Weng Mao-Wen, Hsiao Yi-Min, Chiou Hui-Ling, Yang Shun-Fa, Hsieh Yih-Shou, Cheng Ya-Wen, Yang Chieh-Hsiang, Ko Jiunn-Liang
Institute of Medical and Molecular Toxicology, Chung Shan Medical University, 110, Sec. 1, Chien-Kuo N. Road, Taichung, Taiwan 40203, ROC.
DNA Repair (Amst). 2005 Apr 4;4(4):493-502. doi: 10.1016/j.dnarep.2004.12.006. Epub 2005 Jan 19.
Cellular detoxification is important for the routine removal of environmental and dietary carcinogens. Glutathione S-transferases (GST) are major cellular phase II detoxification enzymes. MRC-5 cells have been found to exhibit significantly higher GST activity than human H1355 cells. This study investigates whether GST-M2 activity acts as a critical determinant of the target dose of carcinogenic benzo[a]pyrene-diolepoxide (BPDE) and whether it has an effect on MDM2 splicing in the two cell lines. We used RT-PCR to clone Mu-class GST cDNA. Two forms of GST coming from the cell lines were characterized as GST-M2 (from MRC-5 cells) and GST-M4 (from H1355 cells). Nested-PCR showed that BPDE-induced MDM2 splicing had occurred in the H1355 cell line but not in normal MRC-5 cells. Furthermore, using nested-PCR and competitive ELISA, we found that in H1355 cells modified to stably overexpress GST-M2, splicing was abolished and BPDE adducts appeared in low abundance. In conclusion, exogenously overexpressed GST-M2 was effective in reducing BPDE-induced DNA damage in H1355 cells. The catalytic activity of GST-M2 may play an important future role in lowering the incidence of BPDE-induced DNA damage.
细胞解毒对于日常清除环境和饮食中的致癌物很重要。谷胱甘肽S-转移酶（GST）是主要的细胞II期解毒酶。已发现MRC-5细胞的GST活性明显高于人H1355细胞。本研究调查GST-M2活性是否是致癌性苯并[a]芘-二环氧物（BPDE）靶剂量的关键决定因素，以及它是否对这两种细胞系中的MDM2剪接有影响。我们使用逆转录聚合酶链反应（RT-PCR）克隆Mu类GST cDNA。来自细胞系的两种GST形式被鉴定为GST-M2（来自MRC-5细胞）和GST-M4（来自H1355细胞）。巢式PCR显示BPDE诱导的MDM2剪接发生在H1355细胞系中，而在正常MRC-5细胞中未发生。此外，使用巢式PCR和竞争性酶联免疫吸附测定（ELISA），我们发现，在稳定过表达GST-M2的H1355细胞中，剪接被消除，且BPDE加合物以低丰度出现。总之，外源性过表达的GST-M2可有效减少H1355细胞中BPDE诱导的DNA损伤。GST-M2的催化活性可能在降低BPDE诱导的DNA损伤发生率方面发挥重要作用。
