MECHANISMS OF RESISTANCE TO TUMOR PROMOTERS

肿瘤促进剂是与启动剂协同作用的化合物 在哺乳动物细胞中诱导肿瘤形成的致癌物。佛波醇酯 是激活蛋白激酶C(PKC)、蛋白质的肿瘤促进剂 丝氨酸/苏氨酸激酶依赖于钙、二酰甘油、 和磷脂的活性。细胞对佛波醇酯的暴露也 结果MAP和S6两个信使-- 独立的丝氨酸/苏氨酸激酶激活的PKC下游 活性受磷酸化的调节。S6激酶在以下情况下被激活 被MAP激酶磷酸化。MAP的作用机制 激酶未知；酪氨酸和苏氨酸上的磷酸化 残留物是必需的。不同的细胞系对 佛波酯是研究其作用机理的有用工具。 肿瘤促进剂。野生型EL4小鼠胸腺瘤细胞，但不是变异型 EL4细胞通过启动白细胞介素2对佛波酯作出反应 抄写。佛波酯导致MAP和S6迅速升高 野生型细胞中的激酶活性，而变异细胞中不存在。这些 激酶分别代表ERK和RSK基因产物。野生型 细胞，但不是变异细胞，会产生一种激活因子作为反应 到佛波尔酯。该因子增强了两种蛋白的磷酸化。 ERK和RSK激酶在细胞中孵育时机制未知 两种激酶都存在。这项提议调查了这样一种假设 下游激酶的激活是介导这一过程的重要途径。 对肿瘤促进剂的反应。提案的部分内容涉及 ERK和RSK激酶的激活机制及其作用 介导转录激活的激酶和PKC-2途径 依赖于磷脂的水解。具体目标如下。 1)PKC的激活与PKC的激活有关的机制 ERK和RSK将被研究：a)产生的激活因子 对佛波酯反应的野生型EL4细胞将被分离出来， 纯化和表征，b)cdc2激酶复合体的作用 关于ERK和RSK的磷酸化将进一步研究，c) 完整野生型与野生型蛋白质磷酸化的差异 对佛波酯反应的变异EL4细胞进行检测。2) EL4细胞对佛波酯产生抗性的机制 待研究：a)额外的佛波酯抗性变异细胞系 将开发，b)将确定PKC亚型的表达 野生型和变异型细胞系，c)佛波酯对ERK的影响， 将评估两种细胞类型的RSK和激活因子，d)佛波醇 酯诱导的Fos和Jun的磷酸化将在野外进行比较- 类型和变异细胞，e)磷脂酰胆碱的水解性反应 将在这些细胞系中分析佛波酯。总而言之， 拟议的工作使用细胞和生化方法的组合 研究肿瘤促进剂的作用机制，并确定 蛋白激酶C在T细胞活化中的作用所获得的信息将提供 这是未来癌症治疗和预防方法的基础。

Tumor promoters are compounds that act synergistically with initiating carcinogens to induce tumor formation in mammalian cells. Phorbol esters are tumor promoters that activate protein kinase C (PKC), protein serine/threonine kinases that are dependent on calcium, diacylglycerol, and phospholipid for activity. Exposure of cells to phorbol esters also results in the activation of MAP kinase and S6 kinase, two messenger- independent serine/threonine kinases activated downstream of PKC whose activities are regulated by phosphorylation. S6 kinase is activated when phosphorylated by the MAP kinase. The mechanism of action of the MAP kinase is unknown; phosphorylation on both tyrosine and threonine residues is required. Cell lines that vary in their responsiveness to phorbol esters are useful tools in the study of the mechanism of action of tumor promoters. Wild-type EL4 murine thymoma cells, but not variant EL4 cells, respond to phorbol ester by initiating interleukin-2 transcription. Phorbol ester causes a rapid increase in MAP and S6 kinase activities in wild-type cells but not in variant cells. These kinases represent the ERK and RSK gene products, respectively. Wild-type cells, but not variant cells, produce an activating factor in response to phorbol ester. this factor enhances the phosphorylation of both the ERK and RSK kinases by an unknown mechanism when incubated in the presence of both kinases. This proposal investigates the hypothesis that the activation of downstream kinases is an essential pathway in mediating responses to tumor promoters. Portions of the proposal deal with the mechanism of activation of the ERK and RSK kinases, the roles of these kinases in mediating transcriptional activation, and pathways for PKC- dependent phospholipid hydrolysis. The specific aims are as follows. 1) The mechanism by which activation of PKC is linked to activation of ERK and RSK will be investigated: a) The activating factor produced by wild-type EL4 cells in response to phorbol ester will be isolated, purified, and characterized, b) The effects of the cdc2 kinase complex on the phosphorylation of ERK and RSK will be further investigated, c) Differences in protein phosphorylation between intact wild-type and variant EL4 cells in response to phorbol ester will be examined. 2) The mechanisms by which EL4 cells develop resistance to phorbol ester will be studied: a) Additional phorbol ester-resistant variant cell lines will be developed, b) Expression of PKC isoforms will be determined in wild-type and variant cell lines, c) The effects of phorbol ester on ERK, RSK and activating factor will be assessed in both cell types, d) Phorbol ester-induced phosphorylation of FOS and jun will be compared in wild- type and variant cells, e) Hydrolysis of phosphatidylcholine in response to phorbol ester will be analyzed in these cell lines. In summary, the proposed work uses a combination of cellular and biochemical approaches to examine the mechanism of action of tumor promoters and to define the role of PKC in T-cell activation. The information gained will provide a basis for future approaches to cancer therapy and prevention.