Suzuki M, Sugimoto Y, Tsukahara S, Okochi E, Gottesman M M, Tsuruo T
Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 170, Japan.
Clin Cancer Res. 1997 Jun;3(6):947-54.
Drug resistance genes can protect normal hematopoietic cells from the toxicity of anticancer agents. Because chemotherapeutic agents are often used in combination in current clinical protocols, coexpression of two different drug resistance genes should be useful in protecting normal bone marrow cells from the hematotoxicities caused by combination chemotherapy. In this study, we have combined the human multidrug resistance gene (MDR1) and human O6-methylguanine DNA methyltransferase (MGMT) gene as drug resistance genes. For the coexpression of two drug resistance genes, we have constructed two bicistronic retrovirus vectors. One vector is Ha-MDR-IRES-MGMT, in which translation of the MDR1 cDNA is cap-dependent and MGMT translation is dependent on an internal ribosome entry site (IRES). The other is Ha-MGMT-IRES-MDR, which has cap-dependent MGMT translation and IRES-dependent MDR1 translation. MGMT-negative HeLa derivative (MR) cells transduced with these retroviruses showed resistance to vincristine (from MDR1) and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosou rea (ACN; from MGMT). Cells transduced with Ha-MDR-IRES-MGMT showed higher resistance to vincristine and lower resistance to ACNU than those transduced with Ha-MGMT-IRES-MDR. In any case, the resistance levels of cells transduced with either vector were high enough to select transduced cells with vincristine or ACNU. The expression levels of P-glycoprotein or MGMT in the transduced cells determined by FACS and Western blot analysis correlated well with the extent of resistance to vincristine and ACNU, respectively. All of the MGMT-transduced cells expressed higher amounts of MGMT than the MGMT-expressing parental cell line HeLa S3. Murine bone marrow cells transduced with Ha-MDR-IRES-MGMT and selected with vincristine also showed simultaneous resistance to vincristine and ACNU. These results suggest that bicistronic retroviral vectors allow the functional coexpression of two different types of drug resistance genes. This strategy could be applicable to any combination of drug resistance genes.
耐药基因可保护正常造血细胞免受抗癌药物的毒性作用。由于目前临床方案中常常联合使用化疗药物，两种不同耐药基因的共表达对于保护正常骨髓细胞免受联合化疗所致的血液毒性应是有用的。在本研究中，我们将人多药耐药基因（MDR1）和人O6-甲基鸟嘌呤DNA甲基转移酶（MGMT）基因作为耐药基因进行了组合。为实现两种耐药基因的共表达，我们构建了两种双顺反子逆转录病毒载体。一种载体是Ha-MDR-IRES-MGMT，其中MDR1 cDNA的翻译依赖于帽子结构，而MGMT的翻译依赖于内部核糖体进入位点（IRES）。另一种是Ha-MGMT-IRES-MDR，其MGMT的翻译依赖于帽子结构，而MDR1的翻译依赖于IRES。用这些逆转录病毒转导的MGMT阴性的HeLa衍生细胞（MR）对长春新碱（来自MDR1）和1-(4-氨基-2-甲基-5-嘧啶基)甲基-3-(2-氯乙基)-3-亚硝基脲（ACNU；来自MGMT）显示出耐药性。与用Ha-MGMT-IRES-MDR转导的细胞相比，用Ha-MDR-IRES-MGMT转导的细胞对长春新碱表现出更高的耐药性，而对ACNU的耐药性较低。无论如何，用任一载体转导的细胞的耐药水平都足以用长春新碱或ACNU筛选出转导细胞。通过流式细胞术（FACS）和蛋白质免疫印迹分析确定的转导细胞中P-糖蛋白或MGMT的表达水平分别与对长春新碱和ACNU的耐药程度密切相关。所有转导了MGMT的细胞均比表达MGMT的亲代细胞系HeLa S3表达更高水平的MGMT。用Ha-MDR-IRES-MGMT转导并用长春新碱筛选的小鼠骨髓细胞也对长春新碱和ACNU同时表现出耐药性。这些结果表明，双顺反子逆转录病毒载体可实现两种不同类型耐药基因的功能性共表达。该策略可应用于任何耐药基因组合。
