In principle, a decline in base excision repair (BER) efficiency with age should lead to genomic instability and ultimately contribute to the onset of the aging phenotype. Although multiple studies have indicated a negative link between aging and BER, the change of BER efficiency with age in humans has not been systematically analyzed. Here, with foreskin fibroblasts isolated from 19 donors between 20 and 64 y of age, we report a significant decline of BER efficiency with age using a newly developed GFP reactivation assay. We further observed a very strong negative correlation between age and the expression levels of SIRT6, a factor which is known to maintain genomic integrity by improving DNA double strand break (DSB) repair. Our mechanistic study suggests that, similar to the regulatory role that SIRT6 plays in DNA DSB repair, SIRT6 regulates BER in a PARP1-depdendent manner. Moreover, overexpression of SIRT6 rescues the decline of BER in aged fibroblasts. In summary, our results uncovered the regulatory mechanisms of BER by SIRT6, suggesting that SIRT6 reactivation in aging tissues may help delay the process of aging through improving BER.

Failing to repair DNA double-strand breaks by either nonhomologous end joining (NHEJ) or homologous recombination (HR) poses a threat to genome integrity, and may have roles in the onset of aging and age-related diseases. Recent work indicates an age-related decrease of NHEJ efficiency in mouse models, but whether NHEJ and HR change with age in humans and the underlying mechanisms of such a change remain uncharacterized. Here, using 50 eyelid fibroblast cell lines isolated from healthy donors at the age of 16-75 years, we demonstrate that the efficiency and fidelity of NHEJ, and the efficiency of HR decline with age, leading to increased IR sensitivity in cells isolated from old donors. Mechanistic analysis suggests that decreased expression of XRCC4, Lig4 and Lig3 drives the observed, age-associated decline of NHEJ efficiency and fidelity. Restoration of XRCC4 and Lig4 significantly promotes the fidelity and efficiency of NHEJ in aged fibroblasts. In contrast, essential HR-related factors, such as Rad51, do not change in expression level with age, but Rad51 exhibits a slow kinetics of recruitment to DNA damage sites in aged fibroblasts. Further rescue experiments indicate that restoration of XRCC4 and Lig4 may suppress the onset of stress-induced premature cellular senescence, suggesting that improving NHEJ efficiency and fidelity by targeting the NHEJ pathway holds great potential to delay aging and mitigate aging-related pathologies. Cell Death and Differentiation (2016) 23, 1765-1777; doi:10.1038/cdd.2016.65; published online 8 July 2016Aging in mammals is a complex biological process, characterized by several major hallmarks.1,2 Of all the features associated with aging, a gradual destabilization of genome integrity is perhaps the most fundamental as increased genomic instability may lead to other age-associated phenotypes such as cellular senescence and stem cell exhaustion. Indeed, for the past several decades, numerous studies have indicated that DNA mutations and chromosomal rearrangements gradually accumulate with age.3-6 Of all types of DNA lesions, which may contribute to the gradual loss of genetic information during aging, DNA double-strand breaks (DSBs) are the most hazardous to cells as unrepaired or inappropriately repaired DSBs can cause insertions, deletions and chromosomal rearrangements. Using different analysis approaches, several studies have demonstrated that aging is often associated with the accumulation of DNA DSBs in various organs and tissues in mammals such as mice and humans. [7][8][9][10][11] Moreover, a recent study provides direct evidence that an induction of DNA DSBs in genomes causes aging in mouse livers.12 However, why DNA DSBs accumulate with age remains an open question. A number of studies indicate that it may be a consequence of a progressing imbalance between DNA damage and the efficiency of the molecular machinery that catalyzes DNA repair. 7,9,11 Two major pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR) evolved to repair DNA DSBs. NHEJ is...

Aging refers to the physical and functional decline of the tissues over time that often leads to age-related degenerative diseases. Accumulating evidence implicates that the senescence of neural stem cells (NSCs) is of paramount importance to the aging of central neural system (CNS). However, exploration of the underlying molecular mechanisms has been hindered by the lack of proper aging models to allow the mechanistic examination within a reasonable time window. In the present study, we have utilized a hydroxyurea (HU) treatment protocol and effectively induced postnatal subventricle NSCs to undergo cellular senescence as determined by augmented senescence-associated-β-galactosidase (SA-β-gal) staining, decreased proliferation and differentiation capacity, increased G0/G1 cell cycle arrest, elevated reactive oxygen species (ROS) level and diminished apoptosis. These phenotypic changes were accompanied by a significant increase in p16, p21 and p53 expression, as well as a decreased expression of key proteins in various DNA repair pathways such as xrcc2, xrcc3 and ku70. Further proteomic analysis suggests that multiple pathways are involved in the HU-induced NSC senescence, including genes related to DNA damage and repair, mitochondrial dysfunction and the increase of ROS level. Intriguingly, compensatory mechanisms may have also been initiated to interfere with apoptotic signaling pathways and to minimize the cell death by downregulating Bcl2-associated X protein (BAX) expression. Taken together, we have successfully established a cellular model that will be of broad utilities to the molecular exploration of NSC senescence and aging.

To avoid cell cycle arrest or apoptosis, rapidly proliferating cancer cells have to promote DNA double strand break (DSB) repair to fix replication stress induced DSBs. Therefore, developing drugs blocking homologous recombination (HR) and nonhomologous end joining (NHEJ) - 2 major DSB repair pathways - holds great potential for cancer therapy. Over the last few decades, much attention has been paid to explore drugs targeting DSB repair pathways for cancer therapy. Here, using 2 well-established reporters for analyzing HR and NHEJ efficiency, we found that both HR and NHEJ are elevated in hepatoma cell lines Hep3B and HuH7 compared with normal liver cell lines Chang liver and QSG-7701. Our further study found that Harmine, a natural compound, negatively regulates HR but not NHEJ by interfering Rad51 recruitment, resulting in severe cytotoxicity in hepatoma cells. Furthermore, NHEJ inhibitor Nu7441 markedly sensitizes Hep3B cells to the anti-proliferative effects of Harmine. Taken together, our study suggested that Harmine holds great promise as an oncologic drug and combination of Harmine with a NHEJ inhibitor might be an effective strategy for anti-cancer treatment.

The homologous recombination (HR) pathway is a promising target for cancer therapy as it is frequently upregulated in tumors. One such strategy is to target tumors with cancer-specific, hyperactive promoters of HR genes including RAD51 and RAD51C. However, the promoter size and the delivery method have limited its potential clinical applications. Here we identified the ~2.1 kb promoter of XRCC2, similar to ~6.5 kb RAD51 promoter, as also hyperactivated in cancer cells. We found that XRCC2 expression is upregulated in nearly all types of cancers, to a degree comparable to RAD51 while much higher than RAD51C. Further study demonstrated that XRCC2 promoter is hyperactivated in cancer cell lines, and diphtheria toxin A (DTA) gene driven by XRCC2 promoter specifically eliminates cancer cells. Moreover, lentiviral vectors containing XRCC2 promoter driving firefly luciferase or DTA were created and applied to subcutaneous HeLa xenograft mice. We demonstrated that the pXRCC2-luciferase lentivirus is an effective tool for in vivo cancer visualization. Most importantly, pXRCC2-DTA lentivirus significantly inhibited the growth of HeLa xenografts in comparison to the control group. In summary, our results strongly indicate that virus-mediated delivery of constructs built upon the XRCC2 promoter holds great potential for tumor diagnosis and therapy.

The decline in DNA repair capacity contributes to the age-associated decrease in genome integrity in somatic cells of different species. However, due to the lack of clinical samples and appropriate tools for studying DNA repair, whether and how age-associated changes in DNA repair result in a loss of genome integrity of human adult stem cells remains incompletely characterized. Here, we isolated 20 eyelid adipose-derived stem cell (ADSC) lines from healthy individuals (young: 10 donors with ages ranging 17-25 years; old: 10 donors with ages ranging 50-59 years). Using these cell lines, we systematically compared the efficiency of base excision repair (BER) and two DNA double-strand break (DSB) repair pathways-nonhomologous end joining (NHEJ) and homologous recombination (HR)-between the young and old groups.Surprisingly, we found that the efficiency of BER but not NHEJ or HR is impaired in aged human ADSCs, which is in contrast to previous findings that DSB repair declines with age in human fibroblasts. We also demonstrated that BER efficiency is negatively associated with tail moment, which reflects a loss of genome integrity in human ADSCs. Mechanistic studies indicated that at the protein level XRCC1, but not other BER factors, exhibited age-associated decline. Overexpression of XRCC1 reversed the decline of BER efficiency and genome integrity, indicating that XRCC1 is a potential therapeutic target for stabilizing genomes in aged ADSCs. K E Y W O R D Sadipose-derived stem cells, base excision repair, genome integrity, human aging, XRCC1