Quantitative fluorescence in situ hybridization for investigation of telomere length dynamics in the pituitary gland using samples from 128 autopsied patients.

In order to investigate the population dynamics of telomere status, we measured the telomere lengths of glandular cells in the adenohypophysis (AH) and pituicytes, a type of glial cell, in the neurohypophysis (NH) of 128 autopsied humans (65 men, 63 women, 0 and 102 years) using our original quantitative fluorescence in situ hybridization (Q-FISH) method. Telomeres in the AH shortened with aging in both men and women, but those of pituicytes did not. Pituicyte telomeres were significantly longer in women than in men. The data suggest that telomeres shorten with age in the AH, whereas those in pituicytes maintain a constant length throughout life. Comparison of pituicyte telomere lengths among 5 generations, <18, 18-69, 70-79, 80-89, and >90 years, revealed a tendency for telomeres to be longer in individuals in their 80 s and 90 s than in those in their 70 s. These findings lend support to the widely held notion that humans with longer telomeres may have a longer life span, and shed light on the biology of pituitary gland in terms of telomere length dynamics, as well contributing to the development of bioengineered hormone-producing cell replacement strategies and regenerative therapies.

Changes in telomere length with aging in human neurons and glial cells revealed by quantitative fluorescence in situ hybridization analysis.

AIM: The telomere is a structure present at the ends of chromosomes, and is known to shorten with aging and successive rounds of cell division. However, very little is known about telomere attrition in post-mitotic cells, such as neurons. METHODS: Using our originally developed quantitative fluorescence in situ hybridization method, we analyzed age-dependent alterations of telomere length in three types of cells in the human cerebrum: neurons and glial cells in both the gray and white matter. RESULTS: In adults, telomeres were significantly longer in neurons than in glial cells, whereas in infants, telomere lengths did not differ among the three cell types. No aging-related telomere attrition was evident in neurons. However, the telomeres of glial cells were shorter in older individuals than in younger individuals, and attrition was more rapid in the white matter than in the gray matter. CONCLUSIONS: The present results suggest that the telomeres of neurons remain stable throughout life, whereas telomeres in white matter glial cells become significantly shorter with age. Examination of adults showed no significant correlation between telomere length and age in the three cell types. Although the present study was cross-sectional, the results suggest that telomere shortening before adolescence contributes to the significant decrease of telomere length in white matter glial cells. The present findings in normal cerebral tissues will be informative for future studies of telomere stability in the diseased brain. Geriatr Gerontol Int 2018; 18: 1507-1512.

Correction to: Telomere length of gallbladder epithelium is shortened in patients with congenital biliary dilatation: measurement by quantitative fluorescence in situ hybridization.

In the original publication of this article, Fig. 5 was published with incorrect color of the approximate lines of CBD and Control.

Telomere length of gallbladder epithelium is shortened in patients with congenital biliary dilatation: measurement by quantitative fluorescence in situ hybridization.

BACKGROUND: Congenital biliary dilatation (CBD) is a congenital malformation involving both dilatation of the extrahepatic bile duct and pancreaticobiliary maljunction. Persistent reflux of pancreatic juice injures the biliary tract mucosa, resulting in chronic inflammation and higher rates of carcinogenesis in the biliary tract, including the gallbladder. Telomeres are repetitive DNA sequences located at the ends of chromosomes. Chromosomal instability due to telomere dysfunction plays an important role in the carcinogenesis of many organs. This study was performed to determine whether excessive shortening of telomeres occurs in the gallbladder mucosa of patients with CBD. METHODS: Resected gallbladders were obtained from 17 patients with CBD, ten patients with cholecystolithiasis without pancreatic juice reflux, and 17 patients with normal gallbladders (controls) (median age of each group of patients: 37, 50, and 53 years, respectively). The telomere lengths of the gallbladder epithelium were measured by quantitative fluorescence in situ hybridization using tissue sections, and the normalized telomere-to-centromere ratio (NTCR) was calculated. RESULTS: The NTCRs in the CBD, cholecystolithiasis, and control groups were 1.24 [interquartile range (IQR) 1.125-1.52], 1.96 (IQR 1.56-2.295), and 1.77 (IQR 1.48-2.53), respectively. The NTCR in the CBD group was significantly smaller than that in the cholecystolithiasis and control groups (p = 0.003 and 0.004, respectively), even in young patients. CONCLUSIONS: Our findings indicate that telomere shortening in the gallbladder mucosa plays an important role in the process of carcinogenesis in patients with CBD. These results support the recommendation of established guidelines for prophylactic surgery in patients with CBD because CBD is a premalignant condition with excessive telomere shortening.

Telomere attrition in beta and alpha cells with age.

We have reported telomere attrition in ß and α cells of the pancreas in elderly patients with type 2 diabetes, but it has not been explored how the telomere lengths of these islet cells change according to age in normal subjects. To examine the telomere lengths of ß and α cells in individuals without diabetes across a wide range of ages, we conducted measurement of the telomere lengths of human pancreatic ß and α cells obtained from 104 autopsied subjects without diabetes ranging in age from 0 to 100 years. As an index of telomere lengths, the normalized telomere-centromere ratio (NTCR) was determined for ß (NTCRß) and α (NTCRα) cells by quantitative fluorescence in situ hybridization (Q-FISH). We found NTCRß and NTCRα showed almost the same levels and both decreased according to age (p < 0.001 for both). NTCRs decreased more rapidly with age and were more widely distributed (p = 0.036 for NTCRß, p < 0.001 for NTCRα) in subjects under 18 years of age than in subjects over 18 years. There was a positive correlation between NTCRß and NTCRα only among adult subjects (p < 0.001). In conclusion, the telomeres of ß and α cells become shortened with normal aging process.

Changes of telomere status with aging: An update.

Accumulated data have shown that most human somatic cells or tissues show irreversible telomere shortening with age, and that there are strong associations between telomere attrition and aging-related diseases, including cancers, diabetes and cognitive disorders. Although it has been largely accepted that telomere attrition is one of the major causes of aging-related disorders, critical aspects of telomere biology remain unresolved, especially the lack of standardized methodology for quantification of telomere length. Another frustrating issue is that no potentially promising methods for safe prevention of telomere erosion, or for telomere elongation, have been devised. Here, we review several methods for quantification of telomere length currently utilized worldwide, considering their advantages and drawbacks. We also summarize the results of our recent studies of human cells and tissues, mainly using quantitative fluorescence in situ hybridization and Southern blotting, including those derived from patients with progeria-prone Werner syndrome and trisomy 21, and several strains of induced pluripotent stem cells. We discuss the possible merits of using telomere shortness as an indicator, or a new marker, for diagnosis of precancerous states and aging-related disorders. In addition, we describe newly found factors that are thought to impact telomere dynamics, providing a new avenue for examining the unsolved issues related to telomere restoration and maintenance.

Telomere attrition and restoration in the normal teleost Oryzias latipes are linked to growth rate and telomerase activity at each life stage.

Telomere shortening occurs when cells divide, both in vitro and in vivo. On the other hand, telomerase is able to maintain telomere length in cells by adding TTAGGG repeats to the ends of telomeres. However, the interrelationships existing among telomere length, telomerase activity and growth in vertebrates remain to be clarified. In the present study we measured telomere length (terminal restriction fragment length), telomerase activity and body growth of Oryzias latipes from the embryo stage until senescence. During the rapid growth stage (age 0-7 months), telomeres shortened in parallel with decreasing telomerase activity. Then, during adolescence (age 7 months - 1 year), telomeres lengthened quickly as growth slowed and telomerase activity increased. In the adult stage (age 1-4 years) characterized by little growth, telomerase activity decreased gradually and telomeres shortened. Our data indicate that telomere attrition and restoration are linked to growth and telomerase activity, and suggest that critical loss of telomere homeostasis is associated with mortality in this animal.

Gradual telomere shortening and increasing chromosomal instability among PanIN grades and normal ductal epithelia with and without cancer in the pancreas.

A large body of evidence supports a key role for telomere dysfunction in carcinogenesis due to the induction of chromosomal instability. To study telomere shortening in precancerous pancreatic lesions, we measured telomere lengths using quantitative fluorescence in situ hybridization in the normal pancreatic duct epithelium, pancreatic intraepithelial neoplasias (PanINs), and cancers. The materials employed included surgically resected pancreatic specimens without cancer (n = 33) and with invasive ductal carcinoma (n = 36), as well as control autopsy cases (n = 150). In comparison with normal ducts, telomere length was decreased in PanIN-1, -2 and -3 and cancer. Furthermore, telomeres were shorter in cancer than in PanIN-1 and -2. Telomere length in cancer was not associated with histological type, lesion location, or cancer stage. PanINs with or without cancer showed similar telomere lengths. The incidences of atypical mitosis and anaphase bridges, which are morphological characteristics of chromosomal instability, were negatively correlated with telomere length. The telomeres in normal duct epithelium became shorter with aging, and those in PanINs or cancers were shorter than in age-matched controls, suggesting that telomere shortening occurs even when histological changes are absent. Our data strongly suggest that telomere shortening occurs in the early stages of pancreatic carcinogenesis and progresses with precancerous development. Telomere shortening and chromosomal instability in the duct epithelium might be associated with carcinogenesis of the pancreas. Determination of telomere length in pancreatic ductal lesions may be valuable for accurate detection and risk assessment of pancreatic cancer.

Arm-specific telomere dynamics of each individual chromosome in induced pluripotent stem cells revealed by quantitative fluorescence in situ hybridization.

We have reported that telomere fluorescence units (TFUs) of established induced pluripotent stem cells (iPSCs) derived from human amnion (hAM933) and fetal lung fibroblasts (MRC-5) were significantly longer than those of the parental cells, and that the telomere extension rates varied quite significantly among clones without chromosomal instability, although the telomeres of other iPSCs derived from MRC-5 became shorter as the number of passages increased along with chromosomal abnormalities from an early stage. In the present study we attempted to clarify telomere dynamics in each individual chromosomal arm of parental cells and their derived clonal human iPSCs at different numbers of passages using quantitative fluorescence in situ hybridization (Q-FISH). Although no specific arm of any particular chromosome appeared to be consistently shorter or longer than most of the other chromosomes in any of the cell strains, telomere elongation in each chromosome of an iPSC appeared to be random and stochastic. However, in terms of the whole genome of any specific cell, the telomeres showed overall elongation associated with iPSC generation. We have thus demonstrated the specific telomere dynamics of each individual chromosomal arm in iPSCs derived from parental cells, and in the parental cells themselves, using Q-FISH.

ß-cell telomere attrition in diabetes: inverse correlation between HbA1c and telomere length.

CONTEXT: Although accelerated ß-cell telomere shortening may be associated with diabetes that shows a dramatically increased incidence with aging, ß-cell telomere length in diabetes has never been explored. OBJECTIVE: The objective of the present study was to examine telomere length in the ß-cells of patients with diabetes. DESIGN AND PATIENTS: We determined telomere length in ß- and α-cells of pancreases obtained at autopsy from 47 patients with type 2 diabetes and 51 controls, all older than 60 years. MAIN OUTCOME MEASURE: The normalized telomere to centromere ratio (NTCR), an index of telomere length, was determined for ß- (NTCRß) and α- (NTCRα) cells by quantitative fluorescence in situ hybridization. RESULTS: The NTCRß was reduced by 27% ± 25% and NTCRα by 15% ± 27% in the patients with diabetes relative to the controls (P < .01 for both). Importantly, the degree of shortening was significantly (P < .01) greater in ß-cells than in α-cells. The histogram of NTCR distribution was significantly skewed to the left in the patients with diabetes relative to the controls for both ß- and α-cells, indicating preferential depletion of longer-telomere islet cells. Glycated hemoglobin was negatively correlated with ß-cell telomere length, and the telomeres were significantly shorter in patients who had used hypoglycemic agents than in those who had not. CONCLUSION: The telomeres of ß-cells are shortened in patients with type 2 diabetes. There may be a vicious cycle involving ß-cell telomere attrition and sustained hyperglycemia.

Q-FISH measurement of hepatocyte telomere lengths in donor liver and graft after pediatric living-donor liver transplantation: donor age affects telomere length sustainability.

Along with the increasing need for living-donor liver transplantation (LDLT), the issue of organ shortage has become a serious problem. Therefore, the use of organs from elderly donors has been increasing. While the short-term results of LDLT have greatly improved, problems affecting the long-term outcome of transplant patients remain unsolved. Furthermore, since contradictory data have been reported with regard to the relationship between donor age and LT/LDLT outcome, the question of whether the use of elderly donors influences the long-term outcome of a graft after LT/LDLT remains unsettled. To address whether hepatocyte telomere length reflects the outcome of LDLT, we analyzed the telomere lengths of hepatocytes in informative biopsy samples from 12 paired donors and recipients (grafts) of pediatric LDLT more than 5 years after adult-to-child LDLT because of primary biliary atresia, using quantitative fluorescence in situ hybridization (Q-FISH). The telomere lengths in the paired samples showed a robust relationship between the donor and grafted hepatocytes (r = 0.765, p = 0.0038), demonstrating the feasibility of our Q-FISH method for cell-specific evaluation. While 8 pairs showed no significant difference between the telomere lengths for the donor and the recipient, the other 4 pairs showed significantly shorter telomeres in the recipient than in the donor. Multiple regression analysis revealed that the donors in the latter group were older than those in the former (p = 0.001). Despite the small number of subjects, this pilot study indicates that donor age is a crucial factor affecting telomere length sustainability in hepatocytes after pediatric LDLT, and that the telomeres in grafted livers may be elongated somewhat longer when the grafts are immunologically well controlled.

Quantitative fluorescence in situ hybridization measurement of telomere length in skin with/without sun exposure or actinic keratosis.

Chromosomal and genomic instability due to telomere dysfunction is known to play an important role in carcinogenesis. To study telomere shortening in the epidermis surrounding actinic keratosis, we measured telomere lengths of basal, parabasal, and suprabasal cells in epidermis with actinic keratosis (actinic keratosis group, n = 18) and without actinic keratosis (sun-protected, n = 15, and sun-exposed, n = 13 groups) and in actinic keratosis itself as well as in dermal fibroblasts in the 3 groups, using quantitative fluorescence in situ hybridization. Among the 3 cell types, telomeres of basal cells were not always the longest, suggesting that tissue stem cells are not necessarily located among basal cells. Telomeres of basal cells in the sun-exposed group were shorter than those in the sun-protected group. Telomeres in the background of actinic keratosis and in actinic keratosis itself and those of fibroblasts in actinic keratosis were significantly shorter than those in the controls. Our findings demonstrate that sun exposure induces telomere shortening and that actinic keratosis arises from epidermis with shorter telomeres despite the absence of any histologic atypia.

Short telomeres and chromosome instability prior to histologic malignant progression and cytogenetic aneuploidy in papillary urothelial neoplasms.

PURPOSE: Evaluation of the relationships existing among 3 histologic types of urothelial tumors, chromosomal instability, and telomere length. PATIENTS AND METHODS: We examined 37 consecutive cases of papillary urothelial neoplasm, from which 26 (70.3%) were suitable for karyotype analysis, comprising 7 papillary urothelial neoplasms of low malignant potential (PUNLMPs), 10 low-grade papillary urothelial carcinomas (PUCs), and 9 high-grade PUCs. We performed karyotype and anaphase bridge analyses, and measured telomere lengths by quantitative fluorescence in situ hybridization. RESULTS: PUNLMPs were always diploid and had anaphase bridges. Low-grade PUCs showed diploidy (n = 2), hypoploidy (n = 4) and polyploidy (n = 4), and high-grade PUCs showed diploidy (n = 1) and polyploidy (n = 8); both had anaphase bridges. The incidence of anaphase bridges did not differ significantly between PUNLMPs and high-grade PUCs (P = 0.105). The telomere lengths of PUNLMP, low-grade PUC, and high-grade PUC, expressed as mean telomere fluorescence units (TFU) ± SD, were 7906 ± 3197, 4893 ± 1567, and 3299 ± 1406, respectively. The differences among the 3 groups were significant. However, 42.9% of the PUNLMPs had shorter telomeres than the mean value for low-grade PUCs, and 30.0% of the low-grade PUCs had shorter telomeres than those for high-grade PUCs. There was an inverse correlation between telomere length and the incidence of anaphase bridges. CONCLUSIONS: PUNLMP appears to progress to low-grade PUC and high-grade PUC in association with telomere shortening and chromosomal instability. Our data suggest that critically shortened telomeres cause chromosomal instability during progression of papillary urothelial neoplasms.

Association of telomere shortening in myocardium with heart weight gain and cause of death.

We attempted to clarify myocardial telomere dynamics using samples from 530 autopsied patients using Southern blot analysis. Overall regression analysis demonstrated yearly telomere reduction rate of 20 base pairs in the myocardium. There was a significant correlation between myocardial telomere and aging. Moreover, regression analyses of telomere and heart weight yielded a telomere reduction rate of 3 base pairs per gram, and a small but significant correlation between telomere reduction and heart weight was demonstrated. Hearts of autopsied patients who had died of heart disease were significantly heavier than those of patients who had died of cancer or other diseases, and heart disease was significantly more correlated with myocardial telomere shortening than cancer or other diseases. Here we show that telomeres in myocardial tissue become shortened with aging and heart disease, and that heart disease was associated with a gain of heart weight and telomere shortening in the myocardium.

Investigation of telomere length dynamics in induced pluripotent stem cells using quantitative fluorescence in situ hybridization.

Here we attempted to clarify telomere metabolism in parental cells and their derived clonal human induced pluripotent stem cells (iPSCs) at different passages using quantitative fluorescence in situ hybridization (Q-FISH). Our methodology involved estimation of the individual telomere lengths of chromosomal arms in individual cells within each clone in relation to telomere fluorescence units (TFUs) determined by Q-FISH. TFUs were very variable within the same metaphase spread and within the same cell. TFUs of the established iPSCs derived from human amnion (hAM933 iPSCs), expressed as mean values of the median TFUs of 20 karyotypes, were significantly longer than those of the parental cells, although the telomere extension rates varied quite significantly among the clones. Twenty metaphase spreads from hAM933 iPSCs demonstrated no chromosomal instability. The iPSCs established from fetal lung fibroblasts (MRC-5) did not exhibit telomere shortening and chromosomal instability as the number of passages increased. However, the telomeres of other iPSCs derived from MRC-5 became shorter as the number of passages increased, and one (5%) of 20 metaphase spreads showed chromosomal abnormalities including X trisomy at an early stage and all 20 showed abnormalities including X and 12 trisomies at the late stage.

Accelerated in vivo epidermal telomere loss in Werner syndrome.

Many data pertaining to the accelerated telomere loss in cultured cells derived from Werner syndrome (WS), a representative premature aging syndrome, have been accumulated. However, there have been no definitive data on in vivo telomere shortening in WS patients. In the present study, we measured terminal restriction fragment (TRF) lengths of 10 skin samples collected from extremities of 8 WS patients aged between 30 and 61 years that had been surgically amputated because of skin ulceration, and estimated the annual telomere loss. Whereas the values of TRF length in younger WS patients (in their thirties) were within the normal range, those in older WS patients were markedly shorter relative to non­WS controls. Regression analyses indicated that the TRF length in WS was significantly shorter than that in controls (p < 0.001). Furthermore, we found that TRF lengths in muscle adjacent to the examined epidermis were also significantly shorter than those of controls (p = 0.047). These data demonstrate for the first time that in vivo telomere loss is accelerated in systemic organs of WS patients, suggesting that abnormal telomere erosion is one of the major causes of early onset of age­related symptoms and a predisposition to sarcoma and carcinoma in WS.

Changes of telomere length with aging.

We reviewed our methodology and results of telomere measurements, with reference to telomere length and aging. Human tissues always showed telomere shortening with age, except for the brain and myocardium. Yearly rates of telomere length reduction in various tissues were mostly within the range 20-60 bp, and thus compatible with that expected from only one round of mitosis. It was suggested that when telomeres were found to be longer in any specific organ in a given individual, then the other organs in that individual would also have longer telomeres. Using the quantitative fluorescence in situ hybridization (Q-FISH) method for telomere measurement, we were able to measure the telomere lengths of various cell types within tissues. Here we summarize the results obtained for various cell types in the stomach, tongue and breast. Our Q-FISH method using our original software program "Tissue Telo" is excellent for measuring telomere lengths using tissue sections and PNA probes.

Telomere shortening in Barrett's mucosa and esophageal adenocarcinoma and its association with loss of heterozygosity.

OBJECTIVE: Telomere shortening is thought to be associated with genetic instability. The purpose of this study was to measure telomere length in a series of Barrett's adenocarcinomas (BAs), focusing on the telomere/centromere fluorescent intensity ratio (TCR) with tissue quantitative fluorescent in situ hybridization (Q-FISH). MATERIAL AND METHODS: A total of 11 cases of BA were evaluated for upper esophagus (UE), lower esophagus (LE), Barrett's mucosa (BM), BA, and gastric cardiac mucosa (GC). Q-FISH was performed using two kinds of peptide nucleic acid probe, specific for telomeres and centromeres. The sections were analyzed with a CCD camera and original software (Tissue Telo) for measuring TCR. In addition, Laser Capture Microdissection and GeneScan were implemented for evaluation of genetic instability. RESULTS: The TCR values in BM and, to a lesser extent, BA were significantly lower than those in the other tissues, particularly in heterozygosity (LOH)-positive cases. However, no significant difference was evident between microsatellite instability (MSI)-positive and -negative groups. CONCLUSIONS: In our study of BA series, telomere length appeared to change with the degree of histological atypia, with decreases linked to LOH.

Basal cells have longest telomeres measured by tissue Q-FISH method in lingual epithelium.

We investigated the telomere lengths of individual cell types in lingual mucosa using an improved tissue quantitative fluorescence in situ hybridization (Q-FISH) method. Our tissue Q-FISH method compensates for partially cut nuclei in a tissue section by using the telomere:centromere ratio (TCR). We normalized our TCR measurements (NTCR) using a section from a block of cultured cells placed on the same slide, thus improving the accuracy and reproducibility of the results. Normal lingual mucosa was obtained from 21 autopsied individuals. Immunohistochemistry showed positivity mainly for p27, p63, and CK19 in basal cells, and for Ki-67 in parabasal cells. Q-FISH revealed that NTCR was significantly highest in basal cells and lowest in prickle cells, and also that telomere length regressed at a certain rate in each cell type, firstly. Significant correlations of NTCR among the three epithelial cell types were demonstrated. The present findings appear to support the theory that stem cells exist in the basal layer of the lingual epithelium. The reduction of telomere length with age and in each cell layer is consistent with the telomere biology theory of cell proliferation and differentiation in oral mucosa.