Leontiou Chrysoula, Lakey Jeremy H, Lightowlers Robert, Turnbull Rozalind M, Austin Caroline A
Institute for Cell and Molecular Bioscience, The Medical School, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK.
Mol Pharmacol. 2006 Jan;69(1):130-9. doi: 10.1124/mol.105.015933. Epub 2005 Oct 20.
The anti cancer drug methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide (mAMSA) targets human DNA topoisomerase IIbeta. We report here the first selection with mAMSA of resistant human topoisomerase IIbeta. Random mutagenesis of human DNA topoisomerase IIbeta cDNA, followed by selection in yeast for resistance to mAMSA, identified betaP732L. This mutant was 10-fold less sensitive to mAMSA and cross-resistant to other chemotherapeutic agents such as etoposide, ellipticine, methyl N-(4'-(9-acridinylamino)-2-methoxy-phenyl) carbamate hydrochloride (mAMCA), methyl N-(4'-(9-acridinylamino)-phenyl) carbamate hydrochloride (AMCA), and doxorubicin. betaP732L is functional but has reduced strand passage activities and altered DNA binding compared with the wild-type protein. It has drastically altered cleavage properties compared with the wild-type enzyme. It cleaved a 40-base pair (bp) DNA substrate in the presence of magnesium but at positions different from that of the wild-type protein. More striking is that betaP732L was unable to cleave the 40-bp DNA substrate, a 500-bp linear substrate, or a 4.3-kilobase supercoiled substrate in the presence of calcium ions. This is the first report of a topoisomerase II mutation abolishing the ability of calcium to support DNA cleavage. This provides evidence for metal ion requirement for the phosphoryltransfer reaction of topoisomerase II and a possible mechanism for drug resistance.
抗癌药物N-（4'-（9-吖啶基氨基）-3-甲氧基苯基）甲磺酰胺（mAMSA）作用于人类DNA拓扑异构酶IIβ。我们在此报告首次筛选出对mAMSA具有抗性的人类拓扑异构酶IIβ。对人类DNA拓扑异构酶IIβ cDNA进行随机诱变，随后在酵母中筛选对mAMSA的抗性，鉴定出βP732L。该突变体对mAMSA的敏感性降低了10倍，并且对其他化疗药物如依托泊苷、玫瑰树碱、N-（4'-（9-吖啶基氨基）-2-甲氧基苯基）氨基甲酸甲酯盐酸盐（mAMCA）、N-（4'-（9-吖啶基氨基）-苯基）氨基甲酸甲酯盐酸盐（AMCA）和阿霉素具有交叉抗性。与野生型蛋白相比，βP732L具有功能，但链通过活性降低且DNA结合发生改变。与野生型酶相比，其切割特性发生了巨大变化。在镁存在的情况下，它能切割一个40碱基对（bp）的DNA底物，但切割位置与野生型蛋白不同。更引人注目的是，在钙离子存在的情况下，βP732L无法切割40-bp的DNA底物、500-bp的线性底物或4.3千碱基的超螺旋底物。这是关于拓扑异构酶II突变消除钙离子支持DNA切割能力的首次报道。这为拓扑异构酶II磷酸转移反应对金属离子的需求提供了证据，并为耐药性提供了一种可能的机制。
