Leyland-Jones B R, Townsend A J, Tu C P, Cowan K H, Goldsmith M E
Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Cancer Res. 1991 Jan 15;51(2):587-94.
Studies have suggested that the alpha class glutathione S-transferase (GST) may protect cells from the chemotherapeutic drugs chlorambucil and melphalan. In order to further define the function of human alpha class GST, a complementary DNA which encodes it was ligated into an expression vector under the direction of the human metallothionein-IIA promoter and stably transfected into human MCF-7 breast cancer cells in conjunction with the G418-selectable plasmid pSV2neo. Clonal cell lines were identified which expressed increased levels of GST enzyme activity (2.2- to 5.6-fold). The transfected cell lines also had increased peroxidase activity using cumene hydroperoxide as the substrate (1.9- to 3.8-fold) which is consistent with the intrinsic peroxidase activity of alpha class GSTs. Southern blot analysis indicated that genomic DNA from these cells contained a fragment indistinguishable from the transfected alpha class GST complementary DNA (850 base pairs); Northern blot analysis of total cellular RNA indicated that these cells contained appropriately sized alpha class GST RNA (980 nucleotides); and Western blot analysis indicated that, while MCF-7 cells contained no detectable alpha class GST protein, the transfected cells contained markedly elevated levels of alpha class GST but no detectable mu or pi class GST. These alpha class GST transfected cells had increased resistance to ethacrynic acid (2.1- to 3.0-fold). However, the transfected cells failed to show any increased resistance measured at the drug dosage which inhibited 50% of the colony formation to the chemotherapeutic drugs chlorambucil, melphalan, Adriamycin, or cisplatin under conditions of either continuous or 1-h drug exposure. Neither was there any change in sensitivity to the cytotoxins benzo(a)pyrene, benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti), or 1-chloro-2,4-dinitrobenzene. These studies indicate that expression of this human alpha class GST by itself in MCF-7 human breast cancer cells does not confer resistance to the chemotherapeutic drugs tested under the conditions used in these studies.
研究表明，α类谷胱甘肽S-转移酶（GST）可能保护细胞免受化疗药物苯丁酸氮芥和美法仑的影响。为了进一步明确人α类GST的功能，将编码它的互补DNA连接到一个在人金属硫蛋白-IIA启动子指导下的表达载体中，并与G418-可选择质粒pSV2neo一起稳定转染到人MCF-7乳腺癌细胞中。鉴定出了克隆细胞系，其表达的GST酶活性水平升高（2.2至5.6倍）。以氢过氧化异丙苯为底物时，转染细胞系的过氧化物酶活性也升高了（1.9至3.8倍），这与α类GST的固有过氧化物酶活性一致。Southern印迹分析表明，这些细胞的基因组DNA含有一个与转染的α类GST互补DNA（850个碱基对）无法区分的片段；对总细胞RNA的Northern印迹分析表明，这些细胞含有大小合适的α类GST RNA（980个核苷酸）；Western印迹分析表明，虽然MCF-7细胞中未检测到α类GST蛋白，但转染细胞中α类GST水平显著升高，而未检测到μ或π类GST。这些转染了α类GST的细胞对依他尼酸的抗性增加（2.1至3.0倍）。然而，在连续或1小时药物暴露条件下，在抑制50%集落形成的药物剂量下，转染细胞对化疗药物苯丁酸氮芥、美法仑、阿霉素或顺铂未显示出任何抗性增加。对细胞毒素苯并(a)芘、苯并(a)芘-反式-7,8-二氢二醇-9,10-环氧化物（反式）或1-氯-2,4-二硝基苯的敏感性也没有任何变化。这些研究表明，在这些研究使用的条件下，在MCF-7人乳腺癌细胞中单独表达这种人α类GST并不能赋予对所测试化疗药物的抗性。
