P16/INK4A is a specific inhibitor of the cyclin D1-dependent kinases cdk4 and cdk6. Normally, in the absence of p16, cyclin D1/cdk4 and /cdk6 complexes phosphorylate and inactivate the pRb, permitting E2F-dependent transcription of genes that encode a set of proteins necessary to initiate chromosome replication. The high frequency of mutation, deletion, and promoter silencing of the gene encoding p16/INK4A. The high frequency of mutation, deletion, and promoter silencing of the gene epithelium has defined p16 as a tumor suppressor in stratified squamous cell carcinoma epithelia.. Humans and mice that inherit a heterozygous or homozygous loss of function mutation in the p16 gene are predisposed to a variety of spontaneous and carcinogen-induced cancers, but they undergo normal development and form structurally and functionally normal stratified squamous epithelia. These results confirm the tumor suppression function of p16 and also are consistent with the finding that p16 protein is not expressed as a feature of normal stratified squamous epithelial renewal or differentiation. Importantly, the point during neoplastic progression toward SCC at which p16 protein becomes expressed and functions as a tumor suppressor has remained unknown. We and others have discovered several years ago that induction of p16, independent of telomere status, is responsible for the senescence arrest of normal human keratinocytes in culture, suggesting the possibility that excessive or spatially abnormal cell growth triggers p16 expression in vivo. In order to test this hypothesis, we examined 73 human skin and oral mucosal and oral mucosal biopsy specimens, immunohistochemically. As described in a manuscript we have recently submitted for publication, we found that p16 is not expressed by keratinocytes in benign hyperplastic lesions but, instead, is expressed heterogeneously in a high percentage of dysplastic and carcinoma and carcinoma in situ lesions and very consistently in areas of microinvasion and in superficial margins of invasive SCC. P16-positive cells in these regions proved also to express the gamma2 chain of laminin 5, which had been identified previously as a marker of invasion in some carcinomas. We found that normal keratinocytes undergoing senescence arrest in culture coordinately expressed p16 and gamma2, as well, and this was frequently associated with conversion to a highly motile state. Keratinocytes at the edges of wounds made in confluent early passage cultures also co-expressed p16 and gamma2, accompanying their migration to fill the wound. These results have identified the point during neoplastic progression in stratified squamous epithelia at which the tumor suppressor p16 is expressed and suggest that normal epithelia may use the same mechanism to generate non-dividing, motile cells for wound repair. We propose to extend our observations by characterizing p16 and gamma2 expression in acute and chronic clinical wound specimens, in early stage, experimentally induced tumors and in experimental wounds in mice and by using human cell culture model systems to ask a series of specific questions about mechanisms that induce the p16/gamma2/hypermotile state in keratinocytes. Define more precisely the setting of p16/gamma2 expression in skin wounds. a. Examine clinical skin specimens and experimental mouse skin tissue immunohistochemically to test the hypothesis that p16/gamma2 induction occurs at the edge of healthy wounds but not at the edge of chronic, non-healing wounds. b. Using an organ culture model, produce incisional and punch wounds in specimens of human skin and identify substratum and soluble factors that affect keratinocyte migration and p16 and gamma2 expression. 2. Test the hypothesis that expression of p16 itself triggers induction of laminin 5 gamma2 expression and hypermotility. 3. Identify candidate genes involved in the p16/gamma2 response by identifying the family of genes that are expressed in culture by young keratinocytes, but not by exponentially growing keratinocytes.