REGULATION OF TOPOISOMERASE II-DRUG-DNA TERNARY COMPLEX

说明：（申请人摘要）本申请的基本主题 是确定位点特异性磷酸化不足的功能作用， 拓扑异构酶II在DNA可切割复合物形成中的作用。申请人 假设拓扑异构酶II α磷酸化（170 kDa）和/或拓扑异构酶II β蛋白依赖于改变 在细胞内游离钙，这影响依托泊苷稳定的DNA 可裂解复合物形成和细胞毒性。实验将 在敏感或渐进性阿霉素耐药HL-60中进行 肿瘤模型系统为了检测细胞内钙离子的影响， 拓扑异构酶II α或拓扑异构酶磷酸化的瞬变 II β，敏感或耐药HL-60人白血病细胞将被 用细胞内钙缓冲剂1，2-二（O- 氨基苯氧基）乙烷-N，N，N '，N'，-四乙酸四（乙酰氧基甲基） 酯（BAPTA-AM）并用[（32）P]-正磷酸标记。使用 拓扑异构酶II α或拓扑异构酶II β免疫沉淀物 申请人将确定依托泊苷稳定化DNA的选择性 通过条带耗竭和位点特异性改变， 拓扑异构酶II β蛋白的磷酸化 磷酸肽的胰蛋白酶作图。磷酸化位点在 拓扑异构酶II α和拓扑异构酶II β蛋白， 受BAPTA-AM治疗的影响将由两个 方法：（a）Edman测序（32）P标记的肽， 高效液相色谱法（HPLC）;和（B）磷酸肽 液相色谱-电喷雾离子化作图和测序 串联质谱法（LC-ESIMS/MS）。拓扑异构酶II α和 磷酸受体位点突变的拓扑异构酶II β蛋白 将在抑制剂敏感性（ISE 2）突变株中表达， 酵母或通过瞬时转染细胞来评估功能性 位点特异性磷酸化的作用，通过表征野生型或 突变体稳定的DNA可切割复合物的形成和药物敏感性。 拟用拓扑异构酶II α和拓扑异构酶II β进行的研究 将提供有关钙的功能作用的新信息， 磷酸化在可裂解复合物的形成，并在长期内， 了解耐药性演变的机制， 拓扑异构酶II抑制剂。

DESCRIPTION: (Applicant's Abstract) The basic theme of this application is to establish the functional role of site specific hypophosphorylation of topoisomerase II in DNA cleavable complex formation. The applicant hypothesizes that phosphorylation of the topoisomerase II alpha (170 kDa) and/or topoisomerase II beta protein is dependent on alterations in intracellular free calcium and this affects etoposide stabilized DNA cleavable complex formation and cytotoxicity. Experiments will be carried out in sensitive or progressively adriamycin resistant HL-60 tumor model systems. To test the effect of intracellular calcium transients on phosphorylation of topoisomerase II alpha or topoisomerase II beta, sensitive or resistant HL-60 human leukemia cells will be treated without or with the intracellular calcium buffer 1,2-bis (o- aminophenoxy) ethane-N,N,N',N', -tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM) and labeled with [(32)P]-orthophosphoric acid. Using immunoprecipitates of topoisomerase II alpha or topoisomerase II beta the applicant will determine selectivity of etoposide stabilized DNA cleavable complex by band depletion and site specific alterations in the phosphorylation of topoisomerase II beta protein by two dimensional tryptic mapping of phosphopeptides. The phosphorylation sites in the topoisomerase II alpha and topoisomerase II beta protein which are affected by treatment with BAPTA-AM will be determined by two approaches: (a) Edman sequencing of (32)P labeled peptides separated by high performance liquid chromatography (HPLC); and (b) phosphopeptide mapping and sequencing by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESIMS/MS). Topoisomerase II alpha and topoisomerase II beta protein with mutations in phosphoacceptor sites will be expressed in an inhibitor sensitive (ISE2) mutant strain of yeast or by transient transfection of cells to evaluate the functional role of site specific phosphorylation by characterizing wild-type or mutant stabilized DNA cleavable complex formation and drug sensitivity. Studies proposed with topoisomerase II alpha and topoisomerase II beta will provide novel information on the functional role of calcium and phosphorylation in cleavable complex formation, and in the long term an understanding of mechanisms in the evolution of drug resistance to topoisomerase II inhibitors.