Meredith M J, Dodson M L
Cancer Res. 1987 Sep 1;47(17):4576-81.
Cell lines established from donors with the inherited disorder ataxia-telangiectasia (A-T) exhibit exceptional sensitivity to ionizing radiation and chemicals known to produce increased levels of intracellular H2O2, suggesting a deficiency in glutathione-dependent detoxication reactions. Glutathione (GSH) biosynthesis in fibroblast and lymphoblast cultures derived from individuals known to be clinically unaffected, homozygous, or heterozygous for A-T was assessed. Following GSH depletion by diethylmaleate, fibroblasts (GM 3492) from a clinically unaffected individual resynthesized GSH at a rate approximately twice that observed in fibroblasts from known heterozygotes (GM 3488 and GM 3489). Unrelated A-T homozygote fibroblast lines GM 3487B and GM 5823 resynthesized GSH only very slowly. GM 3492 cells repleted intracellular GSH by 6 h after depletion, the heterozygote lines by 18 h. The A-T homozygotes replaced only 30% of the intracellular GSH pool by 24 h. A lymphoblast cell line from the A-T homozygote (GM 3189) also exhibited slow resynthesis after depletion. However, if these cells were permeabilized by treatment with digitonin, GSH synthesis proceeded at a rate exceeding synthesis in permeabilized or untreated normal lymphoblasts (GM 3323). The first enzyme in GSH synthesis, gamma-glutamylcysteine synthetase, was found to be elevated about 2.7-fold in A-T homozygote fibroblasts, suggesting that a substrate for GSH synthesis may be rate limiting. A-T homozygote lymphoblasts contained about 2-fold more gamma-cystathionase activity over other cell lines tested suggesting increased flux through the transsulfuration pathway for cysteine production in response to reduced cysteine supply. Transport of cysteine and cystine was found to be 8- and 5-fold slower in A-T homozygotes that did not affect fibroblasts while glutamate and methionine transport Vmax did not differ among the cell lines tested. These experiments demonstrate that cells from A-T homozygotes are deficient in cysteine transport, thus limiting GSH resynthesis after a depleting challenge such as radiation or GSH-depleting xenobiotic compounds.
从患有遗传性共济失调毛细血管扩张症（A-T）的供体建立的细胞系，对电离辐射和已知会使细胞内过氧化氢水平升高的化学物质表现出异常的敏感性，这表明谷胱甘肽依赖性解毒反应存在缺陷。评估了来自临床未受影响、A-T纯合子或杂合子个体的成纤维细胞和淋巴母细胞培养物中的谷胱甘肽（GSH）生物合成。在用马来酸二乙酯耗尽GSH后，来自临床未受影响个体的成纤维细胞（GM 3492）重新合成GSH的速率约为已知杂合子（GM 3488和GM 3489）成纤维细胞的两倍。无关的A-T纯合子成纤维细胞系GM 3487B和GM 5823重新合成GSH的速度非常缓慢。GM 3492细胞在耗尽后6小时内补充了细胞内GSH，杂合子系则在18小时内补充。A-T纯合子在24小时内仅替换了细胞内GSH池的30%。来自A-T纯合子的淋巴母细胞系（GM 3189）在耗尽后也表现出缓慢的重新合成。然而，如果用洋地黄皂苷处理使这些细胞通透，GSH合成的速率超过通透或未处理的正常淋巴母细胞（GM 3323）。GSH合成中的第一种酶，γ-谷氨酰半胱氨酸合成酶，在A-T纯合子成纤维细胞中升高了约2.7倍，这表明GSH合成的一种底物可能是限速因素。A-T纯合子淋巴母细胞中的γ-胱硫醚酶活性比其他测试细胞系高约2倍，这表明在半胱氨酸供应减少的情况下，通过转硫途径产生半胱氨酸的通量增加。发现A-T纯合子中半胱氨酸和胱氨酸的转运速度分别慢8倍和5倍，这对成纤维细胞没有影响，而谷氨酸和蛋氨酸的转运Vmax在测试的细胞系中没有差异。这些实验表明，A-T纯合子的细胞半胱氨酸转运存在缺陷，因此在诸如辐射或消耗GSH的外源性化合物等消耗性挑战后，限制了GSH的重新合成。
