Kishida Yugo, Natsume Atsushi, Toda Hiroshi, Toi Yuki, Motomura Kazuya, Koyama Hiroko, Matsuda Keiji, Nakayama Osamu, Sato Makoto, Suzuki Masaaki, Kondo Yutaka, Wakabayashi Toshihiko
Department of Neurosurgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.
Tumour Biol. 2012 Apr;33(2):373-81. doi: 10.1007/s13277-012-0319-1. Epub 2012 Jan 26.
The DNA repair protein O (6)-methylguanine-DNA methyltransferase (MGMT, AGT) is a determinant of the resistance of tumor cells to alkylating anticancer agents that target the O(6) position of guanine. MGMT promoter methylation in tumors is regarded as the most common predictor of the responsiveness of glioblastoma to alkylating agents. However, MGMT promoter methylation status has been investigated mainly by methylation-specific PCR, which is a qualitative and subjective assay. In addition, the actual enzymatic activities associated with the methylation status of MGMT have not been explored. In the present study, MGMT promoter methylation in glioblastomas was quantified by bisulfite pyrosequencing, and its correlation with enzymatic activity was determined using a novel quantitative assay for studying the functional activity of MGMT. MGMT enzymatic activity was assessed using fluorometrically labeled oligonucleotide substrates containing MGMT-specific DNA lesions and capillary electrophoresis to detect and quantify these lesions. In comparison with existing traditional assays, this assay was equally sensitive but less time consuming and easier to perform. MGMT promoter methylation was assessed in 41 glioblastomas by bisulfite pyrosequencing, and five samples with different values were chosen for comparison with enzymatic assays. Bisulfite pyrosequencing using primers designed to work in the upstream promoter regions of MGMT demonstrated high quantitative capability and reproducibility in triplicate measurements. In comparative studies, MGMT promoter methylation values obtained by bisulfite pyrosequencing were inversely proportional to the measured enzymatic activity. The present results indicate that the quantification of MGMT methylation by bisulfite pyrosequencing represents its enzymatic activity and thus, its therapeutic responsiveness to alkylating agents.
DNA修复蛋白O(6)-甲基鸟嘌呤-DNA甲基转移酶（MGMT，AGT）是肿瘤细胞对靶向鸟嘌呤O(6)位的烷化剂抗癌药物耐药性的决定因素。肿瘤中的MGMT启动子甲基化被认为是胶质母细胞瘤对烷化剂反应性的最常见预测指标。然而，MGMT启动子甲基化状态主要通过甲基化特异性PCR进行研究，这是一种定性且主观的检测方法。此外，与MGMT甲基化状态相关的实际酶活性尚未得到探索。在本研究中，通过亚硫酸氢盐焦磷酸测序对胶质母细胞瘤中的MGMT启动子甲基化进行定量，并使用一种研究MGMT功能活性的新型定量检测方法确定其与酶活性的相关性。使用含有MGMT特异性DNA损伤的荧光标记寡核苷酸底物和毛细管电泳来检测和定量这些损伤，从而评估MGMT酶活性。与现有的传统检测方法相比，该检测方法同样灵敏，但耗时更少且更易于操作。通过亚硫酸氢盐焦磷酸测序对41例胶质母细胞瘤中的MGMT启动子甲基化进行评估，并选择5个具有不同值的样本与酶活性检测进行比较。使用设计用于在MGMT上游启动子区域工作的引物进行亚硫酸氢盐焦磷酸测序，在三次重复测量中显示出高定量能力和可重复性。在比较研究中，通过亚硫酸氢盐焦磷酸测序获得的MGMT启动子甲基化值与测得的酶活性成反比。目前的结果表明，通过亚硫酸氢盐焦磷酸测序对MGMT甲基化进行定量代表了其酶活性，进而代表了其对烷化剂的治疗反应性。
