Mean Terminal Restriction Fragment Research Articles

Telomere-associated genes and telomeric lncRNAs are biomarker candidates in lung squamous cell carcinoma (LUSC)

In the past decade, research efforts were made to identify molecular biomarkers useful as therapeutic targets in Non-Small Cell Lung Cancer (NSCLC), the most frequent type of lung carcinoma. NSCLC presents different histological subtypes being the most prevalent LUSC (Lung Squamous Cell Cancer) and LUAD (Lung Adenocarcinoma), and only a subset of LUAD patients' present tumors expressing known targetable genetic alterations. Telomeres and its components, including telomerase, the enzyme that replenishes telomeres, have been considered potential cancer biomarkers due to their crucial role in cell proliferation and genome stability. Our study aims to quantify expression changes affecting telomere-associated genes and ncRNAs associated with telomere regulation and maintenance in NSCLC. We first assessed the transcriptome (RNA-Seq) data of NSCLC patients from The Cancer Genome Atlas (TCGA) and then we tested the expression of telomere-associated genes and telomeric ncRNAs (TERC, telomerase RNA component, and TERRA, telomere repeat-containing RNA) in Brazilian NCSLC patient samples by quantitative RT-PCR, using matched normal adjacent tissue samples as the control. We also estimated the mean size of terminal restriction fragments (TRF) of some Brazilian NSCLC patients using telomeric Southern blot. The TCGA analysis identified alterations in the expression profile of TERT and telomere damage repair genes, mainly in the LUSC subtype. The study of Brazilian NSCLC samples by RT-qPCR showed that LUSC and LUAD express high amounts of TERT and that although the mean TRF size of tumor samples was shorter compared to normal cells, telomeres in NSCLC are probably maintained by telomerase. Also, the expression analysis of Brazilian NSCLC samples identified statistically significant alterations in the expression of genes involved with telomere damage repair, as well as in TERC and TERRA, mainly in the LUSC subtype. We, therefore, concluded that telomere maintenance genes are significantly deregulated in NSCLC, representing potential biomarkers in the LUSC subtype.

Age-associated telomere shortening in Thoroughbred horses.

Telomeres are genetically conserved repetitive terminal DNA that protect against genomic instability and shorten with ageing. Here, we reveal the leukocyte telomere length of Equus caballus by measuring terminal restriction fragments (TRFs) using Southern Blot analysis in a cohort of 43 Thoroughbred horses (age: 24 h-25 years). Heterogeneous TRFs were observed in each animal and large inter-animal variation in mean TRF was observed (range: 10.5-18.7 kbp). Mean TRFs were inversely correlated with age (r = -0.47). The estimated yearly rate of telomere attrition was 134 bp. Horses should be considered as an alternative animal model to investigate environmental and lifestyle factors that regulate telomeres and promote healthy ageing.

Telomere length is independently associated with age, oxidative biomarkers, and sport training in skeletal muscle of healthy adult males

In skeletal muscle, which mainly contains postmitotic myonuclei, it has been suggested that telomere length remains roughly constant throughout adult life, or shortens in response to physiopathological conditions in muscle diseases or in the elderly. However, telomere length results from both the replicative history of a specific tissue and the exposure to environmental, DNA damage-related factors, therefore the predictive biological significance of telomere measures should combine the analysis of the various interactive factors. In the present study, we analysed any relationship between telomere length [mean and minimum terminal restriction fragment (TRF) length] chronological age, oxidative damage (4-HNE, protein carbonyls), catalase activity, and heat shock proteins expression (αB-crystallin, Hsp27, Hsp90) in semitendinous muscle biopsies of 26 healthy adult males between 20 and 50 years of age, also exploring the influence of regular exercise participation. The multiple linear regression analysis identified age, 4-HNE, catalase, and training status as significant independent variables associated with telomere length and jointly accounting for ∼30–36% of interindividual variation in mean and/or minimum TRF length. No association has been identified between telomere length and protein carbonyl, αB-crystallin, Hsp27, and Hsp90, as well as between age and the variables related to stress response. Our results showed that skeletal muscle from healthy adults displays an age-dependent telomere attrition and that oxidised environment plays an age-independent contribution, partially influenced by exercise training.

Effects of donor cells' sex on nuclear transfer efficiency and telomere lengths of cloned goats.

The aim of this study was to investigate the effects of donor cells' sex on nuclear transfer efficiency and telomere length of cloned goats from adult skin fibroblast cells. The telomere length of somatic cell cloned goats and their offspring was determined by measuring their mean terminal restriction fragment (TRF) length. The result showed that (i) reconstructed embryos with fibroblast cells from males Boer goats obtained significantly higher kids rate and rate of live kids than those of female embryos and (ii) the telomere lengths of four female cloned goats were shorter compared to their donor cells, but five male cloned goats had the same telomere length with their donor cells, mainly due to great variation existed among them. The offspring from female cloned goats had the same telomere length with their age-matched counterparts. In conclusion, the donor cells' sex had significant effects on nuclear transfer efficiency and telomere lengths of cloned goats.

Changes in telomere length distribution in low-dose X-ray-irradiated human umbilical vein endothelial cells.

Ionizing radiation (IR) is known to be a cause of telomere dysfunction in tumor cells; however, very few studies have investigated X-ray-related changes in telomere length and the telomerase activity in normal human cells, such as umbilical vein endothelial cells (HUVECs). The loss of a few hundred base pairs from a shortened telomere has been shown to be important with respect to cellular senescence, although it may not be detected according to traditional mean telomere length [assessed as the terminal restriction fragment (TRF)] analyses. In the present study, a continuous time window from irradiation was selected to examine changes in the telomere length, including the mean TRF length, percentage of the telomere length, telomerase activity, apoptotic rate, and survival rate in HUVECs from the first day to the fourth day after the administration of a 0.5-Gy dose of irradiation. The mean TRF length in the irradiated HUVECs showed shorter telomere length in first 3 days, but they were not statistically significant. On the other hand, according to the percentage analysis of the telomere length, a decreasing tendency was noted in the longer telomere lengths (9.4-4.4 kb), with a significant increase in the shortest telomeres (4.4-2.3 kb) among the irradiated cells versus the controls from the first day to the third after irradiation; no significant differences were noted on the fourth day. These results suggest that the shortest telomeres are sensitive to the late stage of radiation damage. The proliferation of irradiated cells was suppressed after IR in contrast to the non-irradiated cells. The apoptotic rate was elevated initially both in IR- and non-IR-cells, but that of IR-cells was maintained at an elevated level thereafter in contrast to that of non-IR-cells decreasing promptly. Therefore, a 0.5-Gy dose of IR induces persistent apoptosis leading to an apparent growth arrest of the normal HUVECs.

The study on telomere length for age estimation in a Thai population.

Age is one of the key parameters in establishing a physical characteristic profile of an individual. For biological evidence left in crime scenes such as blood, saliva, hair, etc, the evidence owner's age can be determined only by DNA extracted from these materials. Previous researches have found that there are certain DNA regions with specialized characteristic and function called telomere being able to predict age. The present study was to determine the correlation between telomere length and age as well as the effect of sex on the correlation and to create linear regression equation for age estimation in Thai population for forensic purposes. Blood samples obtained from unrelated healthy Thai fresh cadavers without anatomical organ abnormalities were used in this study. All cadaver subjects underwent the postmortem examination in jurisdiction of the Department of Forensic Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, and Institute of Forensic Medicine, Police General Hospital. Fifty blood samples from both sexes of all ages divided into 6 groups for equal age distribution (0-11, 12-23, 24-35, 36-47, 48-59, and 60 years old and older) were collected for a total of 100 samples. The extracted genomic DNA samples were then subjected to telomere length estimation by terminal restriction fragment (TRF) assay. The results showed that the mean TRF length was inversely correlated with age (r = -0.625), and sex did not have a statistically significant influence on the association between age and mean TRF length (P > 0.05). The obtained linear regression equation was y = 113.538 ± 9.604 - 0.012 × (R = 0.391; P < 0.001). However, the correlation was too low to be used for age estimation with high certainty and a possible reason for this in part would be the postmortem DNA degradation at some level, even using fresh cadaver blood, and other biological factors such as ethnicity and DNA sources. Roughly, those individuals who had a mean TRF length longer than 6.3 kilobase (kb), between 5.5 and 6.3 kb, and shorter than 5.5 kb aged younger than 28 years, 30 to 44 years, and older than 46 years, respectively (P < 0.01). As a preliminary study, this study highlighted that telomere length could act as a useful biomarker of aging in human population and might be used for rough age estimation in a Thai population. However, further studies with a larger sample size and/or in living human bloods as well as other cell types are recommended to support the results of this study.

Telomere Shortening in the Colonial CoralAcropora digitiferaDuring Development

To test whether telomere length can be used in estimating the age of colonial corals, we used terminal restriction fragment (TRF) length analysis to compare the telomere lengths of the coral Acropora digitifera at three developmental stages: sperm, planula larvae, and polyps of adult colonies. We also compared the mean TRF lengths between branches at the center and periphery of tabular colonies of A. digitifera. A significant difference was observed in the mean TRF lengths in sperm, planulae, and polyps. The mean TRF length was longest in sperm and shortest in polyps from adult colonies. These results suggest that telomere length decreases during coral development and may be useful for estimating coral age. However, the mean TRF length of branches at the center of a table-form colony tended to be longer than that of peripheral branches, although this difference was not statistically significant. This suggests that both the chronological age of polyps and cell proliferation rate influence telomere length in polyps, and that estimating coral age based on telomere length is not a simple endeavor.

Telomere Length Differences upon Keratinization and its Variations in Normal Human Epidermal Keratinocytes

Telomeres are the physical ends of chromosomes, and are cleaved with each cell division. Telomere loss results from not only incomplete DNA replication (“end replication problem”), followed by the processing of chromosome, but also exposure to various DNA damaging agents. Human epidermis is constantly replaced through replication at the basal layer, followed by upward movement and terminal differentiation at the suprabasal layer. The objective of this study was to assess differences in telomere length between basal and suprabasal keratinocytes, and variations of telomere lengths among basal cells. We investigated terminal restriction fragment (TRF) length in fractions of epidermal cells by Southern blot analysis. Time-dependent trypsin treatment to epidermis divided into basal cell rich and suprabasal cell rich fractions. In the five skin samples from various sites of the body, the mean TRF length were 5,932 ± 470 (range 5,182-6,630) bp in basal cell rich fraction showing 21.5% ± 5.8% (range 12.6-28.6%) of keratin 10 (K10)-positivity, which is marker of keratinizing suprabasal cell, and 5,320 ± 640 (range 4,495-6,212) bp in suprabasal cell rich fraction showing 78.8% ± 4.0% (range 72.8-83.0%) of K10-positivity. It was calculated that K10 positive cells have 1,091 ± 963 (range 248-2,650) bp shorter telomeres compared to K10 negative cells. In addition, fluorescence in situ hybridization (FISH) study showed that columnar groups of keratinocytes with statistically longer telomere in each nucleus than the other epidermal cells in the vicinity occupied occasionally between the tip of dermal papilla and the bottom of rete ridge. The present study showed that the keratinizing suprabasal cells have shorter telomeres compared to basal cells. FISH study showed that some groups of epidermal cells having different replicative histories among the groups of epidermal cells in the vicinity.

Expression profile of telomere‐associated genes in multiple myeloma

To further contribute to the understanding of multiple myeloma, we have focused our research interests on the mechanisms by which tumour plasma cells have a higher survival rate than normal plasma cells. In this article, we study the expression profile of genes involved in the regulation and protection of telomere length, telomerase activity and apoptosis in samples from patients with monoclonal gammopathy of undetermined significance, smouldering multiple myeloma, multiple myeloma (MM) and plasma cell leukaemia (PCL), as well as several human myeloma cell lines (HMCLs). Using conventional cytogenetic and fluorescence in situ hybridization studies, we identified a high number of telomeric associations (TAs). Moreover, telomere length measurements by terminal restriction fragment (TRF) assay showed a shorter mean TRF peak value, with a consistent correlation with the number of TAs. Using gene expression arrays and quantitative PCR we identified the hTERT gene together with 16 other genes directly involved in telomere length maintenance: HSPA9, KRAS, RB1, members of the Small nucleolar ribonucleoproteins family, A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins, and 14-3-3 family. The expression levels of these genes were even higher than those in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), which have unlimited proliferation capacity. In conclusion, the gene signature suggests that MM tumour cells are able to maintain stable short telomere lengths without exceeding the short critical length, allowing cell divisions to continue. We propose that this could be a mechanism contributing to MM tumour cells expansion in the bone marrow (BM).

Long Telomere Length of White Blood Cells Following Umbilical Cord Blood Transplant (UCBT): Is Hematopoiesis Younger in UCBT Recipients Compared to Healthy Age-Matched Controls?

AbstractAbstract ▪4094▪This icon denotes a clinically relevant abstract Background:Telomeres are linear DNA structures at the ends of chromosomes that help to maintain genome stability. In the absence of effective telomere-protecting mechanisms, chromosomes lose a few base pairs (bp) of their telomeric DNA at each mitotic division and thus telomere length (TL) is also considered a marker of cell aging. In the hematopoietic system, TL of both early and committed progenitors progressively shortens with age, as in other highly replicating tissues. Moreover, many studies demonstrated that high proliferative stresses, including bone marrow (BM) regeneration after transplantation, cause a non-physiological TL shortening. Indeed, recent studies suggest that post-autograft TL correlates with TL of grafted material (Ruella et al, 2010). In order to address this issue, TL has been evaluated in a series of adult subjects engrafted with umbilical cord blood (UCB), a graft source characterized by cells harboring very long telomeres. Aim of the study was to verify whether despite the high replicative stress during engraftment, UCB hematopoietic stem cells may renew the recipient BM, maintaining the biologic characteristics of the graft. Patients and Methods:TL was evaluated on peripheral blood (PB) cells from 20 adult recipients of umbilical cord blood transplant (UCBT). They had been transplanted due to acute leukemia (n=12), multiple myeloma (n=2), non-Hodgkin’s Lymphoma (n=3) or chronic myeloproliferative disease (=3). TL analysis was performed in absence of signs of the previous disease and at complete hematological recovery, at a median of 9 mos. (range 1–73) since UCBT. At TL analysis, cell blood counts revealed median values of 7,540 white blood cells/μL (range 2,870-18,800), 11.9 gr% of hemoglobin (range 8.3–14), and 144,000 platelets/μL (range 41,000–351,000). TL was evaluated on both whole PB cells and separated cell populations, i.e. MNC and granulocytes, obtained following Ficoll gradient cell separation. TL was also determined on a series of 47 UCB samples, including samples from cord blood units employed as graft material for four patients of the present series. TL was determined on DNA extracts by Southern blot analysis, according to well established procedures. Briefly, DNA fragments were separated by agarose gel electrophoresis. The Telomere Length Assay Kit (Roche Diagnostic) was used for the hybridization phase and for each telomere smear, mean terminal restriction fragment (TRF) lengths and the point of maximum signal intensity defining the highest concentration of telomere repeats were calculated using Quantity One software (Bio-Rad Laboratories, UK). Results:Overall, TL in PB cells following UCBT showed a rapid fall compared to TL values that characterize cord blood cells. In our series of 47 UCB samples, the median TL-TRF on MNC is 9,171 bp (range 6,573-13,695), whereas MNC from our UCBT recipients showed a median TL-TRF of 8,689 bp (range 5,982-11,149) (p=0,055). When values were distributed according to the age of UCBT recipients, TL remained higher than TL obtained from a large series of hundreds of normal control subjects. Indeed, median TL-TRF on MNC from healthy controls is 6,951 bp (range 4,375-10,467), significantly shorter compared to MNC from UCBT recipients, as shown in Figure 1. Similar results were obtained when TL was assessed on whole PB cells and on separated granulocytes. In fact, the most marked difference in TL between UCBT recipients and normal controls was observed on granulocytes. This was particularly clear among subjects aged >40 yrs. As shown in Figure 2, median TL-TRF on granulocytes from UCBT recipients aged 40–65 yrs. was 9,138 bp (range 7,644 – 11,138) compared to 6,857 bp (range 5,773 – 10,946) of granulocytes from age-matched normal controls. Overall, PB cells from subjects grafted with UCB showed a TL slightly longer than in the youngest control population of 0–20 yrs. of age. Lastly, no difference in TL was observed according to time elapsed since transplant, with analogous TL values in subjects evaluated at less or more than 6 months since UCBT. Conclusions:The results indicate that despite the high replicative stress during BM engraftment, UCBT regenerate a hematopoietic system characterized by long TL. This suggests that in the transplant setting the use of young donor cells such as UCB may replace patient BM with not only a healthy but also a biologically young hematopoietic system. [Display omitted] [Display omitted] Disclosures:Rambaldi:Hoffman-La Roche: Consultancy, Honoraria.

Telomere length is a prognostic factor for overall survival in colorectal cancer

The aim of this study was to determine whether telomere length is an independent prognostic factor for the prevention and survival of colorectal cancer. Terminal restriction fragment (TRF) length was determined by Southern blot in tumours and paired normal tissue samples from 147 patients with sporadic colorectal cancer who had undergone surgery. The TRF length ratio (TRFLR) was determined as the ratio between the length of the patient's tumour and normal tissue.The classification and regression tree technique was used to determine optimal cut-off values (≤ 1 or > 1). Mean TRF length was 6.79 Kbp (1.19-13.99) in tumour tissue and 7.81 Kbp (3.63-15.70) in normal mucosa (P < 0.001). Mean TRFLR was 0.88. Telomere length and telomere length ratio were not correlated with any clinicopathological factors. In univariate analysis, overall survival was related to N stage (lymph node +/-; P = 0.002), TNM classification (P = 0.019) and TRFLR (≤ 1 or > 1; P = 0.014). In multivariate analysis, overall survival was significantly associated with TRFLR and N stage. Colorectal cancer patients with TRFLR ≤ 1 and negative lymph node involvement had a higher overall survival rate. Telomere length ratio is an independent prognostic factor for survival in colorectal cancer patients, and the telomere lengths in the normal and tumour mucosa of the same patient present with parallel behaviour.

Sequencing and analysis of chromosomal extremities of Trypanosoma rangeli in comparison with Trypanosoma cruzi lineages

The aim of this study was to investigate the genetic variability of sequences present in the chromosome ends of Trypanosoma rangeli strains defined by the presence (+) or absence (-) of KP1 minicircles, and to compare the mean terminal restriction fragment (TRF) lengths to those of Trypanosoma cruzi populations representative of groups TcI, TcII, TcIV, and TcVI. Southern blots containing RsaI-digested genomic DNA of T. rangeli KP1(+) strains, T. rangeli KP1(-) strains, and T. cruzi strains were probed with the previously described subtelomeric sequences (170 bp) of T. rangeli and with telomeric hexamer repeats. Mean TRF length analysis showed that the chromosome ends of T. rangeli are distinctly organized, with TRFs ranging from 1.3 to 9 kb for KP1(+) strains and from 0.3 to 5.0 kb for KP1(-) strains. In T. cruzi, TRF length ranged from 0.2 to 9 kb and no association with the genotype of the parasite could be established. Sequence analysis of the 170-bp amplicons revealed the occurrence of sequence polymorphisms in the subtelomeric region between and within KP1(+) and KP1(-) strains. The GTT triplet was detected in all KP1(+) strains, except for strain Cas4, but not in any of the KP1(-) strains. The dendrogram constructed by alignment of all T. rangeli strains showed the division into two main groups, mainly related to the presence or absence of the KP1 minicircle. In conclusion, the present results extend the genotype differences demonstrated by kDNA and karyotype analysis in T. rangeli to the chromosome ends of the parasite.

Analysis of telomere length in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a well defined lymphoid neoplasm genetically characterized by the t(11;14)(q13;q32). Telomeres play an essential role in preserving chromosomal integrity and genomic stability; their shortening can lead to telomere dysfunction and chromosomal instability, a critical factor in cancer development. In this study, telomere length (TL) measured by terminal restriction fragments (TRF) assay in DNA samples of tumor cells from 20 patients with MCL was evaluated. Results were correlated with clinical, morphologic and cytogenetic characteristics. In all cases, the presence of the CCND1/IGH@ rearrangement was confirmed by fluorescence in situ hybridization and/or PCR analysis. TL in total MCL patients revealed a mean TRF value (4.51 +/- 0.79 kb) significantly shorter than those observed in controls (7.49 +/- 1.94 kb) (P < 0.001); 30% of patients had TL shorter than 4.0 kb. TRF length was not associated with patients age (P = 0.07; r = 0.17) nor with sex (females: 4.33 +/- 0.51 kb and males: 4.57 +/- 0.85 kb; P = 0.63). No significant differences were found between patients studied at diagnosis (13) (4.44 +/- 0.81 kb) respect to those analyzed at relapse (7) (4.63 +/- 0.82 kb) (P = 0.53). In addition, we compared patients with (4.84 +/- 1.09 kb) and without (4.40 +/- 0.68 kb) complex karyotypes (P = 0.45) and cases with typical morphology (4.48 +/- 0.79 kb) vs. blastoid variant (4.63 +/- 1.04 kb) (P = 0.83), and no significant differences between them were found. Although the number of cases of our series is not large, our results showed that TL reduction in MCL is independent of the clinical characteristics, morphology and karyotype.

Estimation of Human Age According to Telomere Shortening in Peripheral Blood Leukocytes of Tibetan

In most normal somatic cells, the terminal restriction fragments (TRF) length and age are inversely correlated, which can be used to determine individual age. However, very little is known about the quantitative relationship between human telomeres and age. The aim of the present study was to investigate age-, gender-, and ethnicity-related changes in telomere length in human peripheral blood leukocytes (PBLs). Changes with age in telomere lengths were assessed by Southern blotting. The results shown that telomeres shorten in human PBLs in an age-dependent manner (r = -0.913, P < 0.01). The formula for age estimation according to telomere shortening was Y = -16.539X + 236.287 (Y: age, year; X: mean TRF length, kb). We analyzed the mean TRF length in males and females and found that males had shorter telomeres than females. Moreover, we compared the TRF length of Tibetan and Han population in China and found that telomere length did not differ between 2 populations. We conclude that estimation of human age according to telomere shortening in PBLs is a novel method especially when there is no morphologic information, furthermore, the gender must be considered when age estimation is carried out based on telomere shortening.

Diagonal Earlobe Crease are Associated With Shorter Telomere in Male Japanese Patients With Metabolic Syndrome A Pilot Study

Diagonal earlobe crease (ELC) have been proposed as a marker of generalized atherosclerosis, so in the present study it was investigated whether individuals with ELC have a shortened telomere, which correlates with an accelerated cell turnover and premature aging, leading to atherosclerosis. The mean terminal restriction fragment (TRF) was determined by Southern blot hybridization in the peripheral blood cells of 34 male Japanese patients with metabolic syndrome (MetS) who were under 70 years of age with (n=17) and without (n=17) bilateral ELC, and assessed the relationship of ELC to atherosclerotic cardiovascular disease (AVD). The results showed that the TRF was shorter in the MetS patients with ELC in comparison to age- and risk-factor-matched MetS patients without ELC (7.6+/-1.1 kbp vs 8.6+/-1.2 kbp; P<0.05). ELC were present in 13 patients in the AVD group (n=18), but only 4 patients in the non-AVD group (n=16) had ELC (72.2% and 25% respectively; P<0.05). These findings suggest that ELC is a useful dermatological indicator of an accelerated aging process, as suggested by excessive telomere loss, and might be a useful indirect marker of high-risk patients.

Age-Related Differences Between Unrestricted Somatic Stem Cells from Cord Blood and Bone Marrow Derived Mesenchymal Stroma Cells.

During the past years haematopoietic stem cells from unrelated umbilical cord blood have been increasingly used for treatment of leukaemia and genetic diseases. Human cord blood also contains a non-haematopoietic, adherently growing, CD45 negative, Oct4, nanog and Sox2 negative cell population with intrinsic multipotent differentiation potential, described as Unrestricted Somatic Stem Cells (USSC) (Kögler et al. 2004, Kögler et al. 2005, Kögler et al. 2006, Sensken et al. 2007, Liedtke et al. 2007, Liedtke et al.2008, Greschat et al. 2008, Ghodsizad A et al. 2008, Trapp et al. 2008). USSC can be cultivated GMP-grade reaching 1×109 cells in Passage 4–5 (correlating to 26,5 – 28 cumulative population doublings) without losing multipotency. To better understand and characterise USSC, we carried out in vitro studies concerning age-related changes in USSC-lines from human cord blood and in mesenchymal stroma cells derived from human bone marrow (BM-MSC). Telomerase activity and telomeres are involved in cell proliferation as well as the regulation of cell senescence (Lansdorp 2008). In this study 7 USSC-lines and 9 BM-MSC were analyzed for proliferation and senescence at different population doublings (PD). In vitro USSC have a higher proliferation capacity and accordingly a comparably more extended lifespan than BM-MSC. They undergo about 35 to 45 CPD whereas mean level of CPD from BM-MSC reaches 25. Telomere length of 12 USSC, 5 BM-MSC and 5 clonal populations of USSC were calculated after several PD and telomerase activity was measured. Mean terminal restriction fragment's (TRF's) length calculated from USSC after 31 CPD averages 9.7 kbp whereas mean telomere length from BM-MSC decreases already after 20 CPD to 7.9 kbp. After 47 CPD in clonal USSC populations mean TRF's length is 6.1 kbp. Telomerase activity was analysed with RT-PCR and real time PCR. In contrast to previous publications telomerase activity was detected neither in BM-MSC (Parsch et al.) 2003 nor in USSCs (Manca et al. 2008). The percentage of senescent cells after the same number of CPD is significantly higher in BM-MSC than in USSC. About 80% of senescence was observed in BM-MSC but only 10% senescence in USSCs after 26 CPD. Wnt signalling is mandatory for self-renewal, cell proliferation and differentiation of haematopoietic stem cells (Reya et al. 2003). In primitive MSC populations Wnt signalling regulates mesenchymal lineage specification (Etheridge et al. 2004). For analysing the role of Wnt pathway in USSC development quantitative PCR Arrays have been carried out profiling the expression of 84 genes related to Wnt-mediated signal transduction. 8 different USSC populations have been analysed. In all USSCs factors essential for canonical and also non-canonical Wnt-signalling are present. Poor proliferating USSC with high adipogenic differentiation potential show a stronger expression of Wnt-signalling inhibitors like SFRP1, DKK1 and CXXC4. Based on age-related characteristics, USSC from cord blood are a much better source for regeneration compared to their adult MSC counterpart from bone marrow.

Telomere length and obesity

To assess the telomere length in apparently healthy obese and normal-weight subjects. Seventy-six Caucasian subjects were chosen including 53 children (age 8.2+/-3.5 years) and 23 adults (age 40.5+/-8.4 years). Among these, 22 (12 children and 10 adults) were obese with a body mass index (BMI, kg/m2)>2 SD above the norm. Bioelectrical impedance analysis (BIA), measured with a multiple frequency analyzer, was used to estimate body composition. DNA extraction from white blood cells was used to estimate the telomere length by detection of terminal restriction fragments (TRF). No difference was found between the TRF lengths of obese and normal children. Obese adults had shorter TRF lengths than adults who were not obese (mean TRF length difference, -884.5; 95% confidence intervals -1727 to -41.8; t=2.183; df=17; p<0.041). Obese adults have shorter telomeres than their normal-weight counterparts, while this phenomenon is not present in childhood.

Telomere length and radiosensitivity in human fibroblast clones immortalized by ectopic telomerase expression

Telomeres, the ends of eukaryotic chromosomes, have a variable length among individuals and cell types. While studies in telomerase-deficient mice and cells showed an inverse correlation between telomere length and radiosensitivity, it is less clear whether this remains true in telomerase-proficient cells. To gain insight into this topic, we studied radiosensitivity in three telomerase immortalized fibroblast clones derived from the same cell line and characterized by different telomere length. In two clones, cen3tel4 and cen3tel5, the mean terminal restriction fragment length was approximately 13 and 10 kb, respectively and in the third clone, cen3pci16, it was approximately 4 kb, which is lower than in senescent fibroblasts. To test radiosensitivity, we determined survival to gamma-rays and the induction of chromosomal aberrations after irradiation. Neither the LD50, the gamma-ray dose that reduces survival to 50%, nor the frequency of aberrations detected in the three cell lines showed an inverse correlation with telomere length. In particular, the cen3pci16 cells, which have very short telomeres, did not show a higher sensitivity to irradiation or a greater frequency of chromosomal abnormalities compared to the other two cell lines. Our results suggest that, in the presence of telomerase activity, short telomeres are stabilized and do not cause an increase in radiosensitivity.

The Association Between Physical Activity in Leisure Time and Leukocyte Telomere Length

Physical inactivity is an important risk factor for many aging-related diseases. Leukocyte telomere dynamics (telomere length and age-dependent attrition rate) are ostensibly a biological indicator of human aging. We therefore tested the hypothesis that physical activity level in leisure time (over the past 12 months) is associated with leukocyte telomere length (LTL) in normal healthy volunteers. We studied 2401 white twin volunteers, comprising 2152 women and 249 men, with questionnaires on physical activity level, smoking status, and socioeconomic status. Leukocyte telomere length was derived from the mean terminal restriction fragment length and adjusted for age and other potential confounders. Leukocyte telomere length was positively associated with increasing physical activity level in leisure time (P< .001); this association remained significant after adjustment for age, sex, body mass index, smoking, socioeconomic status, and physical activity at work. The LTLs of the most active subjects were 200 nucleotides longer than those of the least active subjects (7.1 and 6.9 kilobases, respectively; P= .006). This finding was confirmed in a small group of twin pairs discordant for physical activity level (on average, the LTL of more active twins was 88 nucleotides longer than that of less active twins; P= .03). A sedentary lifestyle (in addition to smoking, high body mass index, and low socioeconomic status) has an effect on LTL and may accelerate the aging process. This provides a powerful message that could be used by clinicians to promote the potentially antiaging effect of regular exercise.

Is telomere length in peripheral blood lymphocytes correlated with cancer susceptibility or radiosensitivity?

Mean terminal restriction fragment (TRF) lengths in white blood cells (WBCs) have been previously found to be associated with breast cancer. To assess whether this marker could be used as a test for breast cancer susceptibility in women, TRF length was measured in 72 treated female breast cancer patients and 1696 unaffected female controls between the ages of 45 and 77 from the Twin Research Unit at St Thomas' Hospital, as well as 140 newly diagnosed breast cancer cases and 108 mammographically screened unaffected controls from Guy's Hospital. Mean TRF was also tested for correlation with chromosome radiosensitivity and apoptotic response in the Guy's Hospital patients. After adjusting for age, smoking and body mass index, there was no significant difference in TRF lengths between the treated breast cancer patients and unaffected controls (P=0.71). A positive correlation between age-adjusted apoptotic response and mean TRF in newly diagnosed untreated breast cancer patients (P=0.008) was identified but no significant difference in TRF lengths between breast cancer patients and unaffected controls was detected (P=0.53). This suggests that TRF lengths in WBC, is not a marker of breast cancer susceptibility and does not vary significantly between affected women before and after treatment.