Wang H, Jiang Z g, Wong Y W, Dalton W S, Futscher B W, Chan V T
Department of Obstetrics and Gynecology, The Ohio State University College of Medicine and James Cancer Hospital and Research Institute, Columbus, Ohio, 43210, USA.
Biochem Biophys Res Commun. 1997 Aug 18;237(2):217-24. doi: 10.1006/bbrc.1997.7115.
Decreased topoisomerase II (Topo II) activity results in resistance to antineoplastic agents targeting this enzyme. Dox1V derived from human multiple myeloma RPMI 8226 demonstrated a 4-fold resistance to doxorubicin in the absence of MDR1 overexpression or topo II mutations (Futscher B.W., Foley N., Gleason-Guzman M., Meltzer P.S., Sullivan D.M., and Dalton W.S., Int'l. J. Cancer, 66: 520-5, 1996.). Consistent with its drug resistant phenotype, a 2- to 3-fold decrease in topo II expression was identified. To investigate the molecular basis for decreased topo II expression in Dox1V, a semi-quantitative analysis of Topo II activity, protein level and mRNA transcript were performed. The results demonstrated that reduced Topo II activity is due to a decreased mRNA level. Southern blot and sequencing experiments revealed wild-type sequence of the topo II promoter in the drug resistant cells. Transient gene expression assays demonstrated that topo II is transcriptionally down-regulated in Dox1V independent of the promoter sequence of the endogenous alleles. Instead, the activity of a ubiquitous transcription factor CP-1 (NF-Y) interacting with the topo II promoter is decreased. The decrease in CP-1/NF-Y activity in Dox1V is correlated well with the decrease in topo II transcriptional activity, transcript level, Topo II protein and enzyme activity. Therefore, transcriptional down-regulation resulted from a reduced CP-1/NF-Y activity is responsible for decreased topo II expression in Dox1V cells.
拓扑异构酶II（Topo II）活性降低会导致对靶向该酶的抗肿瘤药物产生耐药性。源自人多发性骨髓瘤RPMI 8226的Dox1V在未发生多药耐药基因1（MDR1）过表达或拓扑异构酶II突变的情况下，对阿霉素表现出4倍的耐药性（Futscher B.W., Foley N., Gleason-Guzman M., Meltzer P.S., Sullivan D.M., and Dalton W.S., 《国际癌症杂志》，66: 520 - 525, 1996年）。与其耐药表型一致，拓扑异构酶II的表达降低了2至3倍。为了研究Dox1V中拓扑异构酶II表达降低的分子基础，对拓扑异构酶II的活性、蛋白质水平和mRNA转录本进行了半定量分析。结果表明，拓扑异构酶II活性降低是由于mRNA水平下降所致。Southern印迹和测序实验揭示了耐药细胞中拓扑异构酶II启动子的野生型序列。瞬时基因表达分析表明，在Dox1V中拓扑异构酶II的转录下调与内源性等位基因的启动子序列无关。相反，与拓扑异构酶II启动子相互作用的普遍存在的转录因子CP - 1（核因子Y，NF - Y）的活性降低。Dox1V中CP - 1/NF - Y活性的降低与拓扑异构酶II转录活性、转录本水平、拓扑异构酶II蛋白和酶活性的降低密切相关。因此，CP - 1/NF - Y活性降低导致的转录下调是Dox1V细胞中拓扑异构酶II表达降低的原因。
