Development of gene therapy against drug-resistant brain tumors using nitrosourea-resistant rat brain-tumor model

氯乙基氢脲(CENU)是目前治疗脑肿瘤最有效的化疗药物之一。然而，获得性耐药已成为脑肿瘤患者治疗中面临的严峻问题。耐药的主要机制是06‘甲基鸟嘌呤DNA甲基转移酶(MGMT)在肿瘤细胞中的募集。以前，我们通过逆转录病毒转导MGMT CDNA进入9L大鼠脑肿瘤细胞，建立了亚硝脲耐药的动物模型(抗癌研究19：5313-5318,1999)。在这项研究中，我们利用这个模型评估了反义序列和核酶转导的有效性。反义序列的逆转录病毒或质粒转导不能增强L-(4-氨基-2-甲基‘5-嘧啶)甲基-3-(2-氯乙基)-3j亚硝脲(ACNU)对脑肿瘤耐药细胞的敏感性。为了提高这种方法的效率，构建了针对MGMT基因的反义序列或核酶的腺病毒载体，然后用这些病毒感染表达MGMT的胶质瘤细胞并测定其敏感性。腺病毒转导反义RNA和核酶在体外下调MGMT的转录和表达。在体外和体内对亚硝脲也有明显的敏感性。然而，这种影响微乎其微。这些数据表明，MGMJT的不完全耗竭不足以克服耐药性，要完全逆转耐药，需要进行额外的优化。

ChloroethyHtorourea (CENU) is one of the most potent chemotherapeutic agents for brain tumors. However, acquired resistance to the drug has become aserious problem in the treatment of brain tumor patients. The main mechanism of the resistance is a recruitment of the 06'methylguanine DNA methyltransferase (MGMT) in tumor cells. Previously, we established an animal model resistant to nitrosourea (Anticancer Research 19: 5313-5318, 1999) by retroviral transduction of MGMT CDNA into 9L rat brain tumor cells. In this study, we evaluate the efficacies of antisense sequences and ribozyme transduction by plasmid, retroviral, or adenoviral vector utilizing this model. Retroviral or plasmid DNA transduction of an antisense sequence did not confer the sensitivity of l-(4-amino-2-methyl'5-pyrimidinyl) methyll-3-(2-chloroethyl)-3jnitrosourea (ACNU) to the resistant brain tumor cells. In order to increase the potency of this approach, adenoviral vectors encodingantisense sequences or ribozyme to MGMT mRNA were constructed, then MGMT-expressing glioma cells were infected with these viruses and sensitivities were quantified. The adenoviral transfer of antisense RNA and ribozyme down-regulated the transcription and expression of MGMT in vitro. It also conferred significant sensitivity to nitrosourea in vitro and in vivo. However, theeffect was minimal. These data suggest that incomplete depletion of MGMJT is not sufficient to overcome the resistance and that additional optimization will be fequired for the complete reversion of drug resistance.