Beketic-Oreskovic L, Durán G E, Chen K G, Dumontet C, Sikic B I
Department of Medicine, Stanford University School of Medicine, CA, USA.
J Natl Cancer Inst. 1995 Nov 1;87(21):1593-602. doi: 10.1093/jnci/87.21.1593.
Various mechanisms can contribute to cellular resistance to doxorubicin. These include expression of the multidrug transporter P-glycoprotein (product of the mdr1 gene [also known as PGY1], Mrp (multidrug resistance-associated protein), the p110 major vault protein, altered glutathione metabolism, and altered levels or activity of topoisomerase II (Topo II). We reported recently that single-step treatment of human MES-SA sarcoma cells with 40 nM doxorubicin resulted in selection of spontaneous mutants at a rate of 1.8 x 10(-6) per cell generation. All individually selected mutants manifested the multidrug-resistant phenotype, related to activation of the mdr1 gene.
Luria and Delbrück fluctuation analysis was performed with MES-SA cells to determine the mutation rate and the nature and mechanisms of resistance after single-step selection with doxorubicin in the presence of the cyclosporin PSC 833, a potent modulator of multidrug resistance.
Ten flasks were seeded with 2000 cells/flask and grown to confluent populations of approximately 8 x 10(6) cells. After reseeding in 96-well plates, the populations were treated with 40 nM doxorubicin and 2 microM PSC 833 for 3 weeks. Surviving colonies were scored, individually harvested, and propagated. The drug-resistant phenotype was assessed by the tetrazolium dye (MTT) cytotoxicity assay and by monitoring cellular glutathione content and radiolabeled drug accumulation. Coupled reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate mdr1, MRP, Topo II alpha, and Topo II beta gene expression. Topo II, P-glycoprotein, and p110 levels were examined by immunoblotting or immunocytochemistry. Topo II activity was assessed by decatenation of kinetoplast DNA, and etoposide-induced cleavable complex formation was studied by the potassium-sodium dodecyl sulfate precipitation assay.
Mutations were detected at a rate of 2.5 x 10(-7) per cell generation. Analysis of variance indicates that spontaneous mutations, rather than changes in cellular function, conferred resistance to doxorubicin and PSC 833. None of the isolated clones expressed mdr1 messenger RNA or P-glycoprotein, and none exhibited an increase in MRP expression. No alterations were found in cellular glutathione content, intracellular accumulations of daunorubicin and etoposide, levels of p110 protein, or levels of Topo II beta transcripts. However, a significant decrease in Topo II alpha messenger RNA and protein was found in all examined clones, as well as decreased Topo II catalytic activity and reduced cleavable complex formation in the presence of etoposide.
PSC 833 co-selection reduced the mutation rate for doxorubicin-selected resistance by 10-fold and suppressed the emergence of mdr1 mutants. Survival of cells exposed to doxorubicin and PSC 833 occurs by selection of spontaneously arising mutants that exhibit altered Topo II alpha expression.
Our results suggest that treatment with multidrug resistance modulators such as PSC 833 together with multidrug resistance-related cytotoxins may suppress the activation of mdr1 and prevent the emergence of resistant cancer cell clones with the multidrug-resistant phenotype.
多种机制可导致细胞对阿霉素产生耐药性。这些机制包括多药转运蛋白P-糖蛋白（mdr1基因[也称为PGY1]的产物）、多药耐药相关蛋白（Mrp）、p110主要穹窿蛋白的表达，谷胱甘肽代谢改变，以及拓扑异构酶II（Topo II）水平或活性改变。我们最近报道，用40 nM阿霉素对人MES-SA肉瘤细胞进行单步处理，导致自发突变体的选择率为每细胞代1.8×10⁻⁶。所有单独选择的突变体均表现出多药耐药表型，与mdr1基因的激活有关。
对MES-SA细胞进行卢里亚-德尔布吕克波动分析，以确定在强力多药耐药调节剂环孢菌素PSC 833存在的情况下，用阿霉素进行单步选择后的突变率以及耐药的性质和机制。
将10个培养瓶中各接种2000个细胞，培养至约8×10⁶个细胞的汇合群体。在重新接种到96孔板后，用40 nM阿霉素和2 μM PSC 833处理这些群体3周。对存活的菌落进行计数，单独收获并传代。通过四唑盐染料（MTT）细胞毒性试验以及监测细胞谷胱甘肽含量和放射性标记药物积累来评估耐药表型。采用逆转录-聚合酶链反应（RT-PCR）评估mdr1、MRP、Topo IIα和Topo IIβ基因表达。通过免疫印迹或免疫细胞化学检测Topo II、P-糖蛋白和p110水平。通过动质体DNA解连环评估Topo II活性，并通过十二烷基硫酸钠钾沉淀试验研究依托泊苷诱导的可裂解复合物形成。
检测到的突变率为每细胞代2.5×10⁻⁷。方差分析表明，自发突变而非细胞功能变化赋予了对阿霉素和PSC 833的耐药性。分离出的克隆均未表达mdr1信使RNA或P-糖蛋白，也没有一个表现出MRP表达增加。在细胞谷胱甘肽含量、柔红霉素和依托泊苷的细胞内积累、p110蛋白水平或Topo IIβ转录本水平方面未发现改变。然而，在所有检测的克隆中均发现Topo IIα信使RNA和蛋白显著减少，同时在依托泊苷存在的情况下Topo II催化活性降低且可裂解复合物形成减少。
PSC 833共选择使阿霉素选择的耐药突变率降低了10倍，并抑制了mdr1突变体的出现。暴露于阿霉素和PSC 833的细胞通过选择自发产生的表现出Topo IIα表达改变的突变体而存活。
我们的结果表明，用PSC 833等多药耐药调节剂与多药耐药相关细胞毒素联合治疗可能会抑制mdr1的激活，并防止出现具有多药耐药表型的耐药癌细胞克隆。
