CELLULAR AND BIOCHEMICAL EFFECTS OF TUMOR PROMOTERS

目的是为了继续我们对生物化学和分子生物学的研究。 肿瘤促进过程的基础机制。因为 有证据表明，酶蛋白激酶C(PKC)在 生长控制和调节佛波酯肿瘤促进剂的作用， 我们将专注于这个酶系统。来自几个国家的研究 实验室，包括我们自己的实验室，表明哺乳动物的基因组编码 这种酶的几种异构体。我们的主要战略将是利用基因 大鼠6成纤维细胞系衍生细胞系的制备技术 稳定过表达PKC的β1、γ或epsilon亚型。这些 细胞系将被用来确定可能的生化差异 在这些异构体之间，评估它们在生长控制和 与癌基因的协同作用，并开发更具特异性的PKC抑制剂。 使用完整细胞的32P标记，结合二维凝胶 电泳法和免疫沉淀法，我们将尝试鉴定 这些细胞中磷酸化的主要蛋白质底物及其可能的 这些磷酸蛋白在介导各种异构体效应中的作用 PKC的基本原理。特定区域的突变将被引入不同的区域 以评估这些结构域的生化功能和它们的 在生长控制中的作用。一个具体的项目将探索PKC在 人类T细胞的激活与人类免疫缺陷的复制 病毒。我们还将分离该基因的全长cdna序列。 TPA-R1，其表达被TPA抑制，并确定是否 该序列的过度表达抑制了转化的表型。一个 长期目标是开发出过度表达的转基因小鼠品系 PKC的特定异构体和突变形式，以确定该酶的作用 系统在完整动物的多阶段致癌过程中。我们是 希望从这些研究中获得的见解和模型系统 这将为检测提供更合理的基础 在我们的环境中潜在的肿瘤促进剂，并导致新的策略 癌症预防。

The purpose is to continue our studies on the biochemical and molecular mechanisms that underlie the process of tumor promotion. Because of the evidence that the enzyme protein kinase C (PKC) plays a central role in growth control and mediates the action of the phorbol ester tumor promoters, we will concentrate on this enzyme system. Studies from several laboratories, including our own, indicate that the mammalian genome encodes several isoforms of this enzyme. Our major strategy will be to use genetic techniques to develop derivatives of the Rat 6 fibroblast cell line that stably overexpress the beta 1, gamma, or epsilon isoforms of PKC. These cell lines will be used to determine possible biochemical differences between these isoforms, to assess their effects on growth control and synergy with oncogenes, and to develop more specific inhibitors of PKC. Using 32P-labelling of intact cells, coupled with two dimensional gel electrophoresis and immunoprecipitation methods, we will attempt to identify the major protein substrates phosphorylated in these cells and the possible roles of these phosphoproteins in mediating the effects of various isoforms of PKC. Region-specific mutations will be introduced into different domains of PKC beta 1 to assess the biochemical functions of these domains and their roles in growth control. A specific project will explore the role of PKC in activation of human T cells and in replication of the human immunodeficiency virus. We will also isolate a full length cDNA sequence for the gene TPA-R1, whose expression is inhibited by TPA, and determine whether overexpression of this sequence suppresses the transformed phenotype. A long term goal is to develop transgenic strains of mice that overexpress specific isoforms and mutant forms of PKC, to define the role of this enzyme system in the multistage carcinogenic process in the intact animal. We are hopeful that the insights obtained from these studies, and the model systems that are developed, will provide a more rational basis for detecting potential tumor promoters in our environment and lead to new strategies of cancer prevention.