Versantvoort C H, Withoff S, Broxterman H J, Kuiper C M, Scheper R J, Mulder N H, de Vries E G
Department of Medical Oncology, Free University Hospital, Amsterdam, The Netherlands.
Int J Cancer. 1995 May 4;61(3):375-80. doi: 10.1002/ijc.2910610317.
Previous studies have shown that the in vitro-selected adriamycin-resistant human small-cell lung-carcinoma cell line GLC4-ADR150 displays multidrug resistance as the result of 3-fold decreased DNA-topoisomerase II (topo II) activity and a 6-fold reduction in adriamycin accumulation. Not the MDR1 gene, but the MRP gene, was over-expressed in this cell line. The aim of our study was to establish which of these drug-resistance-associated factors are already involved in drug resistance occurring at early steps of selection with adriamycin. To address this question, changes in expression of topo II alpha/topo II beta, MRP and drug accumulation were measured along with adriamycin resistance (from 2- to 10- to 150-fold) and in a partial revertant cell line (10-fold resistant). Topo II alpha and II beta mRNA and protein levels were decreased in the resistant sub-lines, except in the 10-fold-resistant cell line. Cellular daunorubicin accumulation was decreased 1.2- to 5-fold with increasing resistance. MRP mRNA was over-expressed in all resistant sub-lines, with a marked increase in the 10-fold-resistant cells (level of expression as high as in the GLC4-ADR150 cells). Expression of an ATP-binding 190-kDa membrane protein and Western-blot analysis with anti-MRP anti-serum ASPKE, was in accordance with the expression of MRP mRNA in all cell lines. Expression of MRP mRNA and protein, however, was not proportional with the decrease in drug accumulation in all resistant sub-lines. This study also shows that drug accumulation, topo II and MRP expression were all changed at the earliest stage of resistance development of GLC4 cells upon adriamycin selection.
先前的研究表明，体外筛选出的阿霉素耐药人小细胞肺癌细胞系GLC4 - ADR150表现出多药耐药性，这是DNA拓扑异构酶II（拓扑II）活性降低3倍以及阿霉素蓄积减少6倍的结果。在该细胞系中过表达的不是MDR1基因，而是MRP基因。我们研究的目的是确定这些与耐药相关的因素中哪些已经参与了阿霉素早期筛选过程中出现的耐药性。为解决这个问题，我们测定了拓扑IIα/拓扑IIβ、MRP的表达变化以及药物蓄积情况，并与阿霉素耐药性（从2倍到10倍再到150倍）以及一个部分回复细胞系（10倍耐药）进行了比较。除了10倍耐药细胞系外，耐药亚系中拓扑IIα和IIβ的mRNA及蛋白水平均降低。随着耐药性增加，细胞柔红霉素蓄积减少了1.2至5倍。MRP mRNA在所有耐药亚系中均过表达，在10倍耐药细胞中显著增加（表达水平与GLC4 - ADR150细胞一样高）。一种190 kDa的ATP结合膜蛋白的表达以及用抗MRP抗血清ASPKE进行的蛋白质印迹分析与所有细胞系中MRP mRNA的表达一致。然而，在所有耐药亚系中，MRP mRNA和蛋白的表达与药物蓄积的减少并不成比例。这项研究还表明，在阿霉素筛选GLC4细胞耐药性发展的最早阶段，药物蓄积、拓扑II和MRP表达均发生了变化。
