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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:427-434 (2006)
© 2006 The Gerontological Society of America

Age-Related Loss of the DNA Repair Response Following Exposure to Oxidative Stress

Diane C. Cabelof1,2,, Julian J. Raffoul, Yubin Ge, Holly Van Remmen, Larry H. Matherly and Ahmad R. Heydari

1 Developmental Therapeutics Program and 2 Department of Nutrition and Food Science, Wayne State University, Detroit, Michigan.
3 Department of Cellular and Structural Biology and the Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio.
4 South Texas Veterans Health Care System, San Antonio.
5 Department of Pharmacology, Wayne State University School of Medicine, Detroit.

Address correspondence to Diane C. Cabelof, PhD, Barbara Ann Karmanos Cancer Institute, 110 East Warren Avenue, Wayne State University School of Medicine, Detroit, MI 48201. E-mail: d.cabelof{at}wayne.edu


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Young (4- to 6-month-old) and aged (24- to 28-month-old) mice were exposed to 2-nitropropane (2-NP), a DNA oxidizing agent, and the ability to induce DNA polymerase ß (ß-pol) and AP endonuclease (APE) was determined. In contrast to the inducibility of these gene products in response to oxidative damage in young mice, aged mice showed a lack of inducibility of ß-pol and APE. APE protein level and endonuclease activity were both reduced 40% (p <.01) in response to 2-NP. Accordingly, the accumulation of DNA repair intermediates in response to 2-NP differed with age. Young animals accumulated 3'OH-containing DNA strand breaks, whereas the aged animals did not. A role for p53 in the difference in DNA damage response with age is suggested by the observation that the accumulation of p53 protein in response to DNA damage in young animals was absent in the aged animals. Our results are consistent with a reduced ability to process DNA damage with age.

THE base excision repair (BER) pathway is critical in the maintenance of genomic integrity, both with respect to endogenously arising DNA damage and induced damage, when the offending agent generates BER substrates. Loss of BER activity reduces the ability to tolerate DNA damage, as evidenced by the increased mutational response to DMS (dimethyl sulfate) in aged animals and in animals heterozygous for ß-pol by gene-targeted deletion (1,2). As aging results in an approximate 50% reduction in in vitro BER capacity and a corresponding reduction in levels of ß-pol protein and transcripts (1,3), we have argued that the loss of ß-pol is responsible for the decrease in ability to tolerate DNA damage. However, we and others have also established that BER is inducible in response to DNA damage (4–7). Thus, the purpose of the present work is to determine whether the DNA damage inducibility of ß-pol is maintained in aged animals.

Many laboratories have reported on the decline in genomic integrity observed with age (8–11), and it is tempting to suggest that a reduced ability to repair DNA damage is, in part, responsible for this phenotype. Previous reports of the age-related loss in BER capacity have focused on the basal expression of BER genes (1,12). In this study, we look at the stress response to carcinogen exposure vis-à-vis BER. In response to exogenously induced stress, a multitude of cellular reactions mediated by complex signaling pathways ultimately result in effective repair of DNA damage, but the ability of aged animals to mount an appropriate DNA repair response has not been extensively tested. Certainly the increased accumulation of mutations in aged animals following carcinogen exposure (1) suggests an inability to repair the DNA damage. Indeed, Kaneko and colleagues (7) have shown that ß-pol inducibility in response to ionizing radiation (IR) is attenuated with age, supporting our hypothesis that the DNA damage response is affected by aging.

Here we have exposed young (4- to 6-month-old) and aged (24- to 28-month-old) mice to 2-nitropropane (2-NP), a hepatocarcinogen that induces oxidative DNA damage, to evaluate the effect of age on the ability 2-NP to induce two key enzymes within the BER pathway, ß-pol and AP endonuclease (APE). Generally ß-pol is considered to be the rate-determining enzyme in the BER pathway by virtue of its deoxyribophosphodiesterase (dRPase) activity (13). There is evidence that both ß-pol (14) and APE (15) are key enzymes in the response to oxidative stress. Loss of APE activity and/or ß-pol is associated with accumulation of toxic DNA repair intermediates (2,14,16), and the effect of age on the accumulation of DNA single-strand breaks has likewise been determined here.

In addition, we have evaluated the critical role that p53 may play in the DNA damage response by measuring the ability of aged animals to accumulate p53 protein. Wild-type p53 stimulates BER activity in vitro (17). In vivo, gene-targeted disruption of p53 results in a clear, dose-dependent loss of ß-pol transcript as p53 gene dosage decreases (N. Anyangwe, D. C. Cabelof, A. R. Heydari, 2005, unpublished observations). This loss of ß-pol in response to p53 null and/or mutant status has also been shown to incapacitate the BER response to methyl methanesulfonate (18). Thus, at both the basal level and in response to DNA damaging agents, evaluation of the effect of aging on the p53 response to carcinogen exposure is potentially informative vis-à-vis the ability to mount an appropriate DNA repair response. We provide evidence that carcinogen-induced activation of p53 may be dysregulated with age. This provides a possible basis for better understanding the mechanism by which the DNA damage response is altered with age.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Animals
Experiments were performed in young (4- to 6-month-old) and old (24- to 28-month-old) male specific pathogen–free mice in accordance with the National Institutes of Health (NIH) guidelines for the use and care of laboratory animals. The animal protocol was approved by the Wayne State University Animal Investigation Committee. Mice were maintained on a 12-hour light/dark cycle and were fed a standard mouse laboratory chow and water ad libitum. Animals were subjected to an i.p. injection of 100 mg/kg body weight 2-NP (CAS registry number 79-46-9; Sigma-Aldrich, St. Louis, MO) or olive oil vehicle. After 24 hours, mice were killed by CO2 asphyxiation and cervical dislocation. Organs were flash frozen in liquid nitrogen and stored at –70°C.

Isolation of Nuclear Extract
Nuclear proteins were isolated using the CelLytic NuCLEAR Extraction Kit (Sigma-Aldrich), a method that disrupts cells with hypotonic buffer allowing the cytoplasmic fraction to be removed while the nuclear proteins are released by a high salt buffer. All samples and tubes were handled and chilled on ice, and all solutions were prepared fresh according to the manufacturer's protocol. The extract was snap frozen in liquid nitrogen and stored at –70°C. To remove salt, the nuclear extract was dialyzed against 1 L of dialysis buffer (20 mM Tris-HCl, pH 8.0; 100 mM KCl; 10 mM NaS2O5; 0.1 mM dithiothreitol; 0.1 mM phenylmethylsulfonylfluoride; and Pepstain A at 1 µg/ml) for 4–6 hours at 4°C using Slide-A-Lyzer MINI Dialysis System (Pierce, Rockford, IL). The dialyzed nuclear extracts were flash frozen in liquid nitrogen and stored at –70°C. Protein concentrations of the nuclear extracts were determined according to Bradford using Protein Assay Kit I (Bio-Rad, Hercules, CA).

Western Blot Analyses
Western analysis was performed using liver nuclear extracts (100 µg) subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a Hybond enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ) using a Bio-Rad semidry transfer apparatus. The membranes were stained with MemCode (Pierce) to visualize the equal transfer of protein to the membrane. Western blot analysis was accomplished using manufacturer-recommended dilutions of anti-sera developed against p53 (pAB 240; Santa Cruz Biotechnology, Santa Cruz, CA), ß-pol (Ab-1 Clone 18S; NeoMarkers, Fremont, CA), and Ape/Ref1 (Novus Biologicals, Littleton, CO). The bands were detected and quantified using a ChemiImager System (AlphaInnotech, San Leandro, CA) after incubation in SuperSignal West Pico Chemiluminescent Substrate (Pierce). The data are expressed as the integrated density value (I.D.V.) of the band per µg of protein loaded. Western blots for ß-pol protein levels in young control/treated and aged control/treated samples were done on separate membranes to allow for different blotting conditions, as the signal for young 2-NP–treated samples overwhelms the signal for aged samples. To control for potential differences between experiments we also blotted young and old untreated samples. The results for old/control and old/treated are expressed relative to young based on the effect of aging observed in the control blot.

APE Activity Assay
The 5'-endonuclease activity of APE was analyzed using a quantitative in vitro assay that measures the incision of a 26-mer duplex oligonucleotide substrate containing a tetrahydrofuran (F) AP site as previously described (30). A 26-mer oligonucleotide (5'-AATTCACCGGTACCFTCTAGAATTCG-3') was 5'-end-labeled and annealed to an equimolar amount of the complementary strand (5'-CGAATTCTAGAGGGTACCGGTGAATT-3'). Reaction mixtures contained 2.5 pmol of a radio-end-labeled double-strand AP DNA substrate, 100 ng of nuclear extract, 50 mM HEPES (pH 7.5), 50 mM KCl, 10 mM MgCl2, 2 mM dithiothreitol, bovine serum albumin at 1 µg/ml, and 0.05% Triton X-100. The samples were incubated for 15 minutes at 37°C and stopped by the addition of 50 mM EDTA. Reaction products were run on a 15% denaturing sequencing gel. Endonuclease activity (presence of a 14-mer band) was visualized and quantified using a Molecular Imager System (Bio-Rad) by calculating the relative amount of the 14-mer oligo product with the unreacted 26-mer substrate (product/[product + substrate]). The data are expressed as machine counts per ng of protein.

Random Oligonucleotide Primed Synthesis Assay
DNA for the random oligonucleotide primed synthesis (ROPS) assay was isolated using Qiagen (Valencia, CA) gravity tip columns as described in the manufacturer's protocol. This method generates large fragments of DNA (up to 150 kb) while minimizing shearing. The relative number of 3'OH group–containing DNA strand breaks was quantified using a Klenow(exo–) incorporation assay based on the ability of Klenow to initiate DNA synthesis from a 3'OH (19). DNA was heat-denatured at 100°C for 5 minutes, and 0.25 µg of DNA was added to 15 µl of a Klenow reaction buffer (0.5 mM thymidylate triphosphate [dTTP], 0.5 mM deoxyguanylate triphosphate [dGTP] and 0.5 mM deoxyadenylate triphosphate [dATP]); 0.33 µM deoxycytidylate triphosphate (dCTP), 5 units Klenow(exo–) (New England Biolabs, Beverly, MA) with 10X Klenow buffer per manufacturer protocol, and 5 µCi [{alpha}-32P]dCTP (3000 Ci/mmole; PerkinElmer Life Sciences, Boston, MA). Reaction mixtures were incubated at 16°C for 30 minutes, and the reaction was stopped with the addition of 25 µl of 12.5 mM EDTA, pH 8.0. Samples were spotted (5 µl) onto scored and numbered Whatman (Florham, NJ) DE81 chromatography paper and allowed to air dry. The chromatography paper was then washed 5 times for 5 minutes each time in 0.5 M Na2HPO4 (dibasic) to remove unincorporated [{alpha}-32P]dCTP, then rinsed twice briefly in water and allowed to air dry. The paper was cut and placed into scintillation vials with 2.5 ml of ScintiVerse cocktail (Fisher Scientific, Pittsburgh, PA). Incorporation of [{alpha}-32P]dCTP was quantified using a Packard scintillation counter.

Real-Time Reverse Transcription–Polymerase Chain Reaction Quantitation of ß-pol Transcripts
Total RNAs were isolated from the livers of untreated and 2-NP–treated young and aged mice with Trizol reagent (Invitrogen, Carlsbad, CA). Complementary DNAs (cDNAs) were synthesized from 2 µg of RNA using random hexamer primers and a reverse transcription–polymerase chain reaction (RT–PCR) kit (PerkinElmer), and were purified with the QIAquick PCR Purification Kit (Qiagen). ß-pol transcripts, 18S RNA, and ß-actin transcripts were quantitated with a LightCycler real-time PCR machine (Roche, Indianapolis, IN). PCRs contained 2 µl of purified cDNA, 4 mM MgCl2, 0.5 µM each sense and antisense primers, and 2 µl of FastStart DNA Master SYBR Green I enzyme–SYBR reaction mix (Roche). Primer sequences for ß-pol, ß-actin, and 18S RNA transcripts are detailed in Table 1. For all amplifications, PCR conditions consisted of an initial denaturing step of 99°C for 10 minutes, followed by 35–55 cycles of 96°C for 10 seconds, 62°C for 10 seconds, and 72°C for 5 seconds, with a melting curve analysis from 40°C to 99°C to confirm specificity. External standards were prepared by amplification of cDNAs for ß-pol, 18S RNA, and ß-actin using the above primers. The amplicons were cloned into pGEM-T Easy Vector, and the vectors were linearized with ApaI and used to prepare external standard curves. ß-pol transcripts were normalized to both 18S RNA and ß-actin. Results are expressed as mean values from two to three separate experiments using the same cDNA preparations from 10 animals per experimental group.


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  		Table 1. Primer Sequences for Real-Time Reverse Transcription–Polymerase Chain Reaction.

 
Statistical Analysis
Statistical significance between means was determined using analysis of variance (ANOVA) followed by the Fisher's least significant difference test where appropriate (20). A p value less than.05 was considered statistically significant.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 References
 
The primary objective of this study was to determine the effect of aging on the DNA damage response, in particular, the response to oxidative DNA damage. 2-NP is a procarcinogen that requires activation by an aryl sulfotransferase (21). The activation of 2-NP has been reported to be unaffected by aging (22–24) such that initial levels of DNA damage between the young and aged mice should be similar. Thus, the DNA damage/repair response should likewise be similar. We previously reported that, at a basal level, livers from aged mice express significantly lower levels of ß-pol transcript and protein than do those of their younger counterparts (1). In this study, we compared the response to carcinogen exposure in young (4- to 6-month-old) versus aged (24- to 28-month-old) mice with respect to the inducibility of ß-pol and APE. Both gene products are essential components of the BER response to oxidative damage. We have previously demonstrated that ß-pol protein abundance increases in response to 100 mg/kg 2-NP (4), establishing ß-pol as a stress-response gene inducible by DNA damage. To address the possibility that this change in ß-pol may occur at the transcriptional level, we determined the abundance of ß-pol transcripts by real-time PCR. In response to 2-NP exposure, young animals expressed about 50% greater levels of ß-pol transcripts (Figure 1, p <.05), suggesting that the DNA damage inducibility of ß-pol is, at least in part, transcriptionally regulated.


Figure 01
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  		Figure 1. Induction of ß-pol transcript in response to 2-nitropropane (2-NP) in young animals is absent in aged animals. Complementary DNAs (cDNAs) were prepared from RNA isolated from liver tissue of young (4- to 6-month-old) and aged (24- to 28-month-old) mice injected with 2-NP and their noninjected counterparts as described in Methods. ß-pol transcript levels were determined by real-time polymerase chain reaction analysis and were normalized to ß-actin. Data are presented as mean (± standard error of the mean [SEM]) from 10 animals in each group. *Significantly different from control at p <.05

 
An important question is whether the DNA damage response vis-à-vis the BER pathway is maintained with age. To address this concern, we have measured the response of two key genes within the BER pathway to 2-NP exposure. Although we expected to see some attenuation of the transcriptional response to 2-NP with age, we were surprised to observe a total attenuation of this effect on ß-pol transcript levels (Figure 1). Relative ß-pol transcript levels were normalized to both 18S ribosomal RNA (rRNA) levels and ß-actin messenger RNA (mRNA) levels, with nearly identical results. Collectively, these data suggest that with aging, there is a loss of transcriptional activation of ß-pol in response to DNA damage. We extended this analysis to the protein level and found that, in accordance with the transcript data, ß-pol protein levels were upregulated in young animals but were completely unaffected by 2-NP exposure in the aged animals (Figure 2). Aged data are expressed relative to young data based on direct comparisons between young and aged ß-pol levels (data not shown) consistent with previously reported findings (1). The graininess of the ß-pol blot exemplifies the need to blot the young and aged samples separately, because extended incubations and exposures are necessitated by very low expression levels of ß-pol in aged liver.


Figure 02
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  		Figure 2. Induction of ß-pol protein in response to 2-nitropropane (2-NP) in young animals is absent in aged animals. Nuclear extracts were isolated from liver tissue of young (4- to 6-month-old) and aged (24- to 28-month-old) mice injected with 2-NP and their noninjected counterparts as described in Methods. The level of the 39 kd ß-pol protein in 100 µg of nuclear extract was determined by western blot analysis using an antibody against ß-pol protein and a SuperSignal West Pico Chemiluminescent Substrate (Pierce). The relative level of ß-pol protein was quantified using an Alpha Innotech MultiImage system, and the data were normalized based on the amount of protein loaded on each gel. Values for aged animals are expressed relative to young values. Values represent an average (± standard error of the mean [SEM]) for data obtained from 10 animals in each group. C = control; 2-NP = 2-NP injected; M = Santa Cruz marker. *Significantly different from control at p <.05

 
In response to oxidative stress, the APE gene product has roles in both BER and redox regulation. APE has previously been shown to be induced in response to oxidative stress (25,26), and we have also demonstrated this effect in young animals in response to 2-NP (J. J. Raffoul, D. C. Cabelof, A. R. Heydari, 2005, unpublished observations). Unlike ß-pol (see above), APE shows a more robustly aberrant response to DNA damage in aged animals. Nuclear levels of APE protein as well as APE endonuclease activity were significantly reduced (40%, p <.01) upon exposure to 2-NP (Figure 3). Thus, our data suggest a major redistribution of APE in stressed, aged animals.


Figure 03
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  		Figure 3. Effect of 2-nitropropane (2-NP) on APE1 expression and activity in aged animals. A, Level of the 37 kd AP endonuclease 1 (APE1) protein in 200 µg of liver nuclear extract was determined by western blot analysis. Data are expressed as the integrated density value (I.D.V.) of the band per µg of protein loaded. B, 5'-endonuclease activity of APE1 was analyzed using a 32P-end-labeled 26-mer duplex AP-DNA substrate incubated with liver nuclear extract. The product of APE1 endonuclease activity was visualized and quantified using a Molecular Imager System by calculating the relative amount of the 14b product with the unreacted 26b substrate (product/[product + substrate]). Data are expressed as machine counts per ng of protein. Values from each experiment represent an average (± standard error of the mean [SEM]) for data obtained from 10 animals in each group. M = molecular weight standard; C = control, no injection; 2-NP = 100 mg/kg intraperitoneal injection of 2-nitropropane; NC = negative control, AP-DNA substrate incubated in the absence of nuclear extract. *Significantly different from control at p <.05

 
In response to oxidative DNA damage, BER generates transient DNA strand breaks that persist until repair is complete. We previously demonstrated by Comet assay that 2-NP results in a twofold increase in the level of DNA single-strand breaks (2). Here we used the ROPS assay to measure the relative number of DNA single-strand breaks containing 3'OH groups in young and aged mice. We found that there was a nearly twofold increase in the number of 3'OH groups in DNA from young mice treated with 2-NP (Figure 4, p <.01). We expected that the aged animals exposed to 2-NP would experience an even greater increase in single-strand breaks, as we have previously shown that a loss of ß-pol results in increased persistence of these DNA repair intermediates (2). It is surprising that we observed a small but significant decline in the level of 3'OH-containing breaks in DNA from aged mice exposed to 2-NP (Figure 4, p <.01). These data suggest that 2-NP may result in an accumulation of 3' blocking lesions (i.e., a reduction in 3'OH groups) that is consistent with a loss of APE protein and activity in aged animals in response to 2-NP.


Figure 04
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  		Figure 4. Induction of 3'OH containing DNA single-strand breaks in response to 2-nitropropane (2-NP) in young animals is absent in aged animals. Liver DNA was isolated from young (4- to 6-month-old) and aged (24- to 26-month-old) mice using gravity tip extraction columns, and the number of 3'OH-containing breaks was measured using the random oligonucleotide primed synthesis (ROPS) assay as described in Methods. Data are expressed as machine counts per minute corresponding to the level of [{alpha}-32P] deoxycytidylate triphosphate (dCTP) incorporation as quantified by a Packard scintillation counter. Values represent an average (± standard error of the mean [SEM]) for data obtained from at least 10 animals in each group. *Significantly different from control p <.01

 
To address the potential mechanism by which regulation of BER genes could occur in older mice, we measured the levels of p53 protein in response to 2-NP exposure. In Figure 5, we verify that DNA damage by 2-NP resulted in a twofold upregulation of p53 (p <.01) in younger animals. The older animals, however, not only failed to exhibit increased p53 protein, but actually showed a 16% reduction in p53 protein levels in response to 2-NP (Figure 5, p <.01).


Figure 05
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  		Figure 5. Induction of p53 protein in response to 2-nitropropane (2-NP) in young animals is absent in aged animals. Nuclear extracts were isolated from liver tissue of young (4- to 6-month-old) and aged (24- to 28-month-old) mice injected with 2-NP and their noninjected counterparts as described in Methods. The level of the 53 kd p53 protein in 100 µg of nuclear extract was determined by western blot analysis using an antibody against p53 protein and a SuperSignal West Pico Chemiluminescent Substrate (Pierce). The relative level of p53 protein was quantified using an Alpha Innotech MultiImage system, and the data were normalized based on the amount of protein loaded on each gel. Values represent an average (± standard error of the mean [SEM]) for data obtained from 10 animals in each group. C = control; 2-NP = 2-NP injected; M = Santa Cruz marker. *Significantly different from control at p <.01

 

   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
With aging comes a reduced ability to respond to stress. Here we demonstrate that, at the level of three critical DNA repair proteins (ß-pol, APE, and p53), the DNA damage response is altered in aged animals exposed to a carcinogen, 2-NP, known to induce an oxidative stress response in young animals. This finding is in agreement with data from Kaneko and colleagues (7) who reported an attenuation of ß-pol inducibility in aged animals exposed to IR. However, one important difference between results with 2-NP and IR is that 2-NP (unlike IR) does not induce the DNA double-strand breaks. This may be important in that the primary response to 2-NP will be a BER response, whereas the critical survival response to IR is double-strand break repair. Whereas BER is likely responsible for repair of some IR-induced damage (free radical–induced oxidized bases), we have found that heterozygous loss of ß-pol does not increase sensitivity to high doses of IR (2).

However, a potential problem with our use of 2-NP relates to its activation by aryl sulfotransferase and the possibility that age-related differences in enzyme activity may occur. However, aryl sulfotransferase levels have been reported to remain constant with age in male rodents (22–24). This finding suggests that initial levels of DNA damage in response to 2-NP should be similar in young and aged animals, and increases our confidence that young and aged mice will exhibit similar sensitivities to this DNA-damaging agent in our experiments. However, we cannot completely rule out the possibility that other age-related factors may affect the initial levels of DNA damage. As evidence for similar DNA damage responses, preliminary microarray data show that the induction of growth-related oncogene is the same in both young and aged mice exposed to 2-NP (10.6-fold in young, p <.0001; 10.7-fold in aged, p <.0001) (D. C. Cabelof, A. Dombkowski, 2005, unpublished observations). Growth-related oncogene is transcriptionally induced by exposure to diesel exhaust (of which 2-NP is a component) (27), and importantly, is induced in a dose-dependent manner in response to DNA-damaging agents (28).

We previously reported that basal expression of ß-pol protein, mRNA, and enzymatic activity declines significantly with age in all tissues studied (1). In this work we were interested in determining the potential age-dependent changes in the DNA damage inducibility of ß-pol. In response to a dose of 2-NP that induces ß-pol protein and transcripts in young animals, no upregulation was observed in aged animals. Edwards and colleagues (29) demonstrated by transcriptional profiling an age-related decrease in oxidative stress–inducible pathways in mouse heart. Here we report that this loss of response, at least in part, involves the rate-determining enzyme in BER. Our finding is consistent with results of Kaneko and colleagues (7), yet suggests that this loss of ß-pol inducibility is the result of decreased ß-pol transcripts, possibly due to decreased transcriptional activation.

The loss of basal ß-pol inducibility of ß-pol in aged animals is striking in that aged, treated mice express less than 30% of the level of ß-pol protein and transcript as do the young, 2-NP–treated mice. This is particularly critical because we have shown that, in young mice, ß-pol levels are adequate to protect against mutations induced by DMS whereas aged animals are not protected from DMS-induced mutations (1). Importantly, these results resemble those for ß-pol levels in the ß-pol heterozygous knockout mouse, in which a 50% reduction of ß-pol increases the mutagenicity of DMS (2). Because the young ß-pol heterozygous knockout mice do not develop spontaneous tumors, we suggest that a threshold level for protection against mutagenesis and cancer may exist. Aged ß-pol heterozygous mice express only 25% of the amount of ß-pol as do the young, wild-type mice and experience an increased incidence of spontaneous tumors (D. C. Cabelof, S. H. Wilson, A. Richardson, A. R. Heydari, 2005, unpublished observations). This phenotype mimics that seen here with respect to ß-pol levels in older mice treated with 2-NP, and supports the possibility that DNA-damaging agents have greater carcinogenic potential in aged animals as a result of a loss of ß-pol.

Another critical protein in the BER pathway, especially the response to oxidative stress, is APE. Subcellular localization of APE has been shown to be altered with age, such that a 2-fold increase in APE levels is observed in the cytoplasmic fraction of the cell, and a 2-fold decrease is observed in the nucleus (30). We have likewise shown a similar effect of aging on compartmentalization of APE (J. J. Raffoul, D. C. Cabelof, A. R. Heydari, 2005, unpublished observations). However, conflicting reports from Cho and colleagues (31) and Intano and colleagues (12) find increased APE nuclear levels with age. Although this question of age-related changes in APE localization may still be open, our data clearly demonstrate that, in response to 2-NP, aged animals sustain a significant loss in nuclear APE levels. Importantly, not only is there less APE protein available in the nucleus to process the DNA damage in aged animals exposed to 2-NP, there is also lower APE activity. This finding establishes a strong correlation between APE functionality, protein levels, and activity. This loss of APE activity in the aged animals is potentially informative with respect to the types of DNA repair intermediates that accumulate in response to 2-NP.

In young animals, DNA single-strand breaks are induced nearly 2-fold by 2-NP, as measured by both the Comet (2) and the ROPS assays. We previously reported that a loss of ß-pol increases the initial level of these strand breaks and results in a greater persistence of the breaks, as well (2). Therefore, we expected that the reduced expression of ß-pol in the aged animals would likewise increase the level of single-strand breaks. As such, we were surprised to see a decline in the level of strand breaks in the older animals. Ivancsits and colleagues (32) reported that aged fibroblasts sustained higher levels of both double- and single-strand breaks than did young fibroblasts in response to an extremely low frequency-electromagnetic field (ELF-ELM), as measured by neutral and alkaline comet assays. Therefore, it is likely that our data from the older animals do not represent an actual decline in the overall level of DNA single-strand breaks, but rather represent a decline in the level of 3'OH-containing strand breaks.

There are at least two mechanisms by which 3'-blocking lesions can persist as BER intermediates. When BER is initiated by a monofunctional glycosylase such as uracil DNA glycosylase (UDG), APE incises the DNA backbone and creates an appropriate 3'OH terminus. However, when BER is initiated by a bifunctional glycosylase, 3'OH termini are only generated upon additional processing. Bifunctional glycosylases that incise the DNA by ß-elimination (Ogg1, Nth1) generate a 3' phospho-{alpha},ß unsaturated deoxyribose (33,34) that is processed by APE. Bifunctional glycosylases that work through ß,{delta} elimination (Neil, Fpg) generate 3' phosphate residues (35) that are processed by polynucleotide kinase (PNK) (36) or APE. This is exciting, because the loss of APE protein and Ape endo activity in the nucleus could very well explain the reduction in 3'OH-containing DNA strand breaks. That is, we might expect that a loss of APE protein would result in reduced end-trimming by this protein. In support of this notion, we have shown a similar reduction in 3'OH-containing breaks in Ape heterozygous mice in response to both 2-NP and folate deficiency (J. J. Raffoul, D. C. Cabelof, A. R. Heydari, 2005, unpublished observations). It is interesting to consider that the inability to liberate a 3'OH may alter the DNA damage signal.

p53 is a critical component of the DNA damage response pathways and has been shown to play roles in both nucleotide excision repair (37) and BER (17,18,38). Wild-type p53 can stimulate BER activity in vitro (17), and loss of p53 or mutated p53 is accompanied by a complete loss of ß-pol protein and an inability to mount a BER response to methylmethane sulfonate (MMS) (18). In our laboratory, we have focused on the role of gene-targeted disruption of p53 on ß-pol transcript abundance and have found a dose-dependent loss of ß-pol as p53 gene dosage decreases (N. Anyangwe, D. C. Cabelof, A. R. Heydari, 2005, unpublished observations). That is, p53 protein abundance directly alters ß-pol transcript abundance.

Previous studies with various p53 mouse models have suggested a direct role for p53 in the aging process (39). On the basis of these reports, and to begin to understand the mechanism by which ß-pol regulation is altered with age, we looked to p53. Multiple mechanisms regulate p53 activity, including (but not limited to) multiple posttranslational events that are believed to impact p53 protein stability. Ultimately it will be important to investigate the effect of aging on phosphorylation, acetylation, and ubiquitination events in response to stress. Presently, we have investigated only total accumulation of p53 in the nucleus. Notably, there is evidence that p53 activity is dependent on concentration of total p53, not solely on phosphorylation status, and that p53 stabilization and activation can occur in the absence of phosphorylation (40). We demonstrate that accumulation of p53 is altered in response to 2-NP exposure in an age-dependent manner. We not only observed a complete loss of p53 induction in response to 2-NP in aged mice, we observed a statistically significant 16% decline in p53 in the aged animals, a striking contrast with the nearly twofold increased p53 observed in the young animals. As a key player in the regulation of DNA damage responses, this reduction in p53 levels in aged animals exposed to 2-NP is consistent with the loss of ß-pol inducibility observed in aged animals. Furthermore, it suggests that this effect may extend beyond that of BER alone, such that other p53-dependent responses to DNA damage (i.e., cell cycle arrest, nucleotide excision repair, and apoptosis pathways) may also be altered with age.


   Acknowledgments
Top
 Abstract
 Methods
 Results
 Discussion
 References
 
This work was supported by National Institutes of Health (NIH) Grant 1R21-DK62256 (A.R.H.), NIH Grant 1F32-ES013643 (D.C.C.), and NIH/ National Institute of Environmental Health Sciences (NIEHS) Grant ES06639 (D.C.C.) and by a grant from the American Cancer Society (H.V.R.).

We thank Dr. MingJun Liu for his assistance in developing the standards used for real-time RT-PCR.


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Decision Editor: James R. Smith, PhD

Received June 27, 2005

Accepted October 17, 2005


   References
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 Abstract
 Methods
 Results
 Discussion
 References
 

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