Respiration-deficient Cells Research Articles

TNFα Enhances the DNA Single-Strand Breakage Induced by the Short-Chain Lipid Hydroperoxide Analogue tert-Butylhydroperoxide via Ceramide-Dependent Inhibition of Complex III Followed by Enforced Superoxide and Hydrogen Peroxide Formation

Treatment of U937 cells with nontoxic concentrations of TNFalpha increased the DNA strand scission induced by a short-chain lipid hydroperoxide analogue, tert-butylhydroperoxide. The following lines of evidence suggest that the enhancing effects of TNFalpha are mediated by inhibition of complex III and by the ensuing formation of superoxides and hydrogen peroxide: (a) the effects of TNFalpha were mimicked by the complex III inhibitor antimycin A; (b) the effects of TNFalpha, or antimycin A, were abolished by the complex I inhibitor rotenone, or by myxothiazol, an agent which inhibits the electron flow from the reduced coenzyme Q to cytochrome c(1) and therefore prevents ubisemiquinone formation; (c) the effects of TNFalpha, or antimycin A, were not observed in respiration-deficient cells; and (d) the effects of TNFalpha, or antimycin A, were sensitive to catalase. The TNFalpha-dependent inhibition of complex III appears to be mediated by ceramide. Three lines of evidence support this inference: (a) a synthetic cell-permeable ceramide analogue reproduced all the effects of TNFalpha, (b) TNFalpha promoted the formation of ceramide via a mechanism sensitive to inhibition of sphingomyelinases by tricyclodecan-9-yl-xanthogenate and imipramine, and (c) the TNFalpha-mediated enhancement of the tert-butylhydroperoxide-induced DNA-damaging response was prevented under conditions in which ceramide formation was inhibited.

Roles of phosphatidylethanolamine and of its several biosynthetic pathways in Saccharomyces cerevisiae.

Three different pathways lead to the synthesis of phosphatidylethanolamine (PtdEtn) in yeast, one of which is localized to the inner mitochondrial membrane. To study the contribution of each of these pathways, we constructed a series of deletion mutants in which different combinations of the pathways are blocked. Analysis of their growth phenotypes revealed that a minimal level of PtdEtn is essential for growth. On fermentable carbon sources such as glucose, endogenous ethanolaminephosphate provided by sphingolipid catabolism is sufficient to allow synthesis of the essential amount of PtdEtn through the cytidyldiphosphate (CDP)-ethanolamine pathway. On nonfermentable carbon sources, however, a higher level of PtdEtn is required for growth, and the amounts of PtdEtn produced through the CDP-ethanolamine pathway and by extramitochondrial phosphatidylserine decarboxylase 2 are not sufficient to maintain growth unless the action of the former pathway is enhanced by supplementing the growth medium with ethanolamine. Thus, in the absence of such supplementation, production of PtdEtn by mitochondrial phosphatidylserine decarboxylase 1 becomes essential. In psd1Delta strains or cho1Delta strains (defective in phosphatidylserine synthesis), which contain decreased amounts of PtdEtn, the growth rate on nonfermentable carbon sources correlates with the content of PtdEtn in mitochondria, suggesting that import of PtdEtn into this organelle becomes growth limiting. Although morphological and biochemical analysis revealed no obvious defects of PtdEtn-depleted mitochondria, the mutants exhibited an enhanced formation of respiration-deficient cells. Synthesis of glycosylphosphatidylinositol-anchored proteins is also impaired in PtdEtn-depleted cells, as demonstrated by delayed maturation of Gas1p. Carboxypeptidase Y and invertase, on the other hand, were processed with wild-type kinetics. Thus, PtdEtn depletion does not affect protein secretion in general, suggesting that high levels of nonbilayer-forming lipids such as PtdEtn are not essential for membrane vesicle fusion processes in vivo.

Mitochondrial respiratory mutants of Saccharomyces cerevisiae accumulate glycogen and readily mobilize it in a glucose-depleted medium

Mutant strains of Saccharomyces cerevisiae defective in respiration have been reported to be unable to store glycogen, as revealed by the iodine-staining method. In this report, it is shown that in contrast to this claim, mitochondrial respiratory mutants accumulated even more glycogen than wild-type cells during the fermentative growth on glucose. However, as soon as glucose was exhausted in the medium, these mutants readily and completely mobilized their glycogen content, contrary to wild-type cells which only transiently degraded this polymer. The mobilization of glycogen was a specific trait resulting from a defect in mitochondrial function that could not be suppressed by mutations in the cAMP- and Pho85 protein kinase-dependent nutrient-sensing pathways, and by other mutations which favour glycogen synthesis. To account for this mobilization, it was found that respiration-defective cells not only contained a less active glycogen synthase, but also a more active glycogen phosphorylase. Since glucose 6-phosphate (Glc6P) is a potent inhibitor of the phosphorylation and an activator of the dephosphorylation processes of glycogen synthase and glycogen phosphorylase, it is suggested that the drop in Glc6P observed at the onset of glucose depletion in respiration-deficient cells triggers this rapid and sustained glycogen mobilization. It is also proposed that this degradation provides the energy for the viability of respiratory mutants in glucose-starved medium.

The mechanism of the nitric oxide-mediated enhancement of tert-butylhydroperoxide-induced DNA single strand breakage.

1. Caffeine (Cf) enhances the DNA cleavage induced by tert-butylhydroperoxide (tB-OOH) in U937 cells via a mechanism involving Ca2+-dependent mitochondrial formation of DNA-damaging species (Guidarelli et al., 1997b). Nitric oxide (NO) is not involved in this process since U937 cells do not express the constitutive nitric oxide synthase (cNOS). 2. Treatment with the NO donors S-nitroso-N-acetyl-penicillamine (SNAP, 10 microM), or S-nitrosoglutathione (GSNO, 300 microM), however, potentiated the DNA strand scission induced by 200 microM tB-OOH. The DNA lesions generated by tB-OOH alone, or combined with SNAP, were repaired with superimposable kinetics and were insensitive to anti-oxidants and peroxynitrite scavengers but suppressed by iron chelators. 3. SNAP or GSNO did not cause mitochondrial Ca2+ accumulation but their enhancing effects on the tB-OOH-induced DNA strand scission were prevented by ruthenium red, an inhibitor of the calcium uniporter of mitochondria. Furthermore, the enhancing effects of both SNAP and GSNO were identical to and not additive with those promoted by the Ca2+-mobilizing agents Cf or ATP. 4. The SNAP- or GSNO-mediated enhancement of the tB-OOH-induced DNA cleavage was abolished by the respiratory chain inhibitors rotenone and myxothiazol and was not apparent in respiration-deficient cells. 5. It is concluded that, in cells which do not express the enzyme cNOS, exogenous NO enhances the accumulation of DNA single strand breaks induced by tB-OOH via a mechanism involving inhibition of complex III.

Mitochondrial Respiratory Mutants in Yeast Inhibit Glycogen Accumulation by Blocking Activation of Glycogen Synthase

Control of glycogen synthase activity by protein phosphorylation is important for regulating the synthesis of glycogen. In this report, we describe a regulatory linkage between the ability of yeast cells to respire and activation of glycogen synthase. Strains containing respiration-deficient mutations in genes such as COQ3, required for the synthesis of coenzyme Q, were reduced in their ability to accumulate glycogen in response to limiting glucose. This lowered glycogen accumulation results from inactivation of the rate-determining enzyme, glycogen synthase (Gsy2p). Reduced glycogen synthase activity is coincident with lowered glucose 6-phosphate and ATP levels in the respiration-deficient cells deprived of glucose. Alanine substitutions of three previously characterized phosphorylation sites in Gsy2p, Ser-650, Ser-654, or Thr-667, each suppressed the glycogen defect in cells unable to respire, suggesting that inactivation of this enzyme is mediated by phosphorylation of these residues. Inactivation of glycogen synthase requires the RAS signaling pathway that controls cAMP-dependent protein kinase and is independent of Pho85p previously identified as a Gsy2p kinase. These results suggest that yeast cells unable to shift from a fermentative to a respiratory metabolic regimen block accumulation of glycogen by inactivating Gsy2p through protein phosphorylation.

Mechanism of the antimycin A-mediated enhancement of t-butylhydroperoxide-induced single-strand breakage in DNA.

Inhibitors of complex III increased the DNA strand scission induced by t-butylhydroperoxide (tB-OOH) and cumene hydroperoxide but did not affect DNA damage induced by H2O2. The hypothesis that these effects are selectively linked to inhibition of the electron transport from cytochrome b to cytochrome c1 is validated by the following observations: (1) two complex III inhibitors, antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide, enhanced the tB-OOH-induced DNA cleavage over the same concentration range as that in which inhibition of oxygen consumption was observed; (2) the complex III inhibitor-mediated enhancement of tB-OOH-induced DNA damage was abolished by the complex I inhibitor rotenone or by glucose omission, and (3) the enhancing effects of antimycin A were not observed in respiration-deficient cells. The mechanism whereby the complex III inhibitors potentiate DNA cleavage promoted by tB-OOH was subsequently investigated with intact as well as permeabilized cells. H2O2, produced at the level of mitochondria via a Ca2+-dependent process, was found to account for the enhancing effects of antimycin A.

Stimulation of Oxygen Consumption Promotes Mitochondrial Calcium Accumulation, a Process Associated with, and Causally Linked to, Enhanced Formation of tert-Butylhydroperoxide-Induced DNA Single-Strand Breaks

The NADH-linked substrates pyruvate, L-glutamine, and beta-hydroxybutyrate, while enhancing the rate of oxygen consumption, also increased the formation of DNA single-strand breaks induced by tert-butylhydroperoxide in intact U937 cells. A cause-effect relationship between these two parameters was established by showing that: (a) rotenone, an inhibitor of complex I, abolished respiration and prevented the enhancement of the DNA-damaging response under all the above circumstances; (b) the membrane-impermeant, complex I-activating substrate L-malate gave similar results in permeabilized cells; and (c) none of the NADH-linked substrates affected the DNA-damaging response to tert-butylhydroperoxide in respiration-deficient cells. Stimulation of electron transport potentiated the DNA-cleaving ability of tert-butylhydroperoxide via a process involving enforced mitochondrial calcium accumulation in the absence of a discernible elevation in the cytosolic concentration of free Ca2+. Finally, mitochondrial calcium was found to promote the mitochondrial formation of DNA-damaging levels of hydrogen peroxide. In conclusion, the data herein presented define a previously unexpected role of respiratory substrates in the control of the deleterious effects of an organic hydroperoxide at the level of genomic DNA. The enhanced DNA cleavage mediated by NADH-linked substrates in response to tert-butylhydroperoxide would appear to depend on a sequence of events involving stimulation of electron transport, mitochondrial accumulation of Ca2+, and mitochondrial formation of DNA-damaging levels of hydrogen peroxide via a Ca(2+)-dependent process.

Activation of CPP32-like protease in tumor necrosis factor-induced apoptosis is dependent on mitochondrial function.

Mitochondria have been implicated in apoptosis, however, the precise mechanisms whereby mitochondria exert their effect are not clear. To gain further insights, we generated a panel of cells from ML-1a cells that were rendered respiration deficient by ethidium bromide treatment. Two respiration-deficient clones were subsequently reconstituted by fusion with platelets. Respiration-deficient clones were resistant to TNF-induced apoptosis, whereas ML-1a and reconstituted clones were sensitive. In contrast, inhibition of proliferation and induction of differentiation by TNF were still observed in respiration deficient clones, suggesting a selective requirement of respiration in TNF-induced apoptosis. Furthermore the apoptosis machinery is not completely altered in respiration-deficient cells because they underwent apoptosis after staurosporine treatment. Next, we showed that apoptosis induced by TNF and staurosporine were blocked by z-DEVD-CH2F, an inhibitor of CPP32-like cysteine protease, suggesting the involvement of CPP32-like protease in both apoptosis signaling pathways. Interestingly, TNF activated CPP32-like protease in the parental and reconstituted clones but not in respiration-deficient clones, and staurosporine in all clones. Thus, the apoptosis signaling block in respiration-deficient clones is located at a step before CPP32-like protease activation, which can be bypassed by staurosporine.

Regulation of intracellular osmotic pressure and some factors that influence the promotion of glycerol synthesis in a respiration-deficient mutant of the salt-tolerant yeast Zygosaccharomyces rouxii during salt stress.

The accumulation of glycerol and inorganic ions were examined in a respiration-deficient (RD) mutant isolated from the salt-tolerant yeast Zygosaccharomyces rouxii for 3 h after salt stress due to 1 M NaCl. After the start of salt stress, intracellular levels of glycerol continued to increase for up to 3 h, while the levels of Na + and Cl - ions in cells reached maximum values within 1 h and then decreased gradually. Increases in intracellular concentrations of solutes resulted in an osmotic pressure that was almost equivalent to the external osmotic pressure within 2 h after salt stress. The RD strain had the same ability to tolerate salt as the wild-type strain. Therefore, we used the RD strain to examine the mechanism in the glycolytic pathway that is responsible for the promotion of glycerol synthesis that is induced by NaCl. When exposed to medium with 1 M NaCl, RD cells diverted about one-sixteenth of the amount of ethanol that was produced in the medium without NaCl to the production of glycerol. This result suggests the presence of factors that mediate a change from the normal metabolism of glucose to the promotion of glycerol synthesis in response to external NaCl. The specific activities of glycerol-3-phosphate dehydrogenase (GPDH) in extracts of cells grown with and without 1 M NaCl were very low in reaction mixtures with NADH or NADPH, although the cellular activity of alcohol dehydrogenase (ADH) was high and was repressed by external Nacl. This result indicates that the pathway involving GPDH makes only a small contribution to the synthesis of glycerol and that an alternative pathway functions for the synthesis in Z. rouxii. The addition of sodium sulfite, which binds to acetaldehyde, and of glycidol, an inhibitor of triosephosphate isomerase (TPI), to the medium promoted the synthesis of glycerol in RD strain. These results suggest the possibility that the extra NADH resulted from the binding of sulfite to acetaldehyde, or the inhibition of ADH and/or TPI under the NaCl-stressed condition lead to the promotion of glycerol synthesis by Z. rouxii.

Involvement of oxidants and oxidant-generating enzyme(s) in tumour-necrosis-factor-α-mediated apoptosis: role for lipoxygenase pathway but not mitochondrial respiratory chain

Cellular signalling by the inflammatory cytokine tumour necrosis factor alpha (TNF alpha) has been suggested to involve generation of low levels of reactive oxygen species (ROS). Certain antioxidants and metal chelators can inhibit cytotoxicity and gene expression in response to TNF alpha in numerous cell types. However, neither the source nor function of TNF alpha-induced oxidant generation is known. Using specific inhibitors, we ruled out involvement of several oxidant-generating enzymes [cyclo-oxygenase (indomethacin), cytochrome P-450 (metyrapone), nitric oxide synthase (NG-methyl-L-arginine), NADPH oxidase (iodonium diphenyl), xanthine oxidase (allopurinol), ribonucleotide reductase (hydroxyurea)] in TNF alpha-mediated apoptosis of the murine fibrosarcoma line, L929. We also demonstrated no role for mitochondrial-derived radicals/respiratory chain in the lytic pathway using specific inhibitors/uncouplers (rotenone, KCN, carboxin, fluoroacetate, antimycin, malonate, carbonyl cyanide p-trifluoromethoxyphenylhydrazone) and chloramphenicol-derived respiration-deficient cells. Significant ROS (H2O2, O2-.) generation was not observed in response to TNF alpha in L929 cells using four separate assays. Also, prevention of intracellular H2O2 removal by inhibition of catalase did not potentiate TNF alpha-mediated cell death. These data suggest that neither H2O2 nor O2-. plays a direct role in TNF alpha cytotoxicity. Finally, we suggest a central role for lipoxygenase in TNF alpha-mediated lysis. Three inhibitors of this radical-generating signalling pathway, including an arachidonate analogue (5,8,11,14-eicosatetraynoic acid), could protect cells against TNF alpha. The inhibitor nordihydroguaiaretic acid is also a radical scavenger, but it could not protect cells from ROS toxicity at concentrations that effectively prevented TNF alpha killing. Therefore protection by nordihydroguaiaretic acid cannot be due to scavenging of cytotoxic H2O or O2-.. The lipoxygenase product, (12S)-hydroxyeicosatetraenoic acid, was also significantly protective. As this analogue can act as a substrate for certain lipoxygenases, this effect may be due to prevention of generation of physiological products.

Differential Glycosylation of the Glucose Transporter Coincides with Enhanced Sugar Transport in Respiration Deficient Cells

Sugar transport regulation was characterized in terms of the expression and subcellular distribution of the glucose transporter (GT) in the V79 hamster fibroblast cell line and in a respiration deficient mutant (G14) of the V79 cell line. Comparison of GT content in V79 and G14 cells and cell fractions revealed that the 3-fold elevation in basal sugar transport observed in the G14 cell line did not coincide with any significant difference in either the whole cell or plasma membrane GT content when compared to the V79 parental cell line. Determination of delta-antibody binding to intact cell monolayers supported the finding that the two cell lines demonstrate equivalent plasma membrane GT content. Further, D-glucose inhibitable cytochalasin B binding to total cell membranes indicates that additional, unrecognized GT isoforms do not occur in either cell line. A higher average molecular weight GT was detected in the G14 cell line, and treatment of GT enriched preparations with endoglycosidase F established that the G14 cell line exhibits a hyperglycosylated form of the same core GT protein expressed in V79. These results suggest that an enhancement of the intrinsic activity of the GT expressed in G14 is responsible for its increased sugar transport capabilities and this may be related to differences in GT post-translational processing.

DNA-mediated transfer of complex I genes into three different respiration-deficient Chinese hamster mutant cell lines with defects in complex I of electron transport chain.

We have used genomic DNA from human or mouse cells as a calcium phosphate precipitate to transfect three different respiration-deficient Chinese hamster mutant cell lines with defects in complex I of the electron transport chain. Transformants were selected in DMEM containing galactose, a medium in which respiration-deficient cells do not grow. Evidence for the DNA-mediated transformation of these respiration-deficient cells with a putative complex I gene includes: the clones are respiration-positive and respire at rates comparable to those of wild-type human, hamster, or mouse cells; the clones have rotenone-sensitive NADH oxidase activities, indicating a functional complex I of the electron transport chain; and the clones appear to be true transformants, as demonstrated by hybridization and Southern blot analyses. These experiments provide the basis for the isolation and subsequent characterization of several of the genes involved with complex I of the mammalian electron transport chain.

On auxotrophy for pyrimidines of respiration‐deficient chick embryo cells

Chick embryo cells treated with chloramphenicol are inherently resistant to the growth-inhibitory effect of the drug when cultured in the presence of tryptose phosphate broth. The cells were found to be auxotrophic for pyrimidines and the presence in the broth of compounds of pyrimidine origin is demonstrated by chromatographic procedures and mass spectral analyses. They are in the form of ribonucleosides, ribonucleotides and pyrimidine-containing oligoribonucleotides. To understand the mechanism responsible for pyrimidine auxotrophy, the activity of enzymes involved in the pyrimidine biosynthetic pathway was determined. Measurement of the conversion of dihydroorotic acid to orotic acid in cell-free extracts revealed that chloramphenicol-treated chick embryo cells are deficient in dihydroorotate dehydrogenase activity. The data in vitro are supported by studies on the nutritional requirements of the respiration-deficient cells and by the incorporation in vivo of labelled dihydroorotic acid into the acid-insoluble fraction of the cells. Although the activity of the dehydrogenase in vitro is decreased by 95%, the enzyme is present in chloramphenicol-treated cells and its activity is unmasked by the artificial electron acceptor menadione. A study of the activity of other enzymes of the pyrimidine biosynthetic pathway demonstrated that their activity is comparable to that in control cells. The present results indicate that auxotrophy for pyrimidines results from the inhibition of the flow of electrons along the mitochondrial electron transport chain.

Induction of rho − mutations in yeast Saccharomyces cerevisiae by ethanol

The kinetics of the killing effect of ethanol was studied at 6-30% concentrations. Ploidy of cells, deficiency of the excision-repair system or holding under no-growth conditions did not influence survival. Ethanol at 24% increased, in the wild strain, the number of respiration-deficient cells from a spontaneous level of 0.4% up to nearly half of all survivors. Genetic analysis showed the mitochondrial nature of induced respiration-deficient mutants (or rho-). The influence of yeast resistance to some antibiotics was studied on rho- mutagenesis, both spontaneous and induced by ethanol. Neomycin-resistant strains were characterized by a significantly lower level of these mutations than were neomycin-sensitive strains.

Action of acriflavine on growth and mutation in yeast: I. Nitrogen sources affecting the mutation induction

Abstract Enrichment by aspartic acid, leucine or glutamic acid of an acriflavine-containing medium increased the frequency of respiration-deficient cells in a yeast culture. These amino acids increased the induction rate of respiration deficiency (RD) in relation to generation of cells, but did not favor multiplication of the mutant cells over normal cells. The effect of concentration of these amino acids and acriflavine on the mutation rate was studied kinetically by analogy with enzyme kinetics. The results have prompted the suggestion that acriflavine (Ac) reacts at least twice and amino acid (A) reacts once in the induction mechanism; leucine reacts in the order Ac-A-Ac, glutamate in the order A-Ac-Ac and Ac-A-Ac, and aspartate in the same manner as glutamate but to a lesser degree in the A-Ac-Ac order.

Heritable glycogen-storage deficiency in yeast and its induction by ultra-violet light.

SUMMARY: Cultures of several diploid strains of Saccharomyces cerevisiae were found to contain about 0.5% of glycogen-deficient cells. Ultraviolet irradiation increased the mutant frequency to about 5%, while 40% of the population survived. The mutants were detected by plating cultures on nutrient agar containing 1% glucose and staining 3-day colonies with iodine; normal colonies became brown due to the stored glycogen whereas the mutants remained white. The size of the mutant colonies was normal. Most of the mutants have remained stable during subculture. Fractionation of the cellular carbohydrate confirmed that the mutants were deficient in glycogen. Glucose concentration in the growth medium had a marked influence on glycogen storage. When the mutants were grown on nutrient agar containing 8% glucose, glycogen was stored and the mutants could not be distinguished by iodine-staining from the wild type. Glycogen-deficient mutants contained higher than usual proportions of respiration-deficient cells. Respiration-deficient colonies, whether derived from glycogen-deficient or from normal cultures contained appreciably less glycogen than those of the wild-type yeast.

The action of acriflavine on Brewer's yeasts.

SUMMARY: The absorption of acriflavine by strains of Saccharomyces carlsbergensis and S. cerevisiae occurred at approximately the same rate and extent and was greatest in the range pH 4-5. In this range, the proportion of respiration-deficient cells produced by acriflavine treatment was also maximal. Furthermore, under more alkaline conditions, the acriflavine exerted a pronounced toxic effect. At pH 4-5, the proportion of mutant cells produced was about 50 times greater in strains of S. cerevisiae than in those of S. carlsbergensis, while fewer cells were killed. With S. cerevisiae the production of mutants was rapid and in certain strains exposure for 1 hr. to 50 μg. acriflavine/ml. led to about one-third of the cells mutating, while after 4 hr., all the cells were respiration-deficient. In contrast, exposure of strains of S. carlsbergensis for 24 hr. at this concentration of acriflavine induced only about one-tenth of the cells to mutate. The mutants took up no measurable amount of oxygen when examined in Warburg respirometers. As compared with the parent cultures, there was no difference in the ability of the cells to utilize carbohydrates and to undergo flocculation.

Ultraviolet Induction of Respiration-deficient Variants of Saccharomyces and Their Stability During Vegetative Growth

Ultraviolet radiation induces respiration-deficient variants with high frequency in both haploid and tetraploid yeasts. Variant colonies arising after irradiation, when overlaid with triphenyl tetrazolium chloride agar, can be classified as (1) “whole colony” and (2) “variegated or sectored.” The heterogeneity in respiratory phenotype of the latter type of colonies is not detectable by colony morphology prior to overlaying with tetrazolium agar. The variegated types may explain some cases of “reversion” of respirationdeficient cells to wild type. Whole colony variants can be grouped into two classes (vI and vII). The vI variants are similar to spontaneously occurring variants and fail to revert to wild type, whereas the vII variants generally revert to wild type at a low frequency. The vI type is produced most frequently and presumably results from nongenic radiation damage, while the less frequently occurring vI type results from nuclear damage.