Antioxidant defences and homeostasis of reactive oxygen speciesin different human mitochondrial DNA-depleted cell lines Lodovica Vergani1, Maura Floreani2, Aaron Russell3, Mara Ceccon1,
Trang 1Antioxidant defences and homeostasis of reactive oxygen species
in different human mitochondrial DNA-depleted cell lines
Lodovica Vergani1, Maura Floreani2, Aaron Russell3, Mara Ceccon1, Eleonora Napoli4, Anna Cabrelle5, Lucia Valente2, Federica Bragantini1, Bertrand Leger3and Federica Dabbeni-Sala2
1
Dipartimento di Scienze Neurologiche and2Dipartimento di Farmacologia e Anestesiologia, Universita` di Padova, Padova, Italy;
3
Clinique Romande de Re´adaptation SUVA Care, Sion, Switzerland;4E.Medea Scientific Institute, Conegliano Research Centre, Conegliano, Italy;5Dipartimento di Medicina Clinica, Universita` di Padova, c/o Istituto Veneto di Medicina Molecolare, Padova, Italy
Three pairs of parental (q+) and established mitochondrial
DNA depleted (q0) cells, derived from bone, lung and muscle
were used to verify the influence of the nuclear background
and the lack of efficient mitochondrial respiratory chain on
antioxidant defences and homeostasis of intracellular
reactive oxygen species (ROS) Mitochondrial DNA
deple-tion significantly lowered glutathione reductase activity,
glutathione (GSH) content, and consistently altered the
GSH2: oxidized glutathione ratio in all of the q0cell lines,
albeit to differing extents, indicating the most oxidized redox
state in bone q0cells Activity, as well as gene expression and
protein content, of superoxide dismutase showed a decrease
in bone and muscle q0cell lines but not in lung q0cells GSH
peroxidase activity was four times higher in all three q0cell
lines in comparison to the parental q+, suggesting that
this may be a necessary adaptation for survival without a
functional respiratory chain Taken together, these data suggest that the lack of respiratory chain prompts the cells to reduce their need for antioxidant defences in a tissue-specific manner, exposing them to a major risk of oxidative injury In fact bone-derived q0cells displayed the highest steady-state level of intracellular ROS (measured directly by 2¢,7¢-di-chlorofluorescin, or indirectly by aconitase activity) com-pared to all the other q+and q0cells, both in the presence or absence of glucose Analysis of mitochondrial and cytosolic/ iron regulatory protein-1 aconitase indicated that most ROS of bone q0 cells originate from sources other than mitochondria
Keywords: A549 q0cells; antioxidant defences; 143 q0cells; reactive oxygen species; rhabdomyosarcoma q0cells
Cellular reactive oxygen species (ROS), such as superoxide
anions (OÆ2)
1 , and hydrogen peroxide (H2O2), have long
been held to be harmful by-products of life in an aerobic
environment ROS are potentially toxic because they are
highly reactive and modify several types of cellular
macro-molecules Lipid, protein and DNA damage can lead to
cytotoxicity and mutagenesis [1] Therefore, cells have evolved elaborate defence systems to counteract the effects
of ROS These include both nonenzymatic (glutathione, pyridine nucleotides, ascorbate, retinoic acid, thioredoxin and tocopherol) and enzymatic (such as superoxide dis-mutases, catalase, glutathione peroxidase and peroxi-redoxin) pathways, which limit the rate of oxidation and thereby protect cells from oxidative stress [1,2] Notwith-standing, evidence is emerging that ROS also act as signals
or mediators in many cellular processes, such as cell pro-liferation, differentiation, apoptosis, and senescence [3–5] The redox environment of a cell may alter the balance between apoptosis and mitosis by affecting gene expression and enzyme activity [6] Consequently, cellular redox state is increasingly accepted as a key mediator of multiple meta-bolic, signalling and transcriptional pathways essential for normal function and cell survival or programmed cell death [3–6]
Mitochondria are certainly the major cellular site for oxygen reduction and hence the site with the greatest potential for ROS formation An estimated 0.4–0.8% [7]
to 2–4% [8] of the total oxygen consumed during electron transport is reduced not to water by cytochrome c oxidase but rather to superoxide by complexes I, and III of the respiratory chain [1,7,8] ROS production increases when respiratory flux is depressed by a high ATP/ADP ratio, high electronegativity of auto-oxidizable redox carriers in
Correspondence to L Vergani, Dipartimento di Scienze Neurologiche,
Universita` di Padova, c/o Istituto Veneto di Medicina Molecolare,
Via Orus 2, 35129 Padova, Italy Fax: +39 049 7923271,
Tel.: +39 049 7923219, E-mail: lodovica.vergani@unipd.it
Abbreviations: CS, citrate synthase; CuZnSOD, copper zinc
super-oxide dismutase; DCF, 2¢,7¢-dichlorofluorescin; DTT, 1,4-dithio- DL
-threitol; GSH, glutathione; GSSG, oxidized glutathione; GPx, GSH
peroxidase; GR, GSSG reductase; GST, GSH transferase; H 2
-DCF-DA, 2¢,7¢-dichlorofluorescin-diacetate; IRP-1, iron regulatory
protein-1; LDH, lactate dehydrogenase; MFI, mean log fluorescence intensity;
MnSOD, manganese superoxide dismutase; MPA, metaphosphoric
acid; mt, mitochondrial; NBT, nitroblue tetrazolium; PMRS, plasma
membrane oxidoreductase system; PBN,
N-tert-butyl-a-phenyl-nitrone; ROS, reactive oxygen species; SOD, superoxide dismutase.
Enzymes: catalase (EC 1.11.1.6); GSH peroxidase (EC 1.11.1.9);
GSSG reductase (EC 1.8.1.7); GSH transferase (EC 2.5.1.18); Mn
superoxide dismutase, CuZn superoxide dismutase, superoxide
dismutase (EC 1.15.1.1).
(Received 26 April 2004, revised 16 July 2004, accepted 23 July 2004)
Trang 2complex I and III, or a rise in oxygen tension (state 4
respiration) Defects in respiratory complexes [9] and
normal aging [10] also lead to increased mitochondrial
ROS production A recent study [11] indicates that
mitochondrial ROS homeostasis plays a key role in the
life and death of eukaryotic cells, as mitochondria not
only respond to ROS but also release ROS in response to
a number of pro-apoptotic stimuli However,
mitochon-dria are not the sole source of cellular ROS ROS also
form in the cytosol and in peroxisomes as by-products of
specific oxidases [7,10] The plasma membrane
oxido-reductase system (PMRS) also influences cellular redox
state [12,13]
Mitochondria are partially autonomous organelles; they
possess DNA, which contributes essential proteins to the
oxidative phosphorylation system In vitro mammalian
cells can be depleted entirely of their mitochondrial DNA,
creating so-called q0 cells [14,15] Rho0 cells lack a
functional electron transport chain and appear incapable
of generating ATP from mitochondria Moreover, it is still
a debated question [16] whether or not q0 cells may
generate ROS at the mitochondrial level Therefore, q0
cells may require alternative mechanisms for energy supply
and for maintenance of an appropriate redox environment
[17,18] Analysis of q0 cells has provided insights into
oxygen metabolism [13,17,19–21] and the role of
mito-chondria in redox signalling during apoptosis [22,23]
Redox-sensitive signalling and sensitivity to oxidative
stress depend on the cell type and its antioxidant systems,
due to differential tissue expression of nuclear genes [24]
There are no reports that compare antioxidant defences
and ROS homeostasis between mitochondrial
(mt)DNA-depleted cells with different nuclear backgrounds In this
study, soluble and enzymatic antioxidant systems and
ROS steady-state level were characterized in three tumour
cell lines derived from bone (osteosarcoma, 143B), muscle
(rhabdomyosarcoma, RD) and lung (adenocarcinoma,
A549) and in the respective q0cells: 143Bq0(bone), RDq0
(muscle) and A549q0 (lung) cells This approach was
undertaken to investigate the effect of the absence of
electron transport chain on cellular redox homeostasis,
with the hypothesis that ROS levels could be altered in
consequence of the ablation of an efficient respiratory
chain We aimed to verify: (a) if q0 status requires
antioxidant defence systems as efficient as those of normal
q+ cells; (b) if nuclear background influences redox
homeostatis in the different cell lines, precursors of
cytoplasmic hybrids (cybrids), that are useful tool for
studies of mtDNA segregation [25,26]
Experimental procedures
Materials
All reagents and enzymes were from Sigma NaCl/Pifrom
Oxoid had the following composition: NaCl 8 gÆL)1, KCl
0.2 gÆL)1, Na2HPO4 1.15 gÆL)1 and KH2PO4 0.2 gÆL)1
(pH 7.3) Tissue culture reagents were purchased from
Gibco-Invitrogen Co Reverse transcription was performed
using the Stratascript enzyme (Stratagene)
2¢,7¢-Dichloro-fluorescin-diacetate (H2-DCF-DA) was from Molecular
Probes
Cell lines and culture conditions The q+wild-type osteosarcoma cells (143B) and the q0cells derived from 143B were a gift from G Attardi (Division of Biology, California Institute of Technology, Pasadena, CA, USA)
2 [14], RD and RDq0cells were established by Vergani
et al [27], lung carcinoma (A549) and the derived q0cells were a gift from I J Holt (MRC, Dunn Human Nutrition Unit, Cambridge, UK)
Dulbecco’s modified Eagle’s medium containing 4.5 gÆL)1 glucose, 110 mgÆL)1 pyruvate, supplemented with 10% (v/v)
4 fetal bovine serum, 100 unitsÆmL)1 penicillin, and 0.1 mgÆmL)1streptomycin, at 37C in a humidified atmo-sphere of 5% CO2 The medium for q0cells was additionally supplemented with 50 lgÆmL)1 uridine The absence of mtDNA in these three cell lines was reconfirmed at several time points throughout the study by PCR as described previously [14,25,27] Routinely, 2· 106 q+ or q0 cells were seeded on 100 mm diameter plates and harvested after 42–48 h of culture during the period of exponential growth Subcellular fraction preparation
In some experiments regarding aconitase reactivation (see below), 40· 106 cells suspended in 0.8 mL were treated with digitonin (0.5 mgÆmL)1) in NaCl/Pifor 15 min on ice The samples were centrifuged at 17 000 g for 15 min at
4C, the supernatant (cytosolic fraction) and the pellet (mitochondria-enriched fraction), as well as the whole cells, were recovered, immediately frozen in liquid N2and stored
at)80 C Aliquots, kept at )80 C for up to 2 weeks, were thawed immediately before the assay, as reported previously [28] As markers of cytosolic and mitochondria-enriched fractions, lactate dehydrogenase (LDH) [29] and citrate synthase (CS) [30] activities were assayed in total cells and in cytosolic and mitochondria-enriched fractions, respectively
In mitochondria-enriched fractions CS activity was twice the value found in the whole cells, whereas cytosolic contamination, checked by measuring LDH, ranged from
10 to 30% In the cytosolic fractions the contamination of mitochondria, checked by measuring CS activity, was about 10% of the value found in whole cells
Antioxidant defences Glutathione and oxidized glutathione amounts Cellular glutathione (GSH) and oxidized glutathione (GSSG) levels were measured enzymatically by using a modification of the procedure of Anderson, as described [31,32] The assay is based on the determination of a chromophoric product, 2-nitro-5-thiobenzoic acid, resulting from the reaction of 5,5¢-dithiobis-(2-nitrobenzoic acid) with GSH In this reaction, GSH is oxidized to GSSG, which is then reconverted to GSH in the presence of glutathione reductase and NADPH The rate of 2-nitro-5-thiobenzoic acid formation is measured spectrophotometrically at 412 nm The cells (about 5–6· 106 cells) were washed once with NaCl/Pi and treated with 6% (v/v) metaphosphoric acid (MPA) (1 mLÆdish)1) at room temperature After 10 min the acid extract was collected, centrifuged for 5 min at
18 000 g at 4C and processed The cellular debris remaining on the plate were solubilized with 0.5 KOH
Trang 3and assayed for their protein content [33] For total
glutathione determination, the above acid extract was
diluted (1 : 6) in 6% (v/v) MPA; thereafter to 0.1 mL of
supernatant, 0.75 mL 0.1M potassium phosphate, 5 mM
EDTA buffer pH 7.4, 0.05 mL 10 mM
5,5¢-dithiobis-2-nitrobenzoic acid (prepared in 0.1Mphosphate buffer) and
0.08 mL 5 mM NADPH were added After a 3 min
equilibration period at 25C, the reaction was started by
the addition of 2 U glutathione reductase (type III, Sigma,
from bakers yeast, diluted in 0.1M phosphate/EDTA
buffer) Product formation was recorded continuously at
412 nm (for 3 min at 25C) with a Shimadzu UV-160
spectrophotometer The total amount of GSH in the
samples was determined from a standard curve obtained
by plotting known amounts (from 0.05 to 0.4 lgÆmL)1) of
GSH vs the rate of change of absorbance at 412 nm GSH
standards were prepared daily in 6% (v/v) MPA and diluted
in phosphate/EDTA buffer pH 7.4 For GSSG
measure-ment, soon after preparation the supernatant of acid extract
was treated for derivatization with 2-vinylpiridine at room
temperature for 60 min In a typical experiment, 0.15 mL of
supernatant was treated with 3 lL of undiluted
2-vinyl-pyridine Nine microliters of triethanolamine were also
added, the mixture was vigorously mixed, and the pH was
checked; it was generally between 6 and 7 After 60 min,
0.1 mL aliquots of the samples were assayed by means of
the procedure described above for total GSH measurement
The amount of GSSG was quantified from a standard curve
obtained by plotting known amounts of GSSG (from 0.05
to 0.20 lgÆmL)1) vs the rate of change of absorbance GSH
present in the samples was calculated as the difference
between total glutathione and GSSG levels
Antioxidant enzyme activities GSH peroxidase (GPx),
GSSG reductase (GR), catalase, superoxide dismutase
(SOD) and GSH transferase (GST) activities were measured
in monolayer cells (about 2–3· 106 cells), washed three
times with NaCl/Pi before treatment directly on the dish
with 0.25M sucrose, 10 mM Tris/HCl pH 7.5, 1 mM
EDTA, 0.5 mM phenylmethanesulfonyl fluoride, 0.5 mM
1,4-dithio-DL-threitol (DTT) and 0.1% (v/v) Nonidet
(named solution A), to obtain complete lysis of intracellular
organelles Cells were then scraped from the plate and the
samples were centrifuged for 30 min at 105 000 g Protein
content measurements [33] and enzymatic assays were
carried out on the clear supernatant fractions
Total GPx activity was measured according to the
coupled enzyme procedure with glutathione reductase, as
described [34], using cumene hydroperoxide as substrate
The enzymatic activity was monitored by following the
disappearance of NADPH at 340 nm for 3 min at 25C
The incubation medium (final volume 1 mL) had the
composition 50 mMKH2PO4pH 7.0, 3 mMEDTA, 1 mM
KCN, 1 mM GSH, 0.1 mM NADPH, 2 U glutathione
reductase and 300 lg protein After a 3 min equilibration
period at 25C, the reaction was started by the addition of
0.1 mM cumene hydroperoxide dissolved in ethanol The
specific activity was calculated by using an extinction molar
coefficient obtained by a standard curve of NADPH
between 0.02 and 0.1 lmolesÆmL)1 and GPx activity
protein)1Æmin)1
GR activity was measured according to the method of Carlberg & Mannervik [35], by following the rate of oxidation of NADPH by GSSG at 340 nm for 3 min at
25C The reaction mixture (final volume 1 mL) contained 0.1M KH2PO4 pH 7.6, 0.5 mM EDTA, 1 mM GSSG, 0.1 mMNADPH, and 300 lg protein The specific activity was calculated by using an extinction molar coefficient obtained by a standard curve of NADPH between 0.02 and 0.1 lmolesÆmL)1and GR activity was expressed in nmoles NADPH consumedÆmg protein)1Æmin)1
Total catalase activity was assayed according to the method of Aebi [36] Activity was measured by monitoring, for 30 s at 25C, the decomposition of 10 mM H2O2 at
240 nm in a medium (final volume 1 mL) consisting of
50 mM phosphate buffer pH 7.0 and 100 lg proteins Catalase activity was expressed as unitsÆmg protein)1, assuming that 1 unit of catalase decomposes 1 lmole of
H2O2Æmin)1 For SOD activity assay a 0.6 mL aliquot of cell lysate was sonicated on ice (2· 30 s) and centrifuged for 30 min
at 105 000 g The supernatant was collected and dialysed overnight in cold double-distilled water
interference substances [37] Enzymatic assays were carried out according to the method of Oberlay & Spitz [38], with minor modifications Briefly, in 1 mL 50 mM KH2PO4
pH 7.8 and 0.1 mM EDTA, a superoxide-generating sys-tem (0.15 mMxanthine plus 0.02 U xanthine oxidase) was used together with 50 lM nitroblue tetrazolium (NBT) to monitor superoxide formation by following the changes in colorimetric absorbance at 560 nm for 5 min at 25C The catalytic activities of the samples were evaluated as their ability to inhibit the rate of NBT reduction; increasing amounts of proteins (5–150 lg) were added to each sample until maximum inhibition was obtained SOD activity was expressed as unitsÆmg protein)1, with 1 unit of SOD activity being defined as the amount of proteins causing half-maximal inhibition of the rate of NBT reduction GST activity was assayed in the supernatant of cell lysates, as described [39] Briefly, 150 lg protein were incubated in 50 mM KH2PO4 pH 6.5, 1 mM GSH and 0.25 mM 1-chloro-2,4-dinitrobenzene The reaction was followed for 2 min at 37C at 340 nm, and GST activity was calculated using an extinction coefficient of 9.6 mM )1Æcm)1[39]
Reverse transcription and quantitative PCR RNA (5 lg) was reverse transcribed to cDNA using random hexamer primers and the Stratascript enzyme Quantitative PCR was performed using an MX3000p thermal cycler system and Brilliant SYBER Green QPCR Master Mix (Stratagene) The conditions for the amplifica-tion of copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD) and the nor-malization gene, ribosomal 36B4, were as follows One denaturation step at 90C for 10 min, 40 cycles consisting
of denaturation at 90C for 30 s, annealing at 56 C for
60 s for CuZnSOD and MnSOD and 60C for 36B4, elongation at 72C for 60 s At the end of the PCR the samples were subjected to melting curve analysis All reactions were performed in triplicate The primer sequences were CuZnSOD [40], sense 5¢-GCGACGAAG
Trang 4GCCGTGTGCGTGC-3¢, antisense 5¢-ACTTTCTTCATT
TCCACCTTTGCC-3¢; MnSOD [40], sense 5¢-CTTCA
GCCTGCACTGAAGTTCAAT-3¢, antisense 5¢-CTGAA
GGTAGTAAGCGTGCTCCC-3¢; 36B4, sense 5¢-GTGA
TGTGCAGCTGATCAAGACT-3¢, antisense 5¢-GATGA
CCAGCCCAAAGGAGA-3¢
Western blot analysis
Cells were lysed in the same buffer as used for the enzyme
activity assay An equal amount of protein (40 lgÆlane)1)
for each sample was separated by SDS/PAGE (12%
acrylamide) and transferred to nitrocellulose membrane
The membrane was blocked in 5% (w/v) nonfat dry milk in
60.02M Tris/HCl pH 7.5, 0.137M NaCl, and 0.1% (v/v)
Tween-20 for 3 h at room temperature After overnight
incubation at 4C in 1 : 1000 of primary antibodies
to CuZnSOD (Santa Crutz) or MnSOD (Stressgen
Bio-technology), membranes were probed with horseradish
peroxidase-conjugated secondary antibody (Amersham
Biosciences) Bound antibody was visualized using an
ECL reagent (Amersham Biosciences) Densitometric
ana-lysis of Western blot signal was performed usingIMAGE
-MASTER VDS-CL (Amersham Pharmacia Biotech) and
IMAGE-MASTER TOTALLABv1.11 software
ROS measurement
Aconitase determination Aconitase activity was measured
as described previously [41] on 1· 106 cells or on the
subcellular fractions obtained as reported above The
samples were dissolved in 0.1% (v/v) Triton X-100 and
incubated for 15 min at 30C in 50 mMTris/HCl pH 7.4,
0.6 mMMgCl2, 0.4 mMNADP, 5 mMNa citrate To start
the assay, 2 U isocitrate dehydrogenase were added
and activity was measured by monitoring absorbance at
340 nm for 15 min Reactivation of aconitase was
obtained by adding 50 lMDTT, 20 lMNa2S and 20 lM
Fe(NH4)2(SO4)2 directly into the cuvette, just before
spectrophotometric determination [41]
DCF fluorescence Direct detection of intracellular
steady-state levels of ROS was carried out on living cells using
2¢,7¢-dichlorofluorescin-diacetate (H2-DCF-DA) [42–44] The
probe is de-acetylated inside the cell The subsequent
oxidation by intracellular oxidants yields a fluorescent
product, 2¢,7¢-dichlorofluorescin (DCF) Cells were collected
by trypsinization and centrifuged for 5 min at 800 g The
pellet was incubated in tissue-culture medium with 5 lM
H2-DCF-DA for 30 min at 37C Cells were washed and
then suspended (1· 106 per mL) in medium (standard
growth conditions) or in NaCl/Pi for 90 min (stress
conditions) A FACSCalibur analyser (Becton-Dickinson
Immunocytometry Systems) equipped with a 488 Argon
laser was used for measurements of intracellular
fluores-cence Dead cells were excluded by electronically gating data
on the basis of forward- vs side-scatter profiles; a minimum
of 1· 104cells of interest were analysed further
Logarith-mic detectors were used for the FL-1 fluorescence channel
necessary for DCF detection Mean log fluorescence
intensity (MFI) values were obtained by the CELLQUEST
software program (Becton-Dickinson)
Results The steady-state levels of intracellular ROS depends on the balance between rates of ROS generation and detoxifica-tion A crucial role in determining ROS cellular homeostasis
is played by the antioxidant defence systems Therefore soluble (GSH and GSSG) and enzymatic defences (GPx,
GR, SOD, catalase and GST) were characterized on three human tumour cell lines, with (q+) and without (q0) mtDNA GSH concentration was significantly decreased in all three mtDNA depleted cell lines compared to parental lines with mtDNA; the decrease in GSH content was most pronounced in bone 143B q0cells (Fig 1) GSSG was also lower in q0cells compared with q+, but only statistically significant in bone-derived cells (Fig 1) The percentage of
Fig 1 GSH and GSSG concentrations and ratio of GSH 2 : GSSG in
q+and q0cells from osteosarcoma (bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) Values are expressed as means ± SD of at least three assays carried out in duplicate Significant differences from respective q+value at: *P < 0.05; **P < 0.01.
Trang 5mitochondrial GSH in respect to total GSH was similar in
all tested q+and q0cell lines, ranging from 2.7 to 5% (data
not shown) To assess the cellular redox state we measured
the GSH2: GSSG ratio which is considered a good index of
this parameter [45] MtDNA loss was associated with an
alteration in this ratio with q0cells having a more oxidized
redox state than q+ cells However the change was
statistically significant only in bone-derived q0cells
More-over, the different values found in bone, muscle and lung q0
cells were all significantly different (P < 0.05) from each
other; in fact the GSH2: GSSG ratio of bone 143Bq0cells is
about one-half of that in muscle RDq0cells and even three
to four times lower than that measured in lung A549q0cells
GPx and GR are crucial antioxidant defences as GPx
transforms H2O2to H2O by coupling the oxidation of GSH
to GSSG and GR mediates the reduction of GSSG to GSH
In the three cell lines tested, mtDNA loss was associated
with a four-fold increase in GPx activity and a significant
decrease in GR activity (Fig 2) Moreover Fig 2 shows
that the absolute values of GPx and GR activity were
considerably higher in lung q0 cells than in other q0 cells
(Fig 2) Catalase activity was assessed in q+and q0cells;
our findings show that such activity was not affected by
mtDNA depletion (data not shown)
Activity, gene expression and protein content of SOD
were studied Total SOD activity was decreased in bone and
muscle q0 cells compared with their parental q+ lines
(Fig 3), whereas there were no significant differences in the activity and expression levels in lung q+ and q0 cells (Figs 3–5) Quantitative PCR (Fig 4) and Western blot (Fig 5) analysis were carried out to evaluate the relative contribution of MnSOD and CuZnSOD Both analyses confirmed that bone q0 cells had significantly lower expression of CuZnSOD than the other cells In muscle-derived cell lines mtDNA ablation reduced the expression and protein amount of mitochondrial MnSOD but not of cytosolic CuZnSOD (Figs 4 and 5) Densitometric analysis
of Western blot was in line with the results of quantitative PCR (data not shown)
Glutathione S-transferase (GST) enzymes metabolize xenobiotics as well as aldehydes, endogenously produced during lipid peroxidation, by conjugation with GSH Moreover, some GSTs also show glutathione-peroxidase-like activity [1] GST activity was decreased to a similar extent in bone- and muscle-derived q0cells, compared with the parental q+ cells, but the absolute value was signifi-cantly higher in bone than in muscle q0cells No differences were evident in lung q+and q0cell lines (Fig 6) To check the ability of the antioxidant defences to balance ROS generation, indirect and direct measurements of intracellular steady state levels of ROS were performed Indirect measurements were carried out by assessing the aconitase activity Aconitase is a four iron–sulfur cluster (Fe–S)-containing hydratase, present in various subcellular compartments (i.e mitochondria and cytosol) which is inactivated by OÆ2 [41] In the cytosol, loss of aconitase activity results in the conversion of this enzyme to the iron regulatory protein-1 (IRP-1), that serves to regulate iron homeostasis [46], and mitochondrial aconitase inactivation serves as a protective response to oxidative stress [46] Aconitase activity was measured in q+and q0 cell lines under basal culture conditions and after 18 h of treatment with the ROS spin-trapping N-tert-butyl-a-phenylnitrone (PBN) [47,48] Figure 7 shows a trend of increasing aconitase activity in almost all PBN-treated cell lines The increase was most marked in bone q+and q0cells (more
Fig 2 GPx and GR activities in q + and q 0 cells from osteosarcoma
(bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) Values
are expressed as means ± SD of at least three assays carried out in
duplicate Significant differences from respective q+ value at:
**P < 0.01; ***P < 0.001.
Fig 3 Total SOD activity in q+andq° cells from osteosarcoma (bone),
rhabdomyosarcoma (muscle) and lung carcinoma (lung) Values are expressed as means ± SD of at least three assays carried out in duplicate Significant differences from respective q + value at:
***P < 0.001.
Trang 6than five-fold) and in muscle q0 cells, suggesting that the
OÆ2 level was higher in these cells than in lung q0 cells
Both mitochondrial [28,46] and cytosolic IRP-1/aconitase
activities [46] are reactivated in the presence of reducing
agents and free Fe2+carrier–donor [41] Therefore, in an
attempt to localize OÆ2 production, we assessed aconitase
reactivation in these subcellular fractions Reactivated
aconitase showed a dramatic increase in cytosolic fractions
of bone q0cells (Fig 8), whereas in mitochondria-enriched
fractions there were no significant differences
Lastly, by means of the DCF technique coupled to flow
cytometric analysis, intracellular fluorescence was measured
as an index of steady-state levels of ROS under basal and
stress conditions (Fig 9, Table 1) In the presence of glucose
and 10% serum (standard growth conditions), the
fluores-cence measured in q0 cells was lower than that in the
parental cell lines containing mtDNA The decrease was
substantial in lung (90%) and muscle (40%) cells but was
less evident in bone (less than one-third) (Table 1) When
the cells were incubated in NaCl/Pi for 90 min, the
intracellular fluorescence signal dramatically increased in
all cases (Fig 9, Table 1) The increases, in comparison to
the signals observed in standard growth conditions, were
consistently greater in q0than in q+cells, yet the extent of the increase varied considerably between the three q0lines
In bone and lung q0cells the increases were 17- and 39-fold, respectively However only in bone q0 cells was DCF oxidation significantly higher compared to the value of the respective q+cell line (Table 1)
Discussion Our analysis of three pairs of q+and q0cells, derived from bone, muscle and lung, indicates that these cells differ significantly both in their antioxidant defences and intra-cellular ROS homeostasis The antioxidant system is
Fig 5 Western blotting analysis of CuZnSOD and MnSOD in q + and
q0cells from osteosarcoma (bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) Total cell extract was resolved by SDS/PAGE and blotted onto nitrocellulose The membrane was cut in strips, corres-ponding to the different molecular masses of MnSOD, CuZnSOD and actin, the last acting as an internal standard, and incubated with the corresponding antibody Forty micrograms of cell protein extract was loaded in each lane The blots depicted are representative of three separate experiments.
Fig 6 GST activity in q +
and q 0
cells from osteosarcoma (bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) Values are ex-pressed as means ± SD of at least three assays carried out in dupli-cate Significant differences from respective q + value at: **P < 0.01.
Fig 4 Quantitative real-time PCR of CuZnSOD and MnSOD in q +
and q 0 cells from osteosarcoma (bone), rhabdomyosarcoma (muscle) and
lung carcinoma (lung) mRNA values of CuZnSOD and MnSOD are
normalized for ribosomal 36B4 gene and are expressed as
means ± SD of three assays in triplicate in arbitrary units (A.U.).
Significant differences from respective q+value at: *P < 0.05.
Trang 7profoundly affected by mtDNA depletion in a tissue
specific-manner, probably as a response to a decreased
need of efficient antioxidant machinery
Antioxidant defences of parental q+ cell lines
The parental (q+) A549 cells, derived from type II human
alveolar epithelial cells [49], are provided with the highest
GSH content and GSH2: GSSG ratio (Fig 1), and the
highest GPx, GR (Fig 2) and SOD (Fig 3) activities in
comparison with bone and muscle derived q+cells This
very efficient ROS defence system may be related to the high
oxygen tension normally present in the lung and explains
the great resistance of these cells to apoptosis, after exposure
to high oxygen concentrations [50] By contrast, bone
(143B)- and muscle derived (RD)- cells are similar in their
low content of GSH (only one-half of that present in A549)
and poor GPx activity (Figs 1 and 2); however, RD cells
differ significantly in GR activity and in particular in
activity, gene expression and protein content of SOD
(Figs 3–5)
Antioxidant defences of q0cell lines
GSH-GSSG and GR We measured GSH and GSSG in
exponentially growing cells, as GSH content changes in the
growth and lag phases [51] In all q0cells studied, GSH was
significantly lower than in the respective parental cells, with
the lowest GSH level in bone-derived q0cells, and significant
differences in the GSH2: GSSG ratios among the different
q0 cells (Fig 1) The intracellular content of GSH is the
result of balance between its synthesis and consumption
GSH synthesis is a two-step ATP-requiring process
cata-lysed by cytosolic c-glutamylcysteine synthetase (c-GCS)
and GSH synthetase and is regulated (feedback-inhibited)
by GSH itself [52] We neither directly measured these
activities in our q0cells nor did we find reports on this topic
in the literature, but we did find a very low amount of ATP (data not shown) in all of the q0cells compared with the respective parental q+cells The smaller GSH pool in q0 cells (reduced GSH and GSSG) suggests that it could be due
to reduced synthesis rather than to enhanced utilization in cells with low amounts of ATP In fact if the lower level of GSH in q0cells was due to its extensive consumption in the GPx pathway or to a direct interaction with ROS, we should find increased GSSG In our experimental condi-tions we found that GSSG levels in all q0cell lines were not increased, but rather decreased, although GR activity was significantly decreased in all q0 cells (Fig 2) However, it cannot be excluded that GSSG is actively secreted from the cells subjected to an oxidative stress [52]
maintain cellular redox environment [45] Therefore our data could indicate that mtDNA-depleted cells need less
Fig 8 Aconitase reactivation Aconitase activity was assayed in mit-ochondrial and cytosolic fractions of q + and q 0 from osteosarcoma (bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) Reactivation was achieved in presence of reducing agents (DTT) and
Fe 2+ carrier–donor [Fe(NH 4 ) 2 (SO 4 ) 2 ], as described in Experimental procedures, and is expressed as percentage of basal value Basal values (nmolesÆmin)1Æmg protein)1) of mitochondrial aconitase activity were:
in bone q +
¼ 3.26 ± 1.87 (4); bone q 0
¼ 2.36 ± 0.93 (4); muscle
q +
¼ 8.77 ± 0.57 (3); muscle q 0
¼ 2.08 ± 0.19 (3); lung q +
¼ 8.46 ± 4.12 (3); lung q0¼ 4.88 ± 0.59 (3) Basal cytosolic aconitase
in bone q +
¼ 1.64 ± 0.57 (4); bone q 0
¼ 2.81 ± 1.12 (4); muscle
q +
¼ 0.76 ± 0.29 (3); muscle q 0
¼ 1.26 ± 0.53 (3); lung q +
¼ 4.79 ± 0.6 (3); lung q0¼ 4.59 ± 2.27 (3) Significant differences from respective q+value at: *P < 0.05, **P < 0.01.
Fig 7 Aconitase activity in whole cells in absence (–) and presence (+)
of PBN Rho+and q0cells from osteosarcoma (bone),
rhabdomyo-sarcoma (muscle) and lung carcinoma (lung) were cultured in the
ab-sence (–) or the preab-sence (+) of 500 l M PBN for 18 h Aconitase
activity were assayed spectrophotometrically in cell lysate Values are
expressed as means ± SD of at least three assays in duplicate as
nmolesÆmin)1Æmg)1protein –PBN value significantly different from
+PBN value at: *P < 0.05; **P < 0.01; ***P < 0.001.
Trang 8anti-ROS buffer in the form of GSH for loss of ROS
mitochondrial fluctuation and of ROS spike, occurring
when the respiratory chain is active
8
SOD, GST, GPx and catalase With the exception of catalase and GPx activity, depletion
of mtDNA diminished SOD and GST activities in bone-and muscle-derived q0cells but not in lung-derived q0cells (Figs 3–6), where SOD (Figs 3–5) and GST (Fig 6) were unaffected after ablation of the respiratory chain In bone and muscle q0 cells SOD activity decreased (Fig 3) as compared with the respective parental q+cells Expression level analysis revealed that in bone q0 cells CuZnSOD mRNA (Fig 4) and protein content were decreased (Fig 5), whereas in muscle q0cells MnSOD decreased in mRNA and protein amount compared with parental cells (Figs 4 and 5) The decrease of SOD and GST antioxidant enzymes in bone and muscle but not in lung q0cells might be ascribed to different expression–regulation of nuclear genes
as a response to cell type differential redox-sensitive signalling [53]
Catalase activity is unaffected by mtDNA depletion (data not shown) and, interestingly, the activity of GPx was found to be considerably increased in all q0 cells relative to the parental cells (Fig 2) GPx, together with catalase and thioredoxin peroxidase, restricts H2O2 accu-mulation and the consequent production of highly reactive
Bone
Muscle
Lung
Blank Standard growth condition Stressed condition
100 101 102 103 104
100 101 102 103 104
100 101 102 103 104 10 0 10 1 10 2 10 3 10 4
10 0 10 1 10 2 10 3 10 4
10 0 10 1 10 2 10 3 10 4
FL1-H FL1-H
FL1-H FL1-H
FL1-H
FL1-H
Counts Counts
Fig 9 DCF oxidation in cells with and without glucose Rho+and q 0 cells from osteosarcoma (bone), rhabdomyosarcoma (muscle), and lung carcinoma (lung) were collected and loaded with H 2 -DCF-DA Fluorimetric signals of oxidized DCF (excitation, 488 nm; emission, 530 nm) were recorded by cytofluorimeter from cells in presence of glucose (dotted line): standard growth conditions or in absence of glucose (bold line): stressed conditions Blank signal, obtained from cells without H 2 -DCF-DA, was deducted to the reported MFI values The panels are representative of the separate experiments summarized in Table 1.
Table 1 Levels of DCF oxidation in q+and q0cells from osteosarcoma
(bone), rhabdomyosarcoma (muscle) and lung carcinoma (lung) MFI of
the DCF signal was measured by fluorescence activated cell sorting as
arbitrary units in cells in presence of glucose (standard growth
con-ditions) and in absence of glucose (stress concon-ditions) Values are
ex-pressed as mean ± SD as arbitrary units of fluorescence Numbers in
parentheses are the numbers of experiments Significant differences
from respective q + value: *P < 0.05; ***P < 0.001.
Conditions Standard growth Stressa
q 0 142 ± 75 (6) 2500 ± 217 (3)*
q 0 143 ± 4 (3)*** 996 ± 210 (3)
q0 25 ± 2 (3)*** 976 ± 319 (3)
a P < 0.001 vs respective values in standard growth conditions.
Trang 9hydroxyl radicals, for which no physiological defence
system exists [1] In the last few years, the view of hydrogen
peroxide as a merely toxic by-product of cellular
metabo-lism has changed, and it is now recognized as playing an
important role in intracellular signalling [3–5] Fine
regu-lation of redox balance may therefore be a critical function
of peroxidases, catalase and of GPx, in particular [54] GPx
regulates the intracellular hydroperoxides and lipid
hydro-peroxides used as signal transducers of many transcription
factors including nuclear factor-jB [55], AP-1 [56] and
MAP kinases [57] Because catalase is unchanged, the
increased GPx activity of q0 cells may be an essential
cellular adaptation that enables gene expression to function
normally in the absence of mtDNA These findings are
in line with results found in hepatoma-derived Hep1q0
cells [16]
ROS
When DCF signal was assessed as a direct index of ROS, all
of the q0 cells had a reduced intracellular fluorescence
compared to q+cells Bone-derived q0cells had the highest
level of intracellular ROS compared to muscle and lung q0
cells both in standard growth conditions and in stressed
conditions (Fig 9, Table 1) If the current idea, that the
DCF technique mainly determines cellular peroxides [42–
44,58], is accepted it can be hypothesized that q0 cells
accumulate a lower DCF fluorescence signal due to their
high GPx activity (Fig 2) in a tissue-specific manner In
fact, lung q0cells have the lowest DCF oxidation (Fig 9,
Table 1) and the highest GPx activity (Fig 2), whereas
bone- and muscle-derived q0cells have rather similar GPx
activities and similar capacities to eliminate intracellular
oxidants under standard growth conditions Yet, in the
absence of glucose (stress conditions), intracellular levels of
ROS in bone-derived q0cells are 2.5 times those of muscle
q0 cells (Fig 9, Table 1) This may be due to the fact
that among q0 cells, bone q0 cells had the less efficient
antioxidant machinery with the lowest GSH level (Fig 1)
Interestingly, bone-derived q0cells also featured the highest
glucose consumption rate and glucose-6-phosphate
dehy-drogenase activity among the six lines analysed (L Vergani,
unpublished data) Glucose-6-phosphate dehydrogenase is
the rate-limiting enzyme in the pentose phosphate pathway
and a major source of cytosolic NADPH and ribose
phosphate [59] When glucose is scarce, NADPH synthesis
decreases This lead to a decrease in GSH levels as NADPH
is required for GSH regeneration via GR Therefore, our
data suggest that increased generation of intracellular ROS
in bone q0 cells, relative to muscle q0, is due to increased
production of oxidants The high production of ROS in
bone-derived q0 cells is further confirmed by indirect
measurement of ROS obtained by comparing aconitase
activity in standard conditions and after 18 h of incubation
with PBN (Fig 7) In biological systems PBN [60,61], or
N-t-butyl hydroxylamine, a breakdown product of PBN
[47,48], efficiently trap free radicals, such as superoxide
anion (OÆ
2 ) that in turn inactives aconitase [41] The
observed PBN-induced increase in aconitase activity in bone
q+ and q0 cells and in muscle q0 cells (Fig 7) strongly
supports a high presence of OÆ
2 in these cells also in standard growth conditions These data are well related to
the lowest GSH2: GSSG ratio and the most oxidized redox state (Fig 1) A PBN effect on antioxidant enzyme activities may be excluded on the basis of a recent report showing that PBN protects U937 cells against ionizing radiation-induced oxidative damage by altering cellular redox state but not affecting antioxidant enzymes [61]
New and original evidence emerges from the experiments
of reactivation of aconitase activity by reducing agents and Fe(NH4)2(SO4)2, as a Fe2+ carrier–donor [41] Figure 8 shows a dramatic increase in cytosolic IRP-1/aconitase activity in bone q0cells, but not in mitochondria-enriched fractions This finding suggests that in bone q0 cells intracellular oxidants derive chiefly from nonmitochondrial compartments and are therefore not related to a vestige of the respiratory electron transport chain Possible sources of nonmitochondrial oxidants include NADPH oxidases [12], and lipoxygenases, whose action plays a role in signal pathways of growth factor-stimulated bone cell mitogenesis [62], and microsomal redox systems [63] NADPH oxidases are up-regulated in lymphoblastoid q0cells, as a compen-satory phenomenon in maintaining cell viability [18] Our results confirm PMRS as a possible source of ROS in bone cells, as the NADPH oxidase inhibitor diphenyleniodo-nium chloride reduces fluorescence accumulation into bone q+ and q0 cells to 65–70% (data not shown) Another possible explanation for the increased generation
of intracellular oxidants in bone-derived q0cells is the high
O2tension to which cultured cells are exposed compared to the low O2tension of osteoblasts The bulk of intracellular oxidants in bone-derived q0cells is in extra-mitochondrial compartments, corroborating an earlier report which showed q0cells to be sensitive to the ablation of cytosolic SOD [64] Moreover the presence of extramitochondrial ROS in q0cells could explain the similar levels of oxidative DNA damage observed in Hela q0 and the parental q+ cells [65]
In conclusion, our study demonstrates that loss of functional mitochondria, the major cellular site for ROS formation, reduces enzymatic and soluble intracellular antioxidant defences but not ROS flux in the studied q0 cells, and that there are cell line-to-cell line variations in intracellular antioxidant defences and ROS homeostasis In fact among the studied cells, those originating from bone are particularly vulnerable to free radical-induced stress after mtDNA ablation These differences could reflect tissue-specific aspects of intracellular oxidant metabolism, although it is inevitable that some specific features of ROS homeostasis in terminally differentiated tissues such as bone, lung and muscle will have been lost during the transformation process that led to tumour formation The pronounced difference in intracellular homeostasis between lung A549 and bone 143B q0cells may also be germane to mtDNA segregation bias, as selection of mutant and wild-type mtDNA is different in the 143B and A549 cellular backgrounds [25,26]
Acknowledgements
We thank Dr G Attardi for the gift of osteosarcoma q 0 and q + cells, Dr I.J Holt for the gift of lung carcinoma q 0 and q + cells and we are grateful
to Dr Aubrey de Grey for great help in interpreting and discussing the data This work was supported by Telethon grant no 1252.
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