar patterns of DNA methylation in non-transformed B[a]PDE-treated cells vs control using high-throughput microarray-based DNA methylation profiling confirmed by conventional bisulfite-based DNA methylation analysis. The absence of aberrant DNA methylation in our model system within a timeframe that precedes cellular transformation suggests that following carcinogen exposure, other as yet unknown factors (secondary to carcinogen treatment) may help initiate global loss of DNA methylation and region-specific gain of DNA methylation, which can, in turn, contribute to lung cancer development. Unveiling the initiating events that cause aberrant DNA methylation in lung cancer has tremendous public health relevance, as it can help define future strategies for early detection and prevention of this highly lethal disease.
Highlights
Lung cancer is the chief cause of cancer-related mortalities, worldwide [1,2]
We performed an electromobility shift assay [28] to determine the affinity of the MBD2b/MBD3L1 complex for methylated CpG-39 dinucleotides (CpGs) in the presence and absence of benzo[a]pyrene diol epoxide (B[a]PDE)-DNA adducts. The latter was to rule out the possibility that B[a]PDEDNA adduction at methylated CpGs may adversely affect the formation of MBD2b/MBD3L1 complex at these dinucleotides, impeding the methylated-CpG island recovery assay (MIRA) pulldown procedure
For the first time, we have comprehensively investigated the modulation of DNA methylation in normal human cells chronically exposed to a typical smoke-derived carcinogen, B[a]PDE [18]
Summary
The death toll of lung cancer is estimated to reach 1.5 millions in 2010 [2]. Tobacco smoking continues to represent the single most important risk factor for lung cancer development [2]. The initial flurry of research has unraveled many aspects of smoke-derived lung carcinogenesis, the exact underlying mechanism of this malignancy awaits further delineation [3,4]. The gaps in mechanistic knowledge of smoke-associated lung cancer constitute the main obstacle in the management of this disease, which is currently diagnosed mostly at late stages with poor response to surgery, chemotherapy, and/or radiotherapy that leads to high mortality [3]. Elucidation of the underlying mechanism of smoke-induced lung carcinogenesis can help define future strategies for early diagnosis, prognosis, treatment, and prevention of lung cancer [4]
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