From the Dipartimento di Biologia Evoluzionistica
Sperimentale, Università di Bologna, Bologna 40126, Italy,
§ University Department of Clinical Neurosciences, Royal
Free and University College Medical School, Rowland Hill Street,
London, NW3 2PF, United Kingdom, ¶ IRCCS "E.Medea,"
Conegliano, Treviso 31015, Italy, and Dipartimento di Science
Neurologiche, Università di Bologna, Bologna 40123, Italy
Received for publication, October 8, 2002
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ABSTRACT |
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Leber's hereditary optic neuropathy (LHON), a
maternally inherited form of central vision loss, is associated with
mitochondrial DNA pathogenic point mutations affecting different
subunits of complex I. We here report that osteosarcoma-derived
cytoplasmic hybrids (cybrid) cell lines harboring one of the three most
frequent LHON pathogenic mutations, at positions 11778/ND4, 3460/ND1,
and 14484/ND6, undergo cell death when galactose replaces glucose in
the medium, contrary to control cybrids that maintain some growth
capabilities. This is a well known way to produce a metabolic stress,
forcing the cells to rely on the mitochondrial respiratory chain to
produce ATP. We demonstrate that LHON cybrid cell death is apoptotic,
showing chromatin condensation and nuclear DNA laddering. Moreover, we
also document the mitochondrial involvement in the activation of the
apoptotic cascade, as shown by the increased release of cytochrome
c into the cytosol in LHON cybrid cells as compared with
controls. Cybrids bearing the 3460/ND1 and 14484/ND6 mutations
seemed more readily prone to undergo apoptosis as compared with
the 11778/ND4 mutation. In conclusion, LHON cybrid cells forced by the
reduced rate of glycolytic flux to utilize oxidative metabolism are
sensitized to an apoptotic death through a mechanism involving mitochondria.
In 1988, Leber's hereditary optic neuropathy
(LHON)1 has been the first
human pathology to be associated with a mitochondrial DNA (mtDNA) point
mutation (1). However, its pathogenesis remains poorly understood
(2-4). Currently, three most frequent (11778/ND4, 3460/ND1, 14484/ND6)
(2-4) and other rare (14459/ND6, 10663/ND4L, 4171/ND1, 14482/ND6)
(5-8) pathogenic mutations are found in the majority of patients
affected with this maternally inherited form of optic neuropathy. LHON
is due to a massive acute or subacute retinal ganglion cell
death, characteristically leading to central vision loss (2-4). These
pathogenic mutations invariably affect complex I subunits, which
possibly interact with the quinone substrate (9, 10), and a combination
of partial respiratory deficiency and increased oxidative stress is
documented to be the pathological consequence in transmitochondrial
cell systems (11-15). However, the mtDNA pathogenic mutations are a
necessary, but not sufficient, condition to actually develop LHON, as
suggested by the variable penetrance (2, 16). Thus, other
mitochondrial or nuclear genetic factors, as well as environmental
factors, are believed to be necessary for triggering the optic
neuropathy. At the present time, most of these factors are not well
defined. One potential modulator is possibly represented by the mtDNA
background, as indicated by the association of some of the pathogenic
mutations (11778/ND4, 14484/ND6, 10663/ND4L) with a specific
mtDNA haplogroup characteristic of the European population, classified
as J (2, 6, 16).
LHON has many other peculiar and a yet unexplained features.
Among these, we emphasize the age-dependent, highly
tissue-specific, mostly apoplectic, and wave of retinal ganglion cell
death in the absence of classical signs of inflammation (16). Vascular signs, such as microangiopathy and small vessel tortuosity at the optic
nerve head, usually precede the onset of the disease (16). Some of
these features are thought to suggest a prevalent apoptotic mode of
cell death (17), and by analogy with other pathological conditions,
such as glaucoma, in which apoptosis has been directly documented (17,
18).
Growth impairment of transmitochondrial cytoplasmic hybrids (cybrids)
carrying the most frequent LHON pathogenic mutation at position
11778/ND4 has been first reported by Hofhaus et al. (19)
when cells were incubated in a medium containing galactose in place of
glucose (19). This finding was in agreement with previous studies
showing that cultured cells with defective mitochondrial metabolism
often die in glucose-free galactose medium (20, 21). Indeed, in glucose
medium, most of the total ATP yield of proliferating cells is produced
by glycolytic glucose breakdown to lactate, and only a minimal amount
of pyruvate is oxidized to CO2 and water. Galactose can
also enter the glycolytic pathway; however, the restricted flow of
galactose to glucose-6-phosphate determines the formation of very
little lactate since pyruvate, which is formed at a much slower rate,
is further oxidized within the mitochondria (21).
In the present study, we have characterized the loss of cell viability
of osteosarcoma-derived cybrids containing the three main LHON
pathogenic mutations (11778/ND4, 3460/ND1, 14484/ND6) grown in a medium
in which glucose was replaced by galactose. We show that LHON cybrids,
but not control cybrids or the parental osteosarcoma cell line
(143B.TK Materials--
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), orange
acridine, ethidium bromide, and protease inhibitors mixture were
purchased from Sigma. Hoechst-33342 was from Calbiochem. Anti-cytochrome c and secondary antibodies were from Santa
Cruz Biotechnology (Santa Cruz, CA).
Cell Lines and Culture Conditions--
Cybrid cell lines were
constructed using enucleated fibroblasts from two controls and six LHON
probands as mitochondria donors and the osteosarcoma
(143B.TK Cell Viability Assay--
The tetrazolium salt MTT was used to
determine the cell viability, as described in Ref. 26. Cells were
seeded into 24-well dishes and incubated in DMEM galactose medium for
different times, and then MTT was added to the final concentration of
0.5 mg/ml. After 3 h, 5% SDS and 5 mM HCl were added
to solubilize the formazan salt crystals, and dishes were kept
overnight in the incubator. Absorbance of solubilized formazan salts
was measured with a Jasco spectrophotometer V-550 (Tokyo, Japan) at the
wavelength of 570 nm. For each experiment, a calibration curve was
constructed by seeding the cells at concentrations ranging from
104 to 105 and measuring the corresponding
value of MTT absorbance.
Chromatin Condensation and DNA Fragmentation--
Nuclear
morphology was assessed in cells detached from the dishes at the times
indicated, stained with 2 µg/ml Hoechst for 30 min at 37 °C, and
fixed with 3% (w/v) paraformaldehyde. Nuclei were observed with a
digital imaging system composed of an inverted epifluorescence
microscope Nikon Eclipse 300 with a back-illuminated CCD camera
(Princeton Instruments, Trenton, NJ) and acquisition/analysis software
Metamorph (Universal Imaging Corp., Downingtown, PA) (27). Quantitative
determination of apoptotic and necrotic cells was obtained by double
staining with orange acridine and ethidium bromide. Both the dyes
intercalate into DNA; however, only orange acridine can cross the
plasma membrane and stain all the cells, whereas ethidium bromide is
excluded from viable and early apoptotic cells, which have an intact
plasma membrane (28). Briefly, cells were seeded in 6-well plates and
incubated with DMEM galactose medium for different times and then
incubated with 3 µg/ml orange acridine and 15 µg/ml ethidium
bromide and examined with an inverted fluorescence microscope
(Nikon, Tokyo, Japan). Early or late apoptotic cells exhibited green or
orange nuclei with condensed chromatin, respectively. Necrotic cells
showed orange nuclei with intact structure. For analysis of DNA
fragmentation, cells were seeded into 25-cm2 flasks in DMEM
galactose medium for the times indicated and then harvested, and DNA
was extracted as described in Ref. 29. DNA was separated by
electrophoresis in 1% agarose gel and stained with ethidium bromide.
Subcellular Fractionation and Western Blot Analysis--
After
incubation in DMEM galactose medium, cells were harvested from five
75-cm2 flasks, resuspended in 0.5 ml of 200 mM
mannitol, 70 mM sucrose, 1 mM EGTA, 10 mM Hepes (pH 7.6), 100 µl/ml protease inhibitors mixture
and homogenized for 30 strokes with a Dounce homogenizer. This and the
subsequent steps were carried out at 4 °C. Samples were centrifuged
for 10 min at 500 × g, and the resulting supernatant was centrifuged for 20 min at 10,000 × g. The
supernatant (cytosolic fraction) was stored at Growth Capability of Control and LHON Cybrids--
Specific growth
impairment has been reported previously in cells carrying the 11778/ND4
LHON mutation incubated in a medium containing galactose in place of
glucose (19). Here, this observation has been extended to the other
LHON pathogenic 3460/ND1 and 14484/ND6 mutations. The parental
143B.TK
Fig. 1A shows that 143B.TK
Fig. 1, B-D, shows that the metabolic stress provoked a
different effect in all cybrid cell lines bearing LHON
mutations. In particular, a similar reduction of viable cells was
observed in the cybrids bearing the 3640/ND1 and 14484/ND6 mutations,
which were alive in an average of 31 and 37% of cells, respectively, after 1 day of incubation with galactose medium, whereas the effect of
the 11778/ND4 mutation was less severe, found in an average 65% of
cells that were viable after 1 day and 20% of cells that were viable
after 4 days. It seems, therefore, that all cybrids carrying LHON
pathogenic mutations are more sensitive to metabolic stress induced by
growth in galactose medium than control cybrids, although some
differences among the three mutations are also apparent.
Haplogroup Influence on Cell Death--
Both control and LHON
cybrid cell lines were characterized for their mtDNA background, as
shown in Table I. The differences in the mtDNA haplogroup did not
induce any observable variability in growth capabilities of control
cybrids and the 143B.TK Chromatin Condensation and Nuclear DNA Laddering in LHON
Cybrids--
The type of cell death, apoptosis versus
necrosis, induced in LHON cybrids by metabolic stress was first
evaluated by detection of nuclear morphology of cells stained with
Hoechst. In fact, cells undergoing apoptosis are characterized by the
dramatic change that occurs in the nucleus, i.e. chromatin
condensation and nuclear DNA fragmentation (28).
Fig. 2 shows that after 72 h of
incubation in galactose medium, the nuclei of 143B.TK
The nuclear morphology characterized by chromatin condensation is
caused by specific digestion of internucleosomal DNA, leading to the
typical fragmentation or laddering of DNA into small molecular weight
bands, which can be easily evaluated after isolation of nuclear DNA and
separation with agarose gel electrophoresis. In Fig.
4, the three upper panels show
representative gels of DNA isolated from the parental
143B.TK Galactose Medium-induced Release of Cytochrome c in LHON Cybrid
Cells--
To understand whether mitochondria are involved in the
apoptotic process induced in LHON cybrids by metabolic stress, the release of cytochrome c from mitochondria to cytosol has
been detected as a function of incubation time in galactose medium. No
significant release of this protein was observed in the cytosolic fractions obtained from subcellular fractionation of homogenates from
the parental 143B.TK Galactose Medium Induces Cell Death in LHON Cybrids--
The first
important result of this study was the significant growth impairment
documented in cybrids carrying the LHON pathogenic mutations when
incubated in a glucose-free/galactose medium. Under this experimental
condition, the reduced rate of the glycolytic pathway causes the forced
oxidation of pyruvate through the mitochondrial respiratory chain. This
experimental condition has been used previously to identify an impaired
respiratory function (20, 21), and Hofhaus et al. (19) did
report a reduced ability of LHON/11778 cybrids to grow in galactose
medium (19). Our study extended this seminal observation to the whole
range of the main LHON pathogenic mutations, also showing that growth
impairment was somewhat milder in 11778 cybrids as compared with those
carrying the 3460 and 14484 mutations.
An apparent discrepancy between our data and those reported by Hofhaus
et al. (19) concerns the behavior of the 143B parental cell
line and the other control cybrids. In fact, control cells were
reported to have a substantial growth rate in galactose medium, although reduced as compared with their growth in glucose medium (19).
Conversely, we report here that the viability of these cells decreased
after 1 day of incubation and then slightly increased up to 4 days to
further decrease at longer times. This late loss of viability is
likely due to nutrient shortage since it was abolished after replacing
the growth medium (result not shown). We believe that the slow
proliferation rate at the early times might be due to the density of
cell seeding because we observed a significant growth of control cells
in galactose medium when seeded at a lower density (1-2 × 104/cm2 instead of 4 × 104/cm2, result not shown).
The growth impairment of LHON cybrids with a different mtDNA haplogroup
did not show any clear variations that could be related to the
mitochondrial genome background. However, we cannot exclude that some
differences could become observable, increasing the number of cell
clones investigated and properly comparing different haplopgroups
within the same mutational category.
The Type of LHON Cybrid Cell Death Is Apoptotic--
The second
novel result presented in this study is that the type of cell death
occurring in LHON cybrids incubated in galactose medium is apoptotic.
In fact, we show, for the first time, that LHON cybrids, but not
control cybrids and the 143B.TK
Glucose is an essential energy source, and its deprivation or treatment
with the glucose antimetabolite 2-deoxyglucose can lead to arrest in
G0/G1 phase non-transformed cells (31) or to
apoptosis in Myc-transformed cells (32). Furthermore, it has been shown
that restriction of the glycolytic rate by reducing the glucose
concentration in the medium or increasing glucose uptake by
over-expression of GLUT1 promotes or delays, respectively, the
apoptosis induced by growth factor withdrawal (33). Therefore, changes
in the cellular metabolism may be sufficient to cause the commitment to
programmed cell death (34).
However, in our experimental cell system, only LHON mutant cybrids did
undergo apoptosis in galactose medium. We assume that the glycolytic
flux reduction in our system is still compatible with an adequate
availability of metabolites needed for mitochondrial synthesis and
substrates for electron transport sustaining cell survival and
eventually lower rates of growth capabilities. Given that the only
difference between control and LHON cybrids is the presence of the
11778/ND4, 3460/ND1, and 14484/ND6 pathogenic mutations, known to
affect complex I function (3, 9, 10, 12), we believe that this genetic
difference is responsible for the inability of the LHON cybrids to cope
with the metabolic stress.
Mitochondrial Involvement in Apoptosis--
The third relevant
finding emerging from this study is the evidence of mitochondrial
involvement in the apoptotic pathway activated in the death of the LHON
cybrids. This has been clearly demonstrated by the significant release
of cytochrome c from mitochondria to cytosol documented in
all LHON cybrids. Different forms of cellular stress (e.g.
DNA damage, cytokine deprivation, exposure to cytotoxic drugs) have
been reported to promote a pathway for apoptosis typically regulated by
these organelles. In this pathway, the engagement of death-promoting
members of the Bcl-2 family of proteins (Bax, Bid, Bad, Bak, Bok, etc.)
induces their translocation to mitochondria and the subsequent release
of proapoptotic molecules, such as cytochrome c,
apoptosis-inducing factor, and Smac/Diablo (22, 35). This effect is
counteracted by the Bcl-2 survival family of proteins (Bcl-2,
Bcl-XL, etc.), which are anchored to the outer membrane of
mitochondria and which function, at least in part, by blocking the
release of cytochrome c (22, 36, 37). It is of interest that
glucose deprivation during hypoxia of cultured kidney cells has been
reported previously to result in the translocation of Bax from the
cytosol to mitochondria (38). We failed to observe detectable
levels of Bax in cell lysates from 143B.TK Potential Mechanisms for Increased Apoptosis in LHON
Cybrids--
The biochemical consequences of LHON pathogenic mutations
have been incompletely characterized (3). However, we have enough data
indicating that a variable respiratory defect is associated with these
mutations with the possibility of a partial decrease of ATP synthesis.
This may be either due to a reduced release of quinol or due to a less
efficient energy conservation at complex I level, which in turn may be
reflected in changes of membrane potential (
On the other hand, the complex I dysfunction in LHON seems mainly
characterized by the common feature of affecting the interaction with
the quinone substrate, and an increase of reactive oxygen species has
been predicted as a direct consequence (9, 10). Some recent studies are
now supporting this hypothesis. A cellular model developed by
Barrientos and Moraes (39), in which a partial complex I impairment is
obtained both genetically or by rotenone inhibition, showed that
apoptotic cell death was positively correlated with reactive oxygen
species production rather than with a decrease in respiratory chain
function. Moreover, a significant increase of reactive oxygen species
production was observed in a neuronal (NT2) cybrid cell model carrying
the 11778/ND4 and 3460/ND1 LHON mutations only after retinoic
acid-mediated differentiation (14). Data from our own laboratories
confirm these same observations in the osteosarcoma
(143B.TK
A different line of recent experimental evidence may possibly be
relevant for linking the LHON mutations, affecting different NADH dehydrogenase subunits, with apoptosis. A regulatory role for
complex I and ubiquinone analogs has been implicated in the modulation
of the mitochondrial permeability transition pore (40, 41). It is
tempting to speculate that the LHON pathogenic mutations, which affect
the interaction of complex I with the ubiquinone substrates, might also
influence the pore opening with the consequent release of cytochrome
c and activation of the apoptotic cascade.
The findings of the current study are in agreement with and complement
those recently reported using the very same LHON cybrid cell lines but
exposed to a different model of apoptotic activation (24). In fact,
Danielson et al. (24) recently showed that these LHON
cybrids were more sensitive to Fas-induced apoptosis (24). In this
apoptotic pathway, the death receptor-dependent autocatalytic activation of caspase 8 can directly cleave and activate
the downstream caspases, such as caspase-3, -6, and 7 (42). In
addition, caspase-8 also activates these downstream caspases indirectly
by inducing cytochrome c release from mitochondria, as a
consequence of cleavage of the cytosolic protein Bid, generation of
truncated Bid, and its subsequent translocation into the outer mitochondrial membrane (43, 44). Although the contribution of these two
pathways to Fas-induced apoptosis in LHON cybrids has not been
investigated, nevertheless, the increased sensitivity of these cells to
both Fas- and metabolic stress-induced apoptosis suggests that the
complex I mutations can strongly influence cell survival.
The further characterization of the exact steps of the apoptotic
pathway involved in LHON will provide more details on the pathophysiology of this disease and more details in general on the link
between mitochondrial dysfunction induced by complex I mutations and
cell death. This issue is of general interest for a wider category of
neurodegenerative diseases and for possible therapeutic pharmacological
strategies aimed to inhibit or reverse the apoptotic cascade. This
therapeutic approach would provide a hope for those LHON patients
undergoing the acute phase of the disease, when the retinal ganglion
cell loss is thought to occur, thus limiting the retinal ganglion cell
death and the consequent visual loss.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), died of apoptosis. It is widely accepted that
the apoptotic process is triggered through two major mechanisms, one
involving the engagement of plasma membrane-associated death receptors
and another involving the participation of mitochondria (reviewed in
Ref. 22). Given that the only relevant difference between control and
LHON cybrids is the presence of mtDNA mutations, our investigation
focused on the possible direct involvement of these organelles in the
apoptotic death triggered by a metabolic stress.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)-derived 206 cell line as acceptor
rho0 cell line (both 143B.TK
and
rho0 206 were kindly provided by Giuseppe Attardi and
Michael King). Table I lists the cybrid
cell lines used in this study and their origin (11, 15, 23-25). Both
definition of the mtDNA haplogroup and identification of the LHON
pathogenic mutations were performed by PCR/restriction fragment length
polymorphism method carried out as reported previously (24, 25).
Parental and cybrid cell lines were grown in DMEM medium supplemented
with 10% fetal calf serum, 2 mM L-glutamine,
100 units/ml penicillin, 100 µg/ml streptomycin, and 0.1 mg/ml
bromodeoxyuridine. For the experiments, cells were seeded 4 × 105 cells/cm2 and incubated in DMEM
glucose-free medium supplemented with 5 mM galactose, 5 mM sodium pyruvate, and 5% fetal calf serum (DMEM galactose medium) at 37 °C in an incubator with a humidified
atmosphere of 5% CO2.
Lhon mutations and mtDNA haplogroups of the cell lines utilized in the
present study
80 °C. Protein
content of fractions was determined as described in Ref. 30. 80-100
µg of protein of cytosolic fraction were separated by 15% SDS-PAGE
and transferred onto nitrocellulose membrane (Bio-Rad). The membrane
was treated with 5% non-fat milk in Tris-buffered saline-Tween 0.05%
for 1 h and incubated with the cytochrome c primary
antibody diluted 1:500 for 1 h at room temperature.
Antigen-antibody complexes were detected by using horseradish
peroxidase-conjugated secondary antibodies diluted 1:2000 in
Tris-buffered saline-Tween 0.05%, supplemented with 5% non-fat milk
and incubated for 20 min at room temperature. The chemiluminescence
signals were revealed using an ECL Western blotting kit (Amersham
Biosciences) and measured with the Fluo-2 MAX Multimager system
(Bio-Rad).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cell line, two control cybrid cell lines, and two
different cybrid cell lines for each of the three primary LHON
mutations were incubated in galactose medium, and changes in cellular
viability were determined. Results are shown in Fig.
1.
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Fig. 1.
Decreased cell viability of
cybrid cell lines bearing LHON mutations incubated in galactose
medium. Control and LHON cybrids were incubated for the times
indicated in galactose medium and then treated for 3 h with MTT
and overnight with SDS and HCl, as described under "Experimental
Procedures." The mtDNA haplogroups (hp) of parental,
control, and LHON cybrid cell lines (Table I) are indicated in the
inset of each panel. The MTT absorbance value at
time 0 was considered as the 100% value of viable cells. Each data
point is means ± S.E. of 3-5 determinations.
cells and control
cybrids exhibited a very similar behavior: after 1 day of incubation, a
small decrease in the number of viable cells was detected, and then the
number of cells remained steady or even increased up to 3 days of
incubation, and then gradually decreased at longer times. During the
time of the experiments shown in Fig. 1, the growth medium was never changed.
parental cell line in galactose
medium, as already reported (25). Moreover, the presence of different
mtDNA haplogroups in the LHON cybrids, in particular haplogroup J,
which has been associated with the 11778/ND4 and 14484/ND6 mutations
(16), did not show any clear-cut influence on cell death. We refer in
particular to the direct comparison of HPE/HFF cybrids (Fig.
1B) carrying both the 11778/ND4 mutation but differing in
their haplogroup, J and U, respectively. Both HBA and HL180
cybrids carrying the 14484/ND6 mutation and haplogroup J seemed to
present the highest sensitivity to cell death in galactose medium.
However, in this case, we unfortunately lack the direct comparison with
a further cell line carrying the same pathogenic mutation with a
different haplogroup.
cells showed a diffuse fluorescence. Conversely, in cybrids bearing the
pathological LHON mutations, the nuclear chromatin of most cells was
condensed and showed highly fluorescent dense aggregates after 48 h of incubation. Furthermore, quantitative data on the percentage of
apoptotic and necrotic cells were obtained by the acridine
orange/ethidium bromide uptake. Cells with bright green or orange
nuclei with condensed chromatin were identified as early or late
apoptotic cells, respectively. As illustrated in Fig. 3, after 2 days of incubation in
galactose medium, the percentage of apoptotic cells was negligible for
143B.TK
parental cells and for control cybrids, whereas
it was about 40% in cybrids carrying the 11778/ND4 mutation and higher
than 60 and 90% in cybrids carrying the 3460/ND1 and 14484/ND6,
respectively. The percentage of necrotic cells (cells with orange
nuclei with intact structure) was negligible (less than 3%, result not
shown).
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Fig. 2.
Nuclear morphology of
control and LHON cybrids incubated in galactose medium.
143B.TK cells and LHON cybrids were incubated for the
times indicated in galactose medium and loaded with Hoechst, as
described under "Experimental Procedures." Images of cells,
captured by a digital imaging system, are representative of most of
cells in the same coverslip.
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Fig. 3.
Quantification of apoptotic cells in cybrids
incubated in galactose medium. Control and LHON cybrids were
maintained in galactose medium for the times indicated and then
incubated with orange acridine and ethidium bromide and examined with
an inverted fluorescence microscopy. Data are means ± S.D. of at
least three determinations. The percentage of apoptotic cells in all
LHON cybrids was significantly different from control or parental cells
(p < 0.05 after 1 day and p < 0.005 after 2 and 3 days respectively, calculated using the paired Student's
t test).
cell line and from two control cybrid cell lines
incubated for the times indicated in galactose medium. No significant
DNA laddering was observed up to 72 h of incubation. Conversely, a
significant DNA fragmentation was apparent after 48-72 h of incubation
with galactose in the two cybrid cell lines bearing the 11778/ND4
mutation and already after 24 h in the cybrids with the 3460/ND1
and 14484/ND6 mutations.
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Fig. 4.
Time-course of DNA fragmentation of cybrid
cell lines with LHON mutations. Nuclear DNA was isolated from
cells incubated in galactose medium for the times indicated, as
described under "Experimental Procedures." DNA was separated in 1%
agarose gel and stained with ethidium bromide. In each
panel, the lanes were: time 0, 24, 48, and
72 h of incubation in galactose medium. The results are
representative of experiments run at least in triplicate.
cells (Fig.
5). Conversely, a significant cytochrome
c release was determined in cybrids with the 11778/ND4
mutation starting from 24 h and maximal after 48 h. In
cybrids with the 3460/ND1 and 14484/ND6 mutations, a remarkable release
of cytochrome c was already apparent after 16 h of
incubation in galactose medium (Fig. 5).
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Fig. 5.
Release of cytochrome c in
the cytosolic fraction in cybrid cell lines with LHON mutations.
Control and LHON cybrids were incubated in galactose medium for the
times indicated and then harvested, homogenized, and centrifuged, as
detailed under "Experimental Procedures." 50-70 µg protein from
the cytosolic fractions were separated by SDS-PAGE, and Western
blotting was performed with specific antibody against cytochrome
c. Data are representative of three similar
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
parental cell line,
exhibited some typical hallmarks of apoptosis, such as the changes in
nuclear morphology, chromatin condensation, and DNA laddering. From
these results, it is clear that cybrids with the 11778/ND4 mutation
were, again, less sensitive to the metabolic stress in comparison with
the 3460/ND1 and 14484/ND6 mutations.
ostosarcoma
cell line and LHON cybrids2;
therefore, studies are in progress to identify the proapoptic Bcl-2
protein involved in the process of cytochrome c release during galactose medium-induced cell death.
). However, it
is not clear whether compensatory mechanisms, such as complex II
up-regulation, may actually maintain the ATP synthesis or whether
unknown tissue-specific regulatory mechanisms may differentially
influence the level of ATP defect.
)-derived cybrids with LHON mutations (15). Thus,
under certain conditions, the increase of production of reactive oxygen
species could be implicated in triggering a
mitochondrial-mediated apoptotic cell death in addition to the
hypothesis of exclusive bioenergetic impairment.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Gino Cortopassi and Steven Danielson, University of California Davis, for providing some cybrids cell lines and to Dr. Rosario Rizzuto, University of Ferrara, for making available the Cell Imaging Facility.
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FOOTNOTES |
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* This work was supported by grants from PRIN 2001-2002: "Mitochondria in Cellular Pathology" (to M. R.); Progetto Dipartimentale "Approcci molecolari e genetici allo studio delle patologie" (to M. R.), "Progetto Giovani Ricercatori" (to A. G.); and from the University of Bologna; and Telethon Grant n.GGP02323 (to V. C.).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: Dipartimento di
Biologia Evoluzionistica Sperimentale, Università di Bologna, Via
Irnerio 42, 40126 Bologna, Italy. Tel.: 39-051-2091286; Fax: 39-051-242576; E-mail: ghelli@alma.unibo.it.
Published, JBC Papers in Press, November 21, 2002, DOI 10.1074/jbc.M210285200
2 A. Ghelli and C. Zanna, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: LHON, Leber's hereditary optic neuropathy; mtDNA, mitochondrial DNA; cybrid, cytoplasmic hybrids; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMEM, Dulbecco's modified Eagle's medium; TK, thymidine kinase.
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