LIPID TURNOVER & GROWTH OF NORMAL & TUMOR MAMMARY CELLS

本申请的长期目标是阐明 脂质参与正常人生长调节的机制 和肿瘤乳腺上皮细胞（MEC）。 孤立正常 MEC，包埋在胶原凝胶中，增殖和分化， 对生长因子、乳腺激素和18：2（n- 6）。 分析18：2对正常MEC增殖的影响， 发现类二十烷酸，PGE2和HETE，可以协同 在MEC存在的情况下， EGF与胰岛素的关系 其他代理商，如TPA， 锂、cAMP和二（18：2）-磷脂酸可支持生长 在不存在生长因子的基础培养基中的MEC，或 促乳腺激素 这些观察表明， 脂质反应途径参与MEC增殖。 该途径的组分可包括以下物质的代谢物： 磷脂周转、类花生酸、蛋白激酶A和蛋白 激酶C 与正常MEC不同，肿瘤细胞是异质性的 因此，它们对18：2的增殖反应不同。 它 观察到，虽然一些肿瘤细胞依赖于18：2， 为了最大限度地增长，其他人则不是。 有关报告 通过喂食抑制啮齿动物乳腺肿瘤生长（n-3） PUFA可能是18：2和20：4的直接干预 通过饮食（n-3）PUFA代谢。 为了揭开 脂质反应途径的细节，这可能解释了 对于（n-6）和（n-3）PUFA，建议进行以下研究 （a）是否所有激素和非激素生长 MEC的启动子需要（n-6）PUFA的代谢物， 持续的细胞增殖，（B）磷脂产物是否 周转和PI循环中间体参与了 增殖信号的产生，（c）是否有任何差异， 类花生酸、cAMP和 cGMP，表征18：2依赖性 和非依赖性乳腺肿瘤，（d）是否（n-3）PUFA 通过抑制正常和肿瘤MEC的增殖来调节增殖 从20：4（n-6）产生类二十烷酸，最后，（e）是否 激酶A和激酶C与假定的脂质相关， 细胞增殖的反应途径。 的主要部分 工作将在无血清培养系统中进行， 正常和肿瘤小鼠MEC。 对增长的影响将是 在存在或不存在脂质类似物或酶的情况下检查 抑制剂的 将通过暴露细胞来研究脂质周转， 用前体预标记的激动剂。 脂肪（包括 类二十烷酸）和环核苷酸将通过 层析和/或RIA。 无细胞蛋白激酶活性， 80 kDa蛋白的磷酸化和标记的PDBu的结合， 将确定对激动剂的反应。 解决这个问题 项目，我们希望能够了解脂质如何调节 正常和肿瘤乳腺组织的生长， 为今后的预防和治疗措施提供途径。

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.