Formation, intracellular distribution and efflux of glutathione-bimane conjugates in drug-sensitive and -resistant MCF-7 cells
Formation, intracellular distribution and efflux of glutathione-bimane conjugates in drug-sensitive and -resistant MCF-7 cells
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DOI：
10.1007/s002800050633
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发表时间：
1997-06-01
影响因子：
3
通讯作者：
Gamcsik, MP
中科院分区：
文献类型：
--
作者：
Millis, KK;Lesko, SA;Gamcsik, MP
在通过落射荧光和共聚焦显微镜检查之前，在天然人乳腺MCF-7腺癌细胞（MCF-7 wt）和对4-氢过氧环磷酰胺（MCF-7 hc）和阿霉素（MCF-7adr）显示抗性的亚系中测量一氯二亚胺与谷胱甘肽（GSH）的反应速率。在37 ℃下孵育60分钟后，在MCF-7 wt和MCF-7adr细胞系中基本上所有GSH都被缀合，而在MCF-7 hc细胞系中仅80%的GSH被缀合。所有三个线显示显着出口的共轭物从细胞在此期间，与MCF-7adr线显示最快速的流出与85%的共轭物出口在60分钟内。落射荧光显微镜检测到约20%的增加，在所有三个线的核区域的综合荧光强度。然而，共聚焦显微镜检查表明，大多数细胞检查显示出均匀的荧光分布。发现在单层中生长的细胞在核区域中更厚，这表明在来自落射荧光显微镜的图像中观察到的核区域中荧光强度的增加可能来自于来自焦平面外的荧光。用丁硫氨酸亚砜亚胺耗尽GSH，然后用mBCl处理的细胞显示出由该探针的非特异性结合引起的显著荧光强度。这些研究表明，需要测量GSH共轭出口的速度和确定探针的特异性，并强调需要使用共聚焦技术的细胞内荧光分布的定量评价。
The rate of reaction of monochlorobimane with glutathione (GSH) was measured in native human mammary MCF-7 adenocarcinoma cells (MCF-7wt) and sublines displaying resistance to 4-hydroperoxycyclophosphamide (MCF-7hc) and adriamycin (MCF-7adr) prior to examination by epifluorescence and confocal microscopy. After a 60-min incubation period at 37 degrees C, essentially all GSH was conjugated in the MCF-7wt and MCF-7adr cell lines whereas only 80% of the GSH was conjugated in the MCF-7hc line. All three lines displayed significant export of the conjugate from the cell during this period, with the MCF-7adr line displaying the most rapid efflux with 85% of the conjugate exported within 60 min. Epifluorescence microscopy detected an approximately 20% increase in integrated fluorescence intensity in the nuclear region in all three lines. Confocal microscopy however, indicated that most of the cells examined showed a homogeneous fluorescence distribution. The cells grown in monolayers were found to be thicker in the nuclear region suggesting that the observed increase in fluorescence intensity in the nuclear region in the images from epifluorescence microscopy was probably derived from fluorescence from an out-of-focus plane. Cells depleted of GSH with buthionine sulfoximine followed by treatment with mBCl showed significant fluorescence intensity resulting from nonspecific binding of this probe. These studies illustrate the need for measuring the rate of GSH conjugate export and for determining probe specificity, and emphasizes the need for using confocal techniques for the quantitative evaluation of the distribution of intracellular fluorescence.