Nelson E J, Zinkin N T, Hinkle P M
Department of Pharmacology and Physiology and the Cancer Center, University of Rochester School of Medicine and Dentistry, NY 14642, USA.
Cancer Chemother Pharmacol. 1998;42(4):292-9. doi: 10.1007/s002800050820.
Microscopic methods to measure the activity of drug extrusion systems important in multidrug resistance in individual cells were developed.
Multidrug-resistant (MDR) and parental lines of hamster CHO and pituitary GH3 cells were incubated with the acetoxymethylester (AM) forms of several fluorescent calcium-sensing dyes, fura2, indo1 and fluo3. The AM forms of these compounds are hydrolyzed by intracellular esterases and then trapped in cells, and the AM forms of the dyes are excellent substrates for P-glycoprotein (Pgp).
The fluorescent free acid forms of fura2, indol and fluo3 did not accumulate in MDR lines unless a chemosensitizer such as cyclosporin A, R(+)verapamil, quinidine, or progesterone was included during loading to prevent the cells from extruding the AM forms of the dyes before they could be hydrolyzed. Cyclosporin A increased the fluorescence due to intracellularly trapped fura2 free acid from 8- to 20-fold and was maximally effective at < 5 microM. Fluorescence microscopy was employed to measure fura2 free acid accumulation by parental and MDR cell lines using excitation at the Ca2+-insensitive wavelength. When MDR cells were incubated with rhodamine 123 and fura2/AM, no fluorescence was detectable. Cellular fluorescence was dramatically increased by inclusion of cyclosporin A, quinidine, progesterone, or R(+)verapamil. There was no measurable decline in the fura2 free acid fluorescence in 1 h while the fluorescence due to rhodamine 123 diminished rapidly in cells overexpressing Pgp.
These fluorescence methods detect drug-extruding activity in individual cells and therefore have the potential to provide complementary information to studies quantifying protein or mRNA levels of Pgp or other efflux pumps. In addition, they provide a rapid and quantifiable method for screening multidrug resistance reversing agents.
开发用于测量在个体细胞多药耐药中起重要作用的药物外排系统活性的微观方法。
将仓鼠CHO和垂体GH3细胞的多药耐药（MDR）细胞系和亲本细胞系与几种荧光钙敏感染料（fura2、indo1和fluo3）的乙酰氧基甲酯（AM）形式一起孵育。这些化合物的AM形式会被细胞内酯酶水解，然后被困在细胞内，并且染料的AM形式是P-糖蛋白（Pgp）的优良底物。
fura2、indo1和fluo3的荧光游离酸形式不会在MDR细胞系中积累，除非在加载过程中加入化学增敏剂，如环孢素A、R（+）维拉帕米、奎尼丁或孕酮，以防止细胞在染料的AM形式被水解之前将其排出。环孢素A使细胞内捕获的fura2游离酸产生的荧光增加了8至20倍，在浓度<5 microM时效果最佳。使用荧光显微镜通过在Ca2+不敏感波长下激发来测量亲本细胞系和MDR细胞系中fura2游离酸的积累。当MDR细胞与罗丹明123和fura2/AM一起孵育时，未检测到荧光。加入环孢素A、奎尼丁、孕酮或R（+）维拉帕米后，细胞荧光显著增加。在1小时内，fura2游离酸荧光没有可测量的下降，而在过表达Pgp的细胞中，罗丹明123产生的荧光迅速减弱。
这些荧光方法可检测个体细胞中的药物外排活性，因此有可能为量化Pgp或其他外排泵的蛋白质或mRNA水平的研究提供补充信息。此外，它们为筛选多药耐药逆转剂提供了一种快速且可量化的方法。
