PATHWAYS OF INDUCTION OF FUNCTIONAL P53 PROTEIN

功能性 P53 蛋白的诱导途径

• 批准号: 2749973
• 负责人: GEORGE ROBERT STARK
• 金额: $ 19.15万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2749973
• 关键词: DNA binding protein; DNA damage; DNA replication; N phosphonoacetyl L aspartate; RNA splicing; RNase protection assay; aphidicolin; camptothecin; cell growth regulation; flow cytometry; gel mobility shift assay; gene complementation; gene mutation; genetic translation; messenger RNA; molecular cloning; mutant; northern blottings; phosphorylation; posttranslational modifications; protein metabolism; protein structure function; tissue /cell culture; tumor suppressor genes; western blottings

DESCRIPTION (Adapted from Applicant Abstract): Normal mammalian cells respond to DNA damage or arrest of DNA synthesis by activating pathways that protect themselves or the organism. These controls are lost in cancer cells, allowing them to tolerate a high rate of genetic variation. Understanding the pathways involved is an important part of understanding the mechanisms that underlie the generation and evolution of cancer cells. Dr. Stark will investigate pathways of response to DNA damage or arrest of DNA synthesis that lead to induction of the tumor suppressor p53 in a functional form. Mutant cell lines will be obtained in which genes encoding signaling components have been inactivated, and the genes will be cloned by functional complementation. Dr. Stark's lab has established a system for selecting mutant human HT1080 fibrosarcoma cell lines defective in induction of functional p53, based on p53-dependent transcription. Expression of a gene encoding the cell-surface protein cd2 is controlled by a p53-regulated element. There is strong induction of cd2 in response to arrest of DNA synthesis (using N-(phosphonacetyl)-L-aspartate (PALA) or aphidicolin) or DNA damage (using adriamycin, camptothecin or radiation). Two independent mutant cell lines have been obtained in which the induction is defective, using the fluorescence- activated cell sorter (FACS). In Specific Aim 1, Dr. Stark proposes to obtain more mutant cell lines, to place the mutants into complementation groups, and to analyze the phenotypes of each complementation group. (For example, at what level is the formation of functional p53 inhibited? Are cells selected for defective p53 induction in response to PALA also defective in response to DNA damage?) The few specific candidate genes currently available will be investigated to determine if they are defective in the mutants. cDNA or genomic DNA libraries will be introduced to clone genes encoding unknown components by functional complementation. When specific genes are identified, they will be reintroduced into the null background of the mutant cell lines in order to define structure-function relationships in the signaling pathways. In Specific Aim 2, mutant cell lines will be isolated that express cd2 constitutively (as an indication of constitutive p53 activity), without induction. Such cells may be defective in pathways that maintain the p53 protein at a low level in unstimulated cells or that maintain the endogenous low levels of p53 protein in an inactive state. Complementation of the defects with cDNA or genomic DNA libraries, as in Specific Aim 1, should again lead to identification of unknown components.

描述（改编自申请人摘要）：正常哺乳动物细胞 通过激活途径来应对DNA损伤或停滞DNA合成 保护自己或生物体。 这些控件在癌症中丢失 细胞，使它们能够忍受高遗传变异率。 了解所涉及的途径是理解的重要组成部分 癌细胞产生和进化的基础的机制。 Stark博士将调查对DNA损伤的反应途径或逮捕 DNA合成导致肿瘤抑制p53在A中的诱导 功能形式。 将获得突变细胞系的基因编码 信号传导成分已被灭活，基因将被克隆 功能互补。 斯塔克博士的实验室已经建立了一个系统 选择突变的人类HT1080纤维肉瘤细胞系在诱导中有缺陷 基于p53依赖性转录的功能p53。 表达 编码细胞表面蛋白CD2的基因受p53调节的控制 元素。 CD2强烈诱导DNA的停滞 合成（使用N-（磷酸乙酰基）-L-天冬氨酸（PALA）或蚜虫蛋白）或 DNA损伤（使用adrimycin，camptothecin或辐射）。 两个独立 已经获得了诱导有缺陷的突变细胞系 使用荧光激活的细胞分辨率（FACS）。 在特定的目标1中， Stark博士建议获得更多的突变细胞系，以放置突变体 分为互补组，并分析每个表型 互补组。 （例如，在哪个层次上的形成 功能性p53抑制了吗？ 是否被选为有缺陷的p53诱导的细胞 对帕拉的响应也因响应DNA损伤而有缺陷？） 将研究当前可用的候选基因 它们在突变体中有缺陷。 cDNA或基因组DNA库将是 引入了通过功能编码未知组件的克隆基因 互补。 当发现特定基因时，它们将是 重新引入突变细胞系的无效背景，以便 定义信号通路中的结构功能关系。 在 特定的目标2，突变细胞系将被隔离为表达CD2 组成型（作为构成p53活性的指示），没有 就职。 这些细胞在维持p53的途径中可能有缺陷 在未刺激的细胞中低水平的蛋白质或维持内源性的蛋白质 低水平的p53蛋白在不活跃状态下。 互补 与特定AIM 1一样 再次导致识别未知组件。