MitoTracker labeling in primary neuronal and astrocytic cultures: influence of mitochondrial membrane potential and oxidants

Buckman, Jennifer F.; Hernández, Hélène; Kress, Geraldine J.; Votyakova, Tatyana V.; Pal, Sumon K.; Reynolds, Ian J.

doi:10.1016/s0165-0270(00)00340-x

Cited by 210 publications

(158 citation statements)

References 30 publications

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“…The reduced form of the dye is taken up by actively respiring cells. Inside the cells, the dye is oxidized to mitochondria-selective form, which sequesters inside the mitochondrial membrane-matrix compartment and is converted to fluorescent form (extinction 525 nm and emission 576 nm) (Buckman et al, 2001). As shown in Figure 1c, the fluorescence units at emission 576 nm increased linearly for about 10 min after a ten-min lag.…”

Section: Growth Characteristics and Morphology Of Mitochondrial Dna-dmentioning

confidence: 92%

“…The membrane potential was measured as a function of mitochondrial uptake of MitoTracker Orange CM-H 2 TM ROS (50 nM) added to the cell suspension. Fluorescence was monitored in a multiwavelength-excitation dual wavelength-emission Delta RAM PTI spectrofluorometer at 525 nm Excitation and 575 nm Emission (Buckman et al, 2001).…”

Section: Measurement Of Mitochondrial Membrane Potential and Calciummentioning

confidence: 99%

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Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells

Amuthan

Biswas

Ananadatheerthavarada

et al. 2002

Oncogene

231

271

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We have investigated mechanisms of mitochondrial stressinduced phenotypic changes and cell invasion in tumorigenic but poorly invasive human pulmonary carcinoma A549 cells that were partly depleted of mitochondrial DNA (mtDNA). Depletion of mtDNA (genetic stress) caused a markedly lower electron transport-coupled ATP synthesis, loss of mitochondrial membrane potential, elevation of steady state [Ca 2+ ] c , and notably induction of both glycolysis and gluconeogenic pathway enzymes. Markers of tumor invasion, cathepsin L and TGFb1, were overexpressed; calcium-dependent MAP kinases (ERK1 and ERK2) and calcineurin were activated. The levels of anti-apoptotic proteins Bcl2 and Bcl-X L were increased, and the cellular levels of pro-apoptotic proteins Bid and Bax were reduced. Both mtDNA-depleted cells (genetic stress) and control cells treated with carbonyl cyanide m-chlorophenylhydrazone (metabolic stress) exhibited higher invasive behavior than control cells in a Matrigel basement membrane matrix assay system. MtDNA-depleted cells stably expressing anti-sense cathepsin L RNA, TGFb1 RNA, or treated with specific inhibitors showed reduced invasion. Reverted cells with 80% of control cell mtDNA exhibited marker protein levels, cell morphology and invasive property closer to control cells. Our results suggest that the mitochondriato-nucleus signaling pathway operating through increased [Ca 2+ ] c plays an important role in cancer progression and metastasis. Oncogene (2002Oncogene ( ) 21, 7839 -7849. doi:10.1038 Keywords: mitochondria; calcium signaling; cathepsin L; TGFb; tumor invasion; MAP kinases IntroductionThe role of mitochondria in carcinogenesis was initially suggested by Warburg et al. (1926;Warburg, 1956), based on the observation that number of experimentally induced rodent tumors exhibit reduced respiration-coupled oxidative metabolism and increased glycolysis. Since then, altered mitochondrial morphology, as well as changes in mitochondrial enzyme patterns and membrane transport systems, have been described in several tumor types (Zafar et al., 1982). Cell fusion studies (Jonasson et al., 1977;Howell and Sager, 1978;Giguere and Morais, 1981) also suggest that unknown cytoplasmic elements may play roles in carcinogenesis. Fusion between normal cytoplasm and karyoplasts from malignant phenotypes resulted in the ablation of tumorigenicity (Israel and Schaeffer, 1987) and conversely, the cytoplasm of the malignant phenotype could transfer the malignancy to the karyoplasts from normal cells (Israel and Schaeffer, 1988). Studies using mitochondrial DNA (mtDNA)-depleted tumor cells to evaluate the role of mtDNA, and thus mitochondrial function in tumorigenicity have yielded mixed results (Giguere and Morais, 1981;Morais et al., 1994;Cavalli et al., 1997;Cavalli and Liang, 1998;Hofhaus and Gattermann, 1999). Cavalli et al. (1997) found diminished tumor formation by glioblastoma cells following depletion of mtDNA (r 0 cells) with ethidium bromide treatment, while Morais et al. (1994) found increased capacity to p...

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Section: Growth Characteristics and Morphology Of Mitochondrial Dna-dmentioning

confidence: 92%

Section: Measurement Of Mitochondrial Membrane Potential and Calciummentioning

confidence: 99%

Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells

Amuthan

Biswas

Ananadatheerthavarada

et al. 2002

Oncogene

231

271

View full text Add to dashboard Cite

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“…The Δψ M or the mitochondrial mass in fixed cells grown in glass coverslips was estimated as follows (Sugrue et al 1999;Wadia et al 1998;Buckman et al 2001). Three hours after exposure to treatments, the medium in each well was supplemented with 50 nM CMTMR or 50 nM MTGFM and incubated at 37°C 5% CO 2 for 15 min or 30 min, respectively.…”

Section: Immunohistochemistry and Double-fluorescence Labelingmentioning

confidence: 99%

Mitochondrial ATP-sensitive potassium channels enhance angiotensin-induced oxidative damage and dopaminergic neuron degeneration. Relevance for aging-associated susceptibility to Parkinson’s disease

Rodríguez‐Pallares

Parga

Joglar

et al. 2011

AGE

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Recent studies have shown that reninangiotensin system overactivation is involved in the aging process in several tissues as well as in longevity and aging-related degenerative diseases by increasing oxidative damage and inflammation. We have recently shown that angiotensin II enhances dopaminergic degeneration by increasing levels of reactive oxygen species and neuroinflammation, and that there is an aging-related increase in angiotensin II activity in the substantia nigra in rats, which may constitute a major factor in the increased risk of Parkinson's disease with aging. The mechanisms involved in the above mentioned effects and particularly a potential angiotensin-mitochondria interaction have not been clarified. The present study revealed that activation of mitochondrial ATP-sensitive potassium channels [mitoK(ATP)] may play a major role in the angiotensin II-induced effects on aging and neurodegeneration. Inhibition of mitoK(ATP) channels with 5-hydroxydecanoic acid inhibited the increase in dopaminergic cell death induced by angiotensin II, as well as the increase in superoxide/superoxide-derived reactive oxygen species levels and the angiotensin II-induced decrease in the mitochondrial inner membrane potential in cultured dopaminergic neurons. The present study provides data for considering brain renin-angiotensin system and mitoK(ATP) channels as potential targets for protective therapy in agingassociated diseases such as Parkinson's disease.

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“…32 To analyze the mitochondrial activity of the cells we took advantage of two fluorescent markers which, when incorporated into cells, emit fluorescence light, Mitotracker Green representing mitochondrial mass and Mitotracker Orange mitochondrial oxidative capacity, respectively. 33 We have taken the ratio of Mitotracker Orange to Mitotracker Green signal as a mitochondrial metabolic index representing the proportion of the actively respirating mitochondria from the total mitochondrial mass. The results are shown in Figure 5.…”

Section: Survivin Expression In Mdck Cellsmentioning

confidence: 99%

Different responses in transformation of MDCK cells in 2D and 3D culture by v-Src as revealed by microarray techniques, RT-PCR and functional assays

Töyli

Rosberg-Kulha

Capra

et al. 2010

Laboratory Investigation

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Differentiation and transformation of untransformed and ts-Src-transformed canine kidney MDCK cells in 2D and 3D environment were investigated using microarray technique, RT-PCR, confocal microscopy and functional assays. Activated Src induced epithelial-mesenchymal transition (EMT) in 2D environment followed by translocation of junctional proteins to the cytoplasm, without significant changes in protein expression. In 3D environment untransformed MDCK cells formed cell cysts with apical domain facing a lumen, E-cadherin delineating the lateral membranes, ZO-1 at tight junctions and caspase-3 in apoptotic cells captured within the lumen. This was accompanied by reduced expression of an apoptosis inhibitor, survivin and vesicle transport effectors, rab 7 and 8, whereas rab 5 expression increased. In 3D environment activated Src induced changes in expression of over 100 genes as revealed by microarray analysis, mostly involved in cell signaling, division and energy metabolism. Only response in cytoskeletal components was decreased expression of actin and Arp2/3 by v-Src, whereas two p120catenin binding proteins Kaiso and Nanos increased their expression. Concomitantly, apoptosis was inhibited by v-Src resulting in formation of a sphere with epitheloid cells facing extracellular matrix and undifferentiated cells captured within the cluster. This was accompanied by increased expression of apoptosis inhibitor survivin, as revealed by western blotting. Mitochondrial membrane potential in untransformed MDCK cells was lower than in ts-Src-MDCK cells in early days of cluster formation correlating with the induction of apoptosis. Hence, v-Src activation in 3D environment did not induce EMT, but brought about inhibition of apoptosis and increased proliferation where increased expression of survivin and inhibition of the mitochondrial permeability have a role.

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MitoTracker labeling in primary neuronal and astrocytic cultures: influence of mitochondrial membrane potential and oxidants

Cited by 210 publications

References 30 publications

Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells

Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells

Mitochondrial ATP-sensitive potassium channels enhance angiotensin-induced oxidative damage and dopaminergic neuron degeneration. Relevance for aging-associated susceptibility to Parkinson’s disease

Different responses in transformation of MDCK cells in 2D and 3D culture by v-Src as revealed by microarray techniques, RT-PCR and functional assays

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