DNA topoisomerase II expression, stability, and phosphorylation in two VM-26-resistant human leukemic CEM sublines.
DNA topoisomerase II expression, stability, and phosphorylation in two VM-26-resistant human leukemic CEM sublines.
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作者：
M. Chen;W. T. Beck
我们检查了人白血病 CCRF-CEM 细胞和两个替尼泊苷耐药亚系 CEM/VM-1 和 CEM/VM-1-5 中 DNA 拓扑异构酶 II (topo II) 亚型的特征。它们对替尼泊苷的耐药性分别约为 40 至 50 倍和 150 至 > 400 倍，并且对其他复合物稳定拓扑 II 抑制剂的交叉耐药性水平有所增加。这些品系中的 Topo II 活性随其抗性的增加而降低。然而，这两个亚系在拓扑 II α cDNA 中携带两个相同的点突变，最近发现这些点突变赋予转染的酵母细胞对依托泊苷和 m-AMSA 的抗性。尽管这些数据为这种类型的多药耐药性提供了强有力的分子基础，但这些发现本身并不能解释耐药性和交叉耐药性水平的增加，以及与 CEM/VM-1 细胞相比，耐药性最强的 CEM/VM-1-5 细胞中细胞拓扑 II 活性的进一步降低。在本研究中我们发现（1）topo II beta在CEM/VM-1-5细胞中不表达； (2) 仅在 CEM 细胞的核提取物中一致检测到并免疫共沉淀 160 kDa 的蛋白质； (3)当所有三种细胞系的核提取物在37℃下孵育时，60-90分钟时仅在CEM细胞样品中出现140-kDa的免疫反应带，而在CEM/VM-1和CEM/VM-1-5细胞样品中则没有； (4)与CEM细胞相比，两种耐药细胞系中拓扑IIα蛋白的体内磷酸化水平降低了≥2倍。因此，为改变的拓扑 II 相关多药耐药表型选择的细胞系可能在两种拓扑 II 亚型中包含多个改变。（摘要截断为 250 个字）
We have examined features of DNA topoisomerase II (topo II) isoforms in human leukemic CCRF-CEM cells and two teniposide-resistant sublines, CEM/VM-1 and CEM/VM-1-5. They are about 40- to 50-fold and 150- to > 400-fold resistant to teniposide, respectively, and have increased levels of cross-resistance to other complex-stabilizing topo II inhibitors. Topo II activity in these lines is reduced in proportion to their resistance. However, both sublines carry two identical point mutations in topo II alpha cDNA that have recently been found to confer resistance to etoposide and m-AMSA in transfected yeast cells. Although these data provide a strong molecular basis for this type of multidrug resistance, these findings alone cannot explain the increased level of resistance and cross-resistance, and the further decreased cellular topo II activities in the most resistant CEM/VM-1-5 cells compared to CEM/VM-1 cells. In this study we found that (1) topo II beta is not expressed in CEM/VM-1-5 cells; (2) a 160-kDa protein was consistently detected and coimmunoprecipatated only in nuclear extracts of CEM cells; (3) when nuclear extracts from all three cell lines were incubated at 37 degrees C, an immunoreactive band of 140-kDa appeared by 60-90 min only in samples of CEM cells, not in those of CEM/VM-1 and CEM/VM-1-5 cells; and (4) the in vivo phosphorylation level of topo II alpha protein was decreased > or = 2-fold in both resistant cell lines, compared to that of CEM cells. Thus, cell lines selected for the altered topo II-associated multidrug resistance phenotype may contain multiple alterations in both topo II isoforms.(ABSTRACT TRUNCATED AT 250 WORDS)