Molecular Carcinogenesis
Our work is focused on determining the molecular mechanisms of cancer. We have been investigating primarily skin cancer and lung cancer, two types of malignancies for which causative agents are suspected. In our earlier work, a method was developed to map DNA damage (adducts) at the sequence level of various genes in carcinogen-exposed or irradiated human cells. UV irradiation of cells produces DNA damage consisting mostly of cyclobutane pyrimidine dimers and some (6-4) photoproducts. The method is sensitive enough to measure repair rates at single nucleotide resolution. We found that induction of UV dimers and excision repair rates are DNA sequence-dependent. The distribution of cigarette smoke carcinogen-induced DNA lesions has been measured along the p53 tumor suppressor gene. The DNA adduct fingerprint of polycyclic aromatic hydrocarbons (PAH) matched well with the distribution of p53 mutations in lung cancers from smokers providing a molecular link between smoking and lung cancer. The mechanism of this selectivity was found to be a preferential modification and mutagenesis of DNA sequences containing 5-methylcytosine bases at 5'-CpG sequences. These sequences are mutational hotspots in many human cancers and genetic diseases, and we are currently exploring novel mechanisms that may produce these mutations.

In our work on skin carcinogenesis, we observed that sunlight induces pyrimidine dimers preferentially at p53 mutational hotspots that contain 5-methylcytosine in a dipyrimidine sequence, such as 5'-TmC and 5'-CmC. In experiments using transgenic mouse cells with prokaryotic reporter genes, we found that sequences containing 5-methylcytosine are preferentially mutated when sunlight is used for irradiation rather than artificial short wave UV lamps. This may explain why this DNA base is a preferential target for mutations in the p53 gene.

Cancer Epigenetics
Another major focus of our research is on cancer epigenetics. Epigenetic changes are defined as alterations of genes not involving the primary DNA sequence and include events such as DNA methylation and chromatin modification. We have cloned a tumor suppressor gene from the 3p21.3 locus, a chromosomal area that is frequently deleted in lung cancer and many other solid tumors. Loss of expression of this gene, which is named RASSF1A, was correlated with methylation of the CpG island promoter sequence and occurred in 80% of small cell lung cancers and in a large fraction of breast cancers, prostate cancers, melanomas, kidney tumors and several other malignancies. The function of the RASSF1A protein is being characterized using biochemical and genetic approaches. We also investigate epigenetic mechanisms of gene silencing in cancer. A new method, the methylated-CpG island recovery assay (MIRA), was developed and is applied for the determination of genome-wide DNA methylation changes in cancer. Silencing of promoters is often accomplished by an interaction of specific methyl-CpG binding proteins and other transcriptional repressors with the methylated promoter. We have identified and cloned several new human genes homologous to the family of methyl-CpG binding proteins and are currently characterizing their corresponding proteins.