Cai Shanbao, Xu Yi, Cooper Ryan J, Ferkowicz Michael J, Hartwell Jennifer R, Pollok Karen E, Kelley Mark R
Section of Hematology/Oncology, Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, 1044 West Walnut, R4-302C, Indianapolis, IN 46202, USA.
Cancer Res. 2005 Apr 15;65(8):3319-27. doi: 10.1158/0008-5472.CAN-04-3335.
DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore, overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria, committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU, TMZ, and MMS, which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy.
近年来，真核细胞的DNA修复能力得到了广泛研究。哺乳动物细胞已被改造以从异位基因过表达重组核DNA修复蛋白，以评估DNA修复能力增强对基因组稳定性的影响。本研究采用这种方法将O(6)-甲基鸟嘌呤DNA甲基转移酶（MGMT）特异性靶向线粒体，并研究其对暴露于DNA烷化剂后细胞存活的影响。对人类造血细胞系和原代造血CD34(+)定向祖细胞的存活情况进行了监测，因为由于MGMT表达不足，烷基化诱导的DNA损伤的基线修复能力通常较低。当K562细胞用核靶向MGMT（nucl-MGMT）或线粒体靶向MGMT（mito-MGMT）转染时，观察到DNA修复能力增强。此外，mito-MGMT的过表达比nucl-MGMT的过表达对1,3-双（2-氯乙基）-1-亚硝基脲（BCNU）诱导的细胞杀伤具有更强的抗性。mito-MGMT和nucl-MGMT的同时过表达并没有增强单独的mito-MGMT所提供的抗性。mito-MGMT或nucl-MGMT的过表达对甲磺酸甲酯（MMS）和替莫唑胺（TMZ）也赋予了相似水平的抗性，但在两个细胞区室中的同时过表达既没有相加作用也没有协同作用。当人类CD34(+)细胞用将O(6)-苄基鸟嘌呤（6BG）抗性MGMT（MGMT(P140K)）靶向细胞核或线粒体的致癌逆转录病毒载体感染时，来自感染细胞的定向祖细胞对6BG/BCNU或6BG/TMZ具有抗性。这些研究表明，MGMT的线粒体或核靶向可保护造血细胞免受BCNU、TMZ和MMS诱导的细胞杀伤，这与线粒体DNA损伤和核DNA损伤在化疗期间对烷化剂诱导的细胞杀伤贡献相等的可能性一致。
