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(The FASEB Journal. 2000;14:1325-1334.)
© 2000 FASEB

Mechanisms and implications of the age-associated decrease in DNA repair capacity

DAVID GOUKASSIAN1, FATEN GAD1, MINA YAAR, MARK S. ELLER, UMBEREEN S. NEHAL and BARBARA A. GILCHREST2

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Skin cancer incidence is clearly linked to UV irradiation and increases exponentially with age. We studied the rate of removal of thymine dimers and (6–4) photoproducts in UV-irradiated human dermal fibroblasts derived from donors of different ages. There was a significant decrease with aging in the repair rates of both thymine dimers and (6–4) photoproducts (P<0.001). In addition, there was an age-associated decrease in the protein levels of ERCC3, PCNA, RPA, XPA, and p53 that participate in nucleotide excision repair. Moreover, the mRNA levels of XPA, ERCC3, and PCNA were significantly reduced with aging, suggesting that these decreases are often regulated at the mRNA level. Furthermore, with age induction of p53 after UV irradiation was significantly reduced. Taken together, our data suggest that the age-associated decrease in the repair of UV-induced DNA damage results at least in part from decreased levels of proteins that participate in the repair process.—Goukassian, D., Gad, F., Yaar, M., Eller, M. S., Nehal, U. S., Gilchrest, B. A. Mechanisms and implications of the age-associated decrease in DNA repair capacity.


Key Words: aging • DNA damage repair • DNA photoproducts • nucleotide excision repair


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SKIN CANCER ACCOUNTS for at least 40% of all human malignancies, more than 1,000,000 cases annually in the U.S. (1) . 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Primary human dermal fibroblast cultures were prepared from newborn foreskins and specimens of sun-protected normal Caucasian skin from healthy donors of increasing age (21 to 88 years), as described (33) . 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.)


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Because indirect measures of DNA repair capacity suggest a decrease with age (29 , 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 doubling<=3) 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 ).



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  		Figure 1. The rate of removal of thymine dimers and (6–4) photoproducts decreases with aging. A) A representative slot blot from a young adult donor serially hybridized with antibodies against thymine dimers and (6–4) PPs and then 32P-DNA to show the rate of removal of UV-induced photoproducts. Total genomic DNA was collected up to 24 h after irradiation and photoproduct levels were determined as described in Materials and Methods. There is no antibody binding in the preirradiation sample, maximum binding immediately after irradiation, and progressively less binding over time. There is more rapid removal of (6–4) PPs than thymine dimers. Quantitative binding comparisons cannot be made between the two antibodies. B) Quantitative effect of aging on photoproduct removal rates. UV or sham-irradiated genomic DNA from 6 newborn (solid bars), 6 young adults (25–34 years, striped bars), and 6 old adults (63–88 years, open bars) was processed for slot blotting and reacted with anti-(6–4) PPs and anti-thymine dimer antibodies as described. After densitometric analysis of band intensity, the percent of remaining photoproducts for each donor at each time point was determined as the ratio of the amount of bound antibody at that time compared to that immediately after irradiation (time 0), normalized for loading of DNA in each lane. Triplet samples of each time point were processed together, the time 0 was set at 100% and all time points for each donor were calculated as a percent of these values. Each graphed normalized time point value (mean±SE) is the average of six independent experiments (donors). The difference in removal rate for both photoproducts between newborn and adult donors was highly statistically significant P < 0.0001 (ANCOVA).

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).



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  		Figure 2. Constitutive mRNA levels of XPA, ERCC3, and PCNA decrease with age. A) Preconfluent cultures of 5 newborn, 6 young adult (21–49 years), and 6 old adult (63–69 years) fibroblasts were collected at 2 population doublings postprimary culture. Total RNA was isolated and processed for Northern blot analysis, with sequential hybridization to XPA, ERCC3, and PCNA cDNAs. Ethidium bromide-stained gel shows comparable loading of the different lanes. B) Graphic representation of band intensity (mean±SE) as determined by densitometric analysis. There are significant age-associated reductions in mRNA levels of XPA [old adult less than newborn (P<0.038), and less than young adult (P<0.037)], ERCC3 [old adult less than newborn (P<0.003) and less than young adult (P<0.002)], and PCNA [old adult less than newborn (P<0.001) and less than young adult (P<0.26)](ANOVA with Fisher and Bonferroni/Dunn post hoc analysis).

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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  		Figure 3. Constitutive protein levels of ERCC3, p53, PCNA, RPA, and XPA decrease with age. Fibroblasts from newborn, young adult, and old adult donors were collected at three population doublings postprimary cultures, and total protein was isolated and processed in tandem for Western blot analysis, loading 100 µg of total protein per lane. The membrane was cut into strips containing proteins of different molecular weights corresponding to the proteins of interest, and each strip was then reacted with a different antibody directed against the appropriate protein. A) Representative experiments show a decrease in the constitutive levels of all examined repair proteins in old adult donors compared to newborn or young adult donors. B) Graphic representation of band density (mean±SE) for 3 donors in each age group as determined by densitometric analysis. After normalization for loading, regression analysis for values from old adult donors (62, 73 and 88 years) revealed a decrease in the constitutive levels of ERCC3 (black bars, P<0.008), p53 (striped bars, P<0.03), PCNA (gray bars, P<0.055), RPA (white bars, P<0.05), and XPA (hatched bars, P<0.04) compared to cells from young adult donors (21, 33, and 37 years) or newborns, for whom values were set at 100%.

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 ).



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  		Figure 4. Modulation of p53 and DNA repair proteins after UV irradiation in fibroblasts from donors of various ages. Representative data from 1 of 3 independent experiments for each protein are shown. A) Normal human fibroblasts at three population doublings postprimary were UV (+) or sham (-) irradiated with 30 mJ/cm2. Total cellular proteins from paired cultures were collected at 7, 16, and 24 h after irradiation and processed for Western blot analysis (100 µg protein per lane). Coomassie blue-stained gels corresponding to each immunoblot are shown to demonstrate the loading of protein samples. Densitometric analysis performed on blots involving 9 donors (3 newborns and 6 adults aged 21 to 88 years) revealed no consistent modulation in the levels of XPA, PCNA, or ERCC3 after UV irradiation. RPA levels were comparably decreased (by 42 ±10% to 62%±2%, depending on time point and age group) in all age groups 16 h and 24 h after irradiation.

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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies using an indirect measurement of DNA damage repair, restoration of expression of a transfected UV-damaged CAT reporter plasmid, have shown a decline in DNA repair capacity with donor age in human dermal fibroblasts, lymphocytes, and transformed lymphoblastoid cells (29 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.


   ACKNOWLEDGMENTS
 
D.G. and F.G. are supported by National Institutes of Health training grants (T32AG00115 and 5T32AR07562, respectively).


   FOOTNOTES
 
1 D.G. and F.G. contributed equally to this paper.

Received for publication August 25, 1999. Accepted for publication November 11, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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