MECHANISMS OF RESISTANCE TO TUMOR PROMOTERS

肿瘤促进剂的抵抗机制

• 批准号: 2099315
• 负责人: Kathryn E Meier
• 金额: $ 14.24万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2099315
• 关键词: SDS polyacrylamide gel electrophoresis; cell cycle proteins; drug resistance; enzyme activity; gene expression; genetic regulation; interleukin 2; isozymes; laboratory rabbit; laboratory rat; leukocyte activation /transformation; neoplastic cell; oncoproteins; phorbols; phosphatidate; phosphatidylcholines; phosphorylation; protein kinase; protein kinase C; tissue /cell culture; tumor promoters

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.

肿瘤促进剂是与启动协同作用的化合物 致癌物质，以诱导哺乳动物细胞中的肿瘤形成。 佛波醇酯 是激活蛋白激酶C（PKC）、蛋白质 依赖于钙，二酰基甘油， 和磷脂的活性。 将细胞暴露于佛波醇酯也 导致MAP激酶和S6激酶的激活，这两个信使- PKC下游激活的独立丝氨酸/苏氨酸激酶， 活性受磷酸化调节。 S6激酶被激活， 被MAP激酶磷酸化。 MAP的作用机制 激酶未知;酪氨酸和苏氨酸磷酸化 残留物是必需的。 不同的细胞系对 佛波酯是研究作用机制的有用工具 肿瘤促进剂。 野生型EL 4小鼠胸腺瘤细胞，但不是变体 EL 4细胞，通过启动白细胞介素-2对佛波酯应答 转录。 佛波酯引起MAP和S6的快速增加 激酶活性在野生型细胞中而不是在变体细胞中。 这些 激酶分别代表ERK和RSK基因产物。 野生型 细胞，而不是变异细胞，产生一种激活因子， 佛波醇酯。 该因子增强了两种蛋白质的磷酸化， ERK和RSK激酶通过一种未知的机制， 两种激酶的存在。 这项建议调查的假设， 下游激酶的激活是介导 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，这些激酶的作用， 激酶介导的转录激活，以及PKC- 依赖磷脂水解。 具体目标如下。 1)蛋白激酶C的激活与蛋白激酶C的激活有关的机制， ERK和RSK将被研究：a）由ERK和RSK产生的激活因子， 将分离响应佛波醇酯的野生型EL 4细胞， B）cdc 2激酶复合物的作用 将进一步研究ERK和RSK的磷酸化，c） 完整野生型和野生型之间蛋白质磷酸化的差异 将检测响应佛波酯的变体EL 4细胞。 2)的 EL 4细胞对佛波醇酯产生抗性的机制将 a）另外的佛波酯抗性变体细胞系 B）PKC亚型的表达将在 野生型和变异细胞系，c）佛波酯对ERK的作用， 将在两种细胞类型中评估RSK和活化因子。 酯诱导的FOS和jun磷酸化将在野生型中进行比较， 型和变异细胞，e）响应中磷脂酰胆碱的水解 将在这些细胞系中分析佛波酯的活性。 总而言之， 拟议的工作使用细胞和生物化学方法的组合 研究肿瘤促进剂的作用机制，并确定 PKC在T细胞活化中的作用 获得的信息将提供 为未来的癌症治疗和预防方法奠定了基础。