Domin B A, Grill S P, Cheng Y
Cancer Res. 1983 May;43(5):2155-8.
A series of methotrexate (MTX)-resistant human sublines developed by step increases in selected MTX concentrations have been cloned and examined for dihydrofolate reductase (DHFR) content, relative DNA copy number, and sensitivity to MTX. These cloned sublines had increased DHFR levels which were dependent on the presence of MTX in the medium. The increased levels of DHFR in the absence of MTX were stable in all the clones examined for over a year. Antibody immunolocalization on Western blots showed good correlation of the intensity of the immunostained DHFR band with enzyme activities. Relative gene copy number in these sublines was low relative to the DHFR increases and was not dependent on the presence of MTX in the medium. The increase in gene copy number in these sublines did not correlate with either the levels of DHFR or the sensitivity to MTX.
通过逐步提高甲氨蝶呤（MTX）的选定浓度培养出一系列对MTX耐药的人亚系，并对其进行克隆，检测二氢叶酸还原酶（DHFR）含量、相对DNA拷贝数以及对MTX的敏感性。这些克隆亚系的DHFR水平升高，这取决于培养基中MTX的存在。在所有检测的克隆中，无MTX时升高的DHFR水平在一年多的时间里保持稳定。蛋白质免疫印迹上的抗体免疫定位显示，免疫染色的DHFR条带强度与酶活性具有良好的相关性。相对于DHFR的增加，这些亚系中的相对基因拷贝数较低，且不依赖于培养基中MTX的存在。这些亚系中基因拷贝数的增加与DHFR水平或对MTX的敏感性均无关联。
