The focus of this investigation is to show a role of epidermal gangliosides in regulating the proliferation and differentiation of keratinocytes from normal skin and lesional skin of patients with hyperproliferative epidermal disorders. To assess changes in keratinocyte ganglioside content with differentiation, gangliosides will be extracted from cultured cells caused to differentiate by high calcium, 12-0-tetradecanoylphorbol-13-acetate, 1,25-dihydroxyvitamin D3, and the retinoids. Gangliosides will be detected by resorcinol staining of thin-layer chromatography (TLC) plates, and identified by comparison with ganglioside standards and by antibody binding after enzyme treatment of lipids. The effect of supplemental GM3 on EGF receptor phosphorylation, protein kinase C activity, and expression of EGF- inducible genes will be assessed to determine the mechanism of inhibition by GM3 of keratinocyte proliferation. Modified forms of GM3 will be added to cultured keratinocytes to determine the structural requirements for inhibition by GM3. Finally, a sialylated antigen that binds to anti-GM3 antibody and may participate in regulation of keratinocyte proliferation will be further characterized. Treatment of tissue sections with proteases, sialidases and N-glycanase before immunoperoxidase studies with anti-GM3 antibody may further suggest the nature of the stratum corneum antigen. Stratum corneum glycolipids will be extracted, and blinding to antibody will be detected by immunostaining of TLC plates. Glycoproteins will be extracted an the molecular weight of protein that binds to anti-GM3 antibody will be determined by immunoblot analysis. Dr. Paller's recent move to Children's Memorial Hospital has provided her with a fully-equipped laboratory and contact with senior investigators at Northwestern in addition to the continued close collaboration with her sponsor. Dr. Paller has had prolonged post-doctoral training and is now seeking support for further research training to allow her to become an independent investigator.
