EXPRESSION OF A MUTANT-DNA TOPOISOMERASE-II IN CCRF-CEM HUMAN LEUKEMIC-CELLS SELECTED FOR RESISTANCE TO TENIPOSIDE
EXPRESSION OF A MUTANT-DNA TOPOISOMERASE-II IN CCRF-CEM HUMAN LEUKEMIC-CELLS SELECTED FOR RESISTANCE TO TENIPOSIDE
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DOI：
10.1073/pnas.88.17.7654
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发表时间：
1991-09-01
影响因子：
11.1
通讯作者：
SUTTLE, DP
中科院分区：
文献类型：
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作者：
BUGG, BY;DANKS, MK;SUTTLE, DP
人类白血病细胞系CCRF-CEM对替尼平苷(VM-26)耐药亚系的核提取物降低了DNA拓扑异构酶II的催化活性，并降低了形成药物稳定的共价蛋白质-DNA复合体的能力。在DNA解结试验中，耐药细胞系的核提取液中同等活性所需的ATP浓度比亲本系高2至8倍。在络合物形成试验中，当腺苷5‘-[β，伽马亚胺]三磷酸取代三磷酸腺苷时，药物敏感细胞没有明显变化，但对VM-26耐药的细胞减少了50%-65%。综上所述，这些结果表明，ATP结合的改变可能与耐药表型有关。因此，我们确定了拓扑异构酶II序列中与先前确定的核苷酸结合位点一致的区域。以cDNA为模板，通过对聚合酶链式反应产生的DNA片段进行测序，确定了这些位点周围的拓扑异构酶II基因的序列。在与Walker等人描述的共识BATP结合序列相对应的区域中。[Walker，J.E.，Saraste，M.，Runswick，M.J.&Gay，N.J.(1982)EMBO J.1,945-951]，来自两个抗VM-26亚系的cDNA包含一个具有G-->；A碱基改变的改变的序列。这种碱基替换导致449位保守的精氨酸被谷氨酰胺取代。与等位基因特异的寡核苷酸杂交证实，在耐药细胞系中既存在正常序列，也存在变异序列，而在敏感的CEM细胞中仅发现正常序列。一种检测DNA双链中错配碱基的化学错配切割程序没有发现包含拓扑异构酶II完整的ATP结合域的mRNA编码序列的5‘三分之一的其他改变。编码拓扑异构酶II的mRNA与Gln449的存在既与拓扑异构酶II蛋白的存在有关，也与拓扑异构酶II蛋白与ATP的相互作用改变以及对VM-26的细胞毒性的抵抗力增加有关。
Nuclear extracts from teniposide (VM-26)-resistant sublines of the human leukemic cell line CCRF-CEM have decreased levels of DNA topoisomerase II catalytic activity and decreased capacity to form drug-stabilized covalent protein-DNA complexes. The ATP concentration required for equivalent activity in a DNA-unknotting assay is 2- to 8-fold higher in nuclear extracts from drug-resistant cell lines as compared with the parental line. When adenosine 5'-[beta, gamma-imido] triphosphate is substituted for ATP in complex-formation assays, no significant change is seen with drug-sensitive cells, but a 50-65% reduction is seen with VM-26-resistant cells. Collectively, these results indicate that an alteration in ATP binding may be involved in the resistance phenotype. Therefore, we identified regions of the topoisomerase II sequence that conform to previously identified nucleotide-binding sites. Starting with cDNA as the template we determined the sequence of the topoisomerase II mRNA surrounding these sites by sequencing DNA fragments produced by the polymerase chain reaction. In the region corresponding (o the consensus B ATP-binding sequence described by Walker el al. [Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. (1982) EMBO J. 1, 945-951], the cDNA from the two VM-26-resistant sublines contained an altered sequence having a G --> A base change. This base substitution results in the replacement of the conserved arginine at position 449 with a glutamine. Hybridization with allele-specific oligonucleotides confirmed the presence of both the normal and the altered sequence in the resistant cell lines, whereas only the normal sequence was found in the sensitive CEM cells. A chemical mismatch cleavage procedure for the detection of mispaired bases in DNA duplexes identified no other alterations in the 5' third of the mRNA coding sequence, which contains the complete ATP-binding domain of topoisomerase II. The presence of mRNA encoding topoisomerase II with Gln449 correlates both with the presence of a topoisomerase II protein whose interaction with ATP is altered and with increased resistance to the cytotoxicity of VM-26.