1 Department of Biomedical Sciences and Biotechnologies, University of Brescia,
Brescia, Italy
2 Department of Pathology, University of Brescia, Brescia, Italy
3 Department of Experimental and Applied Pharmacology, University of Pavia,
Pavia, Italy
* Author for correspondence (e-mail: memo{at}med.unibs.it )
Accepted 16 May 2002
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Summary |
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Key words: DNA damage, Human, Cell cycle, Reactive oxygen species, p21, GADD45, Bax
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Introduction |
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The contribution of oxidative injury and ROS generation to AD is supported
by a number of neurochemical and neuropathological studies in the brain of AD
patients showing accumulation of iron and aluminium
(Savory et al., 1999;
Campbell and Bondy, 2000
), high
levels of peroxynitrite (Hensley et al.,
1995
; Hensley et al.,
1998
; Aksenov et al.,
2001
), high expression of heme oxygenase
(Takahashi et al., 2000
;
Takeda et al., 2000
) and high
levels of markers of lipid peroxidase activity, such as thiobarbituric
acid-reactive substance (Lovell et al.,
1995
; Marcus et al.,
1998
) and 4-hydroxynonenal
(Lovell et al., 1997
;
Markesbery and Lovell, 1998
).
Moreover, neurotoxicity associated both with Ca2+- mediated
activation of N-Methyl-D-Aspartate-type glutamate receptor and with
ß-amyloid plaques deposition involve free radical generation
(Ciani et al., 1996
;
Hensley et al., 1996
;
Mark et al., 1996
;
Weber, 1999
). Finally,
clinical studies show that oral vitamin E intake or selegiline treatment
delays the progression of the disease in patients with moderate to severe
cognitive impairment (Sano et al.,
1997
). Signs of oxidative stress in AD are also detectable in
peripheral cells. Indeed, fibroblasts from sporadic AD patients show impaired
oxidative metabolism (Gibson et al.,
1996
; Gasparini et al.,
1998
), lymphoblasts from familial AD patients carrying a mutation
in the presenilin-1 gene have low levels of reduced glutathione
(Cecchi et al., 1999
), and
lymphocytes from mice carrying multiple presenilin-1 mutations show an
accumulation of ROS (Eckert et al.,
2001
). All together, these data suggest that brain and peripheral
cells of AD patients are abnormally exposed to ROS. However, although this
condition in the brain may be associated with a neuronal cell loss, no
evidence is available, at least up to now, of a clear sign of pathology in
peripheral organs.
In this study, we evaluated the response of different human skin fibroblast cultures obtained from probable AD patients and non-AD subjects to an acute oxidative injury elicited by H2O2. It is well recognized that one of the main effects following H2O2 exposure is damage to DNA molecules. Cells respond to this event by arresting the cell cycle to allow repair of damaged DNA. The damage and the ability to repair it address the cell fate in terms of re-entry into the cell cycle or inducing apoptosis. Thus, to compare the sensitivity of fibroblasts of AD or non-AD patients to H2O2 exposure, we took into consideration different parameters, including cell viability, the extension of DNA damage and the ability to arrest proliferation and to activate an apoptotic program. We found that fibroblasts from AD patients are more resistant that those from non-AD subjects to H2O2. The protective mechanism involves an impairment of ROS-activated, p53-dependent cell death.
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Materials and Methods |
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The diagnosis of probable AD was made by senior neurologists according to
the criteria developed by National Institute of Neurological and Communicative
Disorders and Stroke (NINCDS) and the Alzheimer's Disease and Related
Disorders Association (ADRDA). All AD patients presented a 1 to 4 year history
of progressive cognitive impairment predominantly affecting memory. Cognitive
status was quantified using the Mini-Mental State Examination (MMSE); the
scores were as follows: AD, 3.6±2.4; non-AD, 25.1±1.7. Three AD
patients were non-testable using the MMSE because of severe disease. The ApoE
genotypes were as follows: two controls were respectively heterozygous E3/E4
and E3/E2 and the others homozygous E3/E3; four AD patients were heterozygous
E3/E4 and four homozygous E3/E3. Thus the overall distribution of E4 alleles
was consistent with the allele frequency indicated by the literature
(Frisoni et al., 1994). A
summary of demographic characteristics of all subjects enrolled in the study
is reported in Table 1.
|
Skin fibroblast cultures
Fibroblast cultures were established as previously described
(Govoni et al., 1993). All
cell lines were frozen at passage two-four in a modified growth medium
containing 90% fetal calf serum (FCS) and 10% dimethylsulfoxide. For the
experiments, cell lines were simultaneously thawed and grown up to passages
nine-twelve. Cells were grown in Eagle's Minimum Essential Medium (GIBCO,
Madison, WI), supplemented with 10% FCS, penicillin (100 U/ml) and
streptomicin (100 µg/ml), non-essential amino acid solution (1% v/v) and
Tricine buffer (GIBCO) (20 mM, pH 7.4) at 37°C in 5% CO2/95%
air. The medium was changed every 3 days. As aging affects the apoptotic cell
response to genotoxic stress (Suh et al.,
2002
), each set of experiments was done using cells from the same
passage, carefully matching AD and control cultures. Culture conditions were
kept constant throughout the experiments.
H2O2 treatment
80% confluent monolayers of cells were exposed to
H2O2. Briefly, culture cells were washed with phosphate
buffer saline (PBS) and treated with 1 mM H2O2 for 15
minutes. After washing, cells were returned to full fresh medium for variable
times according to the experiments. For each cell line the experiments were
repeated at least three times.
Cell viability
Cell viability was evaluated 24 hours after the addition of the cytotoxic
agent to the media by measuring lactate dehydrogenase (LDH) activity using the
Cytoxicity Detection Kit (Boehringer Mannheim) and an ELISA reader (340 ATC,
SLT LabInstruments, NC). Cytotoxicity was evaluated as a percentage of the
maximum amount of releasable LDH enzyme activity, which is determined by
lysing the cells with 1% of TritonX-100.
Chromosomal condensation and DNA fragmentation were determined using the chromatin dye Hoechst 33258. After treatments, cells were stained with 1 µM Hoechst 33258 for 5 minutes. After three rinses with PBS buffer, cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature. Coverglasses were mounted and analysed under fluorescence microscope.
Western blot analysis
Cells were harvested in 80 µl of lysis buffer containing 50 mM Tris-HCl
(pH 7.6), 150 mM NaCl, 5 mM EDTA, 1 mM phenyl methyl sulphonyl fluoride, 0.5
µg/µl leupeptin, 5 µg/µl aprotinin and 1 µg/ml pepstatin.
Samples were sonicated and centrifuged at 15,000 g for 30 minutes at
4°C. The resulting supernatants were isolated and protein content
determined by a conventional method (BCA protein assay Kit, Pierce, Rockford,
IL). 30 µg of protein extracts were electrophoresed on 12% SDS-PAGE and
transferred to nitrocellulose paper (Schleicher and Schuell, Dassel, Germany).
Filters were incubated at room temperature overnight with anti-p53 (1:500)
(Ab240, Neo Markers), anti-p21 (1:200) (F5, Santa Cruz Biotechnology, Santa
Cruz, CA), anti-Bax (1:200) (B9, Santa Cruz Biotechnology, Santa Cruz, CA),
anti-GADD45 (1:200) (C4, Santa Cruz Biotechnology, Santa Cruz, CA) or
anti-tubulin (1:1,500) (Ab3, Neo Markers) antibodies in 3% non-fat dried milk
(Sigma). The secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA)
and a chemiluminescence blotting substrate kit (Boehringer, Mannheim, Germany)
were used for immunodetection. For immunoprecipitation experiments, 150 µg
of protein extracts were resuspended in 500 µl RIPA buffer (140 mM NaCl, 10
mM Tris-HCl, pH 8.1% TritonX-100, 0.1% SDS, 1 mM Na-ortho-vanadate and 1 mM
PMSF) and then incubated with 2 µg/ml of mouse p53 antibody (Ab8, Neo
Markers) at 4°C overnight. Immunocomplexes were collected with
Staphylococcus aureus Protein A suspension and washed five times with
RIPA buffer. Immunoprecipitated p53 was recovered by resuspending the pellets
in loading buffer, and protein was detected by western blotting with rabbit
antibodies against p53 phospho-serine-15 (1:500) (Oncogene) or p53
phospho-serine392 (1:500) (Oncogene). Peroxidase-conjugated goat anti-rabbit
immunoglogulin G and a chemiluminescence blotting substrate kit (Boehringer,
Mannheim, Germany) were used for immunodetection. Evaluation of
immunoreactivity was performed on immunoblots by densitometric analysis using
a KLB 2222-020 Ultra Scan XL laser densitometer.
Immunocytochemistry analysis for 8-hydroxy-deoxyguanosine
(8OH-dG)
The cells were fixed in 75% ethanol at -20°C. The cells were then
treated with RNase (100 µg/ml) in 10 mM Tris buffer (pH 7.5) containing 1
mM EDTA and 0.4 M NaCl at 37°C for 1 hour, followed by an incubation with
proteinase K (10 µg/ml) at room temperature for 7 minutes. After rinsing
with PBS, DNA was denaturated by treatment with 4 N HCl for 7 minutes at room
temperature. The pH was adjusted with 50 mM trizma base for 5 minutes followed
by washing in PBS. Non-specific binding sites were blocked with 10% rabbit
serum for 1 hour at 37°C. Cells were incubated with primary anti-8OH-dG
antibody (1:30) (1F711, Pharmigen) at 4°C overnight. Anti-rabbit
anti-mouse IgG coniugated to biotin and ABC reagents and avidin conjugated to
horseradish peroxide were used. To localize peroxidase, cells were treated
with diaminobenzidine for 10 minutes. After mounting with Permount,
coverglasses were analysed by using a camera adapted to a Nikon microscope
with a x20 objective. The image processing and quantitative analysis of
8OH-dG-positive cells were performed using Image-Pro Plus software (Media
Cybernetics, Silver Spring, MD). Data were expressed as a number of labeled
positive cells in the examined field. The mean profile was obtained from at
least three separate determinations in triplicate for each cell line. The
statistical significance of differences between the values was made by one-way
analysis of variance followed by a Student's t-test.
Measurement of DNA synthesis
Cells were seeded in 24-well culture plates at a density of about
5x104 cells per well. 1 µCi/ml [3H]thymidine
was added to the cells 6 hours before or immediately after the
H2O2 pulse. Cells were then incubated for an additional
time at 37°C. At the end of the incubation, cells were washed with
ice-cold PBS and further incubated for 10 minutes at 4°C with 10%
trichloroacetic acid, followed by 1 N NaOH and 1 N HCl for 20 minutes at room
temperature. The resulting solution was collected and analysed for
radioactivity content.
Flow cytometry
For analysis of cell cycle distribution, both floating and adherent cells
were collected and fixed in 70% ethanol in distilled water and stored at
-20°C. After washing in PBS, the cells were treated with 100 µl of
ribonuclease for 5 minutes at room temperature, stained with 400 µl of
propidium iodide (50 µg/ml) and analysed by flow cytometry using 488 nm
excitation.
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Results |
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The experimental paradigm described above was applied to fibroblasts from probable AD patients. In particular, cell cultures from eight AD patients and eight non-AD subjects at similar number of passages (nine to twelve) were challenged with 15 minutes pulse of 1 mM H2O2, cultured for an additional 24 hours and than evaluated for cell viability. As depicted in Fig. 2, cell death, expressed as percentage of H2O2-induced LDH release over basal, was significant lower in fibroblasts from AD (26.4±3.8) in comparison with those from non-AD patients (41.7±4.6).
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Oxidative DNA damage
One of the major events induced by H2O2 is the
generation of ROS and induction of DNA damage
(Gille et al., 1992). We then
asked whether the low sensitivity of AD-fibroblasts to the oxidative injury
was caused by a diminished capability of ROS to generate oxidative DNA damage.
DNA base oxidation was analysed in both cell groups by using a specific
antibody against 8OH-dG (Shigenaga et al.,
1991
). Fig. 3A
shows a representative immunocytochemistry analysis carried out with 8OH-dG
antibody on AD and non-AD fibroblasts after treatment with
H2O2. Only scattered 8OH-dG-positive nuclei were
observed in untreated non-AD fibroblasts. H2O2 treatment
caused a significant increase in the number of labelled cells within 2 hours.
H2O2-induced base DNA damage was evident also in AD
fibroblasts. Quantitative analysis of DNA damage caused by
H2O2 exposure was evaluated in six AD and six non-AD
cell lines, counting the 8OH-dG-positive cells in at least six different
fields of each sample. As shown in Fig.
3B, H2O2-induced 8OH-dG generation was
similar in AD and non-AD fibroblasts.
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Cell cycle and proliferation
As a normal response to DNA damage, cells arrest the cell cycle. The
ability of AD and non-AD fibroblasts to arrest the cell cycle and thus to
diminish DNA synthesis was evaluated at different time following
H2O2 treatment by measuring [3H]thymidine
uptake. [3H]thymidine was added to the cultures 6 hours before or
immediately after H2O2 pulse; radioactivity incorporated
into the cells was measured at different times (0, 6 and 24 hours) later. No
significant difference in the normal cell proliferation rate was observed in
untreated AD and non-AD fibroblasts (Fig.
4A,B). Six hours after cytotoxic injury, non-AD cells decreased
their proliferation rate, and 24 hours later they completely lost their
ability to synthesize DNA (Fig.
4A). In AD fibroblasts, the H2O2 insult
induced a temporary growth arrest, as visualized by a decreased
[3H]thymidine uptake 6 hours after the insult. Then, cells recover
their ability to synthesize DNA within 24 hours
(Fig. 4B). The different
response of AD- and non-AD fibroblasts to H2O2 can be
easily visualized by comparing the rate of [3H]thymidine uptake in
both cell lines between 6 hours and 24 hours
(Fig. 4B).
|
The ability of AD and non-AD fibroblasts to arrest the cell cycle was also evaluated by measuring the distribution of the cells into the different cell cycle phases using standard DNA content analysis by flow cytometry. The cell cycle pattern of non-AD fibroblasts was compared with that of AD either in basal conditions and at different times following the H2O2 pulse. Fig. 5 depicts representative DNA histograms generated by flow cytometric analysis of non-AD and AD fibroblast cell lines. No differences were evident in cell cycle distribution of untreated AD and non-AD fibroblasts (Table 2). Six hours following the H2O2 pulse, cells from non-AD and AD subjects were enriched in G0/G1 phase. Further, at the same time point, a fraction of cells of both groups were confined in sub-G0 phase, characterizing apoptosis. Twenty hours later, the percentage of non-AD cells in G0/G1 phase further increased showing also an enhancement of cell fraction in sub-G0 phase. On the contrary, at the same time-point, AD fibroblasts shown a pattern of cell cycle distribution comparable with that found in basal conditions. Similar results were obtained in six AD and six non-AD cell lines. As summarized in Table 1, the G0/G1 fraction of non-AD fibroblasts progressively increased from time 0 to 20 hours after H2O2 treatment (47.2%, 55.6% and 60.0% at 0, 6 hour and 20 hours, respectively), whereas the percentage of cells in G2/M significantly diminished over time (41.5%, 10.2% and 7.8% at 0, 6 hours and 20 hours, respectively). By contrast, the G0/G1 fraction of H2O2-treated AD cells showed a slightly increase 6 hours after injury, which was followed by a decrease at 20 hours. Concomitantly, the fraction of G2/M cells decreased after 6 hours and returned to control levels 20 hours after the injury.
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|
p53-dependent apoptotic pathway
The transcription factor p53 is one of the proteins that play an important
role in the cellular response to DNA damage by controlling DNA repair, cell
cycle arrest and apoptosis (for a review, see Almog and Rotter, 1997). The
effect of H2O2 on p53 protein levels and p53-dependent
transcriptional activity in non-AD and AD fibroblast was examined by western
blot analysis. Protein extracts from cells prior to and 2 hours following 1 mM
H2O2 pulse were electrophoresed and immunoblotted with
antibodies against p53 and the p53 target gene products p21, bax and GADD45.
Fig. 6A shows representative
results using fibroblasts from two non-AD (lanes 1-4) and two AD subjects
(lanes 5-8). 2 hours after the H2O2 treatment, levels of
p53, p21, bax and GADD45 increased in both non-AD samples. By contrast,
samples from AD patients showed anomalous results. One sample showed a
H2O2-induced increase in p53 levels that was not
accompanied by similar changes in p21, bax and GADD45
(Fig. 6A, lanes 5-6). In the
other AD sample, H2O2 treatment induced a decrease in
p53 and p21 levels with no changes in bax and GADD45 protein content
(Fig. 6A, lanes 7-8). Data
obtained in fibroblasts from six non-AD and six AD patients were evaluated by
densitometric analysis and normalized as a percentage of the corresponding
basal expression. The results are illustrated in
Fig. 6B. All samples from
non-AD subjects showed a significant increase in p53, p21, bax and GADD45
protein levels after H2O2 treatment. The same treatment
did not elicit significant changes in the levels of p53, p21, bax and GADD45
in fibroblasts from AD patients.
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To gain further insight into the alteration of p53 pathway in AD
fibroblasts, we investigated the possible involvement of upstream regulators
of p53 activation by measuring p53 phosphorylation status at ser15 and ser392
in basal conditions and after H2O2 treatment.
Phosphorylation at N-terminal sites of p53, such as at ser15, releases p53
from MDM2 binding, whereas phosphorylation at C-terminus sites, such as at ser
392, induces p53 DNA binding activity
(Lakin and Jackson, 1999;
Kapoor et al., 2000
). We found
that p53 phosphorylation at ser15 and ser392 in non-AD fibroblasts was low in
resting conditions and significantly increased after the oxidative pulse (a
representative sample is shown in Fig.
7, lines 1-2). Phosphorylation of p53 in AD fibroblasts behaved
quite differently. In one AD case (lane 3-4,
Fig. 7) phosphorylation at
ser15 was high in basal conditions and decreased after
H2O2. In two other AD samples (lines 5-6 and 7-8),
phosphorylation at ser15 was low in basal conditions and increased after
H2O2. However, phosphorylation at ser392 was high in
basal conditions and decreased after H2O2. None of the
AD samples behaved like their non-AD counterpart, at least in terms of pattern
of p53 phosphorylation at ser15 and ser392. These results further underline
the heterogeneity of AD fibroblasts and suggest that the impairment of p53
pathway may involve also upstream regulators.
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Discussion |
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The main result of our study is that H2O2-induced
cell death was less effective in fibroblasts from AD patients. We first asked
whether the low sensitivity of AD-fibroblasts to the oxidative injury was due
to a diminished capability of ROS to generate oxidative DNA damage. This was
suggested by data obtained in similar fibroblast cell lines in which
alteration of some components of the mitochondrial oxidative pathways was
observed (Curti et al., 1997;
Gasparini et al., 1999). To this end, we evaluated the extent of ROS-induced
DNA damage, as measured by 8OH-dG formation. When improperly hydroxylated by
ROS attack, guanine may become an 8-hydroxyl-guanine residue, which can bind
incorrectly to adenine instead of to cytosine and cause a DNA base mismatch
(Shigenaga et al., 1991
).
Post-mortem studies demonstrated that levels of 8OH-dG were elevated in
mitochondrial DNA of AD brain relative to controls
(Mecocci et al., 1994
).
Furthermore, lymphocytes isolated by AD patients had higher levels of 8OH-dG
than controls (Mecocci et al.,
1998
). Our results did not show any difference in the ability of
H2O2 to generate 8OH-dG in fibroblasts from AD and
non-AD patients, suggesting that ROS are similarly generated in terms of the
amount and genotoxic potency in all fibroblast cell lines.
We then asked whether the lower sensitivity of AD fibroblasts to
H2O2 was caused by an impairment of the cellular
response to a genotoxic insult. As a normal response to DNA damage, cells
arrest the cell cycle to allow repair of damaged DNA
(Kastan et al., 1992). In the
present study, cell cycle and cell cycle phase distribution of fibroblasts was
measured by incorporation of [3H]thymidine and flow cytometric
analysis. Both techniques gave similar results: fibroblasts from AD patients
showed a fast recover from the oxidative lesion. In fact, only 20 hours after
the lesion, cell cycle phase distribution of AD fibroblasts was similar to
that of controls. Thus, despite the similar extent of DNA damage, fibroblasts
from AD patients show an accelerated re-entry into the cell cycle and a
diminished induction of apoptosis.
Among the proteins that play an important role in the cellular response to
DNA damage is the tumour suppressor p53
(Ko and Prives, 1996;
Almong and Rotter, 1997
;
Grilli and Memo, 1999
). p53 is
a transcription factor that controls cell cycle arrest, DNA repair and
apoptosis (for a review, see Among and Rotter, 1997). Following exposure to
DNA damaging agents, p53 is phosphorylated at several sites by different
kinases, including casein kinase I and II, cdc2 kinase and the DNA-activated
protein kinase (Lakin and Jackson,
1999
). These post-transcriptional modifications activate p53,
allowing this protein to accumulate into the nucleus, to bind to specific DNA
sequences and to transactivate several genes including effectors of the cell
cycle, such as p21 (El-Deiry et al.,
1993
) and GADD45 (Kastan et
al., 1992
), and cell death, such as bax
(Miyashita et al., 1994
;
Oren, 1994
). Lack of
functional p53 in different cell phenotypes may shorten DNA-damage-induced
cell cycle arrest and results in accelerated proliferation
(Gottlieb and Oren, 1996
).
Moreover, Li-Fraumeni syndrome fibroblasts homozygous for p53 mutation were
several fold more resistant to UV cytotoxicity and exhibited much less
UV-induced apoptosis than normal skin fibroblasts expressing wild-type p53
(Ford and Hanawalt, 1997
;
Delia et al., 1997
). In the
present study, fibroblasts from AD patients appear to have a profound (and
heterogeneous) impairment in the ROS-activated, p53-dependent pathway, which
results in abnormal phosphorylation status in the resting condition and a lack
of activation of p53 or p53-target genes. It is interesting that one AD sample
(line 7-8 in Fig. 6 and line
3-4 in Fig. 7), with high p53
levels in resting conditions, also exhibits signs of phosphorylation at ser15
and an enrichment in G2/M phase. These features are not generalized to all AD
samples. For example, another AD cell line shows moderate p53 expression, high
phosphorylation at ser392 and low G2/M enrichment. These results further
underline the heterogeneity of AD fibroblasts in resting conditions.
p53 belongs to a growing list of transcriptional activators that are
post-transcriptionally regulated by redox modulation
(Hainaut and Milner, 1993;
Sun and Oberley, 1996
;
Verhaegh et al., 1997
). In
fact, as a result of protein oxidation, nine of the twelve cysteine residues
in p53 localized in the central DNA-binding domain can form disulfide bonds,
thus altering the three-dimensional structure of the protein. In this abnormal
conformation, p53 loses the capability to transactivate its target genes
(Parks et al., 1997
). However,
at the present we do not know whether the lack of ROS-activated apoptosis is
the result of an elevated redox status of these cells
(Curti et al., 1997
) or whether
p53 in these fibroblasts is per se in an oxidative conformational state. In
this regard, when cells are exposed repeatedly to low doses of
H2O2, they became resistant to subsequent higher amount
of ROS that would be lethal without pre-treatment
(Janssen et al., 1993
),
suggesting that cells can activate an adaptive genetic program against
oxidative stress.
Finally, it should be noted that ROS-activated, p53-dependent pathway in
fibroblasts from AD patients was completely lacking, whereas the ROS-induced
cell death was decreased only by about 40%. This apparent discrepancy
indicates that ROS may induce cell death by activating different pathways,
including necrosis and apoptosis, and that p53-dependent apoptosis contributes
only partially to the cell death induced by ROS. Preliminary data obtained in
our laboratory suggest that non-AD and AD fibroblasts behaved similarly in
response to cisplatin-induced apoptosis, suggesting that the p53 impairment of
AD fibroblasts may affect specific death pathways
(Seluanov et al., 2001).
The brain is particularly vulnerable to oxidative stress because of its
high energy requirement and high oxygen consumption rate; it is also rich in
peroxidizable fatty acid and has a relative deficit of antioxidant defences
compared with other organs (Floyd,
1999). We cannot speculate at this time on whether the lack of
ROS-activated, p53-dependent apoptosis is also present in the brain of AD
patients. If this is true, it is tempting to suggest that such impairment in
sensing and repairing DNA damage could be responsible for generation of
malfunctioning neurons, that is cells living with altered gene transcription
function. Studies in this direction are now in progress in our laboratory.
In conclusion, this study shows a specific alteration of an intracellular pathway involved in sensing and repairing DNA damage in peripheral cells from AD patients. Whether this is a peripheral sign of the disease requires future investigation.
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Acknowledgments |
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