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Dratwa-Kuzmin M, Lacina P, Wysoczanska B, Kilinska D, Siemaszko J, Sobczyk-Kruszelnicka M, Fidyk W, Solarska I, Nasiłowska-Adamska B, Skowronska P, Bieniaszewska M, Tomaszewska A, Basak G, Giebel S, Bogunia-Kubik K. Telomere length and telomerase reverse transcriptase gene polymorphism as potential markers of complete chimerism and GvHD development after allogeneic haematopoietic stem cell transplantation. J Cancer Res Clin Oncol 2025; 151:109. [PMID: 40082305 PMCID: PMC11906511 DOI: 10.1007/s00432-025-06160-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2025] [Accepted: 03/05/2025] [Indexed: 03/16/2025]
Abstract
INTRODUCTION Telomerase reverse transcriptase (TERT) is a catalytic subunit of telomerase that maintains genome stability by maintaining telomere length (TL). The massive proliferation of donor cells in the recipient's body for engraftment results in accelerated telomere shortening. Genetic variability within the TERT gene affects telomerase activity, and was shown to influence of haematopoietic stem cell transplantation (HSCT) outcome. In the present study, we aimed to analyse the effect of recipient and donor TL and TERT single nucleotide polymorphism (SNP) on the occurrence of post-HSCT complications. METHODS Our study included 120 recipient-donor pairs. TERT promoter (TERTp) SNP (rs2853669) SNP variant was detected with the use of the LightSNiP typing assay employing real-time polymerase chain reaction (PCR) amplifications. Telomere length measurements were performed using qPCR test kits (ScienCell's Absolute Human Telomere Length Quantification qPCR Assay Kit [AHTLQ], Carlsbad, CA, USA). RESULTS The presence of TERTp rs2853669 T allele in the recipient was associated with a higher risk for acute graft-versus-host-disease (aGvHD) manifestation (p = 0.046) and a significantly shorter aGvHD-free survival (p = 0.041). The latter association was further confirmed in a Cox proportional hazards model (p = 0.043). However, no statistically significant association between telomere length and post-transplant complications was observed. Furthermore, we found that shorter TL characterized donors of patients with late complete chimerism at 180 day after HSCT (p = 0.011). CONCLUSION Our results suggest that recipient allele TERTp rs2853669 T is a marker of unfavourable outcome in the context of aGvHD. Shorter TL in donors could be associated with later achievement of complete chimerism.
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Affiliation(s)
- Marta Dratwa-Kuzmin
- Laboratory of Clinical Immunogenetics and Pharmacogenetics, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
| | - Piotr Lacina
- Laboratory of Clinical Immunogenetics and Pharmacogenetics, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
| | - Barbara Wysoczanska
- Laboratory of Clinical Immunogenetics and Pharmacogenetics, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
| | - Dorota Kilinska
- Laboratory of Clinical Immunogenetics and Pharmacogenetics, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
| | - Jagoda Siemaszko
- Laboratory of Clinical Immunogenetics and Pharmacogenetics, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
| | - Malgorzata Sobczyk-Kruszelnicka
- Department of Bone Marrow Transplantation and Hematology-Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland
| | - Wojciech Fidyk
- Department of Bone Marrow Transplantation and Hematology-Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland
| | - Iwona Solarska
- Institute of Hematology and Blood Transfusion Medicine, Warsaw, Poland
| | | | | | - Maria Bieniaszewska
- Department of Hematology and Transplantology, Medical University of Gdansk, Gdansk, Poland
| | - Agnieszka Tomaszewska
- Department of Hematology, Transplantation and Internal Medicine, Medical University of Warsaw, Warsaw, Poland
| | - Grzegorz Basak
- Department of Hematology, Transplantation and Internal Medicine, Medical University of Warsaw, Warsaw, Poland
| | - Sebastian Giebel
- Department of Bone Marrow Transplantation and Hematology-Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland
| | - Katarzyna Bogunia-Kubik
- Laboratory of Clinical Immunogenetics and Pharmacogenetics, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland.
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Gregório C, Thakur S, Camara Rivero R, Márcia Dos Santos Machado S, Cuenin C, Carreira C, White V, Cree IA, Vukojevic K, Glavina Durdov M, Bersch Osvaldt A, Ashton-Prolla P, Herceg Z, Talukdar FR. Telomere length assessment and molecular characterization of TERT gene promoter in periampullary carcinomas. Gene 2023; 873:147460. [PMID: 37150235 DOI: 10.1016/j.gene.2023.147460] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2022] [Revised: 04/14/2023] [Accepted: 05/01/2023] [Indexed: 05/09/2023]
Abstract
Genetic and epigenetic alterations of the telomere maintenance machinery like telomere length and telomerase reverse transcriptase (encoded by TERT gene) are reported in several human malignancies. However, there is limited knowledge on the status of the telomere machinery in periampullary carcinomas (PAC) which are rare and heterogeneous groups of cancers arising from different anatomic sites around the ampulla of Vater. In the current study, we investigated the relative telomere length (RTL) and the most frequent genetic and epigenetic alterations in the TERT promoter in PAC and compared it with tumor-adjacent nonpathological duodenum (NDu). We found shorter RTLs (1.27 vs 1.33, P = 0.01) and lower TERT protein expression (p = 0.04) in PAC tissues as compared to the NDu. Although we did not find any mutation at two reactivating hotspot mutation sites of the TERT promoter, we detected polymorphism in 45% (9/20) of the cases at rs2853669 (T > C). Also, we found a hypermethylated region in the TERT promoter of PACs consisting of four CpGs (cg10896616 with Δβ 7%; cg02545192 with Δβ 9%; cg03323598 with Δβ 19%; and cg07285213 with Δβ 15%). In conclusion, we identified shorter telomeres with DNA hypermethylation in the TERT promoter region and lower TERT protein expression in PAC tissues. These results could be used further to investigate molecular pathology and develop theranostics for PAC.
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Affiliation(s)
- Cleandra Gregório
- Departamento de Genética, Programa de Pós-graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil; Laboratório de Medicina Genômica, Centro de Pesquisa Experimental - Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, Rio Grande do Sul, Brazil
| | - Shefali Thakur
- International Agency for Research on Cancer, Lyon, France; Faculty of Science, Charles University, Prague, Czech Republic
| | - Raquel Camara Rivero
- Departamento de Patologia, Faculdade de Medicina, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil; Serviço de Patologia- Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
| | - Simone Márcia Dos Santos Machado
- Grupo de Vias Biliares e Pâncreas - Cirurgia do Aparelho Digestivo, Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
| | - Cyrille Cuenin
- International Agency for Research on Cancer, Lyon, France
| | | | - Valerie White
- International Agency for Research on Cancer, Lyon, France
| | - Ian A Cree
- International Agency for Research on Cancer, Lyon, France
| | - Katarina Vukojevic
- Department of Anatomy, Histology and Embryology, School of Medicine, University of Split, Split, Croatia
| | | | - Alessandro Bersch Osvaldt
- Serviço de Patologia- Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil; Programa de Pós-graduação em Medicina: Ciências Cirúrgicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
| | - Patricia Ashton-Prolla
- Departamento de Genética, Programa de Pós-graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil; Laboratório de Medicina Genômica, Centro de Pesquisa Experimental - Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, Rio Grande do Sul, Brazil; Serviço de Patologia- Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil
| | - Zdenko Herceg
- International Agency for Research on Cancer, Lyon, France
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3
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Salgado C, Roelse C, Nell R, Gruis N, van Doorn R, van der Velden P. Interplay between TERT promoter mutations and methylation culminates in chromatin accessibility and TERT expression. PLoS One 2020; 15:e0231418. [PMID: 32267900 PMCID: PMC7141627 DOI: 10.1371/journal.pone.0231418] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2019] [Accepted: 03/23/2020] [Indexed: 12/19/2022] Open
Abstract
The telomerase reverse transcriptase (TERT) gene is responsible for telomere maintenance in germline and stem cells, and is re-expressed in 90% of human cancers. CpG methylation in the TERT promoter (TERTp) was correlated with TERT mRNA expression. Furthermore, two hotspot mutations in TERTp, dubbed C228T and C250T, have been revealed to facilitate binding of transcription factor ETS/TCF and subsequent TERT expression. This study aimed to elucidate the combined contribution of epigenetic (promoter methylation and chromatin accessibility) and genetic (promoter mutations) mechanisms in regulating TERT gene expression in healthy skin samples and in melanoma cell lines (n = 61). We unexpectedly observed that the methylation of TERTp was as high in a subset of healthy skin cells, mainly keratinocytes, as in cutaneous melanoma cell lines. In spite of the high promoter methylation fraction in wild-type (WT) samples, TERT mRNA was only expressed in the melanoma cell lines with either high methylation or intermediate methylation in combination with TERT mutations. TERTp methylation was positively correlated with chromatin accessibility and TERT mRNA expression in 8 melanoma cell lines. Cooperation between epigenetic and genetic mechanisms were best observed in heterozygous mutant cell lines as chromosome accessibility preferentially concerned the mutant allele. Combined, these results suggest a complex model in which TERT expression requires either a widely open chromatin state in TERTp-WT samples due to high methylation throughout the promoter or a combination of moderate methylation fraction/chromatin accessibility in the presence of the C228T or C250T mutations.
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Affiliation(s)
- Catarina Salgado
- Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands
| | - Celine Roelse
- Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands
| | - Rogier Nell
- Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands
| | - Nelleke Gruis
- Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands
| | - Remco van Doorn
- Department of Dermatology, Leiden University Medical Center, Leiden, The Netherlands
| | - Pieter van der Velden
- Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands
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Ji N, Shi HQ, Fang XY, Wu ZY. Exploring the interaction of G-quadruplex and porphyrin derivative by single protein nanopore sensing interface. Anal Chim Acta 2020; 1106:126-132. [DOI: 10.1016/j.aca.2020.01.053] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2019] [Revised: 01/18/2020] [Accepted: 01/23/2020] [Indexed: 11/26/2022]
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Roggisch J, Ecke T, Koch S. Molecular identification of telomerase reverse transcriptase (TERT) promotor mutations in primary and recurrent tumors of invasive and noninvasive urothelial bladder cancer. Urol Oncol 2020; 38:77.e17-77.e25. [DOI: 10.1016/j.urolonc.2019.08.007] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2019] [Revised: 08/09/2019] [Accepted: 08/14/2019] [Indexed: 11/26/2022]
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Brynychova I, Zinkova A, Hoffmanova I, Korabecna M, Dankova P. Immunoregulatory properties of cell-free DNA in plasma of celiac disease patients - A pilot study. Autoimmunity 2019; 52:88-94. [PMID: 31056951 DOI: 10.1080/08916934.2019.1608965] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The elevated plasma cell-free DNA (cfDNA) concentrations were repeatedly reported in association with the process of inflammation. The qualitative and quantitative characteristics of plasma cfDNA in active (newly diagnosed) celiac disease patients (CD) have not yet been studied despite the fact that cfDNA of healthy individuals is able to regulate immune response. We determined the total cfDNA concentration and relative content of telomeric sequences in plasma cfDNA in CD (n = 10) and healthy age- and sex-matched controls (HC, n = 10) by quantitative PCR. To obtain the evidence that the observed biological effects are caused solely by cfDNA molecules, we applied the treatment of paired plasma samples with DNase. Using paired samples of plasma (non-treated/native and treated by DNase), we analyzed the contribution of cfDNA to the activation of TLR9 and TNF-α mRNA expression in THP1 monocytic cell line. There were no significant differences in the quantities of plasma cfDNA and relative contents of telomeric sequences in their pools. When we compared the levels of TNF-α mRNA expression in THP1 cells achieved after stimulation with native CD and HC plasma samples, we found significantly (p = .031) higher expression after stimulation with CD samples. We documented also the ability of cfDNA contained in CD plasma samples to stimulate the production of TLR9 mRNA. The TLR9 mRNA expression levels were significantly (p = .014) lowered after cfDNA removal from CD plasma samples. The design of our experiments allowed us to study the effects of cfDNA without its isolation from plasma. cfDNA contained in CD plasma samples differs significantly in its immunoregulatory capacity from cfDNA in HC plasma. The differences are caused neither by different concentrations of cfDNA in plasma samples nor by different relative abundance of telomeric sequences. Further studies are needed to elucidate the role of plasma cfDNA in celiac disease pathogenesis.
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Affiliation(s)
- Iva Brynychova
- a Department of Anthropology and Human Genetics, Faculty of Science , Charles University , Prague , Czech Republic
| | - Alzbeta Zinkova
- b Department of Biology and Medical Genetics, First Faculty of Medicine , Charles University and General University Hospital in Prague , Prague , Czech Republic
| | - Iva Hoffmanova
- c Second Department of Internal Medicine, Third Faculty of Medicine , Charles University , Prague , Czech Republic
| | - Marie Korabecna
- b Department of Biology and Medical Genetics, First Faculty of Medicine , Charles University and General University Hospital in Prague , Prague , Czech Republic
| | - Pavlina Dankova
- a Department of Anthropology and Human Genetics, Faculty of Science , Charles University , Prague , Czech Republic
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7
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Leão R, Lee D, Figueiredo A, Hermanns T, Wild P, Komosa M, Lau I, Mistry M, Nunes NM, Price AJ, Zhang C, Lipman T, Poyet C, Valtcheva N, Oehl K, Coelho H, Sayyid R, Gomes AM, Prado E Castro L, Sweet J, Vinagre J, Apolónio J, Stephens D, Faleiro I, Fadaak K, Richard PO, Kulkarni G, Zlotta AR, Hamilton RJ, Castelo-Branco P, Tabori U. Combined genetic and epigenetic alterations of the TERT promoter affect clinical and biological behavior of bladder cancer. Int J Cancer 2018; 144:1676-1684. [PMID: 30350309 PMCID: PMC6519346 DOI: 10.1002/ijc.31935] [Citation(s) in RCA: 61] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2018] [Revised: 09/13/2018] [Accepted: 09/26/2018] [Indexed: 01/08/2023]
Abstract
In urothelial bladder cancer (UBC), risk stratification remains an important unmet need. Limitless self‐renewal, governed by TERT expression and telomerase activation, is crucial for cancer progression. Thus, telomerase activation through the interplay of mutations (TERTpMut) and epigenetic alterations in the TERT promoter may provide further insight into UBC behavior. Here, we investigated the combined effect of TERTpMut and the TERT Hypermethylated Oncological Region (THOR) status on telomerase activation and patient outcome in a UBC international cohort (n = 237). We verified that TERTpMut were frequent (76.8%) and present in all stages and grades of UBC. Hypermethylation of THOR was associated with higher TERT expression and higher‐risk disease in nonmuscle invasive bladder cancers (NMIBC). TERTpMut alone predicted disease recurrence (HR: 3.18, 95%CI 1.84 to 5.51, p < 0.0001) but not progression in NMIBC. Combined THORhigh/TERTpMut increased the risk of disease recurrence (HR 5.12, p < 0.0001) and progression (HR 3.92, p = 0.025). Increased THOR hypermethylation doubled the risk of stage progression of both TERTpwt and TERTpMut NMIBC. These results highlight that both mechanisms are common and coexist in bladder cancer and while TERTpMut is an early event in bladder carcinogenesis THOR hypermethylation is a dynamic process that contributes to disease progression. While the absence of alterations comprises an extremely indolent phenotype, the combined genetic and epigenetic alterations of TERT bring additional prognostic value in NMIBC and provide a novel insight into telomere biology in cancer. What's new? Telomerase reverse transcriptase (TERT) activation is central to cancer cell immortalization. It acts, however, through relatively unknown mechanisms. In urothelial bladder cancer (UBC) in particular, TERT activation can occur in the presence or absence of mutation, raising questions about alternative activation mechanisms. Our study shows that hypermethylation of the TERT promoter (THOR) plays a key part in UBC, being a dynamic and progressive process, with hypermethylation levels increasing with bladder cancer severity. Moreover, both hypermethylation and TERT promoter mutation contributed to increased telomerase expression. The findings provide insight into telomere biology in UBC and may be applicable to other tumors.
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Affiliation(s)
- Ricardo Leão
- Division of Urology, Department of Surgery, University of Toronto, Toronto, ON, Canada.,Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada.,Faculty of Medicine, University of Coimbra, Coimbra, Portugal.,Department of Urology, Coimbra University Hospital, Coimbra, Portugal
| | - Donghyun Lee
- Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada
| | - Arnaldo Figueiredo
- Faculty of Medicine, University of Coimbra, Coimbra, Portugal.,Department of Urology, Coimbra University Hospital, Coimbra, Portugal
| | - Thomas Hermanns
- Department of Urology, University Hospital Zürich, University of Zürich, Zürich, Switzerland
| | - Peter Wild
- Institute of Pathology and Molecular Pathology, University Hospital Zürich, University of Zürich, Zürich, Switzerland
| | - Martin Komosa
- Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada
| | - Irene Lau
- Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada
| | - Mathew Mistry
- Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada
| | - Nuno Miguel Nunes
- Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada
| | - Aryeh J Price
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA
| | - Cindy Zhang
- Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada
| | - Tatiana Lipman
- Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada
| | - Cédric Poyet
- Department of Urology, University Hospital Zürich, University of Zürich, Zürich, Switzerland
| | - Nadejda Valtcheva
- Institute of Pathology and Molecular Pathology, University Hospital Zürich, University of Zürich, Zürich, Switzerland
| | - Kathrin Oehl
- Institute of Pathology and Molecular Pathology, University Hospital Zürich, University of Zürich, Zürich, Switzerland
| | - Hugo Coelho
- Department of Urology, Coimbra University Hospital, Coimbra, Portugal
| | - Rashid Sayyid
- Division of Urology, Department of Surgery, University of Toronto, Toronto, ON, Canada
| | - Ana Melo Gomes
- Department of Pathology, Coimbra University Hospital, Coimbra, Portugal
| | | | - Joan Sweet
- Department of Pathology, University Health Network, Toronto, ON, Canada
| | - João Vinagre
- Institute for Research and Innovation in Health, (I3S), Institute of Molecular Pathology and Immunology of the University of Porto (Ipatimup), Porto, Portugal
| | - Joana Apolónio
- Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal.,Centre for Biomedical Research (CBMR), University of Algarve, Faro, Portugal.,Algarve Biomedical Center, Faro, Portugal
| | - Derek Stephens
- Biostatistics, Design and Analysis, The Hospital for Sick Children, Toronto, ON, Canada
| | - Inês Faleiro
- Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal.,Centre for Biomedical Research (CBMR), University of Algarve, Faro, Portugal.,Algarve Biomedical Center, Faro, Portugal
| | - Kamel Fadaak
- Division of Urology, Department of Surgery, University of Toronto, Toronto, ON, Canada.,Imam Abdulrahman bin Faisal University, Dammam, Saudi Arabia
| | - Patrick O Richard
- Division of Urology, Department of Surgery, University of Toronto, Toronto, ON, Canada.,Division of Urology, Faculty of Medicine, CHUS, University of Sherbrooke, Sherbrooke, QC, Canada
| | - Girish Kulkarni
- Division of Urology, Department of Surgery, University of Toronto, Toronto, ON, Canada
| | - Alexandre R Zlotta
- Division of Urology, Department of Surgery, University of Toronto, Toronto, ON, Canada
| | - Robert J Hamilton
- Division of Urology, Department of Surgery, University of Toronto, Toronto, ON, Canada
| | - Pedro Castelo-Branco
- Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal.,Centre for Biomedical Research (CBMR), University of Algarve, Faro, Portugal.,Algarve Biomedical Center, Faro, Portugal
| | - Uri Tabori
- Arthur and Sonia Labatt Brain Tumor Research Center, The Hospital for Sick Children, University of Toronto, Toronto, ON, Canada
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Beilfuss J, Camargo CA, Kamycheva E. Serum 25-Hydroxyvitamin D Has a Modest Positive Association with Leukocyte Telomere Length in Middle-Aged US Adults. J Nutr 2017; 147:514-520. [PMID: 28179486 DOI: 10.3945/jn.116.244137] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2016] [Revised: 11/28/2016] [Accepted: 01/13/2017] [Indexed: 11/14/2022] Open
Abstract
Background: Vitamin D deficiency has been linked to all-cause mortality and cancer. However, the biological plausibility of these associations is not well established. Leukocyte telomere length (LTL) shortening is associated with aging and is a hallmark of genomic instability and carcinogenesis.Objective: We aimed to investigate the association between serum 25-hydroxyvitamin D [25(OH)D] concentrations and LTL in the general US population.Methods: We analyzed data from the US NHANES 2001-2002. The study population comprised 1542 younger adults (aged 20-39 y), 1336 middle-aged adults (aged 40-59 y), and 1382 older adults (aged ≥60 y). LTL was measured by using quantitative polymerase chain reaction. Serum 25(OH)D concentrations ≥50 nmol/L were considered optimal. Linear regression, adjusted for age, sex, race/ethnicity, body mass index (BMI), total energy and sugar intakes, calcium intake, socioeconomic status, milk and dietary supplement consumption, and physical activity, was applied to investigate the association between serum 25(OH)D and LTL.Results: In the total population, age, sex, BMI, and non-Hispanic black race/ethnicity were significant predictors of LTL. In the participants aged 40-59 y, an increment in serum 25(OH)D of 10 nmol/L was associated with a 0.03- ± 0.01-kbp longer LTL, adjusted for age, sex, race/ethnicity, and other factors (P = 0.001). In the same age group, 25(OH)D concentrations ≥50 nmol/L were associated with a 0.13- ± 0.04-kbp longer LTL than those for 25(OH)D concentrations <50 nmol/L (P = 0.01). The association was independent of age, sex, race/ethnicity, BMI, and other factors.Conclusions: In a nationally representative population of adults, serum 25(OH)D was positively associated with LTL in middle-aged participants (aged 40-59 y), independently of other factors. These findings suggest that decreased 25(OH)D concentrations are associated with genomic instability, although the clinical impact of this observation remains unclear.
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Affiliation(s)
- Julia Beilfuss
- Endocrine Research Group, Faculty of Health Sciences, Department of Clinical Medicine, University of Tromsoe-The Arctic University of Norway, Tromsoe, Norway.,Medical Clinic, University Hospital of North Norway, Tromsoe, Norway
| | - Carlos A Camargo
- Department of Emergency Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA; and.,Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA
| | - Elena Kamycheva
- Endocrine Research Group, Faculty of Health Sciences, Department of Clinical Medicine, University of Tromsoe-The Arctic University of Norway, Tromsoe, Norway; .,Medical Clinic, University Hospital of North Norway, Tromsoe, Norway.,Department of Emergency Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA; and
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9
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Kamycheva E, Goto T, Camargo CA. Celiac disease autoimmunity is associated with leukocyte telomere shortening in older adults: The U.S. National Health and Nutrition Examination Survey. Exp Gerontol 2017; 89:64-68. [PMID: 28104447 DOI: 10.1016/j.exger.2017.01.003] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2016] [Revised: 12/22/2016] [Accepted: 01/02/2017] [Indexed: 12/13/2022]
Abstract
PURPOSE Telomeres are nucleotide sequences, and their function is to maintain cell surveillance. Exaggeration of the attrition rate of leukocyte telomere length (LTL) may result in genomic instability and tumorigenesis. Celiac disease (CD), an autoimmune inflammation of small intestine, has increasing prevalence in the elderly and may lead to lymphomas and gastrointestinal malignancies. We used nationally-representative datasets from the U.S. National Health and Nutrition Examination Survey (NHANES) to investigate if CD autoimmunity in older adults (age≥50years) is associated with shorter LTL. RESULTS Our study included 3939 subjects, where 25 subjects (mean age 65years) were CD seropositive and 3914 (mean age 64years) were CD seronegative. CD seropositive subjects had shorter LTL than CD seronegative subjects (P<0.001). In the linear regression model, CD seropositivity was significantly associated with 0.25kb pairs decrease in LTL length (P<0.001), adjusted for age, sex, race/ethnicity, serum ferritin and folate, and ratio of family income to poverty. CONCLUSIONS In a nationally-representative population of adults age≥50years, CD seropositivity is significantly associated with shorter LTL, independently of age, sex, race/ethnicity, serum ferritin and folate, and socioeconomic status. This supports the enhanced telomere attrition in of CD seropositive adults.
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Affiliation(s)
- Elena Kamycheva
- Department of Emergency Medicine, Massachusetts General Hospital, Harvard Medical School, 125 Nashua St, Suite 920, Boston, MA 02114, USA; Department of Geriatrics, Medical Clinic, University Hospital of North Norway, Sykehusveien 38, 9038 Tromsoe, Norway; Endocrine Research Group, Faculty of Health Sciences, Department of Clinical Medicine, UiT The Arctic University of Norway, Hansine Hansens Veg 18, 9017 Tromsoe, Norway.
| | - Tadahiro Goto
- Department of Emergency Medicine, Massachusetts General Hospital, Harvard Medical School, 125 Nashua St, Suite 920, Boston, MA 02114, USA; Harvard T.H. Chan School of Public Health, 677 Huntington Avenue, Boston, MA 02115, USA.
| | - Carlos A Camargo
- Department of Emergency Medicine, Massachusetts General Hospital, Harvard Medical School, 125 Nashua St, Suite 920, Boston, MA 02114, USA; Harvard T.H. Chan School of Public Health, 677 Huntington Avenue, Boston, MA 02115, USA.
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Panarese S, Brunetti B, Sarli G. Evaluation of Telomerase in Canine Mammary Tissues by Immunohistochemical Analysis and a Polymerase Chain Reaction-Based Enzyme-Linked Immunosorbent Assay. J Vet Diagn Invest 2016; 18:362-8. [PMID: 16921875 DOI: 10.1177/104063870601800407] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022] Open
Abstract
The enzyme telomerase is considered a potential marker for neoplastic tissue and is used as a diagnostic and prognostic tool in clinical medicine and therapeutics. For this reason, the possible role of telomerase activation in the process of malignant transformation is currently the subject of intense research efforts. The focus of the study reported here was to detect telomerase in 37 canine mammary samples, by comparing two methods: immunohistochemical (IHC) analysis for detecting the catalytic subunit of the enzyme, telomerase reverse transcriptase (TERT), and the telomeric repeat amplification protocol–enzyme-linked immunosorbent assay (TRAP-ELISA), a polymerase chain reaction (PCR)-based technique that uses a colorimetric detection method. Using the TRAP-ELISA, samples were considered positive when they yielded a difference of at least 0.2 absorbance units between the readings at 450 nm versus 690 nm wavelength. On the basis of this criterion, 18 negative and 19 positive cases were obtained. Specific immunohistochemical staining was observed mainly in the nucleoli, to a lesser extent in the nuclei, and rarely in the cytoplasm of epithelial cells. A sample was considered positive when at least 10% of the epithelial cells had specific staining. The Pearson correlation between the TRAP-ELISA and IHC results was significant only when IHC nucleolar ( r = 0.53, P < 0.01) or nuclear ( r = 0.36, P < 0.05) staining or their combination ( r = 0.58, P < 0.01) was considered. Thus, IHC staining of nucleoli and nuclei can be considered as an alternative method to the TRAP-ELISA. The detection of telomerase in normal mammary gland and fibrocystic mastopathy using both methods does not support the idea that telomerase may be used as a specific marker of mammary neoplasia in dogs.
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Affiliation(s)
- Serena Panarese
- Department of Veterinary Public Health and Animal Pathology, Section of General Pathology and Pathologic Anatomy, Faculty of Veterinary Medicine, University of Bologna Via Tolara di Sopra, 50-40064, Ozzano dell'Emilia, Bologna, Italy
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11
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Babizhayev MA, Vishnyakova KS, Yegorov YE. Hormone-brain-aging relationships, broadly reactive with imidazole-containing dipeptides: targeting of telomere attrition as an aging biomarker and dynamic telomerase activity flirting. J Basic Clin Physiol Pharmacol 2014; 26:115-40. [PMID: 25153587 DOI: 10.1515/jbcpp-2014-0045] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2014] [Accepted: 05/10/2014] [Indexed: 11/15/2022]
Abstract
It has been documented that telomere-associated cellular senescence may contribute to certain age-related disorders, and telomere length (TL) may be an informative biomarker of healthy aging. Hormone-brain-aging behavior-modulated telomere dynamics and changes in telomerase activity are consistent elements of cellular alterations associated with changes in proliferative state, and these processes are consequently considered as the new therapeutic drug targets for physiological control with advanced drug delivery and nutritional formulations. We raise and support a therapeutic concept of using nonhydrolyzed forms of naturally occurring neuron-specific imidazole dipeptide-based compounds carnosine and carcinine, making it clinically possible that slowing down the rate of telomere shortening could slow down the human aging process in specific tissues where proliferative senescence is known to occur, with the demonstrated evidence of telomere shortening that appeared to be a hallmark of oxidative stress and disease. Carnosine released from skeletal muscle during exercise may be transported into the hypothalamic tuberomammillary nucleus (TMN) histamine neurons and hydrolyzed. The resulting L-histidine may subsequently be converted into histamine, which could be responsible for the effects of carnosine on neurotransmission and hormone-like antiaging physiological function. The preliminary longitudinal studies of elderly individuals suggest that longer telomeres are associated with better survival, and an advanced oral nutritional support with nonhydrolyzed carnosine (or carcinine and patented compositions thereof) is a useful therapeutic tool for a critical TL maintenance that may fundamentally be applied in the treatment of age-related sight-threatening eye disorders, prolonged life expectancy, increased survival and chronological age of an organism in health control, smoking behavior, and disease. "Our pleasures were simple-they included survival." -Dwight D. Eisenhower, 34th President of the United States, 1953-1961.
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12
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Díaz de la Guardia R, Catalina P, Panero J, Elosua C, Pulgarin A, López MB, Ayllón V, Ligero G, Slavutsky I, Leone PE. Expression profile of telomere-associated genes in multiple myeloma. J Cell Mol Med 2014; 16:3009-21. [PMID: 22947336 PMCID: PMC4393729 DOI: 10.1111/j.1582-4934.2012.01628.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2012] [Accepted: 08/14/2012] [Indexed: 12/31/2022] Open
Abstract
To further contribute to the understanding of multiple myeloma, we have focused our research interests on the mechanisms by which tumour plasma cells have a higher survival rate than normal plasma cells. In this article, we study the expression profile of genes involved in the regulation and protection of telomere length, telomerase activity and apoptosis in samples from patients with monoclonal gammopathy of undetermined significance, smouldering multiple myeloma, multiple myeloma (MM) and plasma cell leukaemia (PCL), as well as several human myeloma cell lines (HMCLs). Using conventional cytogenetic and fluorescence in situ hybridization studies, we identified a high number of telomeric associations (TAs). Moreover, telomere length measurements by terminal restriction fragment (TRF) assay showed a shorter mean TRF peak value, with a consistent correlation with the number of TAs. Using gene expression arrays and quantitative PCR we identified the hTERT gene together with 16 other genes directly involved in telomere length maintenance: HSPA9, KRAS, RB1, members of the Small nucleolar ribonucleoproteins family, A/B subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins, and 14-3-3 family. The expression levels of these genes were even higher than those in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), which have unlimited proliferation capacity. In conclusion, the gene signature suggests that MM tumour cells are able to maintain stable short telomere lengths without exceeding the short critical length, allowing cell divisions to continue. We propose that this could be a mechanism contributing to MM tumour cells expansion in the bone marrow (BM).
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Affiliation(s)
- Rafael Díaz de la Guardia
- Andalusian Public Health System Biobank, Centro de Investigación Biomédica, Consejería de Salud-Universidad de Granada, Granada, Spain.
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13
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Vergel M, Marin JJ, Estevez P, Carnero A. Cellular senescence as a target in cancer control. J Aging Res 2010; 2011:725365. [PMID: 21234095 PMCID: PMC3018654 DOI: 10.4061/2011/725365] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2010] [Accepted: 11/03/2010] [Indexed: 01/04/2023] Open
Abstract
Somatic cells show a spontaneous decline in growth rate in continuous culture. This is not related to elapsed time but to an increasing number of population doublings, eventually terminating in a quiescent but viable state termed replicative senescence. These cells are commonly multinucleated and do not respond to mitogens or apoptotic stimuli. Cells displaying characteristics of senescent cells can also be observed in response to other stimuli, such as oncogenic stress, DNA damage, or cytotoxic drugs and have been reported to be found in vivo. Most tumors show unlimited replicative potential, leading to the hypothesis that cellular senescence is a natural antitumor program. Recent findings suggest that cellular senescence is a natural mechanism to prevent undesired oncogenic stress in somatic cells that has been lost in malignant tumors. Given that the ultimate goal of cancer research is to find the definitive cure for as many tumor types as possible, exploration of cellular senescence to drive towards antitumor therapies may decisively influence the outcome of new drugs. In the present paper, we will review the potential of cellular senescence to be used as target for anticancer therapy.
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Affiliation(s)
- Mar Vergel
- Instituto de Biomedicina de Sevilla, Hospital Universitario virgen del Rocio, 41013 Sevilla, Spain
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14
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Saini N, Srinivasan R, Chawla Y, Sharma S, Chakraborti A, Rajwanshi A. Telomerase activity, telomere length and human telomerase reverse transcriptase expression in hepatocellular carcinoma is independent of hepatitis virus status. Liver Int 2009; 29:1162-70. [PMID: 19627485 DOI: 10.1111/j.1478-3231.2009.02082.x] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
BACKGROUND Telomerase expression and the maintenance of a critical telomere length (TL) in cancer initiation indicates that telomere shortening and telomerase expression initiates cancer by induction of chromosomal instability. METHODS Telomerase activity, TL and human telomerase reverse transcriptase (hTERT) expression were investigated in 58 hepatocellular carcinoma (HCC) and 17 chronic hepatitis patients by the telomerase repeat amplification protocol, Southern blotting and reverse transcriptase-polymerase chain reaction. RESULTS Telomerase was positive in 76% of HCC and 11.8% of chronic hepatitis patients (P<0.0001). The mean telomere length (MTL) in HCC was significantly shorter compared with chronic hepatitis (P<0.0001). The MTL was not significantly different in HCC patients with and without cirrhosis (P=0.77). In hepatitis B virus, hepatitis C virus and non-B non-C-related HCC groups, no differences were found in telomerase activity and MTL (P=0.77). hTERT, a regulator of telomerase, was, however, positive in 81% of HCCs. The correlation between telomerase activity and hTERT mRNA expression was statistically significant (P<0.0001). The MTL in telomerase-positive HCC cases was significantly shorter than the MTL in telomerase-negative cases (P<0.0001). CONCLUSION The majority of HCCs exhibited telomerase activity that correlated well with hTERT expression. MTL in HCC was significantly shorter than chronic hepatitis. It was also found that shorter telomeres are present in telomerase-positive HCC cases. However, no correlation was found between telomerase activity and TL with respect to the viral status in HCC.
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Affiliation(s)
- Nitin Saini
- Department of Hepatology, Postgraduate Institute of Medical Education & Research, Chandigarh, India.
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15
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Miura N, Sato R, Tsukamoto T, Shimizu M, Kabashima H, Takeda M, Takahashi S, Harada T, West JE, Drabkin H, Mejia JE, Shiota G, Murawaki Y, Virmani A, Gazdar AF, Oshimura M, Hasegawa J. A noncoding RNA gene on chromosome 10p15.3 may function upstream of hTERT. BMC Mol Biol 2009; 10:5. [PMID: 19187532 PMCID: PMC2661890 DOI: 10.1186/1471-2199-10-5] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2008] [Accepted: 02/02/2009] [Indexed: 01/05/2023] Open
Abstract
Background We attempted to clone candidate genes on 10p14–15 which may regulate hTERT expression, through exon trapping using 3 BAC clones covering the region. After obtaining 20 exons, we examined the function of RGM249 (RGM: RNA gene for miRNAs) we cloned from primary cultured human hepatocytes and hepatoma cell lines. We confirmed approximately 20 bp products digested by Dicer, and investigated the function of this cloned gene and its involvement in hTERT expression by transfecting the hepatoma cell lines with full-length dsRNA, gene-specific designed siRNA, and shRNA-generating plasmid. Results RGM249 showed cancer-dominant intense expression similar to hTERT in cancer cell lines, whereas very weak expression was evident in human primary hepatocytes without telomerase activity. This gene was predicted to be a noncoding precursor RNA gene. Interestingly, RGM249 dsRNA, siRNA, and shRNA inhibited more than 80% of hTERT mRNA expression. In contrast, primary cultured cells overexpressing the gene showed no significant change in hTERT mRNA expression; the overexpression of the gene strongly suppressed hTERT mRNA in poorly differentiated cells. Conclusion These findings indicate that RGM249 might be a microRNA precursor gene involved in the differentiation and function upstream of hTERT.
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Affiliation(s)
- Norimasa Miura
- Division of Pharmacotherapeutics, Department of Pathophysiological and Therapeutic Science, Faculty of Medicine, Tottori University, Yonago, Tottori 683-8503, Japan.
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16
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Chen YJ, Campbell HG, Wiles AK, Eccles MR, Reddel RR, Braithwaite AW, Royds JA. PAX8 Regulates Telomerase Reverse Transcriptase and Telomerase RNA Component in Glioma. Cancer Res 2008; 68:5724-32. [DOI: 10.1158/0008-5472.can-08-0058] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
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17
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Betts DH, Perrault SD, King WA. Low oxygen delays fibroblast senescence despite shorter telomeres. Biogerontology 2007; 9:19-31. [PMID: 17952625 DOI: 10.1007/s10522-007-9113-7] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2007] [Accepted: 10/09/2007] [Indexed: 01/29/2023]
Abstract
It has been widely accepted that telomere shortening acts as a cell division counting mechanism that beyond a set critical length signals cells to enter replicative senescence. In this study, we demonstrate that by simply lowering the oxygen content of the cell culture environment 10-fold (20-2%) extends the replicative lifespan of fetal bovine fibroblasts at least five-times (30-150 days). Although, low oxygen fibroblasts display a slightly slower rate (P > 0.05) of telomere attrition than their high oxygen counterparts (171 bp versus 182 bp/PD), late passage fibroblasts (>50 PD) that have extended their replicative capacity under low oxygen conditions exhibited significantly (P < 0.05) shorter telomere lengths (11,135 +/- 467 bp) compared to senescent cells (25-34 PD) cultured under high oxygen conditions (14,827 +/- 1173 bp). There was a significant increase (P < 0.05) in chromosomal abnormalities with continual cell division under both high and low oxygen environments, however, fibroblasts displayed a significant reduction (P < 0.001) in chromosomal abnormalities at low oxygen tensions compared to those under 20% oxygen. These apparent protective effects on telomere shortening, delayed senescence and reduced chromosomal aberrations may be attributed to the up-regulation of telomerase activity observed for fibroblasts cultured under low oxygen. These results are consistent with the idea that a critically short telomere length may not be the sole trigger of replicative senescence, but may be regulated by the integrity of telomere structure itself and/or the amount of oxidative DNA damage in the cell.
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Affiliation(s)
- Dean H Betts
- Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada, N1G 2W1.
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18
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Affiliation(s)
- Peter J Hornsby
- Department of Physiology, Sam and Ann Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center, San Antonio, Texas, USA
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19
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Shin JS, Hong A, Solomon MJ, Lee CS. The role of telomeres and telomerase in the pathology of human cancer and aging. Pathology 2006; 38:103-13. [PMID: 16581649 DOI: 10.1080/00313020600580468] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Cellular senescence, the state of permanent growth arrest, is the inevitable fate of replicating normal somatic cells. Postulated to underlie this finite replicative span is the physiology of telomeres, which constitute the ends of chromosomes. The repetitive sequences of these DNA-protein complexes progressively shorten with each mitosis. When the critical length is bridged, telomeres trigger DNA repair and cell cycle checkpoint mechanisms that result in chromosomal fusions, cell cycle arrest, senescence and/or apoptosis. Should senescence be bypassed at such time, continued cell divisions in the face of dysfunctional telomeres and activated DNA repair machinery can result in the genomic instability favourable for oncogenesis. The longevity and malignant progression of the thus transformed cell requires coincident telomerase expression or other means to negate the constitutional telomeric loss. Practically then, telomeres and telomerase may represent plausible prognostic and screening cancer markers. Furthermore, if the argument is extended, with assumptions that telomeric attrition is indeed the basis of cellular senescence and that accumulation of the latter equates to aging at the organismal level, then telomeres may well explain the increased incidence of cancer with human aging.
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Affiliation(s)
- Joo-Shik Shin
- Department of Anatomical Pathology, Royal Prince Alfred Hospital, Camperdown, Australia.
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20
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Hansel DE, Meeker AK, Hicks J, De Marzo AM, Lillemoe KD, Schulick R, Hruban RH, Maitra A, Argani P. Telomere length variation in biliary tract metaplasia, dysplasia, and carcinoma. Mod Pathol 2006; 19:772-9. [PMID: 16557277 DOI: 10.1038/modpathol.3800591] [Citation(s) in RCA: 35] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Biliary tract carcinoma, including carcinoma of the gallbladder, intrahepatic bile ducts (cholangiocarcinoma), and extrahepatic bile ducts, affects 7500 people in the United States annually, and has an overall 32% 5-year survival rate for disease limited to the mucosa, and a dismal 10% 5-year survival for more advanced disease. The identification of factors involved in the pathogenesis and progression of biliary tract carcinoma is critical for devising effective methods of screening and treatment. Recent evidence suggests that reduction of the length of telomeres, which normally help maintain chromosomal stability, may promote the development and progression of a variety of carcinomas. Using a novel, recently validated telomere fluorescence in situ hybridization method, we examined telomere length in normal and inflamed gallbladder epithelium, metaplasia and dysplasia of the gallbladder, and biliary tract carcinoma to determine whether telomere shortening is associated with neoplastic progression in the biliary tract. Although normal and inflamed gallbladder epithelium demonstrated uniform normal telomere lengths, over half of all metaplastic lesions demonstrated shortened telomeres, supporting prior evidence that metaplastic lesions of the gallbladder are pre-neoplastic. Dysplastic epithelium and invasive carcinomas demonstrated almost universally abnormally short telomeres, indicating that telomere shortening occurs at an early, preinvasive stage of cancer development. In addition, invasive adenocarcinoma of the biliary tract frequently demonstrated intratumoral heterogeneity of telomere lengths. We conclude that telomere shortening is a consistent and early finding in the development of biliary tract carcinoma.
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Affiliation(s)
- Donna E Hansel
- Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, MD, USA
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21
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Cerone MA, Autexier C, Londoño-Vallejo JA, Bacchetti S. A human cell line that maintains telomeres in the absence of telomerase and of key markers of ALT. Oncogene 2005; 24:7893-901. [PMID: 16116482 DOI: 10.1038/sj.onc.1208934] [Citation(s) in RCA: 55] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Abstract
In human somatic cells proliferation results in telomere shortening due to the end replication problem and the absence of adequate levels of telomerase activity. The progressive loss of telomeric DNA has been associated with replicative senescence. Maintenance of telomere structure and function is, therefore, an essential requisite for cells that proliferate indefinitely. Human cells that have acquired the immortal phenotype mostly rely on telomerase to compensate for telomere shortening with cell division. However, a certain percentage of immortalized cell lines and human tumors maintain their telomeres by Alternative Lengthening of Telomeres (ALT), a mechanism not fully understood but apparently based on homologous recombination. Here, we report the isolation of an immortal human cell line that is derived from an ALT cell line but maintains telomeres in the absence of key features of ALT and of telomerase. The properties of these cells suggest that the identification of ALT cells may not be reliably based on known ALT markers. This finding is of relevance for discriminating between the mortal and immortal phenotype among telomerase-negative cells in vitro and in vivo, particularly in regard to the development of pharmacological approaches for cancer treatment based on telomerase inhibition.
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Affiliation(s)
- Maria A Cerone
- Department of Anatomy and Cell Biology, McGill University, Quebec, Canada H3A 2B4
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22
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Dikmen ZG, Gellert GC, Jackson S, Gryaznov S, Tressler R, Dogan P, Wright WE, Shay JW. In vivo inhibition of lung cancer by GRN163L: a novel human telomerase inhibitor. Cancer Res 2005; 65:7866-73. [PMID: 16140956 DOI: 10.1158/0008-5472.can-05-1215] [Citation(s) in RCA: 183] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Differential regulation of telomerase activity in normal and tumor cells provides a rationale for the design of new classes of telomerase inhibitors. The telomerase enzyme complex presents multiple potential sites for the development of inhibitors. GRN163L, a telomerase enzyme antagonist, is a lipid-modified 13-mer oligonucleotide N3' --> P5'-thio-phosphoramidate, complementary to the template region of telomerase RNA (hTR). We evaluated both the in vitro and in vivo effects of GRN163L using A549-luciferase (A549-Luc) human lung cancer cells expressing a luciferase reporter. GRN163L (1 micromol/L) effectively inhibits telomerase activity of A549-Luc cells, resulting in progressive telomere shortening. GRN163L treatment also reduces colony formation in soft agar assays. Surprisingly, after only 1 week of treatment with GRN163L, A549-Luc cells were unable to form robust colonies in the clonal efficiency assay, whereas the mismatch control compound had no effect. Finally, we show that in vivo treatment with GRN163L is effective in preventing lung metastases in xenograft animal models. These in vitro and in vivo data support the development of GRN163L as a therapeutic for the treatment of cancer.
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Affiliation(s)
- Z Gunnur Dikmen
- University of Hacettepe, Faculty of Medicine, Department of Biochemistry, Ankara, Turkey
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Abstract
Cellular senescence is a signal transduction program leading to irreversible cell cycle arrest. This growth arrest can be triggered by many different mechanisms including recognition by cellular sensors of DNA double-strand breaks leading to the activation of cell cycle checkpoint responses and recruitment of DNA repair foci. Senescence is initiated by the shortening of telomeres (replicative senescence) or by other endogenous and exogenous acute and chronic stress signals (STASIS: stress or aberrant signaling-induced senescence). The process of carcinogenesis involves a series of changes that allow tumor cells to bypass the senescence program. Nevertheless, tumor cells retain the capacity to undergo senescence. Treatment of tumor cells with many conventional anticancer therapies activates DNA damage signaling pathways, which induce apoptosis in some cells and senescence in others. Overexpression of tumor suppressors or inhibition of oncogenes can also induce rapid senescence in tumor cells. Senescent cells, while not dividing, remain metabolically active and produce many secreted factors, some of which stimulate and others inhibit the growth of tumors. The emerging knowledge about the pathways that lead to senescence and determine the pattern of gene expression in senescent cells may lead to more effective treatments for cancer.
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Affiliation(s)
- Jerry W Shay
- The University of Texas Southwestern Medical Center, Department of Cell Biology, 5323 Harry Hines Boulevard, Dallas, TX 75390-9039, USA
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24
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Cottliar A, Palumbo M, La Motta G, de Barrio S, Crivelli A, Viola M, Gómez JC, Slavutsky I. Telomere length study in celiac disease. Am J Gastroenterol 2003; 98:2727-31. [PMID: 14687824 DOI: 10.1111/j.1572-0241.2003.08720.x] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
OBJECTIVES Telomeres are important structures that are critical for maintaining chromosomal integrity and cell surveillance. The aim of this study was to analyze telomere length in patients with celiac disease (CD), a multifactorial disorder with a strong genetic component that exhibits genomic instability and cancer predisposition, particularly T-cell lymphomas. METHODS Telomere length measured by telomere restriction fragments (TRF) was studied in small intestinal biopsy (SIB) samples and peripheral blood lymphocytes (PBL) from 20 untreated CD patients, distributed according to the clinical form as four asymptomatic, five monosymptomatic, and 11 polysymptomatic individuals. We also analyzed TRF from normal peripheral blood lymphocytes and normal biopsy samples as normal controls. RESULTS TRF evaluation showed a significant telomere shortening in SIB samples from CD patients (4.21 +/- 0.29 Kb) compared to PBL from the same individuals (9.17 +/- 0.35 Kb) (p < 0.0001), independently of clinical form. Mean TRF peak values from normal biopsy samples were significantly higher (8.33 +/- 0.38 Kb) than those observed in CD biopsy samples (p < 0.001). No differences between TRF values in CD-PBL and normal peripheral blood lymphocytes (8.89 +/- 0.37Kb) were found. CONCLUSIONS Our findings in patients with CD, a disorder in which the gluten-induced mucosal injury could accelerate telomere shortening, would increase the process of end-to-end fusions resulting in chromosomal changes, supports the hypothesis that genomic instability and telomere reduction may play a role in the cancer predisposition observed in these patients.
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Affiliation(s)
- Alejandra Cottliar
- Departamento de Genética, Instituto de Investigaciones Hematológicas Mariano R. Castex, Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina
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25
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Cottliar A, Pedrazzini E, Corrado C, Engelberger MI, Narbaitz M, Slavutsky I. Telomere shortening in patients with plasma cell disorders. Eur J Haematol 2003; 71:334-40. [PMID: 14667196 DOI: 10.1034/j.1600-0609.2003.00157.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
OBJECTIVES Telomeres are essential for maintaining chromosomal integrity; their shortening is associated with chromosome instability. The aim of this work was to study telomere length (TL) on bone marrow (BM) cells from patients with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). METHODS Thirty-one MM patients: 12 at diagnosis (D), 11 at relapse (R) and eight at remission (RE) and two cases with MGUS were studied. TL based on terminal restriction fragment (TRF) assay was evaluated. Cytogenetic and molecular cytogenetic analyses were performed. Telomeric associations (TAs) on BM metaphases were also studied. RESULTS TRF analysis in total MM patients showed a mean TRF peak value (5.20 +/- 0.35 kb) shorter than those observed in controls (8.5 +/- 0.5 kb) (P < 0.001). Moreover, TRF at D and R showed a significant telomere shortening (P < 0.001), with TL restored at RE. A strong correlation with the percentage of BM plasma cell infiltration (BMPCI) (rK = -0.540; P = 0.002) was found. Patients with abnormal karyotypes (AK) had significantly shorter TRFs than that observed in MM patients with normal karyotypes (P < 0.05). TRFs in MGUS patients did not differ with respect to controls. TA analysis showed an increased percentage in MM (19.46 +/- 1.98%) with respect to MGUS (6.12 +/- 1.87%) and normal BM cells (2.00 +/- 0.93%) (P < 0.001). CONCLUSIONS MM patients showed a significant reduction in TL (> 60% of BMPCI and AK), suggesting a probable association with clinical evolution. Moreover, our findings support the idea that telomere shortening usually leads to increased frequencies of TAs and chromosome instability.
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Affiliation(s)
- Alejandra Cottliar
- Departamento de Genética, Instituto de Investigaciones Hematológicas Mariano R. Castex, Academia Nacional de Medicina, Buenos Aires, Argentina.
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Zhang X, Chen Z, Chen Y, Tong T. Delivering antisense telomerase RNA by a hybrid adenovirus/ adeno-associated virus significantly suppresses the malignant phenotype and enhances cell apoptosis of human breast cancer cells. Oncogene 2003; 22:2405-16. [PMID: 12717417 DOI: 10.1038/sj.onc.1206317] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Activated telomerase is frequently detected in cancer cells and is able to maintain and stabilize the integrity of telomeres; it also contributes to unlimited divisions in cancer cells. Recently, a new generation of selective anticancer strategies is under development targeting the blockage of telomerase activity either at the protein level or telomerase RNA. Here, we report suppression of the malignant phenotype by the expression of the full-length antisense human telomerase RNA (hTR) delivered by a novel hybrid vector recombining adenovirus and adeno-associated virus (vAd-AAV). The hybrid vector vAd-AAV retained the unique traits from two parental viruses, such as high efficiency of gene transfer in mammalian cells and the ability to integrate into the genomic DNA of host cells. The stable expression of antisense hTR in MCF-7 cells significantly suppressed telomerase activity and progressively shortened telomere length for 30 population doublings (PD30). Expression of antisense hTR leads to a telomere-based growth arrest and the induction of spontaneous apoptosis. Antisense hTR decreased soft agar colony formation and reduced the cell proliferation, leading to exit from the cell cycle at G1 at PD15. The expression of antisense hTR also sensitized MCF-7 cells to apoptosis induced by sodium butyrate or serum starvation. Our study demonstrates that delivering antisense hTR by the hybrid Ad/AAV vector is an effective antineoplastic gene therapeutic strategy, which significantly suppresses the malignant phenotype and enhances apoptosis of human breast cancer cells.
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Affiliation(s)
- Xiaowei Zhang
- Department of Biochemistry and Molecular Biology, Peking University, Health Science Center, Beijing 100083, China
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Chen HJ, Cho CL, Liang CL, Lu K, Lin JW. Implication of telomere length as a proliferation-associated marker in schwannomas. J Surg Oncol 2002; 81:93-100; discussion 100. [PMID: 12355410 DOI: 10.1002/jso.10139] [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: 11/11/2022]
Abstract
BACKGROUND AND OBJECTIVES Some schwannomas in the central nervous system may demonstrate relatively aggressive behavior in pathological findings and clinical course. We evaluate the diagnostic values of telomerase activity and telomere length in the clinicopathological behavior of schwannomas. METHODS Thirty surgical specimens from intracranial and intraspinal schwannomas were analyzed by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) for telomerase activity and terminal restriction fragments (TRFs) using Southern blot for telomere length. Proliferative indices were also studied. RESULTS Telomerase activity could not be detected in all schwannomas. Elongated telomere length (mean 17,101 +/- 259 bp) was found in four specimens (13.3%). Three of these four were found to have mitotic figures, high vascularity, cellularity, and pleomorphism in the pathological findings. The proliferative indices (35) showed correlative high values. One patient died of this disease, and one was found to have recurrence at follow-up evaluation. Those that displayed benign histopathological pictures showed relatively short telomere length (8,866 +/- 271 base pairs) and low proliferative indices (21). These is a significant difference between these two groups (P = 0.001). CONCLUSIONS Elongation of telomere length in schwannomas appears to predict aggressive clinicopathological behavior.
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Affiliation(s)
- Han-Jung Chen
- Department of Neurosurgery, Chang Gung University and Medical Center at Kaohsiung, Taiwan, Republic of China.
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28
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Kunifuji Y, Gotoh S, Abe T, Miura M, Karasaki Y. Down-regulation of telomerase activity by anticancer drugs in human ovarian cancer cells. Anticancer Drugs 2002; 13:595-8. [PMID: 12172504 DOI: 10.1097/00001813-200207000-00005] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Maintenance of telomere length is crucial for survival of cells. Telomerase, an enzyme that is responsible for elongation of shortened telomeres, is active in human germ cells as well as most tumor tissues and experimentally immortalized cells. In contrast, most mature somatic cells in human tissues express undetectable or low telomerase activity, implying the existence of a stringent and negative regulatory mechanism. In this study we report the effects of anticancer drugs on telomerase activity in human cancer cells. In assaying for telomerase activity, we basically followed the original TRAP assay system, but with some modifications. A down-regulation of telomerase activity was found when cells of a human ovarian cancer cell line, A2780, were treated with;cis-diamminedichloroplatinum(II) (CDDP; cisplatin). However, down-regulation of telomerase activity was not found in cells of a cisplatin-resistant cell line, A2780CP, treated with cisplatin. On the other hand, telomerase activity in both the cell lines A2780 and A2780CP was reduced when A2780 or A2780CP was treated with adriamycin, an anthracycline antibiotic having a broad spectrum of antineoplastic activity. The different effects on the telomerase activity of the two types of anticancer drugs may be due the distinct chemical functions of these drugs. The present results may indicate a positive relationship between anticancer effects and down-regulation of telomerase activity by anticancer drugs.
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Affiliation(s)
- Yasumasa Kunifuji
- School of Health Sciences, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan.
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29
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Abstract
Replicative aging is the process by which most normal human cells "count" the number of times they have divided, eventually undergoing a growth arrest termed cellular senescence. This process is dependent on the shortening of telomeres, repeated sequences at the ends of the chromosomes. The loss of telomeric sequences with each cell division eventually induces a growth arrest that has a similar phenotype to that of cells stressed by inadequate culture or other conditions. Experiments over the past several years have identified species in which replicative aging does not occur and many examples in which a failure to proliferate has been misinterpreted as replicative senescence. Insights from these studies now permit a reevaluation of much of the seemingly contradictory data concerning replicative aging. There are good theoretical reasons for believing a limited proliferative capacity contributes to declining tissue homeostasis with increasing age. Although the presence of telomere shortening provides strong circumstantial evidence that replicative aging is occurring in vivo, thus far there is only very limited direct evidence for actual physiological effects of replicative aging.
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Affiliation(s)
- Woodring E Wright
- Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9039, USA.
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30
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Rubin H. The disparity between human cell senescence in vitro and lifelong replication in vivo. Nat Biotechnol 2002; 20:675-81. [PMID: 12089551 DOI: 10.1038/nbt0702-675] [Citation(s) in RCA: 126] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Cultured human fibroblasts undergo senescence (a loss of replicative capacity) after a uniform, fixed number of approximately 50 population doublings, commonly termed the Hayflick limit. It has been long known from clonal and other quantitative studies, however, that cells decline in replicative capacity from the time of explantation and do so in a stochastic manner, with a half-life of only approximately 8 doublings. The apparent 50-cell doubling limit reflects the expansive propagation of the last surviving clone. The relevance of either figure to survival of cells in the body is questionable, given that stem cells in some renewing tissues undergo >1,000 divisions in a lifetime with no morphological sign of senescence. Oddly enough, these observations have had little if any effect on general acceptance of the Hayflick limit in its original form. The absence of telomerase in cultured human cells and the shortening of telomeres at each population doubling have suggested that telomere length acts as a mitotic clock that accounts for their limited lifespan. This concept assumed an iconic character with the report that ectopic expression of telomerase by a vector greatly extended the lifespan of human cells. That something similar might occur in vivo seemed consistent with initial reports that most human somatic tissues lack telomerase activity. More careful study, however, has revealed telomerase activity in stem cells and some dividing transit cells of many renewing tissues and even in dividing myocytes of repairing cardiac muscle. It now seems likely that telomerase is active in vivo where and when it is needed to maintain tissue integrity. Caution is recommended in applying telomerase inhibition to kill telomerase-expressing cancer cells, because it would probably damage stem cells in essential organs and even increase the likelihood of secondary cancers. The risk may be especially high in sun-exposed skin, where there are usually thousands of p53-mutant clones of keratinocytes predisposed to cancer.
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Affiliation(s)
- Harry Rubin
- Department of Molecular and Cell Biology, Life Sciences Addition, University of California, Berkeley, CA 94720-3200, USA.
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31
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Vidal JD, Register TC, Gupta M, Cline JM. Estrogen replacement therapy induces telomerase RNA expression in the macaque endometrium. Fertil Steril 2002; 77:601-8. [PMID: 11872219 DOI: 10.1016/s0015-0282(01)03227-7] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
OBJECTIVE To evaluate the effects of hormonal therapies on the expression of telomerase RNA (TRNA) in the endometrium of ovariectomized female cynomolgus macaques (Macaca fascicularis). DESIGN Randomized long-term experimental trial. SETTING Animal study at an academic research institution. PATIENT(S) Surgically postmenopausal cynomolgus macaques. INTERVENTION(S) Treatments were given in the diet for three years and included conjugated equine estrogens (CEE), CEE + medroxyprogesterone acetate (MPA), and tamoxifen, at clinically relevant doses. MAIN OUTCOME MEASURE(S) Expression of TRNA within the basal glands, basal stroma, superficial glands, and superficial stroma of the endometrium by radiolabeled in situ hybridization. RESULT(S) Conjugated equine estrogens increased glandular TRNA expression, and the addition of MPA decreased this effect. Tamoxifen induced glandular TRNA expression to a lesser degree. Both CEE + MPA and tamoxifen increased stromal TRNA expression. The expression of TRNA in the endometrial glands was always greater than TRNA expression in the stroma. Treatment groups with greater proliferation and progesterone receptor expression also had elevated TRNA; within-group correlations were not significant. No statistically significant difference occurred between the basal and superficial endometrial layers. CONCLUSION(S) These results show for the first time a cell-specific hormonal regulation of TRNA in the primate endometrium, with up-regulation of TRNA by treatments associated with increased risk of endometrial cancer in women.
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Affiliation(s)
- Justin D Vidal
- Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, Virginia, USA
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32
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Abstract
Telomeres are distinctive structures, composed of a repetitive DNA sequence and associated proteins, that cap the ends of linear chromosomes. Telomeres are essential for maintaining the integrity and stability of eukaryotic genomes. In addition, under some circumstances, telomeres can influence cellular gene expression. In mammals, the length, structure, and function of telomeres have been proposed to contribute to cellular and organismal phenotypes associated with cancer and aging. Here, we discuss what is known about the basis for the links between telomeres, aging and cancer, and some of the known and proposed consequences of telomere dysfunction and maintenance for mammalian cells and organisms.
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Affiliation(s)
- Sahn-ho Kim Sh
- Life Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, California, CA 94720, USA
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33
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Hultdin M, Grönlund E, Norrback KF, Just T, Taneja K, Roos G. Replication timing of human telomeric DNA and other repetitive sequences analyzed by fluorescence in situ hybridization and flow cytometry. Exp Cell Res 2001; 271:223-9. [PMID: 11716534 DOI: 10.1006/excr.2001.5391] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The replication timing of telomeres seems to differ between species. Yeast telomeres are late replicating, whereas limited data from very few human cell lines have indicated telomere replication throughout S phase. In the present study a series of permanent cell lines and patient samples was investigated using a flow cytometric approach for telomere length determination based on in situ hybridization using peptide nucleic acid probes and DNA staining. This method permits selective analysis of cells in specific phases of the cell cycle without perturbation of the cell cycle machinery. The timing of replication of telomeric C(3)TA(2) and T(2)AG(3) repeats was found to differ between individual samples and could precede or be concomitant with the replication of bulk DNA. Replication of the T(2)AG(3) strand seemed to occur somewhat later than that of the C(3)TA(2) strand in some samples. (GTG)(n) and other repetitive sequences generally showed a replication pattern similar to that of the bulk of DNA with slightly individual differences, whereas centromeric DNA repeats consistently replicated within a short time frame in late S phase. The apparent variability in replication timing seen for telomeric DNA might suggest individual differences in firing of replication origins.
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Affiliation(s)
- M Hultdin
- Department of Medical Biosciences, Pathology, Umeå University, Umeå, S-90187, Sweden
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34
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Armbruster BN, Banik SS, Guo C, Smith AC, Counter CM. N-terminal domains of the human telomerase catalytic subunit required for enzyme activity in vivo. Mol Cell Biol 2001; 21:7775-86. [PMID: 11604512 PMCID: PMC99947 DOI: 10.1128/mcb.21.22.7775-7786.2001] [Citation(s) in RCA: 143] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Most tumor cells depend upon activation of the ribonucleoprotein enzyme telomerase for telomere maintenance and continual proliferation. The catalytic activity of this enzyme can be reconstituted in vitro with the RNA (hTR) and catalytic (hTERT) subunits. However, catalytic activity alone is insufficient for the full in vivo function of the enzyme. In addition, the enzyme must localize to the nucleus, recognize chromosome ends, and orchestrate telomere elongation in a highly regulated fashion. To identify domains of hTERT involved in these biological functions, we introduced a panel of 90 N-terminal hTERT substitution mutants into telomerase-negative cells and assayed the resulting cells for catalytic activity and, as a marker of in vivo function, for cellular proliferation. We found four domains to be essential for in vitro and in vivo enzyme activity, two of which were required for hTR binding. These domains map to regions defined by sequence alignments and mutational analysis in yeast, indicating that the N terminus has also been functionally conserved throughout evolution. Additionally, we discovered a novel domain, DAT, that "dissociates activities of telomerase," where mutations left the enzyme catalytically active, but was unable to function in vivo. Since mutations in this domain had no measurable effect on hTERT homomultimerization, hTR binding, or nuclear targeting, we propose that this domain is involved in other aspects of in vivo telomere elongation. The discovery of these domains provides the first step in dissecting the biological functions of human telomerase, with the ultimate goal of targeting this enzyme for the treatment of human cancers.
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Affiliation(s)
- B N Armbruster
- Department of Pharmacy and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA
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35
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Miura N, Onuki N, Rathi A, Virmani A, Nakamoto S, Kishimoto Y, Murawaki Y, Kawasaki H, Hasegawa J, Oshimura M, Travis WD, Gazdar AF. hTR repressor-related gene on human chromosome 10p15.1. Br J Cancer 2001; 85:1510-4. [PMID: 11720437 PMCID: PMC2363951 DOI: 10.1054/bjoc.2001.2121] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Somatic cells express genes that suppress telomerase activity and these genes may be inactivated in tumour cells. We postulated that cancer cells acquire immortality by activation of telomerase by the loss of such a gene. We have reported recently that a telomerase repressor gene may be located on 10p15.1 by deletion mapping using microcell-mediated chromosome transfer (MMCT), radiated microcell fusion (RMF), fluorescent in situ hybridization (FISH) and STS analysis. To independently confirm this result, we correlated expression of RNA component of telomerase (hTR) as a marker of telomerase expression by in situ hybridization with allelic loss in pulmonary carcinoid tumours. Unlike most malignant tumours, pulmonary carcinoids (which are low-grade malignant tumours) are heterogeneous for telomerase expression. Loss of 5 closely spaced polymorphic markers on 10p15.1, especially D10S1728, were highly correlated with hTR expression. In an additional experiment, 10p15.1 showed higher and more significant correlation than any region of 3p where it has been predicted as another chromosomal location of telomerase repressor with allelic loss of the region. Our findings strongly suggest that 10p15.1 harbours a gene involved in repression of telomerase RNA component in human somatic cells and each putative repressor (on 3p and 10p) may act independently.
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Affiliation(s)
- N Miura
- Hamon Center for Therapeutic Oncology Research, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8593, USA
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36
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Abstract
Telomeres are the repetitive DNA sequences and specialized proteins that form the distinctive structure that caps the ends of linear chromosomes. Telomeres allow cells to distinguish the chromosome ends from double strand DNA breaks. The telomeric structure prevents the degradation or fusion of chromosome ends, and thus is essential for maintaining the integrity and stability of eukaryotic genomes. In addition, and perhaps less widely appreciated, telomeres may also indirectly influence gene expression. The length, structure and organization of telomeres are regulated by a host of telomere-associated proteins, and can be influenced by basic cellular processes such as cell proliferation, differentiation, and DNA damage. In mammalian cells, telomere length and/or telomere structure have been linked to both cancer and aging. Here, we briefly review what is known about mammalian telomeres and the proteins that associate with them, and discuss the cellular and organismal consequences of telomere dysfunction and the evidence that cells with dysfunctional telomeres can contribute to cancer and aging phenotypes.
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Affiliation(s)
- J Campisi
- Life Sciences Division, Lawrence Berkeley National Laboratory, Mailstop 84-171, 1 Cyclotron Road, Berkeley, CA 94720, USA.
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37
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Abstract
Telomerase, the ribonucleoprotein enzyme that elongates chromosomal ends, or telomeres, is repressed in most normal somatic cells but reactivated in transformed cells to compensate for the progressive erosion of the telomeres during cell divisions. In accordance with this hypothesis, the presence of telomerase activity has been reported in more than 90% of human cancers, whereas most normal tissues or benign tumors contain low or undetectable telomerase activity. Reactivation of telomerase has also been widely reported in endocrine neoplasms and in hormone-related cancers. In the present study, we review the most recent publications on telomerase in these types of tumors. The hormonal regulation of telomerase activity and the possible strategies for cancer therapy based on the inhibition of telomerase has also been discussed.
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Affiliation(s)
- C Orlando
- Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, viale Pieraccini 6, 50139, Florence, Italy.
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38
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Affiliation(s)
- CLAUDIO ORLANDO
- From the Clinical Biochemistry Unit Department of Clinical Physiopathology, University of Florence and Division of Urology, Department of Surgery, University of Pisa, Pisa, Italy
| | - STEFANIA GELMINI
- From the Clinical Biochemistry Unit Department of Clinical Physiopathology, University of Florence and Division of Urology, Department of Surgery, University of Pisa, Pisa, Italy
| | - CESARE SELLI
- From the Clinical Biochemistry Unit Department of Clinical Physiopathology, University of Florence and Division of Urology, Department of Surgery, University of Pisa, Pisa, Italy
| | - MARIO PAZZAGLI
- From the Clinical Biochemistry Unit Department of Clinical Physiopathology, University of Florence and Division of Urology, Department of Surgery, University of Pisa, Pisa, Italy
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39
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Youssef N, Paradis V, Ferlicot S, Bedossa P. In situ detection of telomerase enzymatic activity in human hepatocellular carcinogenesis. J Pathol 2001; 194:459-65. [PMID: 11523054 DOI: 10.1002/path.901] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Telomerase enzymatic activity has been detected in most human malignant tumours including hepatocellular carcinoma. In order to assess the cellular source, the topographic distribution, and the chronology of telomerase re-expression during human liver carcinogenesis, an in situ technique derived from the standard TRAP (telomeric repeat amplification protocol) assay was set up that allowed the detection of telomerase enzyme activity at the cellular level on frozen liver tissue sections. In situ TRAP (ISTRAP) was performed on 27 hepatocellular carcinomas (HCCs) and 57 non-tumour livers, including normal liver without HCC, liver samples adjacent to tumour with and without hepatic cirrhosis, and biopsies of chronic hepatitis. In HCC, telomerase was detected in the nuclei of liver tumour cells in 23/27 cases (85%), with a heterogeneous distribution within the tumour. This signal was also detected in clusters of hepatocytes in 16/26 (61%) samples of liver adjacent to HCC, in 10/23 (43%) cases of chronic viral hepatitis without adjacent HCC, and in scattered nuclei of 2/8 histologically normal livers. Comparison of the results obtained with ISTRAP and standard TRAP assays on tissue extracts suggests a gain in sensitivity with the in situ technique. This study confirms that telomerase is expressed in most HCCs and suggests that focal telomerase reactivation is an early event during human liver carcinogenesis. ISTRAP is a sensitive technique that allows the study of telomerase expression in the morphological context.
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Affiliation(s)
- N Youssef
- Service d'Anatomie Pathologique, Hôpital de Bicêtre, Université Paris Sud, EA 1602, 78 Rue de Général Leclerc, 94275 Le Kremlin-Bicêtre Cedex, France
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40
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Abstract
Reactivation of telomerase, an enzyme which elongates human telomeres, is associated with cell immortilization. In approximately 90% of malignant tumours telomerase activity can be demonstrated, whereas in benign tumours it is mostly absent. Chondrosarcomas are relatively rare malignant cartilaginous neoplasms. A small number of chondrosarcomas located centrally in bone arise secondarily to an enchondroma, while the majority of chondrosarcomas developing from the surface arise within the cartilage cap of an osteochondroma. The histological distinction between a benign lesion and low-grade chondrosarcoma is generally considered difficult. To investigate whether the progression towards chondrosarcoma is characterized by reactivation of telomerase activity, this study determined telomerase activity in ten enchondromas, five osteochondromas, and 37 chondrosarcomas using the TRAP assay. In all tumour samples except one, telomerase activity was absent. By adding tumour lysates to the positive control, an increasing inhibition of telomerase activity was found with an increasing chondroid matrix, suggesting that it may contain inhibitory factors. Inhibition due to endogenous RNAse or Taq-polymerase inhibitors was excluded. The lack of detectable telomerase activity in the high-grade component of a dedifferentiated chondrosarcoma without matrix favours the possibility that telomerase is truly absent. Either its true absence or inhibitory effects disabling telomerase detection exclude the telomerase TRAP assay as a diagnostic tool in the differential diagnosis of benign and low-grade malignant cartilaginous tumours.
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Affiliation(s)
- J V Bovée
- Department of Pathology, Leiden University Medical Center, P.O. Box 9600, L1-Q, 2300 RC Leiden, The Netherlands.
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41
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Affiliation(s)
- A J Davis
- Department of Medical Oncology and Hematology, Princess Margaret Hospital/The Toronto Hospital, Ontario, Canada
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42
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43
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Chen HJ, Liang CL, Lu K, Lin JW, Cho CL. Implication of telomerase activity and alternations of telomere length in the histologic characteristics of intracranial meningiomas. Cancer 2000; 89:2092-8. [PMID: 11066050 DOI: 10.1002/1097-0142(20001115)89:10<2092::aid-cncr9>3.0.co;2-n] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
BACKGROUND Telomerase activity and telomere length have been shown to be involved in the control of cell proliferation and regulation of cell senescence. The expression of telomerase activity may endow cells with the capacity of unlimited proliferation and immortality. The authors examined the telomerase activity and telomere length of intracranial meningiomas to determine the relation between the results and the clinicopathologic behavior of these tumors. METHODS Sixty-two specimens of meningiomas including 13 atypical and malignant tumors were used in this study. Telomerase activity was measured with polymerase chain reaction and enzyme-linked immunosolvent assay. Telomere length was measured by detecting the terminal restriction fragments using Southern blots. RESULTS Detectable telomerase activity was found in 4 of 13 (30.8%) malignant or atypical meningiomas and only 1 in 49 benign meningiomas (P = 0.006). Elongated telomere length was measured in 6 of 13 (46.1%) patients with malignant or atypical meningiomas and only 1 of 48 (2.1%) in those with benign tumors (P = 0.0002). Three of 4 (75%) of malignant or atypical meningiomas with detectable telomerase activity revealed shortened telomere length, and all tumors with elongated telomere length displayed undetectable telomerase activity. The percentage of malignant or atypical meningiomas with detectable telomerase activity or elongated telomere were significantly higher (76.9%) than that of benign tumors (4.0%). The proliferative index was calculated as the percentage of tumor cell nuclei immunoreactive for Ki-67 to total tumor nuclei. The mean values of proliferative index in benign, atypical, and malignant meningiomas were 1.2, 11.0, and 30.0, respectively. CONCLUSIONS The results indicate that telomerase activation may be a critical step in the pathogenesis of malignant or atypical meningioma. Elongation of the telomere length also implicates the high potential for malignant behavior in these tumors.
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Affiliation(s)
- H J Chen
- Division of Neurosurgery, Chang Gung University and Medical Center at Kaohsiung, Kaohsiung Hsien, Taiwan
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44
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Armstrong L, Lako M, Lincoln J, Cairns PM, Hole N. mTert expression correlates with telomerase activity during the differentiation of murine embryonic stem cells. Mech Dev 2000; 97:109-16. [PMID: 11025212 DOI: 10.1016/s0925-4773(00)00423-8] [Citation(s) in RCA: 84] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Telomerase, the enzyme which maintains the ends of linear chromosomes in eukaryotic cells, is found at low levels in somatic stem cells but while this is incapable of preventing the progressive erosion of telomeres occurring as a consequence of cell division, such cells show greater proliferative capacity than normal somatic cells hence examination of telomerase activity in such stem cells is of interest. Our aim in this work was to examine the relationship between expression of the reverse transcriptase component (mTert) of murine telomerase. We report here the insertion of a reporter cassette comprising a segment of the promoter sequence of murine Tert gene coupled to the coding sequence of green fluorescent protein (GFP) into murine embryonic stem (ES) cells and show that this is sufficient for mimicking the expression of mTert. We show that the expression of mTert is very closely linked to telomerase activity and that both are substantially reduced upon differentiation of ES cells into more committed lineages giving us a potential reporter system for the selection and isolation of ES cells possessing different levels of telomerase activity.
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Affiliation(s)
- L Armstrong
- Department of Chemical and Life Sciences, University of Northumbria, NE1 8ST, Newcastle, UK
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45
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Iredale JP, Mann DA. Chromosomes and cirrhosis: all's well that ends well? Hepatology 2000; 32:153-4. [PMID: 10869304 DOI: 10.1002/hep.510320124] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/07/2022]
Affiliation(s)
- J P Iredale
- Liver Group Division of Cell and Molecular Medicine University of Southampton Southampton General Hospital Southampton, UK
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46
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Zhang FX, Zhang XY, Fan DM, Deng ZY, Yan Y, Wu HP, Fan JJ. Antisense telomerase RNA induced human gastric cancer cell apoptosis. World J Gastroenterol 2000; 6:430-432. [PMID: 11819619 PMCID: PMC4688773 DOI: 10.3748/wjg.v6.i3.430] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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Sawa Y, Phillips A, Hollard J, Yoshida S, Braithwaite MW. The in vitro life-span of human periodontal ligament fibroblasts. Tissue Cell 2000; 32:163-70. [PMID: 10855702 DOI: 10.1054/tice.2000.0100] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
The in vitro life-span of human periodontal ligament fibroblasts (PDLF) was studied on clones from periodontium of teeth extracted due to periodontitis and dental caries (69 clones/192 individuals, aged 20-80 years) and from periodontium of teeth extracted for orthodontic reasons (23 clones/26 individuals, aged 15-19 years). In the primary cultures the ratio of the number of cells expressing senescence-associated beta-galactosidase (SA-beta-Gal) to the total number of cells is significantly larger in PDLF (92 clones; 11.1+/-4.9%) than in human gingival fibroblasts (GF) (10 clones; 0.5+/-0.1 %). The finite population doubling numbers (PD) of PDLF are not age-matched and the mean PD of PDLF (7.1+/-2.9) is significantly smaller than GF (28.5+/-3.2), IMR-90 (human lung fibroblasts, 5 clones; 44.3 +/- 2.2), and human osteoblasts (5 clones; 19.7+/-1.4). Comparing the ratio of the number of SA-beta-Gal positive cells to the total number of cells in primary culture, and the finite PD in PDLF cultures: 1) the ratio of 15-19 years old donor group is significantly smaller than in the other donor groups (20-29, 30-39, 40-49, 50-59 and 60-80 years old), and 2) there were no statistically significant differences among the 20-29, 30-39, 40-49 and 50-59 year old donor groups, and the 30-39, 40-49, 50-59 and 60-80 year old donor groups. These findings suggest that the in vitro life-span of PDLF is shorter than other fibroblasts in the connective tissues and that PDLF may undergo senescence in adult clones without relation to donor's age. There may be more aged fibroblasts in periodontium than in other tissues, such as gingiva and lung.
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Affiliation(s)
- Y Sawa
- Department of Oral Anatomy I, Hokkaido University School of Dentistry, Sapporo, Japan.
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48
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Sawa Y, Phillips A, Hollard J, Yoshida S, Braithwaite MW. Impairment of osteocalcin production in senescent periodontal ligament fibroblasts. Tissue Cell 2000; 32:198-204. [PMID: 10855706 DOI: 10.1054/tice.2000.0104] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Osteocalcin production of senescent periodontal ligament fibroblasts (PDLF) with the expression of senescence-associated beta-galactosidase (SA-beta-Gal) was investigated on clones from 50-80 years old donors (n=20) with teeth extracted due to periodontitis and dental caries, and from 15-19 year old donors (n=20) with normal teeth extracted for orthodontic reasons. Immunohistochemically, the nonsenescent PDLF in all cultures in passage 2 showed strong reactivity with anti-osteocalcin. The reactive intensity of PDLF (passage 2, PD 3.0) was significantly stronger in 50-80 year old donor group than in 15-19 year old donor group, suggesting that osteocalcin production of PDLF cultured in early passage is larger in cells from adult population than in cells from adolescent population. In PDLF cultures in passage 2 from 50-80 year old donor, two types of senescent cells were found: one with strong reactivity to anti-osteocalcin and the other with little detectable reactivity. The culture consisted of senescent PDLF (passage 8, PD 14.8) did not include cells which have a detectable reactivity with anti-osteocalcin immunohistochemically and the reactive intensity was significantly weaker in the senescent culture than in the culture in passage 2 by ELISA. This suggests that the production potential of osteocalcin is impaired in PDLF with aging in culture. Further, the reactive intensity with anti-osteocalcin of PDLF in passage 2 deprived of serum for 48 h was 6% of that of cells cultured with serum and the reaction increased after serum stimulation, suggesting that the osteocalcin production in PDLF in early passage is implicated in mitogenic stimulation.
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Affiliation(s)
- Y Sawa
- Department of Oral Anatomy I, Hokkaido University School of Dentistry, Sapporo, Japan.
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Oh S, Song YH, Yim J, Kim TK. Identification of Mad as a repressor of the human telomerase (hTERT) gene. Oncogene 2000; 19:1485-90. [PMID: 10723141 DOI: 10.1038/sj.onc.1203439] [Citation(s) in RCA: 117] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
Activation of telomerase, which has been frequently associated with cellular immortality, may constitute a key step in the development of human cancer. De-repression in the expression of its catalytic subunit hTERT gene has been proposed to directly link to the telomerase activation in tumor cells. Little is known about the mechanism how the hTERT gene is repressed in telomerase-negative mortal cells. This study was conducted, using an expression cloning approach, with the aim of identifying the gene(s) responsible for repressing the hTERT gene expression. Using this genetic screen, we isolated the transcription factor Mad as a repressor. Mutation of its DNA binding sites caused significant de-repression of hTERT promoter activity in mortal cells. This Mad-mediated repression of the hTERT promoter in mortal cells was counteracted by ectopic expression of Myc. The antagonism between Mad and Myc was also observed with an endogenous hTERT promoter. Their potential roles in differential hTERT promoter activities were further supported by the relative amounts of Mad and Myc proteins detected in immortal and mortal cells. Thus, Mad may be a direct negative regulator of hTERT in mortal cells and this repression mechanism can be inhibited by induction of Myc in immortal cells.
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Affiliation(s)
- S Oh
- National Creative Research Initiative Center for Genetic Reprogramming, Institute for Molecular Biology and Genetics, Seoul National University, Seoul 151-742, Korea
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Gómez-Román JJ, Romero AF, Castro LS, Nieto EH, Fernández-Luna JL, Val-Bernal JF. Telomerase activity in pulmonary neuroendocrine tumors: correlation with histologic subtype (MS-0060). Am J Surg Pathol 2000; 24:417-21. [PMID: 10716156 DOI: 10.1097/00000478-200003000-00011] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
The authors measured telomerase activity using the telomeric repeat amplification protocol-enzyme-linked immunosorbent assay method in 13 neuroendocrine pulmonary neoplasms and in non-neoplastic frozen lung samples from the same patients. These cases belonged to the complete neuroendocrine neoplastic spectrum: four typical carcinoids, three atypical carcinoids, four large cell neuroendocrine lung carcinomas, and two small cell lung carcinomas. The authors performed the same assay for 52 non-neoplastic lung tissues from the surgical files in their department (negative controls). They verified the presence (or absence) of neoplastic tissue in every case by looking at one frozen section done in the same tissue used for telomerase assay. The telomerase activity level in non-neoplastic tissues (mean, 182 A450nm U) was similar to that obtained in the typical carcinoids (mean, 104.5 A450nm U). All neuroendocrine tumors but the typical carcinoids showed high levels of telomerase activity (mean, 1,750.8 A450nm U). According to the telomerase hypothesis, typical carcinoid cells are mortal pre-M2 stage cells, but atypical carcinoid, large cell neuroendocrine lung carcinoma, and small cell lung carcinoma cells are immortal post-M2 stage cells. This finding may be of important prognostic significance in these kinds of tumors. Measurement of enzyme activity with a good morphologic control could be necessary in telomerase activity assay.
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Affiliation(s)
- J J Gómez-Román
- Department of Anatomical Pathology, Marqués de Valdecilla, University Hospital, Instituto Nacional de la Salud, Medical Faculty, University of Cantabria, Santander, Spain
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