Dumenco L L, Warman B, Hatzoglou M, Lim I K, Abboud S L, Gerson S L
R.L. Ireland Cancer Center,Case Western Reserve University School of Medicine, Cleveland, Ohio.
Cancer Res. 1989 Nov 1;49(21):6044-51.
Maloney murine leukemia virus-based, replication-defective retroviral vectors containing the neomycin resistance gene (neo) were developed to transfer the Escherichia coli ada gene coding for O6-alkylguanine-DNA alkyltransferase, into mammalian cells. To optimize gene transfer and expression, the following promoters were linked to ada: the Maloney murine leukemia virus promoter within the long-terminal repeat, the Rous sarcoma virus promoter, the thymidine kinase promoter, or the human phosphoglycerate kinase promoter. Sequences were transfected into the helper virus-free retroviral packaging psi-2 cell line. Recombinant retroviruses were tested in CCL-1 cells, which, like most murine tissues, have low levels of alkyltransferase and are sensitive to 1,3-bis(2-chloroethyl)nitrosourea (BCNU), and in NIH-3T3 cells, which are BCNU resistant and have high levels of alkyltransferase. Lines infected with each of the four retroviruses were selected for neo expression and found to have intact proviral integration and ada gene expression. Alkyltransferase activity was greatest with retrovirus containing the Rous sarcoma virus-ada gene; infected NIH-3T3 cells had up to 2300 units of alkyltransferase/mg of protein compared with 151 units/mg of protein in control cells, and infected CCL-1 cells had up to 1231 units/mg of protein compared with 33 units/mg of protein in control cells. CCL-1 cells expressing ada were more resistant to BCNU cytotoxicity than were controls. However, NIH-3T3 cells expressing ada were only slightly more resistant to BCNU than controls, possibly because most of the ada protein was cytoplasmic rather than nuclear as suggested by immunohistochemical stain. These studies establish a series of retroviruses containing the bacterial ada gene, which efficiently infect mammalian cells. ada expression increases nitrosourea resistance in cells with low mammalian alkyltransferase activity.
构建了基于莫洛尼鼠白血病病毒的、复制缺陷型逆转录病毒载体，其含有新霉素抗性基因（neo），用于将编码O6-烷基鸟嘌呤-DNA烷基转移酶的大肠杆菌ada基因转移至哺乳动物细胞。为优化基因转移和表达，将以下启动子与ada连接：长末端重复序列内的莫洛尼鼠白血病病毒启动子、劳氏肉瘤病毒启动子、胸苷激酶启动子或人磷酸甘油酸激酶启动子。将这些序列转染至无辅助病毒的逆转录病毒包装psi-2细胞系。在CCL-1细胞（与大多数鼠组织一样，其烷基转移酶水平低且对1,3-双（2-氯乙基）亚硝基脲（BCNU）敏感）和NIH-3T3细胞（对BCNU有抗性且烷基转移酶水平高）中测试重组逆转录病毒。选择感染了四种逆转录病毒中每一种的细胞系用于neo表达，发现其具有完整的前病毒整合和ada基因表达。含劳氏肉瘤病毒-ada基因的逆转录病毒产生 的烷基转移酶活性最高；感染的NIH-3T3细胞每毫克蛋白质的烷基转移酶活性高达2300单位，而对照细胞为151单位/毫克蛋白质；感染的CCL-1细胞每毫克蛋白质的烷基转移酶活性高达1231单位，而对照细胞为33单位/毫克蛋白质。表达ada的CCL-1细胞比对照细胞对BCNU细胞毒性更具抗性。然而，表达ada的NIH-3T3细胞对BCNU的抗性仅略高于对照细胞，这可能是因为免疫组织化学染色显示大多数ada蛋白位于细胞质而非细胞核中。这些研究建立了一系列含有细菌ada基因的逆转录病毒，其能有效感染哺乳动物细胞。ada表达可增加哺乳动物烷基转移酶活性低的细胞对亚硝基脲的抗性。
