BACKGROUND: During induction of apoptosis, the pro-apoptotic member of the Bcl-2 protein family (Bax) undergoes translocation to the mitochondria. The translocation, which leads to accumulation of Bax in the mitochondrial intermembrane space, appears to be the critical event determining release of cytochrome c to cytosol: the latter event triggers the irreversible steps of apoptosis, namely, the activation of caspases and the initiation of the degradation of many proteins. The aim of this study was to utilize the morphometric capabilities of the laser scanning cytometer (LSC) and adapt this instrument to detect and measure in situ the process of translocation of Bax to mitochondria. METHODS: Human breast carcinoma MCF-7 cells growing on microscope slides were treated with the DNA topoisomerase I inhibitor, camptothecin (CPT). CPT is known to induce apoptosis preferentially of S-phase cells. The cells were fixed and permeabilized on the slides, their DNA was stained with propidium iodide (PI), Bax was detected immunocytochemically with the fluoresceinated antibody, and red and green fluorescence emission was measured by the LSC. RESULTS: Prior to induction of apoptosis, Bax was uniformly and diffusely dispersed in the cell nucleus and cytoplasm. Its translocation and accumulation in mitochondria in cells undergoing apoptosis were detected and measured by the LSC as the increase in intensity of maximal pixel of Bax immunofluorescence. Bivariate analysis of DNA content versus maximal pixel of Bax fluorescence revealed that the CPT-induced Bax translocation into mitochondria was preferential to S-phase cells. Total cellular Bax immunofluorescence measured by flow cytometry, however, was increased in all phases of the cycle without a preference to S-phase cells. CONCLUSION: Changes in abundance and localization of particular proteins that undergo translocation within the cell, leading to their altered local density, may be conveniently detected by the LSC by taking advantage of its morphometric capabilities. Measurement of total cellular Bax immunofluorescence by flow cytometry combined with analysis of its translocation by LSC revealed that apoptosis of S-phase cells induced by CPT was unrelated to overall Bax abundance per cell but correlated with its accumulation in mitochondria. Copyright 2000 Wiley-Liss, Inc.
BACKGROUND: During induction of apoptosis, the pro-apoptotic member of the Bcl-2 protein family (Bax) undergoes translocation to the mitochondria. The translocation, which leads to accumulation of Bax in the mitochondrial intermembrane space, appears to be the critical event determining release of cytochrome c to cytosol: the latter event triggers the irreversible steps of apoptosis, namely, the activation of caspases and the initiation of the degradation of many proteins. The aim of this study was to utilize the morphometric capabilities of the laser scanning cytometer (LSC) and adapt this instrument to detect and measure in situ the process of translocation of Bax to mitochondria. METHODS:Human breast carcinoma MCF-7 cells growing on microscope slides were treated with the DNA topoisomerase I inhibitor, camptothecin (CPT). CPT is known to induce apoptosis preferentially of S-phase cells. The cells were fixed and permeabilized on the slides, their DNA was stained with propidium iodide (PI), Bax was detected immunocytochemically with the fluoresceinated antibody, and red and green fluorescence emission was measured by the LSC. RESULTS: Prior to induction of apoptosis, Bax was uniformly and diffusely dispersed in the cell nucleus and cytoplasm. Its translocation and accumulation in mitochondria in cells undergoing apoptosis were detected and measured by the LSC as the increase in intensity of maximal pixel of Bax immunofluorescence. Bivariate analysis of DNA content versus maximal pixel of Bax fluorescence revealed that the CPT-induced Bax translocation into mitochondria was preferential to S-phase cells. Total cellular Bax immunofluorescence measured by flow cytometry, however, was increased in all phases of the cycle without a preference to S-phase cells. CONCLUSION: Changes in abundance and localization of particular proteins that undergo translocation within the cell, leading to their altered local density, may be conveniently detected by the LSC by taking advantage of its morphometric capabilities. Measurement of total cellular Bax immunofluorescence by flow cytometry combined with analysis of its translocation by LSC revealed that apoptosis of S-phase cells induced by CPT was unrelated to overall Bax abundance per cell but correlated with its accumulation in mitochondria. Copyright 2000 Wiley-Liss, Inc.
Authors: Zbigniew Darzynkiewicz; Frank Traganos; Hong Zhao; H Dorota Halicka; Joanna Skommer; Donald Wlodkowic Journal: Methods Cell Biol Date: 2011 Impact factor: 1.441