Gamma rays induce a p53-independent mitochondrial biogenesis that is counter-regulated by HIF1α.

Mitochondrial biogenesis is an orchestrated process that presides to the regulation of the organelles homeostasis within a cell. We show that γ-rays, at doses commonly used in the radiation therapy for cancer treatment, induce an increase in mitochondrial mass and function, in response to a genotoxic stress that pushes cells into senescence, in the presence of a functional p53. Although the main effector of the response to γ-rays is the p53-p21 axis, we demonstrated that mitochondrial biogenesis is only indirectly regulated by p53, whose activation triggers a murine double minute 2 (MDM2)-mediated hypoxia-inducible factor 1α (HIF1α) degradation, leading to the release of peroxisome-proliferator activated receptor gamma co-activator 1ß inhibition by HIF1α, thus promoting mitochondrial biogenesis. Mimicking hypoxia by HIF1α stabilization, in fact, blunts the mitochondrial response to γ-rays as well as the induction of p21-mediated cell senescence, indicating prevalence of the hypoxic over the genotoxic response. Finally, we also show in vivo that post-radiotherapy mitochondrial DNA copy number increase well correlates with lack of HIF1α increase in the tissue, concluding this may be a useful molecular tool to infer the trigger of a hypoxic response during radiotherapy, which may lead to failure of activation of cell senescence.

The antioxidant function of Bcl-2 preserves cytoskeletal stability of cells with defective respiratory complex I.

Human thyroid carcinoma XTC.UC1 cells harbor a homoplasmic frameshift mutation in the MT-ND1 subunit of respiratory complex I. When forced to use exclusively oxidative phosphorylation for energy production by inhibiting glycolysis, these cells triggered a caspase-independent cell death pathway, which was associated to a significant imbalance in glutathione homeostasis and a cleavage of the actin cytoskeleton. Overexpression of the anti-apoptotic Bcl-2 protein significantly increased the level of endogenous reduced glutathione, thus preventing its oxidation after the metabolic stress. Furthermore, Bcl-2 completely inhibited actin cleavage and increased cell adhesion, but was unable to improve cellular viability. Similar effects were obtained when XTC.UC1 cells were incubated with exogenous glutathione. We hence propose that Bcl-2 can safeguard cytoskeletal stability through an antioxidant function.

Rare mtDNA variants in Leber hereditary optic neuropathy families with recurrence of myoclonus.

OBJECTIVE: To investigate the mechanisms underlying myoclonus in Leber hereditary optic neuropathy (LHON). METHODS: Five patients and one unaffected carrier from two Italian families bearing the homoplasmic 11778/ND4 and 3460/ND1 mutations underwent a uniform investigation including neurophysiologic studies, muscle biopsy, serum lactic acid after exercise, and muscle ((31)P) and cerebral ((1)H) magnetic resonance spectroscopy (MRS). Biochemical investigations on fibroblasts and complete mitochondrial DNA (mtDNA) sequences of both families were also performed. RESULTS: All six individuals had myoclonus. In spite of a normal EEG background and the absence of giant SEPs and C reflex, EEG-EMG back-averaging showed a preceding jerk-locked EEG potential, consistent with a cortical generator of the myoclonus. Specific comorbidities in the 11778/ND4 family included muscular cramps and psychiatric disorders, whereas features common to both families were migraine and cardiologic abnormalities. Signs of mitochondrial proliferation were seen in muscle biopsies and lactic acid elevation was observed in four of six patients. (31)P-MRS was abnormal in five of six patients and (1)H-MRS showed ventricular accumulation of lactic acid in three of six patients. Fibroblast ATP depletion was evident at 48 hours incubation with galactose in LHON/myoclonus patients. Sequence analysis revealed haplogroup T2 (11778/ND4 family) and U4a (3460/ND1 family) mtDNAs. A functional role for the non-synonymous 4136A>G/ND1, 9139G>A/ATPase6, and 15773G>A/cyt b variants was supported by amino acid conservation analysis. CONCLUSIONS: Myoclonus and other comorbidities characterized our Leber hereditary optic neuropathy (LHON) families. Functional investigations disclosed a bioenergetic impairment in all individuals. Our sequence analysis suggests that the LHON plus phenotype in our cases may relate to the synergic role of mtDNA variants.

Caspase-independent death of Leber's hereditary optic neuropathy cybrids is driven by energetic failure and mediated by AIF and Endonuclease G.

Leber's hereditary optic neuropathy (LHON) is associated with mitochondrial DNA point mutations affecting different subunits of complex I. By replacing glucose with galactose in the medium, cybrids harboring each of the three LHON pathogenic mutations (11778/ND4, 3460/ND1, 14484/ND6) suffered a profound ATP depletion over a few hours and underwent apoptotic cell death, which was caspase-independent. Control cybrids were unaffected. In addition to cytochrome c, apoptosis inducing factor (AIF) and endonuclease G (EndoG) were also released from the mitochondria into the cytosol in LHON cybrids, but not in control cells. Exposure of isolated nuclei to cytosolic fractions from LHON cybrids maintained in galactose medium caused nuclear fragmentation, which was strongly reduced by immuno-depletion with anti-AIF and anti-EndoG antibodies. In conclusion, the caspase-independent death of LHON cybrids incubated in galactose medium is triggered by rapid ATP depletion and mediated by AIF and EndoG.

Apoptosis induced by staurosporine in ECV304 cells requires cell shrinkage and upregulation of Cl- conductance.

We show that dysregulation of the Cl- homeostasis mediates the staurosporine-induced apoptotic cell death in human ECV304 cells. A pronounced apoptotic volume decrease (AVD), and an increase in plasma membrane Cl- conductance were early (<1 h) events following staurosporine challenge. Both processes were involved in apoptotic death, as demonstrated by the observation that the Cl- channel blocker phloretin inhibited both the staurosporine-evoked Cl- current and AVD, and preserved cell viability. Prolonged incubation (>2 h) with staurosporine caused a decrease in intracellular pH, which, however, was not required for the progression of the apoptotic process, because inhibitors of proton extrusion pathways, which lowered cytoplasmic pH, failed to inhibit both caspase-3 activation and DNA laddering. Moreover, clamping the cytosolic pH to an alkaline value did not prevent the apoptotic cell death. Collectively, these data demonstrate that staurosporine-mediated apoptosis of ECV304 cells is caused by the upregulation of Cl- channel activity and subsequent AVD, but is independent of intracellular acidification.

Staurosporine induces apoptotic volume decrease (AVD) in ECV304 cells.

Incubation of ECV304 cells with 1 micro M staurosporine (STS) causes apoptotic cell death. In the present study, we investigate whether a significant apoptotic volume decrease (AVD) was apparent during the very early times (1 h) of the apoptotic process. Our data suggest that upregulation of Cl(-) (and possibly K(+)) channels by STS may be a very early primary event required for the subsequent onset of AVD, which results in apoptosis.

The phorbol ester PMA and cyclic AMP activate different Cl(-) and HCO3(-) fluxes in C127 cells expressing CFTR.

In mouse mammary epithelial C127 cells expressing wild-type cystic fibrosis transmembrane conductance regulator (CFTR), chloride efflux, measured with the Cl(-)-sensitive dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), was stimulated by activation of protein kinase A with cyclic AMP elevating agents forskolin plus 3-isobutyl-1-methyl-xanthine (IBMX) and, to a less extent, by activation of protein kinase C with the phorbol 12-myristate 13-acetate (PMA). Conversely, bicarbonate influx, determined by intracellular alkalinization of cells incubated with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluoresceintetraacetoxymethyl ester (BCECF-AM), was stimulated by cyclic AMP elevation, but not by PMA. Patch clamp analysis revealed that PMA activated a Cl(-) current with the typical biophysical characteristics of swelling-activated current and not of CFTR.

Sphingosine-1-phosphate activates phospholipase D in human airway epithelial cells via a G protein-coupled receptor.

Sphingosine-1-phosphate (SPP) acts as a first messenger in immortalized human airway epithelial cells (CFNPE9o(-)), possibly interacting with an Edg family receptor. Expression of the SPP receptors Edg-1 and Edg-3, as well as a low level of Edg-5/H218, was detected in these cells, in agreement with their ability to specifically bind SPP. The related lipids, lysophosphatidic acid and sphingosylphosphorylcholine, were unable to displace SPP from its high affinity binding sites, suggesting that the biological responses to these different lysolipids are mediated by distinct receptors. SPP markedly inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner and caused a remarkable elevation of intracellular calcium, both effects being sensitive to pertussis toxin treatment. Most importantly, SPP stimulated phosphatidic acid formation, which was maximal after 2 min and decreased within 8-10 min. In the presence of butan-1-ol, suppression of SPP-induced phosphatidic acid formation and production of phosphatidylbutanol were found, clearly indicating activation of phospholipase D (PLD). This finding was also confirmed by analysis of the fatty acid composition of phosphatidic acid, showing an increase in the monounsaturated oleic acid only. The decrease of phosphatidic acid level after 8-10 min incubation with SPP was accompanied by a parallel increase of diacylglycerol production, which was abolished in the presence of butan-1-ol. This result indicates that activation of phospholipase D is followed by stimulation of phosphatidate phosphohydrolase activity. Phosphatidic acid formation was insensitive to protein kinase C inhibitors and almost completely inhibited by pertussis toxin treatment, suggesting that SPP activates phospholipase D via a G(i/o) protein-coupled receptor.

HIV-1 nef expression inhibits the activity of a Ca2+-dependent K+ channel involved in the control of the resting potential in CEM lymphocytes.

The HIV-1 Nef protein plays an important role in the development of the pathology associated with AIDS. Despite various studies that have dealt with different aspects of Nef function, the complete mechanism by which it alters the physiology of infected cells remains to be established. Nef can associate with cell membranes, therefore supporting the hypothesis that it might interact with membrane proteins as ionic channels and modify their electrical properties. By using the patch-clamp technique, we found that Nef expression determines a 25-mV depolarization of lymphoblastoid CEM cells. Both charybdotoxin (CTX) and the membrane-permeable Ca2+ chelator BAPTA/AM depolarized the membrane of native cells without modifying that of Nef-transfected cells. These data suggested that the resting potential in native CEM cells is settled by a CTX- and Ca2+-sensitive K+ channel (KCa,CTX), whose activity is absent in Nef-expressing cells. This was confirmed by direct measurements of whole-cell KCa,CTX currents. Single-channel recordings on excised patches showed that a KCa,CTX channel of 35 pS with a half-activation near 400 nM Ca2+ was present in both native and Nef-transfected cells. The measurements of free intracellular Ca2+ were not different in the two cell lines, but Nef-transfected cells displayed an increased Ca2+ content in ionomycin-sensitive stores. Taken together, these results indicate that Nef expression alters the resting membrane potential of the T lymphocyte cell line by inhibiting a KCa,CTX channel, possibly by intervening in the regulation of intracellular Ca2+ homeostasis.

Arachidonic acid release by ionomycin and phorbol ester is similar in C127 epithelial cells expressing wild-type or mutated (delta F508) cystic fibrosis transmembrane conductance regulator.

The Ca2+ ionophore ionomycin induced cytosolic [Ca2+ ]i elevation as well as strong activation of Cl- efflux in mouse mammary epithelial cell lines expressing wild-type or mutated (deletion of phenylalaline 508) cystic fibrosis transmembrane conductance regulator (CFTR) or vector. Ionomycin-induced Cl- efflux was abolished by the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, whereas both activators and inhibitors of phospholipase A2 had no effect, indicating the involvement of Ca2+-dependent Cl- channels. Stimulation of arachidonic acid release by ionomycin and phorbol ester was not significantly different between wild-type or mutated cell lines, whereas vector-transfected cells exhibited a significant higher release, which was shown to be due to larger amount of immunoreactive cytosolic phospholipase A2. These results indicate that phospholipase A2 activity of C127 cells was not influenced by the presence of wild-type or mutated CFTR.

Intracellular calcium mobilization and phospholipid degradation in sphingosylphosphorylcholine-stimulated human airway epithelial cells.

Extracellular sphingosylphosphorylcholine (SPC) caused a remarkable elevation in the intracellular Ca2+ concentration ([Ca2+]i) in immortalized human airway epithelial cells (CFNP9o-). An increase in total inositol phosphates formation was determined; however, the dose responses for [Ca2+]i elevation and inositol phosphates production were slightly different and, furthermore, PMA and pertussis toxin almost completely inhibited [Ca2+]i mobilization by SPC, whereas inositol phosphates production was only partially reduced. The possible direct interaction of SPC with Ca2+ channels of intracellular stores was determined by experiments with permeabilized cells, where SPC failed to evoke Ca2+ release, whereas lysophosphatidic acid was shown to be effective. The level of phosphatidic acid was increased by SPC only in the presence of AACOCF3, a specific inhibitor of phospholipase A2 (PLA2) and blocked by both pertussis toxin and R59022, an inhibitor of diacylglycerol kinase. R59022 enhanced diacylglycerol production by SPC and also significantly reduced [Ca2+]i mobilization. Only polyunsaturated diacylglycerol and phosphatidic acid were generated by SPC. Lastly, SPC caused stimulation of arachidonic acid release, indicating the involvement of PLA2. Taken together, these data suggest that, after SPC stimulation, phospholipase C-derived diacylglycerol is phosphorylated by a diacylglycerol kinase to phosphatidic acid, which is further hydrolysed by PLA2 activity to arachidonic and lysophosphatidic acids. We propose that lysophosphatidic acid might be the intracellular messenger able to release Ca2+ from internal stores.

Role of CFTR and anion exchanger in bicarbonate fluxes in C127 cell lines.

C127 cell lines transfected with wtCFTR, delta F508CFTR or vector were employed to determine HCO3- fluxes in the presence or absence of functional CFTR, using the pH-sensitive dye BCECF. Both cytosolic alkalinization and acidification were due to activity of anion exchanger and were similar in the three cell lines, indicating that expression of CFTR did not influence anion exchanger activity. In C127wt cells only, cAMP elevating agents significantly stimulated HCO3- fluxes, insensitive to the inhibitor of anion exchanger 4,4'-diisothiocyanate dihydrostilbene-2,2'-disulfonic acid, suggesting that activated CFTR directly mediates both HCO3- influx and efflux and therefore can contribute to intracellular and extracellular pH regulation.

Sphingosylphosphorylcholine and sphingosine-1-phosphate mobilize cytosolic calcium through different mechanisms in human airway epithelial cells.

The sphingosine derivatives sphingosylphosphorylcholine (SPC) and sphingosine-1-phosphate (S1P) caused a similar elevation of the intracellular Ca2+ concentration ([Ca2+]i) in an immortalized airway epithelial cell line (CFNP9o-) incubated in Ca(2+)-free medium. The maximal effect was obtained with 2 microM SPC and 0.1 microM S1P and was sensitive to pre-incubation with pertussis toxin, indicating the involvement of a Gi/G(o) type of G protein. In Ca2+ containing medium, [Ca2+]i elevation by SPC was significantly higher than that by S1P, due to the fact that SPC was able to stimulate Mn2+ entry, whereas S1P was ineffective. SPC, but not S1P, caused a dose-dependent production of total inositol phosphates. Conversely, S1P, but not SPC, increased the level of phosphatidic acid. These findings suggest the presence of two distinct receptors, specific for SPC and S1P, respectively. Depletion of intracellular Ca2+ stores by SPC makes cells unable to respond to a subsequent addition of S1P. Conversely, cells do respond to SPC after a challenge with S1P, suggesting that the two receptors likely share one or more intracellular signalling component(s).

Pertussis toxin- and PMA-insensitive calcium mobilization by sphingosine in CFPAC-1 cells: evidence for a phosphatidic acid-dependent mechanism.

In a pancreatic duct adenocarcinoma cell line (CFPAC-1) sphingosine (10 microM) induced both mobilization of intracellular Ca2+ and stimulation of inositol phosphates accumulation. Whereas this latter effect was significantly inhibited by treatment with pertussis toxin or by short-term incubation with phorbol 12-myristate 13-acetate, Ca2+ mobilization was completely insensitive to both treatments. Experiments with permeabilized cells showed that sphingosine or the sphingosine metabolites sphingosine-1-phosphate and sphingosylphosphorylcholine were unable to directly release Ca2+ from internal stores, whereas phosphatidic acid, but not arachidonic acid, was effective. Phosphatidic acid formation was markedly enhanced (2.9-fold over control) by sphingosine, this effect being significantly reduced by preincubation with the diacylglycerol kinase inhibitor R59022. Ca2+ mobilization by sphingosine was also cut down by preincubation with R59022. In conclusion, the results suggest that sphingosine activates phospholipase C through a mechanism functionally coupled through a G protein and under control of PKC. Mobilization of [Ca2+]i by sphingosine is independent of phospholipase C stimulation and likely due to elevation of phosphatidic acid generated by stimulation of diacylglycerol kinase activity.

Sphingosine-induced inhibition of capacitative calcium influx in CFPAC-1 cells.

Sphingosine (10 microM) induced mobilization of intracellular Ca2+ stores in the pancreatic duct adenocarcinoma cell line CFPAC-1. The effect was specific for sphingosine, since the sphingosine analog C2-ceramide had no effect. Sphingosine did not cause Ca2+ entry from extracellular medium, as also shown by following Mn2+ quenching of Fura-2 fluorescence. Furthermore, sphingosine, similarly to the mitochondrial inhibitors rotenone and oligomycin, strongly inhibited the rate of Mn2+ entry triggered by both thapsigargin- and agonist-induced depletion of intracellular stores. The uptake of rhodamine 123, a lipophilic cation which estimates mitochondrial energy level, was reduced by sphingosine to an extent similar to that observed in the presence of mitochondrial inhibitors. It is suggested that impairment of mitochondrial function might be responsible for inhibition of capacitative Ca2+ entry caused by sphingosine.

Histamine activates phospholipase C in human airway epithelial cells via a phorbol ester-sensitive pathway.

In human airway epithelial cell lines 9HTEo- and CFNPE9o, histamine causes a transient elevation of intracellular free calcium concentration ([Ca2+]i) detected by fura 2 fluorescence, which is due to both release from intracellular stores and extracellular Ca2+ entry. The effect of histamine is abolished by the Ca(2+)-ATPase inhibitor thapsigargin. Histamine also stimulates inositol phosphate accumulation. Changes in [Ca2+]i and inositol phosphate production exhibit a similar dose-response relationship for histamine (maximal effect at 10(-4) M), with both phenomena being blocked by the H1 antagonist mepyramine and being insensitive to pertussis toxin treatment. The effects of histamine on phosphoinositide metabolism and [Ca2+]i are abolished by a short-term preincubation with phorbol ester, and this effect is reversed by staurosporine and calphostin C, suggesting a feedback regulation by protein kinase C. The results indicate that human airway epithelial cells contain H1 receptors coupled to phospholipase C through a pertussis toxin-insensitive G protein.

Lack of major mitochondrial bioenergetic changes in cultured skin fibroblasts from aged individuals.

Human skin fibroblasts from young and old donors were cultured in vitro and compared in their mitochondrial morphology and function. A decreased numerical density of mitochondria in the fibroblasts from old individuals was balanced by the increased size of individual mitochondria. The mitochondrial membrane potential, estimated in the intact cells by the difference between the total accumulation ratio of the lipophilic cation tetraphenylphosphonium and the accumulation ratio in presence of uncoupler, was unchanged, as were some mitochondrial enzymatic activities tested in the homogenates. The results point out that the decreased proliferating capacity observed in the fibroblasts from the old subjects was accompanied by a likely decrease of mitochondrial duplication; the decreased energy utilization for cell division balances a possible energetic decline in such way that the steady-state energy status is unchanged.

Activation of Ca(2+)-dependent K+ and Cl- currents by UTP and ATP in CFPAC-1 cells.

Activation of Cl- and K+ conductances by nucleotide receptor-operated mobilization of intracellular Ca2+ was investigated in CFPAC-1 cells with the perforated-patch technique. Adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) caused a dose-dependent fast and transient membrane hyperpolarization. UTP was more effective than ATP. In voltage-clamped cells, two currents with different ionic permeability and kinetics were activated by the nucleotides. The first one was carried by Cl- ions, peaked in the first few seconds after addition of nucleotides, and lasted for 1 +/- 0.3 min. Its amplitude was about 2.7 nA at -100 mV with 100 mumol/l of either ATP or UTP. The second current was carried by K+ ions and was blocked by Cs+. This current peaked more slowly and had a mean duration of 4.6 +/- 0.7 min. Its amplitude was 0.9 nA and 0.5 nA at -20 mV with 100 mumol/l UTP and ATP, respectively. Activation of the nucleotide receptor caused a transient increase in intracellular Ca2+ concentration ([Ca2+]i) that was similar in the presence or absence of extracellular Ca2+. The ED50 for UTP was 24 mumol/l and that for ATP was 94 mumol/l. Depletion of the inositol 1,4,5-trisphosphate-sensitive Ca2+ store by thapsigargin prevented both the nucleotide-induced [Ca2+]i increase and the activation of membrane currents. Addition of 2 mmol/l Ca2+ to thapsigargin-treated cells produced a sustained increase of Cl- and K+ currents, which was reversed by Ca2+ removal. The present study demonstrates that CFPAC-1 cells respond to nucleotide receptor activation with a transient increase in [Ca2+]i that stimulates Ca(2+)-dependent Cl- and K+ currents.(ABSTRACT TRUNCATED AT 250 WORDS)

ATP and A1 adenosine receptor agonists mobilize intracellular calcium and activate K+ and Cl- currents in normal and cystic fibrosis airway epithelial cells.

The effect of purinergic compounds on [Ca2+]i and membrane currents of cell lines derived from the airway epithelium of normal and cystic fibrosis individuals has been investigated. 2-Chloroadenosine (2-CADO), as well as other agonists of the A1 adenosine receptors, causes a transient elevation of cytosolic [Ca2+] that is antagonized by the A1 adenosine receptor antagonist 8-cyclopentyl-1,3 dipropylxanthine (DPCPX). ATP is also effective, but at a lower extent. The [Ca2+]i increase induced by 2-CADO and ATP is abolished by preincubation with phorbol 12-myristate 13-acetate and the Ca(2+)-ATPase inhibitor thapsigargin. This latter result suggests that purinergic agonists mobilize Ca2+ from inositol 1,4,5-trisphosphate-sensitive stores. Pertussis toxin completely inhibits the effect of 2-CADO, whereas only it partially affects that of ATP, suggesting the involvement of different types of G proteins. Perforated patch clamp experiments carried out in both current clamp and voltage clamp modes show that 2-CADO and ATP activate K(+)- and Cl(-)-selective membrane currents, with a mechanism inhibited by preincubation with DPCPX and thapsigargin. These data indicate that activation of adenosine A1 receptor, in a similar way to ATP receptor, causes [Ca2+]i increase and ion channels activation through a transduction mechanism that is not impaired in cystic fibrosis airway epithelial cells.