Intracellular localization and intercellular heterogeneity of the human DNA repair protein O-6 methylguanine-DNA methyltransferase
Intracellular localization and intercellular heterogeneity of the human DNA repair protein O-6 methylguanine-DNA methyltransferase
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DOI：
10.1007/s002800050427
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发表时间：
1996-04-01
影响因子：
3
通讯作者：
Yarosh, DB
中科院分区：
文献类型：
--
作者：
Belanich, M;Randall, T;Yarosh, DB
O-6-甲基鸟嘌呤-DNA 甲基转移酶 (MGMT) 是一种 DNA 修复蛋白，可从 DNA 中去除烷基加合物，可能在肿瘤对烷基化化疗的抵抗中发挥重要作用。使用针对 MGMT 的单克隆抗体和免疫荧光显微镜观察人体细胞和肿瘤组织中的 MGMT，并通过数字图像分析对荧光信号进行量化。 MGMT 在细胞质和细胞核中都有发现，在任一部位，该蛋白都会与烷基化 DNA 碱基发生反应，然后失活并从细胞中丢失。培养物和异种移植物中的细胞系显示核 MGMT 水平呈广泛的正态分布，但人脑肿瘤通常呈偏态分布，其中很大一部分细胞具有高水平 MGMT。 O-6-苄基鸟嘌呤是 MGMT 活性的自杀底物灭活剂，可将人类细胞和小鼠异种移植物中的 MGMT 降低至治疗后 1 小时抗体测定无法检测到的水平。在替莫唑胺治疗 3 小时后从患者身上采集的黑色素瘤标本中，MGMT 水平降低了 70%。这种定量免疫荧光测定可用于监测人类肿瘤中的 MGMT 及其消耗，以改善烷化剂在癌症化疗中的使用。
O-6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl adducts from DNA and may be important in tumor resistance to alkylation chemotherapy. MGMT was visualized in human cells and tumor tissues with monoclonal antibodies against MGMT and immunofluorescence microscopy, and fluorescent signals were quantified by digital image analysis. MGMT was found both in the cytoplasm and the nucleus, and in either locale the protein reacts with alkylated DNA bases and becomes inactivated and lost from the cell. Cell lines in culture and xenografts showed a broad normal distribution of nuclear MGMT levels, but human brain tumors often showed a skewed distribution, with a significant fraction of cells with high levels of MGMT. O-6-Benzylguanine, a suicide substrate inactivator for MGMT activity, reduced MGMT in human cells and in a mouse xenograft to levels undetectable by antibody assay 1 h post-treatment. In melanoma specimens taken from a patient 3 h post-treatment with temozolomide, MGMT levels were reduced by 70%. This quantitative immunofluorescence assay can be used to monitor MGMT and its depletion in human tumors to improve the use of alkylating agents in cancer chemotherapy.