Christmann M, Pick M, Lage H, Schadendorf D, Kaina B
Division of Applied Toxicology, Institute of Toxicology, University of Mainz, Germany.
Int J Cancer. 2001 Apr 1;92(1):123-9.
Acquired resistance to antineoplastic agents is a frequent obstacle in tumor therapy. Malignant melanoma cells are particularly well known for their unresponsiveness to chemotherapy; only about 30% of tumors exhibit a transient clinical response to treatment. In our study, we investigated the molecular mechanism of acquired resistance of melanoma cells (MeWo) to the chloroethylating drug fotemustine. Determination of O(6)-methylguanine-DNA methyltransferase (MGMT) activity showed that MeWo cells that acquired resistance to fotemustine upon repeated treatment with the drug display high MGMT activity, whereas the parental cell line had no detectable MGMT. The resistant cell lines exhibit cross-resistance to other O(6)-alkylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine. Acquired resistance to fotemustine was alleviated by treatment with the MGMT inhibitor O(6)-benzylguanine demonstrating that reactivation of MGMT is the main underlying cause of acquired alkylating drug resistance. As compared with control cells, both MGMT mRNA and MGMT protein were expressed at a high level in fotemustine resistant cells. Southern blot analysis proved that the MGMT gene was not amplified. There was also only an insignificant difference in the CpG methylation pattern of the MGMT promoter whereas a clear hypermethylation in the body of the gene was observed in fotemustine resistant cells. The conclusion that hypermethylation is responsible for reactivation of the MGMT gene gained support by the finding that MGMT activity significantly declined and cells reverted (partially) to the parental sensitive phenotype upon treatment with 5-azacytidine. This is the first report of acquired resistance to a chloroethylating antineoplastic drug of melanoma cells due to gene hypermethylation.
获得性抗肿瘤药物耐药性是肿瘤治疗中常见的障碍。恶性黑色素瘤细胞对化疗无反应尤为出名；只有约30%的肿瘤对治疗表现出短暂的临床反应。在我们的研究中，我们调查了黑色素瘤细胞（MeWo）对氯乙化药物福莫司汀获得性耐药的分子机制。O(6)-甲基鸟嘌呤-DNA甲基转移酶（MGMT）活性测定表明，经该药物反复处理后对福莫司汀产生耐药性的MeWo细胞表现出高MGMT活性，而亲本细胞系未检测到MGMT。耐药细胞系对其他O(6)-烷基化剂，如N-甲基-N'-硝基-N-亚硝基胍表现出交叉耐药性。用MGMT抑制剂O(6)-苄基鸟嘌呤处理可减轻对福莫司汀的获得性耐药，这表明MGMT的重新激活是获得性烷基化药物耐药的主要潜在原因。与对照细胞相比，MGMT mRNA和MGMT蛋白在福莫司汀耐药细胞中均高水平表达。Southern印迹分析证明MGMT基因未扩增。MGMT启动子的CpG甲基化模式也只有微小差异，而在福莫司汀耐药细胞中观察到该基因主体明显的高甲基化。用5-氮杂胞苷处理后MGMT活性显著下降且细胞（部分）恢复为亲本敏感表型，这一发现支持了高甲基化导致MGMT基因重新激活的结论。这是关于黑色素瘤细胞因基因高甲基化而对氯乙化抗肿瘤药物获得性耐药的首次报道。
