Matsumoto Y, Morisaki K, Kunishio K, Nagao S, Takano H, Fojo T
Dept. of Neurological Surgery, Kagawa Medical University.
Gan To Kagaku Ryoho. 1998 Jul;25(8):1145-9.
The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance. To further understand resistance to topoisomerase (topo) II inhibitors, 50 sublines were isolated as single clones from parental cells by exposure to ETP or m-AMSA. Subsequently, a population of cells from each subline was exposed to three-fold higher drug concentrations allowing 16 stable sublines to be established at higher extracellular drug concentration. The frequency and nature of mutations in topo II in the drug selected cell lines have been evaluated. In order to screen a large number of cell lines, an RNase protection assay was developed. Fragments covering the entire coding sequence of topo II was isolated after PCR amplification and subcloned in pGEM3Z vector. Using this approach, mismatches was observed in 13.6% of resistant cell lines (12% of resistant cell lines exposed to lower drug concentrations and 18.8% of resistant cell lines exposed to higher drug concentrations). Our findings suggest that mutations of topo II gene seem to be an important and frequent mechanism of resistance to topo II inhibitors.
所有化疗药物的疗效都受到耐药性出现的限制。为了进一步了解对拓扑异构酶（topo）II抑制剂的耐药性，通过暴露于依托泊苷（ETP）或米托蒽醌（m-AMSA）从亲代细胞中分离出50个亚系作为单克隆。随后，将每个亚系的细胞群体暴露于高三倍的药物浓度下，从而在更高的细胞外药物浓度下建立了16个稳定的亚系。已评估了药物选择细胞系中topo II突变的频率和性质。为了筛选大量细胞系，开发了一种核糖核酸酶保护试验。在PCR扩增后分离出覆盖topo II整个编码序列的片段，并亚克隆到pGEM3Z载体中。使用这种方法，在13.6%的耐药细胞系中观察到错配（12%暴露于较低药物浓度的耐药细胞系和18.8%暴露于较高药物浓度的耐药细胞系）。我们的数据表明，topo II基因突变似乎是对topo II抑制剂耐药的一种重要且常见的机制。
