The long-term objective of this application is to elucidate the mechanism of lipid involvement in growth regulation of normal and tumor mammary epithelial cells (MEC). Isolated normal MEC, embedded in collagen gel, proliferate and differentiate in response to growth factors, mammogenic hormones and 18:2, (n- 6). Analyzing the effects of 18:2 on normal MEC proliferation, it was found that the eicosanoids, PGE2 and HETEs, can synergize with each other in sustaining proliferation of MEC in the presence of EGF in basal medium with insulin. Other agents like TPA, lithium, cAMP and di(18:2)-phosphatidic acid can support growth of MEC in basal medium in the absence of growth factors or mammogenic hormones. These observations indicate the involvement of a lipid-responsive pathway in MEC proliferation. The components of this pathway may include metabolites of phospholipid turnover, eicosanoids, protein kinase A and protein kinase-C. Unlike normal MEC, tumor cells are heterogeneous and, consequently, vary in their proliferative response to 18:2. It was observed that, while some tumor cells are dependent on 18:2 for maximum growth, others are not. The reports concerning inhibition of mammary tumor growth in rodents by feeding (n-3) PUFA may be explained as direct intervention of 18:2 and 20:4 metabolism by dietary (n-3) PUFA. In order to uncover the details of a lipid-responsive pathway which may explain the role of (n-6) and (n-3) PUFA, the following studies are being proposed to determine: (a) whether all hormonal and nonhormonal growth promoters of MEC require metabolites of (n-6) PUFA for sustained cell proliferation, (b) whether products of phospholipid turnover and Pl-cycle intermediates are involved in the generation of proliferative signals, (c) whether any difference in the production of and responsiveness to eicosanoids, cAMP and cGMP, characterize the difference between the 18:2-dependent and -independent mammary tumors, (d) whether (n-3) PUFA regulate proliferation of normal and neoplastic MEC by inhibiting eicosanoid production from 20:4 (n-6) and finally, (e) whether kinase A and kinase-C are associated with the putative lipid- responsive pathway for cell proliferation. A major part of the work will be carried out in serum-free culture system using normal and neoplastic mouse MEC. The effects on growth will be examined in the presence of absence of lipid analogues or enzyme inhibitors. Lipid turnover will be studied by exposing cells, prelabelled with precursors, to agonists. Lipids (including eicosanoids) and cyclic nucleotides will be analyzed by chromatography and/or RIA. cell-free protein kinase activities, phosphorylation of 80 kDa protein and binding of labeled PDBu, in response to agonists, will be determined. Working through this project, we hope to be able to understand how lipids regulate growth of normal and tumor mammary tissues which might provide ways for future preventive and therapeutic measures.
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