Sailer B L, Valdez J G, Steinkamp J A, Darzynkiewicz Z, Crissman H A
Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545, USA.
Exp Cell Res. 1997 Oct 10;236(1):259-67. doi: 10.1006/excr.1997.3717.
Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) is a green fluorescent plant alkaloid that inhibits DNA topoisomerase II activity and possesses pharmacologic activity toward both murine and human leukemias in vivo. In this flow cytometric study, the uptake of ellipticine was monitored as a function of cell volume and cell cycle phase in viable human promyelocytic (HL-60) cells costained with the DNA fluorochrome Hoechst 33342. Uptake of ellipticine was time and dose dependent; however, drug content was quantitatively similar in all phases of the cell cycle when normalized for DNA content or similar to cell size when correlated with cell volume. The fluorescence lifetime values of ellipticine in HL-60 cells, as analyzed by novel flow cytometric analysis, reached a plateau when the intra-cellular ellipticine intensity was still rising with increasing drug concentration. Since the free drug and the different subcellular ellipticine complexes, including DNA and RNA, had different lifetime values, the changes in the lifetime values appear to reflect differing proportions of unbound drug to that bound to different cellular constituents in the cells. Further development of phase-sensitive flow cytometry will provide for multiple lifetime determinations so that quantitation of drugs bound to the different cellular components can be performed along with the simultaneous determination of total drug uptake and cell cycle position. Such analyses should provide useful information for the design of drugs with greater affinity for cytotoxic targets.
椭圆玫瑰树碱（5,11 - 二甲基 - 6H - 吡啶并[4,3 - b]咔唑）是一种绿色荧光植物生物碱，它能抑制DNA拓扑异构酶II的活性，并且在体内对小鼠和人类白血病均具有药理活性。在这项流式细胞术研究中，在与DNA荧光染料Hoechst 33342共染色的存活人早幼粒细胞（HL - 60）细胞中，监测椭圆玫瑰树碱的摄取情况，并将其作为细胞体积和细胞周期阶段的函数。椭圆玫瑰树碱的摄取具有时间和剂量依赖性；然而，当以DNA含量进行归一化时，细胞周期各阶段的药物含量在数量上相似，或者当与细胞体积相关时，药物含量与细胞大小相似。通过新型流式细胞术分析，当细胞内椭圆玫瑰树碱强度仍随药物浓度增加而上升时，HL - 60细胞中椭圆玫瑰树碱的荧光寿命值达到平稳状态。由于游离药物以及包括DNA和RNA在内的不同亚细胞椭圆玫瑰树碱复合物具有不同的寿命值，寿命值的变化似乎反映了细胞中未结合药物与结合到不同细胞成分上的药物的不同比例。相敏流式细胞术的进一步发展将实现多个寿命测定，从而能够在同时测定总药物摄取和细胞周期位置的情况下，对结合到不同细胞成分上的药物进行定量。此类分析应为设计对细胞毒性靶点具有更高亲和力的药物提供有用信息。
