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Jin K, Xu J, Zhang L, Liu Z, Su X, Xu Z, Ding Y, Liu H, Chang Y, Xu L, Wang Z, Zhu Y, Xu J. TERT promoter mutations or protein overexpression define an aggressive subset with favourable immunotherapeutic response in advanced urothelial carcinoma. BMJ ONCOLOGY 2025; 4:e000586. [PMID: 40099003 PMCID: PMC11911668 DOI: 10.1136/bmjonc-2024-000586] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/24/2024] [Accepted: 03/03/2025] [Indexed: 03/19/2025]
Abstract
Objective Telomerase reverse transcriptase (TERT) gene promoter mutation (TPM) is a key non-coding somatic alteration in urothelial carcinoma (UC) that plays a critical role in telomerase activation. Despite its importance, the prognostic value of TPM has shown mixed results in previous studies. Methods and analysis This study included 155 UC patients from two local clinical centres and 1652 patients from four public datasets, along with matched clinical annotation. Immunohistochemistry of TERT and immune-related markers was performed on tissue microarrays, and transcriptomic and genomic data were analysed to evaluate immune microenvironment characteristics and mutational profiles associated with TPM. We assessed the association of TPM or TERT overexpression (OE) with clinical outcomes, genomics and immunological profiles across tumour stages. Results In early-stage UC, TPM or TERT OE was not significantly associated with patient outcomes. However, in advanced urothelial carcinoma (aUC), TPM or TERT OE was linked to markedly worse overall survival (OS) and a poor response to platinum-based chemotherapy. Notably, despite this unfavourable prognosis, these patients exhibited a more favourable response to anti-PD-1/PD-L1 immunotherapy. aUC with TPM or TERT OE was characterised by an immune-evasive microenvironment, including infiltration of exhausted CD8+ T cells and elevated PD-1 and PD-L1 expression. Furthermore, genomic analysis further revealed a higher APOBEC mutational signature and a lower clock-like mutational signature in aUC with TPM or TERT OE. Conclusion In this retrospective study, TPM or TERT OE identifies a more aggressive subset of patients with poor OS and an immune-evasive microenvironment but a better response to immunotherapy in aUC.
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Affiliation(s)
- Kaifeng Jin
- Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China
- NHC Key Laboratory of Glycoconjugate Research, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Jingtong Xu
- Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Lingkai Zhang
- NHC Key Laboratory of Glycoconjugate Research, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Zhaopei Liu
- Department of Urology, Fudan University Shanghai Cancer Center, Shanghai, China
| | - Xiaohe Su
- NHC Key Laboratory of Glycoconjugate Research, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Ziyue Xu
- NHC Key Laboratory of Glycoconjugate Research, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Yawei Ding
- Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Hailong Liu
- Department of Urology, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Yuan Chang
- Department of Urology, Fudan University Shanghai Cancer Center, Shanghai, China
| | - Le Xu
- Department of Urology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Zewei Wang
- Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China
| | - Yu Zhu
- Department of Urology, Fudan University Shanghai Cancer Center, Shanghai, China
| | - Jiejie Xu
- NHC Key Laboratory of Glycoconjugate Research, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, Shanghai, China
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Jablonowski CM, Gil HJ, Pinto EM, Pichavaram P, Fleming AM, Clay MR, Hu D, Morton CL, Pruett-Miller SM, Hansen BS, Chen X, Jones KMD, Liu Y, Ma X, Yang J, Davidoff AM, Zambetti GP, Murphy AJ. TERT Expression in Wilms Tumor Is Regulated by Promoter Mutation or Hypermethylation, WT1, and N-MYC. Cancers (Basel) 2022; 14:cancers14071655. [PMID: 35406427 PMCID: PMC8996936 DOI: 10.3390/cancers14071655] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2022] [Revised: 03/08/2022] [Accepted: 03/23/2022] [Indexed: 11/16/2022] Open
Abstract
Simple Summary The telomerase enzyme adds repetitive genetic sequences to the ends of chromosomes called telomeres to prevent cellular senescence. Gain of telomerase function is one of the hallmarks of human cancer. The telomerase protein is coded by the gene TERT and increased TERT RNA levels have been associated with disease relapse in Wilms tumor, the most common kidney cancer of childhood. This study aimed to determine the mechanisms of increased TERT expression in Wilms tumor. This study found mutations in the TERT promoter, increased methylation of the TERT promoter, and genomic copy number amplifications of TERT as potential mechanisms of TERT activation. Conversely, this study found that inactivating WT1 mutation was associated with low TERT RNA levels and telomerase activity. N-MYC overexpression in Wilms tumor cells resulted in increased TERT promoter activity and TERT transcription. TERT transcription is associated with molecular and histologic subgroups in Wilms tumor and telomere-targeted therapies warrant future investigation. Abstract Increased TERT mRNA is associated with disease relapse in favorable histology Wilms tumor (WT). This study sought to understand the mechanism of increased TERT expression by determining the association between TERT and WT1 and N-MYC, two proteins important in Wilms tumor pathogenesis that have been shown to regulate TERT expression. Three out of 45 (6.7%) WTs and the corresponding patient-derived xenografts harbored canonical gain-of-function mutations in the TERT promoter. This study identified near ubiquitous hypermethylation of the TERT promoter region in WT compared to normal kidney. WTs with biallelic inactivating mutations in WT1 (7/45, 15.6%) were found to have lower TERT expression by RNA-seq and qRT-PCR and lower telomerase activity determined by the telomerase repeat amplification protocol. Anaplastic histology and increased percentage of blastema were positively correlated with higher TERT expression and telomerase activity. In vitro shRNA knockdown of WT1 resulted in decreased expression of TERT, reduced colony formation, and decreased proliferation of WiT49, an anaplastic WT cell line with wild-type WT1. CRISPR-Cas9-mediated knockout of WT1 resulted in decreased expression of telomere-related gene pathways. However, an inducible Wt1-knockout mouse model showed no relationship between Wt1 knockout and Tert expression in normal murine nephrogenesis, suggesting that WT1 and TERT are coupled in transformed cells but not in normal kidney tissues. N-MYC overexpression resulted in increased TERT promoter activity and TERT transcription. Thus, multiple mechanisms of TERT activation are involved in WT and are associated with anaplastic histology and increased blastema. This study is novel because it identifies potential mechanisms of TERT activation in Wilms tumor that could be of therapeutic interests.
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Affiliation(s)
- Carolyn M. Jablonowski
- Department of Surgery, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 133, Memphis, TN 38105, USA; (C.M.J.); (H.J.G.); (P.P.); (A.M.F.); (D.H.); (C.L.M.); (J.Y.); (A.M.D.)
| | - Hyea Jin Gil
- Department of Surgery, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 133, Memphis, TN 38105, USA; (C.M.J.); (H.J.G.); (P.P.); (A.M.F.); (D.H.); (C.L.M.); (J.Y.); (A.M.D.)
| | - Emilia M. Pinto
- Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA; (E.M.P.); (G.P.Z.)
| | - Prahalathan Pichavaram
- Department of Surgery, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 133, Memphis, TN 38105, USA; (C.M.J.); (H.J.G.); (P.P.); (A.M.F.); (D.H.); (C.L.M.); (J.Y.); (A.M.D.)
| | - Andrew M. Fleming
- Department of Surgery, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 133, Memphis, TN 38105, USA; (C.M.J.); (H.J.G.); (P.P.); (A.M.F.); (D.H.); (C.L.M.); (J.Y.); (A.M.D.)
| | - Michael R. Clay
- Department of Pathology, University of Colorado Anschutz, Aurora, CO 80045, USA;
| | - Dongli Hu
- Department of Surgery, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 133, Memphis, TN 38105, USA; (C.M.J.); (H.J.G.); (P.P.); (A.M.F.); (D.H.); (C.L.M.); (J.Y.); (A.M.D.)
| | - Christopher L. Morton
- Department of Surgery, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 133, Memphis, TN 38105, USA; (C.M.J.); (H.J.G.); (P.P.); (A.M.F.); (D.H.); (C.L.M.); (J.Y.); (A.M.D.)
| | - Shondra M. Pruett-Miller
- Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA; (S.M.P.-M.); (B.S.H.)
| | - Baranda S. Hansen
- Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA; (S.M.P.-M.); (B.S.H.)
| | - Xiang Chen
- Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA; (X.C.); (K.M.D.J.); (Y.L.); (X.M.)
| | - Karissa M. Dieseldorff Jones
- Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA; (X.C.); (K.M.D.J.); (Y.L.); (X.M.)
| | - Yanling Liu
- Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA; (X.C.); (K.M.D.J.); (Y.L.); (X.M.)
| | - Xiaotu Ma
- Department of Computational Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA; (X.C.); (K.M.D.J.); (Y.L.); (X.M.)
| | - Jun Yang
- Department of Surgery, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 133, Memphis, TN 38105, USA; (C.M.J.); (H.J.G.); (P.P.); (A.M.F.); (D.H.); (C.L.M.); (J.Y.); (A.M.D.)
| | - Andrew M. Davidoff
- Department of Surgery, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 133, Memphis, TN 38105, USA; (C.M.J.); (H.J.G.); (P.P.); (A.M.F.); (D.H.); (C.L.M.); (J.Y.); (A.M.D.)
- Division of Pediatric Surgery, Department of Surgery, University of Tennessee Health Science Center, Memphis, TN 38105, USA
| | - Gerard P. Zambetti
- Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA; (E.M.P.); (G.P.Z.)
| | - Andrew J. Murphy
- Department of Surgery, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 133, Memphis, TN 38105, USA; (C.M.J.); (H.J.G.); (P.P.); (A.M.F.); (D.H.); (C.L.M.); (J.Y.); (A.M.D.)
- Division of Pediatric Surgery, Department of Surgery, University of Tennessee Health Science Center, Memphis, TN 38105, USA
- Correspondence:
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Thompson CA, Wong JM. Non-canonical Functions of Telomerase Reverse Transcriptase: Emerging Roles and Biological Relevance. Curr Top Med Chem 2020; 20:498-507. [DOI: 10.2174/1568026620666200131125110] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2019] [Revised: 01/10/2020] [Accepted: 01/21/2020] [Indexed: 12/11/2022]
Abstract
Increasing evidence from research on telomerase suggests that in addition to its catalytic telomere
repeat synthesis activity, telomerase may have other biologically important functions. The canonical
roles of telomerase are at the telomere ends where they elongate telomeres and maintain genomic
stability and cellular lifespan. The catalytic protein component Telomerase Reverse Transcriptase
(TERT) is preferentially expressed at high levels in cancer cells despite the existence of an alternative
mechanism for telomere maintenance (alternative lengthening of telomeres or ALT). TERT is also expressed
at higher levels than necessary for maintaining functional telomere length, suggesting other possible
adaptive functions. Emerging non-canonical roles of TERT include regulation of non-telomeric
DNA damage responses, promotion of cell growth and proliferation, acceleration of cell cycle kinetics,
and control of mitochondrial integrity following oxidative stress. Non-canonical activities of TERT primarily
show cellular protective effects, and nuclear TERT has been shown to protect against cell death
following double-stranded DNA damage, independent of its role in telomere length maintenance. TERT
has been suggested to act as a chromatin modulator and participate in the transcriptional regulation of
gene expression. TERT has also been reported to regulate transcript levels through an RNA-dependent
RNA Polymerase (RdRP) activity and produce siRNAs in a Dicer-dependent manner. At the mitochondria,
TERT is suggested to protect against oxidative stress-induced mtDNA damage and promote mitochondrial
integrity. These extra-telomeric functions of TERT may be advantageous in the context of increased
proliferation and metabolic stress often found in rapidly-dividing cancer cells. Understanding
the spectrum of non-canonical functions of telomerase may have important implications for the rational
design of anti-cancer chemotherapeutic drugs.
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Affiliation(s)
- Connor A.H. Thompson
- Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, V6T 1Z3, Canada
| | - Judy M.Y. Wong
- Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, V6T 1Z3, Canada
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Bignoux MJ, Cuttler K, Otgaar TC, Ferreira E, Letsolo BT, Weiss SF. LRP::FLAG Rescues Cells from Amyloid-β-Mediated Cytotoxicity Through Increased TERT Levels and Telomerase Activity. J Alzheimers Dis 2019; 69:729-741. [DOI: 10.3233/jad-190075] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Affiliation(s)
- Monique J. Bignoux
- School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, South Africa
| | - Katelyn Cuttler
- School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, South Africa
| | - Tyrone C. Otgaar
- School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, South Africa
| | - Eloise Ferreira
- School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, South Africa
| | - Boitelo T. Letsolo
- School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, South Africa
| | - Stefan F.T. Weiss
- School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, Wits 2050, Johannesburg, South Africa
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Niu P, Wei Y, Gao Q, Zhang X, Hu Y, Qiu Y, Mu Y, Li K. Male Fertility Potential Molecular Mechanisms Revealed by iTRAQ-Based Quantitative Proteomic Analysis of the Epididymis from Wip1−/− Mice. ACTA ACUST UNITED AC 2019; 23:54-66. [DOI: 10.1089/omi.2018.0155] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Affiliation(s)
- Pengxia Niu
- Pig Genetic Engineering and Germplasm Innovation, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Yinghui Wei
- Pig Genetic Engineering and Germplasm Innovation, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Qian Gao
- Pig Genetic Engineering and Germplasm Innovation, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Xue Zhang
- Pig Genetic Engineering and Germplasm Innovation, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Yanqing Hu
- Pig Genetic Engineering and Germplasm Innovation, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Yiqing Qiu
- Pig Genetic Engineering and Germplasm Innovation, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Yulian Mu
- Pig Genetic Engineering and Germplasm Innovation, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Kui Li
- Pig Genetic Engineering and Germplasm Innovation, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, China
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Wang YC, Lee AS, Lu LS, Ke LY, Chen WY, Dong JW, Lu J, Chen Z, Chu CS, Chan HC, Kuzan TY, Tsai MH, Hsu WL, Dixon RAF, Sawamura T, Chang KC, Chen CH. Human electronegative LDL induces mitochondrial dysfunction and premature senescence of vascular cells in vivo. Aging Cell 2018; 17:e12792. [PMID: 29923368 PMCID: PMC6052487 DOI: 10.1111/acel.12792] [Citation(s) in RCA: 45] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/12/2018] [Indexed: 12/30/2022] Open
Abstract
Dysregulation of plasma lipids is associated with age‐related cardiovascular diseases. L5, the most electronegative subfraction of chromatographically resolved low‐density lipoprotein (LDL), induces endothelial dysfunction, whereas the least electronegative subfraction, L1, does not. In this study, we examined the effects of L5 on endothelial senescence and its underlying mechanisms. C57B6/J mice were intravenously injected with L5 or L1 (2 mg kg−1 day−1) from human plasma. After 4 weeks, nuclear γH2AX deposition and senescence‐associated β‐galactosidase staining indicative of DNA damage and premature senescence, respectively, were increased in the aortic endothelium of L5‐treated but not L1‐treated mice. Similar to that, in Syrian hamsters with elevated serum L5 levels induced by a high‐fat diet, nuclear γH2AX deposition and senescence‐associated β‐galactosidase staining were increased in the aortic endothelium. This phenomenon was blocked in the presence of N‐acetyl‐cysteine (free‐radical scavenger) or caffeine (ATM blocker), as well as in lectin‐like oxidized LDL receptor‐1 (LOX‐1) knockout mice. In cultured human aortic endothelial cells, L5 augmented mitochondrial oxygen consumption and mitochondrial free‐radical production, which led to ATM activation, nuclear γH2AX deposition, Chk2 phosphorylation, and TP53 stabilization. L5 also decreased human telomerase reverse transcriptase (hTERT) protein levels and activity. Pharmacologic or genetic manipulation of the reactive oxygen species (ROS)/ATM/Chk2/TP53 pathway efficiently blocked L5‐induced endothelial senescence. In conclusion, L5 may promote mitochondrial free‐radical production and activate the DNA damage response to induce premature vascular endothelial senescence that leads to atherosclerosis. Novel therapeutic strategies that target L5‐induced endothelial senescence may be used to prevent and treat atherosclerotic vascular disease.
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Affiliation(s)
- Yu-Chen Wang
- Division of Cardiovascular Medicine; Asia University Hospital; Taichung Taiwan
- Department of Biotechnology; Asia University; Taichung Taiwan
- Division of Cardiovascular Medicine; China Medical University Hospital; Taichung Taiwan
| | - An-Sheng Lee
- Department of Medicine; Mackay Medical College; New Taipei City Taiwan
- Cardiovascular Research Laboratory; China Medical University Hospital; Taichung Taiwan
| | - Long-Sheng Lu
- Graduate Institute of Biomedical Materials and Tissue Engineering; College of Biomedical Engineering; Taipei Medical University; Taipei Taiwan
- International Ph.D. Program in Biomedical Engineering; College of Biomedical Engineering; Taipei Medical University; Taipei Taiwan
- Department of Radiation Oncology; Taipei Medical University Hospital; Taipei Taiwan
- Translational Laboratory; Department of Medical Research; Taipei Medical University Hospital; Taipei Taiwan
| | - Liang-Yin Ke
- Department of Medical Laboratory Science and Biotechnology; College of Health Sciences; Kaohsiung Medical University; Kaohsiung Taiwan
- Lipid Science and Aging Research Center; Kaohsiung Medical University; Kaohsiung Taiwan
| | - Wei-Yu Chen
- Department of Medicine; Mackay Medical College; New Taipei City Taiwan
- Graduate Institute of Biomedical Sciences; China Medical University; Taichung Taiwan
| | - Jian-Wen Dong
- Department of Neuro-Oncology; The University of Texas MD Anderson Cancer Center; Houston Texas
| | - Jonathan Lu
- Vascular and Medicinal Research; Texas Heart Institute; Houston Texas
| | - Zhenping Chen
- Department of Surgery; The University of Texas Medical Branch; Galveston Texas
| | - Chih-Sheng Chu
- Lipid Science and Aging Research Center; Kaohsiung Medical University; Kaohsiung Taiwan
- Center for Lipid Biosciences; Kaohsiung Medical University Hospital; Kaohsiung Taiwan
- Division of Cardiology; Department of Internal Medicine; Kaohsiung Medical University Hospital; Kaohsiung Taiwan
| | - Hua-Chen Chan
- Lipid Science and Aging Research Center; Kaohsiung Medical University; Kaohsiung Taiwan
- Center for Lipid Biosciences; Kaohsiung Medical University Hospital; Kaohsiung Taiwan
| | - Taha Y. Kuzan
- Department of Radiology; Marmara University Medical School; Istanbul Turkey
| | - Ming-Hsien Tsai
- Lipid Science and Aging Research Center; Kaohsiung Medical University; Kaohsiung Taiwan
- Center for Lipid Biosciences; Kaohsiung Medical University Hospital; Kaohsiung Taiwan
| | - Wen-Li Hsu
- Lipid Science and Aging Research Center; Kaohsiung Medical University; Kaohsiung Taiwan
| | | | - Tatsuya Sawamura
- Department of Physiology; Shinshu University School of Medicine; Matsumoto, Nagano Japan
| | - Kuan-Cheng Chang
- Division of Cardiovascular Medicine; China Medical University Hospital; Taichung Taiwan
- Cardiovascular Research Laboratory; China Medical University Hospital; Taichung Taiwan
- Graduate Institute of Biomedical Sciences; China Medical University; Taichung Taiwan
| | - Chu-Huang Chen
- Lipid Science and Aging Research Center; Kaohsiung Medical University; Kaohsiung Taiwan
- Vascular and Medicinal Research; Texas Heart Institute; Houston Texas
- Center for Lipid Biosciences; Kaohsiung Medical University Hospital; Kaohsiung Taiwan
- Graduate Institute of Medicine; College of Medicine; Kaohsiung Medical University; Kaohsiung Taiwan
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Cimino-Reale G, Gandellini P, Santambrogio F, Recagni M, Zaffaroni N, Folini M. miR-380-5p-mediated repression of TEP1 and TSPYL5 interferes with telomerase activity and favours the emergence of an "ALT-like" phenotype in diffuse malignant peritoneal mesothelioma cells. J Hematol Oncol 2017; 10:140. [PMID: 28716051 PMCID: PMC5513108 DOI: 10.1186/s13045-017-0510-3] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2017] [Accepted: 07/10/2017] [Indexed: 02/06/2023] Open
Abstract
Background Understanding the molecular/cellular underpinnings of diffuse malignant peritoneal mesothelioma (DMPM), a fatal malignancy with limited therapeutic options, is of utmost importance for the fruitful management of the disease. In this context, we previously found that telomerase activity (TA), which accounts for the limitless proliferative potential of cancer cells, is prognostic for disease relapse and cancer-related death in DMPM patients. Consequently, the identification of factors involved in telomerase activation/regulation may pave the way towards the development of novel therapeutic interventions for the disease. Here, the capability of miR-380-5p, a microRNA negligibly expressed in telomerase-positive DMPM clinical specimens, to interfere with telomerase-mediated telomere maintenance and, hence, with cancer cell growth was assessed on preclinical models of DMPM. Methods DMPM cells were transfected with a miR-380-5p synthetic precursor, and the effects of miRNA replacement were evaluated in terms of growing capability, induction of apoptosis and interference with TA. Reiterated weekly transfections were also performed in order to analyse the phenotype arising upon prolonged miR-380-5p reconstitution in DMPM cells. Results The ectopic expression of miR-380-5p elicited a remarkable inhibition of TA and resulted in DMPM cell growth impairment and apoptosis induction. In particular, we demonstrated for the first time that these effects were the result of a molecular circuitry converging on telomerase associated protein 1 (TEP1), where the miRNA was able to target the gene both directly in unconventional targeting modality and indirectly via p53 accumulation consequent to miRNA-mediated downregulation of testis-specific protein, Y-encoded-like 5 gene. Moreover, miR-380-5p did not cause telomere attrition and cell growth arrest in long-term DMPM transfectants, which in turn showed slightly elongated telomeres and molecular features (e.g. c-circle DNA and reduced expression levels of chromatin remodeler ATRX) resembling an alternative lengthening of telomeres (ALT) phenotype. Conclusions miR-380-5p interferes with TA in DMPM cells by targeting TEP1. Notably, in the long-term setting, miR-380-5p-mediated impairment of TA did not result in telomere attrition. Instead, a phenotype reminiscent of ALT emerged in DMPM cells as possible compensatory pathway that safeguards DMPM cell growth, an event that may be regarded as a potential resistance mechanism to anticancer therapies based on telomerase inhibitors. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0510-3) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Graziella Cimino-Reale
- Molecular Pharmacology Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo, 42 - 20133, Milano, Italy
| | - Paolo Gandellini
- Molecular Pharmacology Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo, 42 - 20133, Milano, Italy
| | - Francesca Santambrogio
- Molecular Pharmacology Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo, 42 - 20133, Milano, Italy
| | - Marta Recagni
- Molecular Pharmacology Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo, 42 - 20133, Milano, Italy
| | - Nadia Zaffaroni
- Molecular Pharmacology Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo, 42 - 20133, Milano, Italy
| | - Marco Folini
- Molecular Pharmacology Unit, Department of Applied Research and Technological Development, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo, 42 - 20133, Milano, Italy.
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Rampazzo E, Bojnik E, Trentin L, Bonaldi L, Del Bianco P, Frezzato F, Visentin A, Facco M, Semenzato G, De Rossi A. Role of miR-15a/miR-16-1 and the TP53 axis in regulating telomerase expression in chronic lymphocytic leukemia. Haematologica 2017; 102:e253-e256. [PMID: 28385779 DOI: 10.3324/haematol.2016.157669] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Affiliation(s)
- Enrica Rampazzo
- Department of Surgery, Oncology and Gastroenterology, Section of Oncology and Immunology, University of Padova
| | - Engin Bojnik
- Department of Surgery, Oncology and Gastroenterology, Section of Oncology and Immunology, University of Padova
| | - Livio Trentin
- Department of Clinical and Experimental Medicine, Hematology Section, University of Padova
| | | | | | - Federica Frezzato
- Department of Clinical and Experimental Medicine, Hematology Section, University of Padova
| | - Andrea Visentin
- Department of Clinical and Experimental Medicine, Hematology Section, University of Padova
| | - Monica Facco
- Department of Clinical and Experimental Medicine, Hematology Section, University of Padova
| | - Gianpietro Semenzato
- Department of Clinical and Experimental Medicine, Hematology Section, University of Padova
| | - Anita De Rossi
- Department of Surgery, Oncology and Gastroenterology, Section of Oncology and Immunology, University of Padova .,Istituto Oncologico Veneto-IRCCS, Padova, Italy
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Adámik M, Kejnovská I, Bažantová P, Petr M, Renčiuk D, Vorlíčková M, Brázdová M. p53 binds human telomeric G-quadruplex in vitro. Biochimie 2016; 128-129:83-91. [PMID: 27422117 DOI: 10.1016/j.biochi.2016.07.004] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2016] [Accepted: 07/11/2016] [Indexed: 01/08/2023]
Abstract
The tumor suppressor protein p53 is a key factor in genome stability and one of the most studied of DNA binding proteins. This is the first study on the interaction of wild-type p53 with guanine quadruplexes formed by the human telomere sequence. Using electromobility shift assay and ELISA, we show that p53 binding to telomeric G-quadruplexes increases with the number of telomeric repeats. Further, p53 strongly favors G-quadruplexes folded in potassium over those formed in sodium, thus indicating the telomeric G-quadruplex conformational selectivity of p53. The presence of the quadruplex-stabilizing ligand, N-methyl mesoporphyrin IX (NMM), increases p53 recognition of G-quadruplexes in potassium. Using deletion mutants and selective p53 core domain oxidation, both p53 DNA binding domains are shown to be crucial for telomeric G-quadruplex recognition.
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Affiliation(s)
- Matej Adámik
- Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, CZ-612 65 Brno, Czech Republic
| | - Iva Kejnovská
- Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, CZ-612 65 Brno, Czech Republic
| | - Pavla Bažantová
- Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, CZ-612 65 Brno, Czech Republic
| | - Marek Petr
- Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, CZ-612 65 Brno, Czech Republic
| | - Daniel Renčiuk
- Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, CZ-612 65 Brno, Czech Republic
| | - Michaela Vorlíčková
- Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, CZ-612 65 Brno, Czech Republic
| | - Marie Brázdová
- Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Kralovopolska 135, CZ-612 65 Brno, Czech Republic.
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10
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Nahta R, Al-Mulla F, Al-Temaimi R, Amedei A, Andrade-Vieira R, Bay S, G. Brown D, Calaf GM, Castellino RC, Cohen-Solal KA, Colacci A, Cruickshanks N, Dent P, Di Fiore R, Forte S, Goldberg GS, Hamid RA, Krishnan H, Laird DW, Lasfar A, Marignani PA, Memeo L, Mondello C, Naus CC, Ponce-Cusi R, Raju J, Roy D, Roy R, P. Ryan E, Salem HK, Scovassi AI, Singh N, Vaccari M, Vento R, Vondráček J, Wade M, Woodrick J, Bisson WH. Mechanisms of environmental chemicals that enable the cancer hallmark of evasion of growth suppression. Carcinogenesis 2015; 36 Suppl 1:S2-S18. [PMID: 26106139 PMCID: PMC4565608 DOI: 10.1093/carcin/bgv028] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2014] [Revised: 09/01/2014] [Accepted: 09/19/2014] [Indexed: 12/18/2022] Open
Abstract
As part of the Halifax Project, this review brings attention to the potential effects of environmental chemicals on important molecular and cellular regulators of the cancer hallmark of evading growth suppression. Specifically, we review the mechanisms by which cancer cells escape the growth-inhibitory signals of p53, retinoblastoma protein, transforming growth factor-beta, gap junctions and contact inhibition. We discuss the effects of selected environmental chemicals on these mechanisms of growth inhibition and cross-reference the effects of these chemicals in other classical cancer hallmarks.
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Affiliation(s)
- Rita Nahta
- *To whom correspondence should be addressed.
| | - Fahd Al-Mulla
- Department of Pathology, Kuwait University, Safat 13110, Kuwait
| | | | - Amedeo Amedei
- Department of Experimental and Clinical Medicine, University of Firenze, 50134 Florence, Italy
| | - Rafaela Andrade-Vieira
- Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada
| | - Sarah Bay
- Program in Genetics and Molecular Biology, Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, GA 30322, USA
| | - Dustin G. Brown
- Department of Environmental and Radiological Health Sciences/Colorado State University/Colorado School of Public Health, Fort Collins, CO 80523-1680, USA
| | - Gloria M. Calaf
- Center for Radiological Research, Columbia University Medical Center, New York, NY 10032, USA
- Instituto de Alta Investigacion, Universidad de Tarapaca, Arica 8097877, Chile
| | - Robert C. Castellino
- Division of Hematology and Oncology, Department of Pediatrics, Children’s Healthcare of Atlanta and Emory University, Atlanta, GA 30322, USA
| | - Karine A. Cohen-Solal
- Department of Medicine/Medical Oncology, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ 08901-1914, USA
| | - Annamaria Colacci
- Center for Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency, Bologna 40126, Italy
| | - Nichola Cruickshanks
- Departments of Neurosurgery and Biochemistry and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 980033, USA
| | - Paul Dent
- Departments of Neurosurgery and Biochemistry and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 980033, USA
| | - Riccardo Di Fiore
- Department of Biological, Chemical, and Pharmaceutical Sciences and Technologies, Polyclinic Plexus, University of Palermo, 90127 Palermo, Italy
| | - Stefano Forte
- Mediterranean Institute of Oncology, 95029 Viagrande, Italy
| | - Gary S. Goldberg
- Graduate School of Biomedical Sciences and Department of Molecular Biology, School of Osteopathic Medicine, Rowan University, Stratford, NJ 08084-1501, USA
| | - Roslida A. Hamid
- Department of Biomedical Science, Faculty of Medicine and Health Sciences, University Putra, Serdang, Selangor 43400, Malaysia
| | - Harini Krishnan
- Graduate School of Biomedical Sciences and Department of Molecular Biology, School of Osteopathic Medicine, Rowan University, Stratford, NJ 08084-1501, USA
| | - Dale W. Laird
- Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A 5C1, Canada
| | - Ahmed Lasfar
- Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, NJ 60503, USA
| | - Paola A. Marignani
- Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada
| | - Lorenzo Memeo
- Mediterranean Institute of Oncology, 95029 Viagrande, Italy
| | - Chiara Mondello
- Institute of Molecular Genetics, National Research Council, 27100 Pavia, Italy
| | - Christian C. Naus
- Department of Cellular & Physiological Sciences, Life Sciences Institute, Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
| | - Richard Ponce-Cusi
- Instituto de Alta Investigacion, Universidad de Tarapaca, Arica 8097877, Chile
| | - Jayadev Raju
- Toxicology Research Division, Bureau of Chemical Safety Food Directorate, Health Products and Food Branch Health Canada, Ottawa, Ontario K1A0K9, Canada
| | - Debasish Roy
- Department of Natural Science, The City University of New York at Hostos Campus, Bronx, NY 10451, USA
| | - Rabindra Roy
- Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC 20057, USA
| | - Elizabeth P. Ryan
- Department of Environmental and Radiological Health Sciences/Colorado State University/Colorado School of Public Health, Fort Collins, CO 80523-1680, USA
| | - Hosni K. Salem
- Urology Dept., kasr Al-Ainy School of Medicine, Cairo University, El Manial, Cairo 12515, Egypt
| | - A. Ivana Scovassi
- Institute of Molecular Genetics, National Research Council, 27100 Pavia, Italy
| | - Neetu Singh
- Advanced Molecular Science Research Centre, King George’s Medical University, Lucknow, UP 226003, India
| | - Monica Vaccari
- Center for Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency, Bologna 40126, Italy
| | - Renza Vento
- Department of Biological, Chemical, and Pharmaceutical Sciences and Technologies, Polyclinic Plexus, University of Palermo, 90127 Palermo, Italy
- Sbarro Institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple University, Philadelphia, PA 19122, USA
| | - Jan Vondráček
- Department of Cytokinetics, Institute of Biophysics AS CR, Brno 612 65, Czech Republic
| | - Mark Wade
- Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia (IIT), Milan 16163, Italy and
| | - Jordan Woodrick
- Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC 20057, USA
| | - William H. Bisson
- Environmental and Molecular Toxicology, Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331, USA
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Xu D, Wei G, Lu P, Luo J, Chen X, Skogerbø G, Chen R. Analysis of the p53/CEP-1 regulated non-coding transcriptome in C. elegans by an NSR-seq strategy. Protein Cell 2014; 5:770-82. [PMID: 24844773 PMCID: PMC4180458 DOI: 10.1007/s13238-014-0071-y] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2014] [Accepted: 04/09/2014] [Indexed: 11/28/2022] Open
Abstract
In recent years, large numbers of non-coding RNAs (ncRNAs) have been identified in C. elegans but their functions are still not well studied. In C. elegans, CEP-1 is the sole homolog of the p53 family of genes. In order to obtain transcription profiles of ncRNAs regulated by CEP-1 under normal and UV stressed conditions, we applied the 'not-so-random' hexamers priming strategy to RNA sequencing in C. elegans, This NSR-seq strategy efficiently depleted rRNA transcripts from the samples and showed high technical replicability. We identified more than 1,000 ncRNAs whose apparent expression was repressed by CEP-1, while around 200 were activated. Around 40% of the CEP-1 activated ncRNAs promoters contain a putative CEP-1-binding site. CEP-1 regulated ncRNAs were frequently clustered and concentrated on the X chromosome. These results indicate that numerous ncRNAs are involved in CEP-1 transcriptional network and that these are especially enriched on the X chromosome in C. elegans.
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Affiliation(s)
- Derong Xu
- Laboratory of Non-coding RNA, Institute of Biophysics, University of Chinese Academy of Sciences, Beijing, 100101 China
- Graduate University of Chinese Academy of Sciences, Beijing, 100080 China
| | - Guifeng Wei
- Laboratory of Non-coding RNA, Institute of Biophysics, University of Chinese Academy of Sciences, Beijing, 100101 China
- Graduate University of Chinese Academy of Sciences, Beijing, 100080 China
| | - Ping Lu
- Key Laboratory of Forest Protection, State Forestry
Administration/Research Institute of Forest Ecology, Environment and
Protection, Chinese Academy of Forestry, Beijing, 100091 China
| | - Jianjun Luo
- Laboratory of Non-coding RNA, Institute of Biophysics, University of Chinese Academy of Sciences, Beijing, 100101 China
| | - Xiaomin Chen
- Laboratory of Non-coding RNA, Institute of Biophysics, University of Chinese Academy of Sciences, Beijing, 100101 China
| | - Geir Skogerbø
- Laboratory of Non-coding RNA, Institute of Biophysics, University of Chinese Academy of Sciences, Beijing, 100101 China
| | - Runsheng Chen
- Laboratory of Non-coding RNA, Institute of Biophysics, University of Chinese Academy of Sciences, Beijing, 100101 China
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12
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Telomerase expression in amyotrophic lateral sclerosis (ALS) patients. J Hum Genet 2014; 59:555-61. [PMID: 25142509 DOI: 10.1038/jhg.2014.72] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2014] [Revised: 07/13/2014] [Accepted: 07/17/2014] [Indexed: 01/05/2023]
Abstract
Telomerase and telomeric complex have been linked to a variety of disease states related to neurological dysfunction. In amyotrophic lateral sclerosis (ALS) patients, telomerase activity, as human telomerase reverse transcriptase (hTERT) expression, has not been characterized yet. Here, for the first time, we characterized telomerase and related pathway in blood sample and spinal cord from ALS patients compared with healthy controls. We found that hTERT expression level was significantly lower in ALS patients and was correlated either to p53 mRNA expression or p21 expression, pointing out the hypothesis that telomerase inhibition could be a pathogenetic contributor to neurodegeneration in ALS. As a consequence of the reduced telomerase activity, we identified shorter telomeres in leukocytes from sporadic ALS patients compared with healthy control group.
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13
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Samassekou O, Bastien N, Lichtensztejn D, Yan J, Mai S, Drouin R. DifferentTP53mutations are associated with specific chromosomal rearrangements, telomere length changes, and remodeling of the nuclear architecture of telomeres. Genes Chromosomes Cancer 2014; 53:934-50. [DOI: 10.1002/gcc.22205] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2014] [Accepted: 07/02/2014] [Indexed: 02/06/2023] Open
Affiliation(s)
- Oumar Samassekou
- Division of Genetics; Department of Pediatrics; Faculty of Medicine and Health Sciences; Université de Sherbrooke; Sherbrooke QC Canada
- Manitoba Institute of Cell Biology; CancerCare Manitoba; Department of Physiology; Faculty of Medicine, University of Manitoba; Winnipeg MB Canada
| | - Nathalie Bastien
- Division of Genetics; Department of Pediatrics; Faculty of Medicine and Health Sciences; Université de Sherbrooke; Sherbrooke QC Canada
| | - Daniel Lichtensztejn
- Manitoba Institute of Cell Biology; CancerCare Manitoba; Department of Physiology; Faculty of Medicine, University of Manitoba; Winnipeg MB Canada
| | - Ju Yan
- Division of Genetics; Department of Pediatrics; Faculty of Medicine and Health Sciences; Université de Sherbrooke; Sherbrooke QC Canada
| | - Sabine Mai
- Manitoba Institute of Cell Biology; CancerCare Manitoba; Department of Physiology; Faculty of Medicine, University of Manitoba; Winnipeg MB Canada
| | - Régen Drouin
- Division of Genetics; Department of Pediatrics; Faculty of Medicine and Health Sciences; Université de Sherbrooke; Sherbrooke QC Canada
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14
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15
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hTERT: Another brick in the wall of cancer cells. MUTATION RESEARCH-REVIEWS IN MUTATION RESEARCH 2013; 752:119-128. [DOI: 10.1016/j.mrrev.2012.12.005] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/24/2012] [Revised: 11/28/2012] [Accepted: 12/10/2012] [Indexed: 01/06/2023]
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16
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Lu W, Zhang Y, Liu D, Songyang Z, Wan M. Telomeres-structure, function, and regulation. Exp Cell Res 2012; 319:133-41. [PMID: 23006819 DOI: 10.1016/j.yexcr.2012.09.005] [Citation(s) in RCA: 170] [Impact Index Per Article: 13.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2012] [Accepted: 09/13/2012] [Indexed: 12/15/2022]
Abstract
In mammals, maintenance of the linear chromosome ends (or telomeres) involves faithful replication of genetic materials and protection against DNA damage signals, to ensure genome stability and integrity. These tasks are carried out by the telomerase holoenzyme and a unique nucleoprotein structure in which an array of telomere-associated proteins bind to telomeric DNA to form special protein/DNA complexes. The telomerase complex, which is comprised of telomeric reverse transcriptase (TERT), telomeric RNA component (TERC), and other assistant factors, is responsible for adding telomeric repeats to the ends of chromosomes. Without proper telomere maintenance, telomere length will shorten with successive round of DNA replication due to the so-called end replication problem. Aberrant regulation of telomeric proteins and/or telomerase may lead to abnormalities that can result in diseases such as dyskeratosis congenita (DC) and cancers. Understanding the mechanisms that regulate telomere homeostasis and the factors that contribute to telomere dysfunction should aid us in developing diagnostic and therapeutic tools for these diseases.
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Affiliation(s)
- Weisi Lu
- State Key Laboratory for Biocontrol, SYSU, Guangzhou, PR China
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18
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A cyano analogue of boswellic acid induces crosstalk between p53/PUMA/Bax and telomerase that stages the human papillomavirus type 18 positive HeLa cells to apoptotic death. Eur J Pharmacol 2011; 660:241-8. [DOI: 10.1016/j.ejphar.2011.03.013] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2010] [Revised: 02/23/2011] [Accepted: 03/17/2011] [Indexed: 02/04/2023]
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Decorsière A, Cayrel A, Vagner S, Millevoi S. Essential role for the interaction between hnRNP H/F and a G quadruplex in maintaining p53 pre-mRNA 3'-end processing and function during DNA damage. Genes Dev 2011; 25:220-5. [PMID: 21289067 DOI: 10.1101/gad.607011] [Citation(s) in RCA: 139] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Following DNA damage, mRNA 3'-end formation is inhibited, contributing to repression of mRNA synthesis. Here we investigated how DNA-damaged cells accomplish p53 mRNA 3'-end formation when normal mechanisms of pre-mRNA 3'-end processing regulation are inhibited. The underlying mechanism involves the interaction between a G-quadruplex structure located downstream from the p53 cleavage site and hnRNP H/F. Importantly, this interaction is critical for p53 expression and contributes to p53-mediated apoptosis. Our results uncover the existence of a specific rescue mechanism of 3'-end processing regulation allowing stress-induced p53 accumulation and function in apoptosis.
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20
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Babizhayev MA, Vishnyakova KS, Yegorov YE. Telomere-dependent senescent phenotype of lens epithelial cells as a biological marker of aging and cataractogenesis: the role of oxidative stress intensity and specific mechanism of phospholipid hydroperoxide toxicity in lens and aqueous. Fundam Clin Pharmacol 2011; 25:139-62. [DOI: 10.1111/j.1472-8206.2010.00829.x] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
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Abstract
1. Oestrogen plays an important role in ageing and ageing-related development. Lack of oestrogen prompts endocrine cell ageing of the ovary, whereas oestrogen overflow impacts on epithelial cell neoplastic development. 2. Recent studies indicate that oestrogen regulates cell proliferative fates by a mechanism of reprogramming the size of telomeres (ends of chromosomes) in the oestrogen target cells. This is achieved by upregulating the telomerase reverse transcriptase (TERT) gene in a temporal and spatial manner. 3. Currently, the relationship between oestrogen and telomerase activity in regulating productive cell development and function remains elusive. A number of lines of evidence suggest that telomerase is a downstream target of oestrogen in oestrogen-dependent reproductive ageing and neoplastic development. 4. The present minireview discusses our current understanding of the mechanisms by which telomerase maintenance of telomere homeostasis mediates oestrogen-induced ageing and tumourigenesis in the ovary under physiological and pathological conditions.
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Affiliation(s)
- He Li
- Department of Immunology, Molecular Signalling Laboratory, Monash University Central Clinical School, AMREP, Melbourne, Victoria 3004, Australia.
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22
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Zhang C, Guo X, Zhang L, Lu Z, Ma N, Cheng Y, Shen F, Zhang B, Wu M, Wei L. Methylation-Related silencing of p14ARF gene correlates with telomerase activity and mRNA expression of human telomerase reverse transcriptase in hepatocellular carcinoma. J Surg Oncol 2008; 98:462-8. [DOI: 10.1002/jso.21131] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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Yang C, Przyborski S, Cooke MJ, Zhang X, Stewart R, Anyfantis G, Atkinson SP, Saretzki G, Armstrong L, Lako M. A key role for telomerase reverse transcriptase unit in modulating human embryonic stem cell proliferation, cell cycle dynamics, and in vitro differentiation. Stem Cells 2008; 26:850-63. [PMID: 18203676 DOI: 10.1634/stemcells.2007-0677] [Citation(s) in RCA: 93] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Embryonic stem cells (ESC) are a unique cell population with the ability to self-renew and differentiate into all three germ layers. Human ESC express the telomerase reverse transcriptase (TERT) gene and the telomerase RNA (TR) and show telomerase activity, but TERT, TR, and telomerase are all downregulated during the differentiation process. To examine the role of telomerase in human ESC self-renewal and differentiation, we modulated the expression of TERT. Upregulation of TERT and increased telomerase activity enhanced the proliferation and colony-forming ability of human ESC, as well as increasing the S phase of the cell cycle at the expense of a reduced G1 phase. Upregulation of TERT expression was associated with increases in CYCLIN D1 and CDC6 expression, as well as hyperphosphorylation of RB. The differentiated progeny of control ESC showed shortening of telomeric DNA as a result of loss of telomerase activity. In contrast, the differentiated cells from TERT-overexpressing ESC maintained high telomerase activity and accumulated lower concentrations of peroxides than wild-type cells, implying greater resistance to oxidative stress. Although the TERT-overexpressing human ESC are able to form teratoma composed of three germ layers in vivo, their in vitro differentiation to all primitive and embryonic lineages was suppressed. In contrast, downregulation of TERT resulted in reduced ESC proliferation, increased G1, and reduced S phase. Most importantly, downregulation of TERT caused loss of pluripotency and human ESC differentiation to extraembryonic and embryonic lineages. Our results indicate for the first time an important role for TERT in the maintenance of human ESC pluripotency, cell cycle regulation, and in vitro differentiation capacity.
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Affiliation(s)
- Chunbo Yang
- North East Institute for Stem Cell Research, Newcastle upon Tyne NE1 3BZ, United Kingdom
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Trkova M, Prochazkova K, Krutilkova V, Sumerauer D, Sedlacek Z. Telomere length in peripheral blood cells of germline TP53 mutation carriers is shorter than that of normal individuals of corresponding age. Cancer 2007; 110:694-702. [PMID: 17567834 DOI: 10.1002/cncr.22834] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
BACKGROUND A decrease in the age at cancer onset and increase in cancer incidence in successive generations in Li-Fraumeni syndrome (LFS) families with germline TP53 mutations have been previously described. In the current study a possible relation was analyzed between telomere length and cancer onset in TP53 mutation carriers. METHODS Telomere length was measured using real-time quantitative polymerase chain reaction (PCR) in 20 carriers of germline TP53 mutations and in 83 unrelated healthy individuals. According to the age at blood sampling, patients and controls were divided into 2 age groups, children and adults. Telomere length was correlated to TP53 mutation status and telomere shortening in patients to the age at cancer onset. A t-test and linear regression were used to analyze the data. RESULTS Compared with healthy controls, telomere length was significantly shorter both in the child (P = .001) and adult (P = .034) germline T53 mutation carriers. Although a statistically significant correlation between telomere shortening and the age at cancer onset was not observed, there was a trend of shorter telomeres in mutation carriers affected in childhood compared with those affected later in life. Neither cancer therapy nor sex differences were likely to affect the results. CONCLUSIONS The findings suggest a possible link between the carriership of a germline TP53 mutation, telomere length, predisposition to early-onset cancer, and anticipation in LFS.
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Affiliation(s)
- Marie Trkova
- Department of Biology and Medical Genetics, Charles University 2nd Medical School and University Hospital Motol, Prague, Czech Republic.
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25
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Shervington A, Patel R, Lu C, Cruickshanks N, Lea R, Roberts G, Dawson T, Shervington L. Telomerase subunits expression variation between biopsy samples and cell lines derived from malignant glioma. Brain Res 2007; 1134:45-52. [PMID: 17196947 DOI: 10.1016/j.brainres.2006.11.093] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2006] [Revised: 11/29/2006] [Accepted: 11/30/2006] [Indexed: 01/12/2023]
Abstract
Although scientific advances have recognised the prognostic power of telomerase activity in different cancers, as yet there has been no investigation regarding the expression variation of telomerase subunits in glioma tissues and cell lines. In this study, a recurrent anaplastic ependymoma and seven glioblastoma biopsy samples, four cell lines and four controls including two normal brain tissues were analysed for telomerase subunit expression profiles together with telomerase activity. Since telomerase activity is linked to tumourgenesis, the genes were analysed with respect to their expression variation. TEP1 was expressed in all glioma cell lines and 70% of glioblastoma tissues, in addition to the control brain tissues. Tankyrase was expressed in 85% of the glioblastoma tissues and was down-regulated in the recurrent anaplastic ependymoma tissue control cell lines. However, it was expressed in the control tissues. Dyskerin was expressed in all cell lines and tissues apart from U87-MG and NHA cells and the recurrent anaplastic ependymoma tissue. As expected, PARP1 and GAPDH showed constitutive expression throughout all cell lines and tissues since both are known to be housekeeping genes. hTERT was expressed in all glioma cell lines and tissues but was absent in the control cells and tissues. Telomerase activity was absent in IPDDC-A2 cells and 57% of the glioblastoma tissues. These results suggest that hTERT expression and not telomerase activity possibly represents a simple and reliable biological diagnostic tool.
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Affiliation(s)
- Amal Shervington
- Department of Biological Sciences, University of Central Lancashire, and Neurosurgery Department, Royal Preston Hospital, UK.
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26
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Fischer G, Tutuncuoglu O, Bakhshandeh M, Masood S. Diagnostic value of telomerase expression in breast fine-needle aspiration biopsies. Diagn Cytopathol 2007; 35:653-5. [PMID: 17854082 DOI: 10.1002/dc.20690] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Fine-needle aspiration biopsy (FNAB) of breast is a minimally invasive sampling procedure with a proven value in the initial evaluation of patients with palpable breast lesions. FNAB is a simple, cost-effective, and relatively nontraumatic procedure that has replaced open surgical biopsy in majority of academic institutions across the world. There are, however, inherent limitations in the ability of FNAB to reliably diagnose small percentage of cases that are difficult to diagnose by cytomorphology alone and require excisional biopsy. This shortcoming may be minimized if the morphology can be complemented by a reliable diagnostic adjunct. This retrospective study was designed to assess the added value of telomerase immunostain in interpretation of breast FNABs. Telomerase is a ribonucleoprotein enzyme that has been shown to be activated in different malignant tumors, including breast cancer. Immunocytochemical detection of this molecular marker on cytologic smears and cellblocks may be helpful for interpretation of FNAB specimens. In our retrospective study, we found that 56% of the malignant breast cases (28/50) showed positive telomerase immunostaining while only 4% of the negative cases (2/50) stained with telomerase (positive predictive value: 93%, negative predictive value: 69%). Expression of telomerase on highly suspicious breast fine-needle aspirations may upgrade the diagnosis to malignancy. However, a negative telomerase cannot exclude the possibility of carcinoma.
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Affiliation(s)
- Gabor Fischer
- Department of Pathology, College of Medicine, University of Florida, Jacksonville, Florida 32209, USA
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Liu JP, Cassar L, Pinto A, Li H. Mechanisms of cell immortalization mediated by EB viral activation of telomerase in nasopharyngeal carcinoma. Cell Res 2006; 16:809-17. [PMID: 17016469 DOI: 10.1038/sj.cr.7310098] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Nasopharyngeal carcinoma (NPC) is a common cancer in Southern China and Southeast Asia. The disease is a poorly differentiated carcinoma without effective cure, and the mechanism underlying its development remains largely unknown. Of several factors identified in NPC aetiology in recent years, Epstein-Barr virus (EBV) infection has emerged to be most important. In almost all NPC cells, EBV uses several intracellular mechanisms to cause oncogenic evolution of the infected cells. One such mechanism by which EBV infection induces cellular immortalization is believed to be through the activation of telomerase, an enzyme that is normally repressed but becomes activated during cancer development. Studies show that greater than 85% of primary NPC display high telomerase activity by mechanisms involving EBV infection, consistent with the notion that EBV is commonly involved in inducing cell immortalization. More recently, different EBV proteins have been shown to activate or inhibit the human telomerase reverse transcriptase gene, by modulating intracellular signalling pathways. These findings suggest a new model with a number of challenges towards our understanding, molecular targeting and therapeutic intervention in NPC.
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Affiliation(s)
- Jun-Ping Liu
- Department of Immunology, Central Eastern Clinical School, Monash University, AMREP, Commercial Road, Prahran, Melbourne, Victoria 3181, Australia.
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28
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Renaud S, Loukinov D, Bosman FT, Lobanenkov V, Benhattar J. CTCF binds the proximal exonic region of hTERT and inhibits its transcription. Nucleic Acids Res 2005; 33:6850-60. [PMID: 16326864 PMCID: PMC1301598 DOI: 10.1093/nar/gki989] [Citation(s) in RCA: 106] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
The expression of the catalytic subunit (hTERT) represents the limiting factor for telomerase activity. Previously, we detected a transcriptional repressor effect of the proximal exonic region (first two exons) of the hTERT gene. To better understand the mechanism involved and to identify a potential repressor, we further characterized this region. The addition of the hTERT proximal exonic region downstream of the hTERT minimal promoter strongly reduced promoter transcriptional activity in all cells tested (tumor, normal and immortalized). This exonic region also significantly inhibited the transcriptional activity of the CMV and CDKN2A promoters, regardless of the cell type. Therefore, the repressor effect of hTERT exonic region is neither cell nor promoter-dependent. However, the distance between the promoter and the exonic region can modulate this repressor effect, suggesting that nucleosome positioning plays a role in transcriptional repression. We showed by electrophoretic mobility shift assay that CCCTC-binding factor (CTCF) binds to the proximal exonic region of hTERT. Chromatin immunoprecipitaion assays confirmed the binding of CTCF to this region. CTCF is bound to hTERT in cells in which hTERT is not expressed, but not in telomerase-positive ones. Moreover, the transcriptional downregulation of CTCF by RNA interference derepressed hTERT gene expression in normal telomerase-negative cells. Our results suggest that CTCF participates in key cellular mechanisms underlying immortality by regulating hTERT gene expression.
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Affiliation(s)
| | - Dmitri Loukinov
- Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institutes of HealthRockville, MD 20892, USA
| | | | - Victor Lobanenkov
- Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, National Institutes of HealthRockville, MD 20892, USA
| | - Jean Benhattar
- To whom correspondence should be addressed. Tel: +41 21 314 7153; Fax: +41 21 314 7115;
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Hu B, Tack DC, Liu T, Wu Z, Ullenbruch MR, Phan SH. Role of Smad3 in the regulation of rat telomerase reverse transcriptase by TGFβ. Oncogene 2005; 25:1030-41. [PMID: 16205635 DOI: 10.1038/sj.onc.1209140] [Citation(s) in RCA: 34] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Telomerase is induced in certain pathological conditions such as cancer and tissue injury and repair. This induction in fibroblasts from injured lung is repressed by transforming growth factor beta (TGFbeta) via yet unknown mechanisms. In this study, the role of Smad3 in the inhibition of telomerase reverse transcriptase (TERT) gene transcription by TGFbeta was investigated. The rat TERT (rTERT) gene promoter was cloned by PCR amplification and fused with a luciferase reporter gene. This construct was used to analyse regulation of promoter activity in fibroblasts isolated from bleomycin-injured lung with induced telomerase activity. The results showed that TGFbeta inhibited rTERT transcription while stimulating Smad3 expression. Interestingly, TGFbeta also inhibited the expression of c-myc. Cotransfection with a Smad3 expressing plasmid further repressed rTERT transcription and c-myc expression, while cotransfection with the corresponding antisense Smad3 construct had the opposite effect. Mutation of an E-box in the rTERT promoter suppressed its activity, which could be further reduced by TGFbeta treatment. In contrast, mutation at a Smad binding element enhanced promoter activity whose inhibition was impaired by TGFbeta treatment. Thus TGFbeta inhibition of rTERT gene expression was directly mediated by Smad3 via the Smad binding element, while c-myc appears to primarily regulate its constitutive or induced expression.
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Affiliation(s)
- B Hu
- Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-0602, USA
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30
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Latonen L, Laiho M. Cellular UV damage responses--functions of tumor suppressor p53. Biochim Biophys Acta Rev Cancer 2005; 1755:71-89. [PMID: 15921859 DOI: 10.1016/j.bbcan.2005.04.003] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2004] [Revised: 04/07/2005] [Accepted: 04/21/2005] [Indexed: 02/06/2023]
Abstract
DNA damage, provoked by ultraviolet (UV) radiation, evokes a cellular damage response composed of activation of stress signaling and DNA checkpoint functions. These are translated to responses of replicative arrest, damage repair, and apoptosis aimed at cellular recovery from the damage. p53 tumor suppressor is a central stress response protein, activated by multiple endogenous and environmental insults, including UV radiation. The significance of p53 in the DNA damage responses has frequently been reviewed in the context of ionizing radiation or other double strand break (DSB)-inducing agents. Despite partly similar patterns, the molecular events following UV radiation are, however, distinct from the responses induced by DSBs and are profoundly coupled with transcriptional stress. These are illustrated, e.g., by the UV damage-specific translocations of Mdm2, promyelocytic leukemia protein, and nucleophosmin and their interactions with p53. In this review, we discuss UV damage-provoked cellular responses and the functions of p53 in damage recovery and cell death.
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Affiliation(s)
- Leena Latonen
- Molecular and Cancer Biology Program and Haartman Institute, University of Helsinki, PO Box 63, FIN-00014 Helsinki, Finland
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31
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Carter RT, Giudice C, Dubielzig RR, Colitz CMH. Telomerase activity with concurrent loss of cell cycle regulation in feline post-traumatic ocular sarcomas. J Comp Pathol 2005; 133:235-45. [PMID: 16154137 DOI: 10.1016/j.jcpa.2005.04.009] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2004] [Accepted: 04/22/2005] [Indexed: 11/21/2022]
Abstract
Paraffin wax-embedded ocular globes of cats with post-traumatic ocular sarcomas were examined for the presence of TERT, the active subunit of telomerase. The latter is a ribonucleoprotein complex essential for immortalization and expressed by most malignant tumours, germ line cells, lens epithelial cells, and some stem cells. Due to the frequent loss of cell cycle control with the increased expression of telomerase activity, post-traumatic ocular sarcomas were also examined for loss of p16 expression and alterations in p53, the findings being related to mitotic score, tumour grade, and proliferating cell nuclear antigen. These sarcomas expressed telomerase at a high frequency (62.5%); in addition, the majority showed alterations in cell cycle control, as evaluated by lack of p16 immunolabelling (66.7%). Alterations in p53 were the sole mechanism by which cell cycle control was dysregulated in only two tumours expressing TERT (13%). These findings suggest that p16, and not p53, represents the primary mechanism by which post-traumatic ocular sarcomas that express telomerase activity escape cell cycle control.
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Affiliation(s)
- R T Carter
- Department of Surgical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706, USA
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32
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N/A, 齐 义, 王 立. N/A. Shijie Huaren Xiaohua Zazhi 2005; 13:1317-1321. [DOI: 10.11569/wcjd.v13.i11.1317] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/26/2023] Open
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33
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Liu X, Yuan H, Fu B, Disbrow GL, Apolinario T, Tomaic V, Kelley ML, Baker CC, Huibregtse J, Schlegel R. The E6AP ubiquitin ligase is required for transactivation of the hTERT promoter by the human papillomavirus E6 oncoprotein. J Biol Chem 2005; 280:10807-16. [PMID: 15655249 DOI: 10.1074/jbc.m410343200] [Citation(s) in RCA: 89] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Most human cancer cells display increased telomerase activity that appears to be critical for continued cell proliferation and tumor formation. The E6 protein of malignancy-associated human papillomaviruses increases cellular telomerase in primary human keratinocytes at least partly via transcriptional activation of the telomerase catalytic subunit, hTERT. In the present study, we investigated whether E6AP, a ubiquitin ligase well known for binding and mediating some of the activities of the E6 oncoprotein, participated in the transactivation of the hTERT promoter. Our results demonstrate that E6 mutants that fail to bind E6AP are also defective for increasing telomerase activity and transactivating the hTERT promoter. More importantly, E6AP knock-out mouse cells and small interfering RNA techniques demonstrated that E6AP was required for hTERT promoter transactivation in both mouse and human cells. Neither E6 nor E6AP bound to the hTERT promoter or activated the promoter in the absence of the partner protein. With all transactivation-competent E6 proteins, induction of the hTERT promoter was dependent upon E box elements in the core promoter. It appears, therefore, that E6-mediated activation of the hTERT promoter requires a complex of E6-E6AP to engage the hTERT promoter and that activation is dependent upon Myc binding sites in the promoter. The recruitment of a cellular ubiquitin ligase to the hTERT promoter during E6-mediated transcriptional activation suggests a role for the local ubiquitination (and potential degradation) of promoter-associated regulatory proteins, including the Myc protein.
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Affiliation(s)
- Xuefeng Liu
- Department of Pathology and Oncology, Georgetown University Medical School, 3900 Reservoir Rd. NW, Washington, DC 20057, USA
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34
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Liu WJ, Zhang YW, Zhang ZX, Ding J. Alternative splicing of human telomerase reverse transcriptase may not be involved in telomerase regulation during all-trans-retinoic acid-induced HL-60 cell differentiation. J Pharmacol Sci 2004; 96:106-14. [PMID: 15467265 DOI: 10.1254/jphs.fp0030600] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Alternative splicing of the human telomerase reverse transcriptase subunit (hTERT) suppresses telomerase activity during the development of human fetal kidney cells into mature cells. Tumor cell differentiation is the process of turning abnormal tumor cells into 'normal' cells accompanied by down-regulation of telomerase activity. However, the precise mechanism of the regulation of telomerase activity in differentiated cells is not fully understood. In this study, we observed the role of alternative splicing of hTERT in the regulation of telomerase activity in all-trans-retinoic acid (ATRA)-induced, differentiated HL-60 cells. ATRA-induced down-regulation of telomerase activity in differentiated HL-60 cells was associated with a decrease in hTERT and an increase in human telomerase-associated protein-1 (hTP1) transcription. Expression of full length variant hTERT alpha+ beta+ mRNA decreased in a dose- and time-dependent manner. The drop of hTERT beta- mRNA was time-dependent. hTERT alpha- and hTERT alpha- beta- mRNA were reduced dramatically after ATRA treatment. In the dose-effect study, hTERT alpha+ beta+ and hTERT beta- maintained a relatively stable ratio when telomerase activity decreased largely from treatment with 1 to 5 microM ATRA. Although the splicing pattern of hTERT mRNA was altered in time-effect research, the change was not related to the ATRA-treated decline of telomerase activity. The expression of alternative splicing variants of hTERT also decreased at the protein level. All these results suggested that alternative splicing of hTERT mRNA may not contribute to the suppression of telomerase activity during ATRA-induced HL-60 leukemia cell differentiation.
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Affiliation(s)
- Wei-Jun Liu
- Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China
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Abstract
Human chromosomes have highly specialized structures at their ends termed telomeres, repetitive, non-coding DNA sequences (5'-TTAGGG-3'), ranging in size from 5 to 20 kb in human cells. These highly specialized structures prevent chromosome ends from being recognized as double-strand DNA breaks, and they also provide protection from destabilizing agents. The mechanism for maintaining telomere integrity is controlled by telomerase, a ribonucleoprotein enzyme that specifically restores telomere sequences lost during replication by using an intrinsic RNA component as a template for polymerization. Telomerase has two core functional components required for its activity: the catalytic subunit of human telomerase reverse transcriptase (hTERT) and a telomerase RNA template (hTR). Telomerase is activated in the majority of immortal cell lines in culture and in most malignant tumors. This review outlines our current understanding of telomerase in breast cancer development and critically evaluates potential utilities in diagnosis, prognosis, and therapy.
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Affiliation(s)
- Atac Baykal
- University of Texas M.D. Anderson Cancer Center, Department of Pathology, Houston, Texas, USA
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36
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Stampfer MR, Garbe J, Nijjar T, Wigington D, Swisshelm K, Yaswen P. Loss of p53 function accelerates acquisition of telomerase activity in indefinite lifespan human mammary epithelial cell lines. Oncogene 2003; 22:5238-51. [PMID: 12917625 DOI: 10.1038/sj.onc.1206667] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
We describe novel effects of p53 loss on immortal transformation, based upon comparison of immortally transformed human mammary epithelial cell (HMEC) lines lacking functional p53 with closely related p53(+) lines. Our previous studies of p53(+) immortal HMEC lines indicated that overcoming the stringent replicative senescence step associated with critically short telomeres (agonescence), produced indefinite lifespan lines that maintained growth without immediately expressing telomerase activity. These telomerase(-) 'conditionally immortal' HMEC underwent an additional step, termed conversion, to become fully immortal telomerase(+) lines with uniform good growth. The very gradual conversion process was associated with slow heterogeneous growth and high expression of the cyclin-dependent kinase inhibitor p57(Kip2). We now show that p53 suppresses telomerase activity and is necessary for the p57 expression in early passage p53(+) conditionally immortal HMEC lines, and that p53(-/-) lines exhibit telomerase reactivation and attain full immortality much more rapidly. A p53-inhibiting genetic suppressor element introduced into early passages of a conditionally immortal telomerase(-) p53(+) HMEC line led to rapid induction of hTERT mRNA, expression of telomerase activity, loss of p57 expression, and quick attainment of uniform good growth. These studies indicate that derangements in p53 function may impact malignant progression through direct effects on the conversion process, a potentially rate-limiting step in HMEC acquisition of uniform unlimited growth potential. These studies also provide evidence that the function of p53 in suppression of telomerase activity is separable from its cell cycle checkpoint function.
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Affiliation(s)
- Martha R Stampfer
- Lawrence Berkeley National Laboratory, Life Sciences Division, Berkeley, CA 94720, USA.
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37
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Yoshino A, Katayama Y, Fukushima T, Watanabe T, Komine C, Yokoyama T, Kusama K, Moro I. Telomerase activity in pituitary adenomas: significance of telomerase expression in predicting pituitary adenoma recurrence. J Neurooncol 2003; 63:155-62. [PMID: 12825819 DOI: 10.1023/a:1023935621976] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Clinical and histopathological evaluations are inadequate for assessing biological aggressiveness and regrowth potential in benign pituitary adenomas. To develop reliable and prognostically informative means of predicting behavior remains an intractable problem. Telomerase, a reverse transcriptase that extends telomere length, may facilitate tumorigenesis and tumor immortality. In the present study, we investigated the telomerase activity of pituitary adenomas, and attempted to assess the value of telomerase expression for predicting their clinical course. In total, 31 (30 patients) benign pituitary adenoma samples including 8 recurrent adenomas were studied. Telomerase expression was evaluated by polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay and telomerase activity levels were quantitated by improved PCR enzyme-linked immunosorbent assay (ELISA). The data were analyzed in relation to clinical course which was reviewed at 4-5.5 years (median follow-up time, 52.5 months) after surgery. The relative values of the telomerase expression for predicting the clinical course were compared with the MIB-1 antigen-based proliferative cell index (PCI) and p53 immunoreactivity which have recently been suggested to correlate with aggressive behavior in pituitary adenomas. Overall, telomerase expression was detected in 13% of the adenomas (4 tumor tissues, 3 patients). These adenomas comprised large, invasive, and functioning adenomas. The number of telomerase-positive adenomas was small; however, the PCI was higher in cases with telomerase expression (4 tumor tissues; mean, 4.2 +/- 2.4%) than in those without it (27 tumor tissues; 1.4 +/- 1.3%) (p = 0.01). One tumor with detectable telomerase expression, which did not undergo additional pharmacological or radiotherapeutic intervention after first surgery, recurred rapidly despite gross total surgical resection, although the PCI of both the primary and recurrent adenomas was not high. Detection of telomerase expression may represent an additional useful means of identifying aggressive behavior, complementing the histopathological evaluation of benign-appearing pituitary adenomas.
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Affiliation(s)
- Atsuo Yoshino
- Department of Neurological Surgery, Nihon University School of Medicine, Tokyo, Japan.
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Abstract
Arthur Kornberg "never met a dull enzyme" (For the Love of Enzymes: The Odyssey of a Biochemist, Harvard University Press, 1989) and telomerase is no exception. Telomerase is a remarkable polymerase that uses an internal RNA template to reverse-transcribe telomere DNA, one nucleotide at a time, onto telomeric, G-rich single-stranded DNA. In the 17 years since its discovery, the characterization of telomerase enzyme components has uncovered a highly conserved family of telomerase reverse transcriptases that, together with the telomerase RNA, appear to comprise the enzymatic core of telomerase. While not as comprehensively understood as yet, some telomerase-associated proteins also serve crucial roles in telomerase function in vivo, such as telomerase ribonudeoprotein (RNP) assembly, recruitment to the telomere, and the coordination of DNA replication at the telomere. A selected overview of the biochemical properties of this unique enzyme, in vitro and in vivo, will be presented.
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Abstract
Human epithelial cells encounter two senescence barriers that enforce a limited proliferative potential. A first barrier is mediated by the retinoblastoma protein, and can be overcome by multiple types of errors, many of which are observed in human cancers. A second, extremely stringent telomere-dependent barrier, is a consequence of repression of telomerase activity. Although relieved by ectopic hTERT expression, the nature of the errors required to overcome this latter barrier during in vivo carcinogenesis have not yet been defined. Attainment of immortality and telomerase reactivation are crucial to human carcinoma development; the derangements responsible for attainment of immortality may be rate-limiting and permissive for further progression to malignancy.
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40
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Kaul SC, Yaguchi T, Taira K, Reddel RR, Wadhwa R. Overexpressed mortalin (mot-2)/mthsp70/GRP75 and hTERT cooperate to extend the in vitro lifespan of human fibroblasts. Exp Cell Res 2003; 286:96-101. [PMID: 12729798 DOI: 10.1016/s0014-4827(03)00101-0] [Citation(s) in RCA: 77] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The lifespan of human foreskin fibroblasts (HFF5), cultured under standard in vitro conditions (including ambient atmospheric oxygen tension), was extended slightly by expression of exogenous mortalin (mot-2)/mthsp70/Grp75, but not by the catalytic subunit of telomerase, hTERT. Together, mot-2 and hTERT permitted bypass of senescence, a substantial extension of lifespan, and possibly immortalization. This is the first demonstration that mot-2 and telomerase can cooperate in the immortalization process.
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Affiliation(s)
- Sunil C Kaul
- National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
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41
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Xu W, Yan M, Sun L, Zheng Z, Liu X. Ref-1 protein enhances the IL-2-stimulated telomerase activity. J Cell Biochem 2003; 88:1120-8. [PMID: 12647295 DOI: 10.1002/jcb.10462] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Telomerase is an important ribonucleoprotein enzyme involved in cellular proliferation and senescence. Activation of telomerase has been detected in a vast majority of human cancer cells. In this article, we demonstrated that Interleukin-2 (IL-2) which is the pivotal cytokine in the immune system could stimulate the activity of telomerase in the cultured BA/F3beta cells. It was also found that the level of IL-2-induced telomerase activity was decreased by the treatment with chemical oxidant in vitro. Since IL-2 stimulation produces a oxidative shift of the intracellular environment, the activation and maintenance of telomerase in this oxidative circumstance requires particular protection. Here we proved the redox factor-1 (Ref-1) protein was involved in this process. The addition of GST-Ref-1 protein increased the level of IL-2-induced telomerase activity in the TRAP assay, while elimination of the endogenous Ref-1 protein by immunodepletion decreased it. Consistent with these in vitro results, IL-2-induced telomerase activity could be enhanced by transient overexpression of Ref-1 protein in BA/F3beta cells. Taken together, these findings proved that Ref-1 protein benefits the activation of telomerase activity in the oxidative microenvironment of the BA/F3beta cells stimulated by IL-2.
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Affiliation(s)
- Weijing Xu
- Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China
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42
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Saldanha SN, Andrews LG, Tollefsbol TO. Analysis of telomerase activity and detection of its catalytic subunit, hTERT. Anal Biochem 2003; 315:1-21. [PMID: 12672407 DOI: 10.1016/s0003-2697(02)00663-2] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The discovery of the enzyme telomerase and its subunits has led to major advances in understanding the mechanisms of cellular proliferation, immortalization, aging, and neoplastic transformation. The expression of telomerase in more than 85% of tumors provides an excellent tool for the diagnosis, prognosis, and treatment of cancer. However, the techniques employed in its detection appear to play a significant role in the interpretation of the results. The telomeric repeat amplification protocol (TRAP assay) has been the standard assay in the detection of telomerase activity and many variations of this technique have been reported. Recent advances in the development of the TRAP assay and the incorporation of techniques that provide a quantitative and qualitative estimate of telomerase activity are assessed in this review. In addition to histological and cytological examination of tissues, distribution patterns of the catalytic subunit of telomerase, hTERT, are frequently used in the prognosis of tumors. The methods involved in the detection of hTERT as a biomarker of cellular transformation are also analyzed.
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Affiliation(s)
- Sabita N Saldanha
- Department of Biology, University of Alabama at Birmingham, 35294-1170, USA
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Shao JC, Wu JF, Wang DB, Qin R, Zhang H. Relationship between the expression of human telomerase reverse transcriptase gene and cell cycle regulators in gastric cancer and its significance. World J Gastroenterol 2003; 9:427-31. [PMID: 12632490 PMCID: PMC4621554 DOI: 10.3748/wjg.v9.i3.427] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [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 investigate the expression of human telomerase reverse transcriptase gene (hTRT) in gastric cancer (GC) and its relevance with cell cycle regulators including P16INK4, cyclin and P53.
METHODS: In situ hybridization (ISH) for hTRT mRNA was performed in 53 cases of gastric cancer and adjacent cancerous tissues. Immunohistochemical staining (S-P method) for hTRT protein, P16INK4, cyclinD1 and P53 was performed in 53 cases of GC and adjacent cancerous tissues.
RESULTS: Of 53 cases of GC, the expression of hTRT mRNA and hTRT protein was significantly higher than the expression of hTRT mRNA and hTRT protein in adjacent canerous tissues (P < 0.01), the positive rates of hTRTmRNA and hTRT protein were 79.2% and 88.6%. There was a stastical difference of the expression of hTRT protein among well differentiated adenocarcinoma, poorly differentiated adenocarcinoma and mucoid carcinoma. And there was a highly significant positive correlation between the expression of hTRT mRNA and hTRT protein (r = 0.625, P < 0.01). However, the expression of hTRT mRNA and its protein in GC were not related with other clinicopathological parameters including gender, age, location and size of neoplasm, invasion depth, lymph node metastasis and clinical stage. There was a significant positive correlation between the expression of hTRT mRNA and cyclinD1 protein (r = 0.350, P < 0.01). There was a significant positive correlation between the expression of cyclinD1 protein and hTRT protein (r = 0.549, P < 0.01), so was between P53 and hTRT protein (r = 0.319, P < 0.05).
CONCLUSION: The expression of hTRT gene is correlated significantly to the specific defects of cell cycle on G1/S check point ; telomerase activity may depend on cell cycle in gastric cancer and it is available to clarify the molecular mechanism of telomerase activity regulation. The expression of hTRT mRNA and hTRT protein in GC is significantly different from the expression of hTRT mRNA and hTRT protein in adjacent cancerous tissue which indicates that these targets are correlated closely to the occurrence of GC and can provide important morphologic index for diagnosis of GC.
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Affiliation(s)
- Jin-Chen Shao
- Department of Pathology, Anhui Medical University, Hefei, 230032, Anhui Province, China.
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Renaud S, Bosman FT, Benhattar J. Implication of the exon region in the regulation of the human telomerase reverse transcriptase gene promoter. Biochem Biophys Res Commun 2003; 300:47-54. [PMID: 12480519 DOI: 10.1016/s0006-291x(02)02775-4] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
The expression of the catalytic subunit (hTERT) represents the limiting factor for telomerase activity. In transfection studies, high level of activity of hTERT promoter is found, whereas low copy numbers of hTERT mRNA are detected in vivo. To explain this discrepancy, a series of vectors containing the hTERT promoter and gene were transiently transfected into HeLa cells. Four important regions were identified. First, the core promoter has bidirectional activity. Second, the distal upstream region (-1821 to -811bp) involved in the splicing of the first intron and could be a key of splicing specificity. Third, the intermediate promoter region (-800 to -300bp) could play an important role in silencing the reverse promoter activity. Fourth, the structural gene (up to +1077) strongly reduced hTERT promoter activity. These results provide the first evidence that the first two exons play a major role in the down-regulation of the hTERT promoter in telomerase-positive cells.
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Affiliation(s)
- Stéphanie Renaud
- Institut de Pathologie, Centre Hospitalier Universitaire Vaudois, Bugnon 25, CH-1011 Lausanne, Switzerland
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Fukushima T, Yoshino A, Katayama Y, Watanabe T, Kusama K, Moro I. Prediction of clinical course of diffusely infiltrating astrocytomas from telomerase expression and quantitated activity level. Cancer Lett 2002; 187:191-98. [PMID: 12359368 DOI: 10.1016/s0304-3835(02)00357-9] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
Abstract
Telomerase, one of the reverse transcriptases that extend telomere length, has been suggested to facilitate tumorigenesis and tumor immortality. Little is yet known, however, concerning the clinical significance of telomerase expression in brain tumors. We found in the present study that telomerase expression may contribute to malignant progression and can be an important indicator of high grade diffusely infiltrating astrocytomas, and its quantitated activity level reflects their growth potential. Telomerase expression and its quantitated activity level are also a good predictor of clinical course as compared with other biological markers and clinical factors.
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Affiliation(s)
- Takao Fukushima
- Department of Neurological Surgery, Nihon University School of Medicine, 30-1 Oyaguchi-Kamimachi, Itabashi-ku, Tokyo 173-8610, Japan
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Yaswen P, Stampfer MR. Molecular changes accompanying senescence and immortalization of cultured human mammary epithelial cells. Int J Biochem Cell Biol 2002; 34:1382-94. [PMID: 12200033 DOI: 10.1016/s1357-2725(02)00047-x] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Limits on the proliferative potential of cultured normal human cells may be consequences of pathways that exist to suppress tumorigenicity. Human mammary epithelial cells (HMEC) employ several mechanisms to prevent unlimited growth. One mechanism may be activated by stress, and is associated with upregulated expression of p16(INK4a). In serum-free medium, some HMEC arise spontaneously which do not express p16. These "post-selection" HMEC are capable of long-term proliferation, but ultimately cease growth when their telomeres become very short. As they approach a growth plateau, termed agonescence, post-selection HMEC populations accumulate chromosome abnormalities. In contrast to the crisis exhibited by cells lacking functional p53, agonescent cells can be maintained as viable cultures. Although transduction of hTERT, the catalytic subunit of telomerase, into post-selection cells can, by itself, efficiently produce immortality and avoid agonescence, the errors that produce telomerase reactivation during carcinogenesis are not known. The block to endogenous telomerase reactivation in HMEC is extremely stringent. However, if one predisposing error is present, the probability greatly increases that additional error(s) required for immortalization may be generated by genomic instability encountered during agonescence. In p53(+) HMEC immortalized after chemical carcinogen exposure, the events involved in overcoming agonescence can be temporally separated from activation of telomerase. We have used the term "conversion" to describe the gradual process that leads to telomerase activation, telomere length stabilization, decreased p57 (KIP2) expression, and increased ability to grow uniformly well in the presence or absence of TGF beta. In the presence of active p53, conversion may represent a rate-limiting step in immortal transformation.
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Affiliation(s)
- Paul Yaswen
- Department of Cell and Molecular Biology, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mailstop 70A-1118, Berkeley, CA 94720, USA.
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Xiang H, Wang J, Mao Y, Liu M, Reddy VN, Li DWC. Human telomerase accelerates growth of lens epithelial cells through regulation of the genes mediating RB/E2F pathway. Oncogene 2002; 21:3784-91. [PMID: 12032846 DOI: 10.1038/sj.onc.1205455] [Citation(s) in RCA: 66] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2001] [Revised: 02/15/2002] [Accepted: 02/21/2002] [Indexed: 11/09/2022]
Abstract
Telomerase is a specialized reverse transcriptase that extends telomeres of eukaryotic chromosomes. The catalytic core of human telomerase is composed of an RNA template known as hTER (human telomerase RNA) and a protein subunit named hTERT (human telomerase reverse transcriptase). We have been studying other functions of the telomerase besides its major role in telomere maintenance. In our previous work, we have demonstrated that the hTERT can functionally interact with a rabbit TER to regulate expression of other genes and also attenuate the induced apoptosis. Here we report that overexpression of hTERT in a human lens epithelial cell line accelerates growth of the transfected lens epithelial cells. Associated with the acceleration of cell growth, expression of p53, p21 and GCIP (Grap2 cyclin-D interacting protein) is downregulated in the hTERT-transfected cells. With the downregulation of p21 and GCIP, the retinoblastoma protein (RB) is completely hyperphosphorylated in the hTERT-transfected cells. As expected, in the presence of RB hyperphosphorylation, the E2F transactivity is upregulated. Inhibition of telomerase activity abolishes the observed growth acceleration and also the related molecular changes. Furthermore, expression of hTERT in telomerase-negative human lens epithelial cells derived from primary cultures also accelerates growth of the transfected cells. Taken together, our results suggest that hTERT, when overexpressed in human lens epithelial cells, accelerates cell growth rate through regulation of RB/E2F pathway and possibly other genes.
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Affiliation(s)
- Hua Xiang
- Department of Molecular Biology, University of Medicine and Dentistry of New Jersey School of Osteopathic Medicine, Stratford, New Jersey, NJ 08084, USA
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Cao Y, Li H, Deb S, Liu JP. TERT regulates cell survival independent of telomerase enzymatic activity. Oncogene 2002; 21:3130-8. [PMID: 12082628 DOI: 10.1038/sj.onc.1205419] [Citation(s) in RCA: 163] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2001] [Revised: 01/19/2002] [Accepted: 02/20/2002] [Indexed: 02/08/2023]
Abstract
Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, plays a pivotal role in the maintenance of telomeres and cell proliferation. Here we report that down-regulation of hTERT induces apoptosis independently of telomerase enzymatic activity in human breast cancer cells. Expression of a hTERT mutant lacking telomerase activity rescues the cells with lowered telomerase without inducing cell death. With similar patterns of subcellular distribution to that of the tumor suppressor protein p53 during mitosis, hTERT interacts with p53 and poly(ADP-ribose) polymerase (PARP). Decreasing p53 expression in intact cells worsens, and increasing p53 prevents, cell death induced by lowering hTERT. Thus, hTERT maintains cell survival and proliferation via both telomerase enzymatic activity-dependent telomere lengthening and enzymatic activity-independent intermolecular interactions involving p53 and PARP.
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Affiliation(s)
- Ying Cao
- Molecular Signaling Laboratory, Baker Medical Research Institute, Commercial Road, Prahran, Victoria 3181, Australia
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Affiliation(s)
- Laura J Mauro
- Department of Animal Science, University of Minnesota, St. Paul, Minnesota 55108, USA
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Affiliation(s)
- Jiamei Yu
- Department of Pathology, Keck School of Medicine, University of Southern California, USC/Norris Comprehensive Cancer Center, Los Angeles, California 90033, USA
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