Sunlight has at least one well-documented role in human skin cancer: mutating genes such as TP53 and PTCH. A TP53 mutation confers apoptosis-resistance. The present project is based on indications that sunlight has a second function -- forcing mutated cells to clonally expand into a precancer. Our working hypothesis is that: i) Normal skin contains small clones of cells mutated in a tumor suppressor gene. ii) These clones exist in equilibrium between clonal expansion and regression. iii) Clonal expansion of apoptosis-resistant cells is driven by sunlight-induced apoptosis of surrounding normal cells. Regression is driven by apoptotic or immunologic mechanisms. iv) These cell-level events significantly affect the time needed to accumulate successive gene alterations. Because skin cancer is so frequent, surpassing all other cancers combined in the southern U.S. and Hawaii, the ebb and flow of mutant clones appears to be a common event in normal-appearing skin. Recent technical advances make such clones directly observable under the light- or confocal microscope, like bacterial colonies on a petri dish. The number of clones indicates the frequency of mutations; a clone's size reflects clone-expanding processes. After a clone is identified immunohistochemically, it can be microdissected for DNA sequencing or double-labeled to study another gene. The sun-exposed skin of normal individuals often contains thousands of TP53-mutated clones. Not all clones need be generated by sunlight. The precedent of piebaldism and related mosaic skin diseases leads us to hypothesize that tumor suppressor genes can also mutate during embryogenesis. The adult patient will then have a circumscribed region of skin on which multiple cancers develop. Therefore, human and mouse will be used to study clones of cells with tumor-suppressor gene mutations in normal-appearing skin. First, we investigate clone dynamics: whether UVB and UVA increase the size and frequency of TP53-mutant clones or actinic keratoses, whether sunscreens safely oppose this process or exacerbate it, whether pheomelanin is a key factor in UVA action, and whether the spontaneous regression of TP53-mutant clones and actinic keratoses is due to an immune or apoptotic mechanism. Second, we investigate clone structure: determining functional properties of TP53-mutant clones, identifying PTCH-mutant clones and the role of TP53 in their clonal expansion, and identifying basal cell carcinoma patients having mosaic mutations in PTCH.
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