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Base excision repair but not DNA double-strand break repair is impaired in aged human adipose-derived stem cells.

ABSTRACT: 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.

SUBMITTER: Zhang H

PROVIDER: S-EPMC6996963 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks (ICLs). Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2 inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel candidate biomarkers for Melphalan sensitivity that will be included in targeted quantitation studies in larger patient cohorts to validate their value in prognosis as well as targets for replacement- or adjuvant therapies.

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Base excision repair but not DNA double-strand break repair is impaired in aged human adipose-derived stem cells.

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