Our long-term objective is to characterize the mechanisms of and normal and pathologic settings of growth arrest signal pathways in stratified squamous epithelia, especially the epidermis and oral mucosa. This project seeks to extend our earlier findings about p16INK4A expression in the limited replicative lifespan of human keratinocytes grown in culture and the settings in which this protein becomes expressed in epithelial tissues in vivo. These studies have revealed that p16 and the precursor form of the basement membrane protein Laminin 5 (Lam5pre) are coexpressed in culture by senescing keratinocytes and in vivo by keratinocytes in late dysplastic/early invasive regions of oral mucosa and epidermis and at the migrating fronts of normal healing skin wounds, p16 and Lam5pre are also coexpressed in culture by early passage keratinocytes at the edges of wounds made in confluent cultures and by keratinocytes plated on surfaces coated with the gamma2 precursor form of Laminin 5, associated with growth arrest and directed hypermotility. We have termed this the """"""""keratinocyte motility/arrest (KMA)"""""""" response. The arrest and motility features of KMA are uncoupled in p16- and p53-deficient keratinocytes and in SCC cells: such cells become hypermotile when plated on Lam5pre, but are not growth-arrested. KMA is also induced in normal keratinocytes growing on control surfaces by treatment with TGFbeta. Very interestingly, KMA induction KMA by Lam5pre is dependent upon activity of the TGFbeta receptor. These results provide the rationale and experimental tools for investigating the molecular mechanisms of the KMA response, aimed at understanding how KMA is activated in wound repair and how its growth arrest component is evaded during neoplastic progression. We propose to use cell culture, expression and siRNA retroviral vectors, organotypic culture and xenograft models, and microscopic, immunocytologic, immunohistologic, and Western blot methods to elucidate the proteins and signal pathways that trigger the KMA hypermotility/growth arrest response, which appears to be a central element in epithelial wound healing and neoplastic progression.
