The function of human HLH-type inhibitor of differentiation, Id-1H and Id-2H that induce abrogation of G1 maturation in mutant p53 expressing cells.

人 HLH 型分化抑制剂、Id-1H 和 Id-2H 的功能，可诱导突变 p53 表达细胞中 G1 成熟的终止。

• 批准号: 11680691
• 负责人: NAKAJIMA Takuma
• 金额: $ 2.37万
• 依托单位: Graduate School, Tokyo Medical and Dental University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680691/
• 关键词: Id; apoptosis; Adenovirus; E1A; p53; cell cycle; transcription; interaction; アポトーシス; HLH蛋白; 癌; アデノウイルスE1A; 細胞周期; DNA複製

Induction of apoptosis by adenovirus E1A in rodent cells is stimulated by wild type (wt) p53 but completely suppressed by mutated p53. The suppression is overcome by coexpression with Id proteins (Ids). The cells expressing E1A and Ids undergo apoptosis after accumulation in S phase, suggesting that S phase events are perturbed by E1A and Ids. The E1A domains required for induction of apoptosis, analyzed by transfection with expression vectors for E1A, Ids and their mutants, followed by flow cytometry, reside in N-terminal (positions 17-38), CR1 and CR2 regions. Interaction of E1A with Ids requires the N-terminal and CR1 regions. The cyclin D1 promoter activity in S phase was reduced severely by E1A and this reduction is caused through CR1 and CR2 regions required for interaction with pRB.Analysis of DNA synthesis in G2/M arrested cells indicated that E1A is capable of inducing>4N cells and this E1A-mediated DNA rereplication is enhanced by coexpression with Id-1H.The E1A domains required for induction of DNA rereplication coincide with those required for apoptosis. To analyze the concrete function of Id proteins, we tried to establish cell lines that carry inducible Id genes. To achieve this purpose, we employed the Tet-Off^<TM> gene expression system (Clontech) since this system had been thought as the best system to suppress the unexpected expression of the gene of interest until induction. However, in spite of great deal of the trials, it has not been successful. It may caused by a severe abrogation of cellular homeostasis induced by a leaky expression of introduced Id genes.

腺病毒E1 A在啮齿类动物细胞中诱导凋亡受野生型（wt）p53刺激，但完全受突变型p53抑制。通过与Id蛋白（Ids）共表达来克服抑制。表达E1 A和Ids的细胞在S期积累后发生凋亡，表明S期事件受到E1 A和Ids的干扰。通过用E1 A、Ids及其突变体的表达载体转染，随后通过流式细胞术分析，诱导细胞凋亡所需的E1 A结构域位于N-末端（位置17-38）、CR 1和CR2区。E1 A与Ids的相互作用需要N-末端和CR 1区域。E1 A可显著降低cyclin D1启动子在S期的活性，这种降低是通过与pRB相互作用所需的CR 1和CR2区域引起的。对G2/M期阻滞细胞DNA合成的分析表明，E1 A能够诱导> 4 N的细胞，并且这种E1 A介导的DNA再复制通过与Id-1共表达而增强。1H.诱导DNA再复制所需的E1 A结构域与细胞凋亡所需的E1 A结构域一致。为了分析Id蛋白的具体功能，我们试图建立携带可诱导Id基因的细胞系。为了实现该目的，我们采用Tet-Off<TM>基因表达系统（Clontech），因为该系统被认为是抑制目的基因的意外表达直至诱导的最佳系统。然而，尽管进行了大量的试验，但并不成功。这可能是由于导入的Id基因的泄漏表达导致细胞内稳态的严重破坏。

项目成果

Shirasuna, K., et al.: "The G10BP-1 gene encoding a GC box binding protein, is a target of Myc and Jun/Fos."Genes to Cells. 4. 277-289 (1999)

Shirasuna, K. 等人：“编码 GC 盒结合蛋白的 G10BP-1 基因是 Myc 和 Jun/Fos 的靶标。”Genes to Cells。