From the Laboratory of Molecular Genetics and
¶ Laboratory of Signal Transduction, NIEHS, National Institutes of
Health, Research Triangle Park, North Carolina 27709 and
§ Cancer Research Institute,
833 91 Bratislava, Slovakia
Received for publication, August 27, 2002, and in revised form, October 15, 2002
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ABSTRACT |
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In order to understand the molecular events
following oxidative stress, which lead to persistence of lesions in the
mtDNA, experiments were performed on normal human fibroblast (NHF)
expressing human telomerase reverse transcriptase (hTERT). The
formation and repair of H2O2-induced DNA
lesions were examined using quantitative PCR. It was found that NHF
hTERTs show extensive mtDNA damage (~4 lesions/10 kb) after exposure
to 200 µM H2O2, which is
partially repaired during a recovery period of 6 h. At the same
time, the nDNA seemed to be completely resistant to damage. Cell
sorting experiments revealed persistent mtDNA damage at 24 h only
in the fraction of cells with low mitochondrial membrane potential
( Reactive oxygen species
(ROS)1 are ubiquitous
toxicants generated primarily by the mitochondrial respiration and by
other biochemical processes within cells (1). In addition to these
sources, ROS are also produced in pathophysiological conditions such as
inflammation and ischemic reperfusion injury (2). Furthermore, certain
environmental pollutants (like pesticides and herbicides), as well as
smoking, can induce ROS either directly or indirectly (1, 3, 4). It is
well known that ROS can damage biomolecules and have been postulated as
an important cause of aging, cancer, and various human degenerative
diseases (3, 5). One of the most widely used compounds to produce
oxidative stress, both in vitro and in vivo
biological studies, is hydrogen peroxide
(H2O2). Unlike other ROS,
H2O2 is not charged and is therefore freely
diffusible within the cell. It can arise spontaneously or be generated
from superoxide by superoxide dismutase. More importantly, in the
presence of metal ions such as copper or iron,
H2O2 can undergo Fenton chemistry and give rise
to the potent hydroxyl radical. This radical is extremely reactive and
can damage different components of the cell (6). The responses of
cultured cells to H2O2 vary according to cell
type and are known to be both time- and, especially,
dose-dependent (for review see Ref. 7). For example, low
concentrations (3-15 µM) of H2O2
were shown to activate a mitogenic response (8), whereas higher doses
can have either cytostatic or cytotoxic effects. It was observed that
concentrations ranging from 50 to 150 µM promote DNA
damage (9, 10) replicative senescence (11-13), sustained p21 levels,
cell cycle arrest, transient elevation of p53 protein (14), and
temporary growth arrest followed by increased resistance to subsequent
oxidative stress (8, 15). In contrast, H2O2
doses of 200 µM and higher were demonstrated to induce
apoptosis (16), necrosis (16, 17), protein oxidation (18), as well as
lesions in both nuclear and mitochondrial genomes (9, 10).
With regard to DNA damage, we and others have demonstrated that the
mtDNA of different cell types and organisms is more prone to oxidative
injury than the nDNA (9, 10, 19-22). Additionally, Yakes and Van
Houten (9) demonstrated that depending on the intensity of the damage
(dose and time of exposure), nuclear and mitochondrial genomes are
differentially damaged and repaired. Although the precise molecular
reason(s) that lead to the persistence of lesions in the mitochondrial
genome is not clear, several explanations have been offered to clarify
the increased vulnerability of the mtDNA to damage. These include the
lack of a compact nucleosome structure as compared with the nDNA, the
limited mitochondrial repair pathways, as well as the proximity of the
mtDNA to the main source of ROS generation (9, 23). More work is needed to understand the molecular events underlying the susceptibility of the
mtDNA to oxidative damage.
Whereas the initial results obtained regarding the kinetics of damage
and repair in both mitochondria and nuclear genomes were based on
SV40-transformed fibroblasts (9), data from the literature raise
concerns as to the suitability of the SV40-transformed cell model. In
fact, a significant number of physiological changes after viral
transformation have been reported. For example, loss of p53
function with consequent alteration in cell cycle control (24)
decreased DNA repair (25, 26), and diminished catalase activity
(27) was found.
In an effort to better understand the basis of the increased
susceptibility and persistent damage observed in mtDNA after oxidative
stress, we have used a non-transformed diploid fibroblast cell line
expressing the telomerase gene (NHF hTERT) to follow the formation and
removal of H2O2-induced lesions. With this
approach it was observed that these cells accumulate large amounts of
mtDNA damage, which can be completely repaired after a 15-min treatment but not a 60-min treatment. Cell sorting experiments revealed that
persistent lesions in the mtDNA correlate with loss of Cells and Cell Culture--
The normal human fibroblasts
transfected with the telomerase gene (NHF hTERT) used throughout this
study were a gift from Dr. Carl Barrett (NCI, National Institutes of
Health). The cells were routinely grown and sub-cultured in
75-cm2 flasks at 37 °C and 5% CO2 in
DMEM/F-12 medium (Invitrogen) supplemented with 10% fetal bovine serum
(HyClone), SAP (0.2 mM serine, 0.1 mM aspartic
acid, and 1.0 mM pyruvate), 50 units/ml
penicillin/streptomycin (Invitrogen), and 800 µg/ml G418 (Invitrogen)
for selection of the stable transfectants.
H2O2 Treatments--
Cells were seeded
in 100-mm Petri dishes 15-18 h prior to the experiments. Immediately
before treatment cells were washed once with DMEM/F-12 without any
supplements, and the conditioned medium was saved for later use.
A 30 mM stock of H2O2 (30% Sigma) was prepared in PBS (Invitrogen) and used to generate the 200 µM solution (in DMEM/F-12 alone) with which the cells
were challenged. Monolayer cultures were exposed to
H2O2 for 15 or 60 min and harvested immediately
or allowed to recover for up to 24 h (in this case, in the
original conditioned medium).
DNA Isolation, QPCR Assay, and DNA Damage Analysis--
High
molecular weight DNA was extracted, and QPCR was performed and analyzed
as described elsewhere (22). Briefly, genomic DNA was isolated, and
specific primers were used to amplify a fragment of the mitochondrial
and/or nDNA. The assay is based on the premise that DNA lesions
(including oxidative damage such as strand breaks, base modifications,
and abasic sites) block the progression of the polymerase so that only
undamaged templates can participate in the PCR. Thus, amplification is
inversely proportional to DNA damage: the more lesions encountered on
the target DNA, the less the amplification. Note that for the
mitochondrial genome both a short (221 bp) and a long fragment (8.9 kb)
are routinely amplified. The rationale underlying this procedure is
that the probability of introducing a lesion in a short segment is low, and therefore amplification of this segment gives an accurate estimation of the copy number of mtDNA in the sample. The data obtained
from the small fragment were subsequently used to normalize the results
of the 8.9-kb target (more details can be obtained in Ref. 22).
Amplification of treated samples was then compared with controls, and
the relative amplification was calculated. These values were next used
to estimate the average number of lesions per 10 kb of the genome,
using a Poisson distribution (22, 28). It is important to mention that
a "negative" number of lesions was obtained when amplification of
the treated sample was higher than that of the matched control. This
phenomenon can occur by means of induced repair activity in the treated
sample (9, 29). Results presented here are the mean of two sets of PCR
for each target gene of at least three different biological experiments. Student's unpaired t test was performed to
evaluate statistical significance.
Mitochondrial Membrane Potential Analysis--
Changes in the
Cell Cycle Analysis with Propidium Iodide--
Cells were
treated as described above and at various times after the treatments
were subjected to cell cycle analysis by flow cytometry using propidium
iodide (PI, Molecular Probes). Briefly, at each time point both control
and treated cells were harvested, washed twice with PBS, and incubated
at room temperature for 15 min with 1 mg/ml RNase, 1 µl of Triton
X-100(Sigma), and 20 µg/ml of PI. Samples were then immediately
analyzed by fluorescence-activated cell sorter.
Caspase Activation Assay--
Measurement of caspase 3 activation was performed fluorimetrically using the Bio-Rad Apopain
Kit, according to instructions of the manufacturer. Briefly, 24 h
after the challenge with H2O2 cells were
harvested and subjected to total protein extraction using CHAPS buffer
with 5 mM dithiothreitol (Cell Signaling) and 1 mM phenylmethylsulfonyl fluoride. After 5 rounds of
quick-freezing (dry ice embedded in ethanol) and thawing (37 °C
bath), protein concentrations were determined, and subsequently, 50 µl of each sample was used in the Apopain assay. Pro-caspase/apopain
activity was monitored using a fluorogenic peptide substrate,
Ac-DEVD-AFC. Apopain enzymatically cleaved the AFC from the peptide and
released it in a free form, which upon exposure to near UV light
produced a blue-green fluorescence (detected at 500-550 nm). A
calibration curve was generated first by measuring fluorescence of AFC
released from a serial dilution of the standard substrate
(AC-DEVD-AFC). A positive control consisting of protein extract of
Jurkat cells treated with a Fas ligand, a model system for apoptosis,
was also included. Duplicate fluorescence readings of the samples were taken every 30 min after reaction was started for up to 3 h until the readings stabilized. Caspase activity was calculated comparing results from the samples to the calibration curve. Data were reported as fluorescence readings.
Sorting Procedure Based on JC-1 Staining and Flow
Cytometry--
Sorting of the normal and depolarized (low H2O2 Detection from Sorted
Cells--
Cells were sorted as described above, and supernatants were
immediately collected by centrifugation and subjected to analysis using
the Amplex Red Kit (Molecular Probes). The assay is based on the
detection of H2O2 using
10-acetyl-7-hydroxiphenoxazine (Amplex Red reagent), a highly stable
and specific probe for this compound. In the presence of horseradish
peroxidase, Amplex Red reacts with H2O2 at a
1:1 stoichiometry, producing a highly fluorescent product, resorufin,
which can be detected using excitation and emission wavelengths of 530 and 590 nm, respectively (34). Briefly, 100 µl of the supernatants
was loaded in a 96-well microtiter plate and incubated for 30 min
(protected from light and at room temperature) in the presence of
phosphate buffer containing 400 µM of Amplex Red reagent
and 2 units/ml horseradish peroxidase. Background fluorescence was
corrected by subtracting the values derived from PBS alone. Controls
including catalase were also included to ensure that the fluorescent
signal was due to H2O2. For each sample, all
measurements were made in duplicate. The mean of those readings was
then used to estimate H2O2 concentration using
a standard H2O2 curve.
The purpose of this study was to investigate the formation and
repair of H2O2-induced lesions in both nuclear
and mitochondrial genomes of normal human fibroblasts transfected with
the telomerase gene. In addition, we also wanted to understand the
apparent loss of DNA repair activity in cells showing persistent mtDNA
damage and to gain some insights into the biological outcome(s) of this permanent damage.
H2O2-induced Damage--
Quantitation of
lesions introduced both in the mitochondrial and nuclear genomes was
performed using QPCR, and the details of the assay have been published
previously (9, 22, 28). Fig.
1A shows the relative
amplification for the mtDNA of NHF hTERT cells after treatment with 200 µM H2O2 and repair periods of up
to 24 h. As can be seen, a 15-min exposure to
H2O2 gives rise to an ~70% decrease in
amplification as compared with the untreated control. Note that
amplification was completely restored after a 1.5-h incubation in
conditioned medium. In contrast, cells challenged for 60 min showed a
marked reduction in amplification, corresponding to ~4 lesions/10 kb.
Unlike the 15-min treatment, the 60-min treatment leads to lesions that
persisted even after long recovery periods (24 h). During the first
6 h, ~50% of the damage from a 60-min treatment could be
repaired. However, no further removal could be observed up to 24 h. These data suggest that the 60-min treatment passed some threshold
of damage, such that some other deleterious events have taken place
inside the mitochondria.
Surprisingly, the nDNA of NHF hTERT cells did not show any decrease in
amplification (when compared with untreated controls) even after a
60-min exposure to H2O2 (Fig. 1B).
This result suggested an absence of lesions detected in the two genomic
regions ( Mitochondrial Membrane Potential as an Indicator of
Apoptosis--
We next sought to determine whether the fraction
of cells that were detaching from the dishes was dying by
apoptotic death. We reasoned that cells with extensive and
persistent mtDNA damage might not be able to maintain the Caspase Activation and Apoptotic Population Detection by
PI--
To confirm that a fraction of the cells in our experiments
were undergoing apoptotic death, other apoptosis-specific assays were
conducted including analysis of caspase-3 activation as well as the
staining of the nDNA with PI.
Caspase 3 activation was followed using a fluorimetric assay as
described under "Experimental Procedures." Fig. 2B shows
that only cells that were exposed for 60 min to
H2O2 had increased caspase-3 activation as
compared with the negative control. DNA staining with PI also indicated
a significant percentage of apoptotic cells beginning at 24 h with
a peak at 72 h (Fig. 2C). Taken together, these data
indicate that a fraction of NHF hTERTs, when exposed to
H2O2 for 60 min, undergoes apoptosis; this
process was exclusively a consequence of extensive damage to the
mitochondrial genome. Like many other studies with fibroblasts, these
same cells did not show DNA laddering at any time monitored (24, 36, 48, and 72 h; data not shown).
Sorting Procedure by Flow Cytometry and JC-1
Staining--
Integrity of the
By using a FACSVantage SE flow cytometer and JC-1, both control and
H2O2-treated cells were sorted into separate
populations based on having low (depolarized cells) or high H2O2 Released from Sorted Cells and
Viability Assessment--
Our current working hypothesis predicts that
a damaged mitochondrion generates more ROS than an intact organelle.
The mtDNA encodes 13 polypeptides involved in the function of the
electron transport chain (ETC). Incomplete flow of electrons could be
caused by the lack of expression of vital mitochondrial genes from a damaged genome and would be expected to give rise to increased levels
of ROS. In this way, a high steady-state level of ROS could be
generated inside the organelle, which may in turn continuously injure
the mtDNA in a feed-forward cascade. Eventually, this vicious cycle
results in loss of mitochondria function and apoptotic cell death. Thus
we next sought to determine whether ROS were being released from the
sorted cells, especially from the population showing both low
Finally, it was important to confirm that the cells with persistent
mtDNA damage were the inviable cells after the treatments. In an effort
to investigate this phenomenon, sterile sorting was conducted, and both
H2O2-treated and non-treated polarized and depolarized populations were re-cultured to access viability. Cells
were monitored for the next 72 h and counted with a Coulter counter. It was found that 80% of the control polarized cells were
able to attach and grow, whereas the portion of treated cells with
normal The increased susceptibility of the mtDNA to
H2O2-induced damage, as compared with the nDNA,
has been documented previously (9, 10, 23); however, the specific
molecular causes of these phenomena are not well understood. It has
been hypothesized that a combination of biological determinants, such
as the proximity of the mtDNA to ROS, the lack of a compact nucleosome
structure to protect the genome, and a paucity of mtDNA
damage-processing pathways relative to those present in the nucleus
play a role (reviewed in Ref. 23). In an attempt to understand these
phenomena better, we have used telomerase-expressing normal diploid
fibroblasts and found that repair of ROS-induced lesions in the
mitochondrial genome depends on a threshold of dose and time and that
persistent mtDNA damage leads to loss of The present study confirmed earlier findings (9, 10),
H2O2 induces a large amount of mtDNA lesions
(Fig. 1A) and little or no nDNA damage (Fig. 1B).
Surprisingly, the mitochondrial genome of hTERT-transfected cells
appeared to be more vulnerable to H2O2 than
previously reported (9, 10, 19-22) for several other cell types,
whereas the nuclear genome of the same cells was completely resistant
to damage. This is likely a reflection of the fact that hTERT-transfected fibroblasts have increased levels of intracellular iron, making the mtDNA more prone to
damage.2 The fact that the
lesions induced in the mitochondrial genome after a 60-min treatment
are repaired up to 6 h (~50% of lesions are removed) agrees
with previous reports (23, 37-39) suggesting that repair in this
organelle is capable of removing oxidative DNA lesions. However, these
data also indicate that removal of damage is clearly dependent on the
total amount of lesions induced over a period of time, after which
additional events take place in the mitochondrion that seem to
compromise repair function.
DNA damage caused by ROS is removed by the base excision repair (BER)
machinery both in the nucleus and in the mitochondria. Although
oxidative stress induces a wide variety of lesions, many are removed
from the mtDNA through the action of a glycosylase with an associated
abasic site-lyase activity, followed by the activity of Trafficking of nuclear encoded proteins from the cytosol to the
mitochondria is achieved by the concerted action of at least four
translocation complexes (see Ref. 36 for review). In this scenario, the
However, one cannot rule out other putative causes that may impair the
overall repair activity in the mitochondria. For example, Graziewicz
et al. (18) showed that the human Alternatively, saturation of repair activity by increased promotion of
lesions, even in the presence of functional repair machinery, could be
responsible for the persistence of lesions in the mitochondrial genome.
Inappropriate electron flow on an already damaged ETC represents a
potential source of additional waves of ROS (9). In this case, a
vicious cycle is established where the basal level of ROS generation as
well as of mtDNA lesions is enhanced, possibly saturating the active
repair system. The fact that the depolarized sorted cells (those that
also show the mtDNA damage) release increased amounts of
H2O2 as compared with the polarized
counterparts (Fig. 4B) is consistent with this model.
It is worth pointing out that regardless of the mechanism(s)
contributing to the maintenance of lesions in the mitochondrial genome,
our experiments indicate that the biological effect of these persistent
lesions is apoptosis (Fig. 2, A-C). Accordingly, Dobson
et al. (44) demonstrated that targeting an
8-oxoguanine-specific glycosylase not only enhances the repair of
menadione-induced mtDNA lesions but also the viability of treated
cells. In addition, it has been proposed that the differential
susceptibility of glial cell types to oxidative stress and
apoptosis correlates with increased oxidative mtDNA damage (45).
Although reports are available showing that
H2O2 induces apoptotic death, the mechanisms
through which this oxygen byproduct promotes apoptosis are still
debated (see Ref. 7 for review). Interestingly, down-regulation of mitochondrially encoded mRNA, rRNA, and DNA was reported in
apoptotic cells (46-48). All these events are consistent with lesions
being present in the mitochondrial genome, as previously proposed by Yakes and Van Houten (9).
A striking finding in this study is the total absence of nDNA damage in
the same cells that show a large number of mtDNA lesions. In fact,
detectable levels of nDNA damage were only observed 24 h after the
treatments, which is when apoptosis has been initiated. Because the
mtDNA showed increased susceptibility to oxidative damage in
hTERT-transfected cells, one might also expect the nDNA to be more
prone to insult. It might be argued that the nuclear genome, unlike the
mitochondrial one, is better protected by histones and, thus, less
accessible to oxidative attack. In addition, the presence of additional
and overlapping repair pathways (such as the nucleotide excision
repair) (49) could account for the lack of damage accumulation. An
appealing alternative relates to the fact that the cells used in this
study are not virus-transformed and, therefore, have intact p53
function. Although p53 has been claimed to interact directly and
promote the activity of the BER machinery (50), we have no evidence
that this is the case in our experiments. One could envision that an
active p53 is necessary and sufficient to decrease nDNA damage
resulting from oxidative stress. Further p53 analysis should help to
shed light onto this problem.
In summary, the findings of the sorting experiments lead us to suggest
a chain of events, involving persistent mtDNA damage, drop in m). Further analysis also showed increased production of
H2O2 by these cells, which subsequently undergo
apoptosis. This work supports a hypothesis for a feed-forward cascade
of reactive oxygen species generation and mtDNA damage and also
suggested a possible mechanism for persistence of lesions in the mtDNA
involving a drop in
m, compromised protein import, secondary
reactive oxygen species generation, and loss of repair capacity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m, increased ROS generation, and cell death. Interestingly, we also show
that the nDNA of the NHF hTERTs seems totally resistant to H2O2-induced damage, suggesting that
SV40-transformation makes the nuclear genome more prone to oxidative injury.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m were measured by flow cytometry using JC-1
(5,5',6,6'-tetrachloro-1,1',3,3'-tetra-ethylbenzimidazolylcarbocyanine iodide; Molecular Probes). JC-1 was chosen because of its specificity regarding the mitochondrial membrane (30-32). At various time points (every 2 h up to 24 h and then at 36 h and 48 h) after
the challenge with H2O2, control and treated
cells were harvested, washed twice with PBS, and incubated with 10 µM JC-1 for 10 min (37 °C; 5% CO2
atmosphere). For each sample, 10,000 cells were examined on an FL-1
(530 nm) versus FL-2 (585 nm) dot plot using a BD
Biosciences FACSort. JC-1 has dual emission depending on the state of
the
m, forming aggregates with a high 585 nm fluorescence in
cells (which indicates a normal
m). Loss of mitochondrial
membrane integrity results in reduction in 585 nm fluorescence with a
concurrent gain in 530 nm fluorescence as the dye shifts from an
aggregate to a monomeric state (33). Therefore, retention of the dye in the cell can be monitored. The data were converted to density plots
using the CellQuest software.
m)
JC-1-stained cells was accomplished using a BD Biosciences FACSVantage
SE. In these experiments, both control and 60-min treated cells were initially gated in forward-scatter versus a side-scatter dot
plot to exclude debris. Sort gates were set up on an FL-1 (530:30 nm) versus FL-2 (575:26 nm) dot plot with increased stringency
for only the depolarized cells. An all-sort cell population (cells gated on the initial forward-scatter versus side-scatter dot
plot) was also collected to control for the introduction of DNA lesions and/or any change in the
m because of the sorting procedure. The
data were converted to density plots using CellQuest software. Individual sorted cell populations were then immediately subjected to
DNA extraction and QPCR analysis or placed back into culture for
viability assays.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The mitochondrial genome of NHF hTERT cells
is extremely sensitive to oxidative stress, whereas the nuclear DNA is
resistant to damage. Kinetics of damage and repair of the
mitochondrial fragment (8.9 kb) of cells treated with 200 µM H2O2 for 15 min or 60 min and
allowed to recover for the times indicated (A). B
depicts relative amplification of the nuclear -globin gene
immediately after H2O2 treatments and at
various times for repair (similar results were obtained for the HPRT
gene, not shown). The decrease in amplification was calculated
comparing treated samples to undamaged control (dashed
line). These values were then used to determine the lesion
frequency using a Poisson distribution as described under
"Experimental Procedures." Data are expressed as the mean ± S.E. of three biological experiments in which two PCRs were performed
per experiment. Student's t test was performed comparing
treated samples (time 0) to untreated control or time 0 to each
recovery period (*, significance stands for p < 0.05;
**, p < 0.01).
-globin and HPRT) studied. These data differ significantly
from the findings of Yakes and Van Houten (9), which demonstrated the
same type of treatment to introduce 0.5 lesions/10 kb into the nuclear
genome of SV40-transformed fibroblasts. Interestingly, we were able to perceive a very low amount of nDNA damage 24 h after the
treatments, which was coupled to the observation that a vast proportion
of cells (~50%, data not shown) were detaching from the dishes.
Together, these results indicate that the biology of a specific
population of cells has changed after H2O2
treatment, culminating in cell death. Moreover, the absence of nDNA
damage suggests that, in this scenario, the cell death is exclusively a
consequence of persistent lesions in the mtDNA.
m
across the inner membrane, believed to play a role in the apoptotic
process (reviewed in Ref. 35). The integrity of the
m was
evaluated using JC-1, a fluorophore recognized as the most specific
probe for the detection of changes in the
m of different cell
types as compared with other available dyes (30-32). Cells were
treated as described under "Experimental Procedures" for 15 or 60 min and were analyzed every 2 h after the exposure to
H2O2. No differences in the
m were observed when comparing the 15-min with the control at any time monitored (Fig. 2A,
upper right panel). However, a decline in the
m was detected
at around 14 h in cells treated with H2O2 for 60 min, with the highest proportion of cells (~70%) with
decreased
m values observed at 24 h (Fig. 2A).
These data suggest that the cells treated for long periods of time with
H2O2 might be undergoing apoptosis.
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Fig. 2.
Markers of apoptosis observed in cells
treated with 200 µM
H2O2 for 15 or 60 min. A, cells
were incubated with JC-1 for 10 min (37 °C) and analyzed by flow
cytometry at various times after treatment (14 and 24 h, for 60 min of treatment; 24 h for 15 min of treatment) to follow the
m. Drop in
m is identified as a change in JC-1 properties
from aggregate to monomeric form, only detected in cells treated for 60 min. B represents the fluorimetric analysis of caspase 3 activation (details of the assay are described under "Experimental
Procedures");
, control;
, cells treated for 15 min;
, cells
treated for 60 min; and
, positive control (Jurkat cells treated
with a Fas ligand). Data representative of three different biological
experiments. C, detection of apoptotic population by means
of PI staining, analyzed by flow cytometry, after 24, 48, and 72 h
of the treatments. R1 gates the apoptotic population, which
is translated in percentage of total events analyzed.
m plays a role not only in
apoptosis but also in trafficking various proteins into the
mitochondria. In fact, nuclear encoded proteins that act in this
organelle have to be targeted, unfolded, and refolded inside the
mitochondrion by different protein complexes attached to the outer and
inner mitochondrial membranes. This import mechanism also depended on the maintenance of a high
m (reviewed in Ref. 36). Considering that repair proteins that act in mitochondria are nuclear encoded, we
next sought to evaluate whether the persistent mtDNA damage observed
only in the cells treated for 60 min correlated with the drop in
m. To determine whether this was the case, a procedure relying on
JC-1 staining and flow cytometry was adopted.
m
values (polarized cells). Fig. 3 shows
the gating scheme used to individually isolate polarized and
depolarized populations. Immediately after sorting, DNA was isolated,
and damage was evaluated with the QPCR assay. Fig.
4A illustrates the results
from the mtDNA damage analysis on the various sorted populations of
cells. Interestingly, only the depolarized population from the treated
cells showed significant mtDNA damage. This is the first evidence that
directly links depolarization of the
m with persistent lesions in
this genome. As described under "Experimental Procedures," an
all-sort cell population where cells passed through the sorter but were
not physically separated was included. Note that no significant
differences in the amount of mtDNA damage were observed between the
depolarized and all-sort treated cells, which is probably due to
stringency of the gated depolarized population that excluded some
depolarized cells (those falling between the two sort gates) that were
included in the all-sort. In addition, it is important to notice that
the majority of the treated (
60%) cells were falling in the
depolarized gate (example depicted in Fig. 3).
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Fig. 3.
Flow cytometric parameters used for
separation of distinct population of cells showing high (polarized) and
low (depolarized) m based on
JC-1 staining. Upper panel represents control cells,
and the bottom panel represents the 60-min treated
populations. Left-hand side of the panels depicts polarized
cells, and the right-hand side represents the depolarized
counterparts.
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Fig. 4.
Persistent mitochondrial DNA damage is
restricted to treated depolarized cells, which also show significantly
increased levels of ROS. A, QPCR analysis of the
mitochondrial target of NHF hTERT cells after the sorting procedure.
Cells were treated as described previously, allowed to repair for
24 h, stained with JC-1 for 10 min, and subjected to flow
cytometric sorting using the FACSVantage SE. Controls included 1) cells
harvested immediately after the treatments (data not shown); 2) aliquot
harvested 24 h after the challenge and kept with JC-1 for the same
period as sorted cells but not submitted to this procedure (data not
shown); and 3) cells that passed through the sorter but no separation
of the two distinct populations conducted (all-sort). Data represent
the amount of lesions per 10 kb in the populations that were sorted
(n = 2). B, H2O2
detection in sorted cells. After sorting, cells were harvested, and the
supernatant was collected for H2O2
measurements. To ensure comparability of results, the ratio between
cell number and volume collected was constant so that the dilution was
the same in all samples (i.e. the volume of flow cytometer
fluid per cell was identical). Aliquots of the supernatant were then
subjected to the Amplex Red coupled to horseradish peroxidase assay and
H2O2 concentration calculated based on a
standard H2O2 curve. Numbers
represent the mean ± S.E. (n = 2). Unpaired
Student's t test applied for statistical significance
comparing depolarized samples to polarized counterparts (p
values < 0.05).
m
and permanent mtDNA damage. As described under "Experimental
Procedures," supernatants of sorted cells were collected and used to
perform the Amplex Red assay, which is specific for
H2O2. As can be observed, treated depolarized cells release significantly more H2O2 than
polarized counterparts 24 h after the treatments (Fig.
4B). It could be argued that this result reflects the
presence of the initially supplied H2O2;
however, the exogenous compound showed a half-life of 30 min in the
presence of NHF hTERT cells. Moreover, H2O2
detection reached background levels by 4 h after the treatments
(data not shown).
m showed ~66% attachment. Only 5% of the depolarized treated cells was able to attach as compared with 13% of the
depolarized controls (data not shown), suggesting that these
populations are mainly composed of non-viable cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
m and subsequent cell death.
-polymerase
and DNA ligase (reviewed in Refs. 23 and 37-39). Because the
mitochondrial genome encodes polypeptides exclusively involved in the
production of ATP through oxidative phosphorylation, BER proteins (as
well as the other 1000 estimated proteins that function in this
organelle) are nuclear encoded and therefore must be targeted to the mitochondrion.
m provides one of the major driving forces for the vectorial
transport of polypeptide chains by these import machines (40). The
results obtained during this study provide evidence that a majority
(~70%) of cells exposed to H2O2 for 60 min
have lost their
m within 24 h of the treatments (Fig.
2A). This led us to hypothesize that the presence of mtDNA
lesions and the loss of
m are intimately linked and may result in
lack of repair activity through a compromised import mechanism. Data to
support this concept were obtained from our sorting experiments (Fig.
4A). In fact, this approach clearly showed that only those cells that have low
m also showed mtDNA damage, suggesting that a
compromised import mechanism was involved in the lack of repair activity in mitochondria.
-polymerase, the only
polymerase present and active in mitochondria, is prone to oxidative
damage. This might also be the case in our experiments. Along the same
line, another BER protein that could be the target of oxidative attack
is the human endonuclease III homologue hNTH1 (41). Although there is
no direct evidence that hNTH1 is sensitive to oxidative damage, one
could envision a mechanism analogous to the inactivation of aconitase.
This protein localizes in the mitochondrial matrix and, like hNTH1, has
an iron-sulfur [4Fe-4S] cluster in its active site. Yan et
al. (42) showed that aconitase is modified and inactivated by
oxidative stress, which is believed to occur because of the release of
one iron atom from the cluster (43). It is worth mentioning that if
oxidation of hNTH1 is taking place in our experiments, one would expect
not only the enzyme to decrease repair efficiency but also to release
iron, which could further contribute to Fenton chemistry and higher the
levels of mtDNA damage. In this context, preliminary data following
mitochondrial protein oxidation revealed that several proteins are
oxidized in this organelle in NHF hTERT cells exposed to
H2O2 (data not shown).
m,
loss of repair activity and apoptosis. Because mtDNA lesions were
observed within 5 min of H2O2 exposure (data
not shown), it is most likely that mtDNA damage is the initial event triggering this cascade. Although our results do not implicate a drop
in the
m as the sole event to impair mitochondrial repair activity, they provide a novel insight to account for the increased susceptibility of the mitochondrial genome to oxidative attack as
compared with the nDNA. An increased understanding of the kinetics of
damage and repair (or lack of repair) in the mitochondrial genome
should provide new insights into the biological/physiological significance of mtDNA damage and, eventually, mutagenesis.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Milan Skorvaga and Bhaskar Mandavilli for fruitful discussions in the laboratory; Dr. Julie Horton for help with the cell counting; Dr. Maria Anna Graziewicz for assistance in the protein oxidation assay, and also Drs. William Copeland, Julie Horton, and Michael Resnick for critical review of the manuscript.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of
Molecular Genetics, NIEHS, 111 Alexander Dr., P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-7752; Fax:
919-541-7593, E-mail: vanhout1@niehs.nih.gov.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M208752200
2 J. H. Santos and B. Van Houten, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
ROS, reactive oxygen
species;
H2O2, hydrogen peroxide;
QPCR, quantitative polymerase chain reaction;
NHF, normal human fibroblast;
hTERT, human telomerase reverse transcriptase;
m, mitochondrial
membrane potential;
ETC, electron transport chain;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PBS, phosphate-buffered saline;
DMEM, Dulbecco's modified Eagle's medium;
BER, base excision repair;
AFC, 7-amino-4-trifluoromethylcoumarin;
PI, propidium iodide;
mtDNA, mito- chondrial DNA;
nDNA, nuclear DNA.
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