Dietary Restriction Ameliorates Age-Related Increase in DNA Damage, Senescence and Inflammation in Mouse Adipose Tissuey.

Ageing is associated with redistribution of fat around the body and saturation of visceral adipose depots. Likewise, the presence of excess fat in obesity or during ageing places extra stress on visceral depots, resulting in chronic inflammation and increased senescence. This process can contribute to the establishment of the metabolic syndrome and accelerated ageing. Dietary restriction (DR) is known to alleviate physiological signs of inflammation, ageing and senescence in various tissues including adipose tissue. OBJECTIVES: Our pilot study aimed to analyse senescence and inflammation parameters in mouse visceral fat tissue during ageing and by short term, late-onset dietary restriction as a nutritional intervention. Design, measurements: In this study we used visceral adipose tissue from mice between 5 and 30 months of age and analysed markers of senescence (adipocyte size, γH2A.X, p16, p21) and inflammation (e.g. IL-6, TNFα, IL-1ß, macrophage infiltration) using immuno-staining, as well as qPCR for gene expression analysis. Fat tissues from 3 mice per group were analysed. RESULTS: We found that the amount of γH2A.X foci as well as the expression of senescence and inflammation markers increased during ageing but decreased with short term DR. In contrast, the increase in amounts of single or aggregated macrophages in fat depots occurred only at higher ages. Surprisingly, we also found that adipocyte size as well as some senescence parameters decreased at very high age (30 months). CONCLUSIONS: Our results demonstrate increased senescence and inflammation during ageing in mouse visceral fat while DR was able to ameliorate several of these parameters as well as increased adipocyte size at 17.5 months of age. This highlights the health benefits of a decreased nutritional intake over a relatively short period of time at middle age.

Short telomere length is associated with impaired cognitive performance in European ancestry cohorts.

The association between telomere length (TL) dynamics on cognitive performance over the life-course is not well understood. This study meta-analyses observational and causal associations between TL and six cognitive traits, with stratifications on APOE genotype, in a Mendelian Randomization (MR) framework. Twelve European cohorts (N=17 052; mean age=59.2±8.8 years) provided results for associations between qPCR-measured TL (T/S-ratio scale) and general cognitive function, mini-mental state exam (MMSE), processing speed by digit symbol substitution test (DSST), visuospatial functioning, memory and executive functioning (STROOP). In addition, a genetic risk score (GRS) for TL including seven known genetic variants for TL was calculated, and used in associations with cognitive traits as outcomes in all cohorts. Observational analyses showed that longer telomeres were associated with better scores on DSST (ß=0.051 per s.d.-increase of TL; 95% confidence interval (CI): 0.024, 0.077; P=0.0002), and MMSE (ß=0.025; 95% CI: 0.002, 0.047; P=0.03), and faster STROOP (ß=-0.053; 95% CI: -0.087, -0.018; P=0.003). Effects for DSST were stronger in APOE ɛ4 non-carriers (ß=0.081; 95% CI: 0.045, 0.117; P=1.0 × 10-5), whereas carriers performed better in STROOP (ß=-0.074; 95% CI: -0.140, -0.009; P=0.03). Causal associations were found for STROOP only (ß=-0.598 per s.d.-increase of TL; 95% CI: -1.125, -0.072; P=0.026), with a larger effect in ɛ4-carriers (ß=-0.699; 95% CI: -1.330, -0.069; P=0.03). Two-sample replication analyses using CHARGE summary statistics showed causal effects between TL and general cognitive function and DSST, but not with STROOP. In conclusion, we suggest causal effects from longer TL on better cognitive performance, where APOE ɛ4-carriers might be at differential risk.

Serum 25-hydroxyvitamin D and cognitive decline in the very old: the Newcastle 85+ Study.

BACKGROUND AND PURPOSE: Studies investigating the association between 25-hydroxyvitamin D [25(OH)D] and cognition in the very old (85+) are lacking. METHODS: Cross-sectional (baseline) and prospective data (up to 3 years follow-up) from 775 participants in the Newcastle 85+ Study were analysed for global (measured by the Standardized Mini-Mental State Examination) and attention-specific (measured by the attention battery of the Cognitive Drug Research test) cognitive performance in relation to season-specific 25(OH)D quartiles. RESULTS: Those in the lowest and highest season-specific 25(OH)D quartiles had an increased risk of impaired prevalent (1.66, 95% confidence interval 1.06-2.60, P = 0.03; 1.62, 95% confidence interval 1.02-2.59, P = 0.04, respectively) but not incident global cognitive functioning or decline in functioning compared with those in the middle quartiles adjusted for sociodemographic, health and lifestyle confounders. Random effects models showed that participants belonging to the lowest and highest 25(OH)D quartiles, compared with those in the middle quartiles, had overall slower (log-transformed) attention reaction times for Choice Reaction Time (lowest, ß = 0.023, P = 0.01; highest, ß = 0.021, P = 0.02), Digit Vigilance Task (lowest, ß = 0.009, P = 0.05; highest, ß = 0.01, P = 0.02) and Power of Attention (lowest, ß = 0.017, P = 0.02; highest, ß = 0.022, P = 0.002) and greater Reaction Time Variability (lowest, ß = 0.021, P = 0.02; highest, ß = 0.02, P = 0.03). The increased risk of worse global cognition and attention amongst those in the highest quartile was not observed in non-users of vitamin D supplements/medication. CONCLUSION: Low and high season-specific 25(OH)D quartiles were associated with prevalent cognitive impairment and poorer overall performance in attention-specific tasks over 3 years in the very old, but not with global cognitive decline or incident impairment.

25-hydroxyvitamin D and increased all-cause mortality in very old women: the Newcastle 85+ study.

OBJECTIVE: To investigate the associations between low and high concentrations of baseline serum 25-hydroxyvitamin D [25(OH)D] and all-cause mortality in very old (≥85 years) men and women over 6 years. DESIGN, SETTING AND SUBJECTS: Prospective mortality data from 775 participants in the Newcastle 85+ Study were analysed for survival in relation to 25(OH)D (season-specific quartiles and predefined cut-off values) and sex using Cox proportional hazards models. The models were fitted to the entire and restricted (nonusers of vitamin D-containing supplements and medication) cohorts. RESULTS: For the entire cohort, mortality was higher in both the lowest and highest 25(OH)D season-specific quartiles [SQ1: hazard ratio (HR) 1.31, 95% confidence interval (CI) 1.01-1.69, P = 0.04; SQ4: HR 1.44, 95% CI 1.12-1.85, P = 0.004] compared with the combined middle quartiles (SQ2 + SQ3), after adjustment for sociodemographic factors. The increased risk for the highest quartile remained significant after further adjustment for lifestyle variables (SQ4: HR 1.37, 95% CI 1.06-1.77, P = 0.02) and was seen only in women in sex-specific analyses. Similarly, in sensitivity analyses with predefined 25(OH)D cut-off values, the highest 25(OH)D concentration (≥75 nmol L(-1) ) was associated with a 2.4-fold increased risk of mortality in women (restricted cohort) after adjusting for all covariates. CONCLUSION: Low and high season-specific 25(OH)D quartiles were associated with increased risks of mortality over 6 years in the very old; this effect was particularly noticeable in women, including those who reported taking vitamin D-containing supplements/medication.

SSEA-1 isolates human endometrial basal glandular epithelial cells: phenotypic and functional characterization and implications in the pathogenesis of endometriosis.

STUDY QUESTION: Can the basal epithelial compartment of the human endometrium be defined by specific markers? SUMMARY ANSWER: Human endometrial epithelial cells from the basalis express nuclear SOX9 and the cell-surface marker SSEA-1, with some cells expressing nuclear ß-catenin. In vitro, primary endometrial epithelial cells enriched for SSEA-1+ show some features expected of the basalis epithelium. WHAT IS KNOWN ALREADY: The endometrial glands of the functionalis regenerate from the basalis gland stumps following menstruation. Endometriosis is thought to originate from abnormal dislocation of the basalis endometrium. In the highly regenerative intestinal epithelium, SOX9 and nuclear ß-catenin are more highly expressed in the intestinal crypt, the stem/progenitor cell region. STUDY DESIGN, SIZE, DURATION: A large prospective observational study analysing full-thickness human endometrial hysterectomy samples from 115 premenopausal women, 15 post-menopausal women and ectopic endometriotic lesions from 20 women with endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Full-thickness endometrium from hysterectomy tissues was analysed by immunohistochemistry for SSEA-1, SOX9 and ß-catenin. Primary human endometrial epithelial cells from short-term cultures were sorted into SSEA1+/- fractions with a cell sorter or magnetic beads and analysed for markers of differentiation and pluripotency and telomere lengths (TLs) using qPCR, telomerase activity [telomere repeat amplification protocol (TRAP)] and growth in 3D culture. MAIN RESULTS AND THE ROLE OF CHANCE: Similar to the intestinal crypt epithelium, human endometrial basal glandular epithelial cells expressed nuclear SOX9 and contained a rare subpopulation of cells with nuclear ß-catenin suggestive of an activated Wnt pathway. The embryonic stem cell-surface marker, SSEA-1, also marked the human endometrial basal glandular epithelial cells, and isolated SSEA-1(+) epithelial cells grown in monolayer showed significantly higher expression of telomerase activity, longer mean TLs, lower expression of genes for steroid receptors and produced a significantly higher number of endometrial gland-like spheroids in 3D culture compared with SSEA-1(-) epithelial cells (P = 0.009). Cells in ectopic endometriosis lesions also expressed SSEA-1 and nuclear SOX9, suggesting that the basalis contributes to ectopic lesion formation in endometriosis following retrograde menstruation. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive study with only short-term culture of the primary human epithelial cells in vitro. WIDER IMPLICATIONS OF THE FINDINGS: The surface marker SSEA1 enriches for an endometrial epithelial cell subpopulation from the basalis. Since the functional endometrium originates from these cells, it is now possible to study basalis epithelium for stem/progenitor cell activity to extend our current understanding of endometrial biology in health and diseases. STUDY FUNDING/COMPETING INTEREST(S): The work included in this manuscript was funded by Wellbeing of Women project grant RG1073 (D.K.H. and C.G.). We also acknowledge the support by National Health and Medical Research Council, RD Wright Career Development Award 465121 and Senior Research Fellowship 1042298, and the Victorian Government's Operation Infrastructure Support Program to C.G. and MRC G0601333 to T.V.Z. All authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Biliary epithelial senescence and plasticity in acute cellular rejection.

Biliary epithelial cells (BEC) are important targets in some liver diseases, including acute allograft rejection. Although some injured BEC die, many can survive in function compromised states of senescence or phenotypic de-differentiation. This study was performed to examine changes in the phenotype of BEC during acute liver allograft rejection and the mechanism driving these changes. Liver allograft sections showed a positive correlation (p < 0.0013) between increasing T cell mediated acute rejection and the number of BEC expressing the senescence marker p21(WAF1/Cip) or the mesenchymal marker S100A4. This was modeled in vitro by examination of primary or immortalized BEC after acute oxidative stress. During the first 48 h, the expression of p21(WAF1/Cip) was increased transiently before returning to baseline. After this time BEC showed increased expression of mesenchymal proteins with a decrease in epithelial markers. Analysis of TGF-ß expression at mRNA and protein levels also showed a rapid increase in TGF-ß2 (p < 0.006) following oxidative stress. The epithelial de-differentiation observed in vitro was abrogated by pharmacological blockade of the ALK-5 component of the TGF-ß receptor. These data suggest that stress induced production of TGF-ß2 by BEC can modify liver allograft function by enhancing the de-differentiation of local epithelial cells.

A life course approach to healthy ageing: the HALCyon programme.

Healthy ageing across the life course (HALCyon) is an interdisciplinary research collaboration that harnesses the power of nine UK cohort studies to discover life course influences on physical and cognitive capability, social and psychological well-being, and underlying biology. In this symposium, HALCyon co-investigators reported the first wave of findings from five of the eight work packages.

Sustained replication in endometrium of women with endometriosis occurs without evoking a DNA damage response.

BACKGROUND: To test our hypothesis that eutopic secretory phase endometrium from women with endometriosis is similar to proliferative phase endometrium from fertile women without endometriosis, we explored the expression of regulators of cell fate across the menstrual cycle. METHODS: Endometrial biopsies were taken from 73 women, comprising 38 women with surgically diagnosed active peritoneal endometriosis (Group 1) and 35 fertile women without endometriosis (Group 2). Nucleolin, proliferating cell nuclear antigen (PCNA), telomerase and histone gamma-H2AX expression was evaluated by immunohistochemistry and mean telomere length (TL) by quantitative PCR. RESULTS: We have immunolocalized nucleolin and gamma-H2AX in the benign premenopausal endometrium for the first time. All markers were present in the proliferative phase endometrium of all women. In Group 2, during the secretory phase, proliferative markers declined with a paradoxical increase in stromal gamma-H2AX. Women in Group 1, however, showed a persistent immunoreactivity for the proliferative markers, while the staining for gamma-H2AX decreased in secretory endometrium (P < 0.05). This difference between groups was significant in both stroma and glands for nucleolin (P < 0.0001), PCNA (P < 0.01) and gamma-H2AX (P < 0.05) in the secretory phase. We showed a positive correlation between mean TL and nucleolin expression (glandular r = 0.37, P = 0.002; stromal r = 0.4, P = 0.001), telomerase immunoreactivity (glandular r = 0.33, P = 0.009; stromal r = 0.4, P = 0.001) and glandular PCNA (r = 0.35, P = 0.004), whereas a negative correlation was seen between mean TL and gamma-H2AX (r = -0.28, P = 0.04). CONCLUSIONS: These findings demonstrate that the state of replication seen in secretory phase endometrium from women with active peritoneal endometriosis is not a simple extension of the proliferative phase.

Endometrial telomerase shows specific expression patterns in different types of reproductive failure.

In order to assess whether markers of cell senescence are related to reproductive failure, the expression of telomerase and telomere length in endometrial biopsies from women with and without reproductive failure were assessed. This pilot study included 45 women of whom 10 had idiopathic recurrent loss of empty gestational sacs, 10 had idiopathic recurrent fetal loss (miscarriage following identification of fetal cardiac activity), 10 had recurrent implantation failure and 15 had two or more normal pregnancies (control group). An endometrial sample was collected during the window of implantation from each woman. The mean endometrial telomere length was determined by quantitative polymerase chain reaction. Telomerase expression was evaluated by immunohistochemistry. The endometria of the control group showed virtually no telomerase immunoreactivity during the window of implantation. However, the immunostaining for telomerase was significantly and differentially increased in various endometrial cellular compartments in women with recurrent reproductive failure (P < 0.05). There were no significant differences in mean telomere length between groups. These data provide a novel insight into the biological correlates of clinical types of recurrent reproductive failure and suggest that specific alterations in the regulation of endometrial cell fate are associated with different types of recurrent reproductive failure.

Endometriosis is associated with aberrant endometrial expression of telomerase and increased telomere length.

BACKGROUND: In order to test our hypothesis that endometriosis is associated with abnormal expression of telomerase and telomere lengthening in endometrium, we assessed endometrial expression of the human telomerase enzyme and telomere length (TL). METHODS: This prospective pilot study, included 29 women with symptomatic, surgically diagnosed endometriosis (Group 1) and 27 healthy, fertile, symptom-free women without endometriosis (Group 2, confirmed by laparoscopy). Seventeen women in Group 1 and 15 women in Group 2 had endometrial biopsies taken on Day 21 +/- 2 of the cycle. A further 12 women in each group were biopsied on Day 26 +/- 2. Telomerase and estrogen receptor beta (ERbeta) expression was evaluated by immunohistochemistry. Mean TL was determined by quantitative PCR. RESULTS: The endometria of fertile healthy women showed either weak or no telomerase immunoreactivity throughout the luteal phase. Immunostaining for telomerase was significantly increased during the implantation window and the premenstrual endometria of women with endometriosis (P < 0.0001). This was associated with a loss of stromal and vascular ERbeta immunostaining (P < 0.05). The mean TL were significantly longer in endometria of women with endometriosis during the implantation window (P = 0.005), indicating the biological relevance of our novel finding of telomerase in benign endometrium. There was positive correlation of the circulating estradiol with peripheral blood TL in women. CONCLUSIONS: We speculate that aberrant endometrial expression of telomerase mediates alterations in cell fate that enhance proliferation, contributing to the pathogenesis of endometriosis.

Telomeres as biomarkers for ageing and age-related diseases.

Telomeres in telomerase-negative cells shorten during DNA replication in vitro due to numerous causes including the inability of DNA polymerases to fully copy the lagging strand, DNA end processing and random damage, often caused by oxidative stress. Short telomeres activate replicative senescence, an irreversible cell cycle arrest. Thus, telomere length is an indicator of replicative history, of the probability of cell senescence, and of the cumulative history of oxidative stress. Telomeres in most human cells shorten during ageing in vivo as well, suggesting that telomere length could be a biomarker of ageing and age-related morbidity. There are two distinct possibilities: First, in a tissue-specific fashion, short telomeres might indicate senescence of (stem) cells, and this might contribute to age-related functional attenuation in this tissue. Second, short telomeres in one tissue might cause systemic effects or might simply indicate a history of high stress and damage in the individual and could thus act as risk markers for age-related disease residing in a completely different tissue. In recent years, data have been published to support both approaches, and we will review these. While they together paint a fairly promising picture, it needs to be pointed out that until now most of the evidence is correlative, that much of it comes from underpowered studies, and that causal evidence for essential pathways, for instance for the impact of cell senescence on tissue ageing in vivo, is still very weak.

Overexpression of telomerase confers growth advantage, stress resistance, and enhanced differentiation of ESCs toward the hematopoietic lineage.

Embryonic stem cells (ESCs) are capable of extended self-renewal and maintenance of pluripotency even after many population doublings. This is supported by high levels of telomerase activity and enhanced antioxidant protection in ESCs, both of which are downregulated during differentiation. To examine the role of telomerase for ESC self-renewal and differentiation, we overexpressed the reverse transcriptase subunit (Tert) of murine telomerase in ESCs. Increased telomerase activity enhances the self-renewal ability of the Tert-overexpressing ESCs, improves their resistance to apoptosis, and increases their proliferation. The differentiated progeny of wild-type ESCs express little Tert and show shortening of telomeric overhangs. In contrast, the progeny of Tert-overexpressing ESCs maintain high telomerase activity, as well as the length of G-rich overhangs. In addition, these cells accumulate lower concentrations of peroxides than wild-type cells, implying greater resistance to oxidative stress. Finally, differentiation toward hematopoietic lineages is more efficient as a result of the continued expression of Tert. Microarray analysis revealed that overexpression of Tert altered expression of a variety of genes required for extended self-renewal and lifespan. Our results suggest that telomerase functions as a "survival enzyme" in ESCs and its differentiated progeny by protecting the telomere cap and by influencing the expression patterns of stress response and defense genes. This results in improved proliferation of ESCs and more efficient differentiation, and these results might have profound consequences for stem cell-replacement therapies.

Lymphocyte telomere dynamics and telomerase activity in inflammatory bowel disease: effect of drugs and smoking.

BACKGROUND: The chromosome instability observed in peripheral blood lymphocytes in ulcerative colitis could be a biomarker of cancer susceptibility. AIM: To determine whether accelerated telomere shortening could explain chromosome instability and assess the effect of drugs and smoking on telomere dynamics in these cells. METHODS: Peripheral blood lymphocytes were isolated from ulcerative colitis, Crohn's disease and non-inflammatory bowel disease control patients. Telomere lengths were measured by quantitative real-time polymerase chain reaction. After activation and cell separation, telomerase activity and human telomerase reverse transcriptase messenger ribonucleic acid were measured by telomerase repeat amplification protocol enzyme-linked immunosorbent serological assay and quantitative real-time polymerase chain reaction, respectively. RESULTS: Age-related telomere loss in peripheral blood lymphocytes was similar in ulcerative colitis, Crohn's disease and control patients. Telomerase activity decreased with age in all groups and correlated positively with telomere length (r = 0.489, P = 0.006). Among Crohn's disease patients, azathioprine was associated with decreased telomerase activity (0.66 vs. 1.54, P = 0.026, P < 0.05) and smoking was associated with decreased human telomerase reverse transcriptase mRNA expression (10.5 vs. 33.3, P = 0.036, P < 0.05). CONCLUSIONS: Telomere shortening is not accelerated and therefore cannot be the cause of the chromosome instability observed in ulcerative colitis peripheral blood lymphocytes. Azathioprine and cigarette smoking modify telomerase expression in these cells.

Human cell senescence as a DNA damage response.

It has been established that telomere-dependent replicative senescence of human fibroblasts is stress-dependent. First, it was shown that telomere shortening, which is a major contributor to telomere uncapping, is stress-dependent to a significant degree. Second, the signalling pathway connecting telomere uncapping and replicative senescence appears to be the same as the one that is activated by DNA damage: uncapped telomeres activate signalling cascades involving the protein kinases ATM, ATR and, possibly, DNA-PK. Furthermore, phosphorylation of histone H2A.X facilitates the formation of DNA damage foci around uncapped telomeres, and this in turn activates downstream kinases Chk1 and Chk2 and, eventually, p53. It appears that this signalling pathway has to be maintained in order to keep cells in a senescent state. Thus, cellular senescence can be regarded as a permanently maintained DNA damage response state. This suggests that antibodies against DNA damage foci components might be useful markers for senescent cells in vivo.

Ribozyme-mediated telomerase inhibition induces immediate cell loss but not telomere shortening in ovarian cancer cells.

Telomerase is a promising target for human cancer gene therapy. Its inhibition allows telomere shortening to occur in cancer cells, which in turn is thought to trigger delayed senescence and/or apoptosis. We tested whether telomerase inhibition might have additional, immediate effects on tumor cell growth. Ovarian cancer cell lines with widely differing telomere lengths were efficiently transduced with an adenovirus expressing a ribozyme directed against the T motif of the catalytic subunit of human telomerase, hTERT. Three days after transduction, telomerase activity was significantly reduced and massive cell loss was induced in mass cultures from all four ovarian cancer cell lines tested, whereas transduction of telomerase-negative human fibroblasts did not attenuate their growth. The kinetics of induction of cell death in cancer cells was not significantly dependent on telomere length, and telomeres did not shorten measurably before the onset of apoptosis. The data suggest the existence of a "fast-track" mechanism by which diminution of telomerase can interfere with cancer cell growth and induce cell death, presumably by apoptosis. This phenomenon might be a consequence of the telomere capping function provided by telomerase in tumor cells. Uncapping of telomeres by ribozyme-mediated inhibition of telomerase bears therapeutic potential for ovarian cancer.

Accelerated telomere shortening in Fanconi anemia fibroblasts--a longitudinal study.

Fanconi anemia (FA) is a fatal inherited disease displaying chromosomal instability, disturbances in oxygen metabolism and a high burden of intracellular radical oxygen species. Oxygen radicals can damage DNA including telomeric regions. Insufficient repair results in single strand breaks that can induce accelerated telomere shortening. In a longitudinal study we demonstrate that telomeric DNA is continuously lost at a higher rate in FA fibroblasts compared to healthy controls. Furthermore, we show that this loss is caused rather by an increased shortening per cell division in regularly replicating cells than by apoptosis.

BJ fibroblasts display high antioxidant capacity and slow telomere shortening independent of hTERT transfection.

Human foreskin BJ fibroblasts are well protected against oxidative stress as shown by their low intracellular peroxide content, low levels of protein carbonyls, and low steady-state lipofuscin content as compared to other primary human fibroblasts. This correlates with a long replicative life span of the parental cells of about 90 population doublings and a telomere-shortening rate of only 15-20 bp/PD. This value might define the upper limit of a telomere-shortening rate that can still be explained by the end replication problem alone. In BJ clones immortalized by transfection with hTERT, the catalytic subunit of telomerase, the same telomere-shortening rate as in parental cells is observed over a long time despite strong telomerase activity. Hyperoxia, which induces oxidative stress and accelerates telomere shortening in a variety of human fibroblast strains, does not do so in BJ cells. It is possible that the high antioxidative capacity of BJ cells, by minimizing the accumulation of genomic damage, is instrumental in the successful immortalization of these cells by telomerase.

Telomeres and replicative senescence: Is it only length that counts?

Telomeres are well established as a major 'replicometer', counting the population doublings in primary human cell cultures and ultimately triggering replicative senescence. However, neither is the pace of this biological clock inert, nor is there a fixed threshold telomere length acting as the universal trigger of replicative senescence. The available data suggest that opening of the telomeric loop and unscheduled exposure of the single-stranded G-rich telomeric overhang might act like a semaphore to signal senescent cell cycle arrest. Short telomere length, telomeric single-strand breaks, low levels of loop-stabilizing proteins, or other factors may trigger this opening of the loop. Thus, both telomere shortening and the ultimate signalling into senescence are able to integrate different environmental and genetic factors, especially oxidative stress-mediated damage, which might otherwise become a thread to genomic stability.

Stress, DNA damage and ageing -- an integrative approach.

Ageing is highly complex, involving multiple mechanisms at different levels. Nevertheless, recent evidence suggests that several of the most important mechanisms are linked via endogenous stress-induced DNA damage caused by reactive oxygen species (ROS). Understanding how such damage contributes to age-related changes requires that we explain how these different mechanisms relate to each other and potentially interact. In this article, we review the contributions of stress-induced damage to cellular DNA through (i) the role of damage to nuclear DNA and its repair mediated via the actions of poly(ADP-ribose) polymerase-1, (ii) the role of damage to telomeric DNA and its contribution to telomere-driven cell senescence, and (iii) the role of damage to and the accumulation of mutations in mitochondrial DNA. We describe how an integrative approach to studying these mechanisms, coupled with computational modelling, may be of considerable importance in resolving some of the complexity of cellular ageing.

Ribozyme cleavage of telomerase mRNA sensitizes breast epithelial cells to inhibitors of topoisomerase.

Telomerase activity is necessary and sufficient for immortality in many cells and hence represents a prime target for antitumor strategies. Here, we show that a hammerhead ribozyme cleaves human telomerase (hTERT) mRNA in vitro. Stable transfection in clones of the human breast tumor line MCF-7 and the immortal breast cell line HBL-100 results in expression of the ribozyme, diminishes the abundance of hTERT mRNA, and inhibits telomerase activity. This led to shortened telomeres, inhibition of net growth, and induction of apoptosis. In HBL-100 mass cultures infected with a ribozyme-expressing adenovirus diminution of hTERT mRNA, attenuation of telomerase activity, inhibition of net growth, and induction of apoptosis was found as well. Attenuation of telomerase activity increased the sensitivity of HBL-100 and MCF-7 clones specifically to inhibitors of topoisomerase. Likewise, expression of exogenous telomerase in originally telomerase-negative human fibroblasts decreased their sensitivity to topoisomerase poisons but not to a number of other cytotoxic drugs. The data validate a ribozyme approach for telomerase inhibition therapy in cancer and suggest that it might be combined advantageously with topoisomerase-directed chemotherapy.