Candidate's Abstract) Environmental insults induce squamous metaplasia in many epithelial tissues and have been shown to predispose these tissues to cancer. The broad objective of this proposal is to define the signal pathways and molecular mechanisms by which normal keratinocytes regulate terminal squamous differentiation. The ultimate purpose is to understand the dysregulation that occurs in squamous metaplasia and carcinoma and define potential targets for prevention and/or intervention. p130, p27KiP1 and p16ink4a are growth inhibitory proteins that are up regulated during the multi-step process of squamous differentiation following commitment to irreversible growth arrest, suggesting they are essential to maintain the quiescent, differentiated state. Only protein levels increase with differentiation-MRNA levels do not change. p27 and p130 are known targets for ubiquitin-mediated degradation. The specific focus is the differentiation-specific mechanisms and signals regulating expression of pl6, p27 and p130. Hypotheses to be tested: 1) Increases in p16 and p130 during differentiation are regulated at the level of protein stability through modulation of ubiquitin-mediated degradation. Methods: Signals known to induce squamous differentiation in normal human epidermal keratinocytes including suspension culture, gamma interferon, calcium or phosphorylation will be studied for their effects on pl6 and p13O half-life, phosphorylation and ubiquitin conjugation and on activation of ubiquinating and deubiquinating enzymes. Alternative mechanisms that regulate levels of these proteins will be examined if necessary. 2) The differentiation-specific signal promoting p16, p27 and p130 up-regulation involves modulation of a map kinase pathway. Activation of erk, jnk and p38 map kinases all change early in the process of squamous differentiation. Methods: Constitutively active and dominant negative proteins and chemical inhibitors that block or enhance map kinase effects will be used to explore the potential role of map kinases in regulating p16, p27 and p130 levels. Alternative signal pathways utilized by inducers of squamous differentiation, including p130kinase, Ca 2+/calmodulin and Stat signals will be explored as deemed necessary. The results from this research will provide tools to evaluate the integrity in squamous cell carcinomas of pathways and mechanisms regulating p16, p27 and p130, in order to identify critical defects in these processes that could promote carcinogenesis and be targets for intervention.
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