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Kwon MJ, Park HY, Lee JS, Kim ES, Kim NY, Nam ES, Cho SJ, Kang HS. Dysregulated microRNA Expression Relevant to TERT Promoter Mutations in Tonsil Cancer-A Pilot Study. Life (Basel) 2023; 13:2090. [PMID: 37895471 PMCID: PMC10608590 DOI: 10.3390/life13102090] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2023] [Revised: 10/10/2023] [Accepted: 10/19/2023] [Indexed: 10/29/2023] Open
Abstract
Tonsillar squamous cell carcinomas (TSCCs) exhibit high rates of human papillomavirus (HPV) positivity. The expression profiles of microRNA (miRNA), which are small RNA molecules that play pivotal roles in biological processes, in TSCC in relation to the HPV status and cancer-related genetic mutations are not well investigated. Herein, we expanded our previous research, which was focused on established clinicopathological and genetic mutational data, to profile miRNA expression in TSCC, aiming to identify clinically relevant targets for early diagnosis and therapeutic intervention. The miRNA profiles were analyzed using the nCounter Nanostring miRNA Expression assay in 22 surgically resected TSCC tissues and their contralateral normal tonsil tissues. The TERT promoter (TERTp) gene was the only relevant candidate gene associated with differentially expressed miRNAs in TSCC. Hierarchical clustering analysis revealed high expression levels of hsa-miR-1285-5p, hsa-miR-1203, hsa-miR-663a, hsa-miR-1303, hsa-miR-33a-5p, and hsa-miR-3615 coupled with low expression levels of hsa-miR-3182, hsa-miR-219a-2-3p, and hsa-miR-767-3p, which were associated with HPV-positive TSCC (p = 0.009). Functional enrichment analysis revealed that these dysregulated miRNAs tended to be involved in protein binding (molecular function) and cellular components (biological processes). Therefore, hsa-miR-1285-5p and hsa-miR-663a may be associated with HPV-positive TERTp-mutated tumors and may serve as potential treatment targets and biomarkers for early detection.
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Affiliation(s)
- Mi Jung Kwon
- Department of Pathology, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang 14068, Republic of Korea;
| | - Ha Young Park
- Department of Pathology, Busan Paik Hospital, Inje University College of Medicine, Busan 47392, Republic of Korea;
| | - Joong Seob Lee
- Department of Otorhinolaryngology-Head & Neck Surgery, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang 14068, Republic of Korea;
| | - Eun Soo Kim
- Department of Radiology, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang 14068, Republic of Korea;
| | - Nan Young Kim
- Hallym Institute of Translational Genomics and Bioinformatics, Hallym University Medical Center, Anyang 14068, Republic of Korea;
| | - Eun Sook Nam
- Department of Pathology, Kangdong Sacred Heart Hospital, Hallym University College of Medicine, Seoul 05355, Republic of Korea; (E.S.N.); (S.J.C.)
| | - Seong Jin Cho
- Department of Pathology, Kangdong Sacred Heart Hospital, Hallym University College of Medicine, Seoul 05355, Republic of Korea; (E.S.N.); (S.J.C.)
| | - Ho Suk Kang
- Department of Internal Medicine, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang 14068, Republic of Korea
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Li Y, Wang C, Ma A, Rani AQ, Luo M, Li J, Liu X, Ma Q. Identification of HPV oncogene and host cell differentiation associated cellular heterogeneity in cervical cancer via single-cell transcriptomic analysis. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.08.10.552878. [PMID: 37645794 PMCID: PMC10462038 DOI: 10.1101/2023.08.10.552878] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/31/2023]
Abstract
Human Papillomaviruses (HPVs) are associated with around 5-10% of human cancer, notably nearly 99% of cervical cancer. The mechanisms HPV interacts with stratified epithelium (differentiated layers) during the viral life cycle, and oncogenesis remain unclear. In this study, we used single-cell transcriptome analysis to study viral gene and host cell differentiation-associated heterogeneity of HPV-positive cervical cancer tissue. We examined the HPV16 genes - E1, E6, and E7, and found they expressed differently across nine epithelial clusters. We found that three epithelial clusters had the highest proportion of HPV-positive cells (33.6%, 37.5%, and 32.4%, respectively), while two exhibited the lowest proportions (7.21% and 5.63%, respectively). Notably, the cluster with the most HPV-positive cells deviated significantly from normal epithelial layer markers, exhibiting functional heterogeneity and altered epithelial structuring, indicating that significant molecular heterogeneity existed in cancer tissues and that these cells exhibited unique/different gene signatures compared with normal epithelial cells. These HPV-positive cells, compared to HPV-negative, showed different gene expressions related to the extracellular matrix, cell adhesion, proliferation, and apoptosis. Further, the viral oncogenes E6 and E7 appeared to modify epithelial function via distinct pathways, thus contributing to cervical cancer progression. We investigated the HPV and host transcripts from a novel viewpoint focusing on layer heterogeneity. Our results indicated varied HPV expression across epithelial clusters and epithelial heterogeneity associated with viral oncogenes, contributing biological insights to this critical field of study.
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Affiliation(s)
- Yingjie Li
- Department of Biomedical Informatics, College of Medicine, The Ohio State University, Columbus, OH, 43210, USA
| | - Cankun Wang
- Department of Biomedical Informatics, College of Medicine, The Ohio State University, Columbus, OH, 43210, USA
| | - Anjun Ma
- Department of Biomedical Informatics, College of Medicine, The Ohio State University, Columbus, OH, 43210, USA
- Pelotonia Institute for Immuno-Oncology, The James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA
| | - Abdul Qawee Rani
- The James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA
| | - Mingjue Luo
- The James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA
| | - Jenny Li
- The James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA
| | - Xuefeng Liu
- The James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA
- The Departments of Pathology, Urology, and Radiation Oncology, College of Medicine, The Ohio State University, Columbus, OH, 43210, USA
| | - Qin Ma
- Department of Biomedical Informatics, College of Medicine, The Ohio State University, Columbus, OH, 43210, USA
- Pelotonia Institute for Immuno-Oncology, The James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA
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3
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Li Y, Wang C, Ma A, Rani AQ, Luo M, Li J, Liu X, Ma Q. Identification of HPV oncogene and host cell differentiation associated cellular heterogeneity in cervical cancer via single-cell transcriptomic analysis. J Med Virol 2023; 95:e29060. [PMID: 37638381 DOI: 10.1002/jmv.29060] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Revised: 08/11/2023] [Accepted: 08/16/2023] [Indexed: 08/29/2023]
Abstract
Human Papillomaviruses (HPVs) are associated with around 5%-10% of human cancer, notably nearly 99% of cervical cancer. The mechanisms HPV interacts with stratified epithelium (differentiated layers) during the viral life cycle, and oncogenesis remain unclear. In this study, we used single-cell transcriptome analysis to study viral gene and host cell differentiation-associated heterogeneity of HPV-positive cervical cancer tissue. We examined the HPV16 genes-E1, E6, and E7, and found they expressed differently across nine epithelial clusters. We found that three epithelial clusters had the highest proportion of HPV-positive cells (33.6%, 37.5%, and 32.4%, respectively), while two exhibited the lowest proportions (7.21% and 5.63%, respectively). Notably, the cluster with the most HPV-positive cells deviated significantly from normal epithelial layer markers, exhibiting functional heterogeneity and altered epithelial structuring, indicating that significant molecular heterogeneity existed in cancer tissues and that these cells exhibited unique/different gene signatures compared with normal epithelial cells. These HPV-positive cells, compared to HPV-negative, showed different gene expressions related to the extracellular matrix, cell adhesion, proliferation, and apoptosis. Further, the viral oncogenes E6 and E7 appeared to modify epithelial function via distinct pathways, thus contributing to cervical cancer progression. We investigated the HPV and host transcripts from a novel viewpoint focusing on layer heterogeneity. Our results indicated varied HPV expression across epithelial clusters and epithelial heterogeneity associated with viral oncogenes, contributing biological insights to this critical field of study.
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Affiliation(s)
- Yingjie Li
- Department of Biomedical Informatics, College of Medicine, The Ohio State University, Columbus, Ohio, USA
| | - Cankun Wang
- Department of Biomedical Informatics, College of Medicine, The Ohio State University, Columbus, Ohio, USA
| | - Anjun Ma
- Department of Biomedical Informatics, College of Medicine, The Ohio State University, Columbus, Ohio, USA
- The James Comprehensive Cancer Center, Pelotonia Institute for Immuno-Oncology, The Ohio State University, Columbus, Ohio, USA
| | - Abdul Qawee Rani
- The James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA
| | - Mingjue Luo
- The James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA
| | - Jenny Li
- The James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA
| | - Xuefeng Liu
- The James Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA
- The Departments of Pathology, Urology, and Radiation Oncology, College of Medicine, The Ohio State University, Columbus, Ohio, USA
| | - Qin Ma
- Department of Biomedical Informatics, College of Medicine, The Ohio State University, Columbus, Ohio, USA
- The James Comprehensive Cancer Center, Pelotonia Institute for Immuno-Oncology, The Ohio State University, Columbus, Ohio, USA
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4
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Yapindi L, Bowley T, Kurtaneck N, Bergeson RL, James K, Wilbourne J, Harrod CK, Hernandez BY, Emerling BM, Yates C, Harrod R. Activation of p53-regulated pro-survival signals and hypoxia-independent mitochondrial targeting of TIGAR by human papillomavirus E6 oncoproteins. Virology 2023; 585:1-20. [PMID: 37257253 PMCID: PMC10527176 DOI: 10.1016/j.virol.2023.05.004] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2023] [Revised: 05/02/2023] [Accepted: 05/11/2023] [Indexed: 06/02/2023]
Abstract
The high-risk subtype human papillomaviruses (hrHPVs) infect and oncogenically transform basal epidermal stem cells associated with the development of squamous-cell epithelial cancers. The viral E6 oncoprotein destabilizes the p53 tumor suppressor, inhibits p53 K120-acetylation by the Tat-interacting protein of 60 kDa (TIP60, or Kat5), and prevents p53-dependent apoptosis. Intriguingly, the p53 gene is infrequently mutated in HPV + cervical cancer clinical isolates which suggests a possible paradoxical role for this gatekeeper in viral carcinogenesis. Here, we demonstrate that E6 activates the TP53-induced glycolysis and apoptosis regulator (TIGAR) and protects cells against oncogene-induced oxidative genotoxicity. The E6 oncoprotein induces a Warburg-like stress response and activates PI3K/PI5P4K/AKT-signaling that phosphorylates the TIGAR on serine residues and induces its hypoxia-independent mitochondrial targeting in hrHPV-transformed cells. Primary HPV + cervical cancer tissues contain high levels of TIGAR, p53, and c-Myc and our xenograft studies have further shown that lentiviral-siRNA-knockdown of TIGAR expression inhibits hrHPV-induced tumorigenesis in vivo. These findings suggest the modulation of p53 pro-survival signals and the antioxidant functions of TIGAR could have key ancillary roles during HPV carcinogenesis.
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Affiliation(s)
- Lacin Yapindi
- Laboratory of Molecular Virology, Department of Biological Sciences and the Dedman College Center for Drug Discovery, Design & Delivery, Southern Methodist University, Dallas, TX, 75275-0376, United States
| | - Tetiana Bowley
- Laboratory of Molecular Virology, Department of Biological Sciences and the Dedman College Center for Drug Discovery, Design & Delivery, Southern Methodist University, Dallas, TX, 75275-0376, United States
| | - Nick Kurtaneck
- Laboratory of Molecular Virology, Department of Biological Sciences and the Dedman College Center for Drug Discovery, Design & Delivery, Southern Methodist University, Dallas, TX, 75275-0376, United States
| | - Rachel L Bergeson
- Laboratory of Molecular Virology, Department of Biological Sciences and the Dedman College Center for Drug Discovery, Design & Delivery, Southern Methodist University, Dallas, TX, 75275-0376, United States
| | - Kylie James
- Laboratory of Molecular Virology, Department of Biological Sciences and the Dedman College Center for Drug Discovery, Design & Delivery, Southern Methodist University, Dallas, TX, 75275-0376, United States
| | - Jillian Wilbourne
- Laboratory of Molecular Virology, Department of Biological Sciences and the Dedman College Center for Drug Discovery, Design & Delivery, Southern Methodist University, Dallas, TX, 75275-0376, United States
| | - Carolyn K Harrod
- Laboratory of Molecular Virology, Department of Biological Sciences and the Dedman College Center for Drug Discovery, Design & Delivery, Southern Methodist University, Dallas, TX, 75275-0376, United States
| | - Brenda Y Hernandez
- Hawaii Tumor Registry, University of Hawaii Cancer Center, Honolulu, HI, 96813, United States
| | | | - Courtney Yates
- Laboratory Animal Resource Center, Southern Methodist University, Dallas, TX, 75275, United States
| | - Robert Harrod
- Laboratory of Molecular Virology, Department of Biological Sciences and the Dedman College Center for Drug Discovery, Design & Delivery, Southern Methodist University, Dallas, TX, 75275-0376, United States.
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5
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Therapeutic Vaccination in Head and Neck Squamous Cell Carcinoma—A Review. Vaccines (Basel) 2023; 11:vaccines11030634. [PMID: 36992219 DOI: 10.3390/vaccines11030634] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2023] [Revised: 02/27/2023] [Accepted: 03/07/2023] [Indexed: 03/16/2023] Open
Abstract
Therapeutic vaccination is one of the most effective immunotherapeutic approaches, second only to immune checkpoint inhibitors (ICIs), which have already been approved for clinical use. Head and neck squamous cell carcinomas (HNSCCs) are heterogenous epithelial tumors of the upper aerodigestive tract, and a significant proportion of these tumors tend to exhibit unfavorable therapeutic responses to the existing treatment options. Comprehending the immunopathology of these tumors and choosing an appropriate immunotherapeutic maneuver seems to be a promising avenue for solving this problem. The current review provides a detailed overview of the strategies, targets, and candidates for therapeutic vaccination in HNSCC. The classical principle of inducing a potent, antigen-specific, cell-mediated cytotoxicity targeting a specific tumor antigen seems to be the most effective mechanism of therapeutic vaccination, particularly against the human papilloma virus positive subset of HNSCC. However, approaches such as countering the immunosuppressive tumor microenvironment of HNSCC and immune co-stimulatory mechanisms have also been explored recently, with encouraging results.
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6
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Li D, Thomas C, Shrivastava N, Gersten A, Gadsden N, Schlecht N, Kawachi N, Schiff BA, Smith RV, Rosenblatt G, Augustine S, Gavathiotis E, Burk R, Prystowsky MB, Guha C, Mehta V, Ow TJ. Establishment of a diverse head and neck squamous cancer cell bank using conditional reprogramming culture methods. J Med Virol 2023; 95:e28388. [PMID: 36477880 PMCID: PMC10168123 DOI: 10.1002/jmv.28388] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2022] [Revised: 10/16/2022] [Accepted: 10/17/2022] [Indexed: 12/14/2022]
Abstract
Most laboratory models of head and neck squamous cell cancer (HNSCC) rely on established immortalized cell lines, which carry inherent bias due to selection and clonality. We established a robust panel of HNSCC tumor cultures using a "conditional reprogramming" (CR) method, which utilizes a rho kinase inhibitor (Y-27632) and co-culture with irradiated fibroblast (J2 strain) feeder cells to support indefinite tumor cell survival. Sixteen CR cultures were successfully generated from 19 consecutively enrolled ethnically and racially diverse patients with HNSCC at a tertiary care center in the Bronx, NY. Of the 16 CR cultures, 9/16 were derived from the oral cavity, 4/16 were derived from the oropharynx, and 3/16 were from laryngeal carcinomas. Short tandem repeat (STR) profiling was used to validate culture against patient tumor tissue DNA. All CR cultures expressed ΔNp63 and cytokeratin 5/6, which are markers of squamous identity. Human papillomavirus (HPV) testing was assessed utilizing clinical p16 staining on primary tumors, reverse transcription polymerase chain reaction (RT-PCR) of HPV16/18-specific viral oncogenes E6 and E7 in RNA extracted from tumor samples, and HPV DNA sequencing. Three of four oropharyngeal tumors were p16 and HPV-positive and maintained HPV in culture. CR cultures were able to establish three-dimensional spheroid and murine flank and orthotopic tongue models. CR methods can be readily applied to all HNSCC tumors regardless of patient characteristics, disease site, and molecular background, providing a translational research model that properly includes patient and tumor diversity.
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Affiliation(s)
- Daniel Li
- Department of Pathology, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
| | - Carlos Thomas
- Department of Pathology, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
| | - Nitisha Shrivastava
- Department of Pathology, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
| | - Adam Gersten
- Department of Pathology, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
| | - Nicholas Gadsden
- Department of Anesthesiology, Columbia University, New York, NY, USA
| | - Nicolas Schlecht
- Department of Pathology, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
- Department of Cancer Prevention & Control, Roswell Park Comprehensive Cancer Center, Buffalo, NY, USA
| | - Nicole Kawachi
- Department of Pathology, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
| | - Bradley A. Schiff
- Department of Otorhinolaryngology - Head and Neck Surgery, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
| | - Richard V. Smith
- Department of Otorhinolaryngology - Head and Neck Surgery, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
- Department of Surgery, Montefiore Medical Center/ Albert Einstein College of Medicine, Bronx, NY USA
| | - Gregory Rosenblatt
- Department of Pathology, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
| | - Stelby Augustine
- Department of Otorhinolaryngology - Head and Neck Surgery, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
| | | | - Robert Burk
- Department of Pediatrics, Montefiore Medical Center/ Albert Einstein College of Medicine, Bronx, NY USA
| | - Michael B. Prystowsky
- Department of Pathology, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
| | - Chandan Guha
- Department of Radiation Oncology, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
| | - Vikas Mehta
- Department of Otorhinolaryngology - Head and Neck Surgery, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
| | - Thomas J Ow
- Department of Pathology, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
- Department of Otorhinolaryngology - Head and Neck Surgery, Montefiore Medical Center / Albert Einstein College of Medicine, Bronx, NY, USA
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7
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Luconi M, Sogorb MA, Markert UR, Benfenati E, May T, Wolbank S, Roncaglioni A, Schmidt A, Straccia M, Tait S. Human-Based New Approach Methodologies in Developmental Toxicity Testing: A Step Ahead from the State of the Art with a Feto-Placental Organ-on-Chip Platform. INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH 2022; 19:15828. [PMID: 36497907 PMCID: PMC9737555 DOI: 10.3390/ijerph192315828] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/24/2022] [Revised: 11/20/2022] [Accepted: 11/25/2022] [Indexed: 06/17/2023]
Abstract
Developmental toxicity testing urgently requires the implementation of human-relevant new approach methodologies (NAMs) that better recapitulate the peculiar nature of human physiology during pregnancy, especially the placenta and the maternal/fetal interface, which represent a key stage for human lifelong health. Fit-for-purpose NAMs for the placental-fetal interface are desirable to improve the biological knowledge of environmental exposure at the molecular level and to reduce the high cost, time and ethical impact of animal studies. This article reviews the state of the art on the available in vitro (placental, fetal and amniotic cell-based systems) and in silico NAMs of human relevance for developmental toxicity testing purposes; in addition, we considered available Adverse Outcome Pathways related to developmental toxicity. The OECD TG 414 for the identification and assessment of deleterious effects of prenatal exposure to chemicals on developing organisms will be discussed to delineate the regulatory context and to better debate what is missing and needed in the context of the Developmental Origins of Health and Disease hypothesis to significantly improve this sector. Starting from this analysis, the development of a novel human feto-placental organ-on-chip platform will be introduced as an innovative future alternative tool for developmental toxicity testing, considering possible implementation and validation strategies to overcome the limitation of the current animal studies and NAMs available in regulatory toxicology and in the biomedical field.
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Affiliation(s)
- Michaela Luconi
- Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy
- I.N.B.B. (Istituto Nazionale Biostrutture e Biosistemi), Viale Medaglie d’Oro 305, 00136 Rome, Italy
| | - Miguel A. Sogorb
- Instituto de Bioingeniería, Universidad Miguel Hernández de Elche, Avenida de la Universidad s/n, 03202 Elche, Spain
| | - Udo R. Markert
- Placenta Lab, Department of Obstetrics, University Hospital Jena, Am Klinikum 1, 07747 Jena, Germany
| | - Emilio Benfenati
- Department of Environmental Health Sciences, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Via Mario Negri 2, 20156 Milan, Italy
| | - Tobias May
- InSCREENeX GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany
| | - Susanne Wolbank
- Ludwig Boltzmann Institut for Traumatology, The Research Center in Cooperation with AUVA, Austrian Cluster for Tissue Regeneration, Donaueschingenstrasse 13, 1200 Vienna, Austria
| | - Alessandra Roncaglioni
- Department of Environmental Health Sciences, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Via Mario Negri 2, 20156 Milan, Italy
| | - Astrid Schmidt
- Placenta Lab, Department of Obstetrics, University Hospital Jena, Am Klinikum 1, 07747 Jena, Germany
| | - Marco Straccia
- FRESCI by Science&Strategy SL, C/Roure Monjo 33, Vacarisses, 08233 Barcelona, Spain
| | - Sabrina Tait
- Centre for Gender-Specific Medicine, Istituto Superiore di Sanità, 00161 Rome, Italy
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8
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Romero-Masters JC, Lambert PF, Munger K. Molecular Mechanisms of MmuPV1 E6 and E7 and Implications for Human Disease. Viruses 2022; 14:2138. [PMID: 36298698 PMCID: PMC9611894 DOI: 10.3390/v14102138] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2022] [Revised: 09/26/2022] [Accepted: 09/27/2022] [Indexed: 11/16/2022] Open
Abstract
Human papillomaviruses (HPVs) cause a substantial amount of human disease from benign disease such as warts to malignant cancers including cervical carcinoma, head and neck cancer, and non-melanoma skin cancer. Our ability to model HPV-induced malignant disease has been impeded by species specific barriers and pre-clinical animal models have been challenging to develop. The recent discovery of a murine papillomavirus, MmuPV1, that infects laboratory mice and causes the same range of malignancies caused by HPVs provides the papillomavirus field the opportunity to test mechanistic hypotheses in a genetically manipulatable laboratory animal species in the context of natural infections. The E6 and E7 proteins encoded by high-risk HPVs, which are the HPV genotypes associated with human cancers, are multifunctional proteins that contribute to HPV-induced cancers in multiple ways. In this review, we describe the known activities of the MmuPV1-encoded E6 and E7 proteins and how those activities relate to the activities of HPV E6 and E7 oncoproteins encoded by mucosal and cutaneous high-risk HPV genotypes.
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Affiliation(s)
- James C. Romero-Masters
- McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA
| | - Paul F. Lambert
- McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, USA
| | - Karl Munger
- Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111, USA
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9
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Miller J, Dakic A, Spurgeon M, Saenz F, Kallakury B, Zhao B, Zhang J, Zhu J, Ma Q, Xu Y, Lambert P, Schlegel R, Riegel AT, Liu X. AIB1 is a novel target of the high-risk HPV E6 protein and a biomarker of cervical cancer progression. J Med Virol 2022; 94:3962-3977. [PMID: 35437795 PMCID: PMC9199254 DOI: 10.1002/jmv.27795] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2022] [Revised: 04/13/2022] [Accepted: 04/17/2022] [Indexed: 11/10/2022]
Abstract
The high-risk human papillomaviruses (HPV-16, -18) are critical etiologic agents in human malignancy, most importantly in cervical cancer. These oncogenic viruses encode the E6 and E7 proteins that are uniformly retained and expressed in cervical cancers and required for maintenance of the tumorigenic phenotype. The E6 and E7 proteins were first identified as targeting the p53 and pRB tumor suppressor pathways, respectively, in host cells, thereby leading to disruption of cell cycle controls. In addition to p53 degradation, a number of other functions and critical targets for E6 have been described, including telomerase, Myc, PDZ-containing proteins, Akt, Wnt, mTORC1, as well as others. In this study, we identified Amplified in Breast Cancer 1 (AIB1) as a new E6 target. We first found that E6 and hTERT altered similar profiling of gene expression in human foreskin keratinocytes (HFK), independent of telomerase activity. Importantly, AIB1 was a common transcriptional target of both E6 and hTERT. We then verified that high-risk E6 but not low-risk E6 expression led to increases in AIB1 transcript levels by real-time RT-PCR, suggesting that AIB1 upregulation may play an important role in cancer development. Western blots demonstrated that AIB1 expression increased in HPV-16 E6 and E7 expressing (E6E7) immortalized foreskin and cervical keratinocytes, and in three of four common cervical cancer cell lines as well. Then, we evaluated the expression of AIB1 in human cervical lesions and invasive carcinoma using immunohistochemical staining. Strikingly, AIB1 showed positivity in the nucleus of cells in the immediate suprabasal epithelium, while nuclei of the basal epithelium were negative, as evident in the Cervical Intraepithelial Neoplasia 1 (CIN1) samples. As the pathological grading of cervical lesions increased from CIN1, CIN2, CIN3 carcinoma in situ and invasive carcinoma, AIB1 staining increased progressively, suggesting that AIB1 may serve as a novel histological biomarker for cervical cancer development. For cases of invasive cervical carcinoma, AIB1 staining was specific to cancerous lesions. Increased expression of AIB1 was also observed in transgenic mouse cervical neoplasia and cancer models induced by E6E7 and estrogen. Knockdown of AIB1 expression in E6E7 immortalized human cervical cells significantly abolished cell proliferation. Taken together, these data support AIB1 as a novel target of HPV E6 and a biomarker of cervical cancer progression.
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Affiliation(s)
- Jonathan Miller
- Department of Pathology, Center for Cell ReprogrammingGeorgetown University Medical SchoolWashingtonDistrict of ColumbiaUSA
| | - Aleksandra Dakic
- Department of Pathology, Center for Cell ReprogrammingGeorgetown University Medical SchoolWashingtonDistrict of ColumbiaUSA
| | - Megan Spurgeon
- McArdle Laboratory for Cancer Research, Department of OncologyUniversity of Wisconsin‐Madison School of Medicine and Public HealthMadisonWisconsinUSA
| | - Francisco Saenz
- Department of Oncology, Lombardi Comprehensive Cancer CenterGeorgetown University Medical SchoolWashingtonDistrict of ColumbiaUSA
| | - Bhaskar Kallakury
- Department of Pathology, Center for Cell ReprogrammingGeorgetown University Medical SchoolWashingtonDistrict of ColumbiaUSA
| | - Bo Zhao
- Department of Medicine, Brigham and Women's HospitalHarvard Medical SchoolBostonMassachusettsUSA
| | - Junran Zhang
- Department of Radiation Oncology, Wexner Medical CenterThe Ohio State UniversityColumbusOhioUSA
- The James Comprehensive Cancer CenterThe Ohio State UniversityColumbusOhioUSA
| | - Jian Zhu
- Department of Pathology, Wexner Medical CenterThe Ohio State UniversityColumbusOhioUSA
| | - Qin Ma
- The James Comprehensive Cancer CenterThe Ohio State UniversityColumbusOhioUSA
- Department of Biomedical Informatics, College of MedicineThe Ohio State UniversityColumbusOhioUSA
| | - Ying Xu
- Computational Systems Biology Lab, Department of Biochemistry and Molecular Biology and Institute of BioinformaticsThe University of GeorgiaAthensGeorgiaUSA
| | - Paul Lambert
- McArdle Laboratory for Cancer Research, Department of OncologyUniversity of Wisconsin‐Madison School of Medicine and Public HealthMadisonWisconsinUSA
| | - Richard Schlegel
- Department of Pathology, Center for Cell ReprogrammingGeorgetown University Medical SchoolWashingtonDistrict of ColumbiaUSA
| | - Anna T. Riegel
- Department of Oncology, Lombardi Comprehensive Cancer CenterGeorgetown University Medical SchoolWashingtonDistrict of ColumbiaUSA
| | - Xuefeng Liu
- Department of Pathology, Center for Cell ReprogrammingGeorgetown University Medical SchoolWashingtonDistrict of ColumbiaUSA
- The James Comprehensive Cancer CenterThe Ohio State UniversityColumbusOhioUSA
- Department of Pathology, Wexner Medical CenterThe Ohio State UniversityColumbusOhioUSA
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10
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Rebucci-Peixoto M, Vienot A, Adotevi O, Jacquin M, Ghiringhelli F, de la Fouchardière C, You B, Maurina T, Kalbacher E, Bazan F, Meynard G, Clairet AL, Fagnoni-Legat C, Spehner L, Bouard A, Vernerey D, Meurisse A, Kim S, Borg C, Mansi L. A Phase II Study Evaluating the Interest to Combine UCPVax, a Telomerase CD4 T H1-Inducer Cancer Vaccine, and Atezolizumab for the Treatment of HPV Positive Cancers: VolATIL Study. Front Oncol 2022; 12:957580. [PMID: 35928870 PMCID: PMC9343837 DOI: 10.3389/fonc.2022.957580] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2022] [Accepted: 06/23/2022] [Indexed: 11/18/2022] Open
Abstract
Background There is a strong rational of using anti-programmed cell death protein-1 and its ligand (anti-PD-1/L1) antibodies in human papillomavirus (HPV)-induced cancers. However, anti-PD-1/L1 as monotherapy induces a limited number of objective responses. The development of novel combinations in order to improve the clinical efficacy of an anti-PD-1/L1 is therefore of interest. Combining anti-PD-1/L1 therapy with an antitumor vaccine seems promising in HPV-positive (+) cancers. UCPVax is a therapeutic cancer vaccine composed of two separate peptides derived from telomerase (hTERT, human telomerase reverse transcriptase). UCPVax is being evaluated in a multicenter phase I/II study in NSCLC (non-small cell lung cancer) and has demonstrated to be safe and immunogenic. The aim of the VolATIL study is to evaluate the combination of atezolizumab (an anti-PD-L1) and UCPVax vaccine in a multicenter phase II study in patients with HPV+ cancers. Methods Patients with HPV+ cancer (anal canal, head and neck, and cervical or vulvar), at locally advanced or metastatic stage, and refractory to at least one line of systemic chemotherapy are eligible. The primary end point is the objective response rate (ORR) at 4 months. Patients will receive atezolizumab every 3 weeks at a fixed dose of 1,200 mg in combination with the UCPVax vaccine at 1 mg subcutaneously. Discussion Anti-cancer vaccines can restore cancer-immunity via the expansion and activation of tumor-specific T cells in patients lacking pre-existing anti-tumor responses. Moreover, preclinical data showed that specific TH1 CD4 T cells sustain the quality and homing of an antigen-specific CD8+ T-cell immunity. In previous clinical studies, the induction of anti-hTERT immunity was significantly correlated to survival in patients with advanced squamous anal cell carcinoma. Thus, there is a strong rational to combine an anti-cancer hTERT vaccine and an immune checkpoint inhibitor to activate and promote antitumor T-cell immunity. This pivotal proof of concept study will evaluate the efficacy and safety of the combination of a telomerase-based TH1 inducing vaccine (UCPVax) and an anti-PD-L1 (atezolizumab) immunotherapy in HPV+ cancers, as well as confirming their synergic mechanism, and settling the basis for a new combination for future clinical trials. Clinical Trial Registration https://www.clinicaltrials.gov/, identifier NCT03946358.
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Affiliation(s)
- Magali Rebucci-Peixoto
- Department of Oncology, Centre Hospitalier Universitaire, Besançon, France
- Clinical Investigational Center, CIC-1431, Centre Hospitalier Universitaire, Besançon, France
| | - Angélique Vienot
- Department of Oncology, Centre Hospitalier Universitaire, Besançon, France
- Clinical Investigational Center, CIC-1431, Centre Hospitalier Universitaire, Besançon, France
- INSERM, EFS BFC, UMR1098 RIGHT, University of Bourgogne Franche-Comté, Besançon, France
| | - Olivier Adotevi
- Department of Oncology, Centre Hospitalier Universitaire, Besançon, France
- Clinical Investigational Center, CIC-1431, Centre Hospitalier Universitaire, Besançon, France
- INSERM, EFS BFC, UMR1098 RIGHT, University of Bourgogne Franche-Comté, Besançon, France
| | - Marion Jacquin
- Clinical Investigational Center, CIC-1431, Centre Hospitalier Universitaire, Besançon, France
- Cancéropôle Est, Strasbourg, France
| | | | | | - Benoit You
- Department of Oncology, Hospices Civils de Lyon, Lyon, France
| | - Tristan Maurina
- Department of Oncology, Centre Hospitalier Universitaire, Besançon, France
| | - Elsa Kalbacher
- Department of Oncology, Centre Hospitalier Universitaire, Besançon, France
| | - Fernando Bazan
- Department of Oncology, Centre Hospitalier Universitaire, Besançon, France
| | - Guillaume Meynard
- Department of Oncology, Centre Hospitalier Universitaire, Besançon, France
| | - Anne-Laure Clairet
- Department of Pharmacy, University Hospital of Besançon, Besançon, France
| | | | - Laurie Spehner
- INSERM, EFS BFC, UMR1098 RIGHT, University of Bourgogne Franche-Comté, Besançon, France
| | - Adeline Bouard
- INSERM, EFS BFC, UMR1098 RIGHT, University of Bourgogne Franche-Comté, Besançon, France
| | - Dewi Vernerey
- Department of Oncology, Centre Hospitalier Universitaire, Besançon, France
- Methodology and Quality of Life in Oncology Unit, Centre Hospitalier Universitaire de Besançon, Besançon, France
| | - Aurélia Meurisse
- Department of Oncology, Centre Hospitalier Universitaire, Besançon, France
- Methodology and Quality of Life in Oncology Unit, Centre Hospitalier Universitaire de Besançon, Besançon, France
| | - Stefano Kim
- Clinical Investigational Center, CIC-1431, Centre Hospitalier Universitaire, Besançon, France
- INSERM, EFS BFC, UMR1098 RIGHT, University of Bourgogne Franche-Comté, Besançon, France
- Department of Oncology, Sanatorio Allende, Cordoba, Argentina
| | - Christophe Borg
- Department of Oncology, Centre Hospitalier Universitaire, Besançon, France
- Clinical Investigational Center, CIC-1431, Centre Hospitalier Universitaire, Besançon, France
- INSERM, EFS BFC, UMR1098 RIGHT, University of Bourgogne Franche-Comté, Besançon, France
| | - Laura Mansi
- Department of Oncology, Centre Hospitalier Universitaire, Besançon, France
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11
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Wang LN, Wang L, Cheng G, Dai M, Yu Y, Teng G, Zhao J, Xu D. The association of telomere maintenance and TERT expression with susceptibility to human papillomavirus infection in cervical epithelium. Cell Mol Life Sci 2022; 79:110. [PMID: 35098380 PMCID: PMC11072999 DOI: 10.1007/s00018-021-04113-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2021] [Revised: 12/05/2021] [Accepted: 12/21/2021] [Indexed: 11/03/2022]
Abstract
The role of telomerase reverse transcriptase (TERT) induction and telomere maintenance in carcinogenesis including cervical cancer (CC) pathogenesis has been well established. However, it remains unclear whether they affect infection of high-risk human papillomavirus (hrHPV), an initiating event for CC development. Similarly, genetic variants at the TERT locus are shown to be associated with susceptibility to CC, but it is unclear whether these SNPs modify the risk for cervical HPV infection. Here we show that in CC-derived HeLa cells, TERT overexpression inhibits, while its depletion upregulates expression of Syndecan-1 (SDC-1), a key component for HPV entry receptors. The TCGA cohort of CC analyses reveals an inverse correlation between TERT and SDC-1 expression (R = -0.23, P = 0.001). We further recruited 1330 females (520 non-HPV and 810 hrHPV-infected) without CC or high-grade cervical intraepithelial neoplasia to analyze telomeres in cervical epithelial cells and SNPs at rs2736098, rs2736100 and rs2736108, previously identified TERT SNPs for CC risk. Non-infected females exhibited age-related telomere shortening in cervical epithelial cells and their telomeres were significantly longer than those in hrHPV-infected group (1.31 ± 0.62 vs 1.19 ± 0.48, P < 0.001). There were no differences in rs2736098 and rs2736100 genotypes, but non-infected individuals had significantly a higher C-allele frequency (associated with higher TERT expression) while lower T-allele levels at rs2736108 compared with those in the hrHPV group (P = 0.020). Collectively, appropriate telomere maintenance and TERT expression in normal cervical cells may prevent CC by modulating hrHPV infection predisposition, although they are required for CC development and progression.
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Affiliation(s)
- Li-Na Wang
- Central Research Laboratory, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250033, People's Republic of China
| | - Li Wang
- Central Research Laboratory, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250033, People's Republic of China
| | - Guanghui Cheng
- Central Research Laboratory, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250033, People's Republic of China
| | - Mingkai Dai
- Central Research Laboratory, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250033, People's Republic of China
| | - Yunhai Yu
- Department of Gynecology, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250033, People's Republic of China
| | - Guoxin Teng
- Department of Pathology, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250033, People's Republic of China
| | - Jingjie Zhao
- Central Research Laboratory, The Second Hospital, Cheeloo College of Medicine, Shandong University, Jinan, 250033, People's Republic of China.
| | - Dawei Xu
- Division of Hematology, Bioclinicum and Center for Molecular Medicine (CMM), Department of Medicine, Karolinska Institutet and Karolinska University Hospital Solna, 171 64, Solna, Sweden.
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12
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Spehner L, Boustani J, Cabel L, Doyen J, Vienot A, Borg C, Kim S. Present and Future Research on Anal Squamous Cell Carcinoma. Cancers (Basel) 2021; 13:3895. [PMID: 34359795 PMCID: PMC8345786 DOI: 10.3390/cancers13153895] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2021] [Revised: 07/14/2021] [Accepted: 07/30/2021] [Indexed: 12/24/2022] Open
Abstract
Squamous cell carcinoma of the anus is an orphan disease, and after more than three decades of no substantial advances in disease knowledge and treatment, it is finally gaining momentum with the arrival of a taxane-based chemotherapy and immunotherapy. Currently, about 20 combination clinical trials with an anti-PD1/L1 are ongoing in localized and advanced stages, in association with radiotherapy, chemotherapy, tumor vaccines, anti-CTLA4, anti-EGFR, or antiangiogenic molecules. Moreover, a new biomarker with high sensitivity and specificity such as HPV circulating tumor DNA (HPV ctDNA) by liquid biopsy, is improving not only the prognostic measurement but also the treatment strategy guidance for this disease. Finally, better understanding of potential targets is reshaping the present and future clinical research in this unique, HPV genotype-16-related disease in the great majority of patients.
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Affiliation(s)
- Laurie Spehner
- Interactions Greffon-Hôte-Tumeur/Ingénierie Cellulaire et Génique Research Unit INSERM UMR1098, University of Bourgogne Franche-Comté, 25020 Besançon, France; (L.S.); (A.V.); (C.B.)
- Department of Medical Oncology, University Hospital of Besançon, 25030 Besançon, France
| | - Jihane Boustani
- Department of Radiotherapy, University Hospital of Besançon, 25030 Besançon, France;
| | - Luc Cabel
- Department of Medical Oncology, Curie Institute, 75005 Paris, France;
| | - Jérôme Doyen
- Department of Medical Oncology, Centre Antoine-Lacassagne, 06189 Nice, France;
| | - Angélique Vienot
- Interactions Greffon-Hôte-Tumeur/Ingénierie Cellulaire et Génique Research Unit INSERM UMR1098, University of Bourgogne Franche-Comté, 25020 Besançon, France; (L.S.); (A.V.); (C.B.)
- Department of Medical Oncology, University Hospital of Besançon, 25030 Besançon, France
- Clinical Investigational Center, INSERM CIC-1431, Centre Hospitalier Universitaire de Besançon, 25030 Besançon, France
| | - Christophe Borg
- Interactions Greffon-Hôte-Tumeur/Ingénierie Cellulaire et Génique Research Unit INSERM UMR1098, University of Bourgogne Franche-Comté, 25020 Besançon, France; (L.S.); (A.V.); (C.B.)
- Department of Medical Oncology, University Hospital of Besançon, 25030 Besançon, France
- Clinical Investigational Center, INSERM CIC-1431, Centre Hospitalier Universitaire de Besançon, 25030 Besançon, France
| | - Stefano Kim
- Interactions Greffon-Hôte-Tumeur/Ingénierie Cellulaire et Génique Research Unit INSERM UMR1098, University of Bourgogne Franche-Comté, 25020 Besançon, France; (L.S.); (A.V.); (C.B.)
- Department of Medical Oncology, University Hospital of Besançon, 25030 Besançon, France
- Clinical Investigational Center, INSERM CIC-1431, Centre Hospitalier Universitaire de Besançon, 25030 Besançon, France
- Department of Oncology and Radiotherapy, Nord Franche Comté Hospital, 25209 Montbéliard, France
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13
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Arras W, Vercammen H, Ní Dhubhghaill S, Koppen C, Van den Bogerd B. Proliferation Increasing Genetic Engineering in Human Corneal Endothelial Cells: A Literature Review. Front Med (Lausanne) 2021; 8:688223. [PMID: 34268324 PMCID: PMC8275833 DOI: 10.3389/fmed.2021.688223] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Accepted: 06/07/2021] [Indexed: 11/13/2022] Open
Abstract
The corneal endothelium is the inner layer of the cornea. Despite comprising only a monolayer of cells, dysfunction of this layer renders millions of people visually impaired worldwide. Currently, corneal endothelial transplantation is the only viable means of restoring vision for these patients. However, because the supply of corneal endothelial grafts does not meet the demand, many patients remain on waiting lists, or are not treated at all. Possible alternative treatment strategies include intracameral injection of human corneal endothelial cells (HCEnCs), biomedical engineering of endothelial grafts and increasing the HCEnC density on grafts that would otherwise have been unsuitable for transplantation. Unfortunately, the limited proliferative capacity of HCEnCs proves to be a major bottleneck to make these alternatives beneficial. To tackle this constraint, proliferation enhancing genetic engineering is being investigated. This review presents the diverse array of genes that have been targeted by different genetic engineering strategies to increase the proliferative capacity of HCEnCs and their relevance for clinical and research applications. Together these proliferation-related genes form the basis to obtain a stable and safe supply of HCEnCs that can tackle the corneal endothelial donor shortage.
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Affiliation(s)
- Wout Arras
- Antwerp Research Group for Ocular Science (ARGOS), Translational Neurosciences, Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Hendrik Vercammen
- Antwerp Research Group for Ocular Science (ARGOS), Translational Neurosciences, Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
| | - Sorcha Ní Dhubhghaill
- Antwerp Research Group for Ocular Science (ARGOS), Translational Neurosciences, Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium.,Department of Ophthalmology, Antwerp University Hospital, Edegem, Belgium.,Netherlands Institute for Innovative Ocular Surgery (NIIOS), Rotterdam, Netherlands
| | - Carina Koppen
- Antwerp Research Group for Ocular Science (ARGOS), Translational Neurosciences, Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium.,Department of Ophthalmology, Antwerp University Hospital, Edegem, Belgium
| | - Bert Van den Bogerd
- Antwerp Research Group for Ocular Science (ARGOS), Translational Neurosciences, Faculty of Medicine and Health Sciences, University of Antwerp, Wilrijk, Belgium
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14
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Vats A, Trejo-Cerro O, Thomas M, Banks L. Human papillomavirus E6 and E7: What remains? Tumour Virus Res 2021; 11:200213. [PMID: 33716206 PMCID: PMC7972986 DOI: 10.1016/j.tvr.2021.200213] [Citation(s) in RCA: 72] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2020] [Revised: 01/29/2021] [Accepted: 02/01/2021] [Indexed: 12/12/2022] Open
Abstract
Decades of research on the human papillomavirus oncogenes, E6 and E7, have given us huge amounts of data on their expression, functions and structures. We know much about the very many cellular proteins and pathways that they influence in one way or another. However, much of this information is quite discrete, referring to one activity examined under one condition. It is now time to join the dots to try to understand a larger picture: how, where and when do all these interactions occur... and why? Examining these questions will also show how many of the yet obscure cellular processes work together for cellular and tissue homeostasis in health and disease.
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Affiliation(s)
- Arushi Vats
- Tumour Virology Group, ICGEB, AREA Science Park, Trieste, 34149, Italy
| | - Oscar Trejo-Cerro
- Tumour Virology Group, ICGEB, AREA Science Park, Trieste, 34149, Italy
| | - Miranda Thomas
- Tumour Virology Group, ICGEB, AREA Science Park, Trieste, 34149, Italy.
| | - Lawrence Banks
- Tumour Virology Group, ICGEB, AREA Science Park, Trieste, 34149, Italy
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15
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Yapindi L, Hernandez BY, Harrod R. siRNA-Inhibition of TIGAR Hypersensitizes Human Papillomavirus-Transformed Cells to Apoptosis Induced by Chemotherapy Drugs that Cause Oxidative Stress. JOURNAL OF ANTIVIRALS & ANTIRETROVIRALS 2021; 13:223. [PMID: 35291688 PMCID: PMC8920475] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
The high-risk subtype Human Papillomaviruses (hrHPVs), including HPV16, HPV18, HPV31, HPV33, and HPV45, infect and oncogenically transform epithelial cells and cause squamous cell carcinomas and adenocarcinomas associated with the development of cervical cancer and subsets of vulvar, vaginal, penile, and anogenital cancers, as well as head-and-neck oropharyngeal carcinomas which often have poor clinical prognoses. Many cancers have been shown to contain elevated levels of the TP53-Induced Glycolysis and Apoptosis Regulator (TIGAR)-a glycolytic enzyme and antioxidant effector which frequently correlates with an aggressive tumor phenotype and serves as a determinant of therapy-resistance. We therefore tested whether siRNA-inhibition of TIGAR protein expression could sensitize HPV18-transformed HeLa cells to genotoxic chemotherapy agents (i.e., cisplatin, etoposide, doxorubicin, and 4-hydroxycyclophosphamide) that induce oxidative stress and DNA-damage. Here we demonstrate that the siRNA-knockdown of TIGAR hypersensitized HeLa cells to low, otherwise sub-inhibitory concentrations of these drugs and markedly induced cellular apoptosis, as compared to a scrambled RNA (scrRNA) oligonucleotide negative control or a non-transformed immortalized human fibroblast cell-line, HFL1. Importantly, these findings suggest that therapeutically inhibiting TIGAR could hypersensitize hrHPV+ cervical tumor cells to low-dosage concentrations of chemotherapy drugs that induce oxidative DNA-damage, which could potentially lead to more favorable clinical outcomes by reducing the adverse side-effects of these anticancer medications and making them more tolerable for patients. Our studies have further shown that siRNA-inhibition of TIGAR sensitizes HPV18+ HeLa cells to apoptosis induced by 4-hydroxycyclophosphamide-a DNA-alkylating agent these cells were reported to have resistance to, alluding to another possible benefit of targeting TIGAR in combinatorial treatment strategies against virus-induced cancers.
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Affiliation(s)
- Lacin Yapindi
- Laboratory of Molecular Virology, Department of Biological Sciences, The Dedman College Center for Drug Discovery, Design and Delivery, Southern Methodist University, Dallas, Texas, 75275-0376, United States
| | | | - Robert Harrod
- Laboratory of Molecular Virology, Department of Biological Sciences, The Dedman College Center for Drug Discovery, Design and Delivery, Southern Methodist University, Dallas, Texas, 75275-0376, United States,Correspondence to: Robert Harrod, Laboratory of Molecular Virology, Department of Biological Sciences, The Dedman College Center for Drug Discovery, Design and Delivery, Southern Methodist University, Dallas, Texas, 75275-0376, United States,
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16
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Kim H, Kwon MJ, Park B, Choi HG, Nam ES, Cho SJ, Min KW, Kim ES, Hwang HS, Hong M, Koo T, Kim HJ. Negative Prognostic Implication of TERT Promoter Mutations in Human Papillomavirus-Negative Tonsillar Squamous Cell Carcinoma Under the New 8th AJCC Staging System. Indian J Surg Oncol 2020; 12:134-143. [PMID: 33994739 PMCID: PMC8119516 DOI: 10.1007/s13193-020-01200-9] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2020] [Accepted: 08/19/2020] [Indexed: 01/19/2023] Open
Abstract
Telomerase reverse transcriptase gene promoter (TERTp) mutation is a potential candidate for pathogenesis and therapeutic target of tonsillar squamous cell carcinomas (TSCCs) in association with human papillomavirus (HPV). Their clinical relevance has not been validated under the new 8th American Joint Committee on Cancer (AJCC) staging system. We analyzed real-time peptide nucleic acid–mediated PCR and sequencing methods (TERTp mutation) and real-time PCR-based assay (HPV) in 80 surgically resected TSCCs. The 8th edition staging system improved the stratification of the early and advanced stages and between T or N categories for overall survival over the 7th edition. TERTp mutation was found in 7.5%, and HPV in 80.0% of the patients. The majority (83.3%) of TERTp mutation cases were HPV-positive TSCCs. Applying the 8th edition staging system, TERTp mutation was an independent factor of poor prognosis for disease-free survival (DFS) in TSCC patients, supporting the clinical significance of TERTp mutation in tonsil cancer. TERTp mutations were also negatively correlated with overall survival and DFS in HPV-negative TSCCs. Conclusively, TERTp mutation provides negative prognostic impact on survival of surgically managed tonsil cancers staged with the AJCC 8th edition.
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Affiliation(s)
- Hyunchul Kim
- Department of Pathology, Dongtan Sacred Heart Hospital, 7, Keunjaebong-gil, Hwaseong-si, Gyeonggi-do 18450 Republic of Korea
| | - Mi Jung Kwon
- Department of Pathology, Hallym University Sacred Heart Hospital, Hallym University, 22, Gwanpyeong-ro 170beon-gil, Dongan-gu, Anyang-si, Gyeonggi-do 14068 Republic of Korea
| | - Bumjung Park
- Department of Otorhinolaryngology-Head and Neck Surgery, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Republic of Korea
| | - Hyo Geun Choi
- Department of Otorhinolaryngology-Head and Neck Surgery, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Republic of Korea
| | - Eun Sook Nam
- Department of Pathology, Kangdong Sacred Heart Hospital, Hallym University College of Medicine, Seoul, 134-701 Republic of Korea
| | - Seong Jin Cho
- Department of Pathology, Kangdong Sacred Heart Hospital, Hallym University College of Medicine, Seoul, 134-701 Republic of Korea
| | - Kyueng-Whan Min
- Department of Pathology, Hanyang University Guri Hospital, Hanyang University College of Medicine, Kyoungchun-ro 153, Guri-si, Gyeonggi-do 11923 Republic of Korea
| | - Eun Soo Kim
- Department of Radiology, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Republic of Korea
| | - Hee Sung Hwang
- Department of Nuclear Medicine, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Republic of Korea
| | - Mineui Hong
- Department of Pathology, Kangnam Sacred Heart Hospital, Hallym University College of Medicine, Daerim 1-Dong, Yeongdeungpo-gu, Seoul, 150-950 Republic of Korea
| | - Taeryool Koo
- Department of Radiation Oncology, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Republic of Korea
| | - Hyo Jung Kim
- Department of Hematological Oncology, Hallym University Sacred Heart Hospital, Hallym University College of Medicine, Anyang, Republic of Korea
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Zhong M, Fu L. Culture and application of conditionally reprogrammed primary tumor cells. Gastroenterol Rep (Oxf) 2020; 8:224-233. [PMID: 32665854 PMCID: PMC7333928 DOI: 10.1093/gastro/goaa023] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/03/2020] [Revised: 04/08/2020] [Accepted: 04/09/2020] [Indexed: 12/24/2022] Open
Abstract
Cancer is still a major public-health problem that threatens human life worldwide and further study needs to be carried out in the basic and preclinical areas. Although high-throughput sequencing technology and individualized precise therapy have made breakthroughs over the years, the high failure rate of clinical translational research has limited the innovation of antitumor drugs and triggered the urgent need for optimal cancer-research models. The development of cancerous cell lines, patient-derived xenograft (PDX) models, and organoid has strongly promoted the development of tumor-biology research, but the prediction values are limited. Conditional reprogramming (CR) is a novel cell-culture method for cancer research combining feeder cells with a Rho-associated coiled-coil kinase (ROCK) inhibitor, which enables the rapid and continuous proliferation of primary epithelial cells. In this review, we summarize the methodology to establish CR model and overview recent functions and applications of CR cell-culture models in cancer research with regard to the study of cancer-biology characterization, the exploration of therapeutic targets, individualized drug screening, the illumination of mechanisms about response to antitumor drugs, and the improvement of patient-derived animal models, and finally discuss in detail the major limitations of this cell-culture system.
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Affiliation(s)
- Mengjun Zhong
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangdong Esophageal Cancer Institute, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, P. R. China
| | - Liwu Fu
- State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Guangdong Esophageal Cancer Institute, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, P. R. China
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18
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Liu X, Wu Y, Rong L. Conditionally Reprogrammed Human Normal Airway Epithelial Cells at ALI: A Physiological Model for Emerging Viruses. Virol Sin 2020; 35:280-289. [PMID: 32557270 PMCID: PMC7298165 DOI: 10.1007/s12250-020-00244-z] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2020] [Accepted: 05/22/2020] [Indexed: 01/08/2023] Open
Abstract
Cancer cell lines have been used widely in cancer biology, and as biological or functional cell systems in many biomedical research fields. These cells are usually defective for many normal activities or functions due to significant genetic and epigenetic changes. Normal primary cell yields and viability from any original tissue specimens are usually relatively low or highly variable. These normal cells cease after a few passages or population doublings due to very limited proliferative capacity. Animal models (ferret, mouse, etc.) are often used to study virus-host interaction. However, viruses usually need to be adapted to the animals by several passages due to tropism restrictions including viral receptors and intracellular restrictions. Here we summarize applications of conditionally reprogrammed cells (CRCs), long-term cultures of normal airway epithelial cells from human nose to lung generated by conditional cell reprogramming (CR) technology, as an ex vivo model in studies of emerging viruses. CR allows to robustly propagate cells from non-invasive or minimally invasive specimens, for example, nasal or endobronchial brushing. This process is rapid (2 days) and conditional. The CRCs maintain their differentiation potential and lineage functions, and have been used for studies of adenovirus, rhinovirus, respiratory syncytial virus, influenza viruses, parvovirus, and SARS-CoV. The CRCs can be easily used for air-liquid interface (ALI) polarized 3D cultures, and these coupled CRC/ALI cultures mimic physiological conditions and are suitable for studies of viral entry including receptor binding and internalization, innate immune responses, viral replications, and drug discovery as an ex vivo model for emerging viruses.
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Affiliation(s)
- Xuefeng Liu
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC, USA.
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA.
| | - Yuntao Wu
- National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, Manassas, VA, 20110, USA
| | - Lijun Rong
- Department of Microbiology and Immunology, University of Illinoi at Chicago, Chicago, IL, 60612, USA
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Vinothkumar V, Arun K, Arunkumar G, Revathidevi S, Ramani R, Bhaskar LV, Murugan AK, Munirajan AK. Association between functional TERT promoter polymorphism rs2853669 and cervical cancer risk in South Indian women. Mol Clin Oncol 2020; 12:485-494. [PMID: 32257207 PMCID: PMC7087481 DOI: 10.3892/mco.2020.2003] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2018] [Accepted: 07/17/2019] [Indexed: 02/07/2023] Open
Abstract
A single nucleotide polymorphism (SNP) rs2853669 (A>G) in the telomerase reverse transcriptase (TERT) promoter has recently been reported in chr5:1,295,349 T>C (T349C), and was shown to be associated with increased cancer risk and poor survival in a specific population. However, at present, the role of this particular SNP with TERT promoter driver mutations and its genetic association with human papilloma virus (HPV) in patients with cervical cancer has not been determined. In the present study, the genetic association of the functional SNP rs2853669 in the presence/absence of TERT promoter hotspot mutations and HPV in patients with cervical cancer of South Indian origin was evaluated. To understand and compare the frequency of the variant allele and its risk association in different cancer types of various populations, the SNP was genotyped in 257 cervical cancer samples and 295 controls, and its associations with TERT promoter hotspot mutations and HPV were analyzed. Furthermore, an extensive search of previously published articles in PubMed, Embase and Web of Science was conducted; a meta-analysis was carried out to elucidate the association of the SNP with different cancer types in global populations. The SNP analysis showed significantly high frequency (41%) of homozygous variant allele rs2853669 (GG) in patients with cervical cancer compared with control samples [Recessive allele model odds ratio (OR)=1.71; 95% CI=1.20-2.43; P=0.003]. No significant interaction was observed between the TERT SNP rs2853669 and HPV status as well as other hotspot TERT promoter (C228T and C250T) mutations determined in our previous study. In addition, the overall meta-analysis revealed a significant association of the SNP rs2853669 with other cancer types in different ethnic populations (OR=1.09; 95% CI=1.03-1.16; P=0.004). The present results suggested that the TERT SNP rs2853669 could play an important role in the risk of cervical cancer in a South Indian population.
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Affiliation(s)
- Vilvanathan Vinothkumar
- Department of Genetics, Dr ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai, Tamil Nadu 600113, India
| | - Kanagaraj Arun
- Department of Genetics, Dr ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai, Tamil Nadu 600113, India
| | - Ganesan Arunkumar
- Department of Genetics, Dr ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai, Tamil Nadu 600113, India
| | - Sundaramoorthy Revathidevi
- Department of Genetics, Dr ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai, Tamil Nadu 600113, India
| | - Rajendren Ramani
- Institute of Social Obstetrics and Government Kasturba Gandhi Hospital for Women and Children, Chennai, Tamil Nadu 600005, India
| | | | - Avaniyapuram Kannan Murugan
- Department of Molecular Oncology, King Faisal Specialist Hospital and Research Centre, Riyadh 11211, Kingdom of Saudi Arabia
| | - Arasambattu Kannan Munirajan
- Department of Genetics, Dr ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai, Tamil Nadu 600113, India
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20
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From squamous intraepithelial lesions to cervical cancer: Circulating microRNAs as potential biomarkers in cervical carcinogenesis. Biochim Biophys Acta Rev Cancer 2019; 1872:188306. [DOI: 10.1016/j.bbcan.2019.08.001] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/28/2019] [Accepted: 08/01/2019] [Indexed: 02/06/2023]
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21
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Chen Y, Val S, Krueger A, Zhong L, Panigrahi A, Nino G, Wolf S, Preciado D. Human primary middle ear epithelial cell culture: A novel in vitro model to study otitis media. Laryngoscope Investig Otolaryngol 2019; 4:663-672. [PMID: 31890886 PMCID: PMC6929573 DOI: 10.1002/lio2.319] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2019] [Revised: 07/29/2019] [Accepted: 09/23/2019] [Indexed: 12/21/2022] Open
Abstract
Objectives Otitis media (OM) is a ubiquitous pediatric disease leading to a significant health care burden. There is no medication beneficial to resolving COM fluid, highlighting the need for research in the field. Crucially, current human middle ear epithelial cell models are transformed cells not recapitulating physiological functions. Herein, we describe a new method to proliferate and differentiate pediatric primary middle ear epithelial cells (pMEEC) from patients as a physiological model for the study of OM. Methods We adapted a cell reprogramming protocol using irradiated fibroblast feeder medium in addition to Rho kinase inhibitor to proliferate pMEEC collected during cochlear implant surgery. Cells were plated on transwell membranes, proliferated with conditionally reprogrammed culture medium, and transferred to air–liquid interface (ALI). Cultures were maintained for 4 weeks at ALI, photos were taken and cell lysates and secretions were collected over time for characterization analysis using quantitative polymerase chain reaction, Western bolt, and proteomics. Keratins, MUC5B and MUC5AC mucins, and beta tubulin (TUBB) were analyzed at the mRNA and protein level. Results Cultures took a mean of 2 weeks to proliferate before transwell plating and forming a tight epithelium at ALI from 2 to 4 weeks. Although mRNA expression of MUC5B, MUC5AC, TUBB, and keratin 5 (KRT5) were variable depending on the differentiation stage and the patient, both TUBB and KRT5 proteins were detected until week 2. Conclusion We demonstrate a novel method to proliferate and differentiate pMEECs that express epithelial markers and that are able to secrete mucins for the study of OM. Level of Evidence NA
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Affiliation(s)
- Yajun Chen
- Sheikh Zayed Center for Pediatric Surgical Innovation and Division of Otolaryngology Children's National Health System Washington District of Columbia U.S.A
| | - Stéphanie Val
- Sheikh Zayed Center for Pediatric Surgical Innovation and Division of Otolaryngology Children's National Health System Washington District of Columbia U.S.A
| | - Anna Krueger
- Sheikh Zayed Center for Pediatric Surgical Innovation and Division of Otolaryngology Children's National Health System Washington District of Columbia U.S.A
| | - Lydia Zhong
- Sheikh Zayed Center for Pediatric Surgical Innovation and Division of Otolaryngology Children's National Health System Washington District of Columbia U.S.A
| | - Aswini Panigrahi
- Center for Cancer and Immunology Research Children's National Health System Washington District of Columbia U.S.A
| | - Gustavo Nino
- Division of Pulmonary Medicine Children's National Health System Washington District of Columbia U.S.A
| | - Seth Wolf
- Division of Pulmonary Medicine Children's National Health System Washington District of Columbia U.S.A
| | - Diego Preciado
- Sheikh Zayed Center for Pediatric Surgical Innovation and Division of Otolaryngology Children's National Health System Washington District of Columbia U.S.A.,Division of Pediatric Otolaryngology Children's National Health System Washington District of Columbia U.S.A
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22
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Palechor-Ceron N, Krawczyk E, Dakic A, Simic V, Yuan H, Blancato J, Wang W, Hubbard F, Zheng YL, Dan H, Strome S, Cullen K, Davidson B, Deeken JF, Choudhury S, Ahn PH, Agarwal S, Zhou X, Schlegel R, Furth PA, Pan CX, Liu X. Conditional Reprogramming for Patient-Derived Cancer Models and Next-Generation Living Biobanks. Cells 2019; 8:E1327. [PMID: 31717887 PMCID: PMC6912808 DOI: 10.3390/cells8111327] [Citation(s) in RCA: 55] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2019] [Revised: 10/14/2019] [Accepted: 10/25/2019] [Indexed: 12/21/2022] Open
Abstract
Traditional cancer models including cell lines and animal models have limited applications in both basic and clinical cancer research. Genomics-based precision oncology only help 2-20% patients with solid cancer. Functional diagnostics and patient-derived cancer models are needed for precision cancer biology. In this review, we will summarize applications of conditional cell reprogramming (CR) in cancer research and next generation living biobanks (NGLB). Together with organoids, CR has been cited in two NCI (National Cancer Institute, USA) programs (PDMR: patient-derived cancer model repository; HCMI: human cancer model initiatives. HCMI will be distributed through ATCC). Briefly, the CR method is a simple co-culture technology with a Rho kinase inhibitor, Y-27632, in combination with fibroblast feeder cells, which allows us to rapidly expand both normal and malignant epithelial cells from diverse anatomic sites and mammalian species and does not require transfection with exogenous viral or cellular genes. Establishment of CR cells from both normal and tumor tissue is highly efficient. The robust nature of the technique is exemplified by the ability to produce 2 × 106 cells in five days from a core biopsy of tumor tissue. Normal CR cell cultures retain a normal karyotype and differentiation potential and CR cells derived from tumors retain their tumorigenic phenotype. CR also allows us to enrich cancer cells from urine (for bladder cancer), blood (for prostate cancer), and pleural effusion (for non-small cell lung carcinoma). The ability to produce inexhaustible cell populations using CR technology from small biopsies and cryopreserved specimens has the potential to transform biobanking repositories (NGLB: next-generation living biobank) and current pathology practice by enabling genetic, biochemical, metabolomic, proteomic, and biological assays, including chemosensitivity testing as a functional diagnostics tool for precision cancer medicine. We discussed analyses of patient-derived matched normal and tumor models using a case with tongue squamous cell carcinoma as an example. Last, we summarized applications in cancer research, disease modeling, drug discovery, and regenerative medicine of CR-based NGLB.
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Affiliation(s)
- Nancy Palechor-Ceron
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA; (N.P.-C.); (E.K.); (A.D.); (V.S.); (H.Y.); (S.C.); (S.A.); (R.S.)
| | - Ewa Krawczyk
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA; (N.P.-C.); (E.K.); (A.D.); (V.S.); (H.Y.); (S.C.); (S.A.); (R.S.)
| | - Aleksandra Dakic
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA; (N.P.-C.); (E.K.); (A.D.); (V.S.); (H.Y.); (S.C.); (S.A.); (R.S.)
| | - Vera Simic
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA; (N.P.-C.); (E.K.); (A.D.); (V.S.); (H.Y.); (S.C.); (S.A.); (R.S.)
| | - Hang Yuan
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA; (N.P.-C.); (E.K.); (A.D.); (V.S.); (H.Y.); (S.C.); (S.A.); (R.S.)
| | - Jan Blancato
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA; (J.B.); (W.W.); (Y.-L.Z.); (P.A.F.)
| | - Weisheng Wang
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA; (J.B.); (W.W.); (Y.-L.Z.); (P.A.F.)
| | - Fleesie Hubbard
- Department of Otorhinolaryngology-Head and Neck Surgery, University of Maryland, Baltimore, MD 21201, USA; (F.H.); (H.D.); (S.S.); (K.C.)
| | - Yun-Ling Zheng
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA; (J.B.); (W.W.); (Y.-L.Z.); (P.A.F.)
| | - Hancai Dan
- Department of Otorhinolaryngology-Head and Neck Surgery, University of Maryland, Baltimore, MD 21201, USA; (F.H.); (H.D.); (S.S.); (K.C.)
| | - Scott Strome
- Department of Otorhinolaryngology-Head and Neck Surgery, University of Maryland, Baltimore, MD 21201, USA; (F.H.); (H.D.); (S.S.); (K.C.)
| | - Kevin Cullen
- Department of Otorhinolaryngology-Head and Neck Surgery, University of Maryland, Baltimore, MD 21201, USA; (F.H.); (H.D.); (S.S.); (K.C.)
| | - Bruce Davidson
- Department of Otorhinolaryngology-Head and Neck Surgery, Georgetown University Medical Center, Washington, DC 20057, USA;
| | - John F. Deeken
- Inova Translational Medicine Institute, Inova Health System, Fairfax, VA 22031, USA;
| | - Sujata Choudhury
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA; (N.P.-C.); (E.K.); (A.D.); (V.S.); (H.Y.); (S.C.); (S.A.); (R.S.)
| | - Peter H. Ahn
- Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 20057, USA;
| | - Seema Agarwal
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA; (N.P.-C.); (E.K.); (A.D.); (V.S.); (H.Y.); (S.C.); (S.A.); (R.S.)
| | - Xuexun Zhou
- iCryobiol and iFuture Technologies, Shanghai 200127, China;
| | - Richard Schlegel
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA; (N.P.-C.); (E.K.); (A.D.); (V.S.); (H.Y.); (S.C.); (S.A.); (R.S.)
| | - Priscilla A. Furth
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA; (J.B.); (W.W.); (Y.-L.Z.); (P.A.F.)
| | - Chong-Xian Pan
- University of California at Davis, Sacramento, CA 95817, USA;
| | - Xuefeng Liu
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA; (N.P.-C.); (E.K.); (A.D.); (V.S.); (H.Y.); (S.C.); (S.A.); (R.S.)
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA; (J.B.); (W.W.); (Y.-L.Z.); (P.A.F.)
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23
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Khorraminejad-Shirazi M, Dorvash M, Estedlal A, Hoveidaei AH, Mazloomrezaei M, Mosaddeghi P. Aging: A cell source limiting factor in tissue engineering. World J Stem Cells 2019; 11:787-802. [PMID: 31692986 PMCID: PMC6828594 DOI: 10.4252/wjsc.v11.i10.787] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/18/2019] [Revised: 05/03/2019] [Accepted: 09/05/2019] [Indexed: 02/06/2023] Open
Abstract
Tissue engineering has yet to reach its ideal goal, i.e. creating profitable off-the-shelf tissues and organs, designing scaffolds and three-dimensional tissue architectures that can maintain the blood supply, proper biomaterial selection, and identifying the most efficient cell source for use in cell therapy and tissue engineering. These are still the major challenges in this field. Regarding the identification of the most appropriate cell source, aging as a factor that affects both somatic and stem cells and limits their function and applications is a preventable and, at least to some extents, a reversible phenomenon. Here, we reviewed different stem cell types, namely embryonic stem cells, adult stem cells, induced pluripotent stem cells, and genetically modified stem cells, as well as their sources, i.e. autologous, allogeneic, and xenogeneic sources. Afterward, we approached aging by discussing the functional decline of aged stem cells and different intrinsic and extrinsic factors that are involved in stem cell aging including replicative senescence and Hayflick limit, autophagy, epigenetic changes, miRNAs, mTOR and AMPK pathways, and the role of mitochondria in stem cell senescence. Finally, various interventions for rejuvenation and geroprotection of stem cells are discussed. These interventions can be applied in cell therapy and tissue engineering methods to conquer aging as a limiting factor, both in original cell source and in the in vitro proliferated cells.
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Affiliation(s)
- Mohammadhossein Khorraminejad-Shirazi
- Student Research Committee, Shiraz University of Medical Sciences, Shiraz 7134845794, Iran
- Cell and Molecular Medicine Student Research Group, School of Medicine, Shiraz University of Medical Sciences, Shiraz 7134845794, Iran
| | - Mohammadreza Dorvash
- Student Research Committee, Shiraz University of Medical Sciences, Shiraz 7134845794, Iran
- Cell and Molecular Medicine Student Research Group, School of Medicine, Shiraz University of Medical Sciences, Shiraz 7134845794, Iran
- Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz 7134814336, Iran
| | - Alireza Estedlal
- Student Research Committee, Shiraz University of Medical Sciences, Shiraz 7134845794, Iran
- Cell and Molecular Medicine Student Research Group, School of Medicine, Shiraz University of Medical Sciences, Shiraz 7134845794, Iran
| | - Amir Human Hoveidaei
- Student Research Committee, Shiraz University of Medical Sciences, Shiraz 7134845794, Iran
| | - Mohsen Mazloomrezaei
- Student Research Committee, Shiraz University of Medical Sciences, Shiraz 7134845794, Iran
- Cell and Molecular Medicine Student Research Group, School of Medicine, Shiraz University of Medical Sciences, Shiraz 7134845794, Iran
| | - Pouria Mosaddeghi
- Student Research Committee, Shiraz University of Medical Sciences, Shiraz 7134845794, Iran
- Cell and Molecular Medicine Student Research Group, School of Medicine, Shiraz University of Medical Sciences, Shiraz 7134845794, Iran
- Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz 7134814336, Iran
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24
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Gupta SM, Mania-Pramanik J. Molecular mechanisms in progression of HPV-associated cervical carcinogenesis. J Biomed Sci 2019; 26:28. [PMID: 31014351 PMCID: PMC6477741 DOI: 10.1186/s12929-019-0520-2] [Citation(s) in RCA: 46] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2018] [Accepted: 04/11/2019] [Indexed: 12/13/2022] Open
Abstract
Cervical cancer is the fourth most frequent cancer in women worldwide and a major cause of mortality in developing countries. Persistent infection with high-risk human papillomavirus (HPV) is a necessary cause for the development of cervical cancer. In addition, genetic and epigenetic alterations in host cell genes are crucial for progression of cervical precancerous lesions to invasive cancer. Although much progress has been made in understanding the life cycle of HPV and it’s role in the development of cervical cancer, there is still a critical need for accurate surveillance strategies and targeted therapeutic options to eradicate these cancers in patients. Given the widespread nature of HPV infection and the type specificity of currently available HPV vaccines, it is crucial that molecular details of the natural history of HPV infection as well as the biological activities of viral oncoproteins be elucidated. A better understanding of the mechanisms involved in oncogenesis can provide novel insights and opportunities for designing effective therapeutic approaches against HPV-associated malignancies. In this review, we briefly summarize epigenetic alterations and events that cause alterations in host genomes inducing cell cycle deregulation, aberrant proliferation and genomic instability contributing to tumorigenesis.
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Affiliation(s)
- Sadhana M Gupta
- Department of Infectious Diseases Biology, National Institute for Research in Reproductive Health, J.M. Street, Parel, Mumbai, 400012, India.
| | - Jayanti Mania-Pramanik
- Department of Infectious Diseases Biology, National Institute for Research in Reproductive Health, J.M. Street, Parel, Mumbai, 400012, India
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25
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Mondal AM, Zhou H, Horikawa I, Suprynowicz FA, Li G, Dakic A, Rosenthal B, Ye L, Harris CC, Schlegel R, Liu X. Δ133p53α, a natural p53 isoform, contributes to conditional reprogramming and long-term proliferation of primary epithelial cells. Cell Death Dis 2018; 9:750. [PMID: 29970881 PMCID: PMC6030220 DOI: 10.1038/s41419-018-0767-7] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2018] [Revised: 05/25/2018] [Accepted: 06/08/2018] [Indexed: 12/12/2022]
Abstract
We previously developed the technique of conditional reprogramming (CR), which allows primary epithelial cells from fresh or cryopreserved specimens to be propagated long-term in vitro, while maintaining their genetic stability and differentiation potential. This method requires a combination of irradiated fibroblast feeder cells and a Rho-associated kinase (ROCK) inhibitor. In the present study, we demonstrate increased levels of full-length p53 and its natural isoform, Δ133p53α, in conditionally reprogrammed epithelial cells from primary prostate, foreskin, ectocervical, and mammary tissues. Increased Δ133p53α expression is critical for CR since cell proliferation is rapidly inhibited following siRNA knockdown of endogenous Δ133p53α. Importantly, overexpression of Δ133p53α consistently delays the onset of cellular senescence of primary cells when cultured under non-CR conditions in normal keratinocyte growth medium (KGM). More significantly, the combination of Δ133p53α overexpression and ROCK inhibitor, without feeder cells, enables primary epithelial cells to be propagated long-term in vitro. We also show that Δ133p53α overexpression induces hTERT expression and telomerase activity and that siRNA knockdown of hTERT causes rapid inhibition of cell proliferation, indicating a critical role of hTERT for mediating the effects of Δ133p53α. Altogether, these data demonstrate a functional and regulatory link between p53 pathways and hTERT expression during the conditional reprogramming of primary epithelial cells.
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Affiliation(s)
- Abdul M Mondal
- Center for Cell Reprograming, Department of Pathology, Georgetown University Medical Center, Georgrtown, WA, 20057, USA
| | - Hua Zhou
- Center for Cell Reprograming, Department of Pathology, Georgetown University Medical Center, Georgrtown, WA, 20057, USA.,Guizhou Medical University, Guiyang, Guizhou, China
| | - Izumi Horikawa
- Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Frank A Suprynowicz
- Center for Cell Reprograming, Department of Pathology, Georgetown University Medical Center, Georgrtown, WA, 20057, USA
| | - Guangzhao Li
- Center for Cell Reprograming, Department of Pathology, Georgetown University Medical Center, Georgrtown, WA, 20057, USA
| | - Aleksandra Dakic
- Center for Cell Reprograming, Department of Pathology, Georgetown University Medical Center, Georgrtown, WA, 20057, USA
| | - Bernard Rosenthal
- Center for Cell Reprograming, Department of Pathology, Georgetown University Medical Center, Georgrtown, WA, 20057, USA
| | - Lin Ye
- Center for Cell Reprograming, Department of Pathology, Georgetown University Medical Center, Georgrtown, WA, 20057, USA.,Shenzhen Eye Hospital, Shenzhen, Guangdong, China
| | - Curtis C Harris
- Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Richard Schlegel
- Center for Cell Reprograming, Department of Pathology, Georgetown University Medical Center, Georgrtown, WA, 20057, USA.
| | - Xuefeng Liu
- Center for Cell Reprograming, Department of Pathology, Georgetown University Medical Center, Georgrtown, WA, 20057, USA. .,Second Xianya Hospital (Adjunct Position), Zhongnan University, Changsha, Huna, China. .,Affiliated Cancer Hospital & Institute (Adjunct Position), Guangzhou Medical University, Guangzhou, Guangdong, China.
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26
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Dakic A, DiVito K, Fang S, Suprynowicz F, Gaur A, Li X, Palechor-Ceron N, Simic V, Choudhury S, Yu S, Simbulan-Rosenthal CM, Rosenthal D, Schlegel R, Liu X. ROCK inhibitor reduces Myc-induced apoptosis and mediates immortalization of human keratinocytes. Oncotarget 2018; 7:66740-66753. [PMID: 27556514 PMCID: PMC5341834 DOI: 10.18632/oncotarget.11458] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2016] [Accepted: 08/11/2016] [Indexed: 01/06/2023] Open
Abstract
The Myc/Max/Mad network plays a critical role in cell proliferation, differentiation and apoptosis and c-Myc is overexpressed in many cancers, including HPV-positive cervical cancer cell lines. Despite the tolerance of cervical cancer keratinocytes to high Myc expression, we found that the solitary transduction of the Myc gene into primary cervical and foreskin keratinocytes induced rapid cell death. These findings suggested that the anti-apoptotic activity of E7 in cervical cancer cells might be responsible for negating the apoptotic activity of over-expressed Myc. Indeed, our earlier in vitro studies demonstrated that Myc and E7 synergize in the immortalization of keratinocytes. Since we previously postulated that E7 and the ROCK inhibitor, Y-27632, were members of the same functional pathway in cell immortalization, we tested whether Y-27632 would inhibit apoptosis induced by the over-expression of Myc. Our findings indicate that Y-27632 rapidly inhibited Myc-induced membrane blebbing and cellular apoptosis and, more generally, functioned as an inhibitor of extrinsic and intrinsic pathways of cell death. Most important, Y-27632 cooperated with Myc to immortalize keratinocytes efficiently, indicating that apoptosis is a major barrier to Myc-induced immortalization of keratinocytes. The anti-apoptotic activity of Y-27632 correlated with a reduction in p53 serine 15 phosphorylation and the consequent reduction in the expression of downstream target genes p21 and DAPK1, two genes involved in the induction of cell death.
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Affiliation(s)
- Aleksandra Dakic
- Department of Pathology, Georgetown University Medical School, Washington, DC 20057, USA.,Center for Cell Reprogramming, Georgetown University Medical School, Washington, DC 20057, USA
| | - Kyle DiVito
- Department of Molecular and Cell Biology and Biochemistry, Georgetown University Medical School, Washington, DC 20057, USA
| | - Shuang Fang
- Department of Pathology, Georgetown University Medical School, Washington, DC 20057, USA.,Center for Cell Reprogramming, Georgetown University Medical School, Washington, DC 20057, USA
| | - Frank Suprynowicz
- Department of Pathology, Georgetown University Medical School, Washington, DC 20057, USA.,Center for Cell Reprogramming, Georgetown University Medical School, Washington, DC 20057, USA
| | - Anirudh Gaur
- Department of Molecular and Cell Biology and Biochemistry, Georgetown University Medical School, Washington, DC 20057, USA
| | - Xin Li
- Department of Biostatistics, Bioinformatics, Georgetown University Medical School, Washington, DC 20057, USA
| | - Nancy Palechor-Ceron
- Department of Pathology, Georgetown University Medical School, Washington, DC 20057, USA.,Center for Cell Reprogramming, Georgetown University Medical School, Washington, DC 20057, USA
| | - Vera Simic
- Department of Pathology, Georgetown University Medical School, Washington, DC 20057, USA.,Center for Cell Reprogramming, Georgetown University Medical School, Washington, DC 20057, USA
| | - Sujata Choudhury
- Department of Pathology, Georgetown University Medical School, Washington, DC 20057, USA.,Center for Cell Reprogramming, Georgetown University Medical School, Washington, DC 20057, USA
| | - Songtao Yu
- Department of Pathology, Georgetown University Medical School, Washington, DC 20057, USA.,Center for Cell Reprogramming, Georgetown University Medical School, Washington, DC 20057, USA
| | - Cynthia M Simbulan-Rosenthal
- Department of Molecular and Cell Biology and Biochemistry, Georgetown University Medical School, Washington, DC 20057, USA
| | - Dean Rosenthal
- Department of Molecular and Cell Biology and Biochemistry, Georgetown University Medical School, Washington, DC 20057, USA
| | - Richard Schlegel
- Department of Pathology, Georgetown University Medical School, Washington, DC 20057, USA.,Center for Cell Reprogramming, Georgetown University Medical School, Washington, DC 20057, USA
| | - Xuefeng Liu
- Department of Pathology, Georgetown University Medical School, Washington, DC 20057, USA.,Center for Cell Reprogramming, Georgetown University Medical School, Washington, DC 20057, USA
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27
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Grayson AK, Hearnden V, Bolt R, Jebreel A, Colley HE, Murdoch C. Use of a Rho kinase inhibitor to increase human tonsil keratinocyte longevity for three-dimensional, tissue engineered tonsil epithelium equivalents. J Tissue Eng Regen Med 2017; 12:e1636-e1646. [PMID: 29048773 DOI: 10.1002/term.2590] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2017] [Revised: 09/18/2017] [Accepted: 10/09/2017] [Indexed: 12/25/2022]
Abstract
The generation of tissue-engineered epithelial models is often hampered by the limited proliferative capacity of primary epithelial cells. This study aimed to isolate normal tonsillar keratinocytes (NTK) from human tonsils, increase the lifespan of these cells using the Rho kinase inhibitor Y-27632 and to develop tissue-engineered equivalents of healthy and infected tonsil epithelium. The proliferation rate of isolated NTK and expression of c-MYC and p16INK4A were measured in the absence or presence of the inhibitor. Y-27632-treated NTK were used to generate tissue-engineered tonsil epithelium equivalents using de-epidermised dermis that were then incubated with Streptococcus pyogenes to model bacterial tonsillitis, and the expression of pro-inflammatory cytokines was measured by cytokine array and ELISA. NTK cultured in the absence of Y-27632 rapidly senesced whereas cells cultured in the presence of this inhibitor proliferated for over 30 population doublings without changing their phenotype. Y-27632-treated NTK produced a multi-layered differentiated epithelium that histologically resembled normal tonsillar surface epithelium and responded to S. pyogenes infection by increased expression of pro-inflammatory cytokines including CXCL5 and IL-6. NTK can be isolated and successfully cultured in vitro with Y-27632 leading to a markedly prolonged lifespan without any deleterious consequences to cell morphology. This functional tissue-engineered equivalent of tonsil epithelium will provide a valuable tool for studying tonsil biology and host-pathogen interactions in a more physiologically relevant manner.
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Affiliation(s)
- Amy K Grayson
- School of Clinical Dentistry, Claremont Crescent, University of Sheffield, UK
| | - Vanessa Hearnden
- Department of Materials Science and Engineering, University of Sheffield, UK
| | - Robert Bolt
- School of Clinical Dentistry, Claremont Crescent, University of Sheffield, UK
| | - Ala Jebreel
- Sheffield Teaching Hospitals NHS Foundation Trust, Royal Hallamshire Hospital, Sheffield, UK
| | - Helen E Colley
- School of Clinical Dentistry, Claremont Crescent, University of Sheffield, UK
| | - Craig Murdoch
- School of Clinical Dentistry, Claremont Crescent, University of Sheffield, UK
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28
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Zhang Y, Dakic A, Chen R, Dai Y, Schlegel R, Liu X. Direct HPV E6/Myc interactions induce histone modifications, Pol II phosphorylation, and hTERT promoter activation. Oncotarget 2017; 8:96323-96339. [PMID: 29221209 PMCID: PMC5707103 DOI: 10.18632/oncotarget.22036] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2017] [Accepted: 09/15/2017] [Indexed: 11/25/2022] Open
Abstract
Human Papillomavirus Viruses (HPVs) are associated with the majority of human cervical and anal cancers and 10-30% of head and neck squamous carcinomas. E6 oncoprotein from high risk HPVs interacts with the p53 tumor suppressor protein to facilitate its degradation and increases telomerase activity for extending the life span of host cells. We published previously that the Myc cellular transcription factor associates with the high-risk HPV E6 protein in vivo and participates in the transactivation of the hTERT promoter. In the present study, we further analyzed the role of E6 and the Myc-Max-Mad network in regulating the hTERT promoter. We confirmed that E6 and Myc interact independently and that Max can also form a complex with E6. However, the E6/Max complex is observed only in the presence of Myc, suggesting that E6 associates with Myc/Max dimers. Consistent with the hypothesis that Myc is required for E6 induction of the hTERT promoter, Myc antagonists (Mad or Mnt) significantly blocked E6-mediated transactivation of the hTERT promoter. Analysis of Myc mutants demonstrated that both the transactivation domain and HLH domain of Myc protein were required for binding E6 and for the consequent transactivation of the hTERT promoter, by either Myc or E6. We also showed that E6 increased phosphorylation of Pol II on the hTERT promoter and induced epigenetic histone modifications of the hTERT promoter. More important, knockdown of Myc expression dramatically decreased engagement of acetyl-histones and Pol II at the hTERT promoter in E6-expressing cells. Thus, E6/Myc interaction triggers the transactivation of the hTERT promoter by modulating both histone modifications, Pol II phosphorylation and promoter engagement, suggesting a novel mechanism for telomerase activation and a new target for HPV- associated human cancer.
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Affiliation(s)
- Yiyu Zhang
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA
| | - Aleksandra Dakic
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA
| | - Renxiang Chen
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA
| | - Yuhai Dai
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA
| | - Richard Schlegel
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA
| | - Xuefeng Liu
- Department of Pathology, Center for Cell Reprogramming, Georgetown University Medical Center, Washington, DC 20057, USA
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29
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Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells. Oncotarget 2017; 8:15267-15282. [PMID: 28146426 PMCID: PMC5362485 DOI: 10.18632/oncotarget.14840] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2016] [Accepted: 01/13/2017] [Indexed: 01/31/2023] Open
Abstract
Herpes simplex virus type 2 (HSV-2) infects human genital mucosa and establishes life-long latent infection. It is unmet need to establish a human cell-based microphysiological system for virus biology and anti-viral drug discovery. One of barriers is lacking of culture system of normal epithelial cells in vitro over decades. In this study, we established human normal vaginal epithelial cell (HNVEC) culture using co-culture system. HNVEC cells were then propagated rapidly and stably in a defined culture condition. HNVEC cells exhibited a normal diploid karyotype and formed the well-defined and polarized spheres in matrigel three-dimension (3D) culture, while malignant cells (HeLa) formed disorganized and nonpolar solid spheres. HNVEC cells had a normal cellular response to DNA damage and had no transforming property using soft agar assays. HNVEC expressed epithelial marker cytokeratin 14 (CK14) and p63, but not cytokeratin 18 (CK18). Next, we reconstructed HNVEC-derived 3D vaginal epithelium using air-liquid interface (ALI) culture. This 3D vaginal epithelium has the basal and apical layers with expression of epithelial markers as its originated human vaginal tissue. Finally, we established an HSV-2 infection model based on the reconstructed 3D vaginal epithelium. After inoculation of HSV-2 (G strain) at apical layer of the reconstructed 3D vaginal epithelium, we observed obvious pathological effects gradually spreading from the apical layer to basal layer with expression of a viral protein. Thus, we established an ex vivo 2D and 3D HSV-2 infection model that can be used for HSV-2 virology and anti-viral drug discovery.
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30
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Telomerase Induction in HPV Infection and Oncogenesis. Viruses 2017; 9:v9070180. [PMID: 28698524 PMCID: PMC5537672 DOI: 10.3390/v9070180] [Citation(s) in RCA: 35] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2017] [Revised: 07/05/2017] [Accepted: 07/07/2017] [Indexed: 12/11/2022] Open
Abstract
Telomerase extends the repetitive DNA at the ends of linear chromosomes, and it is normally active in stem cells. When expressed in somatic diploid cells, it can lead to cellular immortalization. Human papillomaviruses (HPVs) are associated with and high-risk for cancer activate telomerase through the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT). The expression of hTERT is affected by both high-risk HPVs, E6 and E7. Seminal studies over the last two decades have identified the transcriptional, epigenetic, and post-transcriptional roles high-risk E6 and E7 have in telomerase induction. This review will summarize these findings during infection and highlight the importance of telomerase activation as an oncogenic pathway in HPV-associated cancer development and progression.
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31
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Katzenellenbogen RA. Activation of telomerase by HPVs. Virus Res 2017; 231:50-55. [DOI: 10.1016/j.virusres.2016.11.003] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2016] [Revised: 10/27/2016] [Accepted: 11/03/2016] [Indexed: 10/20/2022]
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Abstract
High-risk human papillomaviruses (HPVs) are causative agents of anogenital cancers and a fraction of head and neck cancers. The mechanisms involved in the progression of HPV neoplasias to cancers remain largely unknown. Here, we report that O-linked GlcNAcylation (O-GlcNAc) and O-GlcNAc transferase (OGT) were markedly increased in HPV-caused cervical neoplasms relative to normal cervix, whereas O-GlcNAcase (OGA) levels were not altered. Transduction of HPV16 oncogene E6 or E6/E7 into mouse embryonic fibroblasts (MEFs) up-regulated OGT mRNA and protein, elevated the level of O-GlcNAc, and promoted cell proliferation while reducing cellular senescence. Conversely, in HPV-18-transformed HeLa cervical carcinoma cells, inhibition of O-GlcNAc with a low concentration of a chemical inhibitor impaired the transformed phenotypes in vitro. We showed that E6 elevated c-MYC via increased protein stability attributable to O-GlcNAcylation on Thr58. Reduction of HPV-mediated cell viability by a high concentration of O-GlcNAc inhibitor was partially rescued by elevated c-MYC. Finally, knockdown of OGT or O-GlcNAc inhibition in HeLa cells or in TC-1 cells, a mouse cell line transformed by HPV16 E6/E7 and activated K-RAS, reduced c-MYC and suppressed tumorigenesis and metastasis. Thus, we have uncovered a mechanism for HPV oncoprotein-mediated transformation. These findings may eventually aid in the development of effective therapeutics for HPV-associated malignancies by targeting aberrant O-GlcNAc.
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33
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Kanekura K, Nishi H, Isaka K, Kuroda M. MicroRNA and gynecologic cancers. J Obstet Gynaecol Res 2016; 42:612-7. [PMID: 27098274 DOI: 10.1111/jog.12995] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2015] [Revised: 01/25/2016] [Accepted: 02/18/2016] [Indexed: 12/23/2022]
Abstract
AIM Gynecologic malignancies are serious problems in female health. Here we aim to discuss the involvement of microRNA (miRNA) in the pathogenesis of gynecologic cancers and use of miRNA profiles for diagnosis of diseases. METHODS In order to obtain information needed for this review, we searched the PubMed database with the following keywords: miRNA and ovarian cancer; miRNA and cervical cancer; and miRNA and endometrial cancer. RESULTS Recent explosive investigations in the field have dramatically expanded our knowledge of the roles of miRNA in the pathology of gynecologic malignancies. In ovarian cancer, miRNA participates in the development of drug resistance. In cervical cancer and endometrial cancer, miRNA play essential roles in important oncogenic processes, including cell proliferation, migration and metastasis. miRNA also have high potentials to be used as biomarkers in these diseases. CONCLUSION Further validation of the studies and improvement of the methods will result in the broader use of miRNA in the diagnosis of diseases as well as in understanding of the pathomechanisms of gynecologic cancers.
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Affiliation(s)
- Kohsuke Kanekura
- Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan
| | - Hirotaka Nishi
- Department of Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan
| | - Keiichi Isaka
- Department of Obstetrics and Gynecology, Tokyo Medical University, Tokyo, Japan
| | - Masahiko Kuroda
- Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan
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34
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TERT promoter hot spot mutations are frequent in Indian cervical and oral squamous cell carcinomas. Tumour Biol 2015; 37:7907-13. [DOI: 10.1007/s13277-015-4694-2] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2015] [Accepted: 12/16/2015] [Indexed: 02/06/2023] Open
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35
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Human telomerase reverse transcriptase regulates vascular endothelial growth factor expression via human papillomavirus oncogene E7 in HPV-18-positive cervical cancer cells. Med Oncol 2015; 32:199. [PMID: 26067630 DOI: 10.1007/s12032-015-0649-0] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2015] [Accepted: 06/03/2015] [Indexed: 01/03/2023]
Abstract
Human papillomavirus (HPV) infection induces chronic and precancerous lesions and results in invasive cervical cancer. Human telomerase as well as inflammatory and angiogenic factors such as telomerase reverse transcriptase (hTERT) or vascular endothelial growth factor (VEGF) could play a role in regulating HPV-induced cervical cancer. This study investigated underlying molecular events in HPV-induced HPV-positive cervical cancer through hTERT and VEGF in vitro. Expressions of hTERT, a rate-limiting subunit of telomerase, and VEGF mRNA and proteins were, respectively, assessed by qRT-PCR, ELISA, and TRAP-ELISA in HPV-positive tissue samples and cervical cancer cell lines. To assess hTERT and VEGF secretion, hTERT overexpression and knockdown were conducted in HPV-18-positive Hela cells by hTERT cDNA and shRNA transfection, respectively. Then, the effect of HPV E6 and E7 on VEGF expressions was assessed in HPV-negative cervical cancer cells. Data have shown that VEGF expression levels are associated with hTERT expressions and telomerase activity in HPV-positive cervical cancer tissues and cells. Knockdown of hTERT expression down-regulated VEGF expressions, whereas overexpression of hTERT up-regulated VEGF expressions in HPV-18-positive Hela cells. Furthermore, HPV E7 oncoprotein was necessary for hTERT to up-regulate VEGF expressions in HPV-negative cervical cancer cells. Data from this current study indicate that HPV oncoproteins up-regulated hTERT and telomerase activity and in turn promoted VEGF expressions, which could be a key mechanism for HPV-induced cervical cancer development and progression.
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36
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Telomerase expression in amyotrophic lateral sclerosis (ALS) patients. J Hum Genet 2014; 59:555-61. [PMID: 25142509 DOI: 10.1038/jhg.2014.72] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2014] [Revised: 07/13/2014] [Accepted: 07/17/2014] [Indexed: 01/05/2023]
Abstract
Telomerase and telomeric complex have been linked to a variety of disease states related to neurological dysfunction. In amyotrophic lateral sclerosis (ALS) patients, telomerase activity, as human telomerase reverse transcriptase (hTERT) expression, has not been characterized yet. Here, for the first time, we characterized telomerase and related pathway in blood sample and spinal cord from ALS patients compared with healthy controls. We found that hTERT expression level was significantly lower in ALS patients and was correlated either to p53 mRNA expression or p21 expression, pointing out the hypothesis that telomerase inhibition could be a pathogenetic contributor to neurodegeneration in ALS. As a consequence of the reduced telomerase activity, we identified shorter telomeres in leukocytes from sporadic ALS patients compared with healthy control group.
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37
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PinX1, a novel target gene of p53, is suppressed by HPV16 E6 in cervical cancer cells. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2014; 1839:88-96. [PMID: 24412852 DOI: 10.1016/j.bbagrm.2014.01.004] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/20/2013] [Revised: 12/27/2013] [Accepted: 01/02/2014] [Indexed: 01/14/2023]
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38
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Lipps C, May T, Hauser H, Wirth D. Eternity and functionality – rational access to physiologically relevant cell lines. Biol Chem 2013; 394:1637-48. [DOI: 10.1515/hsz-2013-0158] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2013] [Accepted: 07/11/2013] [Indexed: 01/08/2023]
Abstract
Abstract
In the first 50 years of cell culture, the development of new cell lines was mainly based on trial and error. Due to the understanding of the molecular networks of aging, senescence, proliferation, and adaption by mutation, the generation of new cell lines with physiologic properties has become more systematic. This endeavor has been supported by the availability of new technological achievements and increasing knowledge about the biology of cell differentiation and cell-cell communication. Here, we review some promising developments that are contributing toward this goal. These include molecular tools frequently used for the immortalization process. In addition to these broadly acting immortalization regimens, we focus on the developments of cell type-specific immortalization and on the methodologies of how to control the growth of newly established cell lines.
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39
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Egbuniwe O, Grant AD, Renton T, Di Silvio L. Phenotype-independent effects of retroviral transduction in human dental pulp stem cells. Macromol Biosci 2013; 13:851-9. [PMID: 23765615 DOI: 10.1002/mabi.201300020] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2013] [Revised: 04/05/2013] [Indexed: 01/03/2023]
Abstract
An immortalized human dental pulp stem cell (DPSC) line of an odontoblastic phenotype is established to circumvent the normal programmed senescence and to maintain the cell line's usefulness as a tool for further study of cellular activity. DPSCs are isolated from human dental pulp tissues and transfected using hTERT. The influence of this process on the DPSC phenotype and the mRNA expression of oncogenes involved in cellular senescence is investigated. The results reveal an absence of altered DPSC morphology and phenotype following the exogenous introduction of the hTERT gene, which is coupled with a significant reduction in p16 mRNA expression. This provides insight into how to circumvent in vitro dental pulp stem cell death following the exogenous introduction of hTERT.
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Affiliation(s)
- Obi Egbuniwe
- Biomaterials, Tissue Engineering and Imaging, King's College London, Guy's Hospital, London, England.
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40
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Miller J, Dakic A, Chen R, Palechor-Ceron N, Dai Y, Kallakury B, Schlegel R, Liu X. HPV16 E7 protein and hTERT proteins defective for telomere maintenance cooperate to immortalize human keratinocytes. PLoS Pathog 2013; 9:e1003284. [PMID: 23592995 PMCID: PMC3617164 DOI: 10.1371/journal.ppat.1003284] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2012] [Accepted: 02/15/2013] [Indexed: 12/13/2022] Open
Abstract
Previous studies have shown that wild-type human telomerase reverse transcriptase (hTERT) protein can functionally replace the human papillomavirus type 16 (HPV-16) E6 protein, which cooperates with the viral E7 protein in the immortalization of primary keratinocytes. In the current study, we made the surprising finding that catalytically inactive hTERT (hTERT-D868A), elongation-defective hTERT (hTERT-HA), and telomere recruitment-defective hTERT (hTERT N+T) also cooperate with E7 in mediating bypass of the senescence blockade and effecting cell immortalization. This suggests that hTERT has activities independent of its telomere maintenance functions that mediate transit across this restriction point. Since hTERT has been shown to have a role in gene activation, we performed microarray studies and discovered that E6, hTERT and mutant hTERT proteins altered the expression of highly overlapping sets of cellular genes. Most important, the E6 and hTERT proteins induced mRNA and protein levels of Bmi1, the core subunit of the Polycomb Group (PcG) complex 1. We show further that Bmi1 substitutes for E6 or hTERT in cell immortalization. Finally, tissue array studies demonstrated that expression of Bmi1 increased with the severity of cervical dysplasia, suggesting a potential role in the progression of cervical cancer. Together, these data demonstrate that hTERT has extra-telomeric activities that facilitate cell immortalization and that its induction of Bmi1 is one potential mechanism for mediating this activity. The human papillomaviruses (HPVs) are critical elements in the etiology of cervical cancer, as well as several other human cancers. The E6 protein, in combination with the E7 protein of these viruses, immortalizes epithelial cells and increases the expression of the hTERT protein. In the current study we show that the enzymatic activity of hTERT is not required for cooperating in cell immortalization. We further demonstrate that hTERT proteins increase the expression of the Bmi1 protein, which is also capable of cooperating in cell immortalization. We anticipate that these findings will stimulate new studies of telomerase in HPV biology, cancer etiology, and stem cell reprogramming.
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Affiliation(s)
- Jonathan Miller
- Department of Pathology, Georgetown University Medical Center, Washington, D.C., United States of America
| | - Aleksandra Dakic
- Department of Pathology, Georgetown University Medical Center, Washington, D.C., United States of America
| | - Renxiang Chen
- Department of Pathology, Georgetown University Medical Center, Washington, D.C., United States of America
| | - Nancy Palechor-Ceron
- Department of Pathology, Georgetown University Medical Center, Washington, D.C., United States of America
| | - Yuhai Dai
- Department of Pathology, Georgetown University Medical Center, Washington, D.C., United States of America
| | - Bhaskar Kallakury
- Department of Pathology, Georgetown University Medical Center, Washington, D.C., United States of America
| | - Richard Schlegel
- Department of Pathology, Georgetown University Medical Center, Washington, D.C., United States of America
- * E-mail: (RS); (XL)
| | - Xuefeng Liu
- Department of Pathology, Georgetown University Medical Center, Washington, D.C., United States of America
- * E-mail: (RS); (XL)
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Crooks DR, Jeong SY, Tong WH, Ghosh MC, Olivierre H, Haller RG, Rouault TA. Tissue specificity of a human mitochondrial disease: differentiation-enhanced mis-splicing of the Fe-S scaffold gene ISCU renders patient cells more sensitive to oxidative stress in ISCU myopathy. J Biol Chem 2012; 287:40119-30. [PMID: 23035118 DOI: 10.1074/jbc.m112.418889] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
BACKGROUND ISCU myopathy is a disease caused by muscle-specific deficiency of the Fe-S cluster scaffold protein ISCU. RESULTS MyoD expression enhanced ISCU mRNA mis-splicing, and oxidative stress exacerbated ISCU depletion in patient cells. CONCLUSION ISCU protein deficiency in patients results from muscle-specific mis-splicing as well as oxidative stress. SIGNIFICANCE Oxidative stress negatively influences the mammalian Fe-S cluster assembly machinery by destabilization of ISCU. Iron-sulfur (Fe-S) cluster cofactors are formed on the scaffold protein ISCU. ISCU myopathy is a disease caused by an intronic mutation that leads to abnormally spliced ISCU mRNA. We found that two predominant mis-spliced ISCU mRNAs produce a truncated and short-lived ISCU protein product in multiple patient cell types. Expression of the muscle-specific transcription factor MyoD further diminished normal splicing of ISCU mRNA in patient myoblasts, demonstrating that the process of muscle differentiation enhances the loss of normal ISCU mRNA splicing. ISCU protein was nearly undetectable in patient skeletal muscle, but was higher in patient myoblasts, fibroblasts, and lymphoblasts. We next treated patient cells with pro-oxidants to mimic the oxidative stress associated with muscle activity. Brief hydrogen peroxide treatment or incubation in an enriched oxygen atmosphere led to a marked further reduction of ISCU protein levels, which could be prevented by pretreatment with the antioxidant ascorbate. Thus, we conclude that skeletal muscle differentiation of patient cells causes a higher degree of abnormal ISCU splicing and that oxidative stress resulting from skeletal muscle work destabilizes the small amounts of normal ISCU protein generated in patient skeletal muscles.
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Affiliation(s)
- Daniel R Crooks
- Department of Biochemistry, Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC 20057, USA
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42
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Liu X, Ory V, Chapman S, Yuan H, Albanese C, Kallakury B, Timofeeva OA, Nealon C, Dakic A, Simic V, Haddad BR, Rhim JS, Dritschilo A, Riegel A, McBride A, Schlegel R. ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. THE AMERICAN JOURNAL OF PATHOLOGY 2011; 180:599-607. [PMID: 22189618 DOI: 10.1016/j.ajpath.2011.10.036] [Citation(s) in RCA: 587] [Impact Index Per Article: 41.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/24/2011] [Revised: 10/06/2011] [Accepted: 10/11/2011] [Indexed: 12/20/2022]
Abstract
We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 10(6) cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.
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Affiliation(s)
- Xuefeng Liu
- Department of Pathology, Lombardi Comprehensive Cancer Center, Georgetown University Medical School, Washington, DC 20057, USA
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Egbuniwe O, Idowu BD, Funes JM, Grant AD, Renton T, Di Silvio L. P16/p53 expression and telomerase activity in immortalized human dental pulp cells. Cell Cycle 2011; 10:3912-9. [PMID: 22067611 PMCID: PMC3266118 DOI: 10.4161/cc.10.22.18093] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2011] [Revised: 09/13/2011] [Accepted: 09/14/2011] [Indexed: 01/09/2023] Open
Abstract
INTRODUCTION Residing within human dental pulp are cells of an ectomesenchymal origin which have the potential to differentiate into odontoblast-like cells. These cells have a limited growth potential owing to the effects of cell senescence. This study examines the effects of immortalizing odontoblast-like cells on cell proliferation and mineralization by comparing transformed dental pulp stem cells (tDPSCs) and non-transformed dental pulp stem cells (nDPSCs). RESULTS With the exogenous expression of hTERT, tDPSCs maintained a continued expression of odontogenic markers for cell proliferation and mineralization (ALP, COL-1, DMP-1, DSPP, OCN amd OPN)as did nDPScs. Oncoprotein expression was seen in both groups except for a noted absence of p16 in the tDPSCs. nDPSCs also showed lower levels of total ALP and DNA activity in comparison to tDPSCs when assayed as well as low telomerase activity readings. METHODS Using a retroviral vector, exogenous human telomerase reverse transcriptase (hTERT) was expressed in tDPSCs. Both cell groups were cultured and their telomerase activities is determined using a telomerase quantification assay. Also examined were the expression of genes involved in proliferation and mineralization such as human alkaline phosphatase (ALP), β-actin, collagen 1 (col-1), core binding factor (cbfa-1), dentin matrix protein (DMP-1), dentin sialophosphoprotein (DSPP), GAPDH, hTERT, osteocalcin (OCN), osteopontin (OPN) as well as oncoproteins involved in senescence (p16, p21 and p53) using RT-PCR. DNA and alkaline phosphatase activity was assayed in both cell groups. CONCLUSIONS These results indicate maintainance of odontoblast-like differentiation characteristics after retroviral transformation with hTERT and suggest a possible link with a reduced p16 expression.
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Affiliation(s)
- Obi Egbuniwe
- Biomaterials and Biomimetics; University College London Cancer Institute; London, UK
| | - Bernadine D Idowu
- Biomaterials and Biomimetics; University College London Cancer Institute; London, UK
| | - Juan M Funes
- University College London Cancer Institute; London, UK
| | - Andrew D Grant
- Wolfson Centre for Age-Related Diseases; University College London Cancer Institute; London, UK
| | - Tara Renton
- Department of Oral Surgery; Dental Institute; King's College London; London, UK
| | - Lucy Di Silvio
- Biomaterials and Biomimetics; University College London Cancer Institute; London, UK
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Liu Y, Zhang C, Fan J, Xiao L, Yin B, Zhou L, Xia S. Comprehensive analysis of clinical significance of stem-cell related factors in renal cell cancer. World J Surg Oncol 2011; 9:121. [PMID: 21982273 PMCID: PMC3203043 DOI: 10.1186/1477-7819-9-121] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2011] [Accepted: 10/07/2011] [Indexed: 12/15/2022] Open
Abstract
Background C-MYC, LIN28, OCT4, KLF4, NANOG and SOX2 are stem cell related factors. We detected whether these factors express in renal cell carcinoma (RCC) tissues to study their correlations with the clinical and pathological characteristics. Methods The expressions of c-MYC, LIN28, SOX2, KLF4, OCT4 and NANOG in 30 RCC patients and 5 non-RCC patients were detected with quantitative real-time reverse transcription-PCR (qRT-PCR). The data were analyzed with Wilcoxon signed rank sum test and x2 test. Results In RCC group, c-MYC expression was significantly higher in RCC tissues compared with normal tissues (P < 0.05). The expression levels of OCT4, KLF4, NANOG and SOX2 were significantly lower in RCC tissues compared with normal tissues (P < 0.05). LIN28 expression level was not significant. No difference was observed when it comes to clinical and pathological characteristics such as gender, age, tumor size, cTNM classification and differentiation status (P > 0.05). Also the expression levels of all above factors were not significantly changed in non-RCC group (P > 0.05). Conclusions The present analysis strongly suggests that altered expression of several stem cell related factors may play different roles in RCC. C-MYC may function as an oncogene and OCT4, KLF4, NANOG and SOX2 as tumor suppressors.
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Affiliation(s)
- Yongchao Liu
- Department of Urology, Shanghai First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
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Cannavo I, Benchetrit M, Loubatier C, Michel G, Lemichez E, Giordanengo V. Characterization of a cluster of oncogenic mutations in E6 of a human papillomavirus 83 variant isolated from a high-grade squamous intraepithelial lesion. J Gen Virol 2011; 92:2428-2436. [PMID: 21752964 DOI: 10.1099/vir.0.032888-0] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
We previously isolated human papillomavirus 83 (HPV83m) from a cervical smear. Sequence analysis of E6 and E7 proteins highlighted five mutations located in the second putative zinc-finger region of E6 (E6m), an important domain for protein-protein or protein-DNA interactions. Here, we show that E6m of HPV83m can trigger human primary cell proliferation and anchorage-independent growth properties, similarly to E6 of HPV16, a high-risk HPV (HR-HPV). Interestingly, we demonstrate that, in contrast to E6 of HPV16, E6m corrupts neither p53 stability nor telomerase activity, but acts as a specific modulator of the transcriptional machinery. By studying E6m reversion mutants, we confirmed the importance of the second zinc-finger domain in triggering the observed upregulation of cell growth and of the transcriptional machinery. Reversion of these mutations in E6m (to yield strain E6r) fully abolished the oncogenic potential of E6m, transforming the phenotype of E6 from a high-risk to a low-risk phenotype. Importantly, our data define the importance of a cluster of mutations in the second zinc finger of E6m in increasing the oncogenic potential of HPV83.
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Affiliation(s)
- Isabelle Cannavo
- INSERM, U895, Centre Méditerranéen de Médecine Moléculaire, C3M, Toxines Microbiennes dans la relation hote pathogènes, Nice, France
- Université de Nice-Sophia-Antipolis, UFR Médecine, Nice, France
- Laboratoire de Virologie, Centre Hospitalier Universitaire de Nice, Hôpital Archet 2, Nice, France
| | - Maxime Benchetrit
- Université de Nice-Sophia-Antipolis, UFR Médecine, Nice, France
- Laboratoire d'Anatomo-Pathologie, Centre Hospitalier Universitaire de Nice, Hôpital Archet 2, Nice, France
| | - Céline Loubatier
- INSERM, U895, Centre Méditerranéen de Médecine Moléculaire, C3M, Toxines Microbiennes dans la relation hote pathogènes, Nice, France
- Université de Nice-Sophia-Antipolis, UFR Médecine, Nice, France
| | - Gregory Michel
- INSERM, U895, Centre Méditerranéen de Médecine Moléculaire, C3M, Toxines Microbiennes dans la relation hote pathogènes, Nice, France
| | - Emmanuel Lemichez
- INSERM, U895, Centre Méditerranéen de Médecine Moléculaire, C3M, Toxines Microbiennes dans la relation hote pathogènes, Nice, France
| | - Valérie Giordanengo
- INSERM, U895, Centre Méditerranéen de Médecine Moléculaire, C3M, Toxines Microbiennes dans la relation hote pathogènes, Nice, France
- Université de Nice-Sophia-Antipolis, UFR Médecine, Nice, France
- Laboratoire de Virologie, Centre Hospitalier Universitaire de Nice, Hôpital Archet 2, Nice, France
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Higareda-Almaraz JC, Enríquez-Gasca MDR, Hernández-Ortiz M, Resendis-Antonio O, Encarnación-Guevara S. Proteomic patterns of cervical cancer cell lines, a network perspective. BMC SYSTEMS BIOLOGY 2011; 5:96. [PMID: 21696634 PMCID: PMC3152905 DOI: 10.1186/1752-0509-5-96] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/04/2011] [Accepted: 06/22/2011] [Indexed: 01/24/2023]
Abstract
Background Cervical cancer is a major mortality factor in the female population. This neoplastic is an excellent model for studying the mechanisms involved in cancer maintenance, because the Human Papilloma Virus (HPV) is the etiology factor in most cases. With the purpose of characterizing the effects of malignant transformation in cellular activity, proteomic studies constitute a reliable way to monitor the biological alterations induced by this disease. In this contextual scheme, a systemic description that enables the identification of the common events between cell lines of different origins, is required to distinguish the essence of carcinogenesis. Results With this study, we sought to achieve a systemic perspective of the common proteomic profile of six cervical cancer cell lines, both positive and negative for HPV, and which differ from the profile corresponding to the non-tumourgenic cell line, HaCaT. Our objectives were to identify common cellular events participating in cancer maintenance, as well as the establishment of a pipeline to work with proteomic-derived results. We analyzed by means of 2D SDS-PAGE and MALDI-TOF mass spectrometry the protein extracts of six cervical cancer cell lines, from which we identified a consensus of 66 proteins. We call this group of proteins, the "central core of cervical cancer". Starting from this core set of proteins, we acquired a PPI network that pointed, through topological analysis, to some proteins that may well be playing a central role in the neoplastic process, such as 14-3-3ζ. In silico overrepresentation analysis of transcription factors pointed to the overexpression of c-Myc, Max and E2F1 as key transcription factors involved in orchestrating the neoplastic phenotype. Conclusions Our findings show that there is a "central core of cervical cancer" protein expression pattern, and suggest that 14-3-3ζ is key to determine if the cell proliferates or dies. In addition, our bioinformatics analysis suggests that the neoplastic phenotype is governed by a non-canonical regulatory pathway.
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Affiliation(s)
- Juan Carlos Higareda-Almaraz
- Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Apdo, Postal 565-A, Cuernavaca, Morelos, CP 62210, México
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Liu L, Liu C, Lou F, Zhang G, Wang X, Fan Y, Yan K, Wang K, Xu Z, Hu S, Björkholm M, Xu D. Activation of telomerase by seminal plasma in malignant and normal cervical epithelial cells. J Pathol 2011; 225:203-11. [PMID: 21590772 DOI: 10.1002/path.2914] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2011] [Revised: 03/01/2011] [Accepted: 11/15/2010] [Indexed: 01/11/2023]
Abstract
Seminal fluids are involved in the development of cervical cancer but the underlying mechanism is unclear. Because cellular transformation requires telomerase activation by expression of the telomerase reverse transcriptase (hTERT) gene, we examined the role of seminal fluids in telomerase activation. Significantly elevated hTERT mRNA and telomerase activity were observed in cervical cell lines (HeLa, SiHa and Caski) treated with seminal plasma. Normal cervical epithelial cells expressed minimal levels of hTERT mRNA and telomerase activity, and seminal plasma substantially enhanced both expression and activity. The hTERT promoter activity was similarly increased in seminal plasma-treated HeLa cells and this effect was closely correlated with increased Sp1 expression and binding to the hTERT promoter. Cyclooxygenase-2 (COX-2) was simultaneously increased in HeLa cells exposed to seminal plasma, and blockade of COX-2 induction abolished seminal plasma stimulation of the hTERT promoter activity, hTERT expression and telomerase activity. Prostaglandin E2 (PGE2) mimics the effect of seminal plasma, stimulating Sp1 expression, enhancing Sp1 occupancy on the hTERT promoter and promoter activity. Moreover, tumour growth was robustly enhanced when HeLa cells together with seminal plasma were injected into nude-mice. Taken together, seminal plasma stimulates COX-2-PGE2-Sp1-dependent hTERT transcription, which provides insights into the putative mechanism underlying telomerase activation in cervical epithelial and cancer cells.
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Affiliation(s)
- Li Liu
- Ageing and Health Centre, Nursing School, Shandong University, Jinan, People's Republic of China
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Zheng ZM, Wang X. Regulation of cellular miRNA expression by human papillomaviruses. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2011; 1809:668-77. [PMID: 21616186 DOI: 10.1016/j.bbagrm.2011.05.005] [Citation(s) in RCA: 152] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Subscribe] [Scholar Register] [Received: 03/17/2011] [Revised: 04/20/2011] [Accepted: 05/09/2011] [Indexed: 12/17/2022]
Abstract
High-risk HPV infection leads to aberrant expression of cellular oncogenic and tumor suppressive miRNAs. A large number of these miRNA genes are downstream targets of the transcription factors c-Myc, p53, and E2F and their expression can therefore be modulated by oncogenic HPV E6 and E7. Cervical cancer represents a unique tumor model for understanding how viral E6 and E7 oncoproteins deregulate the expression of the miR-15/16 cluster, miR-17-92 family, miR-21, miR-23b, miR-34a, and miR-106b/93/25 cluster via the E6-p53 and E7-pRb pathways. Moreover, miRNAs may influence the expression of papillomavirus genes in a differentiation-dependent manner by targeting viral RNA transcripts. Cellular miRNAs affecting HPV DNA replication are of great interest and will be a future focus. We are entering an era focusing on miRNA and noncoding RNA, and the studies on HPV and host miRNA interactions will continue shedding more light on our understanding of the HPV life cycle and the mechanistic underpinnings of HPV-induced oncogenesis. This article is part of a Special Issue entitled: "MicroRNAs in viral gene regulation".
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Affiliation(s)
- Zhi-Ming Zheng
- National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
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Zheng ZM. Viral oncogenes, noncoding RNAs, and RNA splicing in human tumor viruses. Int J Biol Sci 2010; 6:730-55. [PMID: 21152115 PMCID: PMC2999850 DOI: 10.7150/ijbs.6.730] [Citation(s) in RCA: 71] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2010] [Accepted: 11/27/2010] [Indexed: 12/13/2022] Open
Abstract
Viral oncogenes are responsible for oncogenesis resulting from persistent virus infection. Although different human tumor viruses express different viral oncogenes and induce different tumors, their oncoproteins often target similar sets of cellular tumor suppressors or signal pathways to immortalize and/or transform infected cells. Expression of the viral E6 and E7 oncogenes in papillomavirus, E1A and E1B oncogenes in adenovirus, large T and small t antigen in polyomavirus, and Tax oncogene in HTLV-1 are regulated by alternative RNA splicing. However, this regulation is only partially understood. DNA tumor viruses also encode noncoding RNAs, including viral microRNAs, that disturb normal cell functions. Among the determined viral microRNA precursors, EBV encodes 25 from two major clusters (BART and BHRF1), KSHV encodes 12 from a latent region, human polyomavirus MCV produce only one microRNA from the late region antisense to early transcripts, but HPVs appears to produce no viral microRNAs.
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Affiliation(s)
- Zhi-Ming Zheng
- Tumor Virus RNA Biology Laboratory, HIV and AIDS Malignancy Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
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Chapman S, Liu X, Meyers C, Schlegel R, McBride AA. Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor. J Clin Invest 2010; 120:2619-26. [PMID: 20516646 DOI: 10.1172/jci42297] [Citation(s) in RCA: 252] [Impact Index Per Article: 16.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2010] [Accepted: 04/07/2010] [Indexed: 01/24/2023] Open
Abstract
Primary human keratinocytes are useful for studying the pathogenesis of many different diseases of the cutaneous and mucosal epithelia. In addition, they can form organotypic tissue equivalents in culture that can be used as epidermal autografts for wound repair as well as for the delivery of gene therapy. However, primary keratinocytes have a finite lifespan in culture that limits their proliferative capacity and clinical use. Here, we report that treatment of primary keratinocytes (originating from 3 different anatomical sites) with Y-27632, a Rho kinase inhibitor, greatly increased their proliferative capacity and resulted in efficient immortalization without detectable cell crisis. More importantly, the immortalized cells displayed characteristics typical of primary keratinocytes; they had a normal karyotype and an intact DNA damage response and were able to differentiate into a stratified epithelium. This is the first example to our knowledge of a defined chemical compound mediating efficient cell immortalization, and this finding could have wide-ranging and profound investigational and medical applications.
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Affiliation(s)
- Sandra Chapman
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland 20892, USA
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