Molecular Biology of Cellular Injury

细胞损伤的分子生物学

• 批准号: 6761681
• 负责人: Albert J Fornace
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6761681
• 关键词: DNA damage; DNA repair; antineoplastics; apoptosis; biological signal transduction; cell cycle; cell growth regulation; cytotoxicity; gene expression; gene induction /repression; genetic regulation; genetically modified animals; laboratory mouse; molecular cloning; p53 gene /protein; tissue /cell culture; tumor suppressor genes; tumor suppressor proteins

A major focus of this research group is the study of the cellular responses to genotoxic stress in mammalian cells. This has included the cloning and characterization of a variety of DNA-damage-inducible (DDI) genes, including the gadd genes, and elucidation of the regulatory mechanisms controlling their expression. Cell-cycle checkpoint activation and growth inhibition are universal responses to genotoxic stress. We gave found that the 5 gadd (growth-arrest and DNA-damage inducible) genes to be coordinately induced by cellular exposure to many DNA-damaging agents and certain other stresses that trigger growth arrest (Mol. Cell. Biol. 1989; 9:4196). Evidence for such responses have been found in all mammalian cells examined to date and indicate that this is a well-conserved stress response(s). In the case of gadd45, this was the first cellular gene found to be regulated by the key tumor suppressor p53 via a pathway that is activated by ionizing radiation (and many other stresses). This pathway also involves ATM, the gene defective in ataxia telangiectasia (Mol. Cell. Biol. 1991; 11:1009; Cell 1992; 71:587). Responses to ionizing radiation have been characterized in a variety of human tumor lines including the regulation of key cell death genes like BAX, BCL2, BCL-X, KILLER/DR5 , TRID/TRAIL-R3 (Oncogene 1998; 17:3287). This laboratory has shown that a functional and physical interaction between p53 and WT1 plays a central role in the regulation of such genes after certain stresses, such as UV radiation (Mol. Cell. Biol. 1998; 18:2768). Interestingly, the same promoter control element, which binds to WT1, has been found to be suppressed by c-Myc, a growth-stimulatory signaling protein (Oncogene 1998; 17:2149). Moreover, deletion of c-Myc has been found to markedly upregulate the gadd45 growth-arrest gene. Both c-myc and Brca1 are implicated in breast cancer, and have been shown to contribute to the regulation of the gadd45 gene. We have also shown that the p38 kinases of the MAP kinase pathway have an important role in activation of p53 after certain stresses (EMBO J. 1999; 18:6845). We have recently applied a functional genomics approach to the study of DDI genes. Using cDNA microarray hybridization, a very complex pattern of responses has been found in various human cells which is dependent on p53 status, apoptotic potential, and a variety of other control factors (Oncogene 1999; 18:3666). Characterization of such responses in tumor cells will be used to elucidate the status of signal transduction pathways and may have predictive value in treatment planning. A second major focus is the characterization of the products encoded by particular stress genes with emphasis on p53-regulated genes. This project involves both a genetic and biochemical approach. Targeted disruption of the gadd45 (Gadd45a) gene has been carried out in mice and characterization of these mice is currently underway. Studies are being expanded to other engineered mice including strains with disruption of p53, cip1waf1 (Cdkna1), and other selected genes. Defects in important parameters, such as genomic stability, growth control, resistance to carcinogenesis, and DNA repair, have been found in gadd45-/- mice (Nature Genetics 1999; 23:176). From analysis of these various "knockout" strains, cip1/waf1, gadd45, and other effector genes contribute to the phenotype of p53-/- mice. Targeted disruption of related DDI genes is currently underway. Using a biochemical approach, this laboratory has already demonstrated interactions between Gadd45 with PCNA, p21Cip1/Waf1, Cdc2, and core histone proteins, and evidence for roles in DNA repair and cell cycle control (Molec. Cell. Biol. 1999; 19:1673. Oncogene 1999; 18:2892. Molec. Cell. Biol. 2000; 20:3705). The goal of these studies is to contribute to the understanding of the function of these and related DDI genes, and their potential as targets for cancer therapy in the future. This project was Z01 BC 07184-08 LBC in FY 97.

该研究小组的主要重点是研究哺乳动物细胞中对遗传毒性应激的细胞反应。这包括克隆和表征多种DNA损伤诱导（DDI）基因，包括GADD基因，以及阐明控制其表达的调节机制。细胞周期检查点激活和生长抑制是对遗传毒性应激的普遍反应。我们发现，5个GADD（生长危机和DNA破坏）基因是通过细胞暴露于许多DNA损害剂和某些其他触发生长停滞的其他压力来协调诱导的（Mol。Biol。1989; 9：4196）。迄今为止，在所有检查的哺乳动物细胞中都发现了这种反应的证据，并表明这是保守的应激反应。在GADD45的情况下，这是第一个细胞基因被发现通过关键肿瘤抑制p53调节的途径，该途径通过电离辐射（以及许多其他应力）激活。该途径还涉及ATM，ATM，基因在telangiyctia telangictia中有缺陷（Mol。Cell。Biol。1991; 11：1009; Cell 1992; 71：587）。对电离辐射的反应已在各种人肿瘤系中进行了表征，包括调节BAX，BCL2，BCL-X，BLCL-X，Killer/DR5，TRID/TRAID-R3（Oncogene 1998; 17：3287）等关键细胞死亡基因。该实验室表明，p53和WT1之间的功能和物理相互作用在某些应力后的调节中起着核心作用，例如紫外线辐射（Mol。Cell。Biol。1998; 18：2768）。有趣的是，已经发现与WT1结合的相同启动子控制元件被C-MYC（一种生长刺激信号传导蛋白）抑制（Oncogene 1998; 17：2149）。此外，已经发现C-MYC的缺失显着上调了GADD45生长暂停基因。 C-MYC和BRCA1都与乳腺癌有关，并已证明有助于调节GADD45基因。我们还表明，在某些应力后，MAP激酶途径的p38激酶在p53的激活中具有重要作用（Embo J. 1999; 18：6845）。我们最近将功能基因组学方法应用于DDI基因的研究。使用cDNA微阵列杂交，在各种人类细胞中发现了非常复杂的响应模式，取决于p53状态，凋亡潜力和各种其他控制因素（Oncogene 1999; 18：3666）。肿瘤细胞中这种反应的表征将用于阐明信号转导途径的状态，并可能在治疗计划中具有预测价值。 第二个主要重点是由特定应力基因编码的产物表征，重点是p53调节的基因。该项目涉及一种遗传和生化方法。在小鼠中已进行了靶向破坏GADD45（GADD45A）基因，目前正在进行这些小鼠的表征。研究正在扩展到其他工程小鼠，包括p53，CIP1WAF1（CDKNA1）和其他选定基因的菌株。在GADD45 - / - 小鼠中发现了重要参数的缺陷，例如基因组稳定性，生长控制，对癌变的抗性和DNA修复（自然遗传学1999; 23：176）。从对这些“敲除”菌株的分析，CIP1/WAF1，GADD45和其他效应基因有助于p53 - / - 小鼠的表型。目前正在进行针对相关DDI基因的目标破坏。 Using a biochemical approach, this laboratory has already demonstrated interactions between Gadd45 with PCNA, p21Cip1/Waf1, Cdc2, and core histone proteins, and evidence for roles in DNA repair and cell cycle control (Molec. Cell. Biol. 1999; 19:1673. Oncogene 1999; 18:2892. Molec. Cell. Biol. 2000; 20:3705).这些研究的目的是为理解这些和相关DDI基因的功能及其作为癌症治疗的潜力做出贡献。 该项目是97财年的Z01 BC 07184-08 LBC。