Kinsella A R, Fox M
Department of Biochemical Genetics, Christie Hospital, Manchester, UK.
Int J Cancer. 1988 Jul 15;42(1):87-93. doi: 10.1002/ijc.2910420117.
Three different 3T6 mouse fibroblast cell clones with intrinsically different sensitivities to methotrexate (MTX) have been isolated from an originally heterogeneous population. When these 3 different clones were exposed to MTX in the presence of the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) the degree of enhancement of recovery of methotrexate resistant (MTXR) colonies was greatest in the most sensitive clone. MTXR colonies isolated and cultured in the presence and absence of MTX and TPA were analysed for dihydrofolic acid reductase (dhfr) levels by flow cytometry after binding of fluorescent methotrexate. None of the 58 clones showed major changes in dhfr levels. Dot-blot analysis of 12 clones indicated no increases in dhfr gene copy number or mRNA levels consistent with gene amplification. Southern analysis of 6 further clones indicated that only 1 clone isolated by multi-step selection had amplified dhfr sequences. TPA-enhanced mouse 3T6 N-phosphonacetyl-L-aspartate (PALA)-resistant colony recovery and mouse 3T3 MTXR colony recovery were also shown, by dot-blot analysis, not to be due to gene amplification. The data indicate that TPA can have a profound effect on drug-resistant colony recovery by mechanisms other than induction of gene amplification.
从最初的异质群体中分离出了三种对甲氨蝶呤（MTX）具有本质上不同敏感性的3T6小鼠成纤维细胞克隆。当这三种不同的克隆在肿瘤启动子12-O-十四烷酰佛波醇-13-乙酸酯（TPA）存在的情况下暴露于MTX时，在最敏感的克隆中，甲氨蝶呤抗性（MTXR）集落恢复的增强程度最大。在存在和不存在MTX和TPA的情况下分离并培养的MTXR集落，在荧光甲氨蝶呤结合后通过流式细胞术分析二氢叶酸还原酶（dhfr）水平。58个克隆中没有一个显示dhfr水平有重大变化。对12个克隆的斑点印迹分析表明，dhfr基因拷贝数或与基因扩增一致的mRNA水平没有增加。对另外6个克隆的Southern分析表明，通过多步选择分离的只有1个克隆具有扩增的dhfr序列。斑点印迹分析还表明，TPA增强的小鼠3T6 N-膦酰乙酰-L-天冬氨酸（PALA）抗性集落恢复和小鼠3T3 MTXR集落恢复不是由于基因扩增。数据表明，TPA可以通过基因扩增以外的机制对耐药集落恢复产生深远影响。
