|
1
|
Lin ZZ, Hu MCT, Hsu C, Wu YM, Lu YS, Ho JAA, Yeh SH, Chen PJ, Cheng AL. Synergistic efficacy of telomerase-specific oncolytic adenoviral therapy and histone deacetylase inhibition in human hepatocellular carcinoma. Cancer Lett 2023; 556:216063. [PMID: 36669725 DOI: 10.1016/j.canlet.2023.216063] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2022] [Revised: 12/08/2022] [Accepted: 01/10/2023] [Indexed: 01/20/2023]
Abstract
The telomerase-specific oncolytic adenovirus Telomelysin and the histone deacetylase inhibitor AR42 have demonstrated anticancer effects in preclinical models of human hepatocellular carcinoma (HCC). However, the clinical development of Telomelysin may be hindered by human antiviral immunity and tumor resistance. Combining oncolytic and epigenetic therapies is a viable approach for treating various cancers. This study investigated the potential synergism of Telomelysin and AR42 and the relevant underlying mechanisms. Telomelysin and AR42 exhibited synergistic antiproliferative effects in human HCC models in vitro and in vivo. Apoptosis induced by Telomelysin was significantly enhanced by AR42 in both PLC5 and Hep3B HCC cells. AR42 treatment unexpectedly attenuated the expression of the coxsackievirus and adenovirus receptor and the mRNA levels of human telomerase reverse transcriptase, which may be positively associated with the cytotoxicity of Telomelysin. Meanwhile, the cellular antiviral interferon response was not altered by AR42 treatment. Further, we found that Telomelysin enhanced Akt phosphorylation in HCC cells. AR42 reduced Telomelysin-induced phospho-Akt activation and enhanced Telomelysin-induced apoptosis. The correlation of Akt phosphorylation with drug-induced apoptosis was validated in HCC cells with upregulated or downregulated Akt signaling. Combination therapy with Telomelysin and AR42 demonstrated synergistic anti-HCC efficacy. Clinical trials investigating this new combination regimen are warranted.
Collapse
Affiliation(s)
- Zhong-Zhe Lin
- Department of Medical Oncology, National Taiwan University Cancer Center, Taipei, Taiwan; Departments of Oncology, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
| | | | - Chiun Hsu
- Department of Medical Oncology, National Taiwan University Cancer Center, Taipei, Taiwan; Departments of Oncology, National Taiwan University Hospital, Taipei, Taiwan; Graduate Institute of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Yao-Ming Wu
- Department of Surgery, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Yen-Shen Lu
- Departments of Oncology, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Ja-An Annie Ho
- Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan
| | - Shiou-Hwei Yeh
- Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Pei-Jer Chen
- Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan; Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Ann-Lii Cheng
- Department of Medical Oncology, National Taiwan University Cancer Center, Taipei, Taiwan; Departments of Oncology, National Taiwan University Hospital, Taipei, Taiwan; Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan; Graduate Institute of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan.
| |
Collapse
|
|
2
|
HCV Proteins Modulate the Host Cell miRNA Expression Contributing to Hepatitis C Pathogenesis and Hepatocellular Carcinoma Development. Cancers (Basel) 2021; 13:cancers13102485. [PMID: 34069740 PMCID: PMC8161081 DOI: 10.3390/cancers13102485] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/02/2021] [Revised: 05/12/2021] [Accepted: 05/17/2021] [Indexed: 12/13/2022] Open
Abstract
Simple Summary According to the last estimate by the World Health Organization (WHO), more than 71 million individuals have chronic hepatitis C worldwide. The persistence of HCV infection leads to chronic hepatitis, which can evolve into liver cirrhosis and ultimately into hepatocellular carcinoma (HCC). Although the pathogenic mechanisms are not fully understood, it is well established that an interplay between host cell factors, including microRNAs (miRNA), and viral components exist in all the phases of the viral infection and replication. Those interactions establish a complex equilibrium between host cells and HCV and participate in multiple mechanisms characterizing hepatitis C pathogenesis. The present review aims to describe the role of HCV structural and non-structural proteins in the modulation of cellular miRNA during HCV infection and pathogenesis. Abstract Hepatitis C virus (HCV) genome encodes for one long polyprotein that is processed by cellular and viral proteases to generate 10 polypeptides. The viral structural proteins include the core protein, and the envelope glycoproteins E1 and E2, present at the surface of HCV particles. Non-structural (NS) proteins consist of NS1, NS2, NS3, NS4A, NS4B, NS5a, and NS5b and have a variable function in HCV RNA replication and particle assembly. Recent findings evidenced the capacity of HCV virus to modulate host cell factors to create a favorable environment for replication. Indeed, increasing evidence has indicated that the presence of HCV is significantly associated with aberrant miRNA expression in host cells, and HCV structural and non-structural proteins may be responsible for these alterations. In this review, we summarize the recent findings on the role of HCV structural and non-structural proteins in the modulation of host cell miRNAs, with a focus on the molecular mechanisms responsible for the cell re-programming involved in viral replication, immune system escape, as well as the oncogenic process. In this regard, structural and non-structural proteins have been shown to modulate the expression of several onco-miRNAs or tumor suppressor miRNAs.
Collapse
|
|
3
|
Shiu TY, Shih YL, Feng AC, Lin HH, Huang SM, Huang TY, Hsieh CB, Chang WK, Hsieh TY. HCV core inhibits hepatocellular carcinoma cell replicative senescence through downregulating microRNA-138 expression. J Mol Med (Berl) 2017; 95:629-639. [PMID: 28258280 DOI: 10.1007/s00109-017-1518-4] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2016] [Revised: 12/23/2016] [Accepted: 02/07/2017] [Indexed: 01/06/2023]
Abstract
Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). HCV core protein is considered as a positive regulator of telomerase activity. In this study, we focused on the deregulated microRNA-138 (miR-138) in HCV-associated HCC. Differential expression of miR-138 was determined by TaqMan quantitative real-time PCR. The target gene of miR-138 was verified by luciferase reporter assay, quantitative real-time PCR, and Western blotting. Moreover, three assays based on telomerase activity, cell proliferation, and senescence-associated β-galactosidase activity were performed. The correlation analysis revealed a significantly negative correlation between miR-138 and telomerase reverse transcriptase (TERT) mRNA expression in HCC. Further, we showed that mature HCV core protein of 173 amino acids, but not full-length form of 191 amino acids, suppressed miR-138 expression. TERT was verified as a direct target of miR-138 in HCC cells. Furthermore, TERT-targeting miR-138 supplementation can prevent HCV core protein from repressing HCC cell replicative senescence. Collectively, HCV core protein can enhance TERT protein expression through downregulating TERT-targeting miR-138 expression, which in turn inhibits HCC cell replicative senescence. This study may further help our understanding on the pathogenic mechanisms of HCV core protein in HCV-associated HCC development. KEY MESSAGE: miR-138 is downregulated in HCV-associated HCC. Mature HCV core protein plays a pathogenic role in suppressing miR-138 expression. Telomerase reverse transcriptase represents a direct target of miR-138 in HCC cells. miR-138 promotes HCC cell senescence, suggesting potential for HCC treatment.
Collapse
Affiliation(s)
- Tzu-Yue Shiu
- Division of Gastroenterology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
| | - Yu-Lueng Shih
- Division of Gastroenterology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
| | - An-Chieh Feng
- Division of General Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
| | - Hsuan-Hwai Lin
- Division of Gastroenterology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
| | - Shih-Ming Huang
- Department and Graduate Institute of Biochemistry, National Defense Medical Center, Taipei, Taiwan
| | - Tien-Yu Huang
- Division of Gastroenterology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
| | - Chung-Bao Hsieh
- Division of General Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
| | - Wei-Kuo Chang
- Division of Gastroenterology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan
| | - Tsai-Yuan Hsieh
- Division of Gastroenterology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan.
| |
Collapse
|
|
4
|
Panarese S, Brunetti B, Sarli G. Evaluation of Telomerase in Canine Mammary Tissues by Immunohistochemical Analysis and a Polymerase Chain Reaction-Based Enzyme-Linked Immunosorbent Assay. J Vet Diagn Invest 2016; 18:362-8. [PMID: 16921875 DOI: 10.1177/104063870601800407] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022] Open
Abstract
The enzyme telomerase is considered a potential marker for neoplastic tissue and is used as a diagnostic and prognostic tool in clinical medicine and therapeutics. For this reason, the possible role of telomerase activation in the process of malignant transformation is currently the subject of intense research efforts. The focus of the study reported here was to detect telomerase in 37 canine mammary samples, by comparing two methods: immunohistochemical (IHC) analysis for detecting the catalytic subunit of the enzyme, telomerase reverse transcriptase (TERT), and the telomeric repeat amplification protocol–enzyme-linked immunosorbent assay (TRAP-ELISA), a polymerase chain reaction (PCR)-based technique that uses a colorimetric detection method. Using the TRAP-ELISA, samples were considered positive when they yielded a difference of at least 0.2 absorbance units between the readings at 450 nm versus 690 nm wavelength. On the basis of this criterion, 18 negative and 19 positive cases were obtained. Specific immunohistochemical staining was observed mainly in the nucleoli, to a lesser extent in the nuclei, and rarely in the cytoplasm of epithelial cells. A sample was considered positive when at least 10% of the epithelial cells had specific staining. The Pearson correlation between the TRAP-ELISA and IHC results was significant only when IHC nucleolar ( r = 0.53, P < 0.01) or nuclear ( r = 0.36, P < 0.05) staining or their combination ( r = 0.58, P < 0.01) was considered. Thus, IHC staining of nucleoli and nuclei can be considered as an alternative method to the TRAP-ELISA. The detection of telomerase in normal mammary gland and fibrocystic mastopathy using both methods does not support the idea that telomerase may be used as a specific marker of mammary neoplasia in dogs.
Collapse
Affiliation(s)
- Serena Panarese
- Department of Veterinary Public Health and Animal Pathology, Section of General Pathology and Pathologic Anatomy, Faculty of Veterinary Medicine, University of Bologna Via Tolara di Sopra, 50-40064, Ozzano dell'Emilia, Bologna, Italy
| | | | | |
Collapse
|
|
5
|
Abstract
Primary liver cancer, mostly hepatocellular carcinoma, remains a difficult-to-treat cancer. Incidence of liver cancer varies geographically and parallels with the geographic prevalence of viral hepatitis. A number of staging systems have been developed, reflecting the heterogeneity of primary liver cancer, regional preferences, and regional variations in resectability or transplant eligibility. Multimodality treatments are available for this heterogeneous malignancy, and there are variations in the management recommendations for liver cancers across specialties and geographic regions. Novel treatment strategies have merged with the advance of new treatment modalities. This work focuses on reviewing the incidence, staging, and treatment of liver cancer.
Collapse
Affiliation(s)
- Chun-Yu Liu
- Department of Medicine, Division of Hematology and Oncology, Taipei Veterans General Hospital, Taipei 112, Taiwan School of Medicine, National Yang-Ming University, Taipei 112, Taiwan Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan
| | - Kuen-Feng Chen
- Department of Medical Research, National Taiwan University College of Medicine, Taipei 112, Taiwan National Center of Excellence for Clinical Trial and Research, National Taiwan University College of Medicine, Taipei 112, Taiwan
| | - Pei-Jer Chen
- Department of Medical Research, National Taiwan University College of Medicine, Taipei 112, Taiwan National Center of Excellence for Clinical Trial and Research, National Taiwan University College of Medicine, Taipei 112, Taiwan Graduate Institute of Molecular Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei 112, Taiwan
| |
Collapse
|
|
6
|
Chen KW, Ou TM, Hsu CW, Horng CT, Lee CC, Tsai YY, Tsai CC, Liou YS, Yang CC, Hsueh CW, Kuo WH. Current systemic treatment of hepatocellular carcinoma: A review of the literature. World J Hepatol 2015; 7:1412-20. [PMID: 26052386 PMCID: PMC4450204 DOI: 10.4254/wjh.v7.i10.1412] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/26/2014] [Revised: 11/29/2014] [Accepted: 03/30/2015] [Indexed: 02/06/2023] Open
Abstract
Hepatocellular carcinoma (HCC) is the fifth most common form of human cancer worldwide and the third most common cause of cancer-related deaths. The strategies of various treatments for HCC depend on the stage of tumor, the status of patient's performance and the reserved hepatic function. The Barcelona Clinic Liver Cancer (BCLC) staging system is currently used most for patients with HCC. For example, for patients with BCLC stage 0 (very early stage) and stage A (early stage) HCC, the curable treatment modalities, including resection, transplantation and radiofrequency ablation, are taken into consideration. If the patients are in BCLC stage B (intermediate stage) and stage C (advanced stage) HCC, they may need the palliative transarterial chemoembolization and even the target medication of sorafenib. In addition, symptomatic treatment is always recommended for patients with BCLC stage D (end stage) HCC. In this review, we will attempt to summarize the historical perspective and the current developments of systemic therapies in BCLC stage B and C in HCC.
Collapse
Affiliation(s)
- Kai-Wen Chen
- Kai-Wen Chen, Department of Internal Medicine, Hualien Armed Forces General Hospital, Hualien 97144, Taiwan
| | - Tzu-Ming Ou
- Kai-Wen Chen, Department of Internal Medicine, Hualien Armed Forces General Hospital, Hualien 97144, Taiwan
| | - Chin-Wen Hsu
- Kai-Wen Chen, Department of Internal Medicine, Hualien Armed Forces General Hospital, Hualien 97144, Taiwan
| | - Chi-Ting Horng
- Kai-Wen Chen, Department of Internal Medicine, Hualien Armed Forces General Hospital, Hualien 97144, Taiwan
| | - Ching-Chang Lee
- Kai-Wen Chen, Department of Internal Medicine, Hualien Armed Forces General Hospital, Hualien 97144, Taiwan
| | - Yuh-Yuan Tsai
- Kai-Wen Chen, Department of Internal Medicine, Hualien Armed Forces General Hospital, Hualien 97144, Taiwan
| | - Chi-Chang Tsai
- Kai-Wen Chen, Department of Internal Medicine, Hualien Armed Forces General Hospital, Hualien 97144, Taiwan
| | - Yi-Sheng Liou
- Kai-Wen Chen, Department of Internal Medicine, Hualien Armed Forces General Hospital, Hualien 97144, Taiwan
| | - Chen-Chieh Yang
- Kai-Wen Chen, Department of Internal Medicine, Hualien Armed Forces General Hospital, Hualien 97144, Taiwan
| | - Chao-Wen Hsueh
- Kai-Wen Chen, Department of Internal Medicine, Hualien Armed Forces General Hospital, Hualien 97144, Taiwan
| | - Wu-Hsien Kuo
- Kai-Wen Chen, Department of Internal Medicine, Hualien Armed Forces General Hospital, Hualien 97144, Taiwan
| |
Collapse
|
|
7
|
Friedman SL. Final and future frontiers. J Hepatol 2014; 61:969-70. [PMID: 25149111 DOI: 10.1016/j.jhep.2014.08.017] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/12/2014] [Accepted: 08/12/2014] [Indexed: 12/04/2022]
Affiliation(s)
- Scott L Friedman
- Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, United States.
| |
Collapse
|
|
8
|
Lin WH, Yeh SH, Yang WJ, Yeh KH, Fujiwara T, Nii A, Chang SSC, Chen PJ. Telomerase-specific oncolytic adenoviral therapy for orthotopic hepatocellular carcinoma in HBx transgenic mice. Int J Cancer 2012; 132:1451-62. [PMID: 22886913 DOI: 10.1002/ijc.27770] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2012] [Accepted: 07/18/2012] [Indexed: 12/27/2022]
Abstract
The telomerase-specific replication-competent oncolytic adenovirus, Telomelysin, was developed for virus-mediated preferential lysis of tumor cells. Its selectivity is derived from a human telomerase reverse transcriptase (hTERT) promoter-driven active viral replication, which occurs in cancer cells with high telomerase activity but not in normal cells lacking such activity. Because the TERT activity is elevated in most cases of hepatocellular carcinoma (HCC), the current study aims to investigate whether Telomelysin can be used for treatment of HCC. The oncolytic effect of Telomelysin has been investigated both in vitro using cell culture and in vivo using an immunocompetent in situ orthotopic HCC model. In this model, HCC developed spontaneously in the liver of HBx transgenic mice, which is pathologically and genetically similar to human HCC. In cell culture assay, Telomelysin lyses HCC cell lines at a low multiplicity of infection (MOI), ranging 0.77-6.35 (MOI [PFU/cell]). In the orthotopic HCC model, Telomelysin showed a potent oncolytic effect on HCC but spared normal liver tissue. Dose escalation analysis identified a safety dose of 1.25 × 10(8) PFU for this model. The effect of multiple injections of Telomelysin was also evaluated in this immunocompetent HCC model. We found that the virus replicates in HCC after a second intratumoral injection despite an immune response induced by the previous injection. This preclinical study shows that Telomelysin can be used for treatment of human HCC at an appropriate dosage and that its tumor-killing activity persists after multiple injections.
Collapse
Affiliation(s)
- Wei-Hsiang Lin
- Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan
| | | | | | | | | | | | | | | |
Collapse
|
|
9
|
Shin JS, Foot T, Hong A, Zhang M, Lum T, Solomon MJ, Soon Lee C. Telomerase expression as a predictive marker of radiotherapy response in rectal cancer: in vitro and in vivo study. Pathology 2012; 44:209-15. [DOI: 10.1097/pat.0b013e3283511cd5] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
|
|
10
|
Tillmann HL, Plentz RR, Begus‐Nahrmann Y, Lechel A, Rudolph LK. Telomeres and Aging, Cancer, and Hepatic Fibrosis. THE LIVER 2009:1105-1119. [DOI: 10.1002/9780470747919.ch68] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
|
|
11
|
Nishikawa T, Nakajima T, Katagishi T, Okada Y, Jo M, Kagawa K, Okanoue T, Itoh Y, Yoshikawa T. Oxidative stress may enhance the malignant potential of human hepatocellular carcinoma by telomerase activation. Liver Int 2009; 29:846-56. [PMID: 19141026 DOI: 10.1111/j.1478-3231.2008.01963.x] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/13/2023]
Abstract
BACKGROUND/AIMS Continuous oxidative stress (OS) plays an important role in the progression of chronic liver diseases and hepatocarcinogenesis through telomere shortening in hepatocytes. However, it has not been established how the OS influences the progression of human hepatocellular carcinomas (HCCs). We examined the correlations of OS with telomere length of cancer cells, telomerase activity and other clinicopathological factors in 68 HCCs. METHODS The level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a marker of OS was examined immunohistochemically and OS was scored in four grades (0-3). The telomere length of cancer cells was measured by quantitative fluorescence in situ hybridization. Telomerase activity was measured by (i) immunodetection of human telomerase reverse transcriptase (hTERT) and (ii) telomere repeat amplification protocol (TRAP) assay. Telomerase related proteins, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Akt, and other clinicopathological factors were also evaluated. RESULTS As the OS grade increased, the average telomere length became significantly shorter in HCCs, especially in the hTERT-negative group. In the state of high-grade OS, hTERT-positive HCC cells showed more proliferative and less apoptotic features compared with hTERT-negative HCC cells. Telomerase activity, as measured by the TRAP assay, was strongly correlated with OS grade in HCCs. Furthermore, a high OS grade was correlated with the downexpression of PTEN and the activation of Akt. CONCLUSIONS Oxidative stress enhanced the malignant potential of HCCs through the activation of telomerase, which raises the possibility of using OS as a marker for assessing the clinical state of HCCs.
Collapse
Affiliation(s)
- Taichiro Nishikawa
- Kyoto Prefectural University of Medicine Graduate School of Medical Science, Molecular Gastroenterology and Hepatology, Kyoto, Japan.
| | | | | | | | | | | | | | | | | |
Collapse
|
|
12
|
Fusaro L, Panarese S, Brunetti B, Zambelli D, Benazzi C, Sarli G. Quantitative Analysis of Telomerase in Feline Mammary Tissues. J Vet Diagn Invest 2009; 21:369-73. [DOI: 10.1177/104063870902100312] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
The purpose of this study was to validate immunohistochemistry (IHC) as an alternative to telomerase repeat amplification protocol (TRAP) analysis to detect telomerase activity. TRAP–enzyme-linked immunosorbent assay (ELISA) reactivity was compared with telomerase reverse transcription (TERT) IHC staining in 22 feline mammary tissues (6 normal mammary glands, 2 dysplastic mammary glands, 1 fibroadenoma, and 13 malignant neoplasms [6 solid mammary carcinomas, 2 squamous-cell carcinomas, 4 tubulopapillary mammary carcinomas, and 1 mammary carcinosarcoma]). TERT IHC staining revealed enzymatic expression in nuclear, nucleolar, cytoplasmic, and combined nuclear and nucleolar staining patterns that were separately quantified by image analysis and expressed as the absolute number (average) of positive cells or percentage of positive cells with respect to overall cellularity. With TERT IHC staining, the absolute number and percentage of cells with positive nuclei and nucleoli within the same cell were the variables with the greatest discrimination between benign and malignant mammary lesions (analysis of variance [ANOVA], average P < 0.0001; percentage P < 0.001). For TRAP-ELISA–positive versus TRAP-ELISA–negative tissues, a positive test result provided greater differentiation between malignant versus benign mammary lesions (ANOVA, average P = 0.00038; percentage P = 0.0022). The same IHC pattern of expression showed a proportional and significant (average P = 0.004; percentage P = 0.002) but low (average R = 0.60; percentage R = 0.63) correlation with TRAP-ELISA by the Pearson test. The correlation coefficients obtained show that IHC and TRAP cannot be considered interchangeable because the 2 methods are more complementary than exclusive.
Collapse
Affiliation(s)
- Laura Fusaro
- Department of Veterinary Public Health and Animal Pathology, Section of General Pathology and Pathologic Anatomy
| | - Serena Panarese
- Department of Veterinary Public Health and Animal Pathology, Section of General Pathology and Pathologic Anatomy
| | - Barbara Brunetti
- Department of Veterinary Public Health and Animal Pathology, Section of General Pathology and Pathologic Anatomy
| | - Daniele Zambelli
- Department of Veterinary Public Health and Animal Pathology, Section of General Pathology and Pathologic Anatomy
- Veterinary Clinical Department, Obstetrical and Gynecological Section Faculty of Veterinary Medicine, University of Bologna, Ozzano dell'Emilia, Bologna, Italy
| | - Cinzia Benazzi
- Department of Veterinary Public Health and Animal Pathology, Section of General Pathology and Pathologic Anatomy
| | - Giuseppe Sarli
- Department of Veterinary Public Health and Animal Pathology, Section of General Pathology and Pathologic Anatomy
| |
Collapse
|
|
13
|
Lepreux S, Doudnikoff E, Aubert I, Bioulac-Sage P, Bloch B, Martin-Negrier ML. Cytoplasmic expression of human telomerase catalytic protein (hTERT) in neutrophils: an immunoelectron microscopy study. Ultrastruct Pathol 2008; 32:178-83. [PMID: 18958789 DOI: 10.1080/01913120802034504] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
Human telomerase comprises a catalytic protein subunit (hTERT) and an RNA subunit (hTR). Telomerase extends chromosome ends in compensation for the attrition of the telomeres during replication. In this work, the authors explore the expression of hTERT and hTR in neutrophils, respectively by immunochemistry techniques and in situ hybridization. hTERT was strongly expressed in neutrophils cytoplasm. The ultrastructural study showed that the gold particles were not associated with specific organelles but scattered in the cytosol. hTR was not expressed. hTERT is expressed in the cytoplasm of neutrophils, but its roles-eventually extratelomeric effects-remain to be elucidated.
Collapse
Affiliation(s)
- Sebastien Lepreux
- Laboratoire d'Histologie-Embryologie, UFR II, Universite Victor Segalen Bordeaux, France.
| | | | | | | | | | | |
Collapse
|
|
14
|
Ellor S, Shupe T, Petersen B. Stem cell therapy for inherited metabolic disorders of the liver. Exp Hematol 2008; 36:716-725. [PMID: 18375039 PMCID: PMC2443696 DOI: 10.1016/j.exphem.2008.02.002] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2007] [Revised: 01/28/2008] [Accepted: 02/06/2008] [Indexed: 12/16/2022]
Abstract
Modern medicine has conquered an enormous spectrum of health concerns, from the neonatal to the geriatric, the chronically ill to the acutely injured. Among the unmet challenges remaining in modern medicine are inborn disorders of metabolism within the liver. Such inherited metabolic disorders (IMDs) often leave an otherwise healthy individual with a crippling imbalance. As the principal regulator of the body's many metabolic pathways, malencoded hepatic enzymes can drastically disrupt homeostasis throughout the entire body. Severe phenotypes are usually detected within the first few days of life, and treatments range from palliative lifestyle modifications to aggressive surgical procedures. While orthotopic liver transplantation is the single last resort "cure" for these conditions, research during the past few years has brought new therapeutic technologies ever closer to the clinic. Stem cells, therapeutic viral vectors, or a combination thereof, are projected to be the next, best, and final cure for IMDs, which is well-reflected by this generation's research initiatives.
Collapse
Affiliation(s)
- Susan Ellor
- Department of Pathology, Immunology and Laboratory Medicine; University of Florida
- The Program for Stem Cell Biology and Regenerative Medicine; University of Florida
| | - Thomas Shupe
- Department of Pathology, Immunology and Laboratory Medicine; University of Florida
- The Program for Stem Cell Biology and Regenerative Medicine; University of Florida
| | - Bryon Petersen
- Department of Pathology, Immunology and Laboratory Medicine; University of Florida
- The Program for Stem Cell Biology and Regenerative Medicine; University of Florida
| |
Collapse
|
|
15
|
Burnworth B, Arendt S, Muffler S, Steinkraus V, Bröcker EB, Birek C, Hartschuh W, Jauch A, Boukamp P. The multi-step process of human skin carcinogenesis: A role for p53, cyclin D1, hTERT, p16, and TSP-1. Eur J Cell Biol 2007; 86:763-80. [PMID: 17198740 DOI: 10.1016/j.ejcb.2006.11.002] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2006] [Revised: 11/06/2006] [Accepted: 11/06/2006] [Indexed: 01/01/2023] Open
Abstract
As proposed by Hanahan and Weinberg (2000. Cell 100, 57-70) carcinogenesis requires crucial events such as (i) genomic instability, (ii) cell cycle deregulation, (iii) induction of a telomere length maintenance mechanism, and (iv) an angiogenic switch. By comparing the expression of p53, cyclin D1, p16, hTERT, and TSP-1 in spontaneously regressing keratoacanthoma (KA) as a paradigm of early neoplasia, with malignant invasive cutaneous squamous cell carcinoma (SCC) as a paradigm of advanced tumour development, we are now able to assign the changes in the expression of these proteins to specific stages and allocate them to defined roles in the multi-step process of skin carcinogenesis. We show that mutational inactivation of the p53 gene, and with that the onset of genomic instability is the earliest event. Individual p53-positive cells are already seen in "normal" skin, and 3/5 actinic keratoses (AKs), 5/22 KAs, and 13/23 SCCs contain p53-positive patches. Cell cycle deregulation was indicated by the overexpression of the cell cycle regulator cyclin D1, as well as by the loss of the cell cycle inhibitor p16. Interestingly, overexpression of cyclin D1 - observed in 80% of KAs and SCCs, respectively - showed a cell cycle-independent function in HaCaT cell transplants on nude mice. Cyclin D1 overexpression was associated with a massive inflammatory response, finally leading to tissue destruction. Loss of the cell cycle inhibitor p16, on the other hand, correlated with SCCs. Thus, it is tempting to suggest that overexpression of cyclin D1 is an early change that in addition to growth stimulation leads to an altered epithelial-mesenchymal interaction, while functional p16 is able to control this deregulated growth and needs to be eliminated for malignant progression. Another requirement for uncontrolled growth is the inhibition of telomere erosion by up-regulating telomerase activity. As measured by hTERT protein expression, all of the KAs and SCCs studied were positive, with a similar distribution of the protein in both groups and an expression pattern resembling that of normal epidermis. Thus, telomerase may not need to be increased significantly in skin carcinomas. Finally, we show that the angiogenesis inhibitor TSP-1 is strongly expressed in most KAs, and mainly by the tumour cells, while in SCCs the generally weak expression is restricted to the tumour-stroma. Furthermore, we provide evidence that the loss of a copy of chromosome 15 is responsible for reduced TSP-1 expression and thereby this aberration contributes to tumour vascularisation (i.e. the angiogenic switch) required for malignant growth.
Collapse
Affiliation(s)
- Bettina Burnworth
- Division of Genetics of Skin Carcinogenesis, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany
| | | | | | | | | | | | | | | | | |
Collapse
|
|
16
|
Saha B, Chaiwun B, Tsao-Wei DD, Groshen SL, Naritoku WY, Atkinson RD, Taylor CR, Imam SA. Telomerase and markers of cellular proliferation are associated with the progression of cervical intraepithelial neoplasia lesions. Int J Gynecol Pathol 2007; 26:214-22. [PMID: 17581401 DOI: 10.1097/01.pgp.0000250146.44592.d2] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
The expression of the catalytic subunit of telomerase protein (human telomerase reverse transcriptase [hTERT]), which is associated with telomerase activity, was evaluated as a potential marker of the high-grade premalignant cervical intraepithelial neoplasia (CIN 2/3) lesions. For comparison, cases of normal cervical squamous mucosa, low-grade CIN1 lesion, and cervical squamous cell carcinoma were included. The hTERT expression was also compared with Ki-67 and topoisomerase II-alpha (TPII-alpha) to determine the proliferative activity of the hTERT-positive dysplastic cells by a quantitative immunohistochemical staining method and was classified as follows: negative, 5% or less; moderate, 6% to 50%; or high, greater than 50% of the positive cells. The hTERT-positive cells were detected in a patchy pattern in the lower parabasal layers and in much of the basal layer in normal squamous mucosa. A similar frequency of Ki-67- or TPII-alpha-positive cells was observed, with the exception of the basal layer cells that were mostly negative. It is worthy to note that the recognizable intact basal layer cells in cases of CIN lesions were also consistently positive for the expression of hTERT, but rarely for Ki-67 or TPII-alpha. The expression of hTERT was detected in a less patchy pattern at a high or moderate percentage of the dysplastic epithelial cells each in 28.5% of cases of CIN1 lesions. A similar frequency, high and moderate percentage combined, of the TPII-alpha-positive dysplastic cell was also observed. In contrast, a high percentage of the hTERT-positive dysplastic cells were detected as diffuse basal or full-length thickness in 87.5% or 95% of cases of CIN2 or CIN3, respectively. A similar frequency of Ki-67 or TPII-alpha expression was observed in the dysplastic cells of CIN3 lesions. The pattern of hTERT-positive malignant cells in squamous cell carcinoma and dysplastic cells in the high-grade CIN lesions, to a greater extent, and dysplastic cells in the low-grade CIN lesion, to a lesser extent, was distinct from that of the normal cervical squamous mucosa. The results suggest that the progressive increase in the hTERT expression, together with the proliferative activity of the dysplastic epithelial cells of the high-grade CIN lesions, represents an early genetic abnormality in cervical pathogenesis.
Collapse
Affiliation(s)
- Baisakhi Saha
- Gene Therapy Program, Huntington Medical Research Institutes, Pasadena, California 91101, USA
| | | | | | | | | | | | | | | |
Collapse
|
|
17
|
Ide T. [Mechanism of cell proliferation--cell cycle, oncogenes, and senescence]. YAKUGAKU ZASSHI 2007; 126:1087-115. [PMID: 17077613 DOI: 10.1248/yakushi.126.1087] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Cell proliferation is regulated through a transition between the G0 phase and cell cycle. We isolated a mammalian temperature-sensitive mutant cell line defective in the function from the G0 phase to cell cycle. Senescent human somatic cells fail to enter into the cell cycle from the G0 phase with stimulation by any growth factor. Telomere shortening was found to be a cause of cellular senescence, and reexpression of telomerase immortalized human somatic cells. Immortalized human somatic cells showed normal phenotypes and were useful not only for basic research but also for clinical and applied fields. The importance of p53 and p21 activation/induction i now well accepted in the signal transduction process from telomere shortening to growth arrest, but the precise mechanism is largely unknown as yet. We found that the MAP kinase cascade and histone acetylase have an important role in the signaling process to express p21. Tumor tissues and cells were found to have strong telomerase activity, while most normal somatic human tissues showed very weak or no activity. Telomerase activity was shown to be a good marker for early tumor diagnosis because significant telomerase activity was detected in very early tumors or even in some precancerous tissues compared with adjacent normal tissues. Telomere/telomerase is a candidate target for cancer chemotherapeutics, and an agent that abrogated telomere functions was found to kill tumor cells effectively by inducing apoptosis whereas it showed no effect on the viability of normal cells.
Collapse
Affiliation(s)
- Toshinori Ide
- Department of Cellular and Molecular Biology, Division of Integrated Medical Science, Graduated School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima City 734-8551, Japan.
| |
Collapse
|
|
18
|
Satra M, Gatselis N, Iliopoulos D, Zacharoulis D, Dalekos GN, Tsezou A. Real-time quantification of human telomerase reverse transcriptase mRNA in liver tissues from patients with hepatocellular cancer and chronic viral hepatitis. J Viral Hepat 2007; 14:41-7. [PMID: 17212643 DOI: 10.1111/j.1365-2893.2006.00769.x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
We determined, for the first time, the human telomerase reverse transcriptase (hTERT) mRNA expression, using real-time quantitative PCR, in liver tissues from patients with hepatocellular cancer (HCC; n = 13), chronic hepatitis B (n = 19) and C (n = 13). Liver tissues from the 45 patients and 17 patients without liver disease in whom liver biopsy was performed during cholecystectomy (control group), were investigated for telomerase activity (TA) and hTERT mRNA expression using the LightCycler technology. TA was detected in all HCC tissues compared with 15.6% of chronic hepatitis (P < 0.001) and none of controls (P < 0.001). TA levels and hTERT mRNA were higher in HCC compared with chronic hepatitis (P < 0.001) and normal livers (P < 0.001). hTERT mRNA expression was correlated with TA (P < 0.05). Chronic hepatitis patients who tested negative for TA and hTERT mRNA had significantly lower disease duration (58 +/- 85 months) compared with those tested positive (144 +/- 50 months; P < 0.05). Detection of TA and quantification of hTERT mRNA expression in liver tissues could be useful and additional markers for HCC diagnosis and may serve as prognostic markers for HCC development in chronic viral hepatitis patients. However, we were not able to draw general conclusions at this moment, as the number of chronic hepatitis patients positive for hTERT mRNA was relatively small. Real-time quantification of hTERT mRNA expression as a diagnostic/prognostic marker in patients with chronic hepatitis B and C and its relationship with hepatocarcinogenesis needs further evaluation.
Collapse
MESH Headings
- Adult
- Aged
- Aged, 80 and over
- Biopsy
- Carcinoma, Hepatocellular/enzymology
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/pathology
- Female
- Hepacivirus
- Hepatitis B virus
- Hepatitis B, Chronic/enzymology
- Hepatitis B, Chronic/genetics
- Hepatitis B, Chronic/pathology
- Hepatitis C, Chronic/enzymology
- Hepatitis C, Chronic/genetics
- Hepatitis C, Chronic/pathology
- Humans
- Liver Neoplasms/enzymology
- Liver Neoplasms/genetics
- Liver Neoplasms/pathology
- Male
- Middle Aged
- RNA, Messenger/biosynthesis
- RNA, Messenger/genetics
- Reverse Transcriptase Polymerase Chain Reaction
- Statistics, Nonparametric
- Telomerase/biosynthesis
- Telomerase/genetics
Collapse
Affiliation(s)
- M Satra
- Department of Biology, Medical School, University of Thessaly, Larissa, Greece
| | | | | | | | | | | |
Collapse
|
|
19
|
Sabah M, Cummins R, Leader M, Kay E. Immunohistochemical Detection of hTERT Protein in Soft Tissue Sarcomas. Appl Immunohistochem Mol Morphol 2006; 14:198-202. [PMID: 16785790 DOI: 10.1097/01.pai.0000156606.04726.d3] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Human telomerase reverse transcriptase (hTERT) is a telomerase catalytic subunit that regulates telomerase activity. Telomerase is expressed in many human cancers and cell lines and is thought to contribute to their immortality. Little is known about the expression of telomerase in non-epithelial tumors. The objective of this study was to evaluate hTERT expression in a wide range of soft tissue sarcomas. A total of 154 cases of different types of soft tissue sarcoma (54 low-grade, 40 intermediate-grade, and 60 high-grade cases) were evaluated for hTERT expression using immunohistochemistry on tissue microarrays. hTERT immunoexpression was detected in 59% of cases; it was observed in 46%, 58%, and 72% of low-grade, intermediate-grade, and high-grade sarcoma cases, respectively. The intensity of staining positively correlated with the grade of the sarcomas: diffuse strong positive nuclear staining was identified in 6, 8, and 30 cases of low-grade, intermediate-grade, and high-grade sarcomas, respectively. These results suggest that telomerase expression is more often detected in highly malignant tumors than in low-grade sarcomas and thus may be a critical mechanism in tumor progression.
Collapse
Affiliation(s)
- Muna Sabah
- Department of Histopathology, Beaumont Hospital and Royal College of Surgeons in Ireland, Dublin, Ireland.
| | | | | | | |
Collapse
|
|
20
|
Cakir C, Gulluoglu MG, Yilmazbayhan D. Cell proliferation rate and telomerase activity in the differential diagnosis between benign and malignant mesothelial proliferations. Pathology 2006; 38:10-5. [PMID: 16484001 DOI: 10.1080/00313020500456017] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
AIMS The differential diagnosis of malignant mesothelioma (MM) from benign mesothelial lesions (BML) based on histopathological criteria is sometimes not satisfying and causes diagnostic problems for histopathologists. We aimed to investigate whether the immunohistochemically determined cell proliferation rate and telomerase activity, using Ki-67 and human telomerase reverse transcriptase (hTERT) immunohistochemistry, respectively, are useful in the differential diagnosis of MM from BML. METHODS Sixty-six cases of MM (33 epithelioid, 30 biphasic and 3 sarcomatoid) and 22 cases of BML (15 reactive mesothelial proliferations and 7 fibrous pleuritis/pericarditis) were included in this study. We evaluated the proliferative activity by Ki-67 and telomerase activity by hTERT immunohistochemistries for each case. RESULTS The mean value of the Ki-67 proliferation index (PI) in MMs was significantly higher than that of BMLs. Biphasic MMs have higher a Ki-67 PI than epithelioid and sarcomatoid types. Ki-67 immunohistochemistry has a sensitivity of 74%, specificity of 86% and positive predictive value of 94% in detecting MM. hTERT immunohistochemistry detected MM with sensitivity and specificity of 68%. CONCLUSION As a result, being cheap and simple methods, Ki-67 and hTERT immunohistochemistries can be used in differentiating malignant and benign mesothelial lesions in routine formalin-fixed, paraffin-embedded material.
Collapse
Affiliation(s)
- Caglar Cakir
- Istanbul University, Istanbul Faculty of Medicine, Department of Pathology, Istanbul, Turkey
| | | | | |
Collapse
|
|
21
|
Wirth T, Kühnel F, Fleischmann-Mundt B, Woller N, Djojosubroto M, Rudolph KL, Manns M, Zender L, Kubicka S. Telomerase-dependent virotherapy overcomes resistance of hepatocellular carcinomas against chemotherapy and tumor necrosis factor-related apoptosis-inducing ligand by elimination of Mcl-1. Cancer Res 2005; 65:7393-402. [PMID: 16103092 DOI: 10.1158/0008-5472.can-04-3664] [Citation(s) in RCA: 52] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Hepatocellular carcinomas (HCC) are drug-resistant tumors that frequently possess high telomerase activity. It was therefore the aim of our study to investigate the potential of telomerase-dependent virotherapy in multimodal treatment of HCC. In contrast to normal liver, HCC xenografts showed high telomerase activity, resulting in tumor-restricted expression of E1A by a telomerase-dependent replicating adenovirus (hTERT-Ad). Neither tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or chemotherapy alone nor the combined treatment with both agents resulted in significant destruction of HCC cells. Application of hTERT-Ad at low titers was also not capable to destroy HCC cells, but telomerase-dependent virotherapy overcame the resistance of HCC against TRAIL and chemotherapy. The synergistic effects are explained by a strong down-regulation of Mcl-1 expression through hTERT-Ad that sensitizes HCC for TRAIL- and chemotherapy-mediated apoptosis. To investigate whether down-regulation of Mcl-1 alone is sufficient to explain synergistic effects observed with virotherapy, Mcl-1 expression was inhibited by RNA interference. Treatment with Mcl-1-siRNA significantly enhanced caspase-3 activity after chemotherapy and TRAIL application, confirming that elimination of Mcl-1 is responsible for the drug sensitization by hTERT-Ad. Consistent with these results, heterologous overexpression of Mcl-1 significantly reduced the sensitization of hTERT-Ad transduced cells against apoptosis-inducing agents. Chemotherapy did not interfere with quantitative hTERT-Ad production in HCC cells. Whereas hTERT-Ad virotherapy alone was only capable to inhibit the growth of Hep3B xenografts, virochemotherapy resulted in vast destruction of the drug-resistant HCC. In conclusion our data indicate that telomerase-dependent virotherapy is an attractive strategy to overcome the natural resistance of HCC against anticancer drugs by elimination of Mcl-1.
Collapse
Affiliation(s)
- Thomas Wirth
- Department of Gastroenterology, Hepatology and Endocrinology, Medical School Hannover, Hannover, Germany
| | | | | | | | | | | | | | | | | |
Collapse
|
|
22
|
Lepreux S, Rebouissou S, Le Bail B, Saric J, Balabaud C, Bloch B, Martin-Négrier ML, Zucman-Rossi J, Bioulac-Sage P. Mutation of TP53 gene is involved in carcinogenesis of hepatic undifferentiated (embryonal) sarcoma of the adult, in contrast with Wnt or telomerase pathways: an immunohistochemical study of three cases with genomic relation in two cases. J Hepatol 2005; 42:424-9. [PMID: 15710230 DOI: 10.1016/j.jhep.2004.10.021] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/30/2004] [Revised: 10/07/2004] [Accepted: 10/26/2004] [Indexed: 12/04/2022]
Abstract
BACKGROUND/AIMS Hepatic undifferentiated (embryonal) sarcoma (HUS) is an exceptional hepatic malignant tumor in adults. Genetic studies were never reported in adult cases. METHODS In this study concerning three cases of HUS occurring in adult, we studied the three classical ways of carcinogenesis i.e. the TP53 (p53), Wnt (CTNNB1/beta-catenin and AXIN1) and telomerase (hTERT) pathways. We studied the expression of p53, beta-catenin and telomerase catalytic subunit hTERT by immunohistochemistry in the three cases; we determined TP53 gene mutation in two cases and the genome-wide allelotype, AXIN1, and CTNNB1/beta-catenin gene mutation in one case. RESULTS Immunohistochemistry showed an overexpression of p53 in more than 80% of tumoral cells; furthermore, mutations of TP53 were observed in two cases, involving the sequence-specific DNA binding domain. In contrast, no mutation was found in CTNNB1/beta-catenin and AXIN1 genes. Tumoral cells did not show hTERT staining nor nuclear expression of beta-catenin. In addition, allelotype analysis in one case showed loss of heterozygosity of chromosome 7p, 11p, 17p, 22q, and allelic imbalance of 1p, 8p, 20q. CONCLUSIONS In this report of HUS in three adult patients, we emphasize the role of TP53 pathway in carcinogenesis of this rare tumor. This point could be of interest for therapeutic strategies.
Collapse
Affiliation(s)
- Sébastien Lepreux
- Service d'Anatomie Pathologique, Hôpital Pellegrin, CHU Bordeaux, 33076 Bordeaux, France
| | | | | | | | | | | | | | | | | |
Collapse
|
|
23
|
Wang SL, Chen WT, Wu MT, Chan HM, Yang SF, Chai CY. Expression of human telomerase reverse transcriptase in thyroid follicular neoplasms: an immunohistochemical study. Endocr Pathol 2005; 16:211-8. [PMID: 16299404 DOI: 10.1385/ep:16:3:211] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Differential diagnosis of follicular adenoma (FA) and follicular carcinoma (FC) of the thyroid can be difficult in the routine practice of surgical pathology because the diagnosis of FC is strictly defined and determined by the presence of capsular and/or vascular invasion by the tumor. These features may be equivocal in the histologic sections. On the other hand, telomerase is expressed in many human cancers and is thought to contribute to their immortality. Human telomerase reverse transcriptase (hTERT) is the major determinant of human telomerase activity, and its expression is suggestive of capacity for unlimited replication. This case-control study examined the expression of hTERT using immunohistochemistry in 36 thyroid FC and 36 FA from patients who were matched by age and sex. The aim was to investigate the value of immunohistochemical staining for hTERT in the differential diagnosis of follicular neoplasms. The results revealed 23 cases of FC and 14 cases of FA that showed high expression of hTERT, with moderate to strong immunoreactivity. The remaining cases showed weak or negative staining. The difference between FA and FC was statistically significant (p < 0.05). In conclusion, immunohistochemical staining for hTERT can be considered an ancillary marker for differential diagnosis of FA and FC.
Collapse
Affiliation(s)
- Sheng-Lan Wang
- Department of Pathology, Kaohsiung Medical University Hospital, Kaohsiung City, Taiwan
| | | | | | | | | | | |
Collapse
|
|
24
|
Leung DTM, Ma CH, Niu H, Liew CT, Tang JTY, Lim PL. Nuclear telomerase is less accessible to antibody probing than known nuclear antigens: retrieval with new immunostaining buffer. Histochem Cell Biol 2004; 123:105-12. [PMID: 15538612 DOI: 10.1007/s00418-004-0721-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/05/2004] [Indexed: 10/26/2022]
Abstract
Telomerase is an important tumor marker but few antibodies to the enzyme have been described or used without difficulty in histochemical detection. Here we report specific detection of the enzyme in cell and tissue preparations using a new monoclonal antibody (mAb 476) and a new antigen-retrieval buffer (Enhancing buffer). When used to detect telomerase under normal immunostaining conditions in HL-60 cells or tissue sections of hepatocellular carcinoma or metastatic choriocarcinoma, unexpectedly, the antibody stained the cytoplasm rather than the nucleus. Nuclear staining, however, was revealed using the Enhancing buffer. Since other nuclear antigens in the HL-60 cell could be stained both ordinarily and in the Enhancing buffer, nuclear telomerase appears to be shrouded by the nuclear matrix or blocked by accessory proteins. The cytoplasmic activity seen in normal buffer but absent largely from the Enhancing buffer may be an artifact or the nascent, "naked" enzyme. With a known cytoplasmic antigen (proteinase-3) chosen arbitrarily for comparison, the antigenicity was found enhanced, instead, by the Enhancing buffer. The mode of action of the Enhancing buffer differs from that of microwave irradiation or the signal amplification (CSA) used by some investigators. The latter was found to enhance the cytoplasmic reactivity rather than the nuclear reactivity of mAb 476.
Collapse
Affiliation(s)
- Danny Tze-Ming Leung
- Clinical Immunology Unit, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, Hong Kong.
| | | | | | | | | | | |
Collapse
|
|
25
|
Sabah M, Cummins R, Leader M, Kay E. Expression of human telomerase reverse transcriptase in gastrointestinal stromal tumors occurs preferentially in malignant neoplasms. Hum Pathol 2004; 35:1231-5. [PMID: 15492990 DOI: 10.1016/j.humpath.2004.07.010] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/18/2023]
Abstract
Telomerase is expressed in many human cancers and cell lines and is thought to contribute to their immortality. To date, little is known about the expression of telomerase in nonepithelial tumors. The objective of this study was to evaluate the expression of human telomerase reverse transcriptase (hTERT) in gastrointestinal stromal tumors (GISTs). Twenty-three GISTs (9 low malignant potential, 10 primary malignant, and 4 intra-abdominal recurrences) were evaluated for hTERT expression by using immunohistochemistry on tissue microarray. Tissue blocks were retrieved, and hematoxylin and eosin stains were performed to evaluate the histological tumor type. All cases were strongly positive for KIT (CD117). Immunohistochemistry for hTERT was performed. Eight of 9 cases of the low malignant potential group were negative for hTERT immunoexpression, whereas all malignant GISTs showed positive staining that varied from weak to strong immunoreactivity. Six of 10 cases of the primary malignant GISTs were strongly positive for hTERT. The remaining cases (4/10) showed weak staining. All recurrent GISTs (4/4) showed strong positive immunostaining for hTERT. One malignant case was weakly positive for hTERT, but its recurrence was strongly positive. These results suggest that hTERT expression occurs preferentially in malignant tumors and that telomerase activity may occur during the progression of GISTs. Immunohistochemical staining for hTERT may be a useful marker for the prognostication of GISTs.
Collapse
Affiliation(s)
- Muna Sabah
- Department of Histopathology, Education and Research Centre, Beaumont Hospital and Royal College of Surgeons in Ireland, Dublin
| | | | | | | |
Collapse
|
|
26
|
Varma V, Cohen C. Immunohistochemical and molecular markers in the diagnosis of hepatocellular carcinoma. Adv Anat Pathol 2004; 11:239-49. [PMID: 15322490 DOI: 10.1097/01.pap.0000131822.31576.9d] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Hepatocellular carcinoma (HCC) has distinct morphologic features and can be identified in the majority of cases by routine hematoxylin and eosin (H&E)-stained formalin-fixed paraffin-embedded sections. However, distinguishing a well-differentiated HCC from normal or regenerative tissue may be very difficult in some cases, particularly in small needle aspiration or core biopsies. Furthermore, some of the unusual morphologic variants, including clear-cell, pleomorphic, and sarcomatoid variants, may be mistaken for metastases. Similarly, metastases from various primary tumors to the liver may be mistaken for primary hepatic tumors. In this overview, we summarize the immunohistochemical and molecular markers that have been developed to address these diagnostic challenges. Among the numerous diagnostic markers studied, pCEA, HepPar 1, CD34, CK 7, CK 19, CK 20, and albumin in situ (ISH) have been found to be valuable in distinguishing HCC from metastatic neoplasms of extrahepatic sites.
Collapse
Affiliation(s)
- Vijay Varma
- Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA.
| | | |
Collapse
|
|
27
|
Lechel A, Manns MP, Rudolph KL. Telomeres and telomerase: new targets for the treatment of liver cirrhosis and hepatocellular carcinoma. J Hepatol 2004; 41:491-7. [PMID: 15336455 DOI: 10.1016/j.jhep.2004.06.010] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Affiliation(s)
- André Lechel
- Department of Gastroenterology, Hepatology, and Endocrinology, Medical School Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
| | | | | |
Collapse
|
|
28
|
Lantuejoul S, Soria JC, Moro-Sibilot D, Morat L, Veyrenc S, Lorimier P, Brichon PY, Sabatier L, Brambilla C, Brambilla E. Differential expression of telomerase reverse transcriptase (hTERT) in lung tumours. Br J Cancer 2004; 90:1222-9. [PMID: 15026805 PMCID: PMC2410220 DOI: 10.1038/sj.bjc.6601643] [Citation(s) in RCA: 78] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Human telomerase reverse transcriptase is a ribonucleoprotein that synthesises telomeric sequences, which decrease at each cell division. In cancer cells, its activity is linked to telomere maintenance leading to unlimited cellular proliferation and immortality. To evaluate the prognostic value of the catalytic subunit telomerase reverse transcriptase (hTERT), we analysed its expression by immunohistochemistry in 122 formalin-fixed lung tumours including 42 squamous cell carcinoma (SCC), 43 adenocarcinoma (ADC), 19 basaloid carcinoma (BC) and 18 small-cell lung carcinoma (SCLC) in comparison with detection of hTERT mRNA by in situ hybridisation and relative telomerase activity by TRAP assay in a subset of tumours. We observed a high concordance between hTERT protein expression and detection of hTERT mRNA and telomerase activity. Telomerase expression varied according to histology (P=0.0002) being significantly lower in ADC than in SCC, BC and SCLC (P<0.0001). Adenocarcinoma and SCC exhibited either a nuclear or a nucleolar staining in contrast with a diffuse nuclear staining observed in most BC and all SCLC (P=0.01). In stage I NSCLC telomerase expression was lower than in other stages (P=0.04), and a nucleolar staining was correlated with a short survival (P=0.03). We concluded that telomerase expression and pattern are distinctive among histopathological classes of lung cancer and convey prognostic influence.
Collapse
Affiliation(s)
- S Lantuejoul
- Service de Pathologie Cellulaire, Institut A Bonniot, CHU Michallon Grenoble, France
- Lung Cancer Research Group INSERM U 578, Institut A Bonniot, CHU Michallon Grenoble, France
| | - J C Soria
- Laboratoire de Radiobiologie et Oncologie DSV-DRR CEA Fontenay aux Roses, France
| | - D Moro-Sibilot
- Lung Cancer Research Group INSERM U 578, Institut A Bonniot, CHU Michallon Grenoble, France
| | - L Morat
- Laboratoire de Radiobiologie et Oncologie DSV-DRR CEA Fontenay aux Roses, France
| | - S Veyrenc
- Service de Pathologie Cellulaire, Institut A Bonniot, CHU Michallon Grenoble, France
- Lung Cancer Research Group INSERM U 578, Institut A Bonniot, CHU Michallon Grenoble, France
| | - P Lorimier
- Service de Pathologie Cellulaire, Institut A Bonniot, CHU Michallon Grenoble, France
- Lung Cancer Research Group INSERM U 578, Institut A Bonniot, CHU Michallon Grenoble, France
| | - P Y Brichon
- Lung Cancer Research Group INSERM U 578, Institut A Bonniot, CHU Michallon Grenoble, France
| | - L Sabatier
- Laboratoire de Radiobiologie et Oncologie DSV-DRR CEA Fontenay aux Roses, France
| | - C Brambilla
- Lung Cancer Research Group INSERM U 578, Institut A Bonniot, CHU Michallon Grenoble, France
| | - E Brambilla
- Service de Pathologie Cellulaire, Institut A Bonniot, CHU Michallon Grenoble, France
- Lung Cancer Research Group INSERM U 578, Institut A Bonniot, CHU Michallon Grenoble, France
- Service de Pathologie Cellulaire, CHU A. Michallon, BP 217 Cedex 9, 38043 Grenoble, France. E-mail:
| |
Collapse
|
|
29
|
Onishi T, Nouso K, Higashi T, Toshikuni N, Nakatsukasa H, Kobayashi Y, Uemura M, Yumoto E, Fujiwara K, Sato S, Nakamura S, Yokoyama J, Hanafusa T, Shiratori Y. Cellular distribution of telomerase reverse transcriptase in human hepatocellular carcinoma. J Gastroenterol Hepatol 2003; 18:1168-74. [PMID: 12974904 DOI: 10.1046/j.1440-1746.2003.03137.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
BACKGROUND AND AIM Telomerase is the enzyme that synthesizes telomeric DNA, and the activation of telomerase is closely related to cellular immortality. Telomerase activity has been reported in many human cancer tissues and is regulated by the expression of human telomerase reverse transcriptase (hTERT). The aim of the present study was to identify hTERT-expressing cells in human liver tissues and evaluate the feasibility of the hTERT promoter for gene therapy of hepatocellular carcinoma (HCC). METHODS The authors examined the cellular distribution of hTERT transcripts in surgically resected HCC by in situ hybridization. RESULTS Among 20 samples, hTERT expression was observed in 15 HCC. Transcripts of hTERT were homogenously distributed in the cytoplasm of HCC cells in nine of 15 cases; six of 15 cases displayed a heterogeneous staining pattern. All poorly differentiated HCC that expressed hTERT showed a homogenous pattern of staining. None of the non-cancerous hepatocytes were positive for the transcripts, but infiltrating lymphocytes were faintly stained. The homogenous expression of hTERT was also observed in the vascular invasion of HCC. CONCLUSIONS The results indicate that most HCC cells express hTERT RNA and that the promoter is a good candidate as a target for gene therapy. However, careful consideration must be taken concerning the potential effects on lymphocytes.
Collapse
Affiliation(s)
- Toru Onishi
- Department of Medicine and Medical Science, Isotope center, Okayama University Graduate School of Medicine and Dentistry, Okayama Citizen's Hospital, Okayama, Japan
| | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
30
|
Kyo S, Masutomi K, Maida Y, Kanaya T, Yatabe N, Nakamura M, Tanaka M, Takarada M, Sugawara I, Murakami S, Taira T, Inoue M. Significance of immunological detection of human telomerase reverse transcriptase: re-evaluation of expression and localization of human telomerase reverse transcriptase. THE AMERICAN JOURNAL OF PATHOLOGY 2003; 163:859-67. [PMID: 12937127 PMCID: PMC1868244 DOI: 10.1016/s0002-9440(10)63446-3] [Citation(s) in RCA: 59] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 05/08/2003] [Indexed: 01/09/2023]
Abstract
Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of telomerase and is a potentially useful diagnostic marker for cancers. There have been few studies in which immunological detection of hTERT has been attempted and its subcellular localization has not been precisely defined. In the present study, we re-evaluated expression and localization of hTERT in cancer and normal cells using a newly developed antibody. Immunohistochemistry revealed that hTERT is expressed in approximately 80% of gynecological cancers, but some premalignant lesions exhibited weak expression of hTERT. Interestingly, not only nuclei but also cytoplasm of cancer cells were positive for hTERT staining. This finding was supported by the results of Western blot analysis of cell lines, in which both nuclear and cytoplasmic extracts exhibited significant hTERT bands. Cytoplasmic hTERT in cancer cells may be functional because the telomeric repeat amplification protocol assay of cytoplasmic extracts showed high levels of telomerase activity. Unexpectedly, not all normal primary cells and telomerase-negative cancer cell lines lacked hTERT expression; some exhibited weak TERT signals. In Western analysis, hTERT signals did not always correlate with telomerase activity of the various cell types. These findings suggest that functional hTERT is expressed in both the nucleus and cytoplasm of cancer cells and that hTERT expression does not strictly reflect telomerase activity. Further analysis is needed to clarify the biological significance of cytoplasmic hTERT.
Collapse
Affiliation(s)
- Satoru Kyo
- Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa 920-8641, Japan.
| | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
31
|
Kojima H, Kaita KDE, Xu Z, Ou JH, Gong Y, Zhang M, Minuk GY. The absence of up-regulation of telomerase activity during regeneration after partial hepatectomy in hepatitis B virus X gene transgenic mice. J Hepatol 2003; 39:262-8. [PMID: 12873824 DOI: 10.1016/s0168-8278(03)00215-0] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
Abstract
BACKGROUND/AIMS Transgenic mice that express HBV X protein (HBx) have increased sensitivity to hepatocarcinogens. In the present study, we hypothesized that HBx interferes with the DNA protective increases in telomerase activity that occur in proliferating hepatocytes. METHODS Male CD-1 mice (4-6/grp) were killed and hepatic telomerase activity measured at 0, 6, 12, 24, 36, 48 h post partial hepatectomy (PHx). Four HBx transgenic mice were killed at 12 h post-PHx when maximum telomerase activity was observed in CD-1 non-transgenic mice. mRNA of the telomerase catalytic subunit; murine telomerase reverse transcriptase (mTERT), was measured by reverse transcription-polymerase chain reaction. Telomerase activity and human TERT (hTERT) were also measured in Chang and PLC/PRF/5 cells following transient transfection with HBx cDNA. RESULTS Telomerase activity peaked at 12 h post-PHx in normal mice, however, in HBx transgenic mice, telomerase activity was significantly lower, both at baseline (P<0.05) and 12 h post-PHx (P<0.01). Following PHx, mTERT mRNA expression remained constant in normal mice but decreased significantly (P<0.01) in HBx transgenic mice. Transfection of HBx in Chang and PLC/PRF/5 cells had no effect on telomerase activity or hTERT mRNA expression. CONCLUSIONS The results of this study suggest that HBx expression may play a role in hepatocellular carcinogenesis by interfering with telomerase activity during hepatocyte proliferation.
Collapse
Affiliation(s)
- Hiroshige Kojima
- Section of Hepatology, Department of Medicine, University of Manitoba, Winnipeg, Man., Canada
| | | | | | | | | | | | | |
Collapse
|
|
32
|
Huang TG, Savontaus MJ, Shinozaki K, Sauter BV, Woo SLC. Telomerase-dependent oncolytic adenovirus for cancer treatment. Gene Ther 2003; 10:1241-7. [PMID: 12858189 DOI: 10.1038/sj.gt.3301987] [Citation(s) in RCA: 96] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Conditionally replicative adenovirus (CRAD) is an attractive anticancer agent as it can selectively replicate in tumor cells. Expression of telomerase reverse transcriptase (TERT) is a unique tumor cell characteristic, being absent in normal postmitotic cells. Thus, we constructed a TERT promoter regulated CRAD for tumor-specific oncolysis by replacing the endogenous adenovirus E1A promoter with that of human TERT (Adv-TERTp-E1A). We showed that its replication was severely attenuated in TERT-negative cells, but that it replicated almost as efficiently as wild-type adenovirus in TERT-positive cells. Accordingly, Adv-TERTp-E1A conferred cytopathicity to TERT-positive, but not TERT-negative, cells. In vivo replication of Adv-TERTp-E1A after local administration into a xenograft model of human hepatocellular carcinoma in nude mice was demonstrated by an increase in adenovirus titers in tumor extracts by several orders of magnitude between 6 h and 3 days postvector injection. Furthermore, significant inhibition of tumor growth with substantial necrotic tumor areas staining positively for adenovirus was observed with Adv-TERTp-E1A, but not with a control replication-deficient adenovirus. There was also the absence of hepatotoxicity in tumor-bearing animals after intratumoral delivery of the CRAD. The results indicate that the TERT promoter-driven CRAD is capable of tumor-selective replication and oncolysis in vitro and in vivo, and can be utilized as an adjuvant treatment agent for cancer.
Collapse
Affiliation(s)
- T-G Huang
- Carl C. Icahn Center for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY, USA
| | | | | | | | | |
Collapse
|
|
33
|
Hisatomi H, Ohyashiki K, Ohyashiki JH, Nagao K, Kanamaru T, Hirata H, Hibi N, Tsukada Y. Expression profile of a gamma-deletion variant of the human telomerase reverse transcriptase gene. Neoplasia 2003; 5:193-7. [PMID: 12869302 PMCID: PMC1502410 DOI: 10.1016/s1476-5586(03)80051-9] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
The human telomerase reverse transcriptase (hTERT) is an essential component of the holoenzyme complex that adds telomeric repeats to the ends of chromosomes. The hTERT transcript has been shown to have two deletion type alternative splicing sites. One deletion site induces the alpha-deletion variant, lacking 36 bp from exon 6, and the other induces the beta-deletion variant, lacking 182 bp from exons 7 and 8. Here, we identified a novel deletion variant of the hTERT transcript in hepatocellular carcinoma cell lines. The deleted transcript was characterized by an in-frame deletion of 189 bp, spanning nucleotides 2710 to 2898, corresponding to the complete loss of exon 11 (gamma-deletion). The region lacking in the gamma-deletion lies within RT motifs D and E, suggesting that it is missing conserved residues from the catalytic core of the protein. Both gamma- and alpha-deletion variants were occasionally detected, but the beta-deletion variant was frequently observed. Our results may provide important information for more detailed studies on the regulation of telomerase activity.
Collapse
Affiliation(s)
- Hisashi Hisatomi
- Center for Molecular Biology and Cytogenetics, SRL Inc., 5-6-50 Shin-machi Hino, Tokyo, Japan.
| | | | | | | | | | | | | | | |
Collapse
|
|
34
|
MESH Headings
- Carcinoma, Hepatocellular/etiology
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/pathology
- Carcinoma, Hepatocellular/prevention & control
- Carcinoma, Hepatocellular/virology
- Cell Adhesion/genetics
- Cell Transformation, Neoplastic
- Cell Transformation, Viral
- Chronic Disease
- Genes, cdc
- Genes, p53
- Growth Substances/physiology
- Hepacivirus/pathogenicity
- Hepatitis B virus/pathogenicity
- Hepatitis B, Chronic/complications
- Hepatitis B, Chronic/virology
- Hepatitis C, Chronic/complications
- Hepatitis C, Chronic/virology
- Humans
- Liver Diseases/complications
- Liver Diseases/pathology
- Liver Neoplasms/etiology
- Liver Neoplasms/genetics
- Liver Neoplasms/pathology
- Liver Neoplasms/prevention & control
- Liver Neoplasms/virology
- Neoplasm Invasiveness/genetics
- Neoplasm Metastasis
- Neovascularization, Pathologic/genetics
- Precancerous Conditions/complications
- Precancerous Conditions/pathology
Collapse
|
|
35
|
Sharma GG, Gupta A, Wang H, Scherthan H, Dhar S, Gandhi V, Iliakis G, Shay JW, Young CSH, Pandita TK. hTERT associates with human telomeres and enhances genomic stability and DNA repair. Oncogene 2003; 22:131-46. [PMID: 12527915 DOI: 10.1038/sj.onc.1206063] [Citation(s) in RCA: 188] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Ectopic expression of telomerase in telomerase-silent cells is sufficient to overcome senescence and to extend cellular lifespan. We show here that the catalytic subunit of human telomerase (hTERT) crosslinks telomeres. This interaction is blocked by the telomere repeat binding factor 1, but not by a dominant negative form of this protein. It is also abolished by destruction of the RNA component of telomerase as well as by mutations in the hTERT protein. Ectopic expression of hTERT leads to transcriptional alterations of a subset of genes and changes in the interaction of the telomeres with the nuclear matrix. This is associated with reduction of spontaneous chromosome damage in G(1) cells, enhancement of the kinetics of DNA repair and an increase in NTP levels. The effect on DNA repair is likely indirect as TERT does not directly affect DNA end rejoining in vitro or meiotic recombination in vivo. The observed effects of hTERT occurred rapidly before any significant lengthening of telomeres was observed. Our findings establish an intimate relationship between hTERT-telomere interactions and alteration in transcription of a subset of genes that may lead to increased genomic stability and enhanced repair of genetic damage. These novel functions of telomerase are distinct from its known effect on telomere length and have potentially important biological consequences.
Collapse
Affiliation(s)
- Girdhar G Sharma
- Radiation and Cancer Biology Division, Washington University School of Medicine, St. Louis, MO 63108, USA
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
36
|
Zhang RG, Guo LX, Wang XW, Xie H. Telomerase inhibition and telomere loss in BEL-7404 human hepatoma cells treated with doxorubicin. World J Gastroenterol 2002; 8:827-31. [PMID: 12378624 PMCID: PMC4656569 DOI: 10.3748/wjg.v8.i5.827] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the effects of doxorubicin on telomerase activity and telomere length in hepatocellular carcinoma.
METHODS: Telomerase activity was assayed with a non-radioisotopic quantitative telomerase repeat amplification protocal-based method. The effect of doxorubicin (DOX) on the growth of BEL-7404 human hepatoma cells was determined by microculture tetrazolium assay. Mean telomere length (terminal restriction fragment) was detected by Southern blot method. The expression of telomerase subunits genes was investigated by RT-PCR. Cell apoptosis and cell cycle distribution were evaluated by flow cytometry.
RESULTS: Telomerase activity was inhibited in a dose and time-dependent manner in BEL-7404 human hepatoma cells treated with DOX for 24, 48 or 72 h in concentrations from 0.156 to 2.5 μM which was crrelated with the inhibition of cell growth. No changes were found in the mRNA expression of three telomerase subunits (hTERT, hTR and TP1) after drug exposure for 72 h with indicated concentrations. The cells treated with DOX showed shortened mean telomere length and accumulated at the G2/M phase. However, there was almost no effects on cell apoptosis by DOX.
CONCLUSION: The telomerase inhibition and the telomere shortening by DOX may contribute to its efficiency in the treatment in hepatocellular carcinoma.
Collapse
Affiliation(s)
- Ru-Gang Zhang
- Department of Biotherapy, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China
| | | | | | | |
Collapse
|
|
37
|
Yan P, Benhattar J, Coindre JM, Guillou L. Telomerase activity and hTERT mRNA expression can be heterogeneous and does not correlate with telomere length in soft tissue sarcomas. Int J Cancer 2002; 98:851-6. [PMID: 11948462 DOI: 10.1002/ijc.10285] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
In a previous study, we showed that telomerase activity (TA) and human telomerase reverse transcriptase (hTERT) mRNA expression were undetectable in benign mesenchymal lesions and low-grade soft tissue sarcomas (STSs), but detectable in about 50% of intermediate-/high-grade STSs. We wondered if this lack of TA or hTERT mRNA expression could be related to the tumor sample examined and if there was a relationship between the former 2 parameters and telomere length. Two separate tumor samples from 37 STSs were examined for telomerase activity, using the telomerase repeat amplification protocol (TRAP) assay and for hTERT mRNA expression, using RT-PCR. Telomere length was determined in each tumor sample, using the terminal restriction fragments (TRF) technique. Significant variations in telomere length, TA and hTERT mRNA expression between 2 samples of the same tumor were observed in 27%, 11% and 27% of STSs, respectively. Telomere length did not correlate with TA or hTERT mRNA expression. Despite great intratumoral heterogeneity in telomere length, short and long telomeres were more often seen in the low/intermediate-grade and high-grade STS categories, respectively. Few STSs that showed a TRF pattern suggestive of alternative lengthening of telomeres (ALT) may contain ALT subpopulations.
Collapse
Affiliation(s)
- Pu Yan
- University Institute of Pathology, Lausanne, Switzerland
| | | | | | | |
Collapse
|
|
38
|
Hoekstra R, Chamuleau RAFM. Recent developments on human cell lines for the bioartificial liver. Int J Artif Organs 2002; 25:182-91. [PMID: 11999190 DOI: 10.1177/039139880202500304] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Most bioartificial liver (BAL) devices contain porcine primary hepatocytes as their biological component. However, alternatives are needed due to xenotransplantation associated risks. Human liver cell lines have excellent growth characteristics and are therefore candidates for application in BAL devices. Tumour-derived cell lines HepG2 and C3A express a variety of liver functions, but some specific liver functions, like ammonia detoxification and ureagenesis are insufficient. Immortalised human hepatocytes might offer better prospects. The balance between immortalisation and transformation with dedifferentiation of cells seems controllable by conditional immortalisation and/or the use of telomerase as immortalising agent. Another promising approach will be the use of embryonic or adult human stem cells. Rodent stem cells have been directed to hepatic differentiation in vitro, which might be applicable to human stem cells. However, both functionality and safety of immortalised human liver cell lines and differentiated stem cells should be improved before successful use in BAL devices becomes reality.
Collapse
Affiliation(s)
- R Hoekstra
- Department of Experimental Hepatology, Academic Medical Center, University of Amsterdam, The Netherlands.
| | | |
Collapse
|
|
39
|
Kotoula V, Hytiroglou P, Pyrpasopoulou A, Saxena R, Thung SN, Papadimitriou CS. Expression of human telomerase reverse transcriptase in regenerative and precancerous lesions of cirrhotic livers. LIVER 2002; 22:57-69. [PMID: 11906620 DOI: 10.1046/j.0106-9543.2001.01594.x] [Citation(s) in RCA: 40] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
BACKGROUND/AIMS The catalytic subunit of human telomerase (hTERT) is known to be expressed in a variety of malignant tumours, including hepatocellular carcinoma (HCC). We studied hTERT expression in regenerative and precancerous lesions arising in cirrhosis. METHODS/RESULTS As shown by in situ hybridisation, hTERT mRNA was absent in normal liver, but present in varying numbers of hepatocytes and HCC cells of diseased livers, as well as in biliary epithelial cells, lymphocytes, sinusoidal-lining cells and tumour endothelial cells. RT-PCR for two hTERT transcript regions demonstrated hTERT expression in 11 out of 15 cirrhotic liver samples, in 20 out of 21 large regenerative nodules/low-grade dysplastic nodules, in 5 out of 5 high-grade dysplastic nodules, and in 4 out of 4 HCCs. The beta-splice variant was identified in all hTERT-positive cases, while the corresponding full-length transcript was found only in 13 out of 29 positive large nodular lesions and in 4 out of 11 positive cirrhotic samples. The full-length transcript was always found in the presence of the beta-splice variant, usually in low relative levels, and tended to correlate with telomerase activity in the samples, while the beta-splice variant did not. CONCLUSIONS This study shows that hTERT re-expression takes place both in hepatic regeneration occurring in cirrhosis and in the early steps of hepatocarcinogenesis, and involves mainly the beta-splice variant of this molecule. Additional regulatory mechanisms may be required for the expression of the full-length hTERT transcript.
Collapse
Affiliation(s)
- Vassiliki Kotoula
- Department of Pathology, Aristotle University Medical School, Thessaloniki, Greece
| | | | | | | | | | | |
Collapse
|
|
40
|
Zhang R, Wang X, Guo L, Xie H. Growth inhibition of BEL-7404 human hepatoma cells by expression of mutant telomerase reverse transcriptase. Int J Cancer 2002; 97:173-9. [PMID: 11774261 DOI: 10.1002/ijc.1597] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Human hepatocellular carcinoma (HCC) is one of the most common malignancies in Asia and Africa. Human telomerase reverse transcriptase (hTERT) is expressed in HCC but absent in normal human liver cells, which is consistent with the expression pattern of telomerase. In the present study, expression of a dominant-negative form of hTERT (DN-hTERT) resulted in inhibition of telomerase activity and decreased mean telomeric length of BEL-7404 human hepatoma cells, whereas expression of wild-type hTERT (WT-hTERT) and control vector had no such effects. Cell growth was inhibited by this mutant (DN-hTERT), which was consistent with the changes in telomerase level. Flattened large cells were found in late generations with the DN-hTERT treatment. When mean telomeric length of DN-hTERT-transfected cells reached a critical length (about 1.7 kb), apoptosis was induced. Tumorigenicity of DN-hTERT-expressing cells was eliminated in vivo. These data indicated that hTERT was essential for the growth of hepatoma cells. hTERT can also be used as an important target for anti-HCC drug screening.
Collapse
Affiliation(s)
- Rugang Zhang
- Department of Biotherapy, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, People's Republic of China
| | | | | | | |
Collapse
|
|
41
|
Faris RA, Konkin T, Halpert G. Liver stem cells: a potential source of hepatocytes for the treatment of human liver disease. Artif Organs 2001; 25:513-21. [PMID: 11493271 DOI: 10.1046/j.1525-1594.2001.025007513.x] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Severe liver injury often leads to the proliferation of oval cells, which differentiate along hepatocytic and biliary lineages. Because oval cells proliferate only when hepatocyte replication is impaired, they are considered to be the progeny of facultative liver stem cells (FLSCs). Identification and isolation of FLSCs has been hampered by the lack of markers that delineate these bipotential progenitors. We hypothesized that transition ductal cells are FLSCs because they are located in a unique anatomical niche sharing tight junctions with a neighboring hepatocyte and another terminal ductular cell. Alternatively, it has been proposed recently that bone marrow-derived stem cells are FLSCs since these cells differentiate along the hepatic lineage following colonization of the liver. The intent of this review is to provide insight into the nature and origin of liver stem cells and to explore the possibility that stem cell technology may lead to the development of clinical modalities for the treatment of human liver disease.
Collapse
Affiliation(s)
- R A Faris
- Department of Pediatric Oncology Research, Rhode Island Hospital, 593 Eddy Street, Providence, RI 02903, U.S.A.
| | | | | |
Collapse
|
|
42
|
Abstract
In 1994 a sensitive method for the detection of telomerase was described. This assay, which was based on the polymerase chain reaction, suggested that telomerase activity was associated with immortal and cancer cells. Since then more than a thousand studies have documented the expression and activity of the enzyme in diseased tissues, primarily tumours. This review gives an overview of the biological significance of telomerase expression and methods for detecting its activity. This is followed by an organ system-based discussion of expression in normal tissues and disease states. We finish with speculation as to the future role of telomerase detection in diagnostic histopathology.
Collapse
Affiliation(s)
- P Matthews
- Department of Pathology, University of Wales College of Medicine, Cardiff, UK.
| | | |
Collapse
|