Induction of HIF-2alpha is dependent on mitochondrial O2 consumption in an O2-sensitive adrenomedullary chromaffin cell line

During low O2 (hypoxia), hypoxia-inducible factor (HIF)-alpha is stabilized and translocates to the nucleus, where it regulates genes critical for survival and/or adaptation in low O2. While it appears that mitochondria play a critical role in HIF induction, controversy surrounds the underlying mechanism(s). To address this, we monitored HIF-2alpha expression and oxygen consumption in an O2-sensitive immortalized rat adrenomedullary chromaffin (MAH) cell line. Hypoxia (2-8% O2) caused a concentration- and time-dependent increase in HIF-2alpha induction, which was blocked in MAH cells with either RNA interference knockdown of the Rieske Fe-S protein, a component of complex III, or knockdown of cytochrome-c oxidase subunit of complex IV, or defective mitochondrial DNA (rho0 cells). Additionally, pharmacological inhibitors of mitochondrial complexes I, III, IV, i.e., rotenone (1 microM), myxothiazol (1 microM), antimycin A (1 microg/ml), and cyanide (1 mM), blocked HIF-2alpha induction in control MAH cells. Interestingly, the inhibitory effects of the mitochondrial inhibitors were dependent on O2 concentration such that at moderate-to-severe hypoxia (6% O2), HIF-2alpha induction was blocked by low inhibitor concentrations that were ineffective at more severe hypoxia (2% O2). Manipulation of the levels of reactive oxygen species (ROS) had no effect on HIF-2alpha induction. These data suggest that in this O2-sensitive cell line, mitochondrial O2 consumption, rather than changes in ROS, regulates HIF-2alpha during hypoxia.     

Hypoxic augmentation of Ca2+ channel currents requires a functional electron transport chain

The incidence of Alzheimer disease is increased following ischemic episodes, and we previously demonstrated that following chronic hypoxia (CH), amyloid beta (Abeta) peptide-mediated increases in voltage-gated L-type Ca(2+) channel activity contribute to the Ca(2+) dyshomeostasis seen in Alzheimer disease. Because in certain cell types mitochondria are responsible for detecting altered O(2) levels we examined the role of mitochondrial oxidant production in the regulation of recombinant Ca(2+) channel alpha(1C) subunits during CH and exposure to Abeta-(1-40). In wild-type (rho(+)) HEK 293 cells expressing recombinant L-type alpha(1C) subunits, Ca(2+) currents were enhanced by prolonged (24 h) exposure to either CH (6% O(2)) or Abeta-(1-40) (50 nm). By contrast the response to CH was absent in rho(0) cells in which the mitochondrial electron transport chain (ETC) was depleted following long term treatment with ethidium bromide or in rho(+) cells cultured in the presence of 1 microm rotenone. CH was mimicked in rho(0) cells by the exogenous production of O2(-.). by xanthine/xanthine oxidase. Furthermore Abeta-(1-40) enhanced currents in rho(0) cells to a degree similar to that seen in cells with an intact ETC. The antioxidants ascorbate (200 microm) and Trolox (500 microm) ablated the effect of CH in rho(+) cells but were without effect on Abeta-(1-40)-mediated augmentation of Ca(2+) current in rho(0) cells. Thus oxidant production in the mitochondrial ETC is a critical factor, acting upstream of amyloid beta peptide production in the up-regulation of Ca(2+) channels in response to CH.     

Apoptotic cell death induced by hydrogen peroxide in NT2 parental and mitochondrial DNA depleted cells

Oxidative stress has been implicated in several pathologies associated with degenerative processes. Mitochondria are involved in cell death by necrosis or apoptosis due to a large load of Ca2+, the formation of reactive oxygen species (ROS), mitochondrial depolarization and the release of cytochrome c that initiates the caspase cascade. Nevertheless, the role of mitochondria in cell death processes induced by hydrogen peroxide (H2O2) has not been fully established. In this study, we analyzed the cytotoxic effect of H2O2 on rho+ human teratocarcinoma (NT2) cells and on mitochondria-DNA depleted rho0 NT2 cells, lacking functional mitochondria. The cells were exposed to H2O2 for 24 h and cell viability was dose-dependently decreased in both cell lines upon H2O2 exposure, although cell susceptibility was higher in rho0 NT2 cells. Moreover a decrease in mitochondrial membrane potential (Deltapsi(m)), mitochondrial cytochrome c release, caspases activation and DNA fragmentation were largely induced by H2O2 and occurred in both cell lines. Nevertheless, increased cell toxicity in rho0 cells upon H2O2 exposure was accompanied by a higher activation of the effector caspases-3 and -6. The data support that, in general, no differences were observed in cells containing functional (rho+) or non-functional (rho0) mitochondria upon H2O2-induced apoptotic cell death.     

Hypoxic but not anoxic stabilization of HIF-1alpha requires mitochondrial reactive oxygen species

The molecular mechanisms by which cells detect hypoxia (1.5% O2), resulting in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) protein remain unclear. One model proposes that mitochondrial generation of reactive oxygen species is required to stabilize HIF-1alpha protein. Primary evidence for this model comes from the observation that cells treated with complex I inhibitors, such as rotenone, or cells that lack mitochondrial DNA (rho(0)-cells) fail to generate reactive oxygen species or stabilize HIF-1alpha protein in response to hypoxia. In the present study, we investigated the role of mitochondria in regulating HIF-1alpha protein stabilization under anoxia (0% O2). Wild-type A549 and HT1080 cells stabilized HIF-1alpha protein in response to hypoxia and anoxia. The rho(0)-A549 cells and rho(0)-HT1080 cells failed to accumulate HIF-1alpha protein in response to hypoxia. However, both rho(0)-A549 and rho(0)-HT1080 were able to stabilize HIF-1alpha protein levels in response to anoxia. Rotenone inhibited hypoxic, but not anoxic, stabilization of HIF-1alpha protein. These results indicate that a functional electron transport chain is required for hypoxic but not anoxic stabilization of HIF-1alpha protein.     

Reactive oxygen species generated at mitochondrial complex III stabilize hypoxia-inducible factor-1alpha during hypoxia: a mechanism of O2 sensing

During hypoxia, hypoxia-inducible factor-1alpha (HIF-1alpha) is required for induction of a variety of genes including erythropoietin and vascular endothelial growth factor. Hypoxia increases mitochondrial reactive oxygen species (ROS) generation at Complex III, which causes accumulation of HIF-1alpha protein responsible for initiating expression of a luciferase reporter construct under the control of a hypoxic response element. This response is lost in cells depleted of mitochondrial DNA (rho(0) cells). Overexpression of catalase abolishes hypoxic response element-luciferase expression during hypoxia. Exogenous H(2)O(2) stabilizes HIF-1alpha protein during normoxia and activates luciferase expression in wild-type and rho(0) cells. Isolated mitochondria increase ROS generation during hypoxia, as does the bacterium Paracoccus denitrificans. These findings reveal that mitochondria-derived ROS are both required and sufficient to initiate HIF-1alpha stabilization during hypoxia.     

Chronic hypoxia upregulates adenosine 2a receptor expression in chromaffin cells via hypoxia inducible factor-2α: role in modulating secretion

Catecholamine (CAT) release from chromaffin tissue plays an essential role in the fetus which develops in a low O₂ environment (hypoxia). To address molecular mechanisms regulating CAT secretion in low O₂, we exposed a fetal chromaffin-derived cell line (MAH cells) to chronic hypoxia (CHox; 2% O₂, 24 h) and assessed gene expression using microarrays, quantitative RT-PCR, and western blot. CHox caused a dramatic ∼12× upregulation of adenosine A2a receptor (A2aR) mRNA, an effect critically dependent upon hypoxia-inducible factor (HIF)-2α which bound the promoter of the A2aR gene. In amperometric studies, acute hypoxia and high K⁺ (30 mM) evoked quantal CAT secretion that was enhanced after CHox, and further potentiated during simultaneous A2aR activation by adenosine. A2aR activation also enhanced stimulus-induced rise in intracellular Ca²⁺ in control, but not HIF-2α-deficient, MAH cells. Thus, A2aR, adenosine, and HIF-2α are key contributors to the potentiation of CAT secretion in developing chromaffin cells during chronic hypoxia.     

The co-presence of Na+/Ca2+-K+ exchanger and Na+/Ca2+ exchanger in bovine adrenal chromaffin cells

We have previously shown that there is high Na(+)/Ca(2+) exchange (NCX) activity in bovine adrenal chromaffin cells. In this study, by monitoring the [Ca(2+)](i) change in single cells and in a population of chromaffin cells, when the reverse mode of exchanger activity has been initiated, we have shown that the NCX activity is enhanced by K(+). The K(+)-enhanced activity accounted for a significant proportion of the Na(+)-dependent Ca(2+) uptake activity in the chromaffin cells. The results support the hypothesis that both NCX and Na(+)/Ca(2+)-K(+) exchanger (NCKX) are co-present in chromaffin cells. The expression of NCKX in chromaffin cells was further confirmed using PCR and northern blotting. In addition to the plasma membrane, the exchanger activity, measured by Na(+)-dependent (45)Ca(2+) uptake, was also present in membrane isolated from the chromaffin granules enriched fraction and the mitochondria enriched fraction. The results support that both NCX and NCKX are present in bovine chromaffin cells and that the regulation of [Ca(2+)](i) is probably more efficient with the participation of NCKX.     

Mode of mitochondrial Ca2+ clearance and its influence on secretory responses in stimulated chromaffin cells

To study the role of mitochondrial Ca(2+) clearance in stimulated cells, changes in free Ca(2+) concentration in the cytosol, [Ca(2+)](c) and that in mitochondria, [Ca(2+)](m) along with secretory responses were observed using chromaffin cells co-loaded with Fura-2 and Rhod-2 in the perfused rat adrenal medulla. When the cells were stimulated with 40 mM K(+) in the perfusate, the duration of [Ca(2+)](m) response markedly increased with prolongation of the stimulation period, exhibiting a mean half-decay time of 21 min with 30s stimulation, whereas its amplitude was not altered with stimulations of 10-30s. A computer simulation analysis showed that such a mode of [Ca(2+)](m) response can be produced if excess Ca(2+) taken up by mitochondria precipitates as calcium phosphate (Pi) salt. In the presence of 5 microM rotenone plus 10 microM oligomycin, a decrease in the duration of [Ca(2+)](m) response and a slight but significant increase (24%) in the secretory response to 30s stimulation with 40 mM K(+) were observed. Simulation analyses suggested that this effect of rotenone may be due to reduction in mitochondrial Ca(2+) uptake induced by rotenone-elicited partial depolarization of the mitochondrial membrane potential. In chromaffin cells transsynaptically stimulated through the splanchnic nerve, the intensity of NAD(P)H autofluorescence changed with time courses similar to those of [Ca(2+)](m) responses. The temporal profiles of those two responses were prolonged in a similar manner by application of an inhibitor of mitochondrial Na(+)/Ca(2+) exchanger, CGP37157. Thus, due to the unique Ca(2+) buffering mechanism, [Ca(2+)](m) responses associated with massive mitochondrial Ca(2+) uptake may occur within a limited concentration range in which Ca(2+)-sensitive dehydrogenases are activated to control the mitochondrial redox state in stimulated chromaffin cells.     

GABA(B) receptor activation augments TASK-1 in MAH cells and mediates autoreceptor feedback during hypoxia

Previously, we demonstrated an autoregulatory feedback loop in the rat carotid body (CB), involving presynaptic GABA(B) receptor-mediated activation of the background K(+) channel TASK-1. Here, we examined the effects of the selective GABA(B) receptor agonist baclofen on K(+) currents in immortalised adrenomedullary chromaffin (MAH) cells, which share the same sympathoadrenal lineage as CB type I cells. Under symmetrical K(+) conditions, 50 microM baclofen enhanced a K(+) current which was linear and reversed close to 0 mV. Under physiological K(+) conditions, baclofen enhanced outward K(+) current and caused membrane hyperpolarisation, effects inhibited by 100 nM CGP 55845. Current enhancement was virtually abolished in the presence of 300 microM Zn(2+), a selective inhibitor of TASK-1. When recording membrane potential from MAH cells in clusters, hypoxic depolarisation was augmented by 100 nM CGP 55845. These data demonstrate that GABA(B) receptors mediate autoreceptor feedback in the adrenal medulla presumably via TASK-1, demonstrating a common autoregulatory feedback pathway in neurosecretory, chemosensitive cells.     

Elevated CO(2) levels cause mitochondrial dysfunction and impair cell proliferation

Elevated CO(2) concentrations (hypercapnia) occur in patients with severe lung diseases. Here, we provide evidence that high CO(2) levels decrease O(2) consumption and ATP production and impair cell proliferation independently of acidosis and hypoxia in fibroblasts (N12) and alveolar epithelial cells (A549). Cells exposed to elevated CO(2) died in galactose medium as well as when glucose-6-phosphate isomerase was knocked down, suggesting mitochondrial dysfunction. High CO(2) levels led to increased levels of microRNA-183 (miR-183), which in turn decreased expression of IDH2 (isocitrate dehydrogenase 2). The high CO(2)-induced decrease in cell proliferation was rescued by α-ketoglutarate and overexpression of IDH2, whereas proliferation decreased in normocapnic cells transfected with siRNA for IDH2. Also, overexpression of miR-183 decreased IDH2 (mRNA and protein) as well as cell proliferation under normocapnic conditions, whereas inhibition of miR-183 rescued the normal proliferation phenotype in cells exposed to elevated levels of CO(2). Accordingly, we provide evidence that high CO(2) induces miR-183, which down-regulates IDH2, thus impairing mitochondrial function and cell proliferation. These results are of relevance to patients with hypercapnia such as those with chronic obstructive pulmonary disease, asthma, cystic fibrosis, bronchopulmonary dysplasia, and muscular dystrophies.     

Low glucose sensitivity and polymodal chemosensing in neonatal rat adrenomedullary chromaffin cells

Glucose is the primary metabolic fuel in mammalian fetuses, yet mammals are incapable of endogenous glucose production until several hours after birth. Thus, when the maternal supply of glucose ceases at birth there is a transient hypoglycemia that elicits a counterregulatory surge in circulating catecholamines. Because the innervation of adrenomedullary chromaffin cells (AMCs) is immature at birth, we hypothesized that neonatal AMCs act as direct glucosensors, a property that could complement their previously established roles as hypoxia and acid hypercapnia sensors. During perforated-patch, whole cell recordings, low glucose depolarized and/or excited a subpopulation of neonatal AMCs; in addition, aglycemia (0 mM glucose) caused inhibition of outward K(+) current, blunted by the simultaneous activation of glibenclamide-sensitive K(ATP) channels. Some cells were excited by each of the three metabolic stimuli, i.e., aglycemia, hypoxia (Po(2) ～30 mmHg), and isohydric hypercapnia (10% CO(2); pH = 7.4). Using carbon fiber amperometry, aglycemia and hypoglycemia (3 mM glucose) induced robust catecholamine secretion that was sensitive to nickel (50 μM and 2 mM) and the L-type Ca(2+) channel blocker nifedipine (10 μM), suggesting involvement of both T-type and L-type voltage-gated Ca(2+) channels. Fura-2 measurements of intracellular Ca(2+) ([Ca(2+)] (i)) revealed that ～42% of neonatal AMCs responded to aglycemia with a significant rise in [Ca(2+)] (i). Approximately 40% of these cells responded to hypoxia, whereas ～25% cells responded to both aglycemia and hypoxia. These data suggest that together with hypoxia and acid hypercapnia, low glucose is another important metabolic stimulus that contributes to the vital asphyxia-induced catecholamine surge from AMCs at birth.     

Detection of reactive oxygen species via endogenous oxidative pentose phosphate cycle activity in response to oxygen concentration: implications for the mechanism of HIF-1alpha stabilization under moderate hypoxia

The oxidative pentose phosphate cycle (OPPC) is necessary to maintain cellular reducing capacity during periods of increased oxidative stress. Metabolic flux through the OPPC increases stoichiometrically in response to a broad range of chemical oxidants, including those that generate reactive oxygen species (ROS). Here we show that OPPC sensitivity is sufficient to detect low levels of ROS produced metabolically as a function of the percentage of O2. We observe a significant decrease in OPPC activity in cells incubated under severe and moderate hypoxia (ranging from <0.01 to 4% O2), whereas hyperoxia (95% O2) results in a significant increase in OPPC activity. These data indicate that metabolic ROS production is directly dependent on oxygen concentration. Moreover, we have found no evidence to suggest that ROS, produced by mitochondria, are needed to stabilize hypoxia-inducible factor 1alpha (HIF-1alpha) under moderate hypoxia. Myxothiazol, an inhibitor of mitochondrial electron transfer, did not prevent HIF-1alpha stabilization under moderate hypoxia. Moreover, the levels of HIF-1alpha that we observed after exposure to moderate hypoxia were comparable between rho0 cells, which lack functional mitochondria, and the wild-type cells. Finally, we find no evidence for stabilization of HIF-1alpha in response to the non-toxic levels of H2O2 generated by the enzyme glucose oxidase. Therefore, we conclude that the oxygen dependence of the prolyl hydroxylase reaction is sufficient to mediate HIF-1alpha stability under moderate as well as severe hypoxia.     

Effects of Hypoxia on the Catecholamine Release, Ca2+ Uptake, and Cytosolic Free Ca2+ Concentration in Cultured Bovine Adrenal Chromaffin Cells

The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K(+)-induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K(+)-induced catecholamine release was inhibited by hypoxia to approximately 40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura-2) gradually increased and reached a plateau of approximately 1.0 microM at 30 min, whereas the level was constant in the control medium (approximately 200 nM). High K(+)-induced increases in [Ca2+]i were inhibited by hypoxia to approximately 30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K(+)-induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K(+)-induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K(+)-induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage-dependent Ca2+ channels.     

Cytochrome c-mediated apoptosis in cells lacking mitochondrial DNA. Signaling pathway involving release and caspase 3 activation is conserved.

Mitochondria serve as a pivotal component of the apoptotic cell death machinery. However, cells that lack mitochondrial DNA (rho(0) cells) retain apparently normal apoptotic signaling. In the present study, we examined mitochondrial mechanisms of apoptosis in rho(0) osteosarcoma cells treated with staurosporine. Immunohistochemistry revealed that rho(0) cells maintained a normal cytochrome c distribution in mitochondria even though these cells were deficient in respiration. Upon staurosporine treatment, cytochrome c was released concomitantly with activation of caspase 3 and loss of mitochondrial membrane potential (Deltapsi(m)). After mitochondrial loss of cytochrome c, rho(0) cells underwent little change in glutathione (GSH) redox potential whereas a dramatic oxidation in GSH/glutathione disulfide (GSSG) pool occurred in parental rho(+) cells. These results show that mitochondrial signaling of apoptosis via cytochrome c release was preserved in cells lacking mtDNA. However, intracellular oxidation that normally accompanies apoptosis was lost, indicating that the mitochondrial respiratory chain provides the major source of redox signaling in apoptosis.     

Mitochondrial metabolism underlies hyperoxic cell damage

Exposure of mammals to hyperoxia causes pulmonary and ocular pathology. Hyperoxic damage and cell death may derive from enhanced intracellular formation of reactive oxygen species (ROS), probably of mitochondrial origin. There is, however, controversy on this point. When wild-type and respiration-deficient (rho(o)) HeLa cells were cultured in 80% O2, wild-type cells stopped growing after 5 days and died thereafter whereas rho(o) cells survived and grew to confluence. This tolerance of rho(o) cells for hyperoxia was not associated with greater resistance to oxidants such as hydrogen peroxide and t-butyl hydroperoxide. Under both 20% and 80% O2, rho(o) cells exhibited substantially decreased ROS production, and, under 80% O2, rho(o) cells showed no suppression of aconitase activity or mitochondrial protein carbonyl formation. Replacement of normal mitochondria in rho(o) cells restored ROS production and susceptibility to hyperoxia. Two other approaches that diminished mitochondrial ROS generation also increased tolerance for hyperoxia. HeLa cells constantly exposed to the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone, which enhances respiration but decreases ROS production, showed preferential survival under 80% O2, as did HeLa cells treated with chloramphenicol, which suppresses both respiration and mitochondrial ROS production. We conclude that interactions between respiring mitochondria and O2 are primarily responsible for hyperoxic cell damage.     

Deletion of mtDNA disrupts mitochondrial function and structure, but not biogenesis

The role of nuclear DNA (nDNA)-encoded proteins in the regulation of mitochondrial fission and fusion has been documented, yet the role of mitochondrial DNA (mtDNA) and encoded proteins in mitochondrial biogenesis remains unknown. Long-term treatment of a lymphoblastoid cell line Molt-4 with ethidium bromide generated mtDNA-deficient rho0 mutants. Depletion of mtDNA in rho0 cells produced functional and morphological changes in mitochondria without affecting the nuclear genome and encoded proteins. Indeed, the gene encoding subunit II of mitochondrial cytochrome c oxidase (COX II), a prototypical mitochondrial gene, was reduced in rho0 mutants blunting the activity of mitochondrial cytochrome coxidase. Yet, the amount of the nuclear beta-actin gene and the activity of citrate synthase, a mitochondrial matrix enzyme encoded by nDNA, remained unaffected in rho0 cells. Loss of mtDNA in rho0 cells was associated with significant distortion of mitochondrial structure, decreased electron density of the matrix and disorganized inner and outer membranes, resulting in the appearance of 'ghost-like' mitochondria. However, the number of mitochondria-like structures was not significantly different between mtDNA-deficient and parental cells. Thus, we conclude that cells lacking mtDNA still generate mitochondrial scaffolds, albeit with aberrant function.     

Mitochondrial localization of catalase provides optimal protection from H2O2-induced cell death in lung epithelial cells

Reactive oxygen species (ROS) can cause cell injury and death via mitochondrial-dependent pathways, and supplementation with antioxidants has been shown to ameliorate these processes. The c-Jun NH(2)-terminal kinase (JNK) pathway has been shown to play a critical role in ROS-induced cell death. To determine if targeting catalase (CAT) to the mitochondria provides better protection than cytosolic expression against H(2)O(2)-induced injury, the following two approaches were taken: 1) adenoviral-mediated transduction was performed using cytosolic (CCAT) or mitochondrial (MCAT) CAT cDNAs and 2) stable cell lines were generated overexpressing CAT in mitochondria (n = 3). Cells were exposed to 250 microM H(2)O(2), and cell survival, mitochondrial function, cytochrome c release, and JNK activity were analyzed. Although all viral transduced cells had a transient twofold increase in CAT activity, MCAT cells had significantly higher survival rates, the best mitochondrial function, and lowest JNK activity compared with CCAT and LacZ controls. The improved protection with MCAT was observed in primary type II lung epithelial cells and in transformed lung epithelial cells. In the three stable cell lines, cell survival directly correlated with extent of mitochondrial localization (r = 0.60572, P < 0.05) and not overall CAT activity (r = -0.45501, P < 0.05). Data indicate that targeting of antioxidants directly to the mitochondria is more effective in protecting lung epithelial cells against ROS-induced injury. This has important implications in antioxidant supplementation trials to prevent ROS-induced lung injury in critically ill patients.     

CD133 is a marker of bioenergetic stress in human glioma

Mitochondria dysfunction and hypoxic microenvironment are hallmarks of cancer cell biology. Recently, many studies have focused on isolation of brain cancer stem cells using CD133 expression. In this study, we investigated whether CD133 expression is regulated by bioenergetic stresses affecting mitochondrial functions in human glioma cells. First, we determined that hypoxia induced a reversible up-regulation of CD133 expression. Second, mitochondrial dysfunction through pharmacological inhibition of the Electron Transport Chain (ETC) produced an up-regulation of CD133 expression that was inversely correlated with changes in mitochondrial membrane potential. Third, generation of stable glioma cells depleted of mitochondrial DNA showed significant and stable increases in CD133 expression. These glioma cells, termed rho(0) or rho(0), are characterized by an exaggerated, uncoupled glycolytic phenotype and by constitutive and stable up-regulation of CD133 through many cell passages. Moreover, these rho(0) cells display the ability to form "tumor spheroids" in serumless medium and are positive for CD133 and the neural progenitor cell marker, nestin. Under differentiating conditions, rho(0) cells expressed multi-lineage properties. Reversibility of CD133 expression was demonstrated by transfering parental mitochondria to rho(0) cells resulting in stable trans-mitochondrial "cybrid" clones. This study provides a novel mechanistic insight about the regulation of CD133 by environmental conditions (hypoxia) and mitochondrial dysfunction (genetic and chemical). Considering these new findings, the concept that CD133 is a marker of brain tumor stem cells may need to be revised.     

Cells depleted of mitochondrial DNA (rho0) yield insight into physiological mechanisms.

A resurgence of interest in mitochondrial physiology has recently developed as a result of new experimental data demonstrating that mitochondria function as important participants in a diverse collection of novel intracellular signaling pathways. Cells depleted of mitochondrial DNA, or rho0 cells, lack critical respiratory chain catalytic subunits that are encoded in the mitochondrial genome. Although rho0 cells contain petit mitochondria, they cannot support normal oxidative phosphorylation and must survive and replicate using ATP derived solely from glycolysis. Without a functional electron transport chain, rho0 cells cannot normally regulate redox potential and their mitochondria appear to be incapable of generating reactive oxygen species. Emerging evidence suggests that these signals are important components in a number of mitochondria-initiated signaling pathways. The present article focuses on how rho0 cells have contributed to an understanding of the role that mitochondria play in distinct physiological pathways involved with apoptosis, glucose-induced insulin secretion, and oxygen sensing.