|
1
|
Narozna M, Latham MC, Gorbsky GJ. Origin of Chromosome 12 Trisomy Surge in Human Induced Pluripotent Stem Cells (iPSCs). BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2024.12.02.626470. [PMID: 39677655 PMCID: PMC11642788 DOI: 10.1101/2024.12.02.626470] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/17/2024]
Abstract
Cultured pluripotent stem cells are unique in being the only fully diploid immortal human cell lines. However, during continued culture, they acquire significant chromosome abnormalities. Chromosome 12 trisomy is the most common whole-chromosome abnormality found during culture of human induced pluripotent stem cells (iPSCs). The conventional paradigm is that trisomy 12 occurs very rarely but provides a proliferative advantage, enabling these cells to outcompete the diploid. Here, we challenge this prevailing model by demonstrating that trisomy 12 arises simultaneously in a very high percentage of diploid cells. Using a single cell line that reproducibly undergoes transition from diploid to trisomy 12, we found that proliferation differences alone do not account for the rapid dominance of trisomic cells. Through careful mapping by fluorescent in-situ hybridization, we identified critical transition passages where trisomic cells first appeared and swiftly gained dominance. Remarkably, single trisomic cells repeatedly emerged de novo from diploid parents. Delving deeper, we discovered an extremely high incidence of chromosome 12 anaphase bridging exclusively during transition passages, along with overrepresentation of chromosome 12 chromatids in micronuclei. These micronuclei fail to replicate during S phase. Subsequently, when these micronucleated cells enter mitosis they contain an unreplicated chromosome 12 chromatids. We also found that nearly 20% of the shorter p arms of chromosome 12 but not the longer q arms exhibited loss of subtelomeric repeats during transition passages. Chromosome 12p arms were exclusively responsible for the bridging observed in anaphase cells. Our findings unveil a novel mechanism of whole-chromosome instability in human stem cells, where chromosome 12p arm-specific segregation errors occur simultaneously in a high percentage of cells. The slight yet significant growth advantage of trisomy 12 cells allows them to persist and eventually dominate the population. Our findings detailing this novel interpretation of the origin of chromosome instability in cultured of human stem cells may have broad implications for understanding the genesis of aneuploidy across diverse biological systems.
Collapse
Affiliation(s)
- Maria Narozna
- Program in Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
| | - Megan C. Latham
- Program in Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
| | - Gary J. Gorbsky
- Program in Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA
- Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA
| |
Collapse
|
|
2
|
Sergeeva SV, Loshchenova PS, Oshchepkov DY, Orishchenko KE. Crosstalk between BER and NHEJ in XRCC4-Deficient Cells Depending on hTERT Overexpression. Int J Mol Sci 2024; 25:10405. [PMID: 39408734 PMCID: PMC11476898 DOI: 10.3390/ijms251910405] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2024] [Revised: 09/21/2024] [Accepted: 09/25/2024] [Indexed: 10/20/2024] Open
Abstract
Targeting DNA repair pathways is an important strategy in anticancer therapy. However, the unrevealed interactions between different DNA repair systems may interfere with the desired therapeutic effect. Among DNA repair systems, BER and NHEJ protect genome integrity through the entire cell cycle. BER is involved in the repair of DNA base lesions and DNA single-strand breaks (SSBs), while NHEJ is responsible for the repair of DNA double-strand breaks (DSBs). Previously, we showed that BER deficiency leads to downregulation of NHEJ gene expression. Here, we studied BER's response to NHEJ deficiency induced by knockdown of NHEJ scaffold protein XRCC4 and compared the knockdown effects in normal (TIG-1) and hTERT-modified cells (NBE1). We investigated the expression of the XRCC1, LIG3, and APE1 genes of BER and LIG4; the Ku70/Ku80 genes of NHEJ at the mRNA and protein levels; as well as p53, Sp1 and PARP1. We found that, in both cell lines, XRCC4 knockdown leads to a decrease in the mRNA levels of both BER and NHEJ genes, though the effect on protein level is not uniform. XRCC4 knockdown caused an increase in p53 and Sp1 proteins, but caused G1/S delay only in normal cells. Despite the increased p53 protein, p21 did not significantly increase in NBE1 cells with overexpressed hTERT, and this correlated with the absence of G1/S delay in these cells. The data highlight the regulatory function of the XRCC4 scaffold protein and imply its connection to a transcriptional regulatory network or mRNA metabolism.
Collapse
Affiliation(s)
- Svetlana V. Sergeeva
- Institute of Cytology and Genetics, Russian Academy of Sciences, Lavrentieva 10, Novosibirsk 630090, Russia; (P.S.L.); (K.E.O.)
- Department of Genetic Technologies, Novosibirsk State University, Pirogova 2, Novosibirsk 630090, Russia
| | - Polina S. Loshchenova
- Institute of Cytology and Genetics, Russian Academy of Sciences, Lavrentieva 10, Novosibirsk 630090, Russia; (P.S.L.); (K.E.O.)
- Department of Genetic Technologies, Novosibirsk State University, Pirogova 2, Novosibirsk 630090, Russia
| | - Dmitry Yu. Oshchepkov
- Institute of Cytology and Genetics, Russian Academy of Sciences, Lavrentieva 10, Novosibirsk 630090, Russia; (P.S.L.); (K.E.O.)
| | - Konstantin E. Orishchenko
- Institute of Cytology and Genetics, Russian Academy of Sciences, Lavrentieva 10, Novosibirsk 630090, Russia; (P.S.L.); (K.E.O.)
- Department of Genetic Technologies, Novosibirsk State University, Pirogova 2, Novosibirsk 630090, Russia
| |
Collapse
|
|
3
|
Takakura Y, Hanayama R, Akiyoshi K, Futaki S, Hida K, Ichiki T, Ishii-Watabe A, Kuroda M, Maki K, Miura Y, Okada Y, Seo N, Takeuchi T, Yamaguchi T, Yoshioka Y. Quality and Safety Considerations for Therapeutic Products Based on Extracellular Vesicles. Pharm Res 2024; 41:1573-1594. [PMID: 39112776 PMCID: PMC11362369 DOI: 10.1007/s11095-024-03757-4] [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: 04/30/2024] [Accepted: 07/28/2024] [Indexed: 08/30/2024]
Abstract
Extracellular vesicles (EVs) serve as an intrinsic system for delivering functional molecules within our body, playing significant roles in diverse physiological phenomena and diseases. Both native and engineered EVs are currently the subject of extensive research as promising therapeutics and drug delivery systems, primarily due to their remarkable attributes, such as targeting capabilities, biocompatibility, and low immunogenicity and mutagenicity. Nevertheless, their clinical application is still a long way off owing to multiple limitations. In this context, the Science Board of the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan has conducted a comprehensive assessment to identify the current issues related to the quality and safety of EV-based therapeutic products. Furthermore, we have presented several examples of the state-of-the-art methodologies employed in EV manufacturing, along with guidelines for critical processes, such as production, purification, characterization, quality evaluation and control, safety assessment, and clinical development and evaluation of EV-based therapeutics. These endeavors aim to facilitate the clinical application of EVs and pave the way for their transformative impact in healthcare.
Collapse
Affiliation(s)
- Yoshinobu Takakura
- Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
| | - Rikinari Hanayama
- WPI Nano Life Science Institute (NanoLSI), Kanazawa University, Kanazawa, Japan.
| | - Kazunari Akiyoshi
- Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, Japan
| | - Shiroh Futaki
- Institute for Chemical Research, Kyoto University, Kyoto, Japan
| | - Kyoko Hida
- Vascular Biology and Molecular Biology, Faculty of Dental Medicine, Hokkaido University, Sapporo, Japan
| | - Takanori Ichiki
- Department of Materials Engineering, School of Engineering, The University of Tokyo, Bunkyō, Japan
| | - Akiko Ishii-Watabe
- Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Kawasaki, Japan
| | - Masahiko Kuroda
- Department of Molecular Pathology, Tokyo Medical University, Shinjuku, Japan
| | - Kazushige Maki
- Pharmaceuticals and Medical Devices Agency, Chiyoda-ku, Japan
| | - Yasuo Miura
- Department of Transfusion Medicine and Cell Therapy, School of Medicine, Fujita Health University, Toyoake, Japan
| | - Yoshiaki Okada
- Department of Transfusion Medicine and Cell Transplantation, Saitama Medical University Hospital, Kawagoe, Japan
| | - Naohiro Seo
- Department of Bioengineering, School of Engineering, The University of Tokyo, Bunkyō, Japan
| | - Toshihide Takeuchi
- Life Science Research Institute, Kindai University, Higashi-osaka, Japan
| | | | - Yusuke Yoshioka
- Department of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Shinjuku, Japan
| |
Collapse
|
|
4
|
Hindle J, Williams A, Kim Y, Kim D, Patil K, Khatkar P, Osgood Q, Nelson C, Routenberg DA, Howard M, Liotta LA, Kashanchi F, Branscome H. hTERT-Immortalized Mesenchymal Stem Cell-Derived Extracellular Vesicles: Large-Scale Manufacturing, Cargo Profiling, and Functional Effects in Retinal Epithelial Cells. Cells 2024; 13:861. [PMID: 38786083 PMCID: PMC11120263 DOI: 10.3390/cells13100861] [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: 04/13/2024] [Revised: 05/07/2024] [Accepted: 05/14/2024] [Indexed: 05/25/2024] Open
Abstract
As the economic burden associated with vision loss and ocular damage continues to rise, there is a need to explore novel treatment strategies. Extracellular vesicles (EVs) are enriched with various biological cargo, and there is abundant literature supporting the reparative and immunomodulatory properties of stem cell EVs across a broad range of pathologies. However, one area that requires further attention is the reparative effects of stem cell EVs in the context of ocular damage. Additionally, most of the literature focuses on EVs isolated from primary stem cells; the use of EVs isolated from human telomerase reverse transcriptase (hTERT)-immortalized stem cells has not been thoroughly examined. Using our large-scale EV-manufacturing platform, we reproducibly manufactured EVs from hTERT-immortalized mesenchymal stem cells (MSCs) and employed various methods to characterize and profile their associated cargo. We also utilized well-established cell-based assays to compare the effects of these EVs on both healthy and damaged retinal pigment epithelial cells. To the best of our knowledge, this is the first study to establish proof of concept for reproducible, large-scale manufacturing of hTERT-immortalized MSC EVs and to investigate their potential reparative properties against damaged retinal cells. The results from our studies confirm that hTERT-immortalized MSC EVs exert reparative effects in vitro that are similar to those observed in primary MSC EVs. Therefore, hTERT-immortalized MSCs may represent a more consistent and reproducible platform than primary MSCs for generating EVs with therapeutic potential.
Collapse
Affiliation(s)
| | - Anastasia Williams
- Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA 20110, USA (K.P.)
| | - Yuriy Kim
- Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA 20110, USA (K.P.)
| | | | - Kajal Patil
- Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA 20110, USA (K.P.)
| | - Pooja Khatkar
- Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA 20110, USA (K.P.)
| | | | - Collin Nelson
- Meso Scale Diagnostics, L.L.C., Rockville, MD 20850, USA (D.A.R.)
| | | | - Marissa Howard
- Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA 20110, USA
| | - Lance A. Liotta
- Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA 20110, USA
| | - Fatah Kashanchi
- Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA 20110, USA (K.P.)
| | - Heather Branscome
- ATCC, Manassas, VA 20110, USA
- Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA 20110, USA (K.P.)
| |
Collapse
|
|
5
|
Primak A, Kalinina N, Skryabina M, Usachev V, Chechekhin V, Vigovskiy M, Chechekhina E, Voloshin N, Kulebyakin K, Kulebyakina M, Grigorieva O, Tyurin-Kuzmin P, Basalova N, Efimenko A, Dzhauari S, Antropova Y, Plyushchii I, Akopyan Z, Sysoeva V, Tkachuk V, Karagyaur M. Novel Immortalized Human Multipotent Mesenchymal Stromal Cell Line for Studying Hormonal Signaling. Int J Mol Sci 2024; 25:2421. [PMID: 38397098 PMCID: PMC10889231 DOI: 10.3390/ijms25042421] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2023] [Revised: 02/05/2024] [Accepted: 02/14/2024] [Indexed: 02/25/2024] Open
Abstract
Multipotent mesenchymal stromal cells (MSCs) integrate hormone and neuromediator signaling to coordinate tissue homeostasis, tissue renewal and regeneration. To facilitate the investigation of MSC biology, stable immortalized cell lines are created (e.g., commercially available ASC52telo). However, the ASC52telo cell line has an impaired adipogenic ability and a depressed response to hormones, including 5-HT, GABA, glutamate, noradrenaline, PTH and insulin compared to primary cells. This markedly reduces the potential of the ASC52telo cell line in studying the mechanisms of hormonal control of MSC's physiology. Here, we have established a novel immortalized culture of adipose tissue-derived MSCs via forced telomerase expression after lentiviral transduction. These immortalized cell cultures demonstrate high proliferative potential (up to 40 passages), delayed senescence, as well as preserved primary culture-like functional activity (sensitivity to hormones, ability to hormonal sensitization and differentiation) and immunophenotype up to 17-26 passages. Meanwhile, primary adipose tissue-derived MSCs usually irreversibly lose their properties by 8-10 passages. Observed characteristics of reported immortalized human MSC cultures make them a feasible model for studying molecular mechanisms, which regulate the functional activities of these cells, especially when primary cultures or commercially available cell lines are not appropriate.
Collapse
Affiliation(s)
- Alexandra Primak
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
| | - Natalia Kalinina
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
| | - Mariya Skryabina
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
| | - Vladimir Usachev
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
| | - Vadim Chechekhin
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
| | - Maksim Vigovskiy
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
| | - Elizaveta Chechekhina
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
| | - Nikita Voloshin
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
| | - Konstantin Kulebyakin
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
- Institute for Regenerative Medicine, Medical Research and Education Center, Lomonosov Moscow State University, 119234 Moscow, Russia
| | - Maria Kulebyakina
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
- Institute for Regenerative Medicine, Medical Research and Education Center, Lomonosov Moscow State University, 119234 Moscow, Russia
| | - Olga Grigorieva
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
- Institute for Regenerative Medicine, Medical Research and Education Center, Lomonosov Moscow State University, 119234 Moscow, Russia
| | - Pyotr Tyurin-Kuzmin
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
| | - Nataliya Basalova
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
- Institute for Regenerative Medicine, Medical Research and Education Center, Lomonosov Moscow State University, 119234 Moscow, Russia
| | - Anastasia Efimenko
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
- Institute for Regenerative Medicine, Medical Research and Education Center, Lomonosov Moscow State University, 119234 Moscow, Russia
| | - Stalik Dzhauari
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
| | - Yulia Antropova
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
| | - Ivan Plyushchii
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
| | - Zhanna Akopyan
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
- Institute for Regenerative Medicine, Medical Research and Education Center, Lomonosov Moscow State University, 119234 Moscow, Russia
| | - Veronika Sysoeva
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
| | - Vsevolod Tkachuk
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
- Institute for Regenerative Medicine, Medical Research and Education Center, Lomonosov Moscow State University, 119234 Moscow, Russia
| | - Maxim Karagyaur
- Faculty of Medicine, Lomonosov Moscow State University, 119234 Moscow, Russia (V.C.); (Z.A.)
- Institute for Regenerative Medicine, Medical Research and Education Center, Lomonosov Moscow State University, 119234 Moscow, Russia
| |
Collapse
|
|
6
|
Irwin RE, Scullion C, Thursby SJ, Sun M, Thakur A, Hilman L, Callaghan B, Thompson PD, McKenna DJ, Rothbart SB, Xu G, Walsh CP. The UHRF1 protein is a key regulator of retrotransposable elements and innate immune response to viral RNA in human cells. Epigenetics 2023; 18:2216005. [PMID: 37246786 PMCID: PMC10228402 DOI: 10.1080/15592294.2023.2216005] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2022] [Accepted: 04/14/2023] [Indexed: 05/30/2023] Open
Abstract
While epigenetic mechanisms such as DNA methylation and histone modification are known to be important for gene suppression, relatively little is still understood about the interplay between these systems. The UHRF1 protein can interact with both DNA methylation and repressive chromatin marks, but its primary function in humans has been unclear. To determine what that was, we first established stable UHRF1 knockdowns (KD) in normal, immortalized human fibroblasts using targeting shRNA, since CRISPR knockouts (KO) were lethal. Although these showed a loss of DNA methylation across the whole genome, transcriptional changes were dominated by the activation of genes involved in innate immune signalling, consistent with the presence of viral RNA from retrotransposable elements (REs). We confirmed using mechanistic approaches that 1) REs were demethylated and transcriptionally activated; 2) this was accompanied by activation of interferons and interferon-stimulated genes and 3) the pathway was conserved across other adult cell types. Restoring UHRF1 in either transient or stable KD systems could abrogate RE reactivation and the interferon response. Notably, UHRF1 itself could also re-impose RE suppression independent of DNA methylation, but not if the protein contained point mutations affecting histone 3 with trimethylated lysine 9 (H3K9me3) binding. Our results therefore show for the first time that UHRF1 can act as a key regulator of retrotransposon silencing independent of DNA methylation.
Collapse
Affiliation(s)
- RE Irwin
- Biomedical Sciences, Ulster University, Coleraine, UK
| | - C Scullion
- Biomedical Sciences, Ulster University, Coleraine, UK
- Precision Nanosystems Inc, Vancouver, BC, Canada
| | - SJ Thursby
- Biomedical Sciences, Ulster University, Coleraine, UK
- State Key Laboratory of Molecular Biology, Shanghai Institutes of Biological Sciences, Shanghai, China
| | - M Sun
- Cellular and Molecular Medicine Program, Division of Oncology, Johns Hopkins School of Medicine, St., Baltimore, MD, USA
| | - A Thakur
- Biomedical Sciences, Ulster University, Coleraine, UK
- Department of Epigenetics, Van Andel Research Institute, Grand Rapids, MI, USA
| | - L Hilman
- Biomedical Sciences, Ulster University, Coleraine, UK
| | - B Callaghan
- Biomedical Sciences, Ulster University, Coleraine, UK
| | - PD Thompson
- Department of Epigenetics, Van Andel Research Institute, Grand Rapids, MI, USA
| | - DJ McKenna
- Biomedical Sciences, Ulster University, Coleraine, UK
| | - SB Rothbart
- Nutrition Innovation Centre for Food and Health, Biomedical Sciences, Ulster University, Coleraine, UK
| | - Guoliang Xu
- Cellular and Molecular Medicine Program, Division of Oncology, Johns Hopkins School of Medicine, St., Baltimore, MD, USA
| | - CP Walsh
- Biomedical Sciences, Ulster University, Coleraine, UK
| |
Collapse
|
|
7
|
Chatree K, Sriboonaied P, Phetkong C, Wattananit W, Chanchao C, Charoenpanich A. Distinctions in bone matrix nanostructure, composition, and formation between osteoblast-like cells, MG-63, and human mesenchymal stem cells, UE7T-13. Heliyon 2023; 9:e15556. [PMID: 37153435 PMCID: PMC10160763 DOI: 10.1016/j.heliyon.2023.e15556] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2022] [Revised: 04/07/2023] [Accepted: 04/13/2023] [Indexed: 05/09/2023] Open
Abstract
Osteoblast-like cells and human mesenchymal stem cells (hMSCs) are frequently employed as osteoprogenitor cell models for evaluating novel biomaterials in bone healing and tissue engineering. In this study, the characterization of UE7T-13 hMSCs and MG-63 human osteoblast-like cells was examined. Both cells can undergo osteogenesis and produce calcium extracellular matrix; however, calcium nodules produced by MG-63 lacked a central mass and appeared flatter than UE7T-13. The absence of growing calcium nodules in MG-63 was discovered by SEM-EDX to be associated with the formation of alternating layers of cells and calcium extracellular matrix. The nanostructure and composition analysis showed that UE7T-13 had a finer nanostructure of calcium nodules with a higher calcium/phosphate ratio than MG-63. Both cells expressed high intrinsic levels of collagen type I alpha 1 chain, while only UE7T-13 expressed high levels of alkaline phosphatase, biomineralization associated (ALPL). High ALP activity in UE7T-13 was not further enhanced by osteogenic induction, but in MG-63, low intrinsic ALP activity was greatly induced by osteogenic induction. These findings highlight the differences between the two immortal osteoprogenitor cell lines, along with some technical notes that should be considered while selecting and interpreting the pertinent in vitro model.
Collapse
Affiliation(s)
- Kamonwan Chatree
- Department of Biology, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand
| | - Patsawee Sriboonaied
- Department of Biology, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand
| | - Chinnatam Phetkong
- Department of Biology, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand
| | - Witoon Wattananit
- Scientific and Technological Equipment Centre, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand
| | - Chanpen Chanchao
- Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok, Thailand
| | - Adisri Charoenpanich
- Department of Biology, Faculty of Science, Silpakorn University, Nakhon Pathom, Thailand
| |
Collapse
|
|
8
|
Guo D, Zhang L, Wang X, Zheng J, Lin S. Establishment methods and research progress of livestock and poultry immortalized cell lines: A review. Front Vet Sci 2022; 9:956357. [PMID: 36118350 PMCID: PMC9478797 DOI: 10.3389/fvets.2022.956357] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2022] [Accepted: 08/10/2022] [Indexed: 11/13/2022] Open
Abstract
An infinite cell line is one of the most favored experimental tools and plays an irreplaceable role in cell-based biological research. Primary cells from normal animal tissues undergo a limited number of divisions and subcultures in vitro before they enter senescence and die. On the contrary, an infinite cell line is a population of non-senescent cells that could proliferate indefinitely in vitro under the stimulation of external factors such as physicochemical stimulation, virus infection, or transfer of immortality genes. Cell immortalization is the basis for establishing an infinite cell line, and previous studies have found that methods to obtain immortalized cells mainly included physical and chemical stimulations, heterologous expression of viral oncogenes, increased telomerase activity, and spontaneous formation. However, some immortalized cells do not necessarily proliferate permanently even though they can extend their lifespan compared with primary cells. An infinite cell line not only avoids the complicated process of collecting primary cell, it also provides a convenient and reliable tool for studying scientific problems in biology. At present, how to establish a stable infinite cell line to maximize the proliferation of cells while maintaining the normal function of cells is a hot issue in the biological community. This review briefly introduces the methods of cell immortalization, discusses the related progress of establishing immortalized cell lines in livestock and poultry, and compares the characteristics of several methods, hoping to provide some ideas for generating new immortalized cell lines.
Collapse
|
|
9
|
Jäger K, Mensch J, Grimmig ME, Neuner B, Gorzelniak K, Türkmen S, Demuth I, Hartmann A, Hartmann C, Wittig F, Sporbert A, Hermann A, Fuellen G, Möller S, Walter M. A conserved long-distance telomeric silencing mechanism suppresses mTOR signaling in aging human fibroblasts. SCIENCE ADVANCES 2022; 8:eabk2814. [PMID: 35977016 PMCID: PMC9385144 DOI: 10.1126/sciadv.abk2814] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/10/2023]
Abstract
Telomeres are repetitive nucleotide sequences at the ends of each chromosome. It has been hypothesized that telomere attrition evolved as a tumor suppressor mechanism in large long-lived species. Long telomeres can silence genes millions of bases away through a looping mechanism called telomere position effect over long distances (TPE-OLD). The function of this silencing mechanism is unknown. We determined a set of 2322 genes with high positional conservation across replicatively aging species that includes known and candidate TPE-OLD genes that may mitigate potentially harmful effects of replicative aging. Notably, we identified PPP2R2C as a tumor suppressor gene, whose up-regulation by TPE-OLD in aged human fibroblasts leads to dephosphorylation of p70S6 kinase and mammalian target of rapamycin suppression. A mechanistic link between telomeres and a tumor suppressor mechanism supports the hypothesis that replicative aging fulfills a tumor suppressor function and motivates previously unknown antitumor and antiaging strategies.
Collapse
Affiliation(s)
- Kathrin Jäger
- Institute of Clinical Chemistry and Laboratory Medicine, Rostock University Medical Center, University of Rostock, Rostock, Germany
- Charité–Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Berlin, Germany
| | - Juliane Mensch
- Institute of Clinical Chemistry and Laboratory Medicine, Rostock University Medical Center, University of Rostock, Rostock, Germany
- Charité–Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Berlin, Germany
| | - Maria Elisabeth Grimmig
- Institute of Clinical Chemistry and Laboratory Medicine, Rostock University Medical Center, University of Rostock, Rostock, Germany
| | - Bruno Neuner
- Charité–Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Anesthesiology and Intensive Care Medicine, Berlin, Germany
| | - Kerstin Gorzelniak
- Unfallkrankenhaus Berlin, Institute of Laboratory Medicine, Berlin, Germany
| | - Seval Türkmen
- LNS Hematooncogenetics, National Center of Genetics Luxembourg, Dudelange, Luxemburg
- Charité–Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Medical Genetics and Human Genetics, Berlin, Germany
| | - Ilja Demuth
- Charité–Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Department of Endocrinology and Metabolism, Berlin, Germany
- Berlin Institute of Health at Charité–Universitätsmedizin Berlin, BCRT - Berlin Institute of Health Center for Regenerative Therapies, Berlin, Germany
| | - Alexander Hartmann
- Institute of Clinical Chemistry and Laboratory Medicine, Rostock University Medical Center, University of Rostock, Rostock, Germany
| | - Christiane Hartmann
- Translational Neurodegeneration Section “Albrecht-Kossel”, Department of Neurology, Rostock University Medical Center, University of Rostock, 18147 Rostock, Germany
| | - Felix Wittig
- Institute of Pharmacology and Toxicology, Rostock University Medical Center, University of Rostock, Rostock, Germany
| | - Anje Sporbert
- Advanced Light Microscopy, Max Delbrück Center for Molecular Medicine, Berlin, Germany
| | - Andreas Hermann
- Translational Neurodegeneration Section “Albrecht-Kossel”, Department of Neurology, Rostock University Medical Center, University of Rostock, 18147 Rostock, Germany
- Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE) Rostock/Greifswald, Rostock, Germany
- Center for Transdisciplinary Neurosciences Rostock (CTNR), University Medical Center Rostock, Rostock, Germany
| | - Georg Fuellen
- Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock University Medical Center, Rostock, Germany
| | - Steffen Möller
- Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock University Medical Center, Rostock, Germany
| | - Michael Walter
- Institute of Clinical Chemistry and Laboratory Medicine, Rostock University Medical Center, University of Rostock, Rostock, Germany
- Charité–Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Berlin, Germany
- Corresponding author.
| |
Collapse
|
|
10
|
Iacomi DM, Rosca AM, Tutuianu R, Neagu TP, Pruna V, Simionescu M, Titorencu I. Generation of an Immortalized Human Adipose-Derived Mesenchymal Stromal Cell Line Suitable for Wound Healing Therapy. Int J Mol Sci 2022; 23:ijms23168925. [PMID: 36012192 PMCID: PMC9408591 DOI: 10.3390/ijms23168925] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2022] [Revised: 07/29/2022] [Accepted: 08/06/2022] [Indexed: 11/25/2022] Open
Abstract
Adipose-derived mesenchymal stromal cells (ADSC) are a promising source for cellular therapy of chronic wounds. However, the limited life span during in vitro expansion impedes their extensive use in clinical applications and basic research. We hypothesize that by introduction of an ectopic expression of telomerase into ADSC, the cells’ lifespans could be significantly extended. To test this hypothesis, we aimed at engineering an immortalized human ADSC line using a lentiviral transduction with human telomerase (hTERT). ADSC were transduced with a third-generation lentiviral system and a hTERT codifying plasmid (pLV-hTERT-IRES-hygro). A population characterized by increased hTERT expression, extensive proliferative potential and remarkable (potent) multilineage differentiation capacity was selected. The properties for wound healing of this immortalized ADSC line were assessed after 17 passages. Their secretome induced the proliferation and migration of keratinocytes, dermal fibroblasts, and endothelial cells similarly to untransduced ADSC. Moreover, they sustained the complete re-epithelialization of a full thickness wound performed on a skin organotypic model. In summary, the engineered immortalized ADSC maintain the beneficial properties of parent cells and could represent a valuable and suitable tool for wound healing in particular, and for skin regenerative therapy in general.
Collapse
Affiliation(s)
- Daniela-Madalina Iacomi
- Cell and Tissue Engineering Laboratory, “Nicolae Simionescu” Institute of Cellular Biology and Pathology, 050568 Bucharest, Romania
| | - Ana-Maria Rosca
- Cell and Tissue Engineering Laboratory, “Nicolae Simionescu” Institute of Cellular Biology and Pathology, 050568 Bucharest, Romania
- Correspondence:
| | - Raluca Tutuianu
- Cell and Tissue Engineering Laboratory, “Nicolae Simionescu” Institute of Cellular Biology and Pathology, 050568 Bucharest, Romania
| | - Tiberiu Paul Neagu
- Clinical Department No. 11, “Carol Davila” University of Medicine and Pharmacy, 050474 Bucharest, Romania
| | - Vasile Pruna
- Cell and Tissue Engineering Laboratory, “Nicolae Simionescu” Institute of Cellular Biology and Pathology, 050568 Bucharest, Romania
| | - Maya Simionescu
- Cell and Tissue Engineering Laboratory, “Nicolae Simionescu” Institute of Cellular Biology and Pathology, 050568 Bucharest, Romania
| | - Irina Titorencu
- Cell and Tissue Engineering Laboratory, “Nicolae Simionescu” Institute of Cellular Biology and Pathology, 050568 Bucharest, Romania
| |
Collapse
|
|
11
|
Thakkar MK, Lee J, Meyer S, Chang VY. RecQ Helicase Somatic Alterations in Cancer. Front Mol Biosci 2022; 9:887758. [PMID: 35782872 PMCID: PMC9240438 DOI: 10.3389/fmolb.2022.887758] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2022] [Accepted: 05/23/2022] [Indexed: 11/13/2022] Open
Abstract
Named the “caretakers” of the genome, RecQ helicases function in several pathways to maintain genomic stability and repair DNA. This highly conserved family of enzymes consist of five different proteins in humans: RECQL1, BLM, WRN, RECQL4, and RECQL5. Biallelic germline mutations in BLM, WRN, and RECQL4 have been linked to rare cancer-predisposing syndromes. Emerging research has also implicated somatic alterations in RecQ helicases in a variety of cancers, including hematological malignancies, breast cancer, osteosarcoma, amongst others. These alterations in RecQ helicases, particularly overexpression, may lead to increased resistance of cancer cells to conventional chemotherapy. Downregulation of these proteins may allow for increased sensitivity to chemotherapy, and, therefore, may be important therapeutic targets. Here we provide a comprehensive review of our current understanding of the role of RecQ DNA helicases in cancer and discuss the potential therapeutic opportunities in targeting these helicases.
Collapse
Affiliation(s)
- Megha K. Thakkar
- Department of Pediatrics, Division of Pediatric Hematology-Oncology, University of California, Los Angeles, Los Angeles, CA, United States
| | - Jamie Lee
- Department of Pediatrics, Division of Pediatric Hematology-Oncology, University of California, Los Angeles, Los Angeles, CA, United States
| | - Stefan Meyer
- Division of Cancer Studies, University of Manchester, Manchester, United Kingdom
- Department of Pediatric Hematology Oncology, Royal Manchester Children’s Hospital and Christie NHS Foundation Trust, Manchester, United Kingdom
| | - Vivian Y. Chang
- Department of Pediatrics, Division of Pediatric Hematology-Oncology, University of California, Los Angeles, Los Angeles, CA, United States
- Childrens Discovery and Innovation Institute, UCLA, Los Angeles, CA, United States
- Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, United States
- *Correspondence: Vivian Y. Chang,
| |
Collapse
|
|
12
|
Kumar N, Theil AF, Roginskaya V, Ali Y, Calderon M, Watkins SC, Barnes RP, Opresko PL, Pines A, Lans H, Vermeulen W, Van Houten B. Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins. Nat Commun 2022; 13:974. [PMID: 35190564 PMCID: PMC8861037 DOI: 10.1038/s41467-022-28642-9] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2021] [Accepted: 02/03/2022] [Indexed: 12/17/2022] Open
Abstract
UV-DDB, consisting of subunits DDB1 and DDB2, recognizes UV-induced photoproducts during global genome nucleotide excision repair (GG-NER). We recently demonstrated a noncanonical role of UV-DDB in stimulating base excision repair (BER) which raised several questions about the timing of UV-DDB arrival at 8-oxoguanine (8-oxoG), and the dependency of UV-DDB on the recruitment of downstream BER and NER proteins. Using two different approaches to introduce 8-oxoG in cells, we show that DDB2 is recruited to 8-oxoG immediately after damage and colocalizes with 8-oxoG glycosylase (OGG1) at sites of repair. 8-oxoG removal and OGG1 recruitment is significantly reduced in the absence of DDB2. NER proteins, XPA and XPC, also accumulate at 8-oxoG. While XPC recruitment is dependent on DDB2, XPA recruitment is DDB2-independent and transcription-coupled. Finally, DDB2 accumulation at 8-oxoG induces local chromatin unfolding. We propose that DDB2-mediated chromatin decompaction facilitates the recruitment of downstream BER proteins to 8-oxoG lesions. Nucleotide excision repair proteins are involved in the repair of UV-induced DNA damage. Here, the authors show that NER proteins, DDB2, XPC, and XPA play a vital role in the 8-oxoguanine repair by coordinating with base excision repair protein OGG1.
Collapse
|
|
13
|
Srinivasan G, Brafman DA. The Emergence of Model Systems to Investigate the Link Between Traumatic Brain Injury and Alzheimer's Disease. Front Aging Neurosci 2022; 13:813544. [PMID: 35211003 PMCID: PMC8862182 DOI: 10.3389/fnagi.2021.813544] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2021] [Accepted: 12/20/2021] [Indexed: 12/12/2022] Open
Abstract
Numerous epidemiological studies have demonstrated that individuals who have sustained a traumatic brain injury (TBI) have an elevated risk for developing Alzheimer's disease and Alzheimer's-related dementias (AD/ADRD). Despite these connections, the underlying mechanisms by which TBI induces AD-related pathology, neuronal dysfunction, and cognitive decline have yet to be elucidated. In this review, we will discuss the various in vivo and in vitro models that are being employed to provide more definite mechanistic relationships between TBI-induced mechanical injury and AD-related phenotypes. In particular, we will highlight the strengths and weaknesses of each of these model systems as it relates to advancing the understanding of the mechanisms that lead to TBI-induced AD onset and progression as well as providing platforms to evaluate potential therapies. Finally, we will discuss how emerging methods including the use of human induced pluripotent stem cell (hiPSC)-derived cultures and genome engineering technologies can be employed to generate better models of TBI-induced AD.
Collapse
Affiliation(s)
| | - David A. Brafman
- School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ, United States
| |
Collapse
|
|
14
|
Van Zundert I, Bravo M, Deschaume O, Cybulski P, Bartic C, Hofkens J, Uji-i H, Fortuni B, Rocha S. Versatile and Robust Method for Antibody Conjugation to Nanoparticles with High Targeting Efficiency. Pharmaceutics 2021; 13:2153. [PMID: 34959436 PMCID: PMC8703776 DOI: 10.3390/pharmaceutics13122153] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2021] [Revised: 12/07/2021] [Accepted: 12/08/2021] [Indexed: 02/07/2023] Open
Abstract
The application of antibodies in nanomedicine is now standard practice in research since it represents an innovative approach to deliver chemotherapy agents selectively to tumors. The variety of targets or markers that are overexpressed in different types of cancers results in a high demand for antibody conjugated-nanoparticles, which are versatile and easily customizable. Considering up-scaling, the synthesis of antibody-conjugated nanoparticles should be simple and highly reproducible. Here, we developed a facile coating strategy to produce antibody-conjugated nanoparticles using 'click chemistry' and further evaluated their selectivity towards cancer cells expressing different markers. Our approach was consistently repeated for the conjugation of antibodies against CD44 and EGFR, which are prominent cancer cell markers. The functionalized particles presented excellent cell specificity towards CD44 and EGFR overexpressing cells, respectively. Our results indicated that the developed coating method is reproducible, versatile, and non-toxic, and can be used for particle functionalization with different antibodies. This grafting strategy can be applied to a wide range of nanoparticles and will contribute to the development of future targeted drug delivery systems.
Collapse
Affiliation(s)
- Indra Van Zundert
- Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium; (I.V.Z.); (M.B.); (P.C.); (J.H.); (H.U.-i.)
| | - Maria Bravo
- Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium; (I.V.Z.); (M.B.); (P.C.); (J.H.); (H.U.-i.)
| | - Olivier Deschaume
- Soft-Matter Physics and Biophysics, Department of Physics and Astronomy, KU Leuven, Celestijnenlaan 200D, Box 2416, 3001 Heverlee, Belgium; (O.D.); (C.B.)
| | - Pierre Cybulski
- Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium; (I.V.Z.); (M.B.); (P.C.); (J.H.); (H.U.-i.)
| | - Carmen Bartic
- Soft-Matter Physics and Biophysics, Department of Physics and Astronomy, KU Leuven, Celestijnenlaan 200D, Box 2416, 3001 Heverlee, Belgium; (O.D.); (C.B.)
| | - Johan Hofkens
- Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium; (I.V.Z.); (M.B.); (P.C.); (J.H.); (H.U.-i.)
| | - Hiroshi Uji-i
- Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium; (I.V.Z.); (M.B.); (P.C.); (J.H.); (H.U.-i.)
- Research Institute for Electronic Science (RIES), Hokkaido University, N20W10, Kita Ward, Sapporo 001-0020, Japan
| | - Beatrice Fortuni
- Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium; (I.V.Z.); (M.B.); (P.C.); (J.H.); (H.U.-i.)
| | - Susana Rocha
- Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium; (I.V.Z.); (M.B.); (P.C.); (J.H.); (H.U.-i.)
| |
Collapse
|
|
15
|
The role of telomerase in the etiology of primary spontaneous pneumothorax. TURK GOGUS KALP DAMAR CERRAHISI DERGISI-TURKISH JOURNAL OF THORACIC AND CARDIOVASCULAR SURGERY 2021; 29:377-383. [PMID: 34589257 PMCID: PMC8462098 DOI: 10.5606/tgkdc.dergisi.2021.20522] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/23/2020] [Accepted: 11/09/2020] [Indexed: 11/25/2022]
Abstract
Background
This study aims to investigate the role of telomerase activity in the risk of primary spontaneous pneumothorax, which is most frequently encountered in the practice of thoracic surgery.
Methods
A total of 61 patients (56 males, 5 females; median age: 29.4 years; range, 17 to 43 years) who underwent treatment for primary spontaneous pneumothorax and 19 age- and sex-matched healthy controls (10 males, 9 females; median age: 29.1 years; range, 23 to 43 years) were included in this prospective study between January 2018 - August 2018. Telomerase activity was evaluated with enzyme-linked immunosorbent assay. The correlation between telomerase activity and clinical and demographic parameters was examined.
Results
The mean serum telomerase level was 3.4±0.6 ng/mL in the primary spontaneous pneumothorax group and 1.9±0.5 ng/mL in the control group, indicating significantly higher levels in the patient group (p<0.001). There was no significant association between the telomerase levels and presence of blebs and/or bullae on thoracic computed tomography, extent of pneumothorax, laterality (right, left, or bilateral), and pack years of cigarette smoking.
Conclusion
Telomerase levels of patients with primary spontaneous pneumothorax are significantly higher than healthy individuals. Future genetic studies may ultimately clarify a potential relationship between primary spontaneous pneumothorax and short telomere syndrome.
Collapse
|
|
16
|
Gandullo-Sánchez L, Ocaña A, Pandiella A. Generation of Antibody-Drug Conjugate Resistant Models. Cancers (Basel) 2021; 13:cancers13184631. [PMID: 34572858 PMCID: PMC8466899 DOI: 10.3390/cancers13184631] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2021] [Revised: 09/07/2021] [Accepted: 09/10/2021] [Indexed: 12/17/2022] Open
Abstract
Simple Summary Antibody-drug conjugates (ADCs) constitute new and effective therapies in cancer. However, resistance is frequently observed in treated patients after a given period of time. That resistance may be present from the beginning of the treatment (primary or de novo resistance) or raise after an initial response to the ADC (secondary resistance). Knowing the causes of those resistances is a necessity in the field as it may help in designing strategies to overcome them. Because of that, it is necessary to develop models that allow the identification of mechanisms of resistance. In this review, we present different approaches that have been used to model ADC resistance in the preclinical setting, and that include the use of established cell lines, patient-derived ex vivo cultures and xenografts primarily or secondarily resistant to the ADC. Abstract In the last 20 years, antibody-drug conjugates (ADCs) have been incorporated into the oncology clinic as treatments for several types of cancer. So far, the Food and Drug Administration (FDA) has approved 11 ADCs and other ADCs are in the late stages of clinical development. Despite the efficacy of this type of drug, the tumors of some patients may result in resistance to ADCs. Due to this, it is essential not only to comprehend resistance mechanisms but also to develop strategies to overcome resistance to ADCs. To reach these goals, the generation and use of preclinical models to study those mechanisms of resistance are critical. Some cells or patient tumors may result in primary resistance to the action of an ADC, even if they express the antigen against which the ADC is directed. Isolated primary tumoral cells, cell lines, or patient explants (patient-derived xenografts) with these characteristics can be used to study primary resistance. The most common method to generate models of secondary resistance is to treat cancer cell lines or tumors with an ADC. Two strategies, either continuous treatment with the ADC or intermittent treatment, have successfully been used to develop those resistance models.
Collapse
Affiliation(s)
- Lucía Gandullo-Sánchez
- Instituto de Biología Molecular y Celular del Cáncer, CSIC, IBSAL and CIBERONC, 37007 Salamanca, Spain;
| | - Alberto Ocaña
- Hospital Clínico San Carlos, 28040 Madrid, Spain;
- Symphogen, DK-2750 Ballerup, Denmark
| | - Atanasio Pandiella
- Instituto de Biología Molecular y Celular del Cáncer, CSIC, IBSAL and CIBERONC, 37007 Salamanca, Spain;
- Correspondence: ; Tel.: +34-923-294-815
| |
Collapse
|
|
17
|
Stefanovic B, Stefanovic L, Manojlovic Z. Imaging of type I procollagen biosynthesis in cells reveals biogenesis in highly organized bodies; Collagenosomes. Matrix Biol Plus 2021; 12:100076. [PMID: 34278289 PMCID: PMC8261018 DOI: 10.1016/j.mbplus.2021.100076] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2021] [Revised: 06/18/2021] [Accepted: 06/18/2021] [Indexed: 10/26/2022] Open
Abstract
Mechanistic aspects of type I procollagen biosynthesis in cells are poorly understood. To provide more insight into this process we designed a system to directly image type I procollagen biogenesis by co-expression of fluorescently labeled full size procollagen α1(I) and one α2(I) polypeptides. High resolution images show that collagen α1(I) and α2(I) polypeptides are produced in coordination in discrete structures on the ER membrane, which we termed the collagenosomes. Collagenosomes are disk shaped bodies, 0.5-1 μM in diameter and 200-400 nm thick, in the core of which folding of procollagen takes place. Collagenosomes are intimately associated with the ER membrane and their formation requires intact translational machinery, suggesting that they are the sites of nascent procollagen biogenesis. Collagenosomes show little co-localization with the COPII transport vesicles, which export type I procollagen from the ER, suggesting that these two structures are distinct. LARP6 is the protein which regulates translation of type I collagen mRNAs. The characteristic organization of collagenosomes depends on binding of LARP6 to collagen mRNAs. Without LARP6 regulation, collagenosomes are poorly organized and the folding of α1(I) and α2(I) polypeptides into procollagen in their cores is diminished. This indicates that formation of collagenosomes is dependent on regulated translation of collagen mRNAs. In live cells the size, number and shape of collagenosomes show little change within several hours, suggesting that they are stable structures of type I procollagen biogenesis. This is the first report of structural organization of type I collagen biogenesis in collagenosomes, while the fluorescent reporter system based on simultaneous imaging of both type I collagen polypeptides will enable the detailed elucidation of their structure and function.
Collapse
Affiliation(s)
- Branko Stefanovic
- Department of Biomedical Sciences and Translational Science Laboratory, College of Medicine, Florida State University, 1115 West Call Street, Tallahassee, FL 32306, USA
| | - Lela Stefanovic
- Department of Biomedical Sciences and Translational Science Laboratory, College of Medicine, Florida State University, 1115 West Call Street, Tallahassee, FL 32306, USA
| | - Zarko Manojlovic
- Keck School of Medicine of University of Southern California, 1450 Biggy Street, NRT 4510, Los Angeles, CA 90033, USA
| |
Collapse
|
|
18
|
Clegg MH, Harris TI, Zhang X, Barney JT, Jones JA, Vargis E. Silkworm Silk Fiber Bundles as Improved In Vitro Scaffolds for Skeletal Muscle. ACS Biomater Sci Eng 2020; 6:6853-6863. [PMID: 33320626 DOI: 10.1021/acsbiomaterials.0c00987] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
To mimic skeletal muscle tissues in vitro, native and transgenic spider silk/silkworm silks were seeded with C2C12 myoblasts to observe if these three-dimensional substrates are preferable to a traditional two-dimensional polystyrene cell culture surface. Silks were wound around an acrylic chassis to produce a novel, three-dimensional cell culture device with suspended muscle fibers that genetically and morphologically resemble native skeletal muscle tissue. The transgenic spider silk/silkworm silk has never before been studied for this application. Genetic expression verified skeletal muscle lineage and differentiation, while fluorescent imaging verified contractile protein synthesis. Genetic analysis also revealed an increase in expression of the Myh2 contractile protein gene on silkworm silks, particularly on the transgenic silk. Mechanical properties and protein secondary structure content of the silks indicated correlation between substrate properties and Myh2 gene expression. This increase in contractile protein gene expression suggests that biologically derived silk substrates that are suspended may be a preferable substrate for in vitro muscle modeling because of the proteinaceous character and mechanical flexibility of the silk.
Collapse
Affiliation(s)
- Matthew H Clegg
- Department of Biological Engineering, Utah State University, Logan, Utah 84322, United States
| | - Thomas I Harris
- Department of Biology, Utah State University, Logan, Utah 84322, United States
| | - Xiaoli Zhang
- Department of Biology, Utah State University, Logan, Utah 84322, United States
| | - Jacob T Barney
- Department of Biological Engineering, Utah State University, Logan, Utah 84322, United States
| | - Justin A Jones
- Department of Biology, Utah State University, Logan, Utah 84322, United States
| | - Elizabeth Vargis
- Department of Biological Engineering, Utah State University, Logan, Utah 84322, United States
| |
Collapse
|
|
19
|
Urwin L, Okurowska K, Crowther G, Roy S, Garg P, Karunakaran E, MacNeil S, Partridge LJ, Green LR, Monk PN. Corneal Infection Models: Tools to Investigate the Role of Biofilms in Bacterial Keratitis. Cells 2020; 9:E2450. [PMID: 33182687 PMCID: PMC7696224 DOI: 10.3390/cells9112450] [Citation(s) in RCA: 42] [Impact Index Per Article: 8.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2020] [Revised: 11/06/2020] [Accepted: 11/07/2020] [Indexed: 12/15/2022] Open
Abstract
Bacterial keratitis is a corneal infection which may cause visual impairment or even loss of the infected eye. It remains a major cause of blindness in the developing world. Staphylococcus aureus and Pseudomonas aeruginosa are common causative agents and these bacterial species are known to colonise the corneal surface as biofilm populations. Biofilms are complex bacterial communities encased in an extracellular polymeric matrix and are notoriously difficult to eradicate once established. Biofilm bacteria exhibit different phenotypic characteristics from their planktonic counterparts, including an increased resistance to antibiotics and the host immune response. Therefore, understanding the role of biofilms will be essential in the development of new ophthalmic antimicrobials. A brief overview of biofilm-specific resistance mechanisms is provided, but this is a highly multifactorial and rapidly expanding field that warrants further research. Progression in this field is dependent on the development of suitable biofilm models that acknowledge the complexity of the ocular environment. Abiotic models of biofilm formation (where biofilms are studied on non-living surfaces) currently dominate the literature, but co-culture infection models are beginning to emerge. In vitro, ex vivo and in vivo corneal infection models have now been reported which use a variety of different experimental techniques and animal models. In this review, we will discuss existing corneal infection models and their application in the study of biofilms and host-pathogen interactions at the corneal surface.
Collapse
Affiliation(s)
- Lucy Urwin
- Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield S10 2RX, UK; (L.R.G.); (P.N.M.)
| | - Katarzyna Okurowska
- Department of Chemical and Biological Engineering, University of Sheffield, Sheffield S1 3JD, UK; (K.O.); (G.C.); (E.K.)
- Sheffield Collaboratorium for Antimicrobial Resistance and Biofilms (SCARAB), University of Sheffield, Sheffield S1 3JD, UK; (S.M.); (L.J.P.)
| | - Grace Crowther
- Department of Chemical and Biological Engineering, University of Sheffield, Sheffield S1 3JD, UK; (K.O.); (G.C.); (E.K.)
- Sheffield Collaboratorium for Antimicrobial Resistance and Biofilms (SCARAB), University of Sheffield, Sheffield S1 3JD, UK; (S.M.); (L.J.P.)
| | - Sanhita Roy
- Prof. Brien Holden Eye Research Centre, LV Prasad Eye Institute, Hyderabad 500034, India; (S.R.); (P.G.)
| | - Prashant Garg
- Prof. Brien Holden Eye Research Centre, LV Prasad Eye Institute, Hyderabad 500034, India; (S.R.); (P.G.)
| | - Esther Karunakaran
- Department of Chemical and Biological Engineering, University of Sheffield, Sheffield S1 3JD, UK; (K.O.); (G.C.); (E.K.)
- Sheffield Collaboratorium for Antimicrobial Resistance and Biofilms (SCARAB), University of Sheffield, Sheffield S1 3JD, UK; (S.M.); (L.J.P.)
| | - Sheila MacNeil
- Sheffield Collaboratorium for Antimicrobial Resistance and Biofilms (SCARAB), University of Sheffield, Sheffield S1 3JD, UK; (S.M.); (L.J.P.)
- Department of Materials Science and Engineering, University of Sheffield, Sheffield S1 3JD, UK
| | - Lynda J. Partridge
- Sheffield Collaboratorium for Antimicrobial Resistance and Biofilms (SCARAB), University of Sheffield, Sheffield S1 3JD, UK; (S.M.); (L.J.P.)
- Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK
| | - Luke R. Green
- Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield S10 2RX, UK; (L.R.G.); (P.N.M.)
| | - Peter N. Monk
- Department of Infection, Immunity and Cardiovascular Disease, University of Sheffield, Sheffield S10 2RX, UK; (L.R.G.); (P.N.M.)
- Sheffield Collaboratorium for Antimicrobial Resistance and Biofilms (SCARAB), University of Sheffield, Sheffield S1 3JD, UK; (S.M.); (L.J.P.)
| |
Collapse
|
|
20
|
Bondos SE, Geraldo Mendes G, Jons A. Context-dependent HOX transcription factor function in health and disease. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2020; 174:225-262. [PMID: 32828467 DOI: 10.1016/bs.pmbts.2020.05.003] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
During animal development, HOX transcription factors determine the fate of developing tissues to generate diverse organs and appendages. The power of these proteins is striking: mis-expressing a HOX protein causes homeotic transformation of one body part into another. During development, HOX proteins interpret their cellular context through protein interactions, alternative splicing, and post-translational modifications to regulate cell proliferation, cell death, cell migration, cell differentiation, and angiogenesis. Although mutation and/or mis-expression of HOX proteins during development can be lethal, changes in HOX proteins that do not pattern vital organs can result in survivable malformations. In adults, mutation and/or mis-expression of HOX proteins disrupts their gene regulatory networks, deregulating cell behaviors and leading to arthritis and cancer. On the molecular level, HOX proteins are composed of DNA binding homeodomain, and large regions of unstructured, or intrinsically disordered, protein sequence. The primary roles of HOX proteins in arthritis and cancer suggest that mutations associated with these diseases in both the structured and disordered regions of HOX proteins can have substantial functional effects. These insights lead to new questions critical for understanding and manipulating HOX function in physiological and pathological conditions.
Collapse
Affiliation(s)
- Sarah E Bondos
- Department of Molecular and Cellular Medicine, Texas A&M University, College Station, TX, United States.
| | - Gabriela Geraldo Mendes
- Department of Molecular and Cellular Medicine, Texas A&M University, College Station, TX, United States
| | - Amanda Jons
- Department of Molecular and Cellular Medicine, Texas A&M University, College Station, TX, United States
| |
Collapse
|
|
21
|
Kim H, Sung J, Kim H, Ryu H, Cho Park H, Oh YK, Lee HS, Oh KH, Ahn C. Expression and secretion of CXCL12 are enhanced in autosomal dominant polycystic kidney disease. BMB Rep 2020. [PMID: 31186083 PMCID: PMC6675246 DOI: 10.5483/bmbrep.2019.52.7.112] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023] Open
Abstract
Autosomal dominant polycystic kidney disease (ADPKD), one of the most common human monogenic diseases (frequency of 1/1000-1/400), is characterized by numerous fluid-filled renal cysts (RCs). Inactivation of the PKD1 or PKD2 gene by germline and somatic mutations is necessary for cyst formation in ADPKD. To mechanistically understand cyst formation and growth, we isolated RCs from Korean patients with ADPKD and immortalized them with human telomerase reverse transcriptase (hTERT). Three hTERT-immortalized RC cell lines were characterized as proximal epithelial cells with germline and somatic PKD1 mutations. Thus, we first established hTERT-immortalized proximal cyst cells with somatic PKD1 mutations. Through transcriptome sequencing and Gene Ontology (GO) analysis, we found that upregulated genes were related to cell division and that downregulated genes were related to cell differentiation. We wondered whether the upregulated gene for the chemokine CXCL12 is related to the mTOR signaling pathway in cyst growth in ADPKD. CXCL12 mRNA expression and secretion were increased in RC cell lines. We then examined CXCL12 levels in RC fluids from patients with ADPKD and found increased CXCL12 levels. The CXCL12 receptor CXC chemokine receptor 4 (CXCR4) was upregulated, and the mTOR signaling pathway, which is downstream of the CXCL12/CXCR4 axis, was activated in ADPKD kidney tissue. To confirm activation of the mTOR signaling pathway by CXCL12 via CXCR4, we treated the RC cell lines with recombinant CXCL12 and the CXCR4 antagonist AMD3100; CXCL12 induced the mTOR signaling pathway, but the CXCR4 antagonist AMD3100 blocked the mTOR signaling pathway. Taken together, these results suggest that enhanced CXCL12 in RC fluids activates the mTOR signaling pathway via CXCR4 in ADPKD cyst growth.
Collapse
Affiliation(s)
- Hyunho Kim
- Center for Medical Innovation, Biomedical Research Institute, Seoul National University Hospital, Seoul 03082, Korea
| | - Jinmo Sung
- Center for Medical Innovation, Biomedical Research Institute, Seoul National University Hospital, Seoul 03082, Korea
| | - Hyunsuk Kim
- Internal Medicine, Hallym University Medical Center, Chuncheon Sacred Heart Hospital, Chuncheon 24253, Korea
| | - Hyunjin Ryu
- Department of Internal Medicine, Seoul National University College of Medicine, Seoul 03080, Korea
| | - Hayne Cho Park
- Department of Internal Medicine, Hallym University Medical Center, Kangnam Sacred Heart Hospital, Seoul 07441, Korea
| | - Yun Kyu Oh
- Department of Internal Medicine, Seoul National University Boramae Medical Center, Seoul 07061, Korea
| | - Hyun-Seob Lee
- Genomics Core Facility, Department of Transdisciplinary Research and Collaboration, Biomedical Research Institute, Seoul National University Hospital, Seoul 03082, Korea
| | - Kook-Hwan Oh
- Department of Internal Medicine, Seoul National University College of Medicine, Seoul 03080, Korea
| | - Curie Ahn
- Department of Internal Medicine, Seoul National University College of Medicine, Seoul 03080, Korea
| |
Collapse
|
|
22
|
Abstract
The ability to use human cells in biomedical research and testing has the obvious advantage over the use of laboratory animals that the need for species extrapolation is obviated, due to the presence of more-relevant morphological, physiological and biochemical properties, including receptors. Moreover, human cells exhibit the same advantages as animal cells in culture in that different cell types can be used, from different tissues, with a wide range of techniques, to investigate a wide variety of biological phenomena in tissue culture. Human cells can also be grown as organotypic cultures to facilitate the extrapolation from cells to whole organisms. Human cell lines have been available for many years on an ad hoc basis from individual researchers, and also from recognised sources, such as the European Collection of Animal Cell Cultures (ECACC) and, in the USA, the Human Cell Culture Centre (HCCC). Such cells have usually been derived from tumours and this has restricted the variety of types of cells available. This problem has been addressed by using primary human cells that can be obtained from a variety of sources, such as cadavers, diseased tissue, skin strips, peripheral blood, buccal cavity smears, hair follicles and surgical waste from biopsy material that is unsuitable for transplantation purposes. However, primary human cells need to be obtained, processed, distributed and handled in a safe and ethical manner. They also have to be made available at the correct time to researchers very shortly after they become available. It is only comparatively recently that the safe and controlled acquisition of surgical waste and non-transplantable human tissues has become feasible with the establishment of several human tissue banks. Recently, the formation of a UK and European centralised network for human tissue supply has been initiated. The problems of short longevity and loss of specialisation in culture are being approached by: a) cell immortalisation to generate a cell type possessing the properties of both primary cells and cell lines; b) the inhibition of intracellular activities resulting in oxidative stress; and c) the use of stem cells, both of embryonic and adult origin.
Collapse
Affiliation(s)
- Robert D. Combes
- FRAME, Russell & Burch House, 96–98 North Sherwood Street, Nottingham NG1 4EE, UK
| |
Collapse
|
|
23
|
Stefanovic B, Manojlovic Z, Vied C, Badger CD, Stefanovic L. Discovery and evaluation of inhibitor of LARP6 as specific antifibrotic compound. Sci Rep 2019; 9:326. [PMID: 30674965 PMCID: PMC6344531 DOI: 10.1038/s41598-018-36841-y] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2018] [Accepted: 11/27/2018] [Indexed: 01/17/2023] Open
Abstract
Fibrosis is characterized by excessive production of type I collagen. Biosynthesis of type I collagen in fibrosis is augmented by binding of protein LARP6 to the 5' stem-loop structure (5'SL), which is found exclusively in type I collagen mRNAs. A high throughput screen was performed to discover inhibitors of LARP6 binding to 5'SL, as potential antifibrotic drugs. The screen yielded one compound (C9) which was able to dissociate LARP6 from 5' SL RNA in vitro and to inactivate the binding of endogenous LARP6 in cells. Treatment of hepatic stellate cells (liver cells responsible for fibrosis) with nM concentrations of C9 reduced secretion of type I collagen. In precision cut liver slices, as an ex vivo model of hepatic fibrosis, C9 attenuated the profibrotic response at 1 μM. In prophylactic and therapeutic animal models of hepatic fibrosis C9 prevented development of fibrosis or hindered the progression of ongoing fibrosis when administered at 1 mg/kg. Toxicogenetics analysis revealed that only 42 liver genes changed expression after administration of C9 for 4 weeks, suggesting minimal off target effects. Based on these results, C9 represents the first LARP6 inhibitor with significant antifibrotic activity.
Collapse
Affiliation(s)
- Branko Stefanovic
- Department of Biomedical Sciences, College of Medicine, Florida State University, 1115 West Call Street, Tallahassee, FL, 32306, USA.
| | - Zarko Manojlovic
- Keck School of Medicine of University of Southern California, 1450 Biggy Street, NRT 4510, Los Angeles, CA, 90033, USA
| | - Cynthia Vied
- Translational Science Laboratory, College of Medicine, Florida State University, 1115 West Call Street, Tallahassee, FL, 32306, USA
| | - Crystal-Dawn Badger
- Translational Science Laboratory, College of Medicine, Florida State University, 1115 West Call Street, Tallahassee, FL, 32306, USA
- Proteomics and Metabolomics Facility, Colorado State University, 401 West Pitkin Street, Fort Collins, CO, 80521, USA
| | - Lela Stefanovic
- Department of Biomedical Sciences, College of Medicine, Florida State University, 1115 West Call Street, Tallahassee, FL, 32306, USA
| |
Collapse
|
|
24
|
Burkard M, Bengtson Nash S, Gambaro G, Whitworth D, Schirmer K. Lifetime extension of humpback whale skin fibroblasts and their response to lipopolysaccharide (LPS) and a mixture of polychlorinated biphenyls (Aroclor). Cell Biol Toxicol 2019; 35:387-398. [PMID: 30627956 PMCID: PMC6757015 DOI: 10.1007/s10565-018-09457-1] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2018] [Accepted: 12/18/2018] [Indexed: 01/02/2023]
Abstract
Marine mammals, such as whales, have a high proportion of body fat and so are susceptible to the accumulation, and associated detrimental health effects, of lipophilic environmental contaminants. Recently, we developed a wild-type cell line from humpback whale fibroblasts (HuWa). Extensive molecular assessments with mammalian wild-type cells are typically constrained by a finite life span, with cells eventually becoming senescent. Thus, the present work explored the possibility of preventing senescence in the HuWa cell line by transfection with plasmids encoding the simian virus large T antigen (SV40T) or telomerase reverse transcriptase (TERT). No stable expression was achieved upon SV40 transfection. Transfection with TERT, on the other hand, activated the expression of telomerase in HuWa cells. At the time of manuscript preparation, the transfected HuWa cells (HuWaTERT) have been stable for at least 59 passages post-transfection. HuWaTERT proliferate rapidly and maintain initial cell characteristics, such as morphology and chromosomal stability. The response of HuWaTERT cells to an immune stimulant (lipopolysaccharide (LPS)) and an immunotoxicant (Aroclor1254) was assessed by measurement of intracellular levels of the pro-inflammatory cytokines interleukin (IL)-6, IL-1β and tumour necrosis factor (TNF)-α. HuWaTERT cells constitutively express IL-6, IL-1β and TNFα. Exposure to neither LPS nor Aroclor1254 had an effect on the levels of these cytokines. Overall, this work supports the diverse applicability of HuWa cell lines in that they display reliable long-term preservation, susceptibility to exogenous gene transfer and enable the study of humpback whale-specific cellular response mechanisms.
Collapse
Affiliation(s)
- Michael Burkard
- School of Environment and Science, Griffith University, Brisbane, QLD, Australia.,Swiss Federal Institute of Aquatic Science and Technology (Eawag), Überlandstrasse 133, CH-8600, Dübendorf, Switzerland
| | - Susan Bengtson Nash
- School of Environment and Science, Griffith University, Brisbane, QLD, Australia
| | - Gessica Gambaro
- Swiss Federal Institute of Aquatic Science and Technology (Eawag), Überlandstrasse 133, CH-8600, Dübendorf, Switzerland
| | - Deanne Whitworth
- School of Veterinary Science, The University of Queensland, Gatton, QLD, Australia.,Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD, Australia
| | - Kristin Schirmer
- Swiss Federal Institute of Aquatic Science and Technology (Eawag), Überlandstrasse 133, CH-8600, Dübendorf, Switzerland. .,Institute of Biogechemistry and Pollutant Dynamics, ETH Zürich, Zürich, Switzerland. .,School of Architecture, Civil and Environmental Engineering, EPF Lausanne, Lausanne, Switzerland.
| |
Collapse
|
|
25
|
Bikkul MU, Faragher RGA, Worthington G, Meinke P, Kerr ARW, Sammy A, Riyahi K, Horton D, Schirmer EC, Hubank M, Kill IR, Anderson RM, Slijepcevic P, Makarov E, Bridger JM. Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson-Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform. Genes Chromosomes Cancer 2019; 58:341-356. [PMID: 30474255 PMCID: PMC6590296 DOI: 10.1002/gcc.22711] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2018] [Revised: 11/06/2018] [Accepted: 11/22/2018] [Indexed: 02/06/2023] Open
Abstract
Immortalizing primary cells with human telomerase reverse transcriptase (hTERT) has been common practice to enable primary cells to be of extended use in the laboratory because they avoid replicative senescence. Studying exogenously expressed hTERT in cells also affords scientists models of early carcinogenesis and telomere behavior. Control and the premature ageing disease—Hutchinson‐Gilford progeria syndrome (HGPS) primary dermal fibroblasts, with and without the classical G608G mutation have been immortalized with exogenous hTERT. However, hTERT immortalization surprisingly elicits genome reorganization not only in disease cells but also in the normal control cells, such that whole chromosome territories normally located at the nuclear periphery in proliferating fibroblasts become mislocalized in the nuclear interior. This includes chromosome 18 in the control fibroblasts and both chromosomes 18 and X in HGPS cells, which physically express an isoform of the LINC complex protein SUN1 that has previously only been theoretical. Additionally, this HGPS cell line has also become genomically unstable and has a tetraploid karyotype, which could be due to the novel SUN1 isoform. Long‐term treatment with the hTERT inhibitor BIBR1532 enabled the reduction of telomere length in the immortalized cells and resulted that these mislocalized internal chromosomes to be located at the nuclear periphery, as assessed in actively proliferating cells. Taken together, these findings reveal that elongated telomeres lead to dramatic chromosome mislocalization, which can be restored with a drug treatment that results in telomere reshortening and that a novel SUN1 isoform combined with elongated telomeres leads to genomic instability. Thus, care should be taken when interpreting data from genomic studies in hTERT‐immortalized cell lines.
Collapse
Affiliation(s)
- Mehmet U. Bikkul
- Genome Engineering and Maintenance NetworkInstitute for Environment, Health and Societies, Brunel University LondonUxbridgeEngland
| | | | - Gemma Worthington
- Genome Engineering and Maintenance NetworkInstitute for Environment, Health and Societies, Brunel University LondonUxbridgeEngland
| | - Peter Meinke
- Friedrich‐Baur‐InstitutKlinikum der Universität MünchenMünchenGermany
- The Wellcome Trust Centre for Cell BiologyInstitute of Cell Biology, and Centre for Translational and Chemical Biology, University of EdinburghEdinburghEngland
| | - Alastair R. W. Kerr
- The Wellcome Trust Centre for Cell BiologyInstitute of Cell Biology, and Centre for Translational and Chemical Biology, University of EdinburghEdinburghEngland
| | - Aakila Sammy
- Genome Engineering and Maintenance NetworkInstitute for Environment, Health and Societies, Brunel University LondonUxbridgeEngland
| | - Kumars Riyahi
- Genome Engineering and Maintenance NetworkInstitute for Environment, Health and Societies, Brunel University LondonUxbridgeEngland
| | - Daniel Horton
- Genome Engineering and Maintenance NetworkInstitute for Environment, Health and Societies, Brunel University LondonUxbridgeEngland
| | - Eric C. Schirmer
- The Wellcome Trust Centre for Cell BiologyInstitute of Cell Biology, and Centre for Translational and Chemical Biology, University of EdinburghEdinburghEngland
| | - Michael Hubank
- Centre for Molecular PathologyThe Royal Marsden HospitalLondonEngland
| | - Ian R. Kill
- Genome Engineering and Maintenance NetworkInstitute for Environment, Health and Societies, Brunel University LondonUxbridgeEngland
| | - Rhona M. Anderson
- Genome Engineering and Maintenance NetworkInstitute for Environment, Health and Societies, Brunel University LondonUxbridgeEngland
| | - Predrag Slijepcevic
- Genome Engineering and Maintenance NetworkInstitute for Environment, Health and Societies, Brunel University LondonUxbridgeEngland
| | - Evgeny Makarov
- Genome Engineering and Maintenance NetworkInstitute for Environment, Health and Societies, Brunel University LondonUxbridgeEngland
| | - Joanna M. Bridger
- Genome Engineering and Maintenance NetworkInstitute for Environment, Health and Societies, Brunel University LondonUxbridgeEngland
| |
Collapse
|
|
26
|
Petkov S, Kahland T, Shomroni O, Lingner T, Salinas G, Fuchs S, Debowski K, Behr R. Immortalization of common marmoset monkey fibroblasts by piggyBac transposition of hTERT. PLoS One 2018; 13:e0204580. [PMID: 30261016 PMCID: PMC6160115 DOI: 10.1371/journal.pone.0204580] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2018] [Accepted: 09/11/2018] [Indexed: 02/07/2023] Open
Abstract
Following a certain type-specific number of mitotic divisions, terminally differentiated cells undergo proliferative senescence, thwarting efforts to expand different cell populations in vitro for the needs of scientific research or medical therapies. The primary cause of this phenomenon is the progressive shortening of the telomeres and the subsequent activation of cell cycle control pathways leading to a block of cell proliferation. Restoration of telomere length by transgenic expression of telomerase reverse transcriptase (TERT) usually results in bypassing of the replicative senescence and ultimately in cell immortalization. To date, there have not been any reports regarding immortalization of cells from common marmoset (Callithrix jacchus), an important non-human primate model for various human diseases, with the use of exogenous human TERT (hTERT). In this study, marmoset fibroblasts were successfully immortalized with transposon-integrated transgenic hTERT and expanded in vitro for over 500 population doublings. Calculation of population doubling levels (PDL) showed that the derived hTERT-transgenic lines had significantly higher proliferation potential than the wild-type fibroblasts, which reached only a maximum of 46 doublings. However, the immortalized cells exhibited differences in the morphology compared with the control fibroblasts and transcriptome analysis also revealed changes in the gene expression patterns. Finally, the karyotypes of all hTERT-transgenic cell lines showed various aberrations such as presence of extra Chromosome 17, isochromosome 21q, or tetraploidy. By single-cell expansion of the least affected monoclonal immortalized line, one sub-clonal line with normal karyotype was established, suggesting the possibility to derive immortal marmoset cells with normal karyotypes. The results of this study are an important step towards the development and optimization of methods for the production of immortalized cells from common marmoset monkeys.
Collapse
Affiliation(s)
- Stoyan Petkov
- Platform Degenerative Diseases, German Primate Center- Leibniz Institute for Primate Research, Göttingen, Germany
- DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany
| | - Tobias Kahland
- Platform Degenerative Diseases, German Primate Center- Leibniz Institute for Primate Research, Göttingen, Germany
| | - Orr Shomroni
- Microarray and Deep-Sequencing Core Facility, University Medical Center Göttingen (UMG), Göttingen, Germany
| | - Thomas Lingner
- Microarray and Deep-Sequencing Core Facility, University Medical Center Göttingen (UMG), Göttingen, Germany
| | - Gabriela Salinas
- Microarray and Deep-Sequencing Core Facility, University Medical Center Göttingen (UMG), Göttingen, Germany
| | - Sigrid Fuchs
- Department of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
| | - Katharina Debowski
- Platform Degenerative Diseases, German Primate Center- Leibniz Institute for Primate Research, Göttingen, Germany
| | - Rüdiger Behr
- Platform Degenerative Diseases, German Primate Center- Leibniz Institute for Primate Research, Göttingen, Germany
- DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany
- * E-mail:
| |
Collapse
|
|
27
|
Hung CLK, Maiuri T, Bowie LE, Gotesman R, Son S, Falcone M, Giordano JV, Gillis T, Mattis V, Lau T, Kwan V, Wheeler V, Schertzer J, Singh K, Truant R. A patient-derived cellular model for Huntington's disease reveals phenotypes at clinically relevant CAG lengths. Mol Biol Cell 2018; 29:2809-2820. [PMID: 30256717 PMCID: PMC6249865 DOI: 10.1091/mbc.e18-09-0590] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
The huntingtin protein participates in several cellular processes that are disrupted when the polyglutamine tract is expanded beyond a threshold of 37 CAG DNA repeats in Huntington’s disease (HD). Cellular biology approaches to understand these functional disruptions in HD have primarily focused on cell lines with synthetically long CAG length alleles that clinically represent outliers in this disease and a more severe form of HD that lacks age onset. Patient-derived fibroblasts are limited to a finite number of passages before succumbing to cellular senescence. We used human telomerase reverse transcriptase (hTERT) to immortalize fibroblasts taken from individuals of varying age, sex, disease onset, and CAG repeat length, which we have termed TruHD cells. TruHD cells display classic HD phenotypes of altered morphology, size and growth rate, increased sensitivity to oxidative stress, aberrant adenosine diphosphate/adenosine triphosphate (ADP/ATP) ratios, and hypophosphorylated huntingtin protein. We additionally observed dysregulated reactive oxygen species (ROS)-dependent huntingtin localization to nuclear speckles in HD cells. We report the generation and characterization of a human, clinically relevant cellular model for investigating disease mechanisms in HD at the single-cell level, which, unlike transformed cell lines, maintains functions critical for huntingtin transcriptional regulation and genomic integrity.
Collapse
Affiliation(s)
- Claudia Lin-Kar Hung
- Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4L8, Canada
| | - Tamara Maiuri
- Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4L8, Canada
| | - Laura Erin Bowie
- Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4L8, Canada
| | - Ryan Gotesman
- Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4L8, Canada
| | - Susie Son
- Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4L8, Canada
| | - Mina Falcone
- Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4L8, Canada
| | - James Victor Giordano
- Center for Genomic Medicine, Harvard Medical School, Boston, MA 02114.,Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114
| | - Tammy Gillis
- Center for Genomic Medicine, Harvard Medical School, Boston, MA 02114.,Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114
| | - Virginia Mattis
- Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048
| | - Trevor Lau
- Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4L8, Canada
| | - Vickie Kwan
- Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4L8, Canada.,Stem Cell and Cancer Research Institute, Faculty of Health Sciences, McMaster University, Hamilton, ON L8S 4L8, Canada
| | - Vanessa Wheeler
- Center for Genomic Medicine, Harvard Medical School, Boston, MA 02114.,Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114
| | - Jonathan Schertzer
- Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4L8, Canada
| | - Karun Singh
- Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4L8, Canada
| | - Ray Truant
- Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8S 4L8, Canada
| |
Collapse
|
|
28
|
Priyadarshini R, Hussain M, Attri P, Kaur E, Tripathi V, Priya S, Dhapola P, Saha D, Madhavan V, Chowdhury S, Sengupta S. BLM Potentiates c-Jun Degradation and Alters Its Function as an Oncogenic Transcription Factor. Cell Rep 2018; 24:947-961.e7. [DOI: 10.1016/j.celrep.2018.06.101] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2017] [Revised: 05/07/2018] [Accepted: 06/25/2018] [Indexed: 12/16/2022] Open
|
|
29
|
Carrascal MA, Silva M, Ramalho JS, Pen C, Martins M, Pascoal C, Amaral C, Serrano I, Oliveira MJ, Sackstein R, Videira PA. Inhibition of fucosylation in human invasive ductal carcinoma reduces E-selectin ligand expression, cell proliferation, and ERK1/2 and p38 MAPK activation. Mol Oncol 2018; 12:579-593. [PMID: 29215790 PMCID: PMC5928367 DOI: 10.1002/1878-0261.12163] [Citation(s) in RCA: 49] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2017] [Revised: 10/09/2017] [Accepted: 11/13/2017] [Indexed: 01/08/2023] Open
Abstract
Breast cancer tissue overexpresses fucosylated glycans, such as sialyl-Lewis X/A (sLeX/A ), and α-1,3/4-fucosyltransferases (FUTs) in relation to increased disease progression and metastasis. These glycans in tumor circulating cells mediate binding to vascular E-selectin, initiating tumor extravasation. However, their role in breast carcinogenesis is still unknown. Here, we aimed to define the contribution of the fucosylated structures, including sLeX/A , to cell adhesion, cell signaling, and cell proliferation in invasive ductal carcinomas (IDC), the most frequent type of breast cancer. We first analyzed expression of E-selectin ligands in IDC tissue and established primary cell cultures from the tissue. We observed strong reactivity with E-selectin and anti-sLeX/A antibodies in both IDC tissue and cell lines, and expression of α-1,3/4 FUTs FUT4, FUT5, FUT6, FUT10, and FUT11. To further assess the role of fucosylation in IDC biology, we immortalized a primary IDC cell line with human telomerase reverse transcriptase to create the 'CF1_T cell line'. Treatment with 2-fluorofucose (2-FF), a fucosylation inhibitor, completely abrogated its sLeX/A expression and dramatically reduced adherence of CF1_T cells to E-selectin under hemodynamic flow conditions. In addition, 2-FF-treated CF1_T cells showed a reduced migratory ability, as well as decreased cell proliferation rate. Notably, 2-FF treatment lowered the growth factor expression of CF1_T cells, prominently for FGF2, vascular endothelial growth factor, and transforming growth factor beta, and negatively affected activation of signal-regulating protein kinases 1 and 2 and p38 mitogen-activated protein kinase signaling pathways. These data indicate that fucosylation licenses several malignant features of IDC, such as cell adhesion, migration, proliferation, and growth factor expression, contributing to tumor progression.
Collapse
Affiliation(s)
- Mylène A. Carrascal
- UCIBIODepartamento Ciências da VidaFaculdade de Ciências e TecnologiaUniversidade Nova de LisboaPortugal
- CEDOCChronic Diseases Research CenterNOVA Medical School/Faculdade de Ciências MédicasUniversidade Nova de LisboaPortugal
| | - Mariana Silva
- CEDOCChronic Diseases Research CenterNOVA Medical School/Faculdade de Ciências MédicasUniversidade Nova de LisboaPortugal
- Departments of Dermatology and MedicineBrigham & Women's HospitalBostonMAUSA
- Harvard Medical SchoolProgram of Excellence in GlycosciencesBostonMAUSA
| | - José S. Ramalho
- CEDOCChronic Diseases Research CenterNOVA Medical School/Faculdade de Ciências MédicasUniversidade Nova de LisboaPortugal
| | - Cláudia Pen
- Centro Hospitalar de Lisboa CentralEPE – Serviço de Anatomia PatológicaLisbonPortugal
| | - Manuela Martins
- Centro Hospitalar de Lisboa CentralEPE – Serviço de Anatomia PatológicaLisbonPortugal
| | - Carlota Pascoal
- UCIBIODepartamento Ciências da VidaFaculdade de Ciências e TecnologiaUniversidade Nova de LisboaPortugal
| | - Constança Amaral
- UCIBIODepartamento Ciências da VidaFaculdade de Ciências e TecnologiaUniversidade Nova de LisboaPortugal
| | | | - Maria José Oliveira
- New Therapies GroupINEB‐Institute for Biomedical EngineeringPortoPortugal
- Instituto de Investigação e Inovação em SaúdeUniversidade do PortoPortugal
| | - Robert Sackstein
- Departments of Dermatology and MedicineBrigham & Women's HospitalBostonMAUSA
- Harvard Medical SchoolProgram of Excellence in GlycosciencesBostonMAUSA
| | - Paula A. Videira
- UCIBIODepartamento Ciências da VidaFaculdade de Ciências e TecnologiaUniversidade Nova de LisboaPortugal
- CEDOCChronic Diseases Research CenterNOVA Medical School/Faculdade de Ciências MédicasUniversidade Nova de LisboaPortugal
- CDG & Allies – PPAIN Congenital Disorders of Glycosylation Professionals and Patient Associations International NetworkCaparicaPortugal
| |
Collapse
|
|
30
|
O'Neill KM, Irwin RE, Mackin SJ, Thursby SJ, Thakur A, Bertens C, Masala L, Loughery JEP, McArt DG, Walsh CP. Depletion of DNMT1 in differentiated human cells highlights key classes of sensitive genes and an interplay with polycomb repression. Epigenetics Chromatin 2018; 11:12. [PMID: 29598829 PMCID: PMC5875016 DOI: 10.1186/s13072-018-0182-4] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2018] [Accepted: 03/21/2018] [Indexed: 11/17/2022] Open
Abstract
Background DNA methylation plays a vital role in the cell, but loss-of-function mutations of the maintenance methyltransferase DNMT1 in normal human cells are lethal, precluding target identification, and existing hypomorphic lines are tumour cells. We generated instead a hypomorphic series in normal hTERT-immortalised fibroblasts using stably integrated short hairpin RNA. Results Approximately two-thirds of sites showed demethylation as expected, with one-third showing hypermethylation, and targets were shared between the three independently derived lines. Enrichment analysis indicated significant losses at promoters and gene bodies with four gene classes most affected: (1) protocadherins, which are key to neural cell identity; (2) genes involved in fat homoeostasis/body mass determination; (3) olfactory receptors and (4) cancer/testis antigen (CTA) genes. Overall effects on transcription were relatively small in these fibroblasts, but CTA genes showed robust derepression. Comparison with siRNA-treated cells indicated that shRNA lines show substantial remethylation over time. Regions showing persistent hypomethylation in the shRNA lines were associated with polycomb repression and were derepressed on addition of an EZH2 inhibitor. Persistent hypermethylation in shRNA lines was, in contrast, associated with poised promoters. Conclusions We have assessed for the first time the effects of chronic depletion of DNMT1 in an untransformed, differentiated human cell type. Our results suggest polycomb marking blocks remethylation and indicate the sensitivity of key neural, adipose and cancer-associated genes to loss of maintenance methylation activity. Electronic supplementary material The online version of this article (10.1186/s13072-018-0182-4) contains supplementary material, which is available to authorized users.
Collapse
Affiliation(s)
- Karla M O'Neill
- Genomic Medicine Research Group, Centre for Molecular Biosciences, School of Biomedical Sciences, Ulster University, Cromore Road, Coleraine, BT52 1SA, UK.,The Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, BT9 7AE, UK
| | - Rachelle E Irwin
- Genomic Medicine Research Group, Centre for Molecular Biosciences, School of Biomedical Sciences, Ulster University, Cromore Road, Coleraine, BT52 1SA, UK
| | - Sarah-Jayne Mackin
- Genomic Medicine Research Group, Centre for Molecular Biosciences, School of Biomedical Sciences, Ulster University, Cromore Road, Coleraine, BT52 1SA, UK
| | - Sara-Jayne Thursby
- Genomic Medicine Research Group, Centre for Molecular Biosciences, School of Biomedical Sciences, Ulster University, Cromore Road, Coleraine, BT52 1SA, UK
| | - Avinash Thakur
- Genomic Medicine Research Group, Centre for Molecular Biosciences, School of Biomedical Sciences, Ulster University, Cromore Road, Coleraine, BT52 1SA, UK.,Terry Fox Laboratory, BC Cancer Research Centre, 675 West 10th Avenue, Room 13-112, Vancouver, BC, V5Z 1L3, Canada
| | - Ciske Bertens
- Genomic Medicine Research Group, Centre for Molecular Biosciences, School of Biomedical Sciences, Ulster University, Cromore Road, Coleraine, BT52 1SA, UK.,Academie Life Science, Engineering & Design, Saxion University, M.H. Tromplaan 28, 7500, Enschede, Netherlands
| | - Laura Masala
- Genomic Medicine Research Group, Centre for Molecular Biosciences, School of Biomedical Sciences, Ulster University, Cromore Road, Coleraine, BT52 1SA, UK.,Department of Obstetrics and Gynecology, University of Sassari, Via Vienne 2, 7100, Sassari, Italy
| | - Jayne E P Loughery
- Genomic Medicine Research Group, Centre for Molecular Biosciences, School of Biomedical Sciences, Ulster University, Cromore Road, Coleraine, BT52 1SA, UK
| | - Darragh G McArt
- Centre for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, BT9 7AE, UK
| | - Colum P Walsh
- Genomic Medicine Research Group, Centre for Molecular Biosciences, School of Biomedical Sciences, Ulster University, Cromore Road, Coleraine, BT52 1SA, UK.
| |
Collapse
|
|
31
|
Li JX, Said A, Ge XG, Wang W, Zhang Y, Jin T. Development and validation of immortalized bovine mammary epithelial cell line as an in vitro model for the study of mammary gland functions. Cytotechnology 2018; 70:67-82. [PMID: 28918563 PMCID: PMC5809642 DOI: 10.1007/s10616-017-0114-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2016] [Accepted: 11/04/2016] [Indexed: 01/17/2023] Open
Abstract
This study aimed to develop a bovine mammary epithelial (BME) cell line model, which provides a possibility to determine functional properties of the bovine mammary gland. The primary cell culture was derived from bovine mammary gland tissues and processed enzymatically to obtain cell colonies with epithelial-like morphology. The cultures of BME cells were purified and optimally cultured at 37 °C in DMEM/F12 medium supplemented with 10% fetal bovine serum. The BME cells were identified as epithelial cell line by the evaluating the expression of keratin-18 using immunofluorescence staining. A novel gene expression system strongly enhances the expression of telomerase, has been used to immortalize BME cell line termed hTBME cell line. Interestingly, telomerase remained active even after over 60 passages of hTBME cell line, required for immortalization of BME cells. In addition, the hTBME cell line was continuously subcultured with a spontaneous epithelial-like morphology, with a great proliferation activity, and without evidence of apoptotic and necrotic effects. Further characterization showed that hTBME cell line can be continuously propagated in culture with constant chromosomal features and without tumorigenic properties. Finally, established hTBME cell line was evaluated for mammary gland specific functions. Our results demonstrated that the hTBME cell line was able to retain functional-morphological structure, and functional differentiation by expression of beta (β)-casein as in the bovine mammary gland in vivo. Taken together, our findings suggest that the established hTBME cell line can serve as a valuable tool for the study of bovine mammary gland functions.
Collapse
Affiliation(s)
- Ji-Xia Li
- College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin, 300384, People's Republic of China
| | - Abdelrahman Said
- Parasitology and Animal Diseases Department, National Research Center, Dokki, Giza, Egypt
- Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK, S7N 5E3, Canada
| | - Xiu-Guo Ge
- College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin, 300384, People's Republic of China
| | - Wenxiu Wang
- Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou, 256600, Shandong, China
| | - Yong Zhang
- Institute of Biotechnology, College of Veterinary Medicine, Northwest A&F University, Yangling, 712100, Shaanxi, People's Republic of China.
| | - Tianming Jin
- College of Animal Science and Veterinary Medicine, Tianjin Agricultural University, Tianjin, 300384, People's Republic of China.
| |
Collapse
|
|
32
|
Kang HY, Choi YK, Jeong YI, Choi KC, Hyun SH, Hwang WS, Jeung EB. Immortalization of Porcine 11β-Hydroxysteroid Dehydrogenase Type 1-Transgenic Liver Cells Using SV40 Large T Antigen. Int J Mol Sci 2017; 18:ijms18122625. [PMID: 29206210 PMCID: PMC5751228 DOI: 10.3390/ijms18122625] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2017] [Revised: 11/30/2017] [Accepted: 12/02/2017] [Indexed: 12/12/2022] Open
Abstract
Cortisol is a steroid hormone essential to the maintenance of homeostasis that is released in response to stress and low blood glucose concentration. Cortisol is converted from cortisone by 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1). It has been reported that too much cortisol or overexpression of HSD11B1 induces obesity and the insulin resistance that accompanies metabolic syndrome in rodent adipose tissue. In our previous study, HSD11B1-transgenic (TG) fibroblasts were established, and a porcine model was generated by SCNT using those fibroblasts. Hepatocytes overexpressing HSD11B1 were obtained from livers of this porcine model and cultured in vitro. However, the primary hepatocytes were found to have a short life span or low proliferation rate. To overcome these problems, the SV40 large T antigen was transduced into primary HSD11B1-TG hepatocytes, and those cells were immortalized. Immortalized HSD11B1-TG hepatocytes showed restored morphology, more rapid proliferation rate, and more expression of HSD11B1 than primary hepatocytes. As well, these cells kept the hepatic characteristics such as gluconeogenic response to cortisone and increased expression of hepatic makers. The immortalized HSD11B1-TG hepatocytes may be useful for studying traits and potential therapeutic drugs for treatment of metabolic disorders induced by overexpression of HSD11B1.
Collapse
Affiliation(s)
- Hee Young Kang
- College of Veterinary Medicine, Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Cheongju, Chungbuk 28644, Korea.
- Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Korea.
| | - Young-Kwon Choi
- College of Veterinary Medicine, Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Cheongju, Chungbuk 28644, Korea.
| | - Yeon Ik Jeong
- Sooam Biotech Research Foundation, 64 Kyunginro, Guro-gu, Seoul 08359, Korea.
| | - Kyung-Chul Choi
- College of Veterinary Medicine, Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Cheongju, Chungbuk 28644, Korea.
| | - Sang-Hwan Hyun
- College of Veterinary Medicine, Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Cheongju, Chungbuk 28644, Korea.
| | - Woo-Suk Hwang
- Sooam Biotech Research Foundation, 64 Kyunginro, Guro-gu, Seoul 08359, Korea.
| | - Eui-Bae Jeung
- College of Veterinary Medicine, Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Cheongju, Chungbuk 28644, Korea.
| |
Collapse
|
|
33
|
Vascular disease-causing mutation, smooth muscle α-actin R258C, dominantly suppresses functions of α-actin in human patient fibroblasts. Proc Natl Acad Sci U S A 2017; 114:E5569-E5578. [PMID: 28652363 DOI: 10.1073/pnas.1703506114] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
The most common genetic alterations for familial thoracic aortic aneurysms and dissections (TAAD) are missense mutations in vascular smooth muscle (SM) α-actin encoded by ACTA2 We focus here on ACTA2-R258C, a recurrent mutation associated with early onset of TAAD and occlusive moyamoya-like cerebrovascular disease. Recent biochemical results with SM α-actin-R258C predicted that this variant will compromise multiple actin-dependent functions in intact cells and tissues, but a model system to measure R258C-induced effects was lacking. We describe the development of an approach to interrogate functional consequences of actin mutations in affected patient-derived cells. Primary dermal fibroblasts from R258C patients exhibited increased proliferative capacity compared with controls, consistent with inhibition of growth suppression attributed to SM α-actin. Telomerase-immortalized lines of control and R258C human dermal fibroblasts were established and SM α-actin expression induced with adenovirus encoding myocardin-related transcription factor A, a potent coactivator of ACTA2 Two-dimensional Western blotting confirmed induction of both wild-type and mutant SM α-actin in heterozygous ACTA2-R258C cells. Expression of mutant SM α-actin in heterozygous ACTA2-R258C fibroblasts abrogated the significant effects of SM α-actin induction on formation of stress fibers and focal adhesions, filamentous to soluble actin ratio, matrix contraction, and cell migration. These results demonstrate that R258C dominantly disrupts cytoskeletal functions attributed to SM α-actin in fibroblasts and are consistent with deficiencies in multiple cytoskeletal functions. Thus, cellular defects due to this ACTA2 mutation in both aortic smooth muscle cells and adventitial fibroblasts may contribute to development of TAAD and proliferative occlusive vascular disease.
Collapse
|
|
34
|
Progerin-Induced Replication Stress Facilitates Premature Senescence in Hutchinson-Gilford Progeria Syndrome. Mol Cell Biol 2017; 37:MCB.00659-16. [PMID: 28483909 DOI: 10.1128/mcb.00659-16] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2016] [Accepted: 04/28/2017] [Indexed: 12/12/2022] Open
Abstract
Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutation in LMNA that produces an aberrant lamin A protein, progerin. The accumulation of progerin in HGPS cells leads to an aberrant nuclear morphology, genetic instability, and p53-dependent premature senescence. How p53 is activated in response to progerin production is unknown. Here we show that young cycling HGPS fibroblasts exhibit chronic DNA damage, primarily in S phase, as well as delayed replication fork progression. We demonstrate that progerin binds to PCNA, altering its distribution away from replicating DNA in HGPS cells, leading to γH2AX formation, ATR activation, and RPA Ser33 phosphorylation. Unlike normal human cells that can be immortalized by enforced expression of telomerase alone, immortalization of HGPS cells requires telomerase expression and p53 repression. In addition, we show that the DNA damage response in HGPS cells does not originate from eroded telomeres. Together, these results establish that progerin interferes with the coordination of essential DNA replication factors, causing replication stress, and is the primary signal for p53 activation leading to premature senescence in HGPS. Furthermore, this damage response is shown to be independent of progerin farnesylation, implying that unprocessed lamin A alone causes replication stress.
Collapse
|
|
35
|
Analysis of post-transcriptional regulation during cancer progression using a donor-derived isogenic model of tumorigenesis. Methods 2017; 126:193-200. [PMID: 28529064 DOI: 10.1016/j.ymeth.2017.05.012] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2017] [Accepted: 05/16/2017] [Indexed: 12/26/2022] Open
Abstract
Post-transcriptional regulation of gene expression by RNA binding proteins (RBPs) and non-coding RNAs plays an important role in global gene expression. Many post-transcriptional regulators are misexpressed and misregulated in cancers, resulting in altered programs of protein biosynthesis that can drive tumor progression. While comparative studies of several RBPs and microRNAs expressed in various cancer types have been reported, a model system that can be used to quantify RBP regulation and functional outcomes during the initiation and early stages of tumorigenesis is lacking. It was previously demonstrated that oncogenic transformation of normal human cells can be induced by expressing hTERT, p53DD, cyclin D1, CDK4R24C, C-MYCT58A and H-RASG12V. Here we describe a user-friendly method for generating this genetically defined model of step-wise tumorigenesis beginning with normal donor-derived human cells. This method immortalizes a donor's normal cells in about a week, reducing the chances of senescence. The entire stable system can be established in less than 12weeks. We then demonstrate the utility of such a system in elucidating the expression of multiple RBPs at an early step of tumor formation. We identify significant changes in the expression levels of transcripts encoding RBPs prior to transformation, suggesting that our described donor-derived isogenic system can provide insight about post-transcriptional regulation during the earliest stages of tumorigenesis in the context of diverse genetic backgrounds.
Collapse
|
|
36
|
Malacaria E, Franchitto A, Pichierri P. SLX4 Prevents GEN1-Dependent DSBs During DNA Replication Arrest Under Pathological Conditions in Human Cells. Sci Rep 2017; 7:44464. [PMID: 28290553 PMCID: PMC5349550 DOI: 10.1038/srep44464] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2016] [Accepted: 02/08/2017] [Indexed: 01/06/2023] Open
Abstract
SLX4 is a versatile protein serving as docking for multiple structure-specific endonucleases during DNA repair, however, little is known about its function at demised replication forks. Using RNAi or FA-P cells complemented with SLX4 mutants that abrogate interaction with MUS81 or SLX1, we show that SLX4 cooperates with MUS81 to introduce DSBs after replication stress but also counteracts pathological targeting of demised forks by GEN1. Such unexpected function of SLX4 is unrelated to interaction with endonucleases, but concerns the physical presence of the protein. Strikingly, ectopic expression of the Holliday junction-binding protein RuvA inhibits DSBs in SLX4-deficient cells by preventing GEN1 chromatin-association, and rescues proliferation and genome integrity upon replication stress. Altogether, our results indicate that SLX4 is crucial to prevent accidental processing of Holliday junction-like intermediates at demised forks also suggesting that spontaneous genome instability in FA-P cells may derive, at least partially, from unscheduled action of GEN1 in S-phase.
Collapse
Affiliation(s)
- Eva Malacaria
- Section of Experimental and Computational Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanità - Viale Regina Elena 299, 00161 Rome Italy
| | - Annapaola Franchitto
- Section of Molecular Epidemiology, Department of Environment and Primary Prevention, Istituto Superiore di Sanità - Viale Regina Elena 299, 00161 Rome Italy
| | - Pietro Pichierri
- Section of Experimental and Computational Carcinogenesis, Department of Environment and Primary Prevention, Istituto Superiore di Sanità - Viale Regina Elena 299, 00161 Rome Italy
| |
Collapse
|
|
37
|
Telomere-associated aging disorders. Ageing Res Rev 2017; 33:52-66. [PMID: 27215853 PMCID: PMC9926533 DOI: 10.1016/j.arr.2016.05.009] [Citation(s) in RCA: 99] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2016] [Revised: 05/17/2016] [Accepted: 05/19/2016] [Indexed: 01/25/2023]
Abstract
Telomeres are dynamic nucleoprotein-DNA structures that cap and protect linear chromosome ends. Several monogenic inherited diseases that display features of human premature aging correlate with shortened telomeres, and are referred to collectively as telomeropathies. These disorders have overlapping symptoms and a common underlying mechanism of telomere dysfunction, but also exhibit variable symptoms and age of onset, suggesting they fall along a spectrum of disorders. Primary telomeropathies are caused by defects in the telomere maintenance machinery, whereas secondary telomeropathies have some overlapping symptoms with primary telomeropathies, but are generally caused by mutations in DNA repair proteins that contribute to telomere preservation. Here we review both the primary and secondary telomeropathies, discuss potential mechanisms for tissue specificity and age of onset, and highlight outstanding questions in the field and future directions toward elucidating disease etiology and developing therapeutic strategies.
Collapse
|
|
38
|
Garcia-Mesa Y, Jay TR, Checkley MA, Luttge B, Dobrowolski C, Valadkhan S, Landreth GE, Karn J, Alvarez-Carbonell D. Immortalization of primary microglia: a new platform to study HIV regulation in the central nervous system. J Neurovirol 2016; 23:47-66. [PMID: 27873219 PMCID: PMC5329090 DOI: 10.1007/s13365-016-0499-3] [Citation(s) in RCA: 131] [Impact Index Per Article: 14.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2016] [Revised: 11/03/2016] [Accepted: 11/09/2016] [Indexed: 12/17/2022]
Abstract
The major reservoirs for HIV in the CNS are in the microglia, perivascular macrophages, and to a lesser extent, astrocytes. To study the molecular events controlling HIV expression in the microglia, we developed a reliable and robust method to immortalize microglial cells from primary glia from fresh CNS tissues and commercially available frozen glial cells. Primary human cells, including cells obtained from adult brain tissue, were transformed with lentiviral vectors expressing SV40 T antigen or a combination of SVR40 T antigen and hTERT. The immortalized cells have microglia-like morphology and express key microglial surface markers including CD11b, TGFβR, and P2RY12. Importantly, these cells were confirmed to be of human origin by sequencing. The RNA expression profiles identified by RNA-seq are also characteristic of microglial cells. Furthermore, the cells demonstrate the expected migratory and phagocytic activity, and the capacity to mount an inflammatory response characteristic of primary microglia. The immortalization method has also been successfully applied to a wide range of microglia from other species (macaque, rat, and mouse). To investigate different aspects of HIV molecular regulation in CNS, the cells have been superinfected with HIV reporter viruses and latently infected clones have been selected that reactive HIV in response to inflammatory signals. The cell lines we have developed and rigorously characterized will provide an invaluable resource for the study of HIV infection in microglial cells as well as studies of microglial cell function.
Collapse
Affiliation(s)
- Yoelvis Garcia-Mesa
- Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106 USA
| | - Taylor R. Jay
- Department of Neurosciences, Case Western Reserve University, Cleveland, OH 44106 USA
| | - Mary Ann Checkley
- Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106 USA
| | - Benjamin Luttge
- Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106 USA
| | - Curtis Dobrowolski
- Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106 USA
| | - Saba Valadkhan
- Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106 USA
| | - Gary E. Landreth
- Department of Neurosciences, Case Western Reserve University, Cleveland, OH 44106 USA
| | - Jonathan Karn
- Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106 USA
| | - David Alvarez-Carbonell
- Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106 USA
| |
Collapse
|
|
39
|
In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells. PLoS One 2016; 11:e0158289. [PMID: 27348615 PMCID: PMC4922589 DOI: 10.1371/journal.pone.0158289] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2016] [Accepted: 06/13/2016] [Indexed: 11/19/2022] Open
Abstract
Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs) can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC) and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.
Collapse
|
|
40
|
Agarwal P, Rupenthal ID. In vitro and ex vivo corneal penetration and absorption models. Drug Deliv Transl Res 2016; 6:634-647. [DOI: 10.1007/s13346-015-0275-6] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/22/2022]
|
|
41
|
Zhang H, Huang Y, Wang L, Yu T, Wang Z, Chang L, Zhao X, Luo X, Zhang L, Tong D. Immortalization of porcine placental trophoblast cells through reconstitution of telomerase activity. Theriogenology 2016; 85:1446-56. [PMID: 26850465 DOI: 10.1016/j.theriogenology.2016.01.006] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2015] [Revised: 01/03/2016] [Accepted: 01/04/2016] [Indexed: 11/26/2022]
Abstract
Placental trophoblast cells (PTCs) play a critical role in histotrophic nutrient absorption, gaseous exchange, endocrine activities, and barrier function between the maternal and fetal systems. Establishment of immortalized porcine PTCs will help us to investigate the potential effects of different viruses on porcine trophoblast. In the present study, primary porcine PTCs were isolated from healthy gilts at Day 30 to Day 50 of gestation through collagenase digestion, percoll gradient centrifugation, and anti-CD9 immunomagnetic negative selection. To provide stable and long lifespan cells, primary PTCs were transfected with human telomerase reverse transcriptase (hTERT) gene. One porcine placental trophoblast cell line, named as hTERT-PTCs, was chosen for characterization. Human telomerase reverse transcriptase-PTCs achieved an extended replicative lifespan without exhibiting any neoplastic transformation signs in vivo or in vitro. The morphologic and key physiological characteristics of the immortalized PTCs were similar to primary PTCs. The immortalized PTCs retained original cell polarity and normal karyotype, expressed trophoblast-specific marker cytokeratin 7 and E-cadherin but did not express vimentin and major histocompatibility complex class I antigens as well as primary PTCs. Human telomerase reverse transcriptase-PTCs secreted low levels of chorionic gonadotrophin β-subunit and placental lactogen that were coincident with primary PTCs. Taken together, our results demonstrated that the porcine PTCs could be immortalized through reconstitution of telomerase activity. The immortalized PTCs maintained its original characteristics and can be used as a model cells line to study the pathologic changes of porcine placental trophoblast in viruses infectious diseases.
Collapse
Affiliation(s)
- Hongling Zhang
- Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P.R. China
| | - Yong Huang
- Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P.R. China
| | - Lili Wang
- Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P.R. China
| | - Tingting Yu
- Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P.R. China
| | - Zengguo Wang
- Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P.R. China
| | - Lingling Chang
- Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P.R. China
| | - Xiaomin Zhao
- Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P.R. China
| | - Xiaomao Luo
- Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P.R. China
| | - Liang Zhang
- Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P.R. China
| | - Dewen Tong
- Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, P.R. China.
| |
Collapse
|
|
42
|
Duraimani S, Schneider RH, Randall OS, Nidich SI, Xu S, Ketete M, Rainforth MA, Gaylord-King C, Salerno JW, Fagan J. Effects of Lifestyle Modification on Telomerase Gene Expression in Hypertensive Patients: A Pilot Trial of Stress Reduction and Health Education Programs in African Americans. PLoS One 2015; 10:e0142689. [PMID: 26571023 PMCID: PMC4646647 DOI: 10.1371/journal.pone.0142689] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2014] [Accepted: 10/25/2015] [Indexed: 12/14/2022] Open
Abstract
BACKGROUND African Americans suffer from disproportionately high rates of hypertension and cardiovascular disease. Psychosocial stress, lifestyle and telomere dysfunction contribute to the pathogenesis of hypertension and cardiovascular disease. This study evaluated effects of stress reduction and lifestyle modification on blood pressure, telomerase gene expression and lifestyle factors in African Americans. METHODS Forty-eight African American men and women with stage I hypertension who participated in a larger randomized controlled trial volunteered for this substudy. These subjects participated in either stress reduction with the Transcendental Meditation technique and a basic health education course (SR) or an extensive health education program (EHE) for 16 weeks. Primary outcomes were telomerase gene expression (hTERT and hTR) and clinic blood pressure. Secondary outcomes included lifestyle-related factors. Data were analyzed for within-group and between-group changes. RESULTS Both groups showed increases in the two measures of telomerase gene expression, hTR mRNA levels (SR: p< 0.001; EHE: p< 0.001) and hTERT mRNA levels (SR: p = 0.055; EHE: p< 0.002). However, no statistically significant between-group changes were observed. Both groups showed reductions in systolic BP. Adjusted changes were SR = -5.7 mm Hg, p< 0.01; EHE = -9.0 mm Hg, p < 0.001 with no statistically significant difference between group difference. There was a significant reduction in diastolic BP in the EHE group (-5.3 mm Hg, p< 0.001) but not in SR (-1.2 mm Hg, p = 0.42); the between-group difference was significant (p = 0.04). The EHE group showed a greater number of changes in lifestyle behaviors. CONCLUSION In this pilot trial, both stress reduction (Transcendental Meditation technique plus health education) and extensive health education groups demonstrated increased telomerase gene expression and reduced BP. The association between increased telomerase gene expression and reduced BP observed in this high-risk population suggest hypotheses that telomerase gene expression may either be a biomarker for reduced BP or a mechanism by which stress reduction and lifestyle modification reduces BP. TRIAL REGISTRATION ClinicalTrials.gov NCT00681200.
Collapse
Affiliation(s)
- Shanthi Duraimani
- Center for Natural Medicine and Prevention, Maharishi University of Management Research Institute, Maharishi Vedic City, Iowa, United States of America
- Department of Physiology and Health, Maharishi University of Management, Fairfield, Iowa, United States of America
- MUM Molecular Biology Laboratory, Maharishi University of Management, Fairfield Iowa, United States of America
| | - Robert H. Schneider
- Center for Natural Medicine and Prevention, Maharishi University of Management Research Institute, Maharishi Vedic City, Iowa, United States of America
- Department of Physiology and Health, Maharishi University of Management, Fairfield, Iowa, United States of America
| | - Otelio S. Randall
- Howard University College of Medicine, Department of Internal Medicine, Division of Cardiology, Washington DC, United States of America
| | - Sanford I. Nidich
- Center for Natural Medicine and Prevention, Maharishi University of Management Research Institute, Maharishi Vedic City, Iowa, United States of America
- Department of Physiology and Health, Maharishi University of Management, Fairfield, Iowa, United States of America
| | - Shichen Xu
- Howard University College of Medicine, Department of Internal Medicine, Division of Cardiology, Washington DC, United States of America
| | - Muluemebet Ketete
- Howard University College of Medicine, Department of Internal Medicine, Division of Cardiology, Washington DC, United States of America
| | - Maxwell A. Rainforth
- Center for Natural Medicine and Prevention, Maharishi University of Management Research Institute, Maharishi Vedic City, Iowa, United States of America
| | - Carolyn Gaylord-King
- Center for Natural Medicine and Prevention, Maharishi University of Management Research Institute, Maharishi Vedic City, Iowa, United States of America
| | - John W. Salerno
- Center for Natural Medicine and Prevention, Maharishi University of Management Research Institute, Maharishi Vedic City, Iowa, United States of America
| | - John Fagan
- Department of Physiology and Health, Maharishi University of Management, Fairfield, Iowa, United States of America
- MUM Molecular Biology Laboratory, Maharishi University of Management, Fairfield Iowa, United States of America
| |
Collapse
|
|
43
|
He S, Li Y, Chen Y, Zhu Y, Zhang X, Xia X, Sun H. Immortalization of pig fibroblast cells by transposon-mediated ectopic expression of porcine telomerase reverse transcriptase. Cytotechnology 2015; 68:1435-45. [PMID: 26341227 DOI: 10.1007/s10616-015-9903-8] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2014] [Accepted: 07/20/2015] [Indexed: 11/27/2022] Open
Abstract
Pigs are the most economically important livestock, but pig cell lines useful for physiological studies and/or vaccine development are limited. Although several pig cell lines have been generated by oncogene transformation or human telomerase reverse transcriptase (TERT) immortalization, these cell lines contain viral sequences and/or antibiotic resistance genes. In this study, we established a new method for generating pig cell lines using the Sleeping Beauty (SB) transposon-mediated ectopic expression of porcine telomerase reverse transcriptase (pTERT). The performance of the new method was confirmed by generating a pig fibroblast cell (PFC) line. After transfection of primary PFCs with the SB transposon system, one cell clone containing the pTERT expression cassette was selected by dilution cloning and passed for different generations. After passage for more than 40 generations, the cell line retained stable expression of ectopic pTERT and continuous growth potential. Further characterization showed that the cell line kept the fibroblast morphology, growth curve, population doubling time, cloning efficiency, marker gene expression pattern, cell cycle distribution and anchorage-dependent growth property of the primary cells. These data suggest that the new method established is useful for generating pig cell lines without viral sequence and antibiotic resistant gene.
Collapse
Affiliation(s)
- Shan He
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China
| | - Yangyang Li
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China
| | - Yang Chen
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China
| | - Yue Zhu
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China
| | - Xinyu Zhang
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China
| | - Xiaoli Xia
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China
| | - Huaichang Sun
- College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, People's Republic of China.
| |
Collapse
|
|
44
|
Choi M, Lee C. Immortalization of Primary Keratinocytes and Its Application to Skin Research. Biomol Ther (Seoul) 2015; 23:391-9. [PMID: 26336577 PMCID: PMC4556197 DOI: 10.4062/biomolther.2015.038] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2015] [Revised: 05/01/2015] [Accepted: 05/06/2015] [Indexed: 01/16/2023] Open
Abstract
As a major component of the epidermal tissue, a primary keratinocyte has served as an essential tool not only for the study of pathogenesis of skin-related diseases but also for the assessment of potential toxicities of various chemicals used in cosmetics. However, its short lifespan in ex vivo setting has been a great hurdle for many practical applications. Therefore, a number of immortalization attempts have been made with success to overcome this limitation. In order to understand the immortalization process of a primary keratinocyte, several key biological phenomena governing its lifespan will be reviewed first. Then, various immortalization methods for the establishment of stable keratinocyte cell lines will be explained. Finally, its application to a three-dimensional skin culture system will be described.
Collapse
Affiliation(s)
- Moonju Choi
- College of Pharmacy, Dongguk University-Seoul, Goyang 410-820, Republic of Korea
| | - Choongho Lee
- College of Pharmacy, Dongguk University-Seoul, Goyang 410-820, Republic of Korea
| |
Collapse
|
|
45
|
Harkness L, Twine NA, Abu Dawud R, Jafari A, Aldahmash A, Wilkins MR, Adjaye J, Kassem M. Molecular characterisation of stromal populations derived from human embryonic stem cells: Similarities to immortalised bone marrow derived stromal stem cells. Bone Rep 2015; 3:32-39. [PMID: 28377964 PMCID: PMC5365211 DOI: 10.1016/j.bonr.2015.07.002] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/01/2015] [Revised: 06/30/2015] [Accepted: 07/14/2015] [Indexed: 01/22/2023] Open
Abstract
Human bone marrow-derived stromal (skeletal) stem cells (BM-hMSC) are being employed in an increasing number of clinical trials for tissue regeneration. A limiting factor for their clinical use is the inability to obtain sufficient cell numbers. Human embryonic stem cells (hESC) can provide an unlimited source of clinical grade cells for therapy. We have generated MSC-like cells from hESC (called here hESC-stromal) that exhibit surface markers and differentiate to osteoblasts and adipocytes, similar to BM-hMSC. In the present study, we used microarray analysis to compare the molecular phenotype of hESC-stromal and immortalised BM-hMSC cells (hMSC-TERT). Of the 7379 genes expressed above baseline, only 9.3% of genes were differentially expressed between undifferentiated hESC-stromal and BM-hMSC. Following ex vivo osteoblast induction, 665 and 695 genes exhibited ≥ 2-fold change (FC) in hESC-stromal and BM-hMSC, respectively with 172 genes common to both cell types. Functional annotation of significantly changing genes revealed similarities in gene ontology between the two cell types. Interestingly, genes in categories of cell adhesion/motility and epithelial–mesenchymal transition (EMT) were highly enriched in hESC-stromal whereas genes associated with cell cycle processes were enriched in hMSC-TERT. This data suggests that while hESC-stromal cells exhibit a similar molecular phenotype to hMSC-TERT, differences exist that can be explained by ontological differences between these two cell types. hESC-stromal cells can thus be considered as a possible alternative candidate cells for hMSC, to be employed in regenerative medicine protocols.
hESC-derived MSC-like cells were compared to immortalised BM-MSC. Comparison was performed using microarrays on non-induced and OB induced cells. Analysis demonstrated close hierarchical relationships and molecular phenotypes. 90.7% of genes were similarly expressed in non-induced cells. 73% of OB induced genes for both cell lines correlated with GO ontology analysis.
Collapse
Affiliation(s)
- Linda Harkness
- Molecular Endocrinology Laboratory, Odense University Hospital, University of Southern Denmark, Odense, Denmark
| | - Natalie A Twine
- Molecular Endocrinology Laboratory, Odense University Hospital, University of Southern Denmark, Odense, Denmark; NSW Systems Biology Initiative, University of New South Wales, Sydney, NSW, Australia
| | - Raed Abu Dawud
- Molecular Embryology and Aging group, Max-Planck Institute for Molecular Genetics (Department of Vertebrate Genomics), Berlin, Germany; Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh, Saudi Arabia; Berlin-Brandenburg Center for Regenerative Therapies, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Berlin, Germany
| | - Abbas Jafari
- Molecular Endocrinology Laboratory, Odense University Hospital, University of Southern Denmark, Odense, Denmark; Danish Stem Cell Centre (DanStem), Institute of Cellular and Molecular Medicine, University of Copenhagen, Denmark
| | - Abdullah Aldahmash
- Molecular Endocrinology Laboratory, Odense University Hospital, University of Southern Denmark, Odense, Denmark; Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh, Saudi Arabia
| | - Marc R Wilkins
- NSW Systems Biology Initiative, University of New South Wales, Sydney, NSW, Australia
| | - James Adjaye
- Molecular Embryology and Aging group, Max-Planck Institute for Molecular Genetics (Department of Vertebrate Genomics), Berlin, Germany; Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh, Saudi Arabia; Institute for Stem Cell Research and Regenerative Medicine, Faculty of Medicine, Heinrich-Heine University Düsseldorf, Düsseldorf, Germany
| | - Moustapha Kassem
- Molecular Endocrinology Laboratory, Odense University Hospital, University of Southern Denmark, Odense, Denmark; Stem Cell Unit, Department of Anatomy, College of Medicine, King Saud University, Riyadh, Saudi Arabia; Danish Stem Cell Centre (DanStem), Institute of Cellular and Molecular Medicine, University of Copenhagen, Denmark
| |
Collapse
|
|
46
|
Saraiva-Pava K, Navabi N, Skoog EC, Lindén SK, Oleastro M, Roxo-Rosa M. New NCI-N87-derived human gastric epithelial line after human telomerase catalytic subunit over-expression. World J Gastroenterol 2015; 21:6526-6542. [PMID: 26074691 PMCID: PMC4458763 DOI: 10.3748/wjg.v21.i21.6526] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/14/2014] [Revised: 02/07/2015] [Accepted: 03/31/2015] [Indexed: 02/06/2023] Open
Abstract
AIM: To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori (H. pylori) infection.
METHODS: Aiming to overcome this limitation, clones of the heterogenic cancer-derived NCI-N87 cell line were isolated, by stably-transducing it with the human telomerase reverse-transcriptase (hTERT) catalytic subunit gene. The clones were first characterized regarding their cell growth pattern and phenotype. For that we measured the clones’ adherence properties, expression of cell-cell junctions’ markers (ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance. The gastric properties of the clones, concerning expression of mucins, zymogens and glycan contents, were then evaluated by haematoxylin and eosin staining, Periodic acid Schiff (PAS) and PAS/Alcian Blue-staining, immunocytochemistry and Western blot. In addition, we assessed the usefulness of the hTERT-expressing gastric cell line for H. pylori research, by performing co-culture assays and measuring the IL-8 secretion, by ELISA, upon infection with two H. pylori strains differing in virulence.
RESULTS: Compared with the parental cell line, the most promising NCI-hTERT-derived clones (CL5 and CL6) were composed of cells with homogenous phenotype, presented higher relative telomerase activities, better adhesion properties, ability to be maintained in culture for longer periods after confluency, and were more efficient in PAS-reactive mucins secretion. Both clones were shown to produce high amounts of MUC1, MUC2 and MUC13. NCI-hTERT-CL5 mucins were shown to be decorated with blood group H type 2 (BG-H), Lewis-x (Lex), Ley and Lea and, in a less extent, with BG-A antigens, but the former two antigens were not detected in the NCI-hTERT-CL6. None of the clones exhibited detectable levels of MUC6 nor sialylated Lex and Lea glycans. Entailing good gastric properties, both NCI-hTERT-clones were found to produce pepsinogen-5 and human gastric lipase. The progenitor-like phenotype of NCI-hTERT-CL6 cells was highlighted by large nuclei and by the apical vesicular-like distribution of mucin 5AC and Pg5, supporting the accumulation of mucus-secreting and zymogens-chief mature cells functions.
CONCLUSION: These traits, in addition to resistance to microaerobic conditions and good responsiveness to H. pylori co-culture, in a strain virulence-dependent manner, make the NCI-hTERT-CL6 a promising model for future in vitro studies.
Collapse
|
|
47
|
Trochet D, Mergui X, Ivkovic I, Porreca RM, Gerbault-Seureau M, Sidibe A, Richard F, Londono-Vallejo A, Perret M, Aujard F, Riou JF. Telomere regulation during ageing and tumorigenesis of the grey mouse lemur. Biochimie 2015; 113:100-10. [PMID: 25882681 DOI: 10.1016/j.biochi.2015.04.002] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2015] [Accepted: 04/03/2015] [Indexed: 01/01/2023]
Abstract
Telomere erosion leading to replicative senescence has been well documented in human and anthropoid primates, and provides a clue against tumorigenesis. In contrast, other mammals, such as laboratory mice, with short lifespan and low body weight mass have different telomere biology without replicative senescence. We analyzed telomere biology in the grey mouse lemur, a small prosimian model with a relative long lifespan currently used in ageing research. We report an average telomere length by telomere restriction fragment (TRF) among the longest reported so far for a primate species (25-30 kb), but without detectable overall telomere shortening with ageing on blood samples. However, we demonstrate using universal STELA (Single Telomere Length Amplification) the existence of short telomeres, the increase of which, while correlating with ageing might be related to another mechanism than replicative senescence. We also found a low stringency of telomerase restriction in tissues and an ease to immortalize fibroblasts in vitro upon spontaneous telomerase activation. Finally, we describe the first grey mouse lemur cancer cell line showing a dramatic telomere shortening and high telomerase activity associated with polyploidy. Our overall results suggest that telomere biology in grey mouse lemur is an exception among primates, with at best a physiologically limited replicative telomere ageing and closest to that observed in small rodents.
Collapse
Affiliation(s)
- Delphine Trochet
- Structure et Instabilité des Génomes, Sorbonne Universités, Muséum National d'Histoire Naturelle, Inserm U 1154, CNRS UMR 7196, CP26, 57 rue Cuvier, 75005 Paris, France
| | - Xénia Mergui
- Structure et Instabilité des Génomes, Sorbonne Universités, Muséum National d'Histoire Naturelle, Inserm U 1154, CNRS UMR 7196, CP26, 57 rue Cuvier, 75005 Paris, France
| | - Ivana Ivkovic
- Structure et Instabilité des Génomes, Sorbonne Universités, Muséum National d'Histoire Naturelle, Inserm U 1154, CNRS UMR 7196, CP26, 57 rue Cuvier, 75005 Paris, France
| | - Rosa Maria Porreca
- Telomeres and Cancer Laboratory, CNRS UMR 3244, Institut Curie, 26 rue d'Ulm, 75248 Paris, France; UPMC Univ. Paris 06, 75005 Paris, France
| | - Michèle Gerbault-Seureau
- Institut de Systématique, Evolution, Biodiversité, Sorbonne Universités, Muséum National d'Histoire Naturelle, UMR 7205 CNRS, UPMC Univ. Paris 06, EPHE, 57 rue Cuvier, 75005 Paris, France
| | - Assitan Sidibe
- Structure et Instabilité des Génomes, Sorbonne Universités, Muséum National d'Histoire Naturelle, Inserm U 1154, CNRS UMR 7196, CP26, 57 rue Cuvier, 75005 Paris, France
| | - Florence Richard
- Institut de Systématique, Evolution, Biodiversité, Sorbonne Universités, Muséum National d'Histoire Naturelle, UMR 7205 CNRS, UPMC Univ. Paris 06, EPHE, 57 rue Cuvier, 75005 Paris, France
| | - Arturo Londono-Vallejo
- Telomeres and Cancer Laboratory, CNRS UMR 3244, Institut Curie, 26 rue d'Ulm, 75248 Paris, France; UPMC Univ. Paris 06, 75005 Paris, France
| | - Martine Perret
- Mécanismes Adaptatifs et Evolution, Muséum National d'Histoire Naturelle, Sorbonne Universités, UMR 7179 CNRS, 1 Avenue du Petit Château, 91800 Brunoy, France
| | - Fabienne Aujard
- Mécanismes Adaptatifs et Evolution, Muséum National d'Histoire Naturelle, Sorbonne Universités, UMR 7179 CNRS, 1 Avenue du Petit Château, 91800 Brunoy, France
| | - Jean-François Riou
- Structure et Instabilité des Génomes, Sorbonne Universités, Muséum National d'Histoire Naturelle, Inserm U 1154, CNRS UMR 7196, CP26, 57 rue Cuvier, 75005 Paris, France.
| |
Collapse
|
|
48
|
Endisha H, Merrill-Schools J, Zhao M, Bristol M, Wang X, Kubben N, Elmore LW. Restoring SIRT6 Expression in Hutchinson-Gilford Progeria Syndrome Cells Impedes Premature Senescence and Formation of Dysmorphic Nuclei. Pathobiology 2015; 82:9-20. [PMID: 25765721 DOI: 10.1159/000368856] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2014] [Accepted: 10/03/2014] [Indexed: 11/19/2022] Open
Abstract
OBJECTIVES Mice overexpressing SIRT6 live longer than wild-type mice while SIRT6 knockout mice exhibit similar degenerative phenotypes as individuals with Hutchinson-Gilford progeria syndrome (HGPS). Thus, we sought to test whether levels of SIRT6 are reduced in cells from individuals with HGPS and whether restored SIRT6 expression may impede premature aging phenotypes. METHODS Levels of endogenous SIRT6 and progerin in HGPS and normal fibroblasts were assessed by Western blotting and immunofluorescence. A tetracycline-inducible system was utilized to test whether progerin causes a rapid reduction in SIRT6 protein. SIRT6 was overexpressed in HGPS cells via lentiviral infection with biological endpoints including senescence-associated β-galactosidase (SA-β-gal) positivity, frequency of nuclear atypia, the number of 53BP1-positive DNA damage foci and growth rates. RESULTS Typical HGPS fibroblasts express lower levels of SIRT6 than fibroblasts from normal and atypical HGPS donors. Experimental induction of progerin did not cause a detectable reduction of SIRT6 protein. However, overexpression of SIRT6 in HGPS cells was associated with a reduced frequency of SA-β-gal positivity, fewer misshapen nuclei, fewer DNA damage foci, and increased growth rates. CONCLUSIONS Typical HGPS fibroblasts exhibit reduced levels of SIRT6 protein via a mechanism that remains to be elucidated. Our findings suggest that restoring SIRT6 expression in HGPS cells may partially impede senescence and the formation of dysmorphic nuclei. © 2015 S. Karger AG, Basel.
Collapse
Affiliation(s)
- Helal Endisha
- Department of Pathology, Virginia Commonwealth University, Richmond, Va., USA
| | | | | | | | | | | | | |
Collapse
|
|
49
|
Myklebust LM, Van Damme P, Støve SI, Dörfel MJ, Abboud A, Kalvik TV, Grauffel C, Jonckheere V, Wu Y, Swensen J, Kaasa H, Liszczak G, Marmorstein R, Reuter N, Lyon GJ, Gevaert K, Arnesen T. Biochemical and cellular analysis of Ogden syndrome reveals downstream Nt-acetylation defects. Hum Mol Genet 2014; 24:1956-76. [PMID: 25489052 PMCID: PMC4355026 DOI: 10.1093/hmg/ddu611] [Citation(s) in RCA: 97] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The X-linked lethal Ogden syndrome was the first reported human genetic disorder associated with a mutation in an N-terminal acetyltransferase (NAT) gene. The affected males harbor an Ser37Pro (S37P) mutation in the gene encoding Naa10, the catalytic subunit of NatA, the major human NAT involved in the co-translational acetylation of proteins. Structural models and molecular dynamics simulations of the human NatA and its S37P mutant highlight differences in regions involved in catalysis and at the interface between Naa10 and the auxiliary subunit hNaa15. Biochemical data further demonstrate a reduced catalytic capacity and an impaired interaction between hNaa10 S37P and Naa15 as well as Naa50 (NatE), another interactor of the NatA complex. N-Terminal acetylome analyses revealed a decreased acetylation of a subset of NatA and NatE substrates in Ogden syndrome cells, supporting the genetic findings and our hypothesis regarding reduced Nt-acetylation of a subset of NatA/NatE-type substrates as one etiology for Ogden syndrome. Furthermore, Ogden syndrome fibroblasts display abnormal cell migration and proliferation capacity, possibly linked to a perturbed retinoblastoma pathway. N-Terminal acetylation clearly plays a role in Ogden syndrome, thus revealing the in vivo importance of N-terminal acetylation in human physiology and disease.
Collapse
Affiliation(s)
- Line M Myklebust
- Department of Molecular Biology, University of Bergen, Bergen N-5020, Norway
| | - Petra Van Damme
- Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium, Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium,
| | - Svein I Støve
- Department of Molecular Biology, University of Bergen, Bergen N-5020, Norway, Department of Surgery, Haukeland University Hospital, N-5021 Bergen, Norway
| | - Max J Dörfel
- Stanley Institute for Cognitive Genomics, Cold Spring Harbor Laboratory, Woodbury, NY 11797, USA
| | - Angèle Abboud
- Department of Molecular Biology, University of Bergen, Bergen N-5020, Norway, Computational Biology Unit, Uni Computing, Uni Research AS, Bergen, Norway
| | - Thomas V Kalvik
- Department of Molecular Biology, University of Bergen, Bergen N-5020, Norway
| | - Cedric Grauffel
- Department of Molecular Biology, University of Bergen, Bergen N-5020, Norway, Computational Biology Unit, Uni Computing, Uni Research AS, Bergen, Norway
| | - Veronique Jonckheere
- Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium, Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium
| | - Yiyang Wu
- Stanley Institute for Cognitive Genomics, Cold Spring Harbor Laboratory, Woodbury, NY 11797, USA, Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA
| | | | - Hanna Kaasa
- Department of Molecular Biology, University of Bergen, Bergen N-5020, Norway
| | - Glen Liszczak
- Program in Gene Expression and Regulation, Wistar Institute, PA 19104, USA, Department of Chemistry, and Department of Biochemistry and Biophysics and Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Ronen Marmorstein
- Program in Gene Expression and Regulation, Wistar Institute, PA 19104, USA, Department of Chemistry, and Department of Biochemistry and Biophysics and Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
| | - Nathalie Reuter
- Department of Molecular Biology, University of Bergen, Bergen N-5020, Norway, Computational Biology Unit, Uni Computing, Uni Research AS, Bergen, Norway
| | - Gholson J Lyon
- Stanley Institute for Cognitive Genomics, Cold Spring Harbor Laboratory, Woodbury, NY 11797, USA, Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA,
| | - Kris Gevaert
- Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium, Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium
| | - Thomas Arnesen
- Department of Molecular Biology, University of Bergen, Bergen N-5020, Norway, Department of Surgery, Haukeland University Hospital, N-5021 Bergen, Norway,
| |
Collapse
|
|
50
|
Childs BG, Baker DJ, Kirkland JL, Campisi J, van Deursen JM. Senescence and apoptosis: dueling or complementary cell fates? EMBO Rep 2014; 15:1139-53. [PMID: 25312810 DOI: 10.15252/embr.201439245] [Citation(s) in RCA: 628] [Impact Index Per Article: 57.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023] Open
Abstract
In response to a variety of stresses, mammalian cells undergo a persistent proliferative arrest known as cellular senescence. Many senescence-inducing stressors are potentially oncogenic, strengthening the notion that senescence evolved alongside apoptosis to suppress tumorigenesis. In contrast to apoptosis, senescent cells are stably viable and have the potential to influence neighboring cells through secreted soluble factors, which are collectively known as the senescence-associated secretory phenotype (SASP). However, the SASP has been associated with structural and functional tissue and organ deterioration and may even have tumor-promoting effects, raising the interesting evolutionary question of why apoptosis failed to outcompete senescence as a superior cell fate option. Here, we discuss the advantages that the senescence program may have over apoptosis as a tumor protective mechanism, as well as non-neoplastic functions that may have contributed to its evolution. We also review emerging evidence for the idea that senescent cells are present transiently early in life and are largely beneficial for development, regeneration and homeostasis, and only in advanced age do senescent cells accumulate to an organism's detriment.
Collapse
Affiliation(s)
- Bennett G Childs
- Departments of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA
| | - Darren J Baker
- Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA
| | - James L Kirkland
- Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA
| | | | - Jan M van Deursen
- Departments of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA Robert and Arlene Kogod Center on Aging, Mayo Clinic, Rochester, MN, USA
| |
Collapse
|