MECHANISMS OF INDUCTION OF SKIN CANCERS BY UV LIGHT

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DESCRIPTION: This revised application seeks 4 years' support for studies of molecular mechanisms by which UV radiation induces skin cancer in mice. The cellular and molecular processes that occur during the initial carcinogen-cell interaction and the onset of tumor growth are largely unknown. Previous studies have demonstrated mutations in the p53 tumor suppressor gene, and to a certain extent, in ras oncogenes in both human and UV-induced mouse skin cancers. It is, however, not known whether these mutations arise early or late during UV skin carcinogenesis. The objectives of the proposal are to identify the temporal changes, at the cellular and molecular level, that occur during UV skin carcinogenesis, and to determine whether these alterations result in deregulated apoptotic cell death in the epidermis. The hypothesis is that alterations in the p53 tumor suppressor gene are early events and that dysregulation of apoptotic cell death and apoptosis regulatory genes contribute to the development and progression of UV-induced skin cancer. The specific aims are: (1) To determine whether the UV-induced ras and p53 mutations are early or late events during skin carcinogenesis; (2) to determine whether dysregulation of apoptosis contributes to the development of UV-induced skin cancer; (3) to determine whether specific genetic alterations present in UV-induced mouse skin tumor cells influence their susceptibility to interferon-gamma- or tumor necrosis factor-induced apoptosis. To these ends, in Aim 1, UV-irradiated mouse skin will be analyzed at progressive time points during skin carcinogenesis for ras and p53 mutations by allele-specific polymerase chain reaction followed by single-strand conformation polymorphism analysis and nucleotide sequencing. In Aim 2 the induction of sunburn (apoptotic) cells in UV-irradiated mouse skin during skin carcinogenesis will be measured in situ by the TUNEL method, and expression of apoptosis regulatory genes such as p53, Bcl-2, and Bax, will be determined by western blotting using specific antibodies. In Aim 3 the relationship between specific genetic alterations present in UV-induced mouse skin cancers and susceptibility to interferon-gamma- or tumor necrosis factor-induced apoptosis will be investigated by MTT dye uptake, DNA fragmentation, and chromatin condensation assays.