Our goal is to study the cellular mechanisms involved in hormone and growth-factor-induced cell proliferation, using primary culture of normal mouse mammary epithelial cells in collagen gels, and maintained in serum free media. Since the actions of these mitogens are mediated through their membrane receptors, membrane phospholipid are intimately involved in the activation and/or regulation of receptor mediated second messenger signal transduction. Identifying the key lipid-dependent events which influence activation of these multiple growth-regulating pathways would provide greater understanding of the mechanism(s) mediating mitogen-receptor signal transduction. The following specific aims are designed to test the hypothesis lipids modulate intracellular mechanisms associated with prolactin- and the EGF-dependent mammary epithelial cell mitogenesis, with the goal of further elucidating the interactions between hormones and lipids in the promotion of mammary carcinogenesis: 1) To determine the effects of specific fatty acids on prolactin- and EGF-induced intracellular Ca2+ mobilization. Elevations in cytosolic Ca2+ levels is a major component of mitogen-receptor mediated second messenger signal transduction. Specific fatty acids have been shown to directly increase intracellular Ca2+ mobilization, whereas modifications in the fatty acid composition of cell and intracellular organelle membranes can alter Ca2+ channels functional activity, cytosolic Ca2+ mobilization, and mammary epithelial cell mitogenic-responsiveness; 2)Determine the effects of specific fatty acids on prolactin- and EGF-induced protein kinase C activation. The actions of various hormones and growth factors are mediated through the activation of protein kinase C, which can then affect the actions of other components involved in cell proliferation and growth. Since protein kinase C activation requires membrane derived phospholipids and diacylglycerol, alterations in membrane lipids could have marked effects on enzyme activity. 3) To determine the effects of specific membrane fatty acid modification on cyclic nucleotide modulation of mitogen- induced mammary epithelial cell proliferation. Mitogen-receptor activation of G-proteins in the cell membrane, stimulate (or inhibit) the production of cyclic nucleotides which mediate the effects hormones. Membrane fatty acids modification can effect G-protein activation of cAMP-dependent pathways and effect prolactin and EGF-induced mammary epithelial cell mitogenesis. Understanding the mechanisms by which lipids modulate the mitogenic response would provide essential information necessary not only for basing effective strategies of dietary modification for use in reducing the risk of breast cancer, but also in clarifying the interactions between genetic and promotional factors in mammary carcinogenesis.
