ANALYSIS OF DRUG RESISTANCE BY FLOW MICROFLUOROCYTOMETRY

自从发现mdr-1基因产物在 多药耐药，大多数关于多药耐药的研究 表型的研究已经在核酸水平上进行。我们有 已建立的检测方法，通过使用 流式细胞术来观察完整的单细胞。存在显著 这种方法的优点，特别是在探索的作用， P-糖蛋白在先天性和获得性多药耐药中的表达 临床标本我们开发的检测方法可以测量 P-糖蛋白表达和阿霉素含量在每个数以千计的 细胞，在收到标本的几个小时内。我们已经开发出一种 双参数技术，使我们能够将表达和 肿瘤样本或细胞系中单个细胞中的阿霉素含量。 使用这种技术，我们已经能够识别出一个亚群， 先天性耐药细胞（高P-糖蛋白表达，低Adr含量） 在CML-胚细胞危机细胞系中， 单参数技术。相反，我们能够检测到 在选择的CML母细胞危象细胞系中的药物敏感亚群， MDR-1基因转染的MCF-7细胞对阿霉素的耐药性。 此外，我们正在进行流式细胞仪分析的其他措施 包括单细胞检测二氢叶酸 荧光甲氨蝶呤还原酶和流式细胞术的应用 用于快速筛选耐药逆转剂。我们将使用 概述了研究多种药物耐药性及其逆转的技术 肿瘤。一个项目，利用独特的多参数功能， 流动分析已开始研究药物的关系， 对N-myc表达的抗性。

Since the discovery of the importance of the mdr-1 gene product in multidrug resistance, most of the studies of the multidrug resistance phenotype have been performed at the nucleic acid level. We have established assays to examine drug resistance at the protein level by using flow cytometry to look at intact single cells. There are significant advantages to this approach, especially in probing the role of P-glycoprotein expression in innate and acquired multidrug resistance in clinical specimens. The assays we have developed allow us to measure P-glycoprotein expression and adriamycin content in each of thousands of cells, within hours of receiving a specimen. We have developed a two-parameter technique that allows us to correlate expression and adriamycin content in individual cells in a tumor specimen or cell line. Using this technique, we have been able to identify a subpopulation of innately resistant cells (high P-glycoprotein expression, low Adr content) in a CML-blast crisis cell line that was not detectable by previously available single parameter techniques. conversely, we were able to detect a drugsensitive subpopulation in a CMLblast crisis cell lines selected for Adr resistance and in MCF-7 cells transfected with the mdr-1 gene. In addition, we are working on flow cytometric analysis of other measures of drug resistance, including single cell detection of dihydrofolate reductase by fluoresceinated methotrexate and on the use of flow cytometry for the rapid screening of resistance reversal agents. We will use the techniques outlined to study drug resistance and its reversal in a variety of tumors. A project utilizing the unique multiparamter capabilities of flow analysis has been initiated to study the relationship of drug resistance to N-myc expression.