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Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts 02118, USA
2Correspondence: Department of Dermatology, Boston University School of Medicine, 609 Albany St., Boston, MA 02118, USA. E-mail: bgilchre{at}bu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: aging • DNA damage repair • DNA photoproducts • nucleotide excision repair
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Incidence is clearly linked to ultraviolet (UV) light
exposure (2)
and increases exponentially with age
(3)
, independent of insolation (4)
. UV
irradiation of the skin creates DNA photoproducts that, when not
promptly removed, can give rise to mutations and skin cancer (5
, 6)
.
The predominant UV-induced photoproducts are cyclobutane pyrimidine
dimers (CPDs), particularly thymine dimers, which account for
~75–80% of total DNA lesions (7)
, and pyrimidine
(6–4) pyrimidone photoproducts [(6–4) PPs] (8
9
10)
.
These are removed by nucleotide excision repair (NER) (11
, 12)
, beginning with photoproduct recognition by the proteins
xeroderma pigmentosum group C (XPC) and xeroderma pigmentosum group A
(XPA) as well as replication protein A (RPA), another NER factor that
functions also as damage recognition protein (13
, 14)
. Of
note, RPA is a multifunctional ssDNA binding protein complex composed
of 70, 34, and 14 kDa subunits. RPA binds to DNA through its 70 kDa
subunit (15)
, whereas the 34 and 14 kDa subunits are
primarily responsible for protein–protein interactions
(16)
. Then incision takes place with the involvement of
endonucleases xeroderma pigmentosum group F protein and xeroderma
pigmentosum group G protein as well as the basic transcription factor
TFIIH, which contains the repair proteins XPD and XPB, also called
excision repair cross-complementing 2 and 3 (ERCC2 and ERCC3),
respectively (17)
. The incision step of NER is probably
the step that provides tight linkage between DNA repair and
transcription. Furthermore, physical and functional interactions of p53
with the TFIIH complex may provide an immediate and direct link for the
role of p53 in the regulation of NER, transcription and cell cycle
(18)
. After incisions, there is excision of the damaged
strand; finally, repair is completed by a complex interaction of
replication factors, DNA polymerase, and DNA ligase (19
, 20)
.
DNA damage also initiates a molecular cascade that prevents mammalian
cells from entering the S phase of the cell cycle while harboring
damaged DNA (21)
. This system depends on activation of the
p53 protein, which accumulates in the nucleus in response to a variety
of DNA damaging agents, including UV irradiation (22
, 23)
.
Activated p53 transcriptionally up-regulates genes that are involved in
DNA repair and/or cell cycle arrest (21
, 24)
, suggesting
that appropriate activation and nuclear accumulation of p53 are
required for the proper repair of DNA damage. Recent evidence from
mouse and human systems indicates the involvement of p53 directly
and/or indirectly in NER (25
26
27)
. This central role in
cell cycle arrest, DNA repair, and transcription of genes encoding NER
proteins has led to the designation of p53 as ‘guardian of the
genome’ (28)
.
An age-associated decrease in DNA damage repair capacity has been
demonstrated using an indirect method that examines repair rate of a
damaged reporter plasmid introduced into peripheral blood T lymphocytes
or primary dermal fibroblasts derived from donors of different ages
(29
30
31)
. Furthermore, compared to age-matched controls,
individuals with a history of basal cell carcinomas at an early age had
decreased DNA repair capacity comparable to that of older adults
(30)
. Moreover, study of the removal rate of thymine
dimers in patients who have developed basal cell carcinoma on
sun-exposed skin compared to age-matched cancer-free group showed more
than 40% reduction in ability to repair UV-induced pyrimidine dimers
(32)
. Similar approaches have shown a reciprocal
age-associated increase in UV-induced DNA mutations
(30)
. These data strongly imply causal relationships among
increasing age, decreased DNA repair capacity, increased mutation rate,
and development of skin cancer.
To further document the effects of aging on the repair of UV-induced DNA damage and explore the mechanisms involved, we first measured the rate of removal of thymine dimers and (6–4) PPs from the DNA of UV-irradiated dermal fibroblasts derived from donors of different ages, a direct measurement of DNA repair. We also determined the constitutive and UV-induced mRNA and protein levels for selected NER proteins as a function of donor age.
We report that there is a significant age-associated decrease in the removal of UV-induced (6–4) PPs and thymine dimers, apparently due at least partly to age-associated decreases in both basal and UV-induced expression of repair proteins, with similar changes reflected at the mRNA level.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Cultures were used at postprimary population
doubling 
3. Cells were maintained in Dulbecco’s modified Eagle
medium supplemented with 10% calf serum (Life Technologies, Inc./BRL,
Gaithersburg, Md.) at 37°C in 7% CO2.
UV irradiation
Cells were irradiated in phosphate-buffered saline (PBS) through
the plastic culture dish cover with a solar simulator (Spectral Energy
Corporation, Westwood, N.J.). The 1 kW xenon arc lamp (XMN 1000–21;
Optical Radiation Corp., Azuza, Calif.) irradiance was adjusted to
8 x 10-5 W/cm2 and
metered with a research radiometer fitted with a UV probe at 285 ± 5 nm (model IL1700 A; International Light, Newburyport, Mass.)
(34
, 35)
. As described (34)
, this protocol
exposes cells to a spectrum of light closely resembling terrestrial
sunlight. Sham-irradiated cultures were handled identically except that
they were shielded with aluminum foil during the irradiation. After
irradiation, cultures were given fresh medium.
DNA isolation and detection of UV photoproducts
At different intervals up to 24 h after irradiation with 50
mJ/cm2, cells were washed with PBS and genomic
DNA was isolated using DNAzol (Molecular Research Center Inc.,
Cincinnati, Ohio) according to the manufacturer’s instruction. One
hundred nanograms of heat-denatured DNA was immobilized on Hybond N
filter (Amersham Life Science, Arlington Heights, Ill.) using a 72-well
Minifold II vacuum filter device as described (36)
.
Because preliminary experiments determined that the time of exposure
for (6–4) PPs detection was 10–15 min and for thymine dimer detection
was a few seconds, membranes were incubated first with 10 µg of mouse
anti-(6–4) PPs monoclonal antibody, followed by horseradish peroxidase
(HRP) -conjugated sheep anti-mouse immunoglobulin G (IgG) (Amersham
Life Science, Little Chalfont, Buchinghamshire, England). Antibody
binding was detected using the ECL detection kit (Amersham Life
Science), followed by autoradiography with Kodak Biomax MS film. Then
membranes were incubated with 2.5 µg of mouse anti-thymine dimer
antibody (Kamiya Biomedical Company, Tukila, Wash.), followed by the
same secondary antibody and the ECL detection kit as described above.
For verification of even loading, membranes were hybridized with excess
32P-labeled, EcoRI-restricted human
genomic DNA (gDNA) (Promega, Madison, Wis.) as described
(37)
. The densitometric readings of the bands representing
antibody binding were normalized using the readings of the
corresponding bands representing 32P-gDNA binding
(38)
.
Western blot analysis
Total cellular proteins were collected in a buffer consisting of
0.25 M Tris HCl (pH-7.5), 0.375 M NaCl, 2.5% sodium deoxycholate, 1%
Triton X-100, 25 mM MgCl2, 1 mM phenylmethyl
sulfonyl flouride, and 0.1 mg/ml aprotinin as described
(39)
. Protein concentrations were determined by the
Bradford method and 100 µg of proteins were processed for Western
blot analysis as described (39)
. Antibody reactions were
performed with 1:100 to 1:250 dilutions of affinity-purified mouse
monoclonal and rabbit polyclonal antibodies. The following antibodies
were used: anti-p53 DO-1 (Ab-6), anti-proliferating cell nuclear
antigen (PCNA) (Ab-2), anti-RPA (Ab-1) (34 kDa subunit) (Oncogene
Science Inc., Cambridge, Mass.), anti-XPA, and anti-ERCC3 (Santa Cruz
Biotechnology, Inc, Santa Cruz, Calif.). HRP-tagged goat anti-mouse IgG
(secondary antibody) was obtained from Bio-Rad Laboratories (Hercules,
Calif.). HRP-linked sheep anti-mouse IgG was obtained from Amersham
Life Science (Arlington Heights, Ill.); HRP-linked human and mouse
serum preadsorbed anti-rabbit IgG was obtained from Santa Cruz
Biotechnology. The secondary antibodies were used at 1:2000 dilution.
Antibody binding was detected by the ECL detection kit (Amersham),
followed by autoradiography (Kodak X-Omatic AR).
Northern blot analysis
Total RNA was isolated from cells with Tri-Reagent (Life
Technologies, Inc./BRL) following the protocol of the manufacturer. RNA
concentrations were determined by absorbance at 260 nm. The purity of
the preparations was determined by the ratio of 260/280 readings, which
were consistently >1.8. Twenty micrograms of RNA from each sample
was electrophoresed on 1% agarose gels containing 2.2 M formaldehyde
as described (40)
. RNA was transferred to nylon membranes
(Hybond-N, Amersham Corp., Arlington Heights, Ill.) and immobilized by
shortwave UV irradiation (UV-Stratalinker 1800, Stratagene, La Jolla,
Calif.). Blots were hybridized with 32P-labeled
PCNA (ATCC #61055), ERCC2 (ATCC #59463), and ERCC3 (ATCC #78620) cDNAs.
XPA cDNA was generated by polymerase chain reaction using primers
complementary to the published human sequence (41)
.
Densitometric analysis
Autoradiographs of Western-, Northern-, and DNA-containing blots
as well as ethidium bromide and Coomassie blue-stained gels were
scanned (Microtek Scan Maker II) into a computer (Massachusetts
Engineering). Band intensity was determined after background
subtraction using the densitometric program ‘Sigma Gel’
(Jandel Scientific, Corte Madera, Calif.). Normalization for
loading of the lanes was determined either by Coomassie blue staining
(for Western blots), ethidium bromide staining (for Northern blots), or
32P-gDNA binding (for slot blots).
Statistical analysis
To determine age-associated differences in the rate of DNA
photoproduct removal, analysis of covariance was used. Protein and mRNA
levels were analyzed by analysis of variance (ANOVA) with Fisher and
Bonferroni/Dunn Post Hoc Analysis and Regression Analysis using the
StatView statistical program (SAS Institute, Inc., Cary, N.C.)
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 42)
, we investigated the effects of
aging on repair, using a direct measure, the rate of DNA photoproduct
removal in UV-irradiated fibroblasts derived from donors of different
ages. We then explored the effects of aging on the constitutive and
UV-induced expression of gene products that participate in DNA repair.
Removal rate for thymine dimers and (6–4) PPs decreases in human
fibroblasts with increasing donor age
Human dermal fibroblasts derived from newborn, young adult (21–34
years old), and old adult (63–88 years old) donors (6 donors/age
group) were irradiated at early passage (population doubling3) with
solar-simulated light as described in Materials and Methods. The
removal rate for UV-induced photoproducts was examined by slot blot
analysis of irradiated DNA at multiple time points over a period of
24 h (Fig. 1A
). There was a significant difference in the rate of removal
of thymine dimers vs. (6–4) PPs, which was affected by age
(P<0.001, general linear model of SPSS). A significant
decrease in the repair rate of thymine dimers was observed between
newborn and either young or old adult cells (P<0.001), but
not between young and old adults (Fig. 1B
). For the more
rapidly excised (6–4) PPs, the removal rate decreased between newborn
and adult fibroblasts (P<0.001) and again between young
adult and old adult fibroblasts (P<0.005) (Fig. 1B
). As well, in accordance with previous publications
(43
, 44)
, in all age groups (6–4) PPs were removed faster
than thymine dimers (P<0.005, ANOVA post hoc analysis by
Fisher and Bonferroni/Dunn). There was no statistically significant
difference in the slope (rate of removal) of (6–4)PPs early (up to
3 h) vs. late (from 3 h on) when all the donors were tested
together (P=0.24) or when each age group was tested alone.
In contrast, there was a statistically significant difference in the
removal of thymine dimers early (up to 3 h) vs. late (from 3 h on) when all donors were tested together (P<0.02, ANOVA,
Huynh-Feldt post hoc analysis). For example, after 3 h 24% ±
10%, 47% ± 11%, and 57% ± 7% of (6–4) PPs remained in newborn
vs. young adult and old adult, respectively, whereas 42% ± 6%, 83%
± 8%, and 86% ± 6% of thymine dimers remained in newborn, young,
and old adult fibroblasts, respectively. From 3 h to 24 h,
there was no further removal of thymine dimers detectable in any age
group (Fig. 1B
).
|
Constitutive mRNA levels for XPA, ERCC3, and PCNA decrease with age
To determine whether the age-associated decrease in photoproduct
removal rate correlates with decreased expression of genes that encode
DNA repair enzymes (11)
, total mRNA from fibroblasts
derived from donors of different ages was isolated, processed for
Northern blotting, and hybridized with XPA, PCNA, ERCC2, and ERCC3
cDNAs (Fig. 2A
). The constitutive mRNA levels of XPA and ERCC3 were
comparable in newborn and young adult cells, but reduced with adult
aging. In the old adult age group (
63 years), XPA was reduced by 78%
± 15% on average compared to young adults (P<0.01) and
the ERCC3 mRNA level was reduced by 64% ± 11% (P<0.002)
(Fig. 2B
). Compared to newborn donors, the constitutive mRNA
level of PCNA was markedly reduced by 60% ± 4% in young and by 80%
± 5% in old adult donors (P<0.03 and
P<0.001, respectively). There was not a significant
reduction in PCNA mRNA in old vs. young adult donors (Fig. 2B
). In the same 17 donors, there was no significant
age-associated change in the constitutive mRNA level of ERCC2 (data not
shown).
|
Constitutive levels of DNA repair proteins decrease with age
To determine whether the age-associated decrease in mRNA levels of
DNA repair enzymes is reflected at the protein level, total cellular
proteins from simultaneously processed triplicate donor pairs of
different ages were processed for Western blotting (Fig. 3A
). The constitutive protein levels of ERCC3, p53, RPA, and
XPA were comparable in newborn and young adult donors (Fig. 3B
). In contrast, the levels of ERCC3, p53, PCNA, RPA, and
XPA were decreased in old adults compared to newborns by 61% ± 10%,
41% ± 29%, 91% ± 3%, 53% ± 21%, and 69% ± 4%, respectively
(P<0.008, P<0.03, P<0.055,
P<0.05, and P<0.04, respectively) (Fig. 3B
). Thus, age-associated decreases of at least 50% were
observed in the constitutive level of all proteins examined.
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During the period of initial repair (24 h), UV irradiation does not
modulate ERCC3, PCNA, or XPA and modestly down-regulates RPA
in fibroblasts
Because UV irradiation can induce NER proteins in prokaryotic
(44)
as well as in eukaryotic cells (24)
, we
examined the effect of our UV irradiation protocol on NER protein
levels in human fibroblasts. Western blot analysis revealed no
consistent modulations in the levels of ERCC3, PCNA, or XPA in
fibroblasts up to 24 h after irradiation, regardless of donor age
(Fig. 4A
). Conversely, RPA level was decreased by approximately half (45±10%
to 62±2%, depending on the time point and age group) (Fig. 4A
).
|
Aging affects p53 protein induction after UV irradiation
Consistent with previous reports using human newborn fibroblasts
(45)
, melanocytes (46)
, and other cell types
(47
, 48)
, Western blot analysis revealed that UV
irradiation up-regulated p53 protein in all donors (Fig. 4A
). However, after 16 h the induction of p53 was
slightly but not significantly lower in adult than in newborn donors,
and within 24 h p53 induction was 56% ± 10% and 69% ± 5%
lower in young and old adult cells, respectively, as compared to
newborn cells (P<0.0001) (Fig. 4B
).
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30
31)
. In the current study we
directly measured the rate of removal of thymine dimers and (6–4) PPs
after UV irradiation of normal dermal fibroblasts. Substantial and
significant differences with age in the rate of removal of (6–4) PPs
were observed between newborns and adults. By 24 h after
irradiation, <5% of the initial (6–4) PPs remained in newborn
fibroblast DNA but ~ 25% remained in the genome of both young
adult and old adult fibroblasts. For thymine dimers, a highly
significant difference in removal was observed between newborns and
young adults (53%±9% vs. 27%±10% of thymine dimers removed at
24 h), with a further significant reduction of approximately
two-thirds between young and old adult cells (27%±10% vs. 9%±8%)
at the same time point, with parallel differences at earlier times.
There appear to be two phases in the removal of both photoproducts: a
rapid phase occurring within the first 3 h and a slower phase
thereafter. Furthermore, for the first 24 h, removal of
photoproducts was substantially slower in aged donors. Previous
studies, though, conducted in hepatocytes from young and old rats,
showed an age-related decrease in the removal of CPDs from the
transcribed strand of the albumin gene (49)
. It was found
that the age-related decrease in CPD removal was due to compromised
coupling of transcription and DNA repair in hepatocytes isolated from
old rats (49)
. Therefore, the age-related decrease in the
repair of the transcribed genes might be due to changes in the
activities of proteins involved in coupling transcription to NER.
TFIIH, a multisubunit protein complex, and some of the subunits that
comprise the core of the complex are required for both transcription
and DNA repair (50)
. An age-associated decrease in the
binding of p53 to several TFIIH-associated factors, including XPB and
XPD, may indeed alter regulation of XPB and XPD by p53
(18)
, restricting formation of the active incision complex
(13)
and hence reducing the effectiveness of NER with age.
In the prokaryotic system, treatment of Escherichia coli
with the antibiotic rifampin [known to lock the RNA polymerase in an
abortive cycle preventing transcription elongation (51)
]
before UV irradiation prevents the up-regulation of repair proteins and
leads to a significant decrease in the rate of removal of CPDs
(44)
. It is possible that levels of one or more of the
human NER proteins are low and become rate limiting after the first
3 h, impeding further photoproduct removal. Excluding RPA, our
data do not reveal any reduction in any NER protein studied up to
16 h after irradiation. Thus, RPA level may be rate limiting or
the functional adequacy of the NER proteins may decrease with time.
Another explanation for slower removal of photoproducts after 3 h
of irradiation is that the majority of the accessible photoproducts,
particularly those in actively transcribed genes, are removed within
the first 3 h, whereas the remaining photoproducts are less
accessible to the NER proteins and hence are removed more slowly.
Previously published DNA damage repair kinetics have given rather
complex results, perhaps due to differences in the cell system (mainly
murine cell lines), dose, and frequency of UV irradiations
(52
53
54
55
56)
. In our studies we used an UV irradiation
spectrum that closely resembles terrestrial sunlight, as described in
Materials and Methods (34)
, and human fibroblasts.
In vivo data obtained after UVB irradiation of hairless mice
(57
, 58)
, rat epidermal keratinocytes in vitro
and in vivo (52)
, and normal human skin showed
similar DNA damage removal kinetics of thymine dimers and (6–4)
photoproducts (55)
. Similar to the rates of photoproduct
removal in our studies, these authors report rapid removal during the
first 4 h and almost no or very little removal of cyclobutane
pyrimidine dimers up to 24 h after irradiation. It has also been
suggested that (6–4) PPs may induce more distortion of the DNA double
helix, rendering it more accessible to the repair enzymes or more
easily recognized (59)
, thus explaining the more rapid
removal of (6–4) PPs vs. thymine dimers. Also, participation of as yet
unidentified enzymes or changes in the activity of enzymes that
participate in NER may influence repair at late times.
The significant age-associated difference in the rate of photoproduct
removal likely reflects, at least in part, the reduced constitutive
mRNA and protein levels of the NER proteins observed in this present
study. In general, age-related changes in mRNA levels reflect similar
changes in the level/activity of the protein encoded by the mRNA
(60)
. We examined the levels of four NER proteins and of
the p53 protein that controls the transcription and/or function of at
least three of those four enzymes (18
, 61
62
63)
. The
constitutive levels of all proteins examined were lower in the older
adult group than in the newborn and young adult groups. However, there
does not appear to be a significant difference in the constitutive
levels of studied NER proteins between newborn and young adults,
although the repair rate of both thymine dimers and (6–4) PPs is
significantly slower in young adult cells than in newborn cells. This
suggests that factors other than enzyme levels, such as enzyme
activity, may also contribute to the difference in repair rate with
aging during the first 24 h after DNA damage. Finally, the
different patterns observed for DNA repair (progressive decrease with
age) vs. NER gene expression at the mRNA and protein levels (comparable
in newborns and young adults but decreased in old adults) may also
simply reflect inter-donor variability, given that different donor cell
lines were used in the two sets of assays.
In bacteria, the response to DNA damage includes the coordinated
induction of more than 20 genes that have a role in DNA repair and
growth control, a phenomenon termed the SOS response (45)
.
Recent work from our laboratory (38)
and from others
(64
, 65)
suggests that human cells also have an SOS-like
response to DNA damage, mediated at least partly by
up-regulation/activation of the p53 protein (38
, 45
, 66)
.
We therefore examined modulations in NER proteins in response to UV
irradiation as a function of donor age. Within the first 24 h
after irradiation, apart from p53 there were no inductions in the NER
proteins studied in any age group. In fact, the levels of the RPA were
reduced at 16 and 24 h. p53 was markedly induced, as expected
(26
, 45)
and the level of induction was comparable in all
age groups at 7 and 16 h. However, 24 h after UV irradiation,
p53 induction was significantly lower in adults than in newborns. It is
unlikely that the observed briefer duration of p53 induction in adult
vs. newborn cells alone can explain the significantly reduced repair
rate between the two groups, because the age-associated decrease in p53
induction was quite modest through at least 16 h and was only
significant 24 h after irradiation. Hence, differences in p53
protein levels with age are difficult to implicate in the different DNA
repair rates observable as early as 1–3 h after irradiation. Of note,
however, p53 activation can occur independent of protein levels
(67
, 68)
, and activation per se was not
studied.
It is known that some NER proteins, notably RPA, require
phosphorylation for activation or to form complexes with other repair
enzymes (14
, 16
, 69)
. Phosphorylation of at least some
proteins has been reported to decrease with aging (70)
,
and slower or less extensive phosphorylation/activation of
rate-limiting NER proteins might explain in part our finding of slower
DNA damage repair in adults vs. newborns.
Our current findings, together with previous reports showing
progressive loss of the ability to repair DNA damage with age both
in vitro and in vivo (71
, 72)
,
correlate well with the increased rate of mutations with aging
(73
, 74)
. Because CPDs, particularly thymine dimers, are
two- to threefold more abundant than (6–4) PPs (75
, 76)
and their removal rate is considerably slower (77)
, CPDs
have been postulated to be more mutagenic (56)
. Our
studies demonstrate virtually no removal of thymine dimers in old adult
donor cells within 24 h, and only 27% ± 10% of thymine dimers
are removed in young adult vs. 53% ± 9% in newborn fibroblasts in
this time, consistent with a contribution to the age-associated
mutation risk (30)
. Other evidence suggests an important
contribution of (6–4) PPs to UV-induced mutagenesis
(78
79
80
81)
, however, and the ~20% of (6–4) PPs that
remain after 24 h in UV-irradiated adult cells vs. the 5% in
newborn cells may also increase the risk of mutations.
Cell lines with inactivating mutations in genes that encode NER
proteins such as XPA, ERCC3, and ERCC2 have severely compromised DNA
damage repair after UV irradiation (82)
and the XP
patients from whom the cells derive are markedly cancer prone
(83)
. Moreover, retroviral-mediated restoration of
wild-type gene expression in DNA repair-deficient cell lines leads to
complete recovery of their repair capacity (84
85
86)
. These
phenomena are highly consistent with our hypothesis that the
age-associated reductions in rate of photoproduct removal results at
least partly from decreased transcription of key DNA repair genes.
Regardless of the precise molecular mechanism(s), the observed
decreased DNA repair rate is likely to contribute to the increased
incidence of skin cancer in the elderly.
A major response of human cells to DNA damage is nuclear accumulation
and activation of the p53 protein (87)
. The role of p53 in
NER is not completely understood; attempts to link p53 to repair and
recovery from DNA damage have explored several possibilities, such as
direct physical interactions between p53 and repair proteins. Indeed,
p53 interacts in vitro with each of the XPB, XPD, and p62
components of TFIIH transcription factor (18
, 88
, 89)
. p53
may regulate repair by trans-activation of genes involved in
repair. For example, p21 and Gadd45 are up-regulated by p53 levels and
interact with PCNA (90
, 91)
, which is required for DNA
repair as well as replication (92)
. Finally, it was
suggested that p53 might promote repair by acting as a sensor of DNA
damage and recruiting TFIIH complex to sites where it is needed in
repair (89)
. In our experiments, there was no
up-regulation of NER protein levels within the first 24 h, arguing
against a contribution of p53’s transcriptional role to early repair
or to the age-associated decrease observed in DNA repair rate. However,
if p53 affects enzyme activation or complex formation, as has been
reported (18
, 63)
, its reduced levels in old adults could
directly affect the efficiency of repair in this time frame. It will be
of interest to more fully elucidate the contribution of decreased p53
levels and/or activity to mutation risk, as recent work suggests
methods of increasing p53 levels and activity in the absence of DNA
damage (38)
with an associated increase in the rate of
removal of DNA photoproducts (38)
, thus potentially
decreasing cancer risk (93)
.
In summary, our results show an age-associated reduction in the removal
rate of UV-induced DNA photoproducts, adding to a rapidly expanding
literature linking DNA repair and aging (94
, 95)
. The
reduced repair is associated with reduced constitutive mRNA and protein
levels of p53 and several NER enzymes. Moreover, with age there is less
induction of p53 after DNA damage. Taken together, our data suggest
that the age-associated decrease in DNA damage repair results at least
partly from decreased availability of many proteins that participate in
this process. Our data also suggest that other, as yet unidentified
factors may contribute to this decline.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication August 25, 1999. Accepted for publication November 11, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 P. K. Reddy Thavanati, K. R. Kanala, A. E. de Dios, and J. M. Cantu Garza Age-Related Correlation Between Antioxidant Enzymes and DNA Damage With Smoking and Body Mass Index J Gerontol A Biol Sci Med Sci, April 1, 2008; 63(4): 360 - 364. [Abstract] [Full Text] [PDF]
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 J.-H. Chen, C. N. Hales, and S. E. Ozanne DNA damage, cellular senescence and organismal ageing: causal or correlative? Nucleic Acids Res., December 3, 2007; 35(22): 7417 - 7428. [Abstract] [Full Text] [PDF]
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V. Gorbunova, A. Seluanov, Z. Mao, and C. Hine Changes in DNA repair during aging Nucleic Acids Res., December 3, 2007; 35(22): 7466 - 7474. [Abstract] [Full Text] [PDF]
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M. A. Lovell and W. R. Markesbery Oxidative DNA damage in mild cognitive impairment and late-stage Alzheimer's disease Nucleic Acids Res., December 3, 2007; 35(22): 7497 - 7504. [Abstract] [Full Text] [PDF]
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 J. E. Adair, S. C. Maloney, G. A. Dement, K. J. Wertzler, M. J. Smerdon, and R. Reeves High-Mobility Group A1 Proteins Inhibit Expression of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A Cancer Res., July 1, 2007; 67(13): 6044 - 6052. [Abstract] [Full Text] [PDF]
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 R. Demirbag, R. Yilmaz, A. Kocyigit, and S. Guzel Effect of Coronary Angiography on Oxidative DNA Damage Observed in Circulating Lymphocytes Angiology, April 1, 2007; 58(2): 141 - 147. [Abstract] [PDF]
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 S. C. Gehen, P. F. Vitiello, R. A. Bambara, P. C. Keng, and M. A. O'Reilly Downregulation of PCNA potentiates p21-mediated growth inhibition in response to hyperoxia Am J Physiol Lung Cell Mol Physiol, March 1, 2007; 292(3): L716 - L724. [Abstract] [Full Text] [PDF]
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 J. Shen, M. Desai, M. Agrawal, D. O. Kennedy, R. T. Senie, R. M. Santella, and M. B. Terry Polymorphisms in nucleotide excision repair genes and DNA repair capacity phenotype in sisters discordant for breast cancer. Cancer Epidemiol. Biomarkers Prev., September 1, 2006; 15(9): 1614 - 1619. [Abstract] [Full Text] [PDF]
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 S. Arad, N. Konnikov, D. A. Goukassian, and B. A. Gilchrest T-oligos augment UV-induced protective responses in human skin FASEB J, September 1, 2006; 20(11): 1895 - 1897. [Abstract] [Full Text] [PDF]
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D. C. Cabelof, Y. Ikeno, A. Nyska, R. A. Busuttil, N. Anyangwe, J. Vijg, L. H. Matherly, J. D. Tucker, S. H. Wilson, A. Richardson, et al. Haploinsufficiency in DNA Polymerase {beta} Increases Cancer Risk with Age and Alters Mortality Rate. Cancer Res., August 1, 2006; 66(15): 7460 - 7465. [Abstract] [Full Text] [PDF]
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 J. Gu, M. R. Spitz, H. Zhao, J. Lin, H.B. Grossman, C. P. Dinney, and X. Wu Roles of tumor suppressor and telomere maintenance genes in cancer and aging--an epidemiological study Carcinogenesis, October 1, 2005; 26(10): 1741 - 1747. [Abstract] [Full Text] [PDF]
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 S Wing and D B Richardson Age at exposure to ionising radiation and cancer mortality among Hanford workers: follow up through 1994 Occup. Environ. Med., July 1, 2005; 62(7): 465 - 472. [Abstract] [Full Text] [PDF]
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 G. Xu, G. Spivak, D. L. Mitchell, T. Mori, J. R. McCarrey, C. A. McMahan, R. B. Walter, P. C. Hanawalt, and C. A. Walter Nucleotide Excision Repair Activity Varies Among Murine Spermatogenic Cell Types Biol Reprod, July 1, 2005; 73(1): 123 - 130. [Abstract] [Full Text] [PDF]
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S. Blankenburg, I. R. Konig, R. Moessner, P. Laspe, K.-M. Thoms, U. Krueger, S. G. Khan, G. Westphal, C. Berking, M. Volkenandt, et al. Assessment of 3 xeroderma pigmentosum group C gene polymorphisms and risk of cutaneous melanoma: a case-control study Carcinogenesis, June 1, 2005; 26(6): 1085 - 1090. [Abstract] [Full Text] [PDF]
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S. Neri, A. Gardini, A. Facchini, F. Olivieri, C. Franceschi, G. Ravaglia, and E. Mariani Mismatch Repair System and Aging: Microsatellite Instability in Peripheral Blood Cells From Differently Aged Participants J Gerontol A Biol Sci Med Sci, March 1, 2005; 60(3): 285 - 292. [Abstract] [Full Text] [PDF]
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 M. A. Mahroos, M. Yaar, T. J. Phillips, J. Bhawan, and B. A. Gilchrest Effect of Sunscreen Application on UV-Induced Thymine Dimers Arch Dermatol, November 1, 2002; 138(11): 1480 - 1485. [Abstract] [Full Text] [PDF]
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A. R. Muotri, M. C.N. Marchetto, M. F. Suzuki, K. Okazaki, C. F.P. Lotfi, G. Brumatti, G. P. Amarante-Mendes, and C. F.M. Menck Low amounts of the DNA repair XPA protein are sufficient to recover UV-resistance Carcinogenesis, June 1, 2002; 23(6): 1039 - 1046. [Abstract] [Full Text] [PDF]
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 M. T. Landi, A. Baccarelli, A. C. Pesatori, M. Hedayati, L. Grossman, M. A. Tucker, and R. E. Tarone RESPONSE: Re: DNA Repair, Dysplastic Nevi, and Sunlight Sensitivity in the Development of Cutaneous Malignant Melanoma J Natl Cancer Inst, May 15, 2002; 94(10): 773 - 774. [Full Text] [PDF]
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 M. L. Smith and Y. R. Seo p53 regulation of DNA excision repair pathways Mutagenesis, March 1, 2002; 17(2): 149 - 156. [Abstract] [Full Text] [PDF]
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 M. L. Hamilton, H. Van Remmen, J. A. Drake, H. Yang, Z. M. Guo, K. Kewitt, C. A. Walter, and A. Richardson Does oxidative damage to DNA increase with age? PNAS, August 17, 2001; (2001) 171202698. [Abstract] [Full Text] [PDF]
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C. H. Jiang, J. Z. Tsien, P. G. Schultz, and Y. Hu The effects of aging on gene expression in the hypothalamus and cortex of mice PNAS, February 13, 2001; 98(4): 1930 - 1934. [Abstract] [Full Text] [PDF]
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M. L. Hamilton, H. Van Remmen, J. A. Drake, H. Yang, Z. M. Guo, K. Kewitt, C. A. Walter, and A. Richardson Does oxidative damage to DNA increase with age? PNAS, August 28, 2001; 98(18): 10469 - 10474. [Abstract] [Full Text] [PDF]
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