MAMSA RESISTANCE AND EXPRESION OF P76 PROTEIN

拟议研究的目的是调查潜在的新的 抗插层的分子和生化机制 试剂mAMSA，特别是具有明显分子量的蛋白质的作用， 体重76,000（p76），发现与原发性相关， 在许多独立来源的细胞中，体外诱导对mAMSA的抗性 人急性髓性白血病（AML）细胞系。 我们建议使用 重组DNA克隆和p76抗体作为检测探针， p76在AML细胞系中对mAMSA耐药的作用。 经过进一步 将p76纯化至均一，我们将测定氨基酸 任何内部肽序列，并产生抗体p76。 基于氨基酸序列，将使用独特的寡核苷酸探针。 构建并用于获得对应于p76的全长cDNA。 的 分离的p76 cDNA将使用合适的载体转染到mAMSA中 抗性突变体 将筛选转染细胞的表型， 使用p76抗体的p76表达;用于药物敏感性和药物敏感性的研究 诱导拓扑异构酶II活性的变化。 的到来 描述了分子探针和p76抗体，我们将筛选白血病 来自处于其疾病的各个阶段的AML患者的样品，即先前的 治疗和化疗过程中与mAMSA和出现 临床耐药性 这项研究的最终目的是阐明 p76在白血病细胞对mAMSA的细胞反应中的作用，以及在白血病细胞对mAMSA的细胞反应中的作用。 mAMSA耐药性的发展，在实验模型和临床 situations.

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.