(Taken from application) Epidermal keratinocytes are normal cells which grow and differentiate in vitro. Calcium is the principal regulator of keratinocyte differentiation in vitro, and appears to play that role in vivo. Although calcium is the major regulator of keratinocyte differentiation, its actions involve protein kinase C (PKC). The calcium response element (CaRE) in genes involved in differentiation (such as the involucrin gene) contain an AP-1 site (the element binding transcription factors regulated by PKC) which is critical for the calcium response of those genes. Squamous carcinoma cells lines (e.g. SCC4) fail to respond to the pro-differentiating actions of calcium. Constructs containing the CaRE. Of the involucrin gene when transfected into SCC4 cells do not respond to calcium or phorbol esters,. Suggesting an abnormality in PKC and/or the AP-1 binding transcriptional regulators through which calcium and phorbol esters regulate gene expression. In this project we will test the following hypothesis. SC cells fail to differentiate in response to calcium because they fail to activate the appropriate set of PKC isozymes and/or the PKC regulated AP-1 transcription factors required for expression of the genes encoding differentiation specific proteins. To test this hypothesis we will accomplish the following specific aims. 1. Determine which members of the PKC family mediate calcium induced differentiation in normal human keratinocytes (NHK), and whether such members are absent, maldistributed, or dysfunctional in SCC 4 cells. This will be accomplished by comparing the PKC isozymes in NHK and SCC 4 cells during different stages of differentiation and evaluating the effect of selectively blocking their production and activity. 2. Determine which members of the fos/jun family regulate INV and TG expression during calcium induced differentiation in NHK, and whether they re absent, maldistributed, or dysfunctional in SCC 4 cells. This will be accomplished by comparing the levels and function of the fos/jun family during calcium and phorbol ester induced differentiation in NHK and SCC4 cells and evaluating the effect of blocking or stimulating their production or activity. 3. Determine whether regulation of AP-1 transcription factors by PKC contributes to calcium induced differentiation in normal keratinocytes, and whether such regulation is altered in SCC 4 cells. This will be accomplished by determining whether altering the production or activity of selected PKC isozymes changes the fos/jun family members during calcium and TPA induced differentiation in NHK versus SCC4 cells and whether phosphorylation of the fos/jun family members is altered in SCC 4 cells. These studies are expected to further our understanding of the reasons SCC fail to differentiate, potentially leading to earlier diagnosis and more effective therapy.
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