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.

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.

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.

Alcoholics show reduced telomere length in the oesophagus.

Telomeres are repetitive G-rich DNA sequences located at the ends of chromosomes. Chromosomal and genomic instability due to telomere dysfunction plays an important role in carcinogenesis. To study telomere shortening in the oesophageal epithelium of alcoholics, we measured the telomere lengths of basal and parabasal cells in comparison with those of non-alcoholics using Q-FISH and our original software, Tissue Telo, and also assessed histological inflammation. Telomeres in basal cells were significantly shorter in alcoholics than in age-matched normal controls. Prominent histological findings of chronic inflammation were not evident in either alcoholics or non-alcoholics. Our finding that telomeres in the oesophageal epithelium are shorter in alcoholics than in non-alcoholics indicates that telomere shortening may be associated with the frequent occurrence of squamous cell carcinoma in alcoholics. Further studies to clarify the reason for the large annual loss of telomere length with rapid turnover or lower telomerase activity in the oesophageal epithelium of alcoholics will be necessary.

Short telomeres in an oral precancerous lesion: Q-FISH analysis of leukoplakia.

OBJECTIVES: A precancerous condition is a lesion that, if left untreated, leads to cancer or can be induced to become malignant. In the oral region, leukoplakia is a lesion that has been regarded as precancerous. In cases of oral carcinoma, we have frequently noticed that a type of leukoplakia histologically demonstrating hyper-orthokeratosis and mild atypia (ortho-keratotic dysplasia; OKD) is often associated with carcinoma, either synchronously or metachronously. Therefore, we consider OKD-type leukoplakia to be a true precancerous lesion. MATERIALS AND METHODS: In an attempt to clarify the relationship between OKD as a precancerous condition in the oral mucosa and telomere length, we estimated telomere lengths in this type of leukoplakia using quantitative fluorescence in situ hybridization, and also quantified the frequency of anaphase-telophase bridges (ATBs) in comparison with squamous cell carcinoma in situ (CIS) and the background tissues of CIS and OKD. RESULTS: Ortho-keratotic dysplasia was frequently associated with squamous cell carcinoma (45.0%) and showed significantly shorter telomeres than normal control epithelium, CIS, or the background of CIS or OKD. The frequency of ATBs was much higher in OKD than in control epithelium or CIS. CONCLUSION: Ortho-keratotic dysplasia appears to be frequently associated with carcinoma, chromosomal instability, and excessively shortened telomeres, not only in the lesion itself but also in the surrounding background. Therefore, when this type of leukoplakia is recognized in the oral region, strict follow-up for oral squamous cell carcinoma is necessary, focusing not only on the areas of leukoplakia, but also the surrounding background.

Hepatocellular telomere length in biliary atresia measured by Q-FISH.

BACKGROUND: Liver transplantation for biliary atresia is indicated whenever a Kasai portoenterostomy is considered unfeasible. However, the timing of liver transplantation in biliary atresia has not been precisely defined. Excessive shortening of hepatocellular telomeres may occur in patients with biliary atresia, and therefore, telomere length could be a predictor of hepatocellular reserve capacity. METHODS: Hepatic tissues were obtained from 20 patients with biliary atresia who underwent LT and 10 age-matched autopsied individuals (mean age, 1.7 and 1.2 years, respectively). Telomere lengths were measured by Southern blotting and quantitative fluorescence in situ hybridization using the normalized telomere-centromere ratio. The correlation between the normalized telomere-centromere ratio for the hepatocytes in biliary atresia and the pediatric end-stage liver disease score was analyzed. RESULTS: The median terminal restriction fragment length of the hepatic tissues in biliary atresia was not significantly different from that of the control (p = 0.425), whereas the median normalized telomere-centromere ratio of hepatocytes in biliary atresia was significantly smaller than that of the control (p < 0.001). Regression analysis demonstrated a negative correlation of the normalized telomere-centromere ratio with the pediatric end-stage liver disease score in biliary atresia (p < 0.001). CONCLUSIONS: Telomere length analysis using quantitative fluorescence in situ hybridization could be an objective indicator of hepatocellular reserve capacity in patients with biliary atresia, and excessive telomere shortening supports the early implementation of liver transplantation.

Q-FISH analysis of telomere and chromosome instability in the oesophagus with and without squamous cell carcinoma in situ.

Chromosomal and genomic instability due to telomere dysfunction is known to play an important role in carcinogenesis. To study telomere dysfunction in the surrounding background epithelium of squamous cell carcinoma in situ (CIS) of the oesophagus, we measured telomere lengths of basal and parabasal cells of epithelia with and without CIS using quantitative fluorescence in situ hybridization (Q-FISH) and our original software, Tissue Telo. Additionally, we assessed histological inflammation and the anaphase bridge index. In non-cancerous epithelium, telomeres in basal cells were significantly longer than those in parabasal cells, whereas CIS showed a homogeneous telomere pattern in the basal and parabasal cells. Telomeres in basal and parabasal cells were significantly shorter in the background with CIS than in epithelium from age-matched normal controls. Significant negative correlation was observed between the normalized telomere : centromere ratio (reflected telomere length) and the anaphase bridge index in non-cancerous epithelia from both normal controls and the CIS background with no histological inflammation. These findings indicate that tissue stem cells may be located among basal cells, and that telomere length distribution in component cell types differs between CIS and non-cancerous epithelium. We have demonstrated conclusively that oesophageal CIS arises from epithelium with short telomeres and chromosomal instability in the absence of histological inflammation.

[Telomere].

We reviewed correlations among telomere length, aging and carcinogenesis. Southern blot analysis showed that telomere shortening occurred with aging in all human tissues except for brain and myocardium. We investigated the telomere lengths of individual cell types in human tissues using a Q-FISH method and an original software package, "Tissue Telo". Q-FISH revealed that in squamous epithelia, NTCR corresponding to telomere length was significantly highest in basal cells and lowest in prickle cells, and that telomere length regressed at a certain rate in each cell type except for fibroblasts. Mean telomere length was significantly less in background tissue squamous epithelia of patients with cancer than in normal controls without cancer. We demonstrated telomere shortening and chromosomal instability in the background and normal control with excessively shortened telomeres. The data suggest that cancer arises from epithelial cells with short telomeres.

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.

The teleost Oryzias latipes shows telomere shortening with age despite considerable telomerase activity throughout life.

Previous studies of telomeres and telomerase have focused mostly on mammals, and data for other vertebrates are limited. We analyzed both telomere length (terminal restriction fragment length) and telomerase activity in a small freshwater teleost fish, the medaka (Oryzias latipes), and found that the telomeres shorten during ageing despite the fact that a considerable amount of telomerase activity is ubiquitously detectable throughout the life of the fish. Since the telomere attrition rate during development was greater than that in adulthood, telomere length is inversely correlated with the increase in body length. The difference in telomere length among medaka individuals was similar to that in humans, and the individual specific differences were evident even at the earliest embryonic stage. Telomerase activity was ubiquitously detectable not only in the body of the embryo but also in the systemic organs of mature individuals throughout their entire life span. These data suggest that telomere attrition during ageing in medaka, which is similar to that in humans, may be a major factor determining their mortality, and that telomere maintenance through strong telomerase activity may be required for the characteristic lifelong continuous growth of this fish.

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.

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.

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.

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.

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.

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.

Telomeric DNA length in cerebral gray and white matter is associated with longevity in individuals aged 70 years or older.

Many studies have demonstrated the association between telomere length in mitotic cells and carcinogenesis and mortality, but little attention has been focused on post-mitotic cells and human life expectancy. We assessed the relationship between telomere length in cerebral gray and white matter and longevity in 72 autopsied Japanese patients aged 0-100 years using Southern blot hybridization. The mean telomere lengths in the gray and white matter were 12.3+/-2.5 kilobase pairs and 11.4+/-2.1 kilobase pairs, respectively. The mean telomere lengths in 60-69 year decadal group were less than those of neonates, and declined further in the 70-79-year age group, but those in groups of further advanced age were longer than in the 70-79 year group (70-79<80-89<90-100 years of age). Thus, the 90-100-year age group possessed significantly longer telomeres than the 70s (p=0.029). Autopsy protocols showed a decrease in the rate of cancer death in individuals in their 80s (p=0.041) and 90s (p=0.017) versus those in their 60s, and in their 80s the mean telomere length in the gray matter from cancer death patients was significantly shorter than that of patients who died of other diseases (p=0.04). These data suggest that innate telomere lengths are maintained very well in the cerebrum, and are associated with longevity. Our study lends indispensable support to the hypothesis that longer telomeres protect the genome from instability (a major cause of carcinogenesis) and are beneficial for longevity.

Telomere length variations in 6 mucosal cell types of gastric tissue observed using a novel quantitative fluorescence in situ hybridization method.

We developed a novel method for evaluating telomere length in 6 cell types of noncancerous and cancerous mucosal tissues from 11 cases of gastric neoplasm using the quantitative fluorescence in situ hybridization method with telomere and centromere peptide nucleic acid probes. Our telomere length estimates were determined from the background-corrected telomere intensity divided by the background-corrected centromere intensity (telomere-to-centromere ratio). Our results indicated that telomere lengths in each of the cases studied were reduced in turn from fibroblasts to fundic gland cells, to glandular neck cells, and then to surface foveolar cells. However, the telomere lengths of intestinalized cells located among fundic glands were not always shorter than those of the other cell types, as reported previously by others. Helicobacter pylori infection was suggested to induce the telomere shortening seen in the fundic glands. Although the mean telomere lengths varied among the 8 gastric cancer cases, correlation of the telomere lengths with the Ki-67 labeling index was established after normalization with the fibroblast measurements. We conclude that our telomere-to-centromere ratio method can reliably estimate the telomere lengths of the 6 cell types in the gastric mucosa and clarifies the relationship between proliferative activity and the telomere length of cancer cells.