Premature Replicative Senescence Research Articles

Perinatal mesenchymal stromal cells of the human decidua restore continence in rats with stress urinary incontinence induced by simulated birth trauma and regulate senescence of fibroblasts from women with stress urinary incontinence.

Stress urinary incontinence (SUI) is a condition that causes the involuntary loss of urine when making small efforts, which seriously affects daily life of people who suffer from it. Women are more affected by this form of incontinence than men, since parity is the main risk factor. Weakening of the pelvic floor tissues is the cause of SUI, although a complete understanding of the cellular and molecular mechanisms of the pathology is still lacking. Reconstructive surgery to strengthen tissue in SUI patients is often associated with complications and/or is ineffective. Mesenchymal stromal cells from the maternal side of the placenta, i.e. the decidua, are proposed here as a therapeutic alternative based on the regenerative potential of mesenchymal cells. The animal model of SUI due to vaginal distention simulating labor has been used, and decidual mesenchymal stromal cell (DMSC) transplantation was effective in preventing a drop in pressure at the leak point in treated animals. Histological analysis of the urethras from DMSC-treated animals after VD showed recovery of the muscle fiber integrity, low or no extracellular matrix (ECM) infiltration and larger elastic fibers near the external urethral sphincter, compared to control animals. Cells isolated from the suburethral connective tissue of SUI patients were characterized as myofibroblasts, based on the expression of several specific genes and proteins, and were shown to achieve premature replicative senescence. Co-culture of SUI myofibroblasts with DMSC via transwell revealed a paracrine interaction between the cells through signals that mediated DMSC migration, SUI myofibroblast proliferation, and modulation of the proinflammatory and ECM-degrading milieu that is characteristic of senescence. In conclusion, DMSC could be an alternative therapeutic option for SUI by counteracting the effects of senescence in damaged pelvic tissue.

Effect of Omega-3 Fatty Acids on Telomeres-Are They the Elixir of Youth?

Telomeres are complexes consisting of tandem repeat DNA combined with associated proteins that play a key role in protecting the ends of chromosomes and maintaining genome stability. They are considered a biological clock, as they shorten in parallel with aging. Furthermore, short telomeres are associated with several age-related diseases. However, the variability in telomere shortening independent of chronological age suggests that it is a modifiable factor. In fact, it is regulated inter alia by genetic damage, cell division, aging, oxidative stress, and inflammation. A key question remains: how can we prevent accelerated telomere attrition and subsequent premature replicative senescence? A number of studies have explored the possible impact of omega-3 fatty acids on telomere shortening. This review summarizes published cross-sectional studies, randomized controlled trials, and rodent studies investigating the role of omega-3 fatty acids in telomere biology. It also covers a broad overview of the mechanism, currently favored in the field, that explains the impact of omega-3 fatty acids on telomeres—the food compound’s ability to modulate oxidative stress and inflammation. Although the results of the studies performed to date are not consistent, the vast majority indicate a beneficial effect of omega-3 fatty acids on telomere length.

Pulmonary Alveolar Stem Cell Senescence, Apoptosis, and Differentiation by p53-Dependent and -Independent Mechanisms in Telomerase-Deficient Mice.

Pulmonary premature ageing and fibrogenesis as in idiopathic pulmonary fibrosis (IPF) occur with the DNA damage response in lungs deficient of telomerase. The molecular mechanism mediating pulmonary alveolar cell fates remains to be investigated. The present study shows that naturally occurring ageing is associated with the DNA damage response (DDR) and activation of the p53 signalling pathway. Telomerase deficiency induced by telomerase RNA component (TERC) knockout (KO) accelerates not only replicative senescence but also altered differentiation and apoptosis of the pulmonary alveolar stem cells (AEC2) in association with increased innate immune natural killer (NK) cells in TERC KO mice. TERC KO results in increased senescence-associated heterochromatin foci (SAHF) marker HP1γ, p21, p16, and apoptosis-associated cleaved caspase-3 in AEC2. However, additional deficiency of the tumour suppressor p53 in the Trp53−/− allele of the late generation of TERC KO mice attenuates the increased senescent and apoptotic markers significantly. Moreover, p53 deficiency has no significant effect on the increased gene expression of T1α (a marker of terminal differentiated AEC1) in AEC2 of the late generation of TERC KO mice. These findings demonstrate that, in natural ageing or premature ageing accelerated by telomere shortening, pulmonary senescence and IPF develop with alveolar stem cell p53-dependent premature replicative senescence, apoptosis, and p53-independent differentiation, resulting in pulmonary senescence-associated low-grade inflammation (SALI). Our studies indicate a natural ageing-associated molecular mechanism of telomerase deficiency-induced telomere DDR and SALI in pulmonary ageing and IPF.

Npl3 stabilizes R-loops at telomeres to prevent accelerated replicative senescence.

Telomere shortening rates must be regulated to prevent premature replicative senescence. TERRA R‐loops become stabilized at critically short telomeres to promote their elongation through homology‐directed repair (HDR), thereby counteracting senescence onset. Using a non‐bias proteomic approach to detect telomere binding factors, we identified Npl3, an RNA‐binding protein previously implicated in multiple RNA biogenesis processes. Using chromatin immunoprecipitation and RNA immunoprecipitation, we demonstrate that Npl3 interacts with TERRA and telomeres. Furthermore, we show that Npl3 associates with telomeres in an R‐loop‐dependent manner, as changes in R‐loop levels, for example, at short telomeres, modulate the recruitment of Npl3 to chromosome ends. Through a series of genetic and biochemical approaches, we reveal that Npl3 binds to TERRA and stabilizes R‐loops at short telomeres, which in turn promotes HDR and prevents premature replicative senescence onset. This may have implications for diseases associated with excessive telomere shortening.

Replicative senescence of hematopoietic cells in patients with idiopathic cytopenia of undetermined significance

We hypothesized that a subset of idiopathic cytopenia of undetermined significance (ICUS) is associated with an increased autonomous proliferation with exhaustion of hematopoiesis. The aim of this study was to investigate the cell turnover rate and replicative history of the bone marrow cells of ICUS patients. To this end, we examined telomere length (TL), proliferation, and apoptosis of the bone marrow cells of ICUS patients and healthy controls (HCs) using telomere quantitative fluorescence in situ hybridization and immunohistochemical staining for Ki-67 and cleaved caspase-3. We also performed targeted sequencing of 88 myeloid-associated genes. A total of 37 patients with ICUS were enrolled in this study, with a median age of 66 years (range: 31–83). TLs were significantly shorter in patients with ICUS than in the HCs (8.8, interquartile range [IQR] 6.8–12.1 vs 18.4, IQR 14.4–22.0, p < 0.0001). Proliferation (Ki-67-positive) and apoptosis (cleaved caspase-3-positive) were significantly increased in patients with ICUS compared to HCs (median = 20.0% vs 5.0%, p = 0.0003; 45.0% vs 22.5%, p = 0.0005, respectively). The shortening of TL and the increased proliferation and apoptotic activity was also prominent in patients with ICUS without mutation and dysplasia than in HCs (p < 0.0001, p < 0.0001, and p = 0.0093, respectively). TL was not associated with mutational profile and clinical characteristics as well in patients with ICUS. To our knowledge, this is the first study to show that ICUS is associated with premature replicative senescence with increased proliferation and apoptosis of bone marrow cells. Further study is needed to address the cause of replicative exhaustion in ICUS patients.

Bmal1-deficient mouse fibroblast cells do not provide premature cellular senescence in vitro

ABSTRACTBmal1 is a core circadian clock gene. Bmal1−/− mice show disruption of the clock and premature aging phenotypes with a short lifespan. However, little is known whether disruption of Bmal1 leads to premature aging at cellular level. Here, we established primary mouse embryonic fibroblast (MEF) cells derived from Bmal1−/− mice and investigated its effects on cellular senescence. Unexpectedly, Bmal1−/− primary MEFs that showed disrupted circadian oscillation underwent neither premature replicative nor stress-induced cellular senescence. Our results therefore uncover that Bmal1 is not required for in vitro cellular senescence, suggesting that circadian clock does not control in vitro cellular senescence.

Triallelic and epigenetic-like inheritance in human disorders of telomerase

AbstractDyskeratosis congenita (DC) and related diseases are a heterogeneous group of disorders characterized by impaired telomere maintenance, known collectively as the telomeropathies. Disease-causing variants have been identified in 10 telomere-related genes including the reverse transcriptase (TERT) and the RNA component (TERC) of the telomerase complex. Variants in TERC and TERT can impede telomere elongation causing stem cells to enter premature replicative senescence and/or apoptosis as telomeres become critically short. This explains the major impact of the disease on highly proliferative tissues such as the bone marrow and skin. However, telomerase variants are not always fully penetrant and in some families disease-causing variants are seen in asymptomatic family members. As a result, determining the pathogenic status of newly identified variants in TERC or TERT can be quite challenging. Over a 3-year period, we have identified 26 telomerase variants (16 of which are novel) in 23 families. Additional investigations (including family segregation and functional studies) enabled these to be categorized into 3 groups: (1) disease-causing (n = 15), (2) uncertain status (n = 6), and (3) bystanders (n = 5). Remarkably, this process has also enabled us to identify families with novel mechanisms of inheriting human telomeropathies. These include triallelic mutations, involving 2 different telomerase genes, and an epigenetic-like inheritance of short telomeres in the absence of a telomerase mutation. This study therefore highlights that telomerase variants have highly variable functional and clinical manifestations and require thorough investigation to assess their pathogenic contribution.

Abstract 1775: High resolution characterization of human hepatocellular cancer (HCC) reveals a novel inactivating mutation in the TGF-β pathway that promotes alcohol induced HCC.

Abstract Background: A crucial factor in protection from liver injury, fibrosis and cancer by agents such as alcohol, aflatoxins and viral hepatitis is the enforcement of genomic stability. However, sensors for genotoxicity leading to aberrant DNA repair remain elusive. Because inactivation of the TGF-β pathway results in DNA damage, alcohol toxicity and HCC, TGF-β has been implicated as a critical promoter of genomic stability and tumor suppression; however, the framework by which this occurs is not known. We performed Whole-genome (WGS) and Exome sequencing (WES) on 6 pairs human HCC samples and identified inactivation of TGF-beta pathway members as a prominent characteristic of alcohol induced HCC, potentially through inactivation of a TGF-b adaptor, β2SP. We therefore hypothesized that the TGF-β/β2SP/Smad3 pathway is crucial for protection against aldehyde genotoxicity through enforcing genomic stability. Materials &amp; Methods: We performed Whole-genome (WGS) and Exome sequencing (WES) on 6 pairs human HCC samples. β2SP mutant mice were treated with alcohol to determine their susceptibility to aldehyde-induced developmental abnormalities. Primary MEFs from β2SP+/+, β2SP+/− and β2SP−/− mice were treated with colcemid for cytogenetic analysis, and they were also treated with multiple DNA damaging agents and γ-irradiation to evaluate Mdc1, NBS1 and Rad51 foci formation. ChIP assays were performed to determine the recruitment of β2SP/Smad3/Smad4 at FancD2 promoter. Results: (1) Through our WGS and WES analyses, we discovered a novel inactivating mutation of TGF-β/β2SP pathway in alcohol-associated HCC. This β2SP mutation results in functional disruption of TGF-β signaling. (2) Alcohol treatment in the absence of β2SP mice results in teratogenicity and spontaneous fetal alcohol-like phenotype (FAS). (3) Loss of β2SP results in premature replicative senescence and marked hypersensitivity to DNA interstrand crosslinking agents, such as mitomycin C (MMC). (4) DNA damage response induces nuclear localization of β2SP in a TGF-β-dependent manner. (5) Suppression of β2SP impairs the recruitment of DNA repair proteins in the nucleus and consequently the repair of DNA double-strand breaks. (6) Loss of β2SP correlates with loss of FANCD2 expression, but not of any of the other 12 Fanconi anemia complementation factors. Moreover, our results indicate that β2SP/Smad3/Smad4 regulate expression of FANCD2 at the transcriptional level. (7) The expression of β2SP and FANCD2 is correlated in alcoholic hepatitis and cirrhotic livers. Conclusions: Through whole genome and Exome sequencing we found a novel inactivating mutation in β2SP which results in loss of TGF-β signaling pathway. Importantly, these results could potentially lead to new therapeutics targeting toxin-induced DNA damage and tumorigenesis. Citation Format: Vivek Shukla, Jian Chen, Jiun-Sheng Chen, Ying Li, Lior Katz, Avijit Majumdar, Lei Li, Walter Hittleman, Xiaoping Su, Junjie Chen, Xifeng Wu, Patrizia Farci, Lopa Mishra. High resolution characterization of human hepatocellular cancer (HCC) reveals a novel inactivating mutation in the TGF-β pathway that promotes alcohol induced HCC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1775. doi:10.1158/1538-7445.AM2013-1775

Insufficient stromal support in MDS results from molecular and functional deficits of mesenchymal stromal cells

Ineffective hematopoiesis is a major characteristic of myelodysplastic syndromes (MDS) causing relevant morbidity and mortality. Mesenchymal stromal cells (MSC) have been shown to physiologically support hematopoiesis, but their contribution to the pathogenesis of MDS remains elusive. We show that MSC from patients across all MDS subtypes (n=106) exhibit significantly reduced growth and proliferative capacities accompanied by premature replicative senescence. Osteogenic differentiation was significantly reduced in MDS-derived MSC, indicated by cytochemical stainings and reduced expressions of Osterix and Osteocalcin. This was associated with specific methylation patterns that clearly separated MDS-MSC from healthy controls and showed a strong enrichment for biological processes associated with cellular phenotypes and transcriptional regulation. Furthermore, in MDS-MSC, we detected altered expression of key molecules involved in the interaction with hematopoietic stem and progenitor cells (HSPC), in particular Osteopontin, Jagged1, Kit-ligand and Angiopoietin as well as several chemokines. Functionally, this translated into a significantly diminished ability of MDS-derived MSC to support CD34+ HSPC in long-term culture-initiating cell assays associated with a reduced cell cycle activity. Taken together, our comprehensive analysis shows that MSC from all MDS subtypes are structurally, epigenetically and functionally altered, which leads to impaired stromal support and seems to contribute to deficient hematopoiesis in MDS.

Abstract 4695: TGF-β enforces genomic instability in liver cancers

Abstract Background: Over 90% of liver cancers, from viral hepatitis to alcohol injury, occur against a background of chronic liver disease and cirrhosis. Genomic stability, G1/S cell cycle arrest through c-Myc and CDK suppression, have been some of the mechanisms proposed in tumor suppression by TGF-β. The Smad3/4 adaptor protein, β2SP, is emerging as potent regulator of tumor genesis by its ability to affect TGF-β tumor suppressor function. Deletion of β2SP results in a dramatic and spontaneous formation of liver cancer and gastrointestinal cancers. Moreover, β2SP+/− and β2SP+/− /Smad3+/− mutant mice phenocopy human Beckwith-Wiedemann syndrome (BWS), a hereditary human cancer stem cell syndrome imprinting disorder with loss of p57, increase in IGF2, and an association with 800 fold increase of cancers that include those of the liver. Delayed liver regeneration and extensive DNA damage was observed in β2SP heterozygote mice. In addition, spectrins have been observed to associate with FANC G and D, DNA interstrand cross links, and Smad3/4 to transcriptionally regulate FANC genes. Hypothesis: We therefore hypothesised that TGF-β is a crucial enforcer of genomic stability, and that β2SP and/or Smad3/4 are key mediators in suppressing liver injury and cancer through modulation of the DNA repair pathway. Materials &amp; Methods: To determine the role of β2SP and Smad3 in the DNA damage response, and whether β2SP and β2SP/Smad3 loss plays a causal role in the downregulation of the Fanc/Brca pathway, we used our mouse models as well as the mouse embryonic fibroblasts. Oxidative DNA damage was induced by H2O2 treatment and assayed tail length analysis. Irradiation was induced by 8Gy IR and DNA was studied by immunofluorescence of DNA damage sensors H2AX, 532BP1, and pChk2. DNA repair was assayed by Mdc1, NBS1, and Rad51 foci formation at different time points after irradiation. Results: β2SP+/− and β2SP+/−/Smad3+/− mice exhibited an increased prevalence of HCC and GI cancer and many phenotypic characteristics observed in BWS patients. Loss of β2SP results in premature replicative senescence with spontaneous DNA damage. β2SP deficiency results in increased sensitivity to exogenous DNA damage and insufficient DNA repair. Depletion of TGF-β adaptor protein β2SP impairs the loading of repair proteins and repair of DNA double-strand breaks. Loss of FancD2 expression in HCC and GI cancer cell lines correlates with loss of β2SP. Moreover, β2SP−/− and β2SP+/−/Smad3+/− cell lines accumulate DNA damage that is further exacerbated by DNA cross-linking agents such as mitomycin C similar to P57 null cells and Fanconi anemia cell lines. Conclusions: TGF-β confers genomic stability through regulation of DNA impairment of the Fanc/Brca DNA damage response and interstrand link repair and inactivation of TGF- ≤ pathway results in alcohol toxicity, cirrhosis and liver cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4695. doi:1538-7445.AM2012-4695

The knock-down of ERCC1 but not of XPF causes multinucleation

Excision repair cross complementing gene 1 (ERCC1) associated with xeroderma pigmentosum group F (XPF) is a heterodimeric endonuclease historically involved in the excision of bulky helix-distorting DNA lesions during nucleotide excision repair (NER) but also in the repair of DNA interstrand crosslinks. ERCC1 deficient mice show severe growth retardation associated with premature replicative senescence leading to liver failure and death at four weeks of age. In humans, ERCC1 is overexpressed in hepatocellular carcinoma and in the late G1 phase of hepatocyte cell cycle. To investigate whether ERCC1 could be involved in human hepatocyte cell growth and cell cycle progression, we knocked-down ERCC1 expression in the human hepatocellular carcinoma cell line Huh7 by RNA interference. ERCC1 knocked-down cells were delayed in their cell cycle and became multinucleated. This phenotype was rescued by ERCC1 overexpression. Multinucleation was not liver specific since it also occurred in HeLa and in human fibroblasts knocked-down for ERCC1. Multinucleated cells arose after drastic defects leading to flawed metaphase and cytokinesis. Interestingly, multinucleation did not appear after knocking-down other NER enzymes such as XPC and XPF, suggesting that NER deficiency was not responsible for multinucleation. Moreover, XPF mutant human fibroblasts formed multinucleated cells after ERCC1 knock-down but not after XPF knock-down. Therefore our results seem consistent with ERCC1 being involved in multinucleation but not XPF. This work reveals a new role for ERCC1 distinct from its known function in DNA repair, which may be independent of XPF. The role for ERCC1 in mitotic progression may be critical during development, particularly in humans.

Genetic Demonstration of a Redundant Role of Extracellular Signal-Regulated Kinase 1 (ERK1) and ERK2 Mitogen-Activated Protein Kinases in Promoting Fibroblast Proliferation

The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinase signaling pathway plays an important role in the proliferative response of mammalian cells to mitogens. However, the individual contribution of the isoforms ERK1 and ERK2 to cell proliferation control is unclear. The two ERK isoforms have similar biochemical properties and recognize the same primary sequence determinants on substrates. On the other hand, analysis of mice lacking individual ERK genes suggests that ERK1 and ERK2 may have evolved unique functions. In this study, we used a robust genetic approach to analyze the individual functions of ERK1 and ERK2 in cell proliferation using genetically matched primary embryonic fibroblasts. We show that individual loss of either ERK1 or ERK2 slows down the proliferation rate of fibroblasts to an extent reflecting the expression level of the kinase. Moreover, RNA interference-mediated silencing of ERK1 or ERK2 expression in cells genetically disrupted for the other isoform similarly reduces cell proliferation. We generated fibroblasts genetically deficient in both Erk1 and Erk2. Combined loss of ERK1 and ERK2 resulted in a complete arrest of cell proliferation associated with G(1) arrest and premature replicative senescence. Together, our findings provide compelling genetic evidence for a redundant role of ERK1 and ERK2 in promoting cell proliferation.

Progeria, the nucleolus and farnesyltransferase inhibitors

HGPS (Hutchinson-Gilford progeria syndrome) is a rare genetic disease affecting children causing them to age and die prematurely. The disease is typically due to a point mutation in the coding sequence for the nuclear intermediate-type filament protein lamin A and gives rise to a dominant-negative splice variant named progerin. Accumulation of progerin within nuclei causes disruption to nuclear structure, causes and premature replicative senescence and increases apoptosis. Now it appears that accumulation of progerin may have more widespread effects than previously thought since the demonstration that the presence and distribution of some nucleolar proteins are also adversely affected in progeria cells. One of the major breakthroughs both in the lamin field and for this syndrome is that many of the cellular defects observed in HGPS patient cells and model systems can be restored after treatment with a class of compounds known as FTIs (farnesyltransferase inhibitors). Indeed, it is demonstrated that FTI-277 is able to completely restore nucleolar antigen localization in treated progeria cells. This is encouraging news for the HGPS patients who are currently undergoing clinical trials with FTI treatment.

Pathogenetic Role of Impaired Telomere Repair in Myelodysplastic Syndrome.

Abstract 2777Poster Board II-753Telomeres are repeated nucleotide sequences at the ends of chromosomes that serve to preserve genetic integrity in hematopoietic progenitors (HPs). Abnormal telomere maintenance resulting from mutations in telomerase or other telomere repair genes have an established pathogenetic role in a subset of acquired and congenital forms of bone marrow failure (BMF). Excess telomere shortening of myeloid elements was reported previously in MDS, however it is unclear whether such abnormalities in telomere maintenance play a pathogenetic role in disease susceptibility or represent a disease specific phenotype arising from accelerated cellular proliferation of the myeloid compartment. Alterations in telomere maintenance and telomerase activity in T cells may offer insight into genetic susceptibility since these cells are not derived from the malignant clone. We examined telomere length and replication history in neutrophils and T cells in newly diagnosed MDS cases (n=66) and compared results to unrelated, age-matched controls (n=63) lacking a prior history of cancer. Telomere length in peripheral blood mononuclear cells was assessed by quantitative real-time PCR (qRT-PCR) and found to be significantly shorter among MDS cases compared to controls. Peripheral blood concentration of the senescence protein stathmin was analyzed in a subset of 48 patients and 48 controls and found to be higher in MDS cases compared to controls after adjustment for age and sex (mean 4.24 pg/ml ± 7.25 in cases vs mean 2.45 pg/ml ± 2.84 in controls, p=0.06) with no change observed with age. Mechanisms of cellular senescence and telomere attrition in T-cells may be related to antigen-driven or homeostatic proliferation. Therefore, telomere length was compared to an independent genetic marker of proliferation, i.e., the concentration of T cell receptor excision circles (TRECs). TRECs are episomal DNA fragments excised during TCR gene rearrangement within the thymus that do not transfer to daughter cells. Therefore, TREC DNA copy number has been shown to be diluted during each round of division and tends to be reduced with age due to impaired generation of new thymic T-cell emigrants. In controls, TREC concentration declined with age consistent with cellular proliferation and loss of thymic function. For every year increase in age, log TREC values decreased by 0.05 DNA copies (p=0.0012). More aggressive proliferation was evident in T cells from cases compared to controls with a 2-fold more rapid decline in TREC copy number each year (0.099 unit decrease in TREC copies per year among cases, p<0.0001; 0.05 copies per year among controls, p=0.012). Changes in TREC levels with age were compared to corresponding changes in telomere length. In cases, TRECs and telomere length in CD4+ and CD8+ T cells tended to be positively correlated suggesting that the rate of telomere repair may be insufficient for the degree of proliferation (CD4: r=0.59, p=0.06 CD8: r=0.50, p-value=0.11). Among controls, no statistically significant associations were observed between age and either CD4 or CD8 telomere length. These results suggest that telomere repair is activated to preserve the proliferative capacity of peripheral T-cells and prolong survival after thymic involution. To determine if T cell proliferative function is altered, antigen-induced proliferation was compared among MDS patients and controls. After stimulation of the T cell receptor, proliferation was reduced in cases compared to controls, supporting pre-mature replicative senescence in MDS. These results suggest that altered T cell function in MDS may arise from accelerated age-related telomere erosion arising from an impairment in telomere repair and high rates of replicative stress. This defect in lymphoid cells may represent a genetic marker of disease susceptibility which merits prospective investigation. Disclosures:No relevant conflicts of interest to declare.

SCID Dogs: Similar Transplant Potential but Distinct Intra-Uterine Growth Defects and Premature Replicative Senescence Compared with SCID Mice

We have previously described DNA-dependent protein kinase (DNA-PKcs) mutations in horses and dogs that result in deficits in V(D)J recombination, DNA repair, and SCID. In this paper, we document substantial developmental growth defects in DNA-PKcs-deficient dogs that are not apparent in SCID mice. Fibroblast cell strains derived from either fetal or adult SCID dogs proliferate poorly in culture and undergo premature replicative senescence, somewhat reminiscent of cells derived from Ku-deficient mice. A limited number of animals have been immune reconstituted (by bone marrow transplantation) so that they can be maintained in a normal environment for long periods. Several of these animals have developed conditions associated with premature ageing at 2-3 years of age, roughly 20% of their expected lifespan. These conditions include intestinal malabsorption and primary neural cell neoplasia. These results suggest that DNA-PKcs deficiency is not tolerated equally in all species, perhaps providing insight into why DNA-PKcs deficiency has not been observed in humans. Finally, this study demonstrates the feasibility of maintaining SCID dogs for extended periods of time and documents their utility for bone marrow transplantation studies and as hosts for the propagation of xenografts. In sum, SCID dogs may present researchers with new possibilities for the development of animal models of human disease.

Cell divisions and mammalian aging: integrative biology insights from genes that regulate longevity

Despite recent progress in the identification of genes that regulate longevity, aging remains a mysterious process. One influential hypothesis is the idea that the potential for cell division and replacement are important factors in aging. In this work, we review and discuss this perspective in the context of interventions in mammals that appear to accelerate or retard aging. Rather than focus on molecular mechanisms, we interpret results from an integrative biology perspective of how gene products affect cellular functions, which in turn impact on tissues and organisms. We review evidence suggesting that mutations that give rise to features resembling premature aging tend to be associated with cellular phenotypes such as increased apoptosis or premature replicative senescence. In contrast, many interventions in mice that extend lifespan and might delay aging, including caloric restriction, tend to either hinder apoptosis or result in smaller animals and thus may be the product of fewer cell divisions. Therefore, it appears plausible that changes in the number of times that cells, and particularly stem cells, divide during an organism's lifespan influence longevity and aging. We discuss possible mechanisms related to this hypothesis and propose experimental paradigms.

Regulation of WRN Protein Cellular Localization and Enzymatic Activities by SIRT1-mediated Deacetylation

Werner syndrome is an autosomal recessive disorder associated with premature aging and cancer predisposition caused by mutations of the WRN gene. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Sir2, an NAD-dependent histone deacetylase, has been proven to extend life span in yeast and Caenorhabditis elegans. Mammalian Sir2 (SIRT1) has also been found to regulate premature cellular senescence induced by the tumor suppressors PML and p53. SIRT1 plays an important role in cell survival promoted by calorie restriction. Here we show that SIRT1 interacts with WRN both in vitro and in vivo; this interaction is enhanced after DNA damage. WRN can be acetylated by acetyltransferase CBP/p300, and SIRT1 can deacetylate WRN both in vitro and in vivo. WRN acetylation decreases its helicase and exonuclease activities, and SIRT1 can reverse this effect. WRN acetylation alters its nuclear distribution. Down-regulation of SIRT1 reduces WRN translocation from nucleoplasm to nucleoli after DNA damage. These results suggest that SIRT1 regulates WRN-mediated cellular responses to DNA damage through deacetylation of WRN.

Angiotensin II–Mediated Oxidative DNA Damage Accelerates Cellular Senescence in Cultured Human Vascular Smooth Muscle Cells via Telomere-Dependent and Independent Pathways

Angiotensin II (Ang II) induces reactive oxygen species (ROS) production by human vascular smooth muscle cells (hVSMCs). ROS have been implicated in the development of both acute stress-induced premature senescence (SIPS) and chronic replicative senescence. Global oxidative DNA damage triggers SIPS and telomere DNA damage accelerates replicative senescence, both mediated via p53. This study tests the hypothesis that DNA is an important target for Ang II-induced ROS leading to senescence via telomere-dependent and independent pathways. DNA damage was quantified using the Comet assay, telomere DNA length by Southern blotting and hVSMC senescence by senescence-associated beta-galactosidase staining. Exposure to Ang II increased DNA damage in hVSMCs within 4 hours. Inhibition by an AT1 receptor antagonist (losartan metabolite: E3174) or catalase, confirmed that Ang II-induced DNA damage was AT1 receptor-mediated, via the induction of ROS. Acute exposure to Ang II resulted in SIPS within 24 hours that was prevented by coincubation with E3174 or catalase. SIPS was associated with increased p53 expression but was not dependent on telomere attrition because overexpression of human telomerase did not prevent Ang II-induced SIPS. Exposure to Ang II over several population doublings accelerated the rate of telomere attrition (by >2-fold) and induced premature replicative senescence of hVSMCs--an effect that was also attenuated by E3174 or catalase. These data demonstrate that Ang II-induced ROS-mediated DNA damage results in accelerated biological aging of hVSMCs via 2 mechanisms: (1) Acute SIPS, which is telomere independent, and (2) accelerated replicative senescence which is associated with accelerated telomere attrition.

Glioma-associated endothelial cells show evidence of replicative senescence

The innately programmed process of replicative senescence has been studied extensively with respect to cancer, but primarily from the perspective of tumor cells overcoming this stringent innate barrier and acquiring the capacity for unlimited proliferation. In this study, we focus on the potential role of replicative senescence affecting the non-transformed endothelial cells of the blood vessels within the tumor microenvironment. Based on the well-documented aberrant structural and functional features of blood vessels within solid tumors, we hypothesized that tumor-derived factors may lead to premature replicative senescence in tumor-associated brain endothelial cells (TuBEC). We show here that glioma tissue, but not normal brain tissue, contains cells that express the signature of replicative senescence, senescence-associated β-galactosidase (SA-β-gal), on CD31-positive endothelial cells. Primary cultures of human TuBEC stain for SA-β-gal and exhibit characteristics of replicative senescence, including increased levels of the cell cycle inhibitors p21 and p27, increased resistance to cytotoxic drugs, increased growth factor production, and inability to proliferate. These data provide the first demonstration that tumor-derived brain endothelial cells may have reached an end-stage of differentiation known as replicative senescence and underscore the need for anti-angiogenic therapies to target this unique tumor-associated endothelial cell population.

Role of telomeres and the senescence response in cancer and aging

Telomeres are protein-DNA structures that cap linear chromosomes, protecting them from degradation and fusion by the cellular DNA repair machinery. Most normal human cells lose telomeric DNA with each round of replication, eventually compromising the protective cap structure and triggering the permanent cell cycle arrest termed cellular senescence. The senescence response is a crucial for tumor suppression because it arrests the proliferation of cells at risk for neoplastic transformation, including the genomic instability caused by dysfunctional telomeres. The senescent phenotype entails myriad changes in gene expression, including the upregulation of several genes encoding secreted factors that can alter the local tissue microenvironment. In characterizing this senescent secretory phenotype, we found that senescent stromal cells secret high levels of matrix metalloproteinases, inflammatory cytokines and growth factors. Using two and three dimensional culture models, we found that senescent stromal cells can disrupt the morphological and functional differentiation of normal epithelial cells and promote aggressive phenotypes in premalignant and malignant epithelial cells. These findings suggest that the accumulation of senescent cells can promote aging phenotypes, including late-life cancer. One example of how dysfunctional telomeres and the ensuing senescent response can drive aging and cancer phenotypes is the Werner syndrome, a premature aging syndrome in humans. Werner syndrome is caused by a deficiency in a RECQ-like DNA helicase, which associates with telomeres during S phase and causes premature replicative senescence when deficient.