Liminga G, Nygren P, Larsson R
Division of Clinical Pharmacology, University Hospital, Uppsala University, Sweden.
Exp Cell Res. 1994 Jun;212(2):291-6. doi: 10.1006/excr.1994.1146.
A microtiter plate-based fluorometric assay for functional measurement of 170-kDa P-glycoprotein (Pgp)-mediated transport using fluorescent calcein as a probe is described. The myeloma RPMI 8226 cell line and two of its doxorubicin-resistant Pgp-expressing sublines, dox40 (high expression) and dox6 (low expression), were used as models. Nonfluorescent calcein acetoxymethyl ester (calcein/AM) was added to the cells and subsequent accumulation of calcein was measured in a 96-well scanning fluorometer after 30 min. There was an inverse relationship between Pgp expression and calcein/AM accumulation, which increased dose-dependently in the presence of cyclosporin A (CsA) and the nonimmunosuppressive analogue SDZ PSC 833 (PSC) in the Pgp-expressing cell lines. PSC appeared to restore uptake more effectively than CsA at low concentrations. Calcein accumulation was also increased in Pgp-expressing cells by the addition of the Pgp substrate vincristine and the metabolic inhibitor potassium cyanide, KCN. No effect was observed in parental cell lines. When parental and dox40 cells were mixed, 10% of dox40 cells could reproducibly be detected. The results indicate that microtiter-plate determination of calcein accumulation is a simple and sensitive method for functional determination of Pgp-mediated drug transport. The method may become useful, not only for preclinical screening for novel and improved resistance modifiers, but also for determination of Pgp activity in individual clinical tumor samples.
描述了一种基于微孔板的荧光测定法，该方法使用荧光素钙作为探针，用于功能性测量170 kDa P-糖蛋白（Pgp）介导的转运。骨髓瘤RPMI 8226细胞系及其两个表达多柔比星耐药Pgp的亚系dox40（高表达）和dox6（低表达）被用作模型。将非荧光的荧光素钙乙酰甲酯（calcein/AM）添加到细胞中，30分钟后在96孔扫描荧光计中测量荧光素钙的后续积累。Pgp表达与calcein/AM积累呈负相关，在表达Pgp的细胞系中，环孢素A（CsA）和非免疫抑制类似物SDZ PSC 833（PSC）存在时，calcein/AM积累呈剂量依赖性增加。在低浓度下，PSC似乎比CsA更有效地恢复摄取。通过添加Pgp底物长春新碱和代谢抑制剂氰化钾（KCN），表达Pgp的细胞中荧光素钙的积累也增加。在亲本细胞系中未观察到影响。当亲本细胞和dox40细胞混合时，可以重复检测到10%的dox40细胞。结果表明，微孔板测定荧光素钙积累是一种简单而灵敏的方法，用于功能性测定Pgp介导的药物转运。该方法不仅可能有助于临床前筛选新型和改进的耐药修饰剂，还可用于测定个体临床肿瘤样本中的Pgp活性。
