ANALYSIS OF DRUG RESISTANCE BY FLOW MICROFLUOROCYTOMETRY

我们已经建立了检测耐药性表达的方法， 使用完整单细胞的流式细胞术分析表型。 那里 是这种方法的显著优点，包括能够 获得了数以万计的细胞的快速半定量数据， 研究临床耐药异质性的能力 标本 开发的技术包括P-糖蛋白分析 表达使用单克隆抗体MRK-16分析， 使用单克隆抗体表达胸苷酸合成酶 由帕特里克约翰逊博士开发，并分析了表达的 叶酸结合蛋白和还原型叶酸转运蛋白的性质， 使用荧光素化的甲氨蝶呤。 在过去的一年里，我们利用这些 技术在协作研究中的各种应用。 这些应用包括检查还原叶酸转运蛋白 乳腺癌细胞系中P-糖蛋白表达 用mdr 1基因和mdr 1基因转染的癌细胞系 蛋白激酶C α;流式细胞术与Western印迹的比较， 北方印迹和RNA原位杂交分析P-糖蛋白。 结肠癌细胞系表达临床相关水平的 P-糖蛋白与耐药性;检测P-糖蛋白的表达 瞬时转染CHO、MCF-7和L细胞中的叶酸结合蛋白 与叶酸结合蛋白表达载体结合;胸苷酸分析 合成酶在结肠癌细胞系中的表达; 人结肠癌细胞处理后的细胞周期时相分布 新型抗代谢药MRPP

We have established assays to examine expression of the drug resistance phenotype using flow cytometric analysis of intact single cells. There are significant advantages to this approach, including the ability to derive rapid, semiquantitative data on tens of thousands of cells and the ability to study the heterogeneity of drug resistance within clinical specimens. The techniques developed include analysis of P-glycoprotein expression using the monoclonal antibody MRK-16 analysis of the expression of thymidylate synthetase using a monoclonal antibody developed by Dr. Patrick Johnson, and analysis of the expression of the folate binding protein and properties of the reduced folate transporter, using fluoresceinated methotrexate. In the past year, we have used these techniques in a wide variety of applications in collaborative studies. These applications include examination of the reduced folate transporter in breast cancer cell lines; expression of P-glycoprotein in breast cancer cell lines transfected with the mdr 1 gene and the gene for protein kinase C alpha; comparison of flow cytometry with Western blot, Northern blot, and RNA in situ for the analysis of P-glycoprotein in colon cancer cell lines expressing clinically relevant levels of P-glycoprotein and drug resistance; examination of the expression of the folate binding protein in CHO, MCF-7, and L cells transiently transfected with a folate binding protein expression vector; analysis of thymidylate synthetase expression in colon cancer cell lines; and analysis of the cell cycle phase distribution after treatment of human colon cancer cells with the novel antimetabolite MRPP.