MAMSA RESISTANCE AND EXPRESION OF P76 PROTEIN

拟议研究的目的是调查潜在的新的 插层抗性的分子和生化机制 试剂mAMSA，特别是具有明显分子的蛋白质的作用 体重76,000(P76)，被发现与主要的， 体外诱导多个独立来源的mAMSA耐药 人急性髓系白血病(AML)细胞系。我们建议使用 重组DNA克隆与P76抗体作为检测探针的相关性 P76在急性髓系白血病细胞系耐药中的作用。在进一步 将P76纯化至均一，我们将测定氨基酸 任何内部多肽的序列，也产生P76的抗体。 根据氨基酸序列，一个独特的寡核苷酸探针将是 构建并获得与P76相对应的全长cDNA。这个 用合适的载体将分离的P76基因导入mAMSA 抗药性突变体。将对转基因细胞的表型进行筛选 使用P76抗体表达P76；用于药物敏感性和药物 诱导拓扑异构酶II活性的变化。随着新世纪的到来 描述分子探针和P76抗体我们将筛查白血病 来自疾病不同阶段的AML患者的样本，即以前 MAMSA的治疗和化疗过程中出现的 临床耐药。这项研究的最终目标是澄清 P76在白血病细胞对mAMSA的细胞反应中的作用 耐甲氧西林金黄色葡萄球菌的实验模型和临床研究 情况。

The objective of the proposed studies is to investigate potentially new molecular and biochemical mechanisms of the resistance to intercalating agent mAMSA, specifically the role of a protein with an apparent molecular weight of 76,000 (p76), found to be consistantly associated with primary, in vitro induced resistance to mAMSA in numerous independently derived human acute myelogenous leukemia (AML) cell lines. We propose to use recombinant DNA clones and antibodies to p76 as detection probes to relate the function of p76 to mAMSA resistance in AML cell lines. After further purification of the p76 to homogeneity we will determine amino acid sequence of any internal peptides and also produce antibodies to p76. Based on the amino acid sequence, a unique oligonucleotide probe will be constructed and used to obtain full length cDNA corresponding to p76. The isolated p76 cDNA will be transfected using a suitable vector into mAMSA resistant mutants. The phenotype of transfected cells will be screened for p76 expression using p76 antibodies; for drug sensitivity and for drug induced changes in the Topoisomerase II activity. With the advent of the described molecular probes and p76 antibodies we will screen leukemic samples from AML patients in various stages of their disease i.e. prior treatment and during the course of chemotherapy with mAMSA and emergence of clinical resistance. The ultimate goal of the study is to clarify the role of p76 in cellular response of leukemic cells to mAMSA and in the development of mAMSA resistance, both in experimental models and clinical situations.