DIACYLGLYCEROLS AND MULTISTAGE CARCINOGENESIS

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Cancer involves intrinsic cellular genetic alterations, however, extrinsic factors, such as diffusible signals from and/or physical interactions with other cell types can produce alterations in intracellular signaling pathways that can alter gene expression and contribute to the growth of a genetically altered cell. In the mouse skin model of carcinogenesis, aberrations in epidermal growth and squamous papilloma growth are accompanied by dramatic alterations in protein kinase C-alpha (PKC) and PKC-beta2. sn-1,2-Diacylglycerols (DAG) are intracellular second messengers and ligand of PKC. In DAG-treated epidermis the down-regulation of PKC-alpha and -beta2 is associated with aberrant growth. Exogenously applied DAG is an effector of the clonal expansion of cells containing mutant Ha-ras in mouse skin and PKC-alpha and -beta2 are selectively down- regulated in mutant Ha-ras papillomas in a promoter-independent manner. Based on these observations it is proposed that a chronic elevation in endogenous DAG in cells within the papilloma results in the down- regulation of PKC-alpha and PKC-beta2 which is permissive for the growth of mutant Ha-ras cells. Recent evidence indicates that within the epidermis PKC-beta2 is exclusively expressed in Langerhans cells (LC) and that the changes in PKC-alpha are occurring within the keratinocyte. Therefore it is hypothesized that these cell specific changes in PKC isoforms alter the function of the respective cell types and/or the interaction between the cell types and represent critical events in hyperplasia, papilloma growth and tumor promotion. We will conduct studies aimed at understanding the function and the biological significance of the down-regulation of PKC-alpha in keratinocytes and PKC-beta2 in LC and the possible altered interaction between these cell types which may be important in tumor promotion and papilloma growth. To accomplish this we will: i) localize and determine the level of PKC-alpha and -beta2 isoforms in basal and suprabasal keratinocytes, LCs, dendritic epidermal T-cells (DETC) in untreated and DAG-treated epidermis and papillomas; ii) construct antisense expression vectors to inhibit the expression of PKC- alpha in keratinocytes and study the ensuing effect on cell growth, differentiation and protein phosphorylation; iii) determine the biological consequences of the down-regulation of PKC-beta2 in LCs with respect to LC migration, antigen recognition/ processing/presentation and regulation of keratinocyte mitogenesis; and iv) determine if a physiologically important pool of cellular- DAG is elevated in mutant Ha-ras papillomas. In summary, we propose that perturbations in lipid homeostasis in mutant Ha-ras papillomas are responsible for the dramatic down-regulation of PKC-alpha in keratinocytes and PKC-beta2 in LC and that these events in different cell types interface to contribute to the growth of mutant Ha-ras papillomas.