Tipnis S R, Blake D G, Shepherd A G, McLellan L I
Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, Scotland, U.K.
Biochem J. 1999 Feb 1;337 ( Pt 3)(Pt 3):559-66.
gamma-Glutamylcysteine synthetase (GCS) is reported to catalyse the rate-limiting step in glutathione biosynthesis, and is a heterodimer composed of a catalytic subunit [heavy subunit (GCSh) of Mr 73000] and a regulatory subunit [light subunit (GCSl) of Mr 31000]. In the present study, we have demonstrated for the first time a potential role for GCSl in resistance towards doxorubicin and cadmium chloride. Addition of recombinant GCSl to HeLa cell extracts in vitro was found to result in an increase in GCS activity of between 2- and 3-fold. Transient transfections of COS-1 cells with the GCSl cDNA cause an increase in GCS activity of approx. 2-fold, and a small but significant (P=0.008) increase in glutathione levels from 126.9+/-4. 2 nmol/mg protein to 178.8+/-19.1 nmol/mg protein. We proceeded to make a HeLa cell line (LN73), which stably overexpresses GCSl. These cells overexpress GCSl approx. 20-fold above basal levels. LN73 was found to have a 2-fold increase in GCS activity (437.3+/-85.2 pmol/min per mg) relative to the control cell line, HL9 (213.4+/-71. 8 pmol/min per mg). In contrast with the transient transfections in COS-1 cells, stable overexpression of GCSl was found not to be associated with an increase in glutathione content. However, when the LN73 and HL9 cells were treated with the glutathione-depleting agent, diethylmaleate, the LN73 cells were found to have an enhanced ability to regenerate glutathione, compared with HL9 cells. The cell lines were treated with various anti-cancer drugs, and their cytotoxicity was examined. No obvious differences in toxicity were observed between the different cell lines following treatment with cisplatin and melphalan. The redox-cycling agent doxorubicin, however, was found to be more toxic (approx. 2-fold) to the HL9 cells than the LN73 cells. When the cells were treated with the carcinogenic transition-metal compound, cadmium chloride, LN73 cells were found to be approx. 3-fold more resistant than HL9 cells.
据报道，γ-谷氨酰半胱氨酸合成酶（GCS）催化谷胱甘肽生物合成中的限速步骤，它是一种异二聚体，由一个催化亚基[分子量为73000的重亚基（GCSh）]和一个调节亚基[分子量为31000的轻亚基（GCSl）]组成。在本研究中，我们首次证明了GCSl在对阿霉素和氯化镉的抗性中具有潜在作用。体外向HeLa细胞提取物中添加重组GCSl后，发现GCS活性增加了2至3倍。用GCSl cDNA瞬时转染COS-1细胞导致GCS活性增加约2倍，并且谷胱甘肽水平从126.9±4.2 nmol/mg蛋白质小幅但显著地（P = 0.008）增加到178.8±19.1 nmol/mg蛋白质。我们接着构建了一个稳定过表达GCSl的HeLa细胞系（LN73）。这些细胞中GCSl的过表达量比基础水平高约20倍。相对于对照细胞系HL9（213.4±71.8 pmol/min每mg），发现LN73的GCS活性增加了2倍（437.3±85.2 pmol/min每mg）。与在COS-1细胞中的瞬时转染不同，发现GCSl的稳定过表达与谷胱甘肽含量的增加无关。然而，当用谷胱甘肽消耗剂马来酸二乙酯处理LN73和HL9细胞时，发现LN73细胞与HL9细胞相比具有更强的谷胱甘肽再生能力。用各种抗癌药物处理这些细胞系，并检测它们的细胞毒性。在用顺铂和马法兰处理后，不同细胞系之间未观察到明显的毒性差异。然而，发现氧化还原循环剂阿霉素对HL9细胞的毒性比对LN73细胞高约2倍。当用致癌过渡金属化合物氯化镉处理细胞时，发现LN73细胞的抗性比HL9细胞高约3倍。
