X-Message-Number: 25559 Date: Sun, 16 Jan 2005 05:25:59 -0800 (PST) From: Doug Skrecky <> Subject: DNA repair and human aging [Keyword to look for: phosphorylated KU 80] Ageing Res Rev. 2004 Apr;3(2):143-51. Role of immune responsiveness and DNA repair capacity genes in ageing. The genetic factors that determine immune responsiveness and DNA repair capacity are reviewed as major elements influencing the life span. Within this framework two sets of new data obtained in mice and humans are reported and discussed. As to the first set, the role of immune response genes was investigated in Biozzi mice genetically selected for high (H) or low (L) antibody response. After 15-20 generations of assortative mating, H and L mice exhibited almost complete intraline homozygosity and interline polymorphism including distinct H-2 haplotypes, such as q in H and s in L mice. In order to analyze the impact of quantitative trait loci (QTL) on the antibody response as well as on the DNA repair capacity in cells of the immune system independently of the selected H-2 haplotype, congenic Hq and Hs as well as Lq and Ls mice were produced and analysed. Both the antibody response and DNA repair capacity were found to be independent of the H-2 haplotype and determined by QTL. As to the second set of data, DNA repair was also studied in irradiated peripheral blood mononuclear cells (PBMC) from ageing humans. The levels of ku 70, ku 80, DNA-PKcs, phosphorylated ku 80 as well as the DNA-binding activity of the ku70/ku 80 heterodimer were determined in the cytoplasmic and nuclear extracts obtained, before and after irradiation, from young and elderly subjects. The results of this study suggest that the decreased DNA repair capacity in PBMC from elderly subjects may be related to impaired migration of the phosphorylated ku 80 from the cytoplasm to the nucleus. This finding helps to elucidate questions related to the impairment of DNA repair during ageing. Mech Ageing Dev. 2003 Apr;124(4):517-24 DNA damage recognition and repair capacities in human naive and memory T cells from peripheral blood of young and elderly subjects. T cells accumulate genetic damage over time but naive cells display higher genomic stability and longer lifespan as compared to memory cells. We found in naive and memory T cells from young and elderly subjects that DNA damage in unirradiated cells is higher in memory than in nai;ve T cells, and is increased by radiation in both cell types. Repair of the radiation-induced DNA damage was much higher in naive than in memory T cells from young subjects but null in both cell types from elderly subjects. Molecular mechanisms involved in DNA damage recognition and repair were analyzed in both cell subsets from young subjects. The intracellular distribution and amount of the DNA-dependent protein kinase (DNA-PK) complex components (ku 70, ku 80, DNA-PKcs), which are involved in the recognition and repair of DNA breaks caused by ionizing radiations, V(D)J recombination and isotype switching, was assessed in naive and memory T cells from young subjects. While the expression of ku 70 and ku 80 was at comparable levels in both T cell subsets, DNA-PKcs, phosphorylated ku 80, and DNA-binding of ku 70/80 were mostly evident in naive but negligible or absent in memory T cells. These findings may account for the higher genomic stability and longer lifespan of naive as compared to memory human T cells from young subjects. Annu Rev Med. 2004 Oct 12; [Epub ahead of print] DNA Repair Defects in Stem Cell Function and Aging. Cellular DNA is under constant challenge by exogenous and endogenous genotoxic stress, which results in both transient and accumulated DNA damage and genomic instability. All cells are equipped with DNA damage response pathways that trigger DNA repair, cell cycle arrest, and, if need be, apoptosis, to eliminate DNA damage or damaged cells. The consequences of these processes for stem cells can be profound: diminution in stem cell pools, or, because of altered gene expression, an increased chance for stem cell differentiation or malignant transformation. Furthermore, a number of DNA repair abnormalities are linked to premature aging syndromes, and these are associated with defects in the stem cell population. The specific DNA repair systems for which there are data regarding the impact of repair defects on stem cell function include O6-alkylguanine DNA alkyltransferase, nucleotide excision repair, base excision repair, mismatch repair, non-homologous DNA end-joining Fanconi's anemia protein complex, and homologous recombination. It has recently become clear that deficiencies of these processes are associated not only with cancer and/or aging but also with stem cell defects. This discovery raises the possibility of a link between aging and stem cell dysfunction. In this review, we provide evidence for a link between DNA repair systems and the maintenance and longevity of stem cells. Proc Natl Acad Sci U S A. 2004 May 18;101(20):7624-9. Epub 2004 Apr 28. DNA end joining becomes less efficient and more error-prone during cellular senescence. Accumulation of somatic mutations is thought to contribute to the aging process. Genomic instability has been shown to increase during aging, suggesting an aberrant function of DNA double-strand break (DSB) repair. Surprisingly, DSB repair has not been examined with respect to cellular senescence. Therefore, we have studied the ability of young, presenescent, and senescent normal human fibroblasts to repair DSBs in transfected DNA by using a fluorescent reporter substrate. We have found that the efficiency of end joining is reduced up to 4.5 fold in presenescent and senescent cells, relative to young cells. Sequence analysis of end junctions showed that the frequency of precise ligation was higher in young cells, whereas end joining in old cells was associated with extended deletions. These results indicate that end joining becomes inefficient and more error-prone during cellular senescence. Furthermore, the ability to use microhomologies for end joining was compromised in senescent cells, suggesting that young and senescent cells may use different end joining pathways. We hypothesize that inefficient and aberrant end joining is a likely mechanism underlying the age-related genomic instability and higher incidence of cancer in the elderly. Exp Gerontol. 2004 Apr;39(4):507-15. Age related microsatellite instability in T cells from healthy individuals. Many immune functions decline with age and may jeopardize the elderly, as illustrated, for example by the significantly higher mortality rate from influenza in old age. Although innate and humoral immunity are affected by aging, it is the T cell compartment, which manifests most alterations. The mechanisms behind these alterations are still unclear, and several explanations have been offered including thymic involution and Telomere attrition leading to cell senescence. Age related accumulation of mutations has been documented and could serve as an additional mechanism of T cell dysfunction. One effective repair mechanism capable of rectifying errors in DNA replications is the mismatch repair (MMR) system. We previously reported a comparative examination of individual DNA samples from blood cells obtained at 10 year intervals from young and old subjects. We showed significantly higher rates of microsatellite instability (MSI), an indicator of MMR dysfunction in older subjects, compared to young. In the present study we confirm this result, using direct automated sequencing and in addition, we demonstrate that as CD8 lymphocytes from aged individuals, undergo repeated population doublings (PDs) in culture, they develop MSI. CD4 clones that also undergo repeated PDs in culture develop significant MSI as well. Elucidation of this previously unexplored facet of lymphocyte dynamics in relation to aging may help identify novel mechanisms of immunosenescence and pathways that could serve as targets for interventions to restore immune function. Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=25559