REGULATION OF ORNITHINE DECARBOXYLASE BY PHORBOL ESTERS

佛波酯对鸟氨酸脱羧酶的调节

• 批准号: 3189976
• 负责人: ANDREW P BUTLER
• 金额: $ 12.21万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-03-01 至 1991-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3189976
• 关键词: binding proteins; biotransformation; ceramides; chromatin; diacylglycerols; endonuclease; enzyme mechanism; gel electrophoresis; gene expression; genetic mapping; genetic transcription; hepatocellular carcinoma; molecular cloning; neoplastic cell culture for noncancer research; nucleic acid hybridization; nucleic acid sequence; nucleoproteins; ornithine; ornithine decarboxylase; phorbols; phospholipase C; phosphorylation; protein kinase C; toxin metabolism; transfection; transposon /insertion element

Knowledge of the mechanisms by which tumor promoting agents, such as 12-0-tetradecanoylphorbol-13-acetate (TPA), cause alterations in specific gene expression is fundamental to an understanding of their biological and tumor-promoting properties. TPA, and other agents which stimulate protein kinase C, may induce transcription of specific genes through phosphorylation of chromatin-associated nuclear proteins. Ornithine decarboxylase (ODC) is rapidly induced by TPA in many different systems. We propose to study the mechanism by which TPA alters transcription of ODC, with a focus on the possible role of chromatin protein phosphorylation. This objective will be pursued through the following specific aims. (1) The relative contribution of changes in transcription and messenger stabilization in the accumulation of ODC mRNA following TPA treatment will be determined. A nuclear transcription assay will be used to determine the rates of transcription of ODC in TPA treated rat H35 hepatoma cells, and the stability of ODC mRNA in these cells will be determined through the use of inhibitors of transcription and DNA-RNA hybridization. The requirement for protein kinase C activation in this process will be evaluated using PKC activators (phospholipase C; diacylglycerols) and inhibitors (palmitoyl carnitine, H7, trifluoroperazine). (2) The distribution of nuclear phosphoproteins and ODC coding sequences will be determined in chromatin fractions separated on the basis of their nuclease accessibility and solubility. (3) A functional ornithine decarboxylase gene will be cloned from rat H35 cells selected for resistance to the ODC inhibitor, difluoromethylornithine. The gene will be manned with restriction endonucleases and by S1 transcript mapping, and regions likely to contain important regulatory sites will be sequenced. (4) DNA sequence elements of the ODC gene required for basal expression and TPA induction will be identified through the use of a transient transfection assay. The sites of DNA- protein interactions within these elements will be located using gel mobility shift and nuclease protection assays, and the role of protein phosphorylation in the specificity of these DNA-protein interactions will be determined.

了解肿瘤促进剂的机制， 例如12-0-四烷酰基-13-乙酸酯（TPA），原因 特定基因表达的改变是 了解其生物学和肿瘤促进特性。 TPA和其他刺激蛋白激酶C的药物可能 通过磷酸化诱导特定基因的转录 染色质相关的核蛋白。 鸟嘌呤脱羧酶 （ODC）在许多不同的系统中被TPA迅速诱导。 我们 建议研究TPA改变转录的机制 ODC的重点是染色质蛋白的可能作用 磷酸化。 这个目标将通过 遵循特定目标。 （1）变化的相对贡献 在累积中的转录和使者稳定中 将确定TPA治疗后的ODC mRNA。 一个 核转录测定法将用于确定 TPA处理的大鼠H35肝癌细胞中ODC的转录，以及 这些细胞中ODC mRNA的稳定性将确定 通过使用转录和DNA-RNA的抑制剂 杂交。 蛋白激酶C活化的需求 该过程将使用PKC激活剂（磷脂酶 c;二酰基甘油）和抑制剂（Palmitoyl Carnitine，H7， 三氟吡嗪）。 （2）核磷蛋白的分布 ODC编码序列将在染色质中确定 分数根据其核酸酶的可及性分开，并且 溶解度。 （3）功能性鸟氨酸脱羧酶基因将是 从被选择以抗ODC的大鼠H35细胞克隆 抑制剂，二氟甲基氨酸。 该基因将与 限制性核酸内切酶和S1成绩单映射，以及 可能包含重要监管地点的地区将是 测序。 （4）需要ODC基因的DNA序列元素 对于基础表达和TPA诱导将通过 瞬时转染测定法的使用。 DNA-的位置 这些元素内的蛋白质相互作用将使用 凝胶迁移率转移和核酸酶保护测定法，以及 这些DNA-蛋白质的蛋白质磷酸化 将确定相互作用。