In situ biochemical demonstration that P-glycoprotein is a drug efflux pump with broad specificity.
In situ biochemical demonstration that P-glycoprotein is a drug efflux pump with broad specificity.
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DOI：
10.1083/jcb.148.5.863
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发表时间：
2000-03-06
期刊：
影响因子：
--
通讯作者：
Simon SM
中科院分区：
文献类型：
--
作者：
Chen Y;Simon SM
p -糖蛋白（Pgp）是研究最多的与抗癌药物耐药相关的蛋白，但其作用机制仍存在争议。Pgp的研究使用了化疗药物选择的细胞系，这些化疗药物可能已经发展出许多耐药机制。为了消除药物选择对了解Pgp作用的混淆影响，我们研究了瞬时转染Pgp绿色荧光蛋白（GFP）融合蛋白的细胞。该方法产生了具有广泛Pgp- gfp表达水平的未选择细胞的混合群体，并允许同时测量活细胞中的Pgp水平和药物积累。结果显示，Pgp-GFP表达与化疗药物积累呈负相关。阻断多重耐药（MDR）的药物和UIC2抗pgp抗体逆转了药物浓度的降低。定量分析显示Pgp-GFP与MDR染料的荧光呈反比线性关系。这表明Pgp水平单独限制主动外排的药物积累；酶、底物或抑制剂分子之间不需要协同作用。此外，Pgp-GFP的表达不会改变细胞ph。我们的研究证明了使用GFP融合蛋白在活细胞中进行定量生物化学的价值。
While P-glycoprotein (Pgp) is the most studied protein involved in resistance to anti-cancer drugs, its mechanism of action is still under debate. Studies of Pgp have used cell lines selected with chemotherapeutics which may have developed many mechanisms of resistance. To eliminate the confounding effects of drug selection on understanding the action of Pgp, we studied cells transiently transfected with a Pgp-green fluorescent protein (GFP) fusion protein. This method generated a mixed population of unselected cells with a wide range of Pgp-GFP expression levels and allowed simultaneous measurements of Pgp level and drug accumulation in living cells. The results showed that Pgp-GFP expression was inversely related to the accumulation of chemotherapeutic drugs. The reduction in drug concentration was reversed by agents that block multiple drug resistance (MDR) and by the UIC2 anti-Pgp antibody. Quantitative analysis revealed an inverse linear relationship between the fluorescence of Pgp-GFP and MDR dyes. This suggests that Pgp levels alone limit drug accumulation by active efflux; cooperativity between enzyme, substrate, or inhibitor molecules is not required. Additionally, Pgp-GFP expression did not change cellular pH. Our study demonstrates the value of using GFP fusion proteins for quantitative biochemistry in living cells.
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影响因子：
2.9
作者：
SUN, XM;SNOWDEN, RT;COHEN, GM
通讯作者：
COHEN, GM
影响因子：
3
作者：
Lan, LB;Ayesh, S;Stein, WD
通讯作者：
Stein, WD
人乳腺肿瘤细胞中的酸化不足及其对化学疗法的影响。
影响因子：
15.3
作者：
Altan, N;Chen, Y;Schindler, M;Simon, S M
通讯作者：
Simon, S M
影响因子：
3.2
作者：
THIEBAUT, F;CURRIER, SJ;WILLINGHAM, MC
通讯作者：
WILLINGHAM, MC
DOI：
10.1002/cyto.990210308
发表时间：
1995-11-01
期刊：
CYTOMETRY
影响因子：
--
作者：
LIZARD, G;FOURNEL, S;REVILLARD, JP
通讯作者：
REVILLARD, JP