MUTANT P53 ACTIVITY AFFECTS OF MYOGENESIS IN CULTURE

突变 P53 活性影响培养中的肌生成

• 批准号: 6481218
• 负责人: SANDRA B SHARP
• 金额: $ 7.33万
• 依托单位: CALIFORNIA STATE UNIVERSITY LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6481218
• 关键词: animal tissue; developmental genetics; gel mobility shift assay; gene expression; gene mutation; genetic regulatory element; human tissue; immunocytochemistry; messenger RNA; myogenesis; northern blottings; nucleic acid sequence; p53 gene /protein; phenotype; polymerase chain reaction; recombinant DNA; regulatory gene; striated muscles; tissue /cell culture; transfection; western blottings

P53 mutations are found in more than 50% of human cancers. It is known that expression of the temperature sensitive p53 missense mutation 135Val inhibits differentiation of myogenic C2 cells, which express wild type p53, but does not affect their ability to exit the cell cycle (S Soddu et al.). In vivo, however, p53 is not required for myogenesis (LA Donehower et al.; T Jacks et al.). The proposed experiments test two hypotheses. The first is that wild type (wt) p53 possesses an activity that contributes to the progress of myogenesis but is not required for withdrawal from the cell cycle. The second is that different mutant forms of p53 possess different abilities to inhibit the progress of myogenesis, either by differential inactivation of various p53 activities, or by a gain of function, independent of the ability to functionally inactivate wt p53. These hypotheses will be addressed by using stable and transient transfections in cells which are null for p53 or in which p53 has been inactivated, either by anti-sense, the Adenoviral E1B-55 kD protein, or a dominant negative p53 mutation. Th effects of p53 missense and denatured conformations of dominant negative p53, will be compared. Resulting phenotypes will be evaluated by microscopy for myotube formation. Identification of phenotypic differences between derivative cell lines and the control lines will provide clues as to the mechanism(s) by which p53 acts. Immunoblot analyses will e used to assess the accumulation and modification of several proteins that have been shown to be important in the progress of myogenesis. Difference libraries will be constructed to facilitate identification of transcriptional events affected by p53. Reporter assays will assess the effects of wt and mutant p53s on promoters activated by myogenesis regulatory and enhancing factors (MRFs and MEF2s). Rescue experiments will be used to determine what molecules other than wt p53 restore the normal phenotype. Knowledge of why a mutant p53 inhibits myogenesis in culture will increase our knowledge both of the activities by which p53 controls growth and differentiation and out of the steps in myogenesis.

在超过50％的人类癌症中发现了p53突变。众所周知，温度敏感的p53错义突变的表达135VAL抑制了表达野生型p53的肌源性C2细胞的分化，但不会影响其退出细胞周期的能力（S Soddu等人）。然而，在体内，肌发生并不需要p53（La Donehower等； T Jacks等人）。提出的实验检验了两个假设。首先是野生型（WT）p53具有有助于肌发生进展的活性，但不需要退出细胞周期。第二个是，p53的不同突变形式具有不同的能力来抑制肌发生的进展，要么通过各种p53活性的差异失活，要么是通过功能的增益，而与功能上失活的WT p53的能力无关。这些假设将通过在p53为null的细胞中使用稳定和瞬态转染来解决，或者通过抗渗透，腺病毒E1B-55 kD蛋白或主要的负p53突变使p53灭活的p53被灭活。将比较p53错义和变性构象的影响。将通过显微镜进行肌管形成评估所得的表型。识别衍生细胞系与控制线之间的表型差异将提供有关p53作用的机制的线索。免疫印迹分析将用于评估几种蛋白质的积累和修饰，这些蛋白在肌发生中很重要。差异库将被构建，以促进识别受p53影响的转录事件。记者分析将评估WT和突变体p53s对肌发生调节和增强因子（MRFS和MEF2S）激活的启动子的影响。救援实验将用于确定除WT p53以外的哪些分子恢复了正常表型。知识为什么突变体p53抑制培养物中的肌发生会增加我们的知识，这既可以控制生长和分化，又是肌发生的步骤。