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Pita JM, Raspé E, Coulonval K, Decaussin-Petrucci M, Tarabichi M, Dom G, Libert F, Craciun L, Andry G, Wicquart L, Leteurtre E, Trésallet C, Marlow LA, Copland JA, Durante C, Maenhaut C, Cavaco BM, Dumont JE, Costante G, Roger PP. CDK4 phosphorylation status and rational use for combining CDK4/6 and BRAF/MEK inhibition in advanced thyroid carcinomas. Front Endocrinol (Lausanne) 2023; 14:1247542. [PMID: 37964967 PMCID: PMC10641312 DOI: 10.3389/fendo.2023.1247542] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/26/2023] [Accepted: 09/26/2023] [Indexed: 11/16/2023] Open
Abstract
Background CDK4/6 inhibitors (CDK4/6i) have been established as standard treatment against advanced Estrogen Receptor-positive breast cancers. These drugs are being tested against several cancers, including in combinations with other therapies. We identified the T172-phosphorylation of CDK4 as the step determining its activity, retinoblastoma protein (RB) inactivation, cell cycle commitment and sensitivity to CDK4/6i. Poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinomas, the latter considered one of the most lethal human malignancies, represent major clinical challenges. Several molecular evidence suggest that CDK4/6i could be considered for treating these advanced thyroid cancers. Methods We analyzed by two-dimensional gel electrophoresis the CDK4 modification profile and the presence of T172-phosphorylated CDK4 in a collection of 98 fresh-frozen tissues and in 21 cell lines. A sub-cohort of samples was characterized by RNA sequencing and immunohistochemistry. Sensitivity to CDK4/6i (palbociclib and abemaciclib) was assessed by BrdU incorporation/viability assays. Treatment of cell lines with CDK4/6i and combination with BRAF/MEK inhibitors (dabrafenib/trametinib) was comprehensively evaluated by western blot, characterization of immunoprecipitated CDK4 and CDK2 complexes and clonogenic assays. Results CDK4 phosphorylation was detected in all well-differentiated thyroid carcinomas (n=29), 19/20 PDTC, 16/23 ATC and 18/21 thyroid cancer cell lines, including 11 ATC-derived ones. Tumors and cell lines without phosphorylated CDK4 presented very high p16CDKN2A levels, which were associated with proliferative activity. Absence of CDK4 phosphorylation in cell lines was associated with CDK4/6i insensitivity. RB1 defects (the primary cause of intrinsic CDK4/6i resistance) were not found in 5/7 tumors without detectable phosphorylated CDK4. A previously developed 11-gene expression signature identified the likely unresponsive tumors, lacking CDK4 phosphorylation. In cell lines, palbociclib synergized with dabrafenib/trametinib by completely and permanently arresting proliferation. These combinations prevented resistance mechanisms induced by palbociclib, most notably Cyclin E1-CDK2 activation and a paradoxical stabilization of phosphorylated CDK4 complexes. Conclusion Our study supports further clinical evaluation of CDK4/6i and their combination with anti-BRAF/MEK therapies as a novel effective treatment against advanced thyroid tumors. Moreover, the complementary use of our 11 genes predictor with p16/KI67 evaluation could represent a prompt tool for recognizing the intrinsically CDK4/6i insensitive patients, who are potentially better candidates to immediate chemotherapy.
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Affiliation(s)
- Jaime M. Pita
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) and Université Libre de Bruxelles (ULB)-Cancer Research Center (U-CRC), Université Libre de Bruxelles (ULB), Brussels, Belgium
| | - Eric Raspé
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) and Université Libre de Bruxelles (ULB)-Cancer Research Center (U-CRC), Université Libre de Bruxelles (ULB), Brussels, Belgium
| | - Katia Coulonval
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) and Université Libre de Bruxelles (ULB)-Cancer Research Center (U-CRC), Université Libre de Bruxelles (ULB), Brussels, Belgium
| | | | - Maxime Tarabichi
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) and Université Libre de Bruxelles (ULB)-Cancer Research Center (U-CRC), Université Libre de Bruxelles (ULB), Brussels, Belgium
| | - Geneviève Dom
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) and Université Libre de Bruxelles (ULB)-Cancer Research Center (U-CRC), Université Libre de Bruxelles (ULB), Brussels, Belgium
| | - Frederick Libert
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) and Université Libre de Bruxelles (ULB)-Cancer Research Center (U-CRC), Université Libre de Bruxelles (ULB), Brussels, Belgium
- BRIGHTCore, Université Libre de Bruxelles (ULB), Brussels, Belgium
| | - Ligia Craciun
- Tumor Bank of the Institut Jules Bordet Comprehensive Cancer Center – Hôpital Universitaire de Bruxelles, Université Libre de Bruxelles (ULB), Brussels, Belgium
| | - Guy Andry
- Department of Head & Neck and Thoracic Surgery, Institut Jules Bordet Comprehensive Cancer Center – Hôpital Universitaire de Bruxelles, Université Libre de Bruxelles (ULB), Brussels, Belgium
| | - Laurence Wicquart
- Tumorothèque du Groupement de Coopération Sanitaire-Centre Régional de Référence en Cancérologie (C2RC) de Lille, Lille, France
| | - Emmanuelle Leteurtre
- Department of Pathology, Univ. Lille, Centre National de la Recherche Scientifique (CNRS), Inserm, Centre Hospitalo-Universitaire (CHU) Lille, UMR9020-U1277-CANTHER-Cancer Heterogeneity, Plasticity and Resistance to Therapies, Lille, France
| | - Christophe Trésallet
- Department of General and Endocrine Surgery - Pitié-Salpêtrière Hospital, Sorbonne University, Assistance Publique des Hôpitaux de Paris, Paris, France
- Department of Digestive, Bariatric and Endocrine Surgery - Avicenne University Hospital, Paris Nord - Sorbonne University, Assistance Publique des Hôpitaux de Paris, Paris, France
| | - Laura A. Marlow
- Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, United States
| | - John A. Copland
- Department of Cancer Biology, Mayo Clinic, Jacksonville, FL, United States
| | - Cosimo Durante
- Department of Translational and Precision Medicine, Sapienza University of Rome, Rome, Italy
| | - Carine Maenhaut
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) and Université Libre de Bruxelles (ULB)-Cancer Research Center (U-CRC), Université Libre de Bruxelles (ULB), Brussels, Belgium
| | - Branca M. Cavaco
- Molecular Endocrinology Group, Unidade de Investigação em Patobiologia Molecular (UIPM), Instituto Português de Oncologia de Lisboa Francisco Gentil (IPOLFG), Lisbon, Portugal
| | - Jacques E. Dumont
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) and Université Libre de Bruxelles (ULB)-Cancer Research Center (U-CRC), Université Libre de Bruxelles (ULB), Brussels, Belgium
| | - Giuseppe Costante
- Departments of Endocrinology and Medical Oncology, Institut Jules Bordet Comprehensive Cancer Center – Hôpital Universitaire de Bruxelles, Université Libre de Bruxelles (ULB), Brussels, Belgium
| | - Pierre P. Roger
- Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM) and Université Libre de Bruxelles (ULB)-Cancer Research Center (U-CRC), Université Libre de Bruxelles (ULB), Brussels, Belgium
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Cheng F, Carroll L, Joglekar MV, Januszewski AS, Wong KK, Hardikar AA, Jenkins AJ, Ma RCW. Diabetes, metabolic disease, and telomere length. Lancet Diabetes Endocrinol 2021; 9:117-126. [PMID: 33248477 DOI: 10.1016/s2213-8587(20)30365-x] [Citation(s) in RCA: 123] [Impact Index Per Article: 30.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/04/2020] [Revised: 10/01/2020] [Accepted: 10/08/2020] [Indexed: 02/06/2023]
Abstract
Telomeres are regions of repetitive nucleotide sequences at the ends of chromosomes. Telomere length is a marker of DNA damage, which is often considered a biomarker for biological ageing, and has also been linked with cardiovascular disease, diabetes, and cancer. Emerging studies have highlighted the role of genetic and environmental factors, and explored the effect of modulating telomere length. We provide an overview of studies to date on diabetes and telomere length, and compare different methods and assays for evaluating telomere length and telomerase activity. We highlight the limitations of current studies and areas that warrant further research to unravel the link between diabetes and telomere length. The value of adding telomere length to clinical risk factors to improve risk prediction of diabetes and related complications also merits further investigation.
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Affiliation(s)
- Feifei Cheng
- Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China; Laboratory for Molecular Epidemiology in Diabetes, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China
| | - Luke Carroll
- NHMRC Clinical Trial Centre, University of Sydney, Sydney, NSW, Australia
| | - Mugdha V Joglekar
- NHMRC Clinical Trial Centre, University of Sydney, Sydney, NSW, Australia; School of Medicine, Western Sydney University, Campbelltown, NSW, Australia
| | | | - Kwun Kiu Wong
- Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China; Laboratory for Molecular Epidemiology in Diabetes, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China
| | - Anandwardhan A Hardikar
- NHMRC Clinical Trial Centre, University of Sydney, Sydney, NSW, Australia; School of Medicine, Western Sydney University, Campbelltown, NSW, Australia.
| | - Alicia J Jenkins
- Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China; NHMRC Clinical Trial Centre, University of Sydney, Sydney, NSW, Australia.
| | - Ronald C W Ma
- Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China; Laboratory for Molecular Epidemiology in Diabetes, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China; Hong Kong Institute of Diabetes and Obesity, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China; Chinese University of Hong Kong-Shanghai Jiao Tong University Joint Research Centre in Diabetes Genomics and Precision Medicine, The Chinese University of Hong Kong, Hong Kong Special Administrative Region, China; NHMRC Clinical Trial Centre, University of Sydney, Sydney, NSW, Australia.
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The Emerging Role of Senescence in Ocular Disease. OXIDATIVE MEDICINE AND CELLULAR LONGEVITY 2020; 2020:2583601. [PMID: 32215170 PMCID: PMC7085400 DOI: 10.1155/2020/2583601] [Citation(s) in RCA: 42] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 12/18/2019] [Accepted: 02/14/2020] [Indexed: 02/07/2023]
Abstract
Cellular senescence is a state of irreversible cell cycle arrest in response to an array of cellular stresses. An important role for senescence has been shown for a number of pathophysiological conditions that include cardiovascular disease, pulmonary fibrosis, and diseases of the skin. However, whether senescence contributes to the progression of age-related macular degeneration (AMD) has not been studied in detail so far and the present review describes the recent research on this topic. We present an overview of the types of senescence, pathways of senescence, senescence-associated secretory phenotype (SASP), the role of mitochondria, and their functional implications along with antisenescent therapies. As a central mechanism, senescent cells can impact the surrounding tissue microenvironment via the secretion of a pool of bioactive molecules, termed the SASP. An updated summary of a number of new members of the ever-growing SASP family is presented. Further, we introduce the significance of mechanisms by which mitochondria may participate in the development of cellular senescence. Emerging evidence shows that extracellular vesicles (EVs) are important mediators of the effects of senescent cells on their microenvironment. Based on recent studies, there is reasonable evidence that senescence could be a modifiable factor, and hence, it may be possible to delay age-related diseases by modulating basic aging mechanisms using SASP inhibitors/senolytic drugs. Thus, antisenescent therapies in aging and age-related diseases appear to have a promising potential.
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Quantitative and integrated proteome and microRNA analysis of endothelial replicative senescence. J Proteomics 2015; 126:12-23. [DOI: 10.1016/j.jprot.2015.05.023] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2015] [Revised: 05/05/2015] [Accepted: 05/20/2015] [Indexed: 12/31/2022]
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Kim J, Piao Y, Pak YK, Chung D, Han YM, Hong JS, Jun EJ, Shim JY, Choi J, Kim CJ. Umbilical cord mesenchymal stromal cells affected by gestational diabetes mellitus display premature aging and mitochondrial dysfunction. Stem Cells Dev 2015; 24:575-86. [PMID: 25437179 DOI: 10.1089/scd.2014.0349] [Citation(s) in RCA: 67] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Human umbilical cord mesenchymal stromal cells (hUC-MSCs) of Wharton's jelly origin undergo adipogenic, osteogenic, and chondrogenic differentiation in vitro. Recent studies have consistently shown their therapeutic potential in various human disease models. However, the biological effects of major pregnancy complications on the cellular properties of hUC-MSCs remain to be studied. In this study, we compared the basic properties of hUC-MSCs obtained from gestational diabetes mellitus (GDM) patients (GDM-UC-MSCs) and normal pregnant women (N-UC-MSCs). Assessments of cumulative cell growth, MSC marker expression, cellular senescence, and mitochondrial function-related gene expression were performed using a cell count assay, senescence-associated β-galactosidase staining, quantitative real-time reverse transcription-polymerase chain reaction, immunoblotting, and cell-based mitochondrial functional assay system. When compared with N-UC-MSCs, GDM-UC-MSCs showed decreased cell growth and earlier cellular senescence with accumulation of p16 and p53, even though they expressed similar levels of CD105, CD90, and CD73 MSC marker proteins. GDM-UC-MSCs also displayed significantly lower osteogenic and adipogenic differentiation potentials than N-UC-MSCs. Furthermore, GDM-UC-MSCs exhibited a low mitochondrial activity and significantly reduced expression of the mitochondrial function regulatory genes ND2, ND9, COX1, PGC-1α, and TFAM. Here, we report intriguing and novel evidence that maternal metabolic derangement during gestation affects the biological properties of fetal cells, which may be a component of fetal programming. Our findings also underscore the importance of the critical assessment of the biological impact of maternal-fetal conditions in biological studies and clinical applications of hUC-MSCs.
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Affiliation(s)
- Jooyeon Kim
- 1 Departments of Pathology, Asan Medical Center, University of Ulsan College of Medicine , Seoul, Korea
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Marthandan S, Priebe S, Hemmerich P, Klement K, Diekmann S. Long-term quiescent fibroblast cells transit into senescence. PLoS One 2014; 9:e115597. [PMID: 25531649 PMCID: PMC4274099 DOI: 10.1371/journal.pone.0115597] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2014] [Accepted: 11/28/2014] [Indexed: 12/16/2022] Open
Abstract
Cellular senescence is described to be a consequence of telomere erosion during the replicative life span of primary human cells. Quiescence should therefore not contribute to cellular aging but rather extend lifespan. Here we tested this hypothesis and demonstrate that cultured long-term quiescent human fibroblasts transit into senescence due to similar cellular mechanisms with similar dynamics and with a similar maximum life span as proliferating controls, even under physiological oxygen conditions. Both, long-term quiescent and senescent fibroblasts almost completely fail to undergo apoptosis. The transition of long-term quiescent fibroblasts into senescence is also independent of HES1 which protects short-term quiescent cells from becoming senescent. Most significantly, DNA damage accumulates during senescence as well as during long-term quiescence at physiological oxygen levels. We suggest that telomere-independent, potentially maintenance driven gradual induction of cellular senescence during quiescence is a counterbalance to tumor development.
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Affiliation(s)
- Shiva Marthandan
- Leibniz-Institute for Age Research- Fritz Lipmann Institute, JenAge (Jena Centre for Systems Biology of Aging), Beutenbergstrasse 11, Jena, Germany
| | - Steffen Priebe
- Leibniz Institute for Natural Product Research and Infection Biology - Hans-Knöll-Institute e.V. (HKI), Jena, Germany
| | - Peter Hemmerich
- Leibniz-Institute for Age Research- Fritz Lipmann Institute, JenAge (Jena Centre for Systems Biology of Aging), Beutenbergstrasse 11, Jena, Germany
| | - Karolin Klement
- Leibniz-Institute for Age Research- Fritz Lipmann Institute, JenAge (Jena Centre for Systems Biology of Aging), Beutenbergstrasse 11, Jena, Germany
| | - Stephan Diekmann
- Leibniz-Institute for Age Research- Fritz Lipmann Institute, JenAge (Jena Centre for Systems Biology of Aging), Beutenbergstrasse 11, Jena, Germany
- * E-mail:
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Klement K, Goodarzi AA. DNA double strand break responses and chromatin alterations within the aging cell. Exp Cell Res 2014; 329:42-52. [PMID: 25218945 DOI: 10.1016/j.yexcr.2014.09.003] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2014] [Revised: 08/28/2014] [Accepted: 09/01/2014] [Indexed: 12/23/2022]
Abstract
Cellular senescence is a state of permanent replicative arrest that allows cells to stay viable and metabolically active but resistant to apoptotic and mitogenic stimuli. Specific, validated markers can identify senescent cells, including senescence-associated β galactosidase activity, chromatin alterations, cell morphology changes, activated p16- and p53-dependent signaling and permanent cell cycle arrest. Senescence is a natural consequence of DNA replication-associated telomere erosion, but can also be induced prematurely by telomere-independent events such as failure to repair DNA double strand breaks. Here, we review the molecular pathways of senescence onset, focussing on the changes in chromatin organization that are associated with cellular senescence, particularly senescence-associated heterochromatin foci formation. We also discuss the altered dynamics of the DNA double strand break response within the context of aging cells. Appreciating how, mechanistically, cellular senescence is induced, and how changes to chromatin organization and DNA repair contributes to this, is fundamental to our understanding of the normal and premature human aging processes associated with loss of organ and tissue function in humans.
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Affiliation(s)
- Karolin Klement
- Southern Alberta Cancer Research Institute, Departments of Biochemistry & Molecular Biology and Oncology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1
| | - Aaron A Goodarzi
- Southern Alberta Cancer Research Institute, Departments of Biochemistry & Molecular Biology and Oncology, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1.
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Abstract
Cellular senescence is a stress response that accompanies stable exit from the cell cycle. Classically, senescence, particularly in human cells, involves the p53 and p16/Rb pathways, and often both of these tumor suppressor pathways need to be abrogated to bypass senescence. In parallel, a number of effector mechanisms of senescence have been identified and characterized. These studies suggest that senescence is a collective phenotype of these multiple effectors, and their intensity and combination can be different depending on triggers and cell types, conferring a complex and diverse nature to senescence. Series of studies on senescence-associated secretory phenotype (SASP) in particular have revealed various layers of functionality of senescent cells in vivo. Here we discuss some key features of senescence effectors and attempt to functionally link them when it is possible.
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Affiliation(s)
- Rafik Salama
- Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, United Kingdom
| | - Mahito Sadaie
- Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
| | - Matthew Hoare
- Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, United Kingdom
| | - Masashi Narita
- Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, United Kingdom
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Sarcoid-derived fibroblasts: links between genomic instability, energy metabolism and senescence. Biochimie 2013; 97:163-72. [PMID: 24148276 DOI: 10.1016/j.biochi.2013.10.010] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2013] [Accepted: 10/10/2013] [Indexed: 12/30/2022]
Abstract
Bovine papillomavirus 1 (BPV-1) is a well recognized etiopathogenetic factor in a cancer-like state in horses, namely equine sarcoid disease. Nevertheless, little is known about BPV-1-mediated cell transforming effects. It was shown that BPV-1 triggers genomic instability through DNA hypomethylation and oxidative stress. In the present study, we further characterized BPV-1-positive fibroblasts derived from sarcoid tumors. The focus was on cancer-like features of sarcoid-derived fibroblasts, including cell cycle perturbation, comprehensive DNA damage analysis, end-replication problem, energy metabolism and oncogene-induced premature senescence. The S phase of the cell cycle, polyploidy events, DNA double strand breaks (DSBs) and DNA single strand breaks (SSBs) were increased in BPV-1-positive cells compared to control fibroblasts. BPV-1-mediated oxidative stress may contribute to telomere dysfunction in sarcoid-derived fibroblasts. Loss of mitochondrial membrane potential and concurrent elevation in intracellular ATP production may be a consequence of changes in energy-supplying pathways in BPV-1-positive cells which is also typical for cancer cells. Shifts in energy metabolism may support rapid proliferation in cells infected by BPV-1. Nevertheless, sarcoid-derived fibroblasts representing a heterogeneous cell fraction vary in some aspects of metabolic phenotype due to a dual role of BPV-1 in cell transformation and oncogene-induced premature senescence. This was shown with increased senescence-associated β-galactosidase (SA-β-gal) activity. Taken together, metabolic phenotypes in sarcoid-derived fibroblasts are plastic, which are similar to greater plasticity of cancer tissues than normal tissues.
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Nakayama Y, Yamaguchi N. Role of cyclin B1 levels in DNA damage and DNA damage-induced senescence. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2013; 305:303-37. [PMID: 23890385 DOI: 10.1016/b978-0-12-407695-2.00007-x] [Citation(s) in RCA: 76] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The cyclin B1-Cdk1 complex is a key regulator of mitotic entry. A large number of proteins are phosphorylated by the cyclin B1-Cdk1 complex prior to mitotic entry. Regulation of the mitotic events is linked to the control of the activity of the cyclin B1-Cdk1 complex to make cells enter mitosis, arrest at G2-phase, or skip mitosis. The roles of cyclin B1 levels in DNA damage are described. The ATM/ATR pathway acts as a molecular switch for regulating cell fates, flipping between cell death via progress into mitosis and polyploidization via sustained G2 arrest upon DNA damage, where cyclin B1 degradation is important for inducing polyploidization. The decrease in cyclin B1 levels that is induced by DNA damage leads to polyploidization in DNA damage-induced senescence. A useful method for monitoring the expression level of cyclin B1 throughout cell cycle progression in living cells is also presented.
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Affiliation(s)
- Yuji Nakayama
- Department of Biochemistry & Molecular Biology, Kyoto Pharmaceutical University, Kyoto, Japan.
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Abstract
The advent of massively parallel sequencing technologies has allowed the characterization of cancer genomes at an unprecedented resolution. Investigation of the mutational landscape of tumours is providing new insights into cancer genome evolution, laying bare the interplay of somatic mutation, adaptation of clones to their environment and natural selection. These studies have demonstrated the extent of the heterogeneity of cancer genomes, have allowed inferences to be made about the forces that act on nascent cancer clones as they evolve and have shown insight into the mutational processes that generate genetic variation. Here we review our emerging understanding of the dynamic evolution of the cancer genome and of the implications for basic cancer biology and the development of antitumour therapy.
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Affiliation(s)
- Lucy R Yates
- Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton CB10 1SA, UK
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Sohn JJ, Schetter AJ, Yfantis HG, Ridnour LA, Horikawa I, Khan MA, Robles AI, Hussain SP, Goto A, Bowman ED, Hofseth LJ, Bartkova J, Bartek J, Wogan GN, Wink DA, Harris CC. Macrophages, nitric oxide and microRNAs are associated with DNA damage response pathway and senescence in inflammatory bowel disease. PLoS One 2012; 7:e44156. [PMID: 22970173 PMCID: PMC3435404 DOI: 10.1371/journal.pone.0044156] [Citation(s) in RCA: 50] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2012] [Accepted: 07/30/2012] [Indexed: 01/08/2023] Open
Abstract
BACKGROUND Cellular senescence can be a functional barrier to carcinogenesis. We hypothesized that inflammation modulates carcinogenesis through senescence and DNA damage response (DDR). We examined the association between senescence and DDR with macrophage levels in inflammatory bowel disease (IBD). In vitro experiments tested the ability of macrophages to induce senescence in primary cells. Inflammation modulating microRNAs were identified in senescence colon tissue for further investigation. METHODOLOGY/PRINCIPAL FINDINGS Quantitative immunohistochemistry identified protein expression by colon cell type. Increased cellular senescence (HP1γ; P = 0.01) or DDR (γH2A.X; P = 0.031, phospho-Chk2, P = 0.014) was associated with high macrophage infiltration in UC. Co-culture with macrophages (ANA-1) induced senescence in >80% of primary cells (fibroblasts MRC5, WI38), illustrating that macrophages induce senescence. Interestingly, macrophage-induced senescence was partly dependent on nitric oxide synthase, and clinically relevant NO• levels alone induced senescence. NO• induced DDR in vitro, as detected by immunofluorescence. In contrast to UC, we noted in Crohn's disease (CD) that senescence (HP1γ; P<0.001) and DDR (γH2A.X; P<0.05, phospho-Chk2; P<0.001) were higher, and macrophages were not associated with senescence. We hypothesize that nitric oxide may modulate senescence in CD; epithelial cells of CD had higher levels of NOS2 expression than in UC (P = 0.001). Microarrays and quantitative-PCR identified miR-21 expression associated with macrophage infiltration and NOS2 expression. CONCLUSIONS Senescence was observed in IBD with senescence-associated β-galactosidase and HP1γ. Macrophages were associated with senescence and DDR in UC, and in vitro experiments with primary human cells showed that macrophages induce senescence, partly through NO•, and that NO• can induce DDR associated with senescence. Future experiments will investigate the role of NO• and miR-21 in senescence. This is the first study to implicate macrophages and nitrosative stress in a direct effect on senescence and DDR, which is relevant to many diseases of inflammation, cancer, and aging.
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Affiliation(s)
- Jane J. Sohn
- Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America
| | - Aaron J. Schetter
- Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America
| | - Harris G. Yfantis
- Pathology and Laboratory Medicine, Baltimore Veterans Affairs Medical Center, and Department of Pathology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America
| | - Lisa A. Ridnour
- Radiation Biology Branch, National Cancer Institute, Bethesda, Maryland, United States of America
| | - Izumi Horikawa
- Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America
| | - Mohammed A. Khan
- Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America
| | - Ana I. Robles
- Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America
| | - S. Perwez Hussain
- Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America
| | - Akiteru Goto
- Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America
| | - Elise D. Bowman
- Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America
| | - Lorne J. Hofseth
- Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, South Carolina, United States of America
| | | | - Jiri Bartek
- Cancer Society Research Center, Copenhagen, Denmark
- Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic
| | - Gerald N. Wogan
- Department of Biological Engineering, Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America
| | - David A. Wink
- Radiation Biology Branch, National Cancer Institute, Bethesda, Maryland, United States of America
| | - Curtis C. Harris
- Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America
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Vizioli MG, Possik PA, Tarantino E, Meissl K, Borrello MG, Miranda C, Anania MC, Pagliardini S, Seregni E, Pierotti MA, Pilotti S, Peeper DS, Greco A. Evidence of oncogene-induced senescence in thyroid carcinogenesis. Endocr Relat Cancer 2011; 18:743-57. [PMID: 21937739 DOI: 10.1530/erc-11-0240] [Citation(s) in RCA: 39] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Oncogene-induced senescence (OIS) is a growth arrest triggered by the enforced expression of cancer-promoting genes and acts as a barrier against malignant transformation in vivo. In this study, by a combination of in vitro and in vivo approaches, we investigate the role of OIS in tumours originating from the thyroid epithelium. We found that expression of different thyroid tumour-associated oncogenes in primary human thyrocytes triggers senescence, as demonstrated by the presence of OIS hallmarks: changes in cell morphology, accumulation of SA-β-Gal and senescence-associated heterochromatic foci, and upregulation of transcription of the cyclin-dependent kinase inhibitors p16(INK4a) and p21(CIP1). Furthermore, immunohistochemical analysis of a panel of thyroid tumours characterised by different aggressiveness showed that the expression of OIS markers such as p16(INK4a), p21(CIP1) and IGFBP7 is upregulated at early stages, and lost during thyroid tumour progression. Taken together, our results suggest a role of OIS in thyroid carcinogenesis.
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Affiliation(s)
- Maria Grazia Vizioli
- Molecular Mechanisms Unit, Department of Experimental Oncology and Molecular Medicine, IRCCS Foundation-Istituto Nazionale dei Tumori, Via G. Amadeo, 42 20133 Milan, Italy
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14
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ROS-generating NADPH oxidase NOX4 is a critical mediator in oncogenic H-Ras-induced DNA damage and subsequent senescence. Oncogene 2011; 31:1117-29. [PMID: 21841825 PMCID: PMC3307059 DOI: 10.1038/onc.2011.327] [Citation(s) in RCA: 239] [Impact Index Per Article: 17.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Activated Ras oncogene induces DNA-damage response by triggering reactive oxygen species (ROS) production and this is critical for oncogene-induced senescence. Until now, little connections between oncogene expression, ROS-generating NADPH oxidases and DNA-damage response have emerged from different studies. Here we report that H-RasV12 positively regulates the NADPH oxidase system NOX4-p22(phox) that produces H(2)O(2). Knocking down the NADPH oxidase with small interference RNA decreases H-RasV12-induced DNA-damage response detected by γ-H2A.X foci analysis. Using HyPer, a specific probe for H(2)O(2), we detected an increase in H(2)O(2) in the nucleus correlated with NOX4-p22(phox) perinuclear localization. DNA damage response can be caused not only by H-RasV12-driven accumulation of ROS but also by a replicative stress due to a sustained oncogenic signal. Interestingly, NOX4 downregulation by siRNA abrogated H-RasV12 regulation of CDC6 expression, an essential regulator of DNA replication. Moreover, senescence markers, such as senescence-associated heterochromatin foci, PML bodies, HP1β foci and p21 expression, induced under H-RasV12 activation were decreased with NOX4 inactivation. Taken together, our data indicate that NADPH oxidase NOX4 is a critical mediator in oncogenic H-RasV12-induced DNA-damage response and subsequent senescence.
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Abstract
Cellular senescence is a specialized form of growth arrest, confined to mitotic cells, induced by various stressful stimuli and characterized by a permanent growth arrest, resistance to apoptosis, an altered pattern of gene expression and the expression of some markers that are characteristic, although not exclusive, to the senescent state. Senescent cells profoundly modify neighboring and remote cells through the production of an altered secretome, eventually leading to inflammation, fibrosis and possibly growth of neoplastic cells. Mammalian aging has been defined as a reduction in the capacity to adequately maintain tissue homeostasis or to repair tissues after injury. Tissue homeostasis and regenerative capacity are nowadays considered to be related to the stem cell pool present in every tissue. For this reason, pathological and patho-physiological conditions characterized by altered tissue homeostasis and impaired regenerative capacity can be viewed as a consequence of the reduction in stem cell number and/or function. Last, cellular senescence is a double-edged sword, since it may inhibit the growth of transformed cells, preventing the occurrence of cancer, while it may facilitate growth of preneoplastic lesions in a paracrine fashion; therefore, interventions targeting this cell response to stress may have a profound impact on many age-related pathologies, ranging from cardiovascular disease to oncology. Aim of this review is to discuss both molecular mechanisms associated with stem cell senescence and interventions that may attenuate or reverse this process.
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16
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Roger PP, van Staveren WCG, Coulonval K, Dumont JE, Maenhaut C. Signal transduction in the human thyrocyte and its perversion in thyroid tumors. Mol Cell Endocrinol 2010; 321:3-19. [PMID: 19962425 DOI: 10.1016/j.mce.2009.11.015] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/28/2009] [Revised: 11/23/2009] [Accepted: 11/27/2009] [Indexed: 11/19/2022]
Abstract
The study of normal signal transduction pathways regulating the proliferation and differentiation of a cell type allows to predict and to understand the perversions of these pathways which lead to tumorigenesis. In the case of the human thyroid cell, three cascades are mostly involved in tumorigenesis: The pathways and genetic events affecting them are described. Caveats in the use of models and the interpretation of results are formulated and the still pending questions are outlined.
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Affiliation(s)
- Pierre P Roger
- I.R.I.B.H.M., Université Libre de Bruxelles, Campus Erasme, Route de Lennik 808, B - 1070 Bruxelles, Belgium
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17
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Preto A, Gonçalves J, Rebocho AP, Figueiredo J, Meireles AM, Rocha AS, Vasconcelos HM, Seca H, Seruca R, Soares P, Sobrinho-Simões M. Proliferation and survival molecules implicated in the inhibition of BRAF pathway in thyroid cancer cells harbouring different genetic mutations. BMC Cancer 2009; 9:387. [PMID: 19878585 PMCID: PMC2776025 DOI: 10.1186/1471-2407-9-387] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2008] [Accepted: 10/31/2009] [Indexed: 01/07/2023] Open
Abstract
Background Thyroid carcinomas show a high prevalence of mutations in the oncogene BRAF which are inversely associated with RAS or RET/PTC oncogenic activation. The possibility of using inhibitors on the BRAF pathway as became an interesting therapeutic approach. In thyroid cancer cells the target molecules, implicated on the cellular effects, mediated by inhibition of BRAF are not well established. In order to fill this lack of knowledge we studied the proliferation and survival pathways and associated molecules induced by BRAF inhibition in thyroid carcinoma cell lines harbouring distinct genetic backgrounds. Methods Suppression of BRAF pathway in thyroid cancer cell lines (8505C, TPC1 and C643) was achieved using RNA interference (RNAi) for BRAF and the kinase inhibitor, sorafenib. Proliferation analysis was performed by BrdU incorporation and apoptosis was accessed by TUNEL assay. Levels of protein expression were analysed by western-blot. Results Both BRAF RNAi and sorafenib inhibited proliferation in all the cell lines independently of the genetic background, mostly in cells with BRAFV600E mutation. In BRAFV600E mutated cells inhibition of BRAF pathway lead to a decrease in ERK1/2 phosphorylation and cyclin D1 levels and an increase in p27Kip1. Specific inhibition of BRAF by RNAi in cells with BRAFV600E mutation had no effect on apoptosis. In the case of sorafenib treatment, cells harbouring BRAFV600E mutation showed increase levels of apoptosis due to a balance of the anti-apoptotic proteins Mcl-1 and Bcl-2. Conclusion Our results in thyroid cancer cells, namely those harbouring BRAFV600Emutation showed that BRAF signalling pathway provides important proliferation signals. We have shown that in thyroid cancer cells sorafenib induces apoptosis by affecting Mcl-1 and Bcl-2 in BRAFV600E mutated cells which was independent of BRAF. These results suggest that sorafenib may prove useful in the treatment of thyroid carcinomas, particularly those refractory to conventional treatment and harbouring BRAF mutations.
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Affiliation(s)
- Ana Preto
- Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200-465, Porto, Portugal.
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18
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Leikam C, Hufnagel A, Schartl M, Meierjohann S. Oncogene activation in melanocytes links reactive oxygen to multinucleated phenotype and senescence. Oncogene 2008; 27:7070-82. [PMID: 18806824 DOI: 10.1038/onc.2008.323] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Contrary to malignant melanoma, nevi are a benign form of melanocytic hyperproliferation. They are frequently observed as precursor lesions of melanoma, but they also feature biochemical markers of senescence. In particular, evidence for oncogene-induced melanocyte senescence as natural means to prevent tumorigenesis has been obtained in nevi with mutated B-Raf(V600E). Here, we demonstrate that strong oncogenic growth factor receptor signalling drives melanocytes into senescence, whereas weaker signals keep them in the proliferative state. Activation of oncogene-induced senescence also produces multinucleated giant cells, a long known histological feature of nevus cells. The protein levels of the senescence mediators, p53 and pRB, and their upstream activators do not correlate with senescence. However, strong oncogene signalling leads to pronounced reactive oxygen stress, and scavenging of reactive oxygen species (ROS) efficiently prevents the formation of multinucleated cells and senescence. Similarly, expression of oncogenic N-RAS results in ROS generation, DNA damage and the same multinuclear senescent phenotype. Hence, we identified oncogenic signalling-dependent ROS production as critical mediator of the melanocytic multinuclear phenotype and senescence, both of them being hallmarks of human nevus cells.
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Affiliation(s)
- C Leikam
- Department of Physiological Chemistry I, Biocenter, Am Hubland, University of Wurzburg, Wurzburg, Germany
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19
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Abstract
Cellular senescence is associated with ageing and cancer in vivo and has a proven tumour-suppressive function. Common to both ageing and cancer is the generation of DNA damage and the engagement of the DNA-damage response pathways. In this Review, the diverse mechanisms that lead to DNA-damage generation and the activation of DNA-damage-response signalling pathways are discussed, together with the evidence for their contribution to the establishment and maintenance of cellular senescence in the context of organismal ageing and cancer development.
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20
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Di Micco R, Fumagalli M, d'Adda di Fagagna F. Breaking news: high-speed race ends in arrest--how oncogenes induce senescence. Trends Cell Biol 2007; 17:529-36. [PMID: 17980599 DOI: 10.1016/j.tcb.2007.07.012] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2007] [Revised: 07/27/2007] [Accepted: 07/27/2007] [Indexed: 12/30/2022]
Abstract
Oncogene activation in normal cells induces a permanent proliferative arrest known as cellular senescence. This phenomenon restrains the expansion of cells that bear an activated oncogene and acts as a powerful tumor-suppressive process. Although the full molecular mechanisms are still being elucidated, it has been observed recently that some oncogenes alter the DNA-replication process and cause DNA-damage accumulation. DNA-damage checkpoint-response activation together with the increased appearance of heterochromatin formation that leads to transcriptional silencing of proliferative genes are, presently, the two main mechanisms known that establish and maintain oncogene-induced senescence. Here, we discuss the most recent advancements in understanding the molecular and cellular mechanisms that control cellular senescence caused by oncogene activation and their impact on cancer studies.
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Affiliation(s)
- Raffaella Di Micco
- IFOM Foundation - FIRC Institute of Molecular Oncology Foundation, via Adamello 16, 20139 Milan, Italy
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21
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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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22
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Bond JA, Gescher AJ, Verschoyle RD, Lemoine NR, Errington R, Wiltshire M, Smith PJ, Wynford-Thomas D. Cytotoxic action of phorbol esters on human pancreatic cancer cells. Int J Cancer 2007; 121:1445-54. [PMID: 17582609 DOI: 10.1002/ijc.22869] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
We previously showed that phorbol esters are cytotoxic to human thyroid epithelial cells expressing a mutant RAS oncogene. Here we explore the generality of this finding using cells derived from pancreatic cancer, which, like thyroid, shows a high frequency of RAS mutation, but is a much greater cause of cancer mortality. The response to phorbol myristate acetate (PMA) and related agents was assessed on a panel of 9 pancreatic cancer cell lines, using a range of assays for cell growth and death in vitro and in vivo. In most lines, PMA induced non-apoptotic cell death which was, surprisingly, independent of its "classic" target, protein kinase C. With 24 hr exposure, the IC(50) in the most sensitive line (Aspc-1) was <1 ng/ml (1.6 nM), with survival undetectable at concentrations >/=>/=16 nM, and after only 1 hr exposure the IC(50) was still
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Affiliation(s)
- Jane A Bond
- Department of Pathology, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom
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23
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Abulaiti A, Fikaris AJ, Tsygankova OM, Meinkoth JL. Ras Induces Chromosome Instability and Abrogation of the DNA Damage Response. Cancer Res 2006; 66:10505-12. [PMID: 17079472 DOI: 10.1158/0008-5472.can-06-2351] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Ras mutations are frequent in thyroid tumors, the most common endocrine malignancy. The ability of Ras to transform thyroid cells is thought to rely on its mitogenic activity. Unexpectedly, acute expression of activated Ras in normal rat thyroid cells induced a DNA damage response, followed by apoptosis. Notably, a subpopulation of cells evaded apoptosis and emerged with features of transformation, including the loss of epithelial morphology, dedifferentiation, and the acquisition of hormone- and anchorage-independent proliferation. Strikingly, the surviving cells showed marked chromosomal instability. Acutely, Ras stimulated replication stress as evidenced by the induction of ataxia telangiectasia mutated and Rad3-related protein kinase (ATR) activity (Chk1 phosphorylation) and of gammaH2A.X, a marker of DNA damage. Despite the activation of a checkpoint, cells continued through mitosis in the face of DNA damage, resulting in an increase in cells harboring micronuclei, an indication of defects in chromosome segregation and other forms of chromosome damage. Cells that survived exposure to Ras continued to exhibit replication stress (ATR activation) but no longer exhibited gammaH2A.X or full activation of p53. When rechallenged with Ras or DNA-damaging agents, the surviving cells were more resistant to apoptosis than parental cells. These data show that acute expression of activated Ras is sufficient to induce chromosomal instability in the absence of other signals, and suggest that Ras-induced chromosomal instability arises as a consequence of defects in the processing of DNA damage. Hence, abrogation of the DNA damage response may constitute a novel mechanism for Ras transformation.
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Affiliation(s)
- Adili Abulaiti
- Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6061, USA
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24
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Ball E, Bond J, Franc B, Demicco C, Wynford-Thomas D. An immunohistochemical study of p16(INK4a) expression in multistep thyroid tumourigenesis. Eur J Cancer 2006; 43:194-201. [PMID: 17046239 DOI: 10.1016/j.ejca.2006.08.025] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2006] [Revised: 08/11/2006] [Accepted: 08/14/2006] [Indexed: 11/20/2022]
Abstract
Normal human thyroid follicular epithelial cells exhibit a very low proliferative rate which in vitro is dramatically increased by RAS oncogene activation, resulting in clones displaying a phenotype consistent with that of a ras-induced follicular adenoma in vivo. Eventual spontaneous cessation of growth of these clones is closely correlated with increasing expression of the tumour suppressor gene p16(INK4a), suggesting that p16 may limit clonal expansion in this tumour model. We therefore hypothesised that p16 expression would also increase in vivo in follicular adenomas, and further that escape from growth control in follicular cancers would be accompanied by loss of p16 expression. This was tested using tissue microarrays, representing multiple stages of thyroid tumourigenesis. Whereas the majority of normal thyroids showed no immunostaining, p16 protein was readily detectable in follicular adenomas. Unexpectedly, however, p16 expression was also observed in follicular and papillary carcinomas. Poorly differentiated (insular) carcinomas showed either very intense staining, or a complete loss of staining. We conclude that loss of p16 is not necessary for malignant transformation in thyroid follicular cells, but that it may form one of two or more events needed for progression to more aggressive forms of thyroid cancer.
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Affiliation(s)
- Elizabeth Ball
- Department of Pathology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK
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25
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Fikaris AJ, Lewis AE, Abulaiti A, Tsygankova OM, Meinkoth JL. Ras triggers ataxia-telangiectasia-mutated and Rad-3-related activation and apoptosis through sustained mitogenic signaling. J Biol Chem 2006; 281:34759-67. [PMID: 16968694 DOI: 10.1074/jbc.m606737200] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Genetic evidence indicates that Ras plays a critical role in the initiation and progression of human thyroid tumors. Paradoxically, acute expression of activated Ras in normal rat thyroid cells induced deregulated cell cycle progression and apoptosis. We investigated whether cell cycle progression was required for Ras-stimulated apoptosis. Ras increased CDK-2 activity following its introduction into quiescent cells. Apoptotic cells exhibited a sustained increase in CDK-2 activity, accompanied by the loss of CDK-2-associated p27. Blockade of Ras-induced CDK-2 activity and S phase entry via overexpression of p27 inhibited apoptosis. Inactivation of the retinoblastoma protein in quiescent cells through expression of HPV-E7 stimulated cell cycle progression and apoptosis, indicating that deregulated cell cycle progression is sufficient to induce apoptosis. Ras failed to induce G1 phase growth arrest in normal rat thyroid cells. Rather, Ras-expressing thyroid cells progressed into S and G2 phases and evoked a checkpoint response characterized by the activation of ATR. Ras-stimulated ATR activity, as evidenced by Chk1 and p53 phosphorylation, was blocked by p27, suggesting that cell cycle progression triggers checkpoint activation, likely as a consequence of replication stress. These data reveal that Ras is capable of inducing a DNA damage response with characteristics similar to those reported in precancerous lesions. Our findings also suggest that the frequent mutational activation of Ras in thyroid tumors reflects the ability of Ras-expressing cells to bypass checkpoints and evade apoptosis rather than to simply increase proliferative potential.
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Affiliation(s)
- Aphrothiti J Fikaris
- Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA
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26
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Abstract
Cellular senescence, a permanent cell cycle arrest, is considered a safeguard mechanism that may prevent aged or abnormal cells from further expansion. Although the term "replicative senescence" stands for the widely accepted model of a terminal growth arrest due to telomere attrition, the significance of "oncogene-inducible senescence" remained an issue of debate over the years. A number of recent studies now show the effect of this acute and telomere-independent form of senescence as a tumor-protective, fail-safe mechanism in vivo that shares conceptual and possibly therapeutic similarities with the genetically encoded apoptosis machinery.
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Affiliation(s)
- Melanie Braig
- Department of Hematology/Oncology, Charité-Universitätsmedizin Berlin, Germany
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27
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Affiliation(s)
- Judy L Meinkoth
- Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
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28
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Motti ML, Califano D, Troncone G, De Marco C, Migliaccio I, Palmieri E, Pezzullo L, Palombini L, Fusco A, Viglietto G. Complex regulation of the cyclin-dependent kinase inhibitor p27kip1 in thyroid cancer cells by the PI3K/AKT pathway: regulation of p27kip1 expression and localization. THE AMERICAN JOURNAL OF PATHOLOGY 2005; 166:737-49. [PMID: 15743786 PMCID: PMC1602368 DOI: 10.1016/s0002-9440(10)62295-x] [Citation(s) in RCA: 75] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Accepted: 12/01/2004] [Indexed: 01/03/2023]
Abstract
Functional inactivation of the tumor suppressor p27(kip1) in human cancer occurs either through loss of expression or through phosphorylation-dependent cytoplasmic sequestration. Here we demonstrate that dysregulation of the PI3K/AKT pathway is important in thyroid carcinogenesis and that p27(kip1) is a key target of the growth-regulatory activity exerted by this pathway in thyroid cancer cells. Using specific PI3K inhibitors (LY294002, wortmannin, and PTEN) and a dominant active AKT construct (myrAKT), we demonstrated that the PI3K/AKT pathway controlled thyroid cell proliferation by regulating the expression and subcellular localization of p27. Results obtained with phospho-specific antibodies and with transfection of nonphosphorylable p27(kip1) mutant constructs demonstrated that PI3K/AKT-dependent regulation of p27(kip1) mislocalization in thyroid cancer cells occurred via phosphorylation of p27(kip1) at T157 and T198 (but not at S10 or T187). Finally, we evaluated whether these results were applicable to human tumors. Analysis of 100 thyroid carcinomas indicated that p27(kip1) phosphorylation at T157/T198 and cytoplasmic mislocalization were preferentially associated with activation of the PI3K/AKT pathway. Thus the PI3/AKT pathway and its effector p27(kip1) play major roles in thyroid carcinogenesis.
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Affiliation(s)
- Maria Letizia Motti
- Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano, Università di Napoli Federico II, via S. Pansini 5, 80131 Napoli, Italy
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29
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Sahin M, Allard BL, Yates M, Powell JG, Wang XL, Hay ID, Zhao Y, Goellner JR, Sebo TJ, Grebe SKG, Eberhardt NL, McIver B. PPARgamma staining as a surrogate for PAX8/PPARgamma fusion oncogene expression in follicular neoplasms: clinicopathological correlation and histopathological diagnostic value. J Clin Endocrinol Metab 2005; 90:463-8. [PMID: 15483076 DOI: 10.1210/jc.2004-1203] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
The PAX8/PPARgamma (PPFP) fusion-oncogene is moderately specific for follicular thyroid carcinomas (FTC). It remains unknown whether this can be translated into improved diagnosis, classification, or outcome prediction. We studied a cohort of well-characterized follicular adenomas (FA), FTC, and Hurthle cell carcinomas (HCC) from patients with complete clinical follow-up, to determine whether PPARgamma immunohistochemistry (as a surrogate of PAX8/PPARgamma expression) helps to distinguish FA from FTC and to assess its diagnostic accuracy as an adjunct to frozen section. We also correlated PPARgamma staining with clinical outcomes to assess its role as a prognostic marker.PPARgamma staining was more common in FTC (31 of 54; 57%) than in HCC (one of 23; 4%) or FA (four of 31; 13%) (P < 0.000001). Adjunctive use of PPARgamma immunohistochemistry improved diagnostic sensitivity of intraoperative frozen section from 84% to 96% (P < 0.05) but reduced specificity from 100% to 90% (P < 0.05). PPARgamma staining was associated with favorable prognostic indicators (female gender, better tumor differentiation, and lesser risk of metastases).PPARgamma staining may be helpful in the differential diagnosis of FA, FTC, and HCC, particularly when diagnostic sensitivity of histomorphology is reduced (e.g. during intraoperative frozen section). PPARgamma staining also shows an association with favorable prognosis and may have a role in risk stratification.
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Affiliation(s)
- Mustafa Sahin
- Department of Medicine, Mayo Clinic and Foundation, 200 First Street SW, Rochester, Minnesota 55905, USA
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30
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Shay JW, Wright WE. Senescence and immortalization: role of telomeres and telomerase. Carcinogenesis 2004; 26:867-74. [PMID: 15471900 DOI: 10.1093/carcin/bgh296] [Citation(s) in RCA: 505] [Impact Index Per Article: 24.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Telomere dynamics are a critical component of both aging and cancer. Telomeres progressively shorten in almost all dividing cells and most human cells do not express or maintain sufficient telomerase activity to fully maintain telomeres. There is accumulating evidence that when only a few telomeres are short, they form end-associations, leading to a DNA damage signal resulting in replicative senescence (a cellular growth arrest, also called the M1 stage). In the absence of cell-cycle checkpoint pathways (e.g. p53 and or p16/Rb), cells bypass M1 senescence and telomeres continue to shorten eventually resulting in crisis (also called the M2 stage). M2 is characterized by many 'uncapped' chromosome ends, end-fusions, chromosome breakage fusion-bridge cycles, mitotic catastrophe and a high fraction of apoptotic cells. In a rare M2 cell, telomerase (a cellular reverse transcriptase) can be reactivated or up-regulated, resulting in indefinite cell proliferation. This cellular immortalization is a potentially rate-limiting step in carcinogenesis that is important for the continuing evolution of most advanced cancers. In this perspective we will present our views on the evidence for telomere dysfunction in aging and in cancer progression. We will argue that telomere shortening in the absence of other alterations may be a potent tumor suppressor mechanism and we will discuss the evidence for and against the major molecular mechanisms proposed to initiate replicative senescence.
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Affiliation(s)
- Jerry W Shay
- University of Texas Southwestern Medical Center at Dallas, Department of Cell Biology, 5323 Harry Hines Boulevard, Dallas, TX 75390-9039, USA.
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31
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Kipling D, Davis T, Ostler EL, Faragher RGA. What can progeroid syndromes tell us about human aging? Science 2004; 305:1426-31. [PMID: 15353794 DOI: 10.1126/science.1102587] [Citation(s) in RCA: 127] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
Human genetic diseases that resemble accelerated aging provide useful models for gerontologists. They combine known single-gene mutations with deficits in selected tissues that are reminiscent of changes seen during normal aging. Here, we describe recent progress toward linking molecular and cellular changes with the phenotype seen in two of these disorders. One in particular, Werner syndrome, provides evidence to support the hypothesis that the senescence of somatic cells may be a causal agent of normal aging.
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Affiliation(s)
- David Kipling
- Department of Pathology, School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK
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Brookes S, Rowe J, Gutierrez Del Arroyo A, Bond J, Peters G. Contribution of p16INK4a to replicative senescence of human fibroblasts. Exp Cell Res 2004; 298:549-59. [PMID: 15265701 DOI: 10.1016/j.yexcr.2004.04.035] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2004] [Revised: 04/23/2004] [Indexed: 11/29/2022]
Abstract
In standard conditions of tissue culture, human fibroblasts undergo a limited number of population doublings before entering a state of irreversible growth arrest termed replicative senescence or M1. The arrest is triggered by a combination of telomere dysfunction and the stresses inflicted by culture conditions and is implemented, at least in part, by the cyclin-dependent kinase inhibitors p21(CIP1) and p16(INK4a). To investigate the role of p16(INK4a), we have studied fibroblasts from members of melanoma prone kindreds with mutations in one or both copies of the CDKN2A locus. The mutations affect the function of p16(INK4a) but not of the alternative product, p14(ARF). The p16(INK4a)-defective fibroblasts have an above average life span, compared to the heterozygous and normal age-matched controls, but they arrest with characteristics typical of senescence. Using agents that are known to bypass M1, such as DNA tumor virus oncoproteins or the Bmi1 transcriptional repressor, we provide evidence that p16(INK4a) defective cells arrest at a stage that is operationally between M1 and M2 (crisis). As well as indicating that p16(INK4a) contributes to but is not essential for replicative senescence of human fibroblasts, our data reveal considerable heterogeneity in the levels and accumulation of p16(INK4a) in different strains.
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Affiliation(s)
- Sharon Brookes
- Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, London WC2A 3PX, UK
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Smith SK, Kipling D. The role of replicative senescence in cancer and human ageing: utility (or otherwise) of murine models. Cytogenet Genome Res 2004; 105:455-63. [PMID: 15237234 DOI: 10.1159/000078219] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2004] [Accepted: 12/04/2003] [Indexed: 11/19/2022] Open
Abstract
Replicative senescence has the potential both to act as an anti-tumour mechanism, and to contribute to age-related changes in tissue function. Studies on human cells have revealed much, both about the nature of cell division counters, some of which utilize the gradual erosion of chromosomal telomeres, and the downstream signalling pathways that initiate and maintain growth arrest in senescence. A powerful test of the hypothesis that senescence is linked to either ageing or tumour prevention now requires a suitable animal model system. Here we overview the current understanding of replicative senescence in human cells, and address to what extent the senescence of murine cells in culture mirrors this phenomenon. We also discuss whether examples of telomere-independent senescence, such as those seen in mouse embryonic fibroblasts (MEFs) and several human cells types, should be viewed not as a consequence of "inadequate growth conditions", but rather as a powerful potential model system to dissect the selective pressures that occur in the early stages of tumour development, ones that we speculate lead to the observed high frequency of abrogation of p16INK4a function in human cancer.
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Affiliation(s)
- S K Smith
- Department of Pathology, University of Wales College of Medicine, Cardiff, UK
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Skinner J, Bounacer A, Bond JA, Haughton MF, deMicco C, Wynford-Thomas D. Opposing effects of mutant ras oncoprotein on human fibroblast and epithelial cell proliferation: implications for models of human tumorigenesis. Oncogene 2004; 23:5994-9. [PMID: 15208687 DOI: 10.1038/sj.onc.1207798] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
Using microinjection of recombinant protein to directly control 'expression' levels, we have compared the proliferative response to ras oncogene activation in two normal cell types--fibroblast and thyroid epithelial cell--which give rise to human tumours with very low and high frequencies of ras mutation respectively. A concentration-dependent stimulation of DNA synthesis was observed in thyrocytes, matched by an almost perfectly reciprocal inhibition in fibroblasts. A concentration-dependent induction of the cyclin-dependent kinase (CDK) inhibitor p21WAF1 was observed in both cell types, but p16Ink4a was induced by ras only in fibroblasts. This difference could not account for the fibroblast specificity of the growth-inhibitory response, however, since proliferation of p16-deficient fibroblasts was also inhibited by mutant ras. We conclude that the striking contrast in proliferative response to ras between fibroblasts and thyroid epithelial cells cannot readily be explained by differential induction of either of the two key CDK inhibitors, p16Ink4a and p21WAF1, but is consistent with a differential ability of p21WAF1 to antagonize ras-induced mitogenic signals in the two cell types. Such tissue-specific differences provide an attractive explanation for the observed specificity of ras mutation for particular human tumour types, and emphasize the inappropriateness of fibroblasts as a model for ras-induced tumorigenesis.
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Affiliation(s)
- Julia Skinner
- Cancer Research UK Laboratories, Department of Pathology, University of Wales College of Medicine, Cardiff CF14 4XN, UK
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Munro J, Barr NI, Ireland H, Morrison V, Parkinson EK. Histone deacetylase inhibitors induce a senescence-like state in human cells by a p16-dependent mechanism that is independent of a mitotic clock. Exp Cell Res 2004; 295:525-38. [PMID: 15093749 DOI: 10.1016/j.yexcr.2004.01.017] [Citation(s) in RCA: 93] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2003] [Indexed: 12/11/2022]
Abstract
We show here that histone deacetylase inhibitors (HDACIs) sodium dibutyrate (SDB) and trichostatin A (TSA) induce a phenotype that has similarities to replicative senescence in human fibroblasts. There was no evidence that SDB accelerated a constitutive cell division counting mechanism as previously suggested because cells pretreated with SDB for three mean population doublings (MPDs) exhibited a similar overall proliferative life span to controls once SDB was withdrawn. SDB-treated cells upregulated the cell cycle inhibitors p21(WAF1) and p16(INK4A), but not p14(ARF), in the same sequential order as in senescence and the cells developed biochemical markers of senescence. However, the mechanism of senescence did not involve telomere dysfunction and there was no evidence for any posttranslational modification of p53. The expression of human papillomavirus (HPV) 16 E6 in human fibroblasts or targeted disruption of the p53 and p21(WAF) genes only weakly antagonized HDACI-induced senescence. However, expression of the E7 gene, which inhibits the function of pRb, cooperated with E6 to block SDB-induced senescence completely and human cells deficient in p16(INK4A) (but not p14(ARF)) were also resistant to SDB-induced senescence, suggesting that the p16(INK4A)/pRb pathway is the major mediator of HDACI-induced senescence in human cells. However, p53-/- mouse fibroblasts were resistant to HDACI-induced senescence, identifying p53 as the major pathway to senescence in this species.
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Affiliation(s)
- June Munro
- Cancer Research UK Beatson Laboratories, Beatson Institute for Cancer Research, Bearsden, Glasgow, G61 1BD UK
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36
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Abstract
Oncogenic stimuli are thought to induce senescence in normal cells in order to protect against transformation and to induce proliferation in cells with altered p53 and/or retinoblastoma (Rb) pathways. In human fibroblasts, RAS initiates senescence through upregulation of the cyclin-dependent kinase inhibitor p16INK4A. We show here that in contrast to cultured fibroblast strains, freshly isolated normal fibroblasts are resistant to RAS-induced senescence and instead show some characteristics of transformation. RAS did not induce growth arrest or expression of senescence-associated beta-galactosidase, and Rb remained hyperphosphorylated despite elevated levels of p16. Instead, RAS promoted anchorage-independent growth of normal fibroblasts, although expression of hTert with RAS increased colony formation and allowed normal fibroblasts to bypass contact inhibition. To test the hypothesis that p16 levels determine how cells respond to RAS, we expressed RAS in freshly isolated fibroblasts that expressed very low levels of p16, in hTert-immortalized fibroblasts that had accumulated intermediate levels of p16, and in IMR90 fibroblasts with high levels of p16. RAS induced growth arrest in cells with higher p16 levels, and this effect was reversed by p16 knockdown in the hTert-immortalized fibroblasts. These findings indicate that culture-imposed stress sensitizes cells to RAS-induced arrest, whereas early passage cells do not arrest in response to RAS.
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Affiliation(s)
- Jennifer A Benanti
- Program in Cancer Biology, Fred Hutchinson Cancer Research Center. Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, Washington 98109-1024, USA
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Gregory Powell J, Wang X, Allard BL, Sahin M, Wang XL, Hay ID, Hiddinga HJ, Deshpande SS, Kroll TG, Grebe SKG, Eberhardt NL, McIver B. The PAX8/PPARγ fusion oncoprotein transforms immortalized human thyrocytes through a mechanism probably involving wild-type PPARγ inhibition. Oncogene 2004; 23:3634-41. [PMID: 15077183 DOI: 10.1038/sj.onc.1207399] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Follicular thyroid carcinoma (FTC) frequently harbors the PAX8/PPARgamma fusion gene (PPFP); however, its oncogenic role and mechanism(s) of action remain undefined. We investigated PPFP's effects on cell growth, apoptosis, cell-cell, and cell-matrix interactions in immortalized human thyroid cells (Nthy-ori 3-1) and NIH 3T3 cells. PPFP expression increased the growth of transient and stable Nthy-ori transfectants ( approximately threefold by 72 h). There was an 8.4% increase of cells in the S+G2/M phase, a 7.8% decrease in cells in the G0+G1 phase and a 66% decline in apoptosis at 72 h. Stable Nthy-ori PPFP transfectants grew in soft agar, and PPFP-transfected NIH 3T3 cells exhibited efficient focus formation, suggesting loss of anchorage-dependent growth and contact inhibition, respectively. Overexpression of PPARgamma in Nthy-ori cells did not recapitulate PPFP's growth effects. Treatment of Nthy-ori cells with an irreversible PPARgamma inhibitor mimicked the growth-promoting effects of PPFP and co-expression of PPFP and PPARgamma blocked PPARgamma transactivation activity. Our data provide functional evidence that PPFP acts as an oncoprotein, whose transforming properties depend in part on inhibition of PPARgamma. Our data suggest that PPFP contributes to malignant transformation during FTC oncogenesis by acting on several cellular pathways, at least some of which are normally regulated by PPARgamma.
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Affiliation(s)
- J Gregory Powell
- Department of Medicine, Division of Endocrinology, Mayo Clinic, Rochester, MN 55906, USA
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Bond J, Jones C, Haughton M, DeMicco C, Kipling D, Wynford-Thomas D. Direct evidence from siRNA-directed "knock down" that p16(INK4a) is required for human fibroblast senescence and for limiting ras-induced epithelial cell proliferation. Exp Cell Res 2004; 292:151-6. [PMID: 14720514 DOI: 10.1016/j.yexcr.2003.09.021] [Citation(s) in RCA: 37] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
The selective pressure for disruption of the cyclin-dependent kinase inhibitor p16(INK4a) in human cancer has been postulated to reflect its role in mediating growth arrest, both in response to telomere erosion (replicative senescence) and to oncogene-induced and other "stress" signals. Given the known species-specific differences in regulation of senescence, we have tested this hypothesis in human, as opposed to rodent, cells by designing a small interfering RNA (siRNA) to knock down p16(INK4a) expression. Transfection of this siRNA into late-passage normal human diploid fibroblasts allowed at least temporary escape from entry into replicative senescence. Furthermore, in our in vitro model of early-stage, RAS-induced thyroid tumorigenesis, sequential transfections with this siRNA allowed outgrowth of small clusters of proliferating epithelial cells, consistent with escape from the spontaneous "senescence", which normally curtails their proliferative response to mutant RAS. These data provide the first direct evidence that p16(INK4a) is necessary for the initiation of both telomere-dependent and telomere-independent senescence in human cells.
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Affiliation(s)
- Jane Bond
- Department of Pathology, University of Wales College of Medicine, Cardiff CF14 4XN, UK
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Pellegrini G, Dellambra E, Paterna P, Golisano O, Traverso CE, Rama P, Lacal P, De Luca M. Telomerase activity is sufficient to bypass replicative senescence in human limbal and conjunctival but not corneal keratinocytes. Eur J Cell Biol 2004; 83:691-700. [PMID: 15679113 DOI: 10.1078/0171-9335-00424] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The human ocular surface is covered by the conjunctival, corneal and limbal stratified epithelia. While conjunctival stem cells are distributed in bulbar and forniceal conjunctiva, corneal stem cells are segregated in the basal layer of the limbus, which is the transitional zone between the cornea and the bulbar conjunctiva. Keratinocyte stem and transient amplifying (TA) cells when isolated in culture give rise to holoclones and paraclones, respectively. Keratinocyte replicative senescence ensues when all holoclones have generated paraclones which express high levels of p16(INK4a). In the present study, we show that enforced telomerase activity induces the bypass of replicative senescence in limbal and conjunctival keratinocytes, without the inactivation of the p16(INK4a)/Rb pathway or the abrogation of p53 expression. hTERT-transduced limbal and conjunctival keratinocytes are capable to respond to both growth inhibitory and differentiation stimuli, since they undergo growth arrest in response to phorbol esters, and activate p53 upon DNA damage. Following a sustained PKC stimulation, occasional clones of p16(INK4a)-negative cells emerge and resume ability to proliferate. Telomerase activity, however, is unable to induce the bypass of senescence in corneal TA keratinocytes cultured under the same conditions. These data support the notion that telomere-dependent replicative senescence is a general property of all human somatic cells, including keratinocytes, and suggest that telomerase activity is sufficient to extend the lifespan only of keratinocytes endowed with high proliferative potentials (which include stem cells), but not of TA keratinocytes.
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Affiliation(s)
- Graziella Pellegrini
- Epithelial Stem Cell Research Center, The Veneto Eye Bank Foundation, Hospital SS Giovanni and Paolo, Venice, Italy
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Abstract
Thyrotropin (TSH) is considered the main regulator of thyrocyte differentiation and proliferation. Thus, the characterization of the different signaling pathways triggered by TSH on these cells is of major interest in order to understand the mechanisms implicated in thyroid pathology. In this review we focus on the different signaling pathways involved in TSH-mediated proliferation and their role in thyroid transformation and tumorigenesis. TSH mitogenic activities are mediated largely by cAMP, which in turn may activate protein kinase (PKA)-dependent and independent processes. We analyze the effects of increased cAMP levels and PKA activity during cell cycle progression and the role of this signaling pathway in thyroid tumor initiation. Alternative pathways to PKA in the cAMP-mediated proliferation appear to involve the small GTPases Rap1 and Ras. We analyze the Ras effectors (PI3K, RalGDS and Raf) that are thought to mediate its oncogenic activity, as well as the ability of Ras to induce apoptosis in thyrocytes. Finally, we discuss the activation of the PLC/PKC cascade by TSH in thyroid cells and the role of this signaling pathway in the TSH-mediated proliferation and tumorigenesis.
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Affiliation(s)
- Marcos Rivas
- Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Arturo Duperier # 4, E-28029 Madrid, Spain
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41
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Portella G, Laezza C, Laccetti P, De Petrocellis L, Di Marzo V, Bifulco M. Inhibitory effects of cannabinoid CB1 receptor stimulation on tumor growth and metastatic spreading: actions on signals involved in angiogenesis and metastasis. FASEB J 2003; 17:1771-3. [PMID: 12958205 DOI: 10.1096/fj.02-1129fje] [Citation(s) in RCA: 134] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Abstract
Stimulation of cannabinoid CB1 receptors by 2-methyl-arachidonyl-2'-fluoro-ethylamide (Met-F-AEA) inhibits the growth of a rat thyroid cancer cell-derived tumor in athymic mice by inhibiting the activity of the oncogene product p21ras. Here we report that Met-F-AEA also blocks the growth of tumors previously induced in nude mice by the s.c. injection of the same rat thyroid carcinoma cells. Met-F-AEA significantly inhibited, in tumors as well as transformed cells, the expression of the vascular endothelial growth factor, an angiogenetic factor known to be up-regulated by p21ras, as well as of one of its receptors, flt-1/VEGFR-1. The levels of the cyclin-dependent kinase inhibitor p27(kip1), which is down-regulated by p21ras, were instead increased by Met-F-AEA. All these effects were antagonized by the selective CB1 receptor antagonist SR141716A. Met-F-AEA inhibited in vitro the growth of a metastasis-derived thyroid cancer cell line more potently than a primary cancer cell line. Therefore, the hypothesis that CB1 receptor stimulation interferes not only with angiogenesis but also with metastatic processes was tested in a widely used model of metastatic infiltration in vivo, the Lewis lung carcinoma (3LL) in C57Bl/6 mice. Three weeks from the paw injection of 3LL cells, Met-F-AEA reduced significantly the number of metastatic nodes, in a way antagonized by SR141716A. Our findings indicate that CB1 receptor agonists might be used therapeutically to retard tumor growth in vivo by inhibiting at once tumor growth, angiogenesis, and metastasis.
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Affiliation(s)
- Giuseppe Portella
- Endocannabinoid Research Group, Istituto di Endocrinologia ed Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, and Dipartimento di Biologia e Patologia Cellulare e Molecolare L. Califano, Università di Napoli Federico II, Italy
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Cheng G, Lewis AE, Meinkoth JL. Ras stimulates aberrant cell cycle progression and apoptosis in rat thyroid cells. Mol Endocrinol 2003; 17:450-9. [PMID: 12554771 DOI: 10.1210/me.2002-0344] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Abundant evidence supports the ability of Ras to stimulate thyroid cell proliferation. Stable expression of activated Ras enhances the sensitivity of thyroid cells to apoptosis. We report that apoptosis is a primary and general response of rat thyroid cells to acute expression of activated Ras in the absence or presence of thyrotropin, insulin, and serum, survival factors for thyroid cells. Ras induced apoptosis in quiescent and cycling cells. Concomitantly, Ras stimulated S phase entry in quiescent cells and enhanced G1/S transition in cycling cells. Ras effects on the cell cycle were characterized by delayed progression through S phase and an apparent failure to proceed through G2/M phase. Unlike thyroid cell mitogens, Ras markedly decreased cyclin D1 expression. Although acute expression of Ras decreased cyclin D1 protein levels, cells selected to survive chronic Ras expression exhibited a selective increase in cyclin D1 expression. In summary, thyroid cells harbor an apoptotic program activated by Ras that outstrips the protective effects of thyrotropin, insulin, and serum. Apoptosis is accompanied by dysregulated cell cycle progression, suggesting that cell death may arise, at least in part, as a consequence of inappropriate proliferative cues.
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Affiliation(s)
- Guanjun Cheng
- Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084, USA
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Ramirez RD, Herbert BS, Vaughan MB, Zou Y, Gandia K, Morales CP, Wright WE, Shay JW. Bypass of telomere-dependent replicative senescence (M1) upon overexpression of Cdk4 in normal human epithelial cells. Oncogene 2003; 22:433-44. [PMID: 12545164 DOI: 10.1038/sj.onc.1206046] [Citation(s) in RCA: 73] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Many stimuli causing 'stress' or DNA damage in cells can produce phenotypes that overlap with telomere-based replicative senescence. In epithelial systems, the p16/RB pathway may function as a stress senescence-signaling pathway independent of telomere shortening. Overexpressing cyclin-dependent kinase 4 (Cdk4) in human epidermal keratinocytes and human mammary epithelial cells not only prevents the p16(INK4a)-associated premature growth arrest due to telomere-independent stress (e.g., inadequate culture conditions), but also bypasses the ensuing telomere-dependent senescence (M1). Overexpressed Cdk4 in epithelial cells induces a dramatic upregulation of p16(INK4a) and milder upregulation of p53 and p21(WAF1), which become unresponsive to UV irradiation. Despite the high levels of these checkpoint factors, Cdk4-overexpressing cells divide in an apparently normal regulated fashion, are able to respond to changes in calcium levels, retain the stem cell phenotype, and fully differentiate and stratify. These results suggest that the differentiation pathways in Cdk4-overexpressing cells remain intact.
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Affiliation(s)
- Ruben D Ramirez
- Hamon Center for Therapeutic Oncology Research, The University of Texas Southwestern Medical Center, Dallas, TX 75390-8593, USA.
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Yawata T, Kamino H, Kugoh H, Katoh M, Nomura N, Oishi M, Horikawa I, Barrett JC, Oshimura M. Identification of a </= 600-kb region on human chromosome 1q42.3 inducing cellular senescence. Oncogene 2003; 22:281-90. [PMID: 12527897 DOI: 10.1038/sj.onc.1206143] [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: 11/09/2022]
Abstract
The introduction of a human chromosome 1 via microcell-mediated chromosome transfer (MMCT) induces the cellular senescence in mouse melanoma B16-F10 cells. The senescent cells maintained still the telomerase activity, which is frequently associated with immortal growth of human cells, suggesting that a telomerase-independent mechanism is involved in the senescence observed in this mouse cell line. To map the senescence-inducing gene to a specific chromosomal region, we took two experimental approaches: identification of a minimal region with the senescence-inducing activity via MMCT of a series of subchromosomal transferrable fragments (STFs), each consisting of a different profile of human chromosome 1-derived regions, and identification of a region commonly deleted from the transferred chromosome 1 in the revertant clones that escaped cellular senescence. These approaches identified a 2.7-3.0 Mb of senescence-inducing region shared among the active STFs and a 2.4-3.0 Mb of commonly deleted region in the revertant clones. These two regions overlapped each other to map the responsible gene at the 450 to 600-kb interval between UniSTS93710 and D1S3542 on chromosome 1q42.3. This study provides essential information and materials for cloning and characterization of a novel senescence-inducing gene that functions in a telomerase-independent pathway, which is likely to be conserved between mice and humans.
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Affiliation(s)
- Toshio Yawata
- Department of Molecular and Cell Genetics, Faculty of Medicine, School of Life Sciences, Tottori University, Japan
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Shen ZY, Xu LY, Chen MH, Shen J, Cai WJ, Zeng Y. Progressive transformation of immortalized esophageal epithelial cells. World J Gastroenterol 2002; 8:976-81. [PMID: 12439909 PMCID: PMC4656402 DOI: 10.3748/wjg.v8.i6.976] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the progressive transformation of immortal cells of human fetal esophageal epithelium induced by human papillomavirus, and to examine biological criteria of sequential passage of cells, including cellular phenotype, proliferative rate, telomerase, chromosome and tumorigenicity.
METHODS: The SHEE cell series consisted of immortalized embryonic esophageal epithelium which was in malignant transformation when cultivated over sixty passages without co-carcinogens. Cells of the 10th, 31st, 60th and 85th passages were present in progressive development after being transfected with HPV. Cells were cultivated in a culture flask and 24-hole cultural plates. Progressive changes of morphology, cell growth, contact-inhibition, and anchorage-dependent growth characteristics were examined by phase contrast microscopy. The cell proliferation rate was assayed by flow cytometry. The modal number of chromosomes was analyzed. HPV18E6E7 was detected by Western blot methods and activities of telomerase were analyzed by TRAP. Tumorigenicity of cells was detected with soft agar plates cultivated and with tumor formation in SCID mice.
RESULTS: In morphological examination the 10th passage cells were in good differentiation, the 60th and 85th passages cells were in relatively poor differentiation, and the 31st passage cells had two distinct differentiations. The characteristics of the 85th and 60th passage cells were weakened at contact-inhibition and anchorage-dependent growth. Karyotypes of four stages of cells belonged to hyperdiploid or hypotriploid, and bimodal distribution of chromosomes appeared in the 31st and 60th passage cells. All of these characteristics combined with a increasing trend. The activities of telomerase were expressed in the latter three passages. Four fourths of SCID mice in the 85th passage cells and one fourth of SCID mice in the 60th passage cells developed tumors, but the cells in the 10th and 31st passage displayed no tumor formation.
CONCLUSION: In continual cultivation of fetal esophageal epithelial cells with transduction of HPV18E6E7, cells from the 10th to the 85th passage were changed gradually from preimmortal, immortal, precancerous to malignantly transformed stages. All of these changes were in a dynamic progressive process. The establishment of a continuous line of esophageal epithelium may provide a in vitro model of carcinogenesis induced by HPV.
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Affiliation(s)
- Zhong-Ying Shen
- Department of Tumor Pathology, Medical College of Shantou University, Guandong Province, China.
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46
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Ferbeyre G. PML a target of translocations in APL is a regulator of cellular senescence. Leukemia 2002; 16:1918-26. [PMID: 12357343 DOI: 10.1038/sj.leu.2402722] [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] [Received: 03/15/2002] [Accepted: 06/07/2002] [Indexed: 02/07/2023]
Abstract
PML is the most frequent fusion partner of the RARalpha in the specific translocations associated with acute promyelocytic leukemia (APL). Models to explain the origin of this leukemia propose a block in cell differentiation due to aberrant repression of retinoic acid responsive genes and/or disruption of the function of the PML-containing nuclear bodies. Recently, PML has been identified as a regulator of replicative senescence and the premature senescence that occurs in response to oncogenic ras. This review discusses the idea that senescence is a general tumor suppressor mechanism related to terminal differentiation and disrupted during the establishment of APL and other cancers. According to this idea the PML-RARalpha fusion protein promotes leukemogenesis not only through repression of retinoic acid responsive genes, but also by way of interfering with several tumor suppressor proteins that cooperate to establish senescence. Retinoids and other drugs effective against APL do so by re-establishment of the senescence program, which also includes features of cell differentiation.
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Affiliation(s)
- G Ferbeyre
- Université de Montréal, Département de Biochimie, Canada
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Vécsey-Semjén B, Becker KF, Sinski A, Blennow E, Vietor I, Zatloukal K, Beug H, Wagner E, Huber LA. Novel colon cancer cell lines leading to better understanding of the diversity of respective primary cancers. Oncogene 2002; 21:4646-62. [PMID: 12096341 DOI: 10.1038/sj.onc.1205577] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2001] [Revised: 03/05/2002] [Accepted: 04/15/2002] [Indexed: 11/09/2022]
Abstract
A major obstacle to obtaining more detailed insights into the diversity of phenotypic and molecular changes occurring in colon cancer cells is the lack of low-passage colon cancer cell lines, which would still closely reflect the phenotype of the colon cancer cells in vivo. Here, we characterize eight novel, low passage number human colon carcinoma cell lines, originating from colorectal cancers extensively characterized in the clinics. All cell lines closely resemble the original tumors with respect to phenotype, markers and detectable genetic changes. Cell morphology and marker expression is highly variable, ranging from fully polarized cells correctly expressing all basolateral epithelial markers, to cells with mesenchymal characteristics and a complete loss of polarity due to delocalization or loss of junction complex proteins. The alterations in phenotype and epithelial marker expression correspond to changes already detectable in the primary tumor in vivo. Seven of the cell lines show chromosomal instability, while one cell line is characterized by microsatellite instability. p53 associated with K-ras mutations were detected in three cell lines. Hitherto non-described E-cadherin mutations were found at both alleles in one cell line whereas in another cell line the E-cadherin protein was down-regulated. A stabilizing beta-catenin mutation (S45F) appears in the same cell line that carried the mutated E-cadherin gene. Six cell lines carried APC mutations, which in five of the lines led to an activated beta-catenin/Tcf/LEF signaling pathway. In accordance with beta-catenin/Tcf/LEF activation, the cell lines show increased migration and invasiveness. Our results show that the characterized, low-passage cell lines mirror the diversity of the individual tumors from which they were derived. Through molecular analyses of these cell lines we demonstrate that tumorgenicity events are much more diverse in human colon cancer than expected, despite the common origin of the tumors from a small patient group with similar tumor grading and clinical prognosis.
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Morris M, Hepburn P, Wynford-Thomas D. Sequential extension of proliferative lifespan in human fibroblasts induced by over-expression of CDK4 or 6 and loss of p53 function. Oncogene 2002; 21:4277-88. [PMID: 12082615 DOI: 10.1038/sj.onc.1205492] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2001] [Revised: 03/13/2002] [Accepted: 03/18/2002] [Indexed: 11/09/2022]
Abstract
Replicative senescence is thought to be a significant barrier to human tumorigenesis, which in human fibroblasts, and many other cell types, can be overcome experimentally by combined loss of function of p53 and Rb 'pathways'. To avoid the confounding pleiotropic effects of HPVE7 frequently used in such studies, here we have employed retroviral vectors over-expressing CDK4 or CDK6 as a more representative model of naturally-occurring mutations targeting the Rb pathway. We show that these can extend fibroblast lifespan by approximately 10 population doublings, ending in a viable senescence-like state which contrasts with the apoptotic end-stage seen with E7. Compared with 'normal' senescence, this growth arrest was, in most cases, not accompanied by any further increase in p21(Waf1) levels but with up to a 19-fold increase in p16(Ink4a). Surprisingly however, this could not explain arrest, since expression of mutant CDK4 and/or CDK6, incapable of binding p16(Ink4a), did not confer any greater lifespan extension than the wild-type CDKs. Subsequent abrogation of p53 function by a second vector, encoding HPVE6, downregulated p21(Waf1) and conferred a second lifespan extension, ending in a crisis-like state, consistent with full escape from senescence. These data: (i) point to a back-up 'senescence' mechanism distinct from induction of p21(Waf1) or p16(Ink4a); and (ii) provide an in vitro model of clonal evolution through successive dysfunction of Rb and p53 pathways in a relevant human cell context.
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Affiliation(s)
- Mark Morris
- Cancer Research Campaign Laboratories, Department of Pathology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK
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Fagin JA. Minireview: branded from the start-distinct oncogenic initiating events may determine tumor fate in the thyroid. Mol Endocrinol 2002; 16:903-11. [PMID: 11981026 DOI: 10.1210/mend.16.5.0838] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022] Open
Abstract
Thyroid follicular neoplasms commonly have aneuploidy, presumably due to chromosomal instability. This property is associated with a greater malignant potential and worse prognosis. Recently, there has been considerable progress in our understanding of mechanisms that may account for chromosomal instability in cancer cells. Many tumors with chromosomal instability have abnormalities in the cell cycle checkpoint that monitors the fidelity of mitosis. Mutations of Bub1 or BubR1, genes coding for kinases involved in mitotic spindle assembly checkpoint signaling, are found in a small subset of aneuploid tumors. Other components of protein complexes responsible for attachment of kinetochores to microtubules, or for cohesion between sister chromatids, may also be subject to alterations during tumor progression. Here, we also discuss the evidence that certain oncogenic events, such as Ras mutations, may predispose cells to chromosomal instability by favoring inappropriate posttranslational changes in mitotic checkpoint components through activation of upstream kinases during tumor initiation or progression.
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Affiliation(s)
- James A Fagin
- Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0547, USA.
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Shen ZY, Xu LY, Li EM, Cai WJ, Chen MH, Shen J, Zeng Y. Telomere and telomerase in the initial stage of immortalization of esophageal epithelial cell. World J Gastroenterol 2002; 8:357-62. [PMID: 11925625 PMCID: PMC4658384 DOI: 10.3748/wjg.v8.i2.357] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To search for the biomarker of cellular immortalization, the telomere length, telomerase activity and its subunits in cultured epithelial cells of human fetal esophagus in the process of immortalization.
METHODS: The transgenic cell line of human fetal esophageal epithelium (SHEE) was established with E6E7 genes of human papillomavirus (HPV) type 18 in our laboratory. Morphological phenotype of cultured SHEE cells from the 6th to 30th passages, was examined by phase contrast microscopy, the telomere length was assayed by Southern blot method, and the activity of telomerase was analyzed by telomeric repeat amplification protocol (TRAP). Expressions of subunits of telomerase, hTR and hTERT, were assessed by RT-PCR. DNA content in cell cycle was detected by flow cytometry. The cell apoptosis was examined by electron microscopy (EM) and TUNEL label.
RESULTS: SHEE cells from the 6th to 10th passages showed cellular proliferation with a good differentiation. From the 12th to the 16th passages, many senescent and apoptotic cells appeared, and the telomere length sharply shortened from 23 kb to 17 kb without expression of hTERT and telomerase activity. At the 20th passage, SHEE cells overcame the senescence and apoptosis and restored their proliferative activity with expression of telomerase and hTERT at low levels, but the telomere length shortened continuously to the lowest of 3 kb. After the 30th passage cells proliferation was restored by increment of cells at S and G2M phase in the cell cycle and telomerase activity expressed at high levels and with maintenance of telomere length.
CONCLUSION: At the early stage of SHEE cells, telomeres are shortened without expression of telomerase and hTERT causing cellular senescence and cell death. From the 20th to the 30th passages, the activation of telomerase and maintenance of telomere length show a progressive process for immortalization of esophageal epithelial cells. The expression of telomerase may constitute a biomarker for detection of immortalization of cells.
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Affiliation(s)
- Zhong-Ying Shen
- Department of Tumor Pathology, Medical College of Shantou University, 22 Xinling Road, Shantou 515031, Guandong Province, China.
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