REGULATION OF TOPOISOMERASE II-DRUG-DNA TERNARY COMPLEX

描述：(申请人摘要)本申请的基本主题 是为了确定位点特异性低磷酸化的功能作用 拓扑异构酶II在DNA裂解复合体形成中的作用。申请人 假设拓扑异构酶IIα(170)的磷酸化 Kda)和/或拓扑异构酶IIβ蛋白依赖于改变 细胞内游离钙离子，这会影响依托泊苷稳定的DNA 可切割的复合体的形成和细胞毒性。实验将是 在对阿霉素敏感或逐渐耐药的HL-60中进行 肿瘤模型系统。为了测试细胞内钙离子的作用 拓扑异构酶IIα或拓扑异构酶的瞬时磷酸化 IIβ，敏感或耐药的HL-60人白血病细胞 不使用或使用细胞内钙缓冲剂1，2-双(o- 氨基苯氧基)乙烷-N，N，N‘，N’，-四乙酸四乙酰氧甲基 酯(BAPTA-AM)，用[(32)P]-正磷酸标记。vbl.使用 拓扑异构酶IIα或拓扑异构酶IIβ免疫沉淀物 申请者将确定依托泊苷稳定的DNA的选择性 可切割复合体的条带耗尽和位点特异性改变 拓扑异构酶IIβ蛋白的二维磷酸化 磷酸多肽的胰酶标记法。细胞内的磷酸化位点 拓扑异构酶IIα和拓扑异构酶IIβ蛋白 受BAPTA-AM治疗的影响将由两个人决定 方法：(A)对~(32)P标记的多肽进行Edman测序 高效液相色谱法；和(B)磷酸多肽 高效液相色谱-电喷雾电离作图测序 串联质谱仪(LC-ESIMS/MS)。拓扑异构酶IIα和 磷酸受体位点突变的拓扑异构酶IIβ蛋白 将在对抑制剂敏感(ISE2)的突变株中表达 酵母或通过瞬时转染法对细胞进行功能评价 位点特异性磷酸化在鉴定野生型OR中的作用 突变体稳定了DNA裂解复合体的形成和药物敏感性。 拓扑异构酶IIα和拓扑异构酶IIβ的研究建议 将提供有关钙和钙的功能作用的新信息 在可切割的复合体形成中的磷酸化，以及在长期和 对甲氧西林耐药机制的认识 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.