RESPONSE TO INJURY IN GAMMAGLUTAMYL CYCLE DEFICIENT MICE

这项建议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(GSH合成的限速酶)重(催化)亚单位的小鼠，在性交后7.5天饮食，完全缺乏GSH；然而，如果在培养液中提供GSH，来自伽马-GCS-HSU缺陷胚胎的细胞系存活下来。这些细胞只含有50-60微米的谷胱甘肽(1-2%的野生型水平)。初步数据表明，从培养液中去除GSH会导致GSH下降到无法检测到的水平，增加羟基自由基的形成，激活JNK/SAPK，并导致细胞死亡。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物质/辐射诱导的，可能是启动氧化应激反应和细胞凋亡的关键触发因素。我们开发的伽马-GCS-HSU缺陷小鼠和细胞，以及我们计划构建的额外的基因敲除小鼠和细胞，为检验关于GSH功能的四个假说提供了一个独特的机会：1.非应激细胞的生存只需要低水平的GSH，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.极低水平/缺失的细胞GSH激活细胞应激反应程序，其中包括JNK/SAPK、p38MAPK和NFkappaB，而这些途径的激活取决于它们降低GSH水平的能力。3.极低水平的细胞GSH缺失会加剧细胞的死亡，而诱导细胞凋亡的化学物质/辐射是通过降低细胞GSH水平来实现的。这一过程包括抑制抗凋亡因子和激活促凋亡因子。4.非应激小鼠的发育和存活只需要低水平的GSH。这些低水平的GSH使小鼠对化学损伤非常敏感。

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.