Transcriptase Telomerase Research Articles

A shorter telomere is the key factor in preventing cultured human mesenchymal stem cells from senescence escape

Mesenchymal stem cells (MSCs) from various animals undergo spontaneous transformation in vitro,establishing some malignant characteristics. However,this phenomenon seems seldom appearing in human (h)MSCs. To address the question whether the hMSCs really do not undergo the spontaneous transformation and why,the present study compared MSCs from two species under the same conditions, the commercialized primary hMSCs whose in vitro life span is very uniform, and the rat (r)MSCs whose spontaneous transformation in vitro is well defined.It was demonstrated that in rMSCs, there were small numbers of re-proliferating cells appearing after a substantial senescent period. These “senescence-escaped”rMSCs were highly proliferative and did not show any sign of growth arrest during the following subcultures upto observed passage 32. Whereas after entering senescence, hMSCs no longer re-proliferated and finally died from apoptosis. Compared with rMSCs, the hMSCs possessed a much shorter telomere, and lacked both telomerase reverse transcriptase expression and telomerase activity. When proliferating from pre-senescent to senescent stages,the hMSCs had a greater loss of relative telomere length(51 % in hMSC vs. 15 % in rMSC), but both cells displayed a similar average telomere shortening per population doubling (0.50 ± 0.06 kb in rMSC vs. 0.49 ± 0.06 kbin hMSC; p > 0.05), indicating that the greater relative shortening of the hMSC telomeres was due to their original shorter length, rather than lack of telomere maintenance mechanisms. In conclusion, the hMSCs do not spontaneously initiate transformation, because they cannot escape senescence. This is particularly due to their much shorter telomere.

Telomerase and Telomere Length in Pulmonary Fibrosis

In addition to its expression in stem cells and many cancers, telomerase activity is transiently induced in murine bleomycin (BLM)-induced pulmonary fibrosis with increased levels of telomerase transcriptase (TERT) expression, which is essential for fibrosis. To extend these observations to human chronic fibrotic lung disease, we investigated the expression of telomerase activity in lung fibroblasts from patients with interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF). The results showed that telomerase activity was induced in more than 66% of IPF lung fibroblast samples, in comparison with less than 29% from control samples, some of which were obtained from lung cancer resections. Less than 4% of the human IPF lung fibroblast samples exhibited shortened telomeres, whereas less than 6% of peripheral blood leukocyte samples from patients with IPF or hypersensitivity pneumonitis demonstrated shortened telomeres. Moreover, shortened telomeres in late-generation telomerase RNA component knockout mice did not exert a significant effect on BLM-induced pulmonary fibrosis. In contrast, TERT knockout mice exhibited deficient fibrosis that was independent of telomere length. Finally, TERT expression was up-regulated by a histone deacetylase inhibitor, while the induction of TERT in lung fibroblasts was associated with the binding of acetylated histone H3K9 to the TERT promoter region. These findings indicate that significant telomerase induction was evident in fibroblasts from fibrotic murine lungs and a majority of IPF lung samples, whereas telomere shortening was not a common finding in the human blood and lung fibroblast samples. Notably, the animal studies indicated that the pathogenesis of pulmonary fibrosis was independent of telomere length.

Abstract 3820: Wnt signaling and telomerase activation in hepatoblastoma.

Abstract Purpose: The abnormal Wnt/β-catenin signaling plays a key role in hepatoblastoma (HBL) development. In HBL, CTNNB1 coding ß-catenin protein with mutated or deleted at hot-spot regions involving exon 3 stimulates this pathway. Previously we reported that the levels of telomerase reverse transcriptase (TERT) mRNA and telomerase activities were correlated with outcomes of the patients with HBL. In this study, we examined the correlation between Wnt/β-catenin signaling and telomerase activation in HBL. And, we investigated the location of β-catenin and TERT in HBL and the expression levels of the Wnt signal genes downstream of ß -catenin. Methods: Tumors derived from 212 HBL cases treated with the JPLT-2 protocol were analyzed for oncogenic mutations (missense mutations and interstitial deletions in the third exon) of the CTNNB1 gene encoding β-catenin and mutations of Wnt signal genes including APC, AXIN1, and 2. Moreover, these tumors were analyzed for the expression levels of TERT (telomerase reverse transcriptase) and the Wnt signal genes downstream of ß -catenin (MYC, cyclin D1 and MMP7) using Real time RT-PCR and immunohistochemistry. Results: Oncogenic mutations of CTNNB1 were detected in 163 cases (76.4%) and more than 80% showed abnormalities of Wnt signal genes. The expression levels of TERT were significantly higher in 49 cases without mutation (P < 0.05). Interestingly, Wnt/β-catenin target genes including MYC, cyclin D1 and MMP7 were significantly activated in the tumors with telomerase activation (P = 0.01). ß -catenin staining distinguished the CTNNB1 mutated tumors from others but cyclin D1 and MMP-7 did not. In advanced HBLs, double stain analysis of TERT and β-catenin showed that both were located in the same site in nucleus, suggesting that both proteins were co-localized in nucleus in hepatoblastoma.Conclusions: Wnt/β-catenin signaling in the HBLs without CNNB1 mutations was activated by telomerase activation. The clinical courses in HBLs with mutated Wnt signal gens and high TERT expression seemed to be unfavorable due to highly activated Wnt/β-catenin signaling. This correlated pathway might become molecular targets for advanced hepatoblastoma. Citation Format: Eiso Hiyama, Yuka Ueda, Arata Kamimatsuse, Yoshiuki Onitake, Kaoru Ogura, Keiko Hiyama. Wnt signaling and telomerase activation in hepatoblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3820. doi:10.1158/1538-7445.AM2013-3820

Abstract 2862: Deregulated GSK3β sustains gastrointestinal cancer cells by modulating hTERT and telomerase

Abstract Purpose: Glycogen synthase kinase-3β (GSK3β) regulates multiple cell signaling pathways and has been implicated in glucose intolerance, neurodegenerative disorders, and inflammation. We investigated the expression, activity, and putative pathologic role of GSK3β in gastrointestinal, pancreatic, and liver cancers. Experimental Design: Colon, stomach, pancreatic, and liver cancer cell lines; nonneoplastic HEK293 cells; and matched pairs of normal and tumor tissues of stomach and colon cancer patients were examined for GSK3β expression and its phosphorylation at serine 9 (inactive form) and tyrosine 216 (active form) by Western immunoblotting and for GSK3β activity by in vitro kinase assay. The effects of small-molecule GSK3β inhibitors and of RNA interference on cell survival, proliferation, and apoptosis were examined in vitro and on human colon cancer cell xenografts in athymic mice. The effects of GSK3β inhibition on human telomerase reverse transcriptase (hTERT) expression and telomerase activity were compared between colon cancer and HEK293 cells. Results: Cancer cell lines and most cancer tissues showed increased GSK3β expression and increased tyrosine 216 phosphorylation and activity but decreased serine 9 phosphorylation compared with HEK293 cells and nonneoplastic tissues. Inhibition of GSK3β resulted in attenuated cell survival and proliferation and increased apoptosis in most cancer cell lines and in HT-29 xenografts in rodents but not in HEK293 cells. GSK3β inhibition in colon cancer cells was associated with decreased hTERT expression and telomerase activity. Conclusion: The results indicate that deregulated GSK3β sustains gastrointestinal cancer cells survival through modulation of hTERT and telomerase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2862. doi:1538-7445.AM2012-2862

A functional tandem-repeats polymorphism in the downstream of TERTis associated with the risk of nasopharyngeal carcinoma in Chinese population

BackgroundIncreases in human telomerase reverse transcriptase (TERT) expression and telomerase activity are frequently seen in nasopharyngeal carcinoma (NPC). Recently, a variable tandem-repeats polymorphism, MNS16A, located in the downstream region of the TERT gene, was identified and reported to have an effect on TERT expression and telomerase activity. We examined whether the functional MNS16A was related to the risk of occurrence or progression of NPC in the Chinese population.MethodsWe genotyped the MNS16A polymorphism in a case-control study of 855 patients with NPC and 1036 cancer-free controls using PCR, and determined genotype by classifying the DNA band of 243 or 272 base pairs (bp) as the short (S) allele and 302 or 333 bp as the long (L) allele. The genetic associations with the risk of NPC were analyzed by logistic regression.ResultsThe MNS16A genotype was not associated with the progression of NPC. However, individuals carrying the S alleles (SL + SS genotype) had a significantly reduced risk of NPC occurrence compared with those carrying the LL genotype (odds ratio (OR) = 0. 71, 95% confidence interval (CI) = 0. 52 to 0. 96, P = 0. 025). Using a immunohistochemical assay on the NPC tissues, the SL genotype carriers were found to have lower TERT expression than the LL genotype carriers (P = 0. 035).ConclusionsOur study indicates that the TERT MNS16A polymorphism may contribute to the risk of NPC onset in Chinese population.

Tolemerase Reverse Transcriptase Gene Expression as a Tumor Marker for Hepatocellular Carcinoma

Problem statement: Hepatocellular carcinoma will emerge as a major fo rm of malignancy in the coming decades. The continuing high incidenc e of hepatocellular carcinoma, suggests that this disease will continue to represent a global health problem far into the future. Different genes encode for the various components of the human telomerase complex. These components include the human Telomerase RNA Component (hTERC) and the Telomerase Catalytic Subunit (hTERT). Correlation between Telomerase Reverse Transcriptase (hTERT) expression and telomerase activity has been reported in cancer patients. This work aimed to cla rify the significance of human Telomerase Reverse Transcriptase (hTERT mRNA) as a potential molecular tumor marker for Hepatocellular Carcinoma (HCC). Approach: The current study included 25 patients of hepatoce llular carcinoma (HCC), 30 patients with liver cirrhosis and 25 age and sex matched individuals with normal laboratory and Image findings as a control group. h TERT mRNA was measured in plasma by Real time PCR in all patients samples in comparison with normal healthy controls. Results: The expression of hTERT mRNA by relative unit was 129.10 ±27.6 with range (67.72-69.6) Vs 5245.87 ±2382.48 (2053-12232.90) Vs 92782.76 ±16158 (61783.25-118596.47) for control Vs cirrhosis Vs HCC group respectively. The hTERT expression was significantly with 699 and 33 fold increase in HCC and cirrhosis groups correspondingl y when compared to that of controls p<0.05. Conclusion: It was suggested that this procedure was highly di scriminating between healthy subjects and cancer patients and strongly support t he idea that a valuable diagnostic test for cancer might be developed using this genetic marker in pla sma. However it needs to be combined with other markers in future studies to be more specific for liver cancer.

Abstract 1454: Telomerase-specific adenoviral labeling and treatment of hepatocellular carcinoma

Abstract We previously constructed telomerase-specific replication-selective adenoviruses OBP-301 (Telomelysin) and OBP-401 which contains the GFP gene (Telomelysin-GFP, TelomeScan). The replication of these adenoviruses is regulated by a human telomerase transcriptase (hTERT) promoter and allows specific targeting and labeling of tumor cells in vivo (Proc. Natl. Acad. Sci. USA, 1-6, 14514-14517, 2009 and Molecular Cancer Therapeutics 8, 3001-3008, 2009). The aim of this study was to validate systemic injection of these telomerase-specific replicating adenoviruses for in vivo hepatocellular carcinoma (HCC) GFP labeling and tumor-cell killing. We assessed the antitumor efficacy and the tumor labeling ability of the adenoviruses in an orthotopic mouse model using the Hep3B-GFP human HCC cell line. Biweekly i.v. treatment of OBP-301 caused a significant inhibition in liver tumor growth and showed a reduction in the number of liver tumor nodules compared to controls. To assess the GFP tumor labeling ability of OBP-401 for HCC, an orthotopic liver tumor model of unlabeled Hep3B was used. After i.v. injection of OBP-401, the liver tumors were labeled with GFP and visualized by fluorescence imaging. These results suggest that the two viruses, OBP-301 and OBP-401, when administrated systemically, are novel anticancer agents and labeling tools for HCC, respectively, with potential for human use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1454.

Expression of E6 oncoprotein of HPV16,cyclin D&lt;SUB&gt;1&lt;/SUB&gt;,and human telomerase transcriptase in nasopharyngeal carcinoma tissues and its significance

Expression of E6 oncoprotein of HPV16,cyclin D&lt;SUB&gt;1&lt;/SUB&gt;,and human telomerase transcriptase in nasopharyngeal carcinoma tissues and its significance

Leukemic cells with increased telomerase activity exhibit resistance to imatinib

Imatinib mesylate (imatinib), previously known as STI571 (Gleevec), is currently utilized in the treatment of chronic myeloid leukemia (CML). However, its effect on telomerase activity and the correlation of this to its observed antitumor effect has yet to be defined. We investigated the effects of this agent on human telomerase reverse transcriptase (hTERT) expression and telomerase activity and found that it significantly down-regulated telomerase activity in both K562 cells and primary leukemic cells. The telomerase activity of primary leukemic cells from CML patients in blastic crisis showed less suppression than that of cells from patients in chronic phase. Additionally, data also demonstrate that inhibition of telomerase was due to the direct action of imatinib on hTERT transcription, rather than an increase in cell death. These results suggest a novel mechanism in the antitumor activity of imatinib and may provide a basis for future development of anti-telomerase therapies, as well as leading to better understanding of the regulation of telomerase in leukemic cells.

Genomic instability is associated with lack of telomerase activation in ovarian cancer

Introduction: Malignant cells are capable of an unlimited number of cell divisions, either through production of telomerase, or through the alternate lengthening of telomere (ALT) mechanism. Yeast cells with genomic instability have been shown to survive in the absence of telomerase by increased recombination events. We hypothesized that ovarian cancers with high microsatellite instability (MSI-H) are more likely to lack telomerase activation. Methods We examined 104 invasive ovarian cancers for MSI with 6 microsatellite markers (BAT25, BAT26, D5S346, D2S123, D17S250, and NME1). Telomerase activity was determined with ELISA, and its subunits human telomerase reverse transcriptase (hTERT) and human telomerase RNA (hTR) by RT-PCR. Statistical analysis was performed with Chi-square and p

Measuring telomerase activity in various human tumors in routine histology and cytology

The aim of this study was to investigate telomerase reactivation, to quantitatively measure the human telomerase reverse transcriptase (hTERT) content and telomerase activity level (TA) in routine histological and cytological samples, and to examine the relationship between these values and morphological factors. We analyzed 86 (35 cytological and 51 histological) lesions which were divided into four main groups: renal tumors, soft tissue tumors, bladder-urine and thyroid gland lesions. The relative expression of mRNA of hTERT was examined by real-time polymerase chain reaction (RT-PCR). Almost all of the renal cell carcinomas showed TA. In soft tissue tumors no correlation was seen between TA and histogenesis, aggressiveness and prognosis. Concerning cytological material a very good correlation was seen between TA and the benign or malignant nature of these tumors (92.3% specificity and 60% sensitivity). Our results indicated that TA can be used beside histology also in cytologic samples, for example in the preoperative differential diagnosis of the thyroid gland lesions and urine samples. Telomerase reactivation may not play an important role in tumorigenesis in STT. Useful observations can be made by concentrating not only on one group of diseases or localization but the unselected analysis of routine diagnostic cases can also be of help both in diagnosis and therapy.

Telomere dysfunction increases cisplatin and ecteinascidin-743 sensitivity of melanoma cells.

The aim of this study was to investigate the role of telomerase function on the chemosensitivity of melanoma cells. To this end, ecteinascidin-743 (ET-743) and cisplatin [cis-diamminedichloroplatinum(II) (CDDP)], two DNA-interacting drugs that invariably cause an arrest in the G(2)/M phase, and 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid (LND), a mitochondria-targeting drug inducing a G(1) block, were used. As experimental model, human melanoma clones showing reduced human telomerase reverse transcriptase (hTERT) expression and telomerase activity and characterized by telomere dysfunction were used. Reconstitution of telomerase activity by exogenous hTERT expression improved telomere function and reduced the sensitivity to CDDP and ET-743 without affecting LND susceptibility. The decreased sensitivity to CDDP and ET-743 was mainly caused by the ability of cells to recover from drug-induced damage, evaluated in terms of both chromosomal lesions and cell survival. The ability of hTERT-reconstituted cells to recover from drug-induced damage was attributable to the restoration of cell cycle progression. In fact, the cells without hTERT restoration remained for a prolonged time in the G(2)/M phase, and this cell cycle alteration made irreversible the drug-induced S-G(2)/M block and led to the activation of apoptotic program. On the contrary, the hTERT-reconstituted cells progressed quickly through the cell cycle, thus acquiring the capacity to recover from drug-induced block and to protect themselves from the G(2)/M phase-specific drug-triggered apoptosis.

Comparison of Human Telomerase Reverse Transcriptase Messenger RNA and Telomerase Activity as Urine Markers for Diagnosis of Bladder Carcinoma

Comparison of Human Telomerase Reverse Transcriptase Messenger RNA and Telomerase Activity as Urine Markers for Diagnosis of Bladder Carcinoma