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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Telomere shortening triggers a p53-dependent cell cycle arrest via accumulation of G-rich single stranded DNA fragments.

It has been repeatedly suspected that telomere shortening might be one possible trigger of the p53-dependent cell cycle arrest, although the mechanism of this arrest remained unclear. Telomeres in human cells under mild oxidative stress accumulate single-strand damage faster than interstitial repetitive sequences. In MRC-5 fibroblasts and U87 glioblastoma cells, which both express wild-type p53, oxidative stress-mediated production of single-strand damage in telomeres is concomitant to the accumulation of p53 and p21 and to cell cycle arrest. This response can be modeled by treatment of cells with short single stranded telomeric G-rich DNA fragments. The arrest is transient in U87 cells. Recovery from it is accompanied by up-regulation of telomerase activity and elongation of telomeres. Overexpression of mutated p53 is sufficient to reverse the phenotype of inhibition as well as the delayed activation of telomerase. These data suggest that the production of G-rich single stranded fragments during the course of telomere shortening is sufficient to trigger a p53 dependent cell cycle arrest.

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.

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.

Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts.

Telomere shortening triggers replicative senescence in human fibroblasts. The inability of DNA polymerases to replicate a linear DNA molecule completely (the end replication problem) is one cause of telomere shortening. Other possible causes are the formation of single-stranded overhangs at the end of telomeres and the preferential vulnerability of telomeres to oxidative stress. To elucidate the relative importance of these possibilities, amount and distribution of telomeric single-strand breaks, length of the G-rich overhang, and telomere shortening rate in human MRC-5 fibroblasts were measured. Treatment of nonproliferating cells with hydrogen peroxide increases the sensitivity to S1 nuclease in telomeres preferentially and accelerates their shortening by a corresponding amount as soon as the cells proliferate. A reduction of the activity of intracellular peroxides using the spin trap alpha-phenyl-t-butyl-nitrone reduces the telomere shortening rate and increases the replicative life span. The length of the telomeric single-stranded overhang is independent of DNA damaging stresses, but single-strand breaks accumulate randomly all along the telomere after alkylation. The telomere shortening rate and the rate of replicative aging can be either accelerated or decelerated by a modification of the amount of oxidative stress. Quantitatively, stress-mediated telomere damage contributes most to telomere shortening under standard conditions.

Accelerated telomere shortening in fibroblasts after extended periods of confluency.

Telomere length in MRC-5 fibroblasts remains constant if the cells are proliferation-inhibited for up to 3 months by confluency. However, the apparent frequency of single-stranded sites in telomeres, measured as sensitivity to degradation by S1 nuclease, increases about fourfold during this extended inhibition of proliferation. After release of the cells, the frequency of telomeric single-stranded sites decreases to control values, and the telomere shortening rate increases about threefold as compared to controls proliferating without inhibition. This acceleration is transitory, the telomere shortening rate decreases to control values after about two population doublings after release. Finally, temporarily arrested fibroblast populations senesce at a lower cumulative population doubling level, but at about the same telomere length, as continuously proliferating controls. The data suggest that metabolic time-dependent single-strand degradation is a major cause of telomere shortening. They support the idea that telomere shortening plays an important role in triggering cellular senescence.

Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts.

One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The aging process on a cellular level can be treated either as the ongoing proliferation until a certain number of cell divisions is reached (the Hayflick limit) or as the aging of nondividing cells, that is, the age-related changes in cells without proliferation. The present investigation was undertaken to reveal the changes in protein turnover, proteasome activity, and protein oxidation status during proliferative senescence. We were able to demonstrate that the activity of the cytosolic proteasomal system declines dramatically during the proliferative senescence of human MRC-5 fibroblasts. Regardless of the loss in activity, it could be demonstrated that there are no changes in the transcription and translation of proteasomal subunits. This decline in proteasome activity was accompanied by an increased concentration of oxidized proteins. Cells at higher proliferation stages were no longer able to respond with increased degradation of endogenous [(35)S]-Met-radiolabeled proteins after hydrogen peroxide- or quinone-induced oxidative stress. It could be demonstrated that oxidized proteins in senescent human MRC-5 fibroblasts are not as quickly removed as they are in young cells. Therefore, our study demonstrates that the accumulation of oxidized proteins and decline in protein turnover and activity of the proteasomal system are not only a process of postmitotic aging but also occur during proliferative senescence and result in an increased half-life of oxidized proteins.

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.

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.