Smoking-induced lung cancer is characterized by an abnormally regulated expression of normal cellular genes resulting from heritable alterations in DNA sequence, e.g. mutations, gene rearrangements, and changes in DNA methylation patterns. Endogenous cytosine methylation controls gene expression by mediating the binding of specific proteins (methyl-CpG binding proteins) to MeCG sites (MeC = 5-methylcytosine) and the recruitment of histone-modifying enzymes that promote chromatin remodeling. However, the exact mechanisms by which the altered methylation patterns arise in lung cancer are not well understood. The presence of MeC induces small, but noticeable changes in DNA structure and dynamics. Previous studies have revealed that MeC is capable of increasing the reactivity of guanine bases in MeCG dinucleotides towards carcinogens. Remarkably, the majority of lung cancer mutational """"""""hot spots"""""""" observed within the p53 tumor suppressor gene are found at endogenously methylated MeCG dinucleotides, e.g. p53 codons 157, 158, 245, 248, and 273. Our long-range goal is to elucidate the molecular mechanisms of smoking-induced lung tumor initiation. The objective of this application is to examine the means by which tobacco carcinogen-DNA adduct formation at MeCG dinucleotides induces genetic and epigenetic changes observed in lung cancer. Our central hypothesis is that endogenous cytosine methylation and the local DNA sequence context control the rates of formation and repair of tobacco carcinogen-DNA adducts. Guided by strong preliminary data, this hypothesis will be tested by pursuing the following four specific aims: 1) Identify the mechanisms by which 5-methylcytosine (MeC) influences the yields of B[a]P diolepoxide-induced N2-guanine adducts within CG dinucleotides. 2) Examine the effects of MeC and its structural analogs on the stereochemistry of N2-guanine adducts induced by B[a]P diolepoxides. 3) Map the distribution of oxidative DNA lesions within p53 and K-ras derived DNA sequences. 4) Examine O6-alkylguanine DNA alkyltransferase-catalyzed repair of NNK-induced O6-guanine lesions at methylated and unmethylated CG dinucleotides. Our approach is innovative, because we will be employing a novel, mass spectrometry based approach developed in our laboratory and novel structural models to identify the effects of endogenous cytosine methylation on reactivity of neighboring guanines towards tobacco carcinogens.
The proposed research is significant because of the widespread human exposure to tobacco products and because of their central role in lung cancer initiation. Our studies will provide an increased understanding of the structural origins of genetic and epigenetic changes associated with lung cancer, which will facilitate the development of cancer treatment and chemoprevention strategies for individuals at risk. An estimated 80-90% of total lung cancer cases are the result of cigarette smoking. Binding of tobacco carcinogens to genomic DNA is considered critical for lung cancer initiation in smokers. The present work will employ a novel, mass spectrometry based approach to investigate the mechanisms by which the local DNA sequence and endogenous methylation of cytosine influence the rates of formation and repair of tobacco carcinogen-DNA adducts. The rationale for these studies is that an increased understanding of the structural origins of genetic and epigenetic changes associated with lung cancer will facilitate the development of cancer treatment and chemoprevention strategies for individuals at risk.
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