Flow cytometric measurement of mitochondrial mass and function: A novel method for assessing chemoresistance
Flow cytometric measurement of mitochondrial mass and function: A novel method for assessing chemoresistance
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DOI：
10.1007/bf02303787
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发表时间：
1998-04-01
影响因子：
3.7
通讯作者：
Anderson, BO
中科院分区：
文献类型：
--
作者：
Mancini, M;Sedghinasab, M;Anderson, BO
背景：化疗药物诱导癌细胞凋亡。不能诱导细胞凋亡的药物可能会降低临床疗效。我们假设（1）化疗药物通过与活性氧化物产生相关的机制诱导线粒体变化和凋亡;（2）抗凋亡蛋白Bcl-2阻止药物诱导的线粒体变化、活性氧（ROS）产生和凋亡;和（3）药物诱导的线粒体变化的测定可以反映给定癌细胞系中的药物特异性化学抗性。产生Bcl-2阴性乳腺癌细胞系SKBr 3的稳定Bcl-2转染子（SKBr 3/Bcl 2 -2）。用Herbimycin A（300 ng/mL）或溶媒（1% DMSO）处理SKBr 3和SKBr 3/Bcl 2 -2细胞。通过BRDU染色评估细胞周期变化。细胞凋亡通过电镜、TUNEL（TdT介导的dUTP-生物素缺口末端标记）染色和DNA片段化的二苯胺测定来确定。通过JC-1荧光的流式细胞术评估来评估线粒体质量和跨膜电位（Δ Psi（m））的变化。结果：Herbimycin处理后SKBr 3和SKBr 3/Bcl 2 -2细胞均出现细胞周期阻滞。然而，SKBr 3细胞，而不是SKBr 3/Bcl 2 -2细胞，经历凋亡。经Herbimycin处理的SKBr 3细胞显示线粒体质量增加（JC-1绿色荧光），而Δ Psi（m）没有相应增加（JC-1红色荧光）。相比之下，经Herbimycin处理的SKBr 3/Bcl 2 -2细胞显示线粒体质量或Δ Psi（m）没有变化。同样，药物处理的SKBr 3细胞，而不是SKBr 3/Bcl 2 -2细胞，表现出增加活性氧（ROS）的生产伴随着发展apoptosis.Conclusion：SKBr 3细胞凋亡表现出线粒体的变化与ROS的生产。Bcl-2转染阻止了这些变化，因为它阻止了细胞凋亡：Ind诱导SKBr 3对Herbimycin的耐药性。流式细胞术测量药物诱导的线粒体变化和ROS产生可能有助于体外评估乳腺癌的化疗敏感性或耐药性。
Background: Chemotherapeutic agents induce apoptosis in cancer cells. Drugs failing to induce apoptosis are likely to have decreased clinical efficacy. We hypothesize that (1) chemotherapeutic agents induce mitochondrial changes and apoptosis through mechanisms associated with reactive oxidant species production; (2) the anti-apoptotic protein Bcl-2 prevents drug-induced mitochondrial changes, reactive oxygen species (ROS) production, and apoptosis; and (3) the assay of drug-induced mitochondrial changes can reflect drug-specific chemoresistance in a given cancer cell line.Methods: A stable Bcl-2 transfectant of the Bcl-2 negative breast cancer cell line SKBr3 was created (SKBr3/Bcl2-2). Both SKBr3 and SKBr3/Bcl2-2 cells were treated with Herbimycin A (300 ng/mL) or vehicle (1% DMSO). Cell cycle changes were assessed by BRDU staining. Apoptosis was determined by electron microscopy, TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining, and diphenylamine assay of DNA fragmentation. Changes in mitochondrial mass and transmembrane potential (Delta Psi(m)) were assessed by flow cytometric assessment of JC-1 fluorescence. Reactive oxygen species production was measured by 2',7'-dichlorodihydrofluorescein diacetate (DCFH) fluorescence.Results: Both SKBr3 and SKBr3/Bcl2-2 cells show cell cycle arrest after Herbimycin treatment. However, SKBr3 cells, but not SKBr3/Bcl2-2 cells, undergo apoptosis. Herbimycin-treated SKBr3 cells show increased mitochondrial mass (JC-1 green fluorescence), with no corresponding increase in Delta Psi(m) (JC-1 red fluorescence). By contrast, Herbimycin-treated SKBr3/Bcl2-2 cells show no change in mitochondrial mass or Delta Psi(m). Similarly, drug-treated SKBr3 cells, but not SKBr3/Bcl2-2 cells, demonstrate increased reactive oxygen species (ROS) production concomitant with the development of apoptosis.Conclusion: SKBr3 cells undergoing apoptosis demonstrate mitochondrial changes associated with ROS production. Bcl-2 transfection prevents these changes because it prevents apoptosis :Ind induces chemoresistance to Herbimycin in SKBr3. Flow cytometric measurement of drug induced mitochondrial changes and ROS production may facilitate in vitro assessment of chemosensitivity or chemoresistance in breast cancer.