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• W2001609847 abstract "Most cultured cells and intact animals die under hyperoxic conditions. However, a strain of HeLa cells that proliferates under 80% O2, termed “HeLa-80,” has been derived from wildtype HeLa cells (“HeLa-20”) by selection for resistance to stepwise increases of oxygen partial pressure. The tolerance of HeLa-80 cells to hyperoxia is not associated with changes in antioxidant defenses or susceptibility to oxidant-mediated killing. Rather, under both 20 and 80% O2, mitochondrial reactive oxygen species (ROS) production is ∼2-fold less in HeLa-80 cells, likely related to a significantly higher cytochrome c oxidase (COX) activity (∼2-fold), which may act to deplete upstream electron-rich intermediates responsible for ROS generation. We now report that in HeLa-80 cells elevated COX activity is associated with a >2-fold increase in the regulatory subunit COX Vb, whereas expression levels of other subunits are very close to wild type. Small interfering RNA against Vb selectively lowers COX Vb expression in HeLa-80 cells, increases mitochondrial ROS generation, decreases COX activity 60–80%, and diminishes viability under 80% (but not 20%) O2. In addition, overexpression of subunit Vb increases COX activity and decreases ROS production in wild-type HeLa-20 cells, along with some increase in tolerance to hyperoxia. Overall, our results indicate that it is possible to make cells tolerant of hyperoxia by manipulation of mitochondrial electron transport. These observations may suggest new pharmaceutical strategies to diminish oxygen-mediated cellular damage. Most cultured cells and intact animals die under hyperoxic conditions. However, a strain of HeLa cells that proliferates under 80% O2, termed “HeLa-80,” has been derived from wildtype HeLa cells (“HeLa-20”) by selection for resistance to stepwise increases of oxygen partial pressure. The tolerance of HeLa-80 cells to hyperoxia is not associated with changes in antioxidant defenses or susceptibility to oxidant-mediated killing. Rather, under both 20 and 80% O2, mitochondrial reactive oxygen species (ROS) production is ∼2-fold less in HeLa-80 cells, likely related to a significantly higher cytochrome c oxidase (COX) activity (∼2-fold), which may act to deplete upstream electron-rich intermediates responsible for ROS generation. We now report that in HeLa-80 cells elevated COX activity is associated with a >2-fold increase in the regulatory subunit COX Vb, whereas expression levels of other subunits are very close to wild type. Small interfering RNA against Vb selectively lowers COX Vb expression in HeLa-80 cells, increases mitochondrial ROS generation, decreases COX activity 60–80%, and diminishes viability under 80% (but not 20%) O2. In addition, overexpression of subunit Vb increases COX activity and decreases ROS production in wild-type HeLa-20 cells, along with some increase in tolerance to hyperoxia. Overall, our results indicate that it is possible to make cells tolerant of hyperoxia by manipulation of mitochondrial electron transport. These observations may suggest new pharmaceutical strategies to diminish oxygen-mediated cellular damage. Although oxygen is essential to aerobic metabolism, excess oxygen is harmful. Hyperoxia-induced lung damage and retinopathy of prematurity occur frequently in premature infants given oxygen as therapy for pulmonary insufficiency (1O'Donovan D.J. Fernandes C.J. Mol. Genet. Metab. 2000; 71: 352-358Crossref PubMed Scopus (72) Google Scholar). Hyperoxia also causes pulmonary damage in adults exposed to high partial pressures of inhaled O2 for more than 48 h. The symptoms include cough, shortness of breath, decreased vital capacity, and increased alveolar-capillary permeability (2Mantell L.L. Lee P.J. Mol. Genet. Metab. 2000; 71: 359-370Crossref PubMed Scopus (119) Google Scholar, 3Slutsky A.S. Chest. 1999; 116: S9-S15Abstract Full Text Full Text PDF PubMed Google Scholar) as well as damage to cardiovascular, nervous, and gastrointestinal systems (4Bostek C.C. AANA. J. 1989; 57: 231-237PubMed Google Scholar, 5Crapo J.D. Barry B.E. Foscue H.A. Shelburne J. Am. Rev. Respir. Dis. 1980; 122: 123-143PubMed Google Scholar, 6Janssen Y.M. Van Houten B. Borm P.J. Mossman B.T. Lab. Investig. 1993; 69: 261-274PubMed Google Scholar, 7Morris A.H. New Horiz. 1994; 2: 19-33PubMed Google Scholar, 8Stogner S.W. Payne D.K. Ann. Pharmacother. 1992; 26: 1554-1562Crossref PubMed Scopus (51) Google Scholar). However, the mechanisms involved in hyperoxic damage are still not completely understood.It is commonly believed that free radicals play a central role in oxygen toxicity, and cellular damage is probably mediated by increased production of ROS 2The abbreviations used are: ROS, reactive oxygen species; COX, cytochrome c oxidase; DCFDA, dihydrodichlorofluorescein diacetate; DMEM, Dulbecco's modified Eagle's medium; HBSS, Hanks' balanced salt solution; HeLa-20, wild-type HeLa cells; HeLa-80, oxygen-tolerant HeLa cells; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; RT, reverse transcription; siRNA, small interfering RNA; NS, non-sense; DCF, dihydrodichlorofluorescein; oligo, oligonucleotide; CCCP, carbonyl cyanide p-chlorophenylhydrazone; FBS, fetal bovine serum. 2The abbreviations used are: ROS, reactive oxygen species; COX, cytochrome c oxidase; DCFDA, dihydrodichlorofluorescein diacetate; DMEM, Dulbecco's modified Eagle's medium; HBSS, Hanks' balanced salt solution; HeLa-20, wild-type HeLa cells; HeLa-80, oxygen-tolerant HeLa cells; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; RT, reverse transcription; siRNA, small interfering RNA; NS, non-sense; DCF, dihydrodichlorofluorescein; oligo, oligonucleotide; CCCP, carbonyl cyanide p-chlorophenylhydrazone; FBS, fetal bovine serum. (9Gerschman R. Gilbert D. Nye S.W. Dwyer P. Fenn W.O. Nutrition. 2001; 17: 162PubMed Google Scholar). This excessive production of ROS likely derives from the mitochondria which, under conditions of high oxygen, exhibit increased “leak” from the electron transport chain (9Gerschman R. Gilbert D. Nye S.W. Dwyer P. Fenn W.O. Nutrition. 2001; 17: 162PubMed Google Scholar, 10Chance B. Sies H. Boveris A. Physiol. Rev. 1979; 59: 527-605Crossref PubMed Scopus (4782) Google Scholar). Our previous results lend support to the importance of mitochondrial respiration and ROS generation in the etiology of hyperoxic cell damage. Using three strategies to diminish mitochondrial ROS production by HeLa cells (rhoo cells, chronic exposure to chloramphenicol, and exposure to the protonophoric uncoupler of respiration, CCCP) we consistently found improved cell survival and growth under hyperoxic conditions (11Li J. Gao X. Qian M. Eaton J.W. Free Radic. Biol. Med. 2004; 36: 1460-1470Crossref PubMed Scopus (61) Google Scholar).In an effort to further understand the nature of hyperoxic damage, we focused on an oxygen-tolerant strain of HeLa cells, which proliferates even under 80% O2 (HeLa-80). This strain was derived from wild-type HeLa cells (HeLa-20) by selection for resistance to stepwise increases of oxygen partial pressure (12Joenje H. Gille J.J. Oostra A.B. van der Valk P. Lab. Investig. 1985; 52: 420-428PubMed Google Scholar). The oxygen tolerant HeLa-80 cells exhibit significantly decreased mitochondrial ROS generation (under both normoxia and hyperoxia). Furthermore, the lessened ROS production probably derives from enhanced activity of the terminal component of electron transport, cytochrome c oxidase (COX) (13Campian J.L. Gao X. Qian M. Eaton J.W. J. Biol. Chem. 2004; 279: 46580-46587Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). We earlier speculated that the net effect of this increased COX activity might be to deplete electron-rich intermediates (such as ubisemiquinone) in the electron transport chain, thereby diminishing the leak of electrons into ROS. In partial support of this, preferential inhibition of COX by treatment with n-methyl protoporphyrin (which selectively diminishes synthesis of heme a that is required for cytochrome c oxidase activity) enhances ROS production and abrogates the oxygen tolerance of the HeLa-80 cells (13Campian J.L. Gao X. Qian M. Eaton J.W. J. Biol. Chem. 2004; 279: 46580-46587Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).Cytochrome c oxidase (also known as complex IV) is the terminal complex of the mitochondrial respiratory chain and is comprised of 13 different subunits encoded by three mitochondrial genes (COX subunits I, II, and III) and 10 nuclear genes (COX subunits IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, and VIII). Given the complex nature of COX, the reasons for increased activity in HeLa-80 cells were unclear; clearly, gain-of-function mutations in all 13 would be very unlikely, but increased expression of some or all of the subunits of COX remained a possibility.We now report that the elevated COX activity in oxygen-tolerant HeLa-80 cells is associated with a >2-fold increase in the regulatory subunit COX Vb, whereas expression levels of other subunits are very close to wild-type HeLa-20 cells. Transfection of COX Vb-specific siRNA into HeLa-80 cells selectively lowers Vb expression, increases mitochondrial ROS generation, decreases COX activity 60–80%, and diminishes viability under 80% (but not 20%) O2. In addition, in wild-type HeLa-20 cells, overexpression of subunit Vb increases COX activity, decreases ROS production, and increases tolerance to hyperoxia. We speculate that manipulations designed to similarly enhance the “downhill” flow of electrons in the electron transport chain may have some utility in the suppression of cell damage caused by hyperoxia and other insults.EXPERIMENTAL PROCEDURESCells and Reagents—A wild-type strain of HeLa cells (HeLa-20) and an oxygen-tolerant strain that grows normally under 80% oxygen (HeLa-80) were generously provided by Dr. Hans Joenje (VU University Medical Center, Amsterdam, The Netherlands). Unless otherwise noted, all reagents were purchased from Sigma. Dulbecco's modified Eagle's medium (DMEM), Opti-MEM, phosphate-buffered saline (PBS), trypsin-EDTA, and fetal bovine serum (FBS) were obtained from Invitrogen. Dihydrodichlorofluorescein diacetate (DCFDA) and dihydroethidium were purchased from Molecular Probes (Eugene, OR). All antibodies, including the primary monoclonal antibodies (mouse anti-human) for cytochrome c oxidase subunits and a secondary antibody (goat anti-mouse IgG), were purchased from Molecular Probes. Oligofectamine was purchased from Invitrogen. FuGENE 6 was obtained from Roche Applied Science. The pcDNA3.1/V5-His TOPO-TA expression vector was kindly provided by Dr. Robert Mitchell (University of Louisville, Louisville, KY).Conditions of Cell Culture—HeLa cells were routinely cultured in DMEM supplemented with 10% (v/v) heat-inactivated FBS under 20% O2 (normoxia) or 80% O2 (hyperoxia) with 5% CO2 at 37 °C. For routine passage, cells were washed with PBS and lifted with 0.05% trypsin, 0.02% EDTA in PBS. Stock cultures were grown in 10-cm cell culture dishes in a Forma Scientific incubator under an atmosphere of 20% O2,5%CO2. Exposure to hyperoxia was performed with cells grown in 10-cm dishes under an atmosphere of 80% O2, 15% N2, and 5% CO2 contained in a specially designed gas-tight modular incubator chamber (Billups-Rothenberg, Inc., Del Mar, CA). Because susceptibility to hyperoxia may vary with cell density (14Jyonouchi H. Sun S.N. Abiru T. Chareancholvanich S. Ingbar D.H. Am. J. Respir. Cell Mol. Biol. 1998; 19: 426-436Crossref PubMed Scopus (55) Google Scholar), the initial cell number was routinely adjusted to ∼1 × 105 per dish in DMEM. The sealed chamber was placed in a standard tissue culture incubator, and the gas was replenished every 48 h. The survival and growth of cells were assessed over a period of 10 days by counting cell numbers in marked sectors of the culture dishes using light microscopy.It is important to note that the oxygen tolerance of HeLa-80 is a stable characteristic. Even after these cells have been continuously passed in normoxic culture for more than 2 months, they retain resistance to 80% O2. To avoid artifacts that might be introduced by the tendency of cell cultures to “drift,” cultures of both cell types were replenished from liquid nitrogen stabilities every 30 days, and the oxygen tolerance of each new culture was checked.Estimation of ROS Production—ROS production was assessed by the oxidation of DCFDA or dihydroethidium. To estimate ROS production with DCFDA, cells were plated onto 48-well plates at an initial density of 2 × 104 cells per well and grown for 3 days. After cells were >90% confluent, they were washed three times with HBSS, and then 0.5 ml of HBSS was replaced per well. Following the addition of 20 μm DCFDA (final concentration), the appearance of DCF fluorescence was followed continuously using a thermostated plate reading spectrofluorometer (Molecular Devices Corp., Sunnyvale, CA), typically for 1 h, at excitation 486 nm and emission 530 nm. Cell protein in each well was measured using the bicinchoninic acid reaction (15Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18447) Google Scholar) (Pierce), and the DCF fluorescence was corrected for variations in total protein between wells.Generation of ROS was also evaluated under both 20 and 80% O2 using the oxidation of dihydroethidium (16Bindokas V.P. Jordan J. Lee C.C. Miller R.J. J. Neurosci. 1996; 16: 1324-1336Crossref PubMed Google Scholar). Cells were plated and grown as for experiments with DCFDA (as mentioned above). When the cells were >90% confluent, fresh medium was added containing 100 μm dihydroethidium. Dihydroethidium oxidation permits estimates of ROS formation over longer periods of time in complete culture medium, and the product, ethidium, is held within the cell via intercalation into nucleic acids. After incubation with dihydroethidium for 4 h, cells were washed three times with HBSS, and ethidium fluorescence was detected at excitation 520 nm and emission 610 nm. Again, the relative fluorescence was corrected for variations in cell protein between individual wells.Real Time Quantitative RT-PCR—Real time quantitative RT-PCR measurements of the levels of mRNA for COX subunits along with control β-actin mRNA were carried out. RNA was prepared as described previously (17Holthofer H. Kretzler M. Haltia A. Solin M.L. Taanman J.W. Schagger H. Kriz W. Kerjaschki D. Schlondorff D. FASEB J. 1999; 13: 523-532Crossref PubMed Scopus (46) Google Scholar). Sequence-specific oligonucleotide primers for the human genes have been tested and published (18Grandjean F. Bremaud L. Robert J. Ratinaud M.H. Biochem. Pharmacol. 2002; 63: 823-831Crossref PubMed Scopus (21) Google Scholar). Results were obtained by measuring the cycle threshold (CT), the first cycle in which there is significant increase in fluorescence above the background, and which correlates to the log-linear phase of PCR amplification. Generally, the runs were stopped at eight cycles after CT. The relative quantification in mRNA levels was evaluated by the ratio between the target gene and the housekeeping gene β-actin. Data calculation was based on the “Delta-Delta method,” using the equation of R (ratio) = 2–(ΔCT sample–ΔCT control) (19Livak K.J. Schmittgen T.D. Methods (Orlando). 2001; 25: 402-408Crossref Scopus (119535) Google Scholar). The identity of the amplified DNA was confirmed by determination of melting temperature.There are two different methods of analyzing data from real time quantitative RT-PCR, absolute quantification and relative quantification. Absolute quantification can be done using a competitive oligonucleotide. Relative quantification involves measurement of the ratio between mRNA levels for the gene of interest versus a housekeeping gene. In the present instance, we used relative quantification because if mRNA levels for an appropriate housekeeping gene are used, it is adequate for investigating proportionate changes in gene expression levels. The caveat is that relative quantification relies on the assumption that the reference gene is unaffected by the experimental conditions.Western Blot Analysis—General conditions of cell culture were as described previously (11Li J. Gao X. Qian M. Eaton J.W. Free Radic. Biol. Med. 2004; 36: 1460-1470Crossref PubMed Scopus (61) Google Scholar). As a semi-quantitative method to determine the levels of COX subunits in HeLa-20 and HeLa-80 cell lines, we performed Western blot analysis on COX subunits from both cell types. Lysates were prepared from near-confluent cultures of HeLa cells using RIPA lysis buffer (Upstate, Charlottesville, VA) with protease inhibitors. Lysates were clarified by centrifugation at 7,000 × g for 4 min at 4 °C. Twenty μg of total cellular protein was loaded on 10–20% SDS-polyacrylamide gels and electrotransferred to polyvinylidene difluoride membranes (Amersham Biosciences). The membranes were blocked with 5% milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 for 1 h, followed by incubation at room temperature 1 h with primary monoclonal antibodies (1:1000 dilution in PBS-T (0.1% Tween 20) buffer) against human COX subunits (Molecular Probes, Eugene, OR). For detection of immunoreactivity, the blots were incubated in a secondary antibody solution (horseradish peroxidase-conjugated goat anti-mouse diluted 1:4000 in PBS containing 0.1% Tween 20 buffer) and developed using an enhanced chemiluminescence, ECL™-Plus Western blotting detection kit (Amersham Biosciences). β-Actin was used as a loading control.COX Assay—Lysates of fresh cells (directly counted and adjusted to 6 × 106 cells/ml) were prepared by suspension in 0.25 m sucrose, 40 mm potassium chloride, 2 mm EGTA, 1 mg/ml bovine serum albumin, and 20 mm Tris-HCl (pH 7.2) and disrupted by 10 1-s bursts from a microtip Fisher model 100 sonic dismembrator at scale 3 (on a scale of 0–10 of the 100-watt maximum power output) (Fisher). The lysate was centrifuged at 2,000 × g for 10 min. The pellet was discarded, and the supernatant was used for COX assays (20Jarreta D. Orus J. Barrientos A. Miro O. Roig E. Heras M. Moraes C.T. Cardellach F. Casademont J. Cardiovasc. Res. 2000; 45: 860-865Crossref PubMed Scopus (159) Google Scholar). Assays contained ∼20 μg of protein and were performed at 37 °C in 200-μl reaction volumes. The assay involved the addition of 30 μm ferrocytochrome c in an isosmotic medium (10 mm KH2PO4 (pH 6.5), 1 mg/ml bovine serum albumin, 0.3 m sucrose) containing 2.5 mm n-dodecyl maltoside to permeabilize mitochondrial membranes (20Jarreta D. Orus J. Barrientos A. Miro O. Roig E. Heras M. Moraes C.T. Cardellach F. Casademont J. Cardiovasc. Res. 2000; 45: 860-865Crossref PubMed Scopus (159) Google Scholar). To confirm that this method specifically detects the oxidase activity, 10 μm antimycin-A was added to a control group to block complex III. The activity was calculated from the rate of decrease in absorbance of ferrocytochrome c at 550 nm (ϵ = 19.1 mm–1 cm–1) (21Kruidering M. Van De Water B. De Heer E. Mulder G.J. Nagelkerke J.F. J. Pharmacol. Exp. Ther. 1997; 280: 638-649PubMed Google Scholar), and results were normalized for protein.siRNA Transfection—Two siRNA duplexes that specifically target COX subunit Vb were designed and obtained from Dharmacon (Lafayette, CO). A non-sense siRNA was used as control. We used two different siRNA duplexes (one against the open reading frame and one against an untranslated region) because some siRNAs may have multiple effects, may not work against the intended mRNA, or may exhibit nonspecific silencing. The first target sequence was AAAGUAGGCUGCAUCUGUGAA. The corresponding siRNA duplex 1 (s1) was 21 nucleotides. Its sense sequence was 5′-AGUAGGCUGCAUCUGUGAAdTdT-3′; the antisense sequence was 5′-UUCACAGAUGCAGCCUACUdTdT-3′. The second target sequence was AACAGUAAAGACUAGCCAUUG. The corresponding siRNA duplex 2 (s2) was also 21 nucleotides. Its sense sequence was 5′-CAGUAAAGACUAGCCAUUGdTdT-3′; the antisense sequence was 5′-CAAUGGCUAGUCUUUACUGdTdT-3′.One day before transfection, HeLa-80 cells were seeded at subconfluent density (8 × 104 cells per well in 6-well cell culture plates). During and after exposure to siRNA, the cultures were fed with enriched DMEM (10% FBS, supplemented with 4 g/liter glucose, 100 μg/ml pyruvate, and 50 μg/ml uridine) because substantial blockade of mitochondrial respiration might result in respiratory insufficiency and cell death. Control cultures were also fed with the same enriched medium. Transfection with siRNA was done with Oligofectamine (Invitrogen), following the manufacturer's guidelines with some modifications. We also carried out Oligofectamine controls with or without nonsense siRNA. Cells were further incubated for 48 h at 37 °C after transfection. To ensure siRNA effectiveness, cells were processed for the evaluation of changes in COX subunit protein expression (Western blot) and mRNA by real time RT-PCR (as above).Effects of Vb siRNA on COX Activity and ROS Production—Two different concentrations of siRNA of COX Vb (20 and 100 nm) were used in the experiments along with Oligofectamine controls with or without non-sense siRNA. Cells were further incubated for 48 h at 37 °C, 5% CO2, 20% O2 after transfection. Then the cells were collected and processed as described above. COX activities were measured, and ROS production was estimated in control cells and siRNA-treated cells (as above).Survival and Growth of siRNA-transfected Cells under Normoxia and Hyperoxia—We cultured control cells and siRNA-treated cells under both 20% O2 and 80% O2 for 6 days. Exposure to hyperoxia was performed as described above. Cells were seeded at a density of ∼10% confluence, and cell numbers were estimated by microscopy (as described above) as well as Alamar Blue reduction as described previously (13Campian J.L. Gao X. Qian M. Eaton J.W. J. Biol. Chem. 2004; 279: 46580-46587Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).Transfection and Overexpression of COX Subunit Vb in Oxygen-sensitive HeLa-20 Cells—Full-length cDNA for COX subunit Vb was obtained by RT-PCR, and the open reading frame was ligated into the pcDNA3.1/V5-His TOPO-TA expression vector. For the PCR amplification reactions, Taq polymerase (Promega, Madison, WI) was employed as follows: denaturation at 94 °C, 30 s; annealing at 56 °C, 30 s; and elongation at 72 °C, 1 min; 30 cycles. A 10-min extension at 72 °C was included after the last cycle to ensure that all PCR products were full length. The recombinant vector was transfected into TOP10 Escherichia coli that was selected for ampicillin resistance. All PCR products and plasmids were analyzed and verified by restriction enzyme analysis and DNA sequencing for the presence and orientation of PCR inserts.Using the TOPO cloning system, we employed the pcDNA3.1/V5-His TOPO TA expression system (Invitrogen) to overexpress Vb in HeLa-20 cells. With this system, a Taq polymerase-amplified PCR product is directly inserted into the expression vector. The vector contains a cytomegalovirus promoter allowing high level expression. The TOPO vector contains both an ampicillin resistance gene (for selection in E. coli) and a neomycin (G418) resistance gene (for selection of stable transfectants). Cells were transfected with verified plasmids containing the entire open reading frame of COX subunit cDNA (2 μg per 1–3 × 105 cells) using FuGENE 6. As a control, cells were also transfected with empty vector pcDNA3.1/V5-His without an insert. The vector containing COX Vb sequence was transfected into oxygen-sensitive HeLa-20 cells. To establish stable cell lines, transfected cells were cultured in a selection medium containing 400 μg/ml neomycin (G418) (Sigma) for 2–4 weeks. Resulting colonies were further selected with 400 μg/ml G418 in order to obtain single stably transfected clones. These cells were cultured in the continuous presence of G418 (400 μg/ml). Several stable clones expressing selected COX subunit and nonexpression clones containing empty vector were selected.Functional Tests of Overexpression of COX Subunit Vb—The level of Vb expression of individual clones was analyzed by Western blot analysis using a specific antibody for COX subunit Vb as described above. COX activity was measured in these clones, and several of these (in which Vb was overexpressed 2–4-fold) were selected for subsequent experiments. ROS production was detected in selected clones under both normoxic and hyperoxic conditions (as above). Survival and growth in hyperoxia were tested by exposure of clones to 80% oxygen. A negative control of oxygen-sensitive HeLa-20 cells and a positive control of oxygen-tolerant HeLa-80 cells were also used in addition to cells treated with the empty vector.Immunostaining of Proliferating Cell Nuclear Antigen—Cell proliferation was evaluated by detecting PCNA in cells grown in 24-well plates under either 20 or 80% oxygen. After 6 days of growth, cells were fixed with fresh 4% paraformaldehyde. After washing with PBS, cells were heated to 95 °C for 30 min in citrate buffer (10 mm sodium citrate, 0.05% Tween 20, pH 6.0). After cooling and PBS washing, the cells were incubated with normal horse serum blocking solution for 30 min, and finally, a peroxidase blocking solution (3% H2O2 in PBS) was added for 10 min to eliminate unspecific peroxidase reaction. Immunostaining was performed by incubating the fixed cells with the primary antibody for 30 min (mouse anti-PCNA, IgG2a, PC10; Vector Laboratories) and subsequently with the secondary antibody for 30 min (Biotinylated Universal Antibody from Vector Laboratories). The revealing reagents were provided by a commercial kit (Vectastain ABC Elite, Vector Laboratories). Finally, diaminobenzidine peroxidase substrate solution (Sigma) was added, and samples were observed by light microscopy immediately.Oxygen Consumption—HeLa cell cultures in 10-cm dishes (>90% confluent) were washed with PBS, detached with trypsin/EDTA, and washed (1,000 × g for 8 min) in complete culture medium. Oxygen consumption was measured using a Gilson oxygraph with a Clark-type oxygen electrode (YSI Inc., Yellow Springs, OH). Respiration rates were measured using 2–3 × 106 cells suspended in a total volume of 3.0 ml of complete culture medium containing 10% FBS and supplemented with 17 mm glutamate at 37 °C. A starting O2 concentration of 240 μm was assumed based on O2 solubility at sea level at 37 °C (22Chappell J.B. Biochem. J. 1964; 90: 225-237Crossref PubMed Scopus (218) Google Scholar).Statistics Analysis—Analysis of differences between groups was done by unpaired two-tailed Student's t test. Analyses between multiple groups were determined by one-way analysis of variance. Results were expressed as means ± 1 S.D.RESULTSCOX Subunit Vb Is Overexpressed in HeLa-80 Cells—We reported previously that the elevated COX activity in oxygentolerant HeLa-80 cells is associated with diminished mitochondrial ROS production and lessened hyperoxic damage (13Campian J.L. Gao X. Qian M. Eaton J.W. J. Biol. Chem. 2004; 279: 46580-46587Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). However, the mechanism(s) through which COX activity was increased remained unknown. COX consists of 13 subunits variously encoded by mitochondrial and nuclear DNA as described above. The larger subunits (I, II, and III) are involved in the catalytic activity of COX, and the smaller subunits are important in its regulation (23Capaldi R.A. Annu. Rev. Biochem. 1990; 59: 569-596Crossref PubMed Scopus (512) Google Scholar). To determine the reason(s) for the increased COX activity in HeLa-80 cells, we carried out analyses of the differential expression of COX subunits in HeLa-20 and HeLa-80 cells by Western blotting analyses. Western blots of 11 of the 13 subunits of COX (antibodies against two of these were not available) indicated that only COX subunit Vb was significantly increased (>2-fold) (Fig. 1). This may be a critical observation because this subunit and its yeast homologue have been indicated as important in the regulation of overall COX activity and proper assembly of COX (24Beauchemin A.M.J. Gottlieb B. Beitel L.K. Elhaji Y.A. Pinsky L. Trifiro M.A. Brain Res. Bull. 2001; 56: 285-297Crossref PubMed Scopus (45) Google Scholar, 25Dowhan W. Bibus C.R. Schatz G. EMBO J. 1985; 4: 179-184Crossref PubMed Scopus (74) Google Scholar, 26Lee I. Bender E. Kadenbach B. Mol. Cell. Biochem. 2002; 234: 63-70Crossref PubMed Scopus (134) Google Scholar, 27Lightowlers R. Chrzanowska-Lightowlers Z. Marusich M. Capaldi R.A. J. Biol. Chem. 1991; 266: 7688-7693Abstract Full Text PDF PubMed Google Scholar). Interestingly, however, the mRNA expression for each of the 13 subunits, including Vb, was similar (data not shown). This is surprising given the elevated levels of Vb protein and suggests either enhanced translation efficiency or increased stability of the protein product (although there are other possible explanations).COX Subunit Vb Is Selectively Suppressed in HeLa-80 Cells by siRNA—siRNA transfection was used as a means to specifically and directly test whether the overexpressed COX Vb in HeLa-80 cells might lead to an overall increase in COX activity. The effects of siRNA on Vb expression were determined following 48 h of exposure to siRNA. As shown in Fig. 2, Western blots revealed significantly decreased COX Vb in siRNA-treated HeLa-80 cells, and the extent of inhibition of expression was dependent on the dose of siRNA. In similar experiments, we also determined by Western blot the expression levels of several other COX subunits (I and II that are mitochondrial products and Va and VIb that are nuclear products). No significant changes in the expression of" @default.
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• W2001609847 date "2007-04-01" @default.
• W2001609847 modified "2023-09-28" @default.
• W2001609847 title "Cytochrome c Oxidase Activity and Oxygen Tolerance" @default.
• W2001609847 cites W1459222285 @default.
• W2001609847 cites W158267464 @default.
• W2001609847 cites W1603367677 @default.
• W2001609847 cites W1652996689 @default.
• W2001609847 cites W1975129989 @default.
• W2001609847 cites W2002857173 @default.
• W2001609847 cites W2027413649 @default.
• W2001609847 cites W2034411287 @default.
• W2001609847 cites W2046910842 @default.
• W2001609847 cites W2051820252 @default.
• W2001609847 cites W2063057532 @default.
• W2001609847 cites W2071452423 @default.
• W2001609847 cites W2093776791 @default.
• W2001609847 cites W2115705971 @default.
• W2001609847 cites W2132826785 @default.
• W2001609847 cites W2133739669 @default.
• W2001609847 cites W2171285875 @default.
• W2001609847 cites W2171789870 @default.
• W2001609847 cites W2176573833 @default.
• W2001609847 cites W4232534952 @default.
• W2001609847 cites W4238953154 @default.
• W2001609847 doi "https://doi.org/10.1074/jbc.m604547200" @default.
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