Mini E, Srimatkandada S, Medina W D, Moroson B A, Carman M D, Bertino J R
Cancer Res. 1985 Jan;45(1):317-24.
A methotrexate-resistant subline, CCRF-CEM/R1, was selected stepwise from the human leukemic lymphoblast T-cell line, CCRF-CEM, and maintained in 0.2 microM methotrexate. The development of resistance to methotrexate (75-fold) was associated with a 20-fold increase of dihydrofolate reductase activity. The affinity of dihydrofolate reductase from the resistant cells for methotrexate did not vary significantly as compared to the enzyme from the parent cells. Southern blot analysis of DNA from parent and CCRF-CEM/R1 cells demonstrated amplification of the dihydrofolate reductase gene in the resistant line. Quantitative dot-blot DNA hybridization demonstrated the presence of about 18 reductase gene copies in the R1 cells. The human dihydrofolate reductase gene contained at least 4 intervening sequences and was about 30 kilobases in size. Northern blot analysis demonstrated an increase in dihydrofolate reductase messenger RNA species, the predominant message was 3.8 kilobases. Cytogenetic analysis of CCRF-CEM/R1 cells revealed an elongated marker chromosome containing a homogeneous staining region not present in the parent line. This chromosome appeared to be derived from chromosome 21.
从人白血病淋巴母细胞T细胞系CCRF-CEM中逐步筛选出甲氨蝶呤耐药亚系CCRF-CEM/R1，并在0.2微摩尔甲氨蝶呤中维持培养。对甲氨蝶呤耐药性的产生（75倍）与二氢叶酸还原酶活性增加20倍相关。与亲本细胞的酶相比，耐药细胞中二氢叶酸还原酶对甲氨蝶呤的亲和力没有显著变化。对亲本细胞和CCRF-CEM/R1细胞的DNA进行Southern印迹分析表明，耐药系中二氢叶酸还原酶基因发生了扩增。定量点杂交DNA分析表明，R1细胞中存在约18个还原酶基因拷贝。人二氢叶酸还原酶基因至少含有4个间隔序列，大小约为30千碱基。Northern印迹分析表明二氢叶酸还原酶信使RNA种类增加，主要信使RNA为3.8千碱基。对CCRF-CEM/R1细胞的细胞遗传学分析显示，有一条伸长的标记染色体，其上含有亲本细胞系中不存在的均匀染色区。这条染色体似乎来源于21号染色体。
