RESPONSE TO INJURY IN GAMMAGLUTAMYL CYCLE DEFICIENT MICE

丙谷氨酰循环缺陷小鼠对损伤的反应

• 批准号: 6041314
• 负责人: Michael Lieberman
• 金额: $ 21.33万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6041314
• 关键词: BCL2 gene /protein; JUN kinase; N methyl N' nitro N nitrosoguanidine; apoptosis; cell line; chemoprevention; cis platinum compound; cysteine; enzyme activity; free radical oxygen; genetically modified animals; glutamates; glutathione; ionizing radiation; laboratory mouse; ligase; methane sulfonate; mitogen activated protein kinase; mitomycin C; nitrosourea; nuclear factor kappa beta; oxidative stress; posttranslational modifications; radiation protection

The overall goal of this proposal is to define the function of glutathione (GSH) in mammals. Mice deficient in the heavy (catalytic) subunit of gamma-glutamyl cysteine synthetase (gamma-GCS-HSU), the rate limiting enzyme in GSH synthesis, diet at post coital day 7.5 and completely lack GSH; however, cell lines derived from gamma-GCS- HSU-deficient embryos survive if GSH is provided in the medium. These cells contain only 50-60 mum GSH (1-2% of wild type levels). Preliminary data indicate that removal of GSH from the medium produces a fall in GSH to undetectable levels, increased hydroxyl radical formation, activation of JNK/SAPK and cell death. These data suggest that very low GSH levels (,1% of wild type cells) whether "spontaneous" or induced by chemicals/radiation may be a key trigger that initiates the oxidative stress response and apoptosis. The gamma-GCS-HSU-deficient mice and cells we have developed and additional knock-out mice and cells we plan to construct provide a unique opportunity to test four hypotheses about GSH function: 1. Only low levels of GSH are required for survival of unstressed cells, but high levels are necessary to protect cells from chemical and radiation injury, and this protection is directly proportional to GSH level. 2. Very low/absent levels of cellular GSH activate a cellular stress response program that includes JNK/SAPK, p38 MAPK and NFkappaB, and activation of these pathways by chemicals radiation depends upon their ability to lower GSH levels. 3.Very low-absent levels of cellular GSH potentiate apoptotic cell death, and chemicals/radiation that induce apoptosis do so by lowering cellular GSH levels. The process involves suppression of anti-apoptotic factors and activation of pro-apoptotic factors. 4. Only low levels of GSH are needed for development and survival of unstressed mice. These low levels of GSH render mice hypersensitive to chemical injury.

该提案的总体目标是确定谷胱甘肽（GSH）在哺乳动物中的功能。缺乏γ-谷氨酰半胱氨酸合成酶（γ-GCS-HSU）（GSH合成的限速酶）重（催化）亚基的小鼠，在交配后第7.5天进食，完全缺乏GSH;然而，如果培养基中提供GSH，则来自γ-GCS-HSU缺陷胚胎的细胞系存活。这些细胞仅含有50-60 μ mGSH（野生型水平的1-2%）。初步数据表明，从培养基中去除GSH会导致GSH下降至检测不到的水平，增加羟基自由基的形成，JNK/SAPK的激活和细胞死亡。这些数据表明，非常低的GSH水平（野生型细胞的1%），无论是“自发的”还是由化学品/辐射诱导的，都可能是引发氧化应激反应和细胞凋亡的关键触发因素。我们已经开发的γ-GCS-HSU缺陷小鼠和细胞以及我们计划构建的其他敲除小鼠和细胞提供了一个独特的机会来测试关于GSH功能的四个假设：只有低水平的GSH是非应激细胞生存所必需的，但高水平的GSH是保护细胞免受化学和辐射损伤所必需的，这种保护与GSH水平成正比。2.非常低/缺乏细胞GSH水平激活细胞应激反应程序，包括JNK/SAPK，p38 MAPK和NF κ B，化学辐射对这些途径的激活取决于它们降低GSH水平的能力。3.非常低水平的细胞GSH缺乏增强凋亡性细胞死亡，并且诱导凋亡的化学品/辐射通过降低细胞GSH水平来实现。该过程涉及抑制抗凋亡因子和激活促凋亡因子。4.只有低水平的谷胱甘肽是需要的发展和生存的非应激小鼠。这些低水平的GSH使小鼠对化学损伤过敏。