Characterization of cells with different mitochondrial membrane potential during apoptosis

Lugli, Enrico; Troiano, Leonarda; Ferraresi, Roberta; Roat, Erika; Prada, Nicole; Nasi, Milena; Pinti, Marcello; Cooper, Edwin L.; Cossarizza, Andrea

doi:10.1002/cyto.a.20188

Cited by 116 publications

(106 citation statements)

References 25 publications

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“…At 24 h after treatment with IFN-a, PLC-P/shRNA and PLC-P/shRNA-NC cells were stained by Annexin V-APC and propidium iodide (PI) (BD Biosciences, Franklin Lakes, NJ, USA), and analysed on a FACS Aria (BD Biosciences). Annexin V-positive and PI-negative cells, considered as early apoptotic cells, were used for the assessment of apoptosis in this study (Lugli et al, 2005).…”

Section: Annexin V Assaymentioning

confidence: 99%

Insulin-like growth factor-binding protein 7 alters the sensitivity to interferon-based anticancer therapy in hepatocellular carcinoma cells

et al. 2010

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BACKGROUND: A striking efficiency of interferon (IFN)-based anticancer therapy for advanced hepatocellular carcinoma (HCC) has been reported. Because its clinical efficiency greatly depends on each patient's local response, prediction of local response is crucial. METHODS: Continuous exposure of IFN-a to parental PLC/PRF/5 cells (PLC-P) and a limiting dilution method resulted in the establishment of IFN-resistant cell clones (PLC-Rs). Microarray analyses of PLC-P and PLC-Rs identified insulin-like growth factor-binding protein 7 (IGFBP7) as one of the most significantly downregulated genes in PLC-Rs. Changes in anticancer effects of IFN-a were examined in HCC cells after genetic manipulation of IGFBP7 expression. The correlation between immunohistochemically determined IGFBP7 expression and the response to IFN-a/5-fluorouracil (5-FU) therapy was investigated in surgically resected HCC specimens. RESULTS: PLC-R cells showed a remarkable downregulation of IGFBP7 and resistance to IFN-a, compared with PLC-P. Parental PLC/ PRF/5 cells transfected with short hairpin RNA against IGFBP7 showed a significant resistance to IFN-a relative to control cells (IC 50 fold increase ¼ 14.38 times). Insulin-like growth factor-binding protein 7 transfection into PLC-R restored sensitivity to IFN-a. In resected specimens, IGFBP7 expression significantly correlated with the response to IFN-a/5-FU therapy. CONCLUSION: IGFBP7 could be a useful predictor of the response to IFN-based therapy in advanced HCC.

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Section: Annexin V Assaymentioning

confidence: 99%

Insulin-like growth factor-binding protein 7 alters the sensitivity to interferon-based anticancer therapy in hepatocellular carcinoma cells

et al. 2010

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“…The gradient drives the synthesis of adenosinetriphosphate (ATP). DC m is a marker of the state of energization of the organelle [12][13][14][15][16][17][18][19][20][21], and can be influenced by the energetic status of the cell depending not strictly on mitochondrial activity alone but also on oxygen consumption, cytosolic ATP production and NADH:NAD 1 (nicotinamide adenine dinucleotide) ratio [12,22]. Bioenergetics is of importance in infectious disease because immune reactions are important consumers of energy [23].…”

Section: Mitochondrial Membrane Potential (Dc M ) As a Marker Of Mitomentioning

confidence: 99%

Mitochondrial membrane potential and apoptosis of blood mononuclear cells in untreated HIV‐1 infected patients

et al. 2009

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ObjectivesInfection with HIV leads to progressive CD4 T-cell loss, resulting in AIDS. Apoptosis is the main mechanism for the loss of infected and bystander cells, but the complex interacting factors inducing and inhibiting apoptosis are not fully understood. Mitochondrial dysfunction is a pivotal step of the apoptotic cascade and can result in reduced mitochondrial membrane potential. MethodsThe mitochondrial membrane potential of peripheral blood mononuclear cells (PBMC) was measured by flow cytometry using the dye JC-1 (Molecular Probes Inc). Apoptotic cells were identified using the Annexin V assay (Becton Dickinson GmbH). ResultsThe mitochondrial membrane potential of PBMC was significantly decreased and apoptotic cell rate was increased in HIV-infected therapy-naïve patients compared with HIV-negative controls. There was a highly significant correlation between the mitochondrial membrane potential and the rate of apoptosis. CD4 cell count was correlated negatively to the apoptotic rate and positively to the mitochondrial membrane potential. ConclusionsThe JC-1 assay is a sensitive tool to detect changes of mitochondrial membrane potential associated with apoptosis in HIV-infected therapy-naïve patients. We could show in vivo that a reduction of mitochondrial membrane potential is correlated to apoptosis of PBMC, CD4 cell count and HIV viral load during HIV infection. IntroductionInfection with HIV leads to progressive CD4 T-cell death, resulting in the development of AIDS. The mechanisms triggering this CD4 T-cell death are still not understood fully, but data indicate that apoptosis plays a major role [1]. There is a correlation between the extent of apoptosis and disease progression [2,3], and highly active antiretroviral therapy is associated with a lower level of CD4 T-cell apoptosis in HIV-1 infected patients [4][5][6][7].Both infected and uninfected CD4 T-cells can die during HIV-1 infection by different cell death pathways, but HIV-1 induced bystander CD4 T-cell demise is now recognized as pivotal to the development of immunodeficiency. Chronically increased activation of the immune system may contribute directly to progressive CD4 T-cell depletion, irrespective of viral load [8,9]. Upon receiving a death signal, a cascade of molecular events resulting in the activation or inactivation of numerous cellular proteins is initiated, leading to morphological and biochemical changes such as plasma membrane blebbing, DNA fragmentation and mitochondrial dysfunction. The regulators of the apoptotic pathway exert their function primarily at the mitochondrion by either preventing or inducing mitochondrial dysfunction [10,11]. Furthermore, there is evidence that resistance to apoptosis in persistently infected cells involves direct modulation of the mitochondrial pathway [1]. The measurement of the mitochondrial membrane potential of peripheral blood mononuclear cells (PBMC) might be a useful tool to [24]. In the early stages of apoptosis, there are changes at the cell surface. One of these plasma membrane al...

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“…Until recently the flow cytometry community has searched for a means to distinguish between cells undergoing apoptosis or oncosis by standard flow cytometry (6,(11)(12)(13)(14). There are a plethora of assays for the study of apoptosis several hours after induction, by measurement of mitochondrial membrane potential, caspases, annexin V binding through to cell death or necrosis as measured by lack of viability and DNA fragmentation.…”

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confidence: 99%

“…Classically caspases become activated during apoptosis; this activation can be detected by fluorogenic caspase peptide substrates (PhiPhiLux) and Fluorochrome-Labeled Inhibitors of Caspases or FLICA, which become fluorogenic when acted on by active caspases (18,19). These fluorescence signals resulting from activation of caspases can be detected flow cytometrically and multiplexed with annexin V, mitochondrial potential and reactive oxygen dyes further subdivisions of apoptosis and necrosis can be studied (5,6,(20)(21)(22). Another approach has been the use of SYTO dyes, originally thought of as nucleic acid binding dyes that could be used to detect apoptosis (23)(24)(25)(26).…”

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confidence: 99%

Real‐time flow cytometry for the kinetic analysis of oncosis

Warnes

Martins

2011

Cytometry Pt A

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The standard method of distinguishing apoptotic and oncotic cells has been by microscopic analysis of nuclei and cell membrane morphology. Thus a rapid test for analyzing large numbers of cells in the study of cell necrobiology has not been possible until the recent advent of the Amnis Image-stream and real-time Lab-on-a-Chip technologies. An interesting difference between apoptosis and oncosis is that they are ATP dependent and independent processes, respectively. Here we describe an assay measuring real-time kinetic changes in the potential differences of the inner mitochondrial membrane (mmp) and the plasma membrane (pmp) in cells immediately before and after the addition of the inducing agent. Live cells were loaded with carbocyanine dye DiIC 1 (5) and bis-oxonol (DiBAC 4 (5)) to measure mmp and pmp in conjunction with annexin V-FITC and DAPI labeling for gating out annexin V binding cells and dead cells respectively. Live cells gave specific membrane signatures in response to apoptotic or oncotic reagents in real-time. Apoptosis showed little change in mmp and pmp signals over the course of 25 min, the mitochondria only showed a slight hyperpolarization. In contrast chemical treatment with oxidative phosphorylation blocker, sodium azide (SA) caused an immediate hyperpolarization spike followed by a complete abrogation of mmp over a 25 min time course. Treatment with SA (1%) also caused plasma membrane depolarization. Likewise detergent (0.01% Triton X-100) treatments also caused abrogation of mmp and depolarization of pmp. Whereas heat shock (428C) treatment showed only a slight mitochondrial membrane potential depolarization. These flow cytometric observations were confirmed by confocal microscopy. This novel real-time kinetic assay measuring mitochondrial and plasma membrane potential changes has important implications in the field of cell necrobiology in that it allows the researcher to differentiate apoptotic and oncotic processes in an immediate manner for the first time. '

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Characterization of cells with different mitochondrial membrane potential during apoptosis

Cited by 116 publications

References 25 publications

Insulin-like growth factor-binding protein 7 alters the sensitivity to interferon-based anticancer therapy in hepatocellular carcinoma cells

Insulin-like growth factor-binding protein 7 alters the sensitivity to interferon-based anticancer therapy in hepatocellular carcinoma cells

Mitochondrial membrane potential and apoptosis of blood mononuclear cells in untreated HIV‐1 infected patients

Real‐time flow cytometry for the kinetic analysis of oncosis

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