Melanoma is the fifth and seventh most commonly diagnosed cancer in America men and women. The molecular and genetic basis for the formation of melanoma is still largely unclear. The majority of melanoma (90%) is of sporadic origin, and only about 10% appears to have familial clustering. Familiar melanoma has been observed to be associated with CDKN2a/ARF and CDK mutations. In contract, a high proportion of the sporadic melanoma shows mutation in the N-RAS and B-RAF gene. B-RAF mutations, in particular mutations in exon 15 were found to be associated with greater than 65% of melanoma. It is of interest to note that more than 90% of B-RAF mutations at exon 15 occur at V599. The predominant mutation signature at V599 is T to A transversion mutation (GTG to GAG), changing valine to aspartic acid. Tandem mutation at GTG(V599) site can account for up to 30 % of the B-RAF mutations observed in primary invasive melanoma. B-RAF mutations also occur at relatively high frequency (70-80%) in melanocytic nevi, but not the surrounding tissue. It is clear that UV exposure is a major etiological risk factor for cutaneous melanoma(CM);however, UV is not sufficient for the generation of the unique B-RAF mutation that is present in high frequencies in melanocytic nevi and melanoma. Genetic and environmental (occupational?) factors are potentially contributing to the development of sporadic melanoma and the appearance of the unique B-RAF mutation. We will focus on identifying the potential environmental and genetic factors that might cooperate with UV that lead to the generation of the unique V599 mutation. Two cell lines will be used for the proposed research, the immortalized normal keratinocytes, HaCat cells and immortalized normal melanocytes, PIG1 cells. These two cell lines will be exposed to various genotoxicants, singly or in combination with UV exposure. The mutation frequency and spectrum at the V599 codon will be determined for the mutants generated in both cell lines. Comparison of the mutation frequency and spectrum at V599 codon generated in these two cell lines is expected to help to delineate the potential agents that will lead to the generation of expected double mutations generated only in melanocytes and not in keratinocytes. In order to determine the potential genetic factors that might be involved, we will use siRNA to knockdown various DNA repair genes in both HaCat and PIG1 cells and determine which DNA repair enzyme plays a role in the generation of the unique V599 mutation. The mutation frequency will be determined by the random mutation assay developed by Loeb's. The mutation spectrum at the V599 codon will be obtained by DNA sequencing of PCR fragments generated from the mutant cells that harbor the V599 mutation. Completion of the proposed research is expected to yield crucial information on the nature of the potential environmental agents that play an essential role in the formation of melanoma. In addition, it will provide significant insight as to the potential mechanism involved in melanoma progression.
-Relevance Clustered DNA base lesions are an important class of DNA damage generated by ionizing radiation. The proposed study will increase our knowledge on the biological consequences of clustered lesion and its contribution of genomic instability.
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