Correlation Between Differentiation of Adrenocortical Zones and Telomere Lengths Measured by Q-FISH.

CONTEXT: Adrenocortical zonation is associated with a markedly complex developmental process, and the pathogenesis and/or etiology of many disorders of adrenocortical zonal development have remained unknown. Cells from the three adrenocortical zones are morphologically and functionally differentiated, and the mature stage of cell development or senescence has been recently reported to be correlated with telomere length. However, the telomere length of each adrenocortical zonal cell has not yet been studied in human adrenal glands. OBJECTIVE: We aimed to study the telomere lengths of adrenocortical parenchymal cells from three different zones of the adrenal glands present during childhood, adolescence, and adulthood. METHODS: Adrenal glands of 30 autopsied subjects, aged between 0 and 68 years, were retrieved from pathology files. The normalized telomere to centromere ratio (NTCR), an index of telomere length, was determined in the parenchymal cells of the zona glomerulosa, zona fasciculata, and zona reticularis (ZR), using quantitative fluorescence in situ hybridization. RESULTS: NTCR of ZR cells was the longest, followed in decreasing order by that of zona glomerulosa and zona fasciculata cells in subjects aged 20 to 68 years, but no substantial differences in NTCR were detected among these three zones in the group <20 years of age. NTCR of ZR increased with age in subjects aged 20 to 68 years, whereas no important age-dependent changes in NTCR were detected in the group <20 years of age. CONCLUSION: The telomere lengths for three zones in adrenal cortex were correlated with their differentiation in adulthood but not in childhood and adolescence.

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.

Pancreatic cancer stem cells: features and detection methods.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a high incidence of distant metastasis and recurrence. Cancer stem cells (CSCs), which are pluripotent, self-renewable, and capable of forming tumors, contribute to PDAC initiation and metastasis and are responsible for resistance to chemotherapy and radiation. Three types of experimental methods are commonly used to identify CSCs: CSC-specific marker detection, a sphere-formation assay that reveals cell proliferation under non-adherent conditions, and detection of side-population (SP) cells that possess high intracellular-to-extracellular pump functions. Several CSC-specific markers have been reported in PDACs, including CD133, CD24, CD44, CXCR4, EpCAM, ABCG2, c-Met, ALDH-1, and nestin. There remains controversy regarding which markers are specific to PDAC CSCs and which are expressed alone or in combination in CSCs. Examining characteristics of isolated CSCs and discovering CSC-specific treatment options are important to improve the prognosis of PDAC cases. This review summarizes CSC-detection methods for PDAC, including CSC-marker detection, the sphere-formation assay, and detection of SP cells.

Reduced expression of the H19 long non-coding RNA inhibits pancreatic cancer metastasis.

H19 is an oncofetal RNA expressed in the developing embryo as well as in bladder, breast, gastric, pancreatic, hepatocellular, and prostate cancers. Recent studies have shown that H19 enhances cancer invasion and metastasis; however, its roles in cancer remain controversial. In the current study, H19 exhibited the second largest increase (82.4-fold) and represented the only non-protein coding gene among 11 genes identified that were elevated over 10-fold in lung-metastasis-derived pancreatic cancer cells compared with their parental cells using a mouse metastatic model. Subsequently, we further clarified the roles of H19 in pancreatic cancer growth and metastasis using in vitro and in vivo techniques. In situ hybridization showed that H19 was detected in 23 of 139 invasive ductal carcinomas (17%), and that H19 expression positively correlated with higher histological grades (P < 0.0001). Overexpression of H19 in PANC-1 pancreatic cancer cells induced higher motilities, whereas H19 inhibition using shRNA and siRNA showed opposite results; however, cell growth rates were not impacted. Intravenous injection of H19 shRNA vector-transfected PANC-1 cells yielded marked inhibition of metastasis in the liver and lungs of immunodeficient mice. These findings suggest that H19 has important roles in pancreatic cancer metastasis, and that inhibition of H19 represents a novel candidate for pancreatic cancer therapy.

Electron microscopic analysis of different cell types in human pancreatic cancer spheres.

Cancer stem cells (CSCs), which are pluripotent and self-renewable, contribute to the initiation and metastasis of cancer, and are responsible for resistance to chemotherapy and radiation. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive type of cancer that is associated with a high incidence of distant metastasis and recurrence. Sphere formation reveals cell proliferation under nonadherent conditions and is commonly used to identify CSCs; measurements of the number, area and volume of the spheres are used to estimate stemness of PDAC cells. However, detailed morphological analysis of such spheres has not been performed. The aim of the present study was to examine the morphology of spheres isolated from PANC-1 human pancreatic cancer cells via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). PANC-1 cells formed round to irregular oblong spheres within 1 week following seeding in ultra-low-attachment plates. These spheres exhibited higher levels of expression of CSC markers, including nestin, sex determining region Y-box 2, and CD44 containing variant exon 9, compared with adherent cells. SEM analysis revealed that the spheres exhibited a grape-like appearance, harboring cancer cells with smooth or rough surfaces. Similarly, TEM analysis detected cancer cells with varying surface types within the spheres: Those with smooth surfaces, irregular large protrusions, protrusions and a small number of microvilli, and those with many microvilli throughout the entire cell surface. These morphological differences among cancer cells may be indicative of different stages in the differentiation process, from CSCs to non-CSCs, within the spheres.

Stemness and anti-cancer drug resistance in ATP-binding cassette subfamily G member 2 highly expressed pancreatic cancer is induced in 3D culture conditions.

The expression of ATP-binding cassette subfamily G member 2 (ABCG2) is related to tumorigenic cancer stem cells (CSC) in several cancers. However, the effects of ABCG2 on CSC-related malignant characteristics in pancreatic ductal adenocarcinoma (PDAC) are not well elucidated. In this study, we compared the characteristics of low (ABCG2-) and high (ABCG2+)-ABCG2-expressing PDAC cells after cell sorting. In adherent culture condition, human PDAC cells, PANC-1, contained approximately 10% ABCG2+ cell populations, and ABCG2+ cells displayed more and longer microvilli compared with ABCG2- cells. Unexpectedly, ABCG2+ cells did not show significant drug resistance against fluorouracil, gemcitabine and vincristine, and ABCG2- cells exhibited higher sphere formation ability and stemness marker expression than those of ABCG2+ cells. Cell growth and motility was greater in ABCG2- cells compared with ABCG2+ cells. In contrast, epithelial-mesenchymal transition ability between ABCG2- and ABCG2+ cells was comparable. In 3D culture conditions, spheres derived from ABCG2- cells generated a large number of ABCG2+ cells, and the expression levels of stemness markers in these spheres were higher than spheres from ABCG2+ cells. Furthermore, spheres containing large populations of ABCG2+ cells exhibited high resistance against anti-cancer drugs presumably depending on ABCG2. ABCG2+ cells in PDAC in adherent culture are not correlated with stemness and malignant behaviors, but ABCG2+ cells derived from ABCG2- cells after sphere formation have stemness characteristics and anti-cancer drug resistance. These findings suggest that ABCG2- cells generate ABCG2+ cells and the malignant potential of ABCG2+ cells in PDAC varies depending on their environments.

Clinicopathological characteristics of distant metastases of adenocarcinoma, squamous cell carcinoma and urothelial carcinoma: An autopsy study of older Japanese patients.

AIM: We aimed to clarify the characteristics of malignancies in older adults focusing on distant metastasis in the whole body. METHODS: We retrospectively evaluated 7710 cases of autopsies (4011 men, 3699 women, median age of 80 years), and analyzed the characteristics of metastasis of adenocarcinoma, squamous cell carcinoma and urothelial carcinoma in each organ. RESULTS: The total number of cases with adenocarcinoma, squamous cell carcinoma or urothelial carcinoma was 2856, and most of them were adenocarcinomas. Among them, 1604 had metastatic lesions, and patients with metastasis were younger than those without metastasis. The major primary organs of adenocarcinoma were the stomach, colon, lung, prostate, gallbladder and pancreas, whereas those for squamous cell carcinoma were the lung, esophagus and uterus. Urothelial carcinoma cases were found in the urinary bladder, kidney and ureter. Metastatic adenocarcinomas mainly originated from the stomach, colon, lung, pancreas and gallbladder. Metastatic squamous cell carcinomas were from the lung, esophagus and uterus, whereas the kidney, bladder and ureter were the primary origins of metastatic urothelial carcinomas. Squamous cell carcinoma showed the highest incidence of metastasis, suggestive of it being of an aggressive phenotype. Furthermore, metastatic ability and the preferred metastatic sites varied among primary organs. CONCLUSIONS: We revealed an accurate incidence and the characteristics of metastatic cancer in a large-scale autopsy study of older Japanese patients from one institution. Identifying these features might prompt screening for malignancies, and consequently improve quality of life for older adults. Geriatr Gerontol Int 2018; 18: 211-215.

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.

Maternal grafts protect daughter recipients from acute cellular rejection after pediatric living donor liver transplantation for biliary atresia.

Some studies have found that gender mismatch between donors and recipients are related to poor graft prognosis after liver transplantation. However, few studies have investigated the impact of gender mismatch on acute cellular rejection (ACR) in pediatric living donor liver transplantation (LDLT). This retrospective study investigated the clinical significance of these factors in ACR after pediatric LDLT. Between November 2001 and February 2012, 114 LDLTs were performed for recipients with biliary atresia (BA) using parental grafts. We performed univariate and multivariate analyses to identify the factors associated with ACR. The donor-recipient classifications included mother donor to daughter recipient (MD; n = 43), mother to son (n = 18), father to daughter (FD; n = 33), and father to son (n = 20) groups. The overall incidence rate of ACR in the recipients was 36.8%. Multivariate analysis showed that gender mismatch alone was an independent risk factor for ACR (P = 0.012). The FD group had a higher incidence of ACR than the MD group (P = 0.002). In LDLT, paternal grafts with gender mismatch were associated with a higher increased incidence of ACR than maternal grafts with gender match. Our findings support the possibility that maternal antigens may have an important clinical impact on graft tolerance in LDLT for patients with BA.