MICRODETECTION ASSAY FOR DRUG RESISTANT TUMORS

耐药肿瘤的微量检测分析

• 批准号: 2376763
• 负责人: WILLIAM T BECK
• 金额: $ 23.56万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-15 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2376763
• 关键词: DNA damage; DNA topoisomerases; P glycoprotein; antineoplastics; apoptosis; cell cycle proteins; digital imaging; fluorescence microscopy; human tissue; immunologic assay /test; laboratory mouse; leukemia; messenger RNA; methylation; multidrug resistance; neoplasm /cancer pharmacology; single photon emission computed tomography; single strand conformation polymorphism; technology /technique development; tissue /cell culture

In recent years, much effort has been directed to understanding mechanisms of clinical anticancer drug resistance. Markers of multidrug resistance (MDR) - overexpression of the mdr1 gene and its product, P- glycoprotein (Pgp) - have been shown to be associated with some clinically resistant tumors. However, only incremental progress has bee made in assessing the role of MDR in clinical drug resistance, likely du in part to the fact that not all drug resistant tumors express Pgp. Indeed, other forms of MDR have been described experimentally that may complicate assays for MDR in clinical specimens. A long-term goal of th proposed studies is the development of microdetection and in situ assays to detect MDR, regardless of the mechanism, in the tumor cells of patients. Our approach has been to characterize biochemical and molecular features of MDR cells and to develop single-cell functional an molecular assays based on these resistance-associated features in order to permit detection of such drug resistant cells in patients' tumors. In this way, we discovered mutations in the topo IIalpha gene associated with an altered topoisomerase II-form of MDR (at MDR) and exploited this finding in a molecular assay (single-strand conformational polymorphism; SSCP) that permitted screening of a large number of cell lines and leukemic specimens for these and other mutations. We are developing single-cell functional assays, based on resistance-associated features, to detect MDR in clinical specimens. In this grant-period, I propose to test the hypotheses that [1] single-cell functional assays for drug resistance can predict the situation in patient's tumors and [2] it is possible to directly image functional drug resistance in tumors in situ. The following specific aims are designed to test these hypotheses: (1) determine in PCR-based and immunologic assays the association of resistance-associated proteins with MDR; (2) develop fluorescence digital image microscopy (FDIM) to quantitate and immunolocalize resistance-associated proteins and to quantitate DNA damage (comet assay and apoptosis in single-cells; (3) develop single photon emission computed tomography (SPECT) methods for functional analysis of Pgp in solid tumors in situ; and (4) apply these findings and assays (SSCP, quantitate PCR, FDIM, comet, SPECT) to patients' tumors.

近年来，已经努力理解很多努力 临床抗癌耐药性的机制。 多药的标记 抗性（MDR） - MDR1基因及其产物的过表达P- 糖蛋白（PGP） - 已显示与某些相关 临床上抗性肿瘤。 但是，只有增量进度才有蜜蜂 在评估MDR在临床耐药性中的作用时可能是DU 部分原因是并非所有耐药性肿瘤表达PGP。 实际上，已经通过实验描述了其他形式的MDR 在临床标本中对MDR的测定复杂。 TH的长期目标 拟议的研究是微调和原位测定的发展 在肿瘤细胞中检测MDR 患者。 我们的方法是表征生化和 MDR细胞的分子特征并发展单细胞功能A 基于这些抗性相关特征的分子测定 允许检测患者肿瘤中这种耐药细胞。 通过这种方式，我们发现了与topo iialpha基因相关的突变 MDR（在MDR上）的拓扑异构酶II形式变化并利用了这一点 在分子测定中发现（单链构象多态性； SSCP）允许筛选大量的细胞系和 这些突变和其他突变的白血病标本。 我们正在发展 基于电阻相关特征的单细胞功能测定 检测临床标本中的MDR。 在这个赠款期间，我建议 测试[1]药物单细胞功能测定的假设 抗性可以预测患者肿瘤的情况，[2]是 可能直接在原位肿瘤中成像功能性耐药性。 以下特定目的旨在检验以下假设：（1） 在基于PCR和免疫学测定中确定 与MDR相关的抗性相关蛋白； （2）发展荧光 数字图像显微镜（FDIM）来定量和免疫定位 抗性相关蛋白质并定量DNA损伤（彗星测定法 和单细胞的凋亡； （3）开发单个光子发射 用于PGP功能分析的计算机断层扫描方法（SPECT） 原位实体瘤； （4）应用这些发现和测定（SSCP， 将PCR，FDIM，彗星和SPECT定量为患者的肿瘤。