Caloric restriction promotes genomic stability by induction of base excision repair and reversal of its age-related decline
Introduction
Caloric restriction (CR) is a powerful experimental tool known to reduce the onset and progression of cancer and to increase mean and maximum life span in laboratory rodents [1], [2]. The underlying mechanism(s) by which CR exerts its anti-tumor and anti-aging effects remain elusive. If CR is ever to become clinically relevant, which seems desirable in light of its well-established benefits, it becomes important to establish the mechanisms by which CR works. Identification of the important anti-tumor or anti-aging targets of CR, which may well be distinct, could eventually provide targets for intervention. One possibility that has experimental support is that CR promotes genomic stability by reducing levels of DNA damage and mutations. To focus on the role of CR in the maintenance and/or promotion of genomic stability is logical as a breakdown in genomic stability is associated with both aging and cancer [1]. However, there are a variety of mechanisms by which genomic integrity can be affected. That is, the level of DNA damage and/or mutations can be affected by changes in carcinogen activation [3], increased detoxification of carcinogens, increased ability to repair DNA, or a combination of these factors.
Over the last two decades, numerous laboratories have addressed the question of whether CR improves genomic stability (for reviews see [4], [5]). As a result, it is well documented that CR reduces the accumulation of nuclear as well as mitochondrial DNA damage [6], [7], [8], [9]. Additionally, CR reduces spontaneous and induced mutation frequency [10], [11]. This protection of genomic integrity by CR suggests a role for DNA repair in the mechanism of CR’s action. In support, studies measuring the rate of unscheduled DNA synthesis (UDS) show higher levels of UDS in CR animals compared to ad libitum-fed animals. [12], [13], [14], [15], [16]. Yet, because UDS neither directly nor selectively measures DNA repair, further investigation into the specifics of how CR affects DNA repair is warranted. Currently, researchers are able to directly investigate specific DNA repair pathways and the expression of key enzymes within these pathways. As such, we have previously demonstrated that CR reduces UV-induced damage in hepatocytes [17], demonstrating that CR enhances nucleotide excision repair (NER) in response to UV exposure. This study addresses the question of base excision repair (BER) capacity in response to CR, both at the basal level and in response to DNA damage. A connection between CR and BER in maintaining genomic stability is possible because CR reduces levels of DNA damage that are processed by the BER pathway. And, having previously determined that BER capacity declines with age, we are interested in determining whether CR will reverse this age-related decline.
Of the varied DNA repair pathways, base excision repair (BER) processes oxidized and alkylated bases, uracil and abasic sites arising in the DNA either endogenously or in response to mutagen exposure. Base modifications that are repaired by the BER pathway are associated with both cancer and aging. Thus, an effect of CR on this pathway could provide some explanation into the life-extending and/or cancer-preventing effects of CR. The BER pathway is a coordinated and sequential series of enzymatic reactions involving removal of a damaged base, incision of the DNA backbone, synthesis of new DNA and removal of the deoxyribose phosphate (dRp) moiety, and ligation, at which point repair is complete. DNA polymerase β (β-pol) is responsible for repair synthesis [18], [19], [20], [21], [22] as well as for dRp removal, the rate-limiting action in the predominant BER pathway [23].
The objective of this research is to determine whether CR affects the capacity of the BER pathway. In this study, we have measured the ability of CR to reverse the previously described age-related decline in BER [24]. We have also measured the effect of CR on β-pol, a rate-limiting enzyme in the BER pathway. In addition to our investigation into the effects of CR in aged animals, we have also measured the effects of CR in young animals with respect to BER capacity and DNA damage protection to determine whether there is a direct up-regulation in BER in response to CR. In light of data [25] demonstrating that many effects of CR are nearly immediate, we were interested in determining whether young animals subjected to CR would experience changes in BER gene expression that might exert a lifelong protective effect. We find that BER and β-pol are significantly upregulated by CR in young animals, supporting the hormesis theory of aging and caloric restriction and potentially signifying β-pol as a stress-response gene. In addition, we are interested in beginning to elucidate whether the life-extending effects of CR and the anti-tumor effects of CR are achieved by distinct mechanisms.
Section snippets
Animals
Male Fischer 344 rats (specific pathogen-free) were obtained at 3 weeks of age from Harlan Sprague–Dawley (Indianapolis, IN). Experiments were performed in young (4–6-month-old) and aged (22–24-month-old) rats in accordance with the NIH guidelines for the use and care of laboratory animals and the animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Texas Health Science Center at San Antonio, the Subcommittee for Animal Studies at Audie L. Murphy
Reversal of age-related decline in BER by caloric restriction
BER capacity declines with age [24], and may help explain the age-related increase in DNA damage and mutations observed by many investigators. Because CR has been shown to cause a reduction in levels of these same types of DNA damage and mutations, we were interested in determining whether CR affected the BER pathway. We have measured the ability of extracts from tissues of CR and ad libitum-fed animals to repair a uracil-containing synthetic oligonucleotide. When the extract initiates repair,
Reversal of 2-NP-induced mutagenesis in young caloric restricted mice
While DMS induces DNA alkylation damage, 2-NP induces a high level of oxidative damage in liver DNA. We have shown that 2-NP increases levels of 8-OHdG four–five-fold and significantly increases mutation frequency [33]. We also found that 2-NP induces β-pol, upregulates BER activity, and is poorly tolerated by β-pol heterozygous knockout mice, demonstrating that the processing of 2-NP-induced damage is accomplished by the BER pathway. Thus, if CR reduces the mutagenicity of 2-NP, we can suggest
Discussion
CR reverses the age-related decline in BER, as well as β-pol activity, protein and mRNA levels. These data are in good agreement with others [34] who have shown that CR enhances total DNA polymerase activity in brain tissue. Our data determine that β-pol is the specific polymerase altered in response to CR. Importantly, β-pol levels have been experimentally determined by us and by others to correlate tightly to BER activity. For example, addition of neutralizing antibody to β-pol in the BER
Acknowledgements
We thank Drs. Samuel H. Wilson and Robert W. Sobol (NIEHS) for their generous gifts of the DNA polymerase β cDNA probe and antibody. This work was supported in part by a grant (AICR 97A113) from American Institute for Cancer Research and a grant (AG14242) from the National Institute on Aging, and by a Pilot Project Program grant from the NIEHS Center grant, ES 06639, Wayne State University. We would like to thank Dr. David Klurfeld for the critical reading of this manuscript.
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- 1
Present address: Department of Radiation Oncology, University of Michigan, Ann Arbor, MI 48109, USA.
- 2
Present address: Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA.