Fraser D J, Brandt T L, Kroll D J
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver 80262, USA.
Mol Pharmacol. 1995 Apr;47(4):696-706.
The cytotoxic efficacy of antitumor drugs targeted at DNA topoisomerase II (topo II) in many cases varies in direct proportion to cellular topo II content. To investigate the transcriptional control of the predominant alpha form of topo II, the 5' flanking region of the human topo II alpha gene (positions -562 to +90) was subcloned into a firefly luciferase reporter plasmid and transiently transfected into HL-60 human leukemia cells, a line capable of monocytic differentiation after treatment with various agents. Early in phorbol-12-myristate-13-acetate (30 nM)-induced differentiation (18-24 hr after treatment), an unexpected 3-5-fold activation of topo II alpha gene promoter activity was observed. Activation was observed in HL-60 cells and U-937 cells, but not in HeLa human cervical carcinoma cells. Sodium butyrate (NaB) (0.4 mM) also led to activation (4-17-fold) of the topo II alpha promoter in HL-60 and U-937 cells. Promoter sequences between position -90 and position +90 mediated the inducing effects of NaB. This NaB-dependent promoter-reporter induction was partly mirrored by a transient approximately 2-fold increase in endogenous topo II alpha enzyme. The stimulus for promoter activation could be partly attributed to a 2-fold increase in DNA synthesis at 16 hr for NaB, but not phorbol-12-myristate-13-acetate. Regardless of the primary stimulus for topo II alpha promoter trans-activation, it could be bypassed by treatment of HL-60 cells with NaB for 48 hr before transfection, revealing the expected 60-70% suppression of topo II alpha promoter activity. Further study of topo II alpha promoter down-regulation later in monocytic differentiation may serve as a model for elucidating the transcriptional mechanisms that may also be exploited by tumor cells expressing intrinsic or acquired resistance to topo II-directed drugs.
许多情况下，靶向DNA拓扑异构酶II（拓扑II）的抗肿瘤药物的细胞毒性功效与细胞拓扑II含量成正比。为了研究拓扑II主要α形式的转录调控，将人拓扑IIα基因的5'侧翼区域（位置-562至+90）亚克隆到萤火虫荧光素酶报告质粒中，并瞬时转染到HL-60人白血病细胞中，该细胞系在用各种试剂处理后能够进行单核细胞分化。在佛波醇-12-肉豆蔻酸酯-13-乙酸酯（30 nM）诱导分化的早期（处理后18-24小时），观察到拓扑IIα基因启动子活性意外地激活了3-5倍。在HL-60细胞和U-937细胞中观察到激活，但在HeLa人宫颈癌细胞中未观察到。丁酸钠（NaB）（0.4 mM）也导致HL-60和U-937细胞中拓扑IIα启动子激活（4-17倍）。-90位至+90位之间的启动子序列介导了NaB的诱导作用。这种依赖NaB的启动子-报告基因诱导部分反映在内源性拓扑IIα酶瞬时增加约2倍上。启动子激活的刺激部分可归因于NaB在16小时时DNA合成增加2倍，但佛波醇-12-肉豆蔻酸酯-13-乙酸酯则不然。无论拓扑IIα启动子反式激活的主要刺激因素如何，在转染前用NaB处理HL-60细胞48小时可以绕过它，显示出拓扑IIα启动子活性预期的60-70%抑制。在单核细胞分化后期对拓扑IIα启动子下调的进一步研究可能作为阐明转录机制的模型，这些机制也可能被对拓扑II导向药物具有内在或获得性抗性的肿瘤细胞所利用。
