Matsumoto Y, Takano H, Fojo T
Division of Clinical Sciences, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Cancer Res. 1997 Nov 15;57(22):5086-92.
The efficacy of all chemotherapeutic agents is limited by the occurrence of drug resistance. For etoposide (VP-16), increased expression of MDR-1 or MRP and alterations in topoisomerase IIalpha have been shown to confer tolerance. To further understand resistance to VP-16, three sublines, designated MCF-7-VP17, ZR-75B-VP13, and MDA-MB-231-VP7, were initially isolated as single clones from parental cells by exposure to VP-16. Subsequently, a population of cells from each subline was exposed to 3-fold higher drug concentrations, allowing stable sublines to be established at higher extracellular drug concentrations. Characterization of the resistant sublines demonstrates the adaptation that occurs with advancing drug concentrations during in vitro selections. Reduced topoisomerase II mRNA and protein levels were observed in the initial isolates. This reduction was accompanied by a decrease in topoisomerase II activity and cellular growth rate and was associated with 6-314-fold resistance to topoisomerase II poisons. With advancing resistance, MRP expression increased and VP-16 accumulation decreased. This adaptation allowed for partial restoration of topoisomerase II activity as a result of increased expression (MCF-7-VP17 and ZR-75B-VP13) or hyperphosphorylation (MDA-MB-231-VP7), with a resultant increase in growth rate. In MDA-MB-231-VP7 cells, hyperphosphorylation coincided with increased casein kinase II mRNA and protein levels, suggesting a role for this kinase in the acquired hyperphosphorylation. In this cell line, hyperphosphorylation mediated the increased activity despite a fall in topoisomerase IIalpha protein levels secondary to an acquired 600-bp deletion in one topoisomerase IIalpha allele, which resulted in reduced protein levels. In all three sublines, high levels of resistance were attained as a result of synergism between the reduced topoisomerase IIalpha levels and MRP overexpression. These studies demonstrate how cellular adaptation to increasing drug pressure occurs and how more than one mechanism can contribute to the resistant phenotype when increasing selecting pressure is applied. Reduced expression of topoisomerase II is sufficient to confer substantial resistance early in the selection process, with synergy from MRP overexpression helping to confer high levels of resistance.
所有化疗药物的疗效都受到耐药性出现的限制。对于依托泊苷（VP - 16），多药耐药蛋白1（MDR - 1）或多药耐药相关蛋白（MRP）的表达增加以及拓扑异构酶IIα的改变已被证明可赋予耐受性。为了进一步了解对VP - 16的耐药性，最初通过暴露于VP - 16从亲代细胞中分离出三个亚系，分别命名为MCF - 7 - VP17、ZR - 75B - VP13和MDA - MB - 231 - VP7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稳定的亚系。耐药亚系的特征表明在体外选择过程中随着药物浓度的增加会发生适应性变化。在最初分离出的细胞中观察到拓扑异构酶II的mRNA和蛋白质水平降低。这种降低伴随着拓扑异构酶II活性和细胞生长速率的下降，并且与对拓扑异构酶II抑制剂产生6 - 314倍的耐药性相关。随着耐药性增加，MRP表达增加而VP - 16积累减少。这种适应性变化由于表达增加（MCF - 7 - VP17和ZR - 75B - VP13）或过度磷酸化（MDA - MB - 231 - VP7）而使拓扑异构酶II活性部分恢复，从而导致生长速率增加。在MDA - MB - 231 - VP7细胞中，过度磷酸化与酪蛋白激酶II的mRNA和蛋白质水平增加同时出现，表明该激酶在获得性过度磷酸化中起作用。在该细胞系中，尽管由于一个拓扑异构酶IIα等位基因中获得性600碱基对缺失导致拓扑异构酶IIα蛋白水平下降，但过度磷酸化介导了活性增加，这导致蛋白质水平降低。在所有三个亚系中，由于拓扑异构酶IIα水平降低和MRP过表达之间的协同作用而获得了高水平的耐药性。这些研究表明细胞如何适应不断增加的药物压力，以及当施加越来越高的选择压力时，不止一种机制如何导致耐药表型。拓扑异构酶II表达降低足以在选择过程早期赋予显著的耐药性，MRP过表达的协同作用有助于赋予高水平的耐药性。
