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Karagiannis TC, Orlowski C, Ververis K, Pitsillou E, Sarila G, Keating ST, Foong LJ, Fabris S, Ngo-Nguyen C, Malik N, Okabe J, Hung A, Mantamadiotis T, El-Osta A. γH2AX in mouse embryonic stem cells: Distribution during differentiation and following γ-irradiation. Cells Dev 2024; 177:203882. [PMID: 37956740 DOI: 10.1016/j.cdev.2023.203882] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2023] [Revised: 10/20/2023] [Accepted: 11/02/2023] [Indexed: 11/15/2023]
Abstract
Phosphorylated histone H2AX (γH2AX) represents a sensitive molecular marker of DNA double-strand breaks (DSBs) and is implicated in stem cell biology. We established a model of mouse embryonic stem cell (mESC) differentiation and examined the dynamics of γH2AX foci during the process. Our results revealed high numbers of γH2AX foci in undifferentiated mESCs, decreasing as the cells differentiated towards the endothelial cell lineage. Notably, we observed two distinct patterns of γH2AX foci: the typical discrete γH2AX foci, which colocalize with the transcriptionally permissive chromatin mark H3K4me3, and the less well-characterized clustered γH2AX regions, which were only observed in intermediate progenitor cells. Next, we explored responses of mESCs to γ-radiation (137Cs). Following exposure to γ-radiation, mESCs showed a reduction in cell viability and increased γH2AX foci, indicative of radiosensitivity. Despite irradiation, surviving mESCs retained their differentiation potential. To further exemplify our findings, we investigated neural stem progenitor cells (NSPCs). Similar to mESCs, NSPCs displayed clustered γH2AX foci associated with progenitor cells and discrete γH2AX foci indicative of embryonic stem cells or differentiated cells. In conclusion, our findings demonstrate that γH2AX serves as a versatile marker of DSBs and may have a role as a biomarker in stem cell differentiation. The distinct patterns of γH2AX foci in differentiating mESCs and NSPCs provide valuable insights into DNA repair dynamics during differentiation, shedding light on the intricate balance between genomic integrity and cellular plasticity in stem cells. Finally, the clustered γH2AX foci observed in intermediate progenitor cells is an intriguing feature, requiring further exploration.
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Affiliation(s)
- Tom C Karagiannis
- Epigenetics in Human Health and Disease Program, Baker Heart and Diabetes Institute, 75 Commercial Road, Prahran, VIC 3004, Australia; Epigenomic Medicine Laboratory at prospED Training, Carlton, VIC 3053, Australia; Department of Clinical Pathology, The University of Melbourne, Parkville, VIC 3010, Australia; Department of Microbiology and Immunology, The University of Melbourne, Parkville, VIC 3010, Australia.
| | - Christian Orlowski
- Epigenetics in Human Health and Disease Program, Baker Heart and Diabetes Institute, 75 Commercial Road, Prahran, VIC 3004, Australia
| | - Katherine Ververis
- Epigenomic Medicine Laboratory at prospED Training, Carlton, VIC 3053, Australia; Department of Clinical Pathology, The University of Melbourne, Parkville, VIC 3010, Australia
| | - Eleni Pitsillou
- Epigenomic Medicine Laboratory at prospED Training, Carlton, VIC 3053, Australia; School of Science, STEM College, RMIT University, VIC 3001, Australia
| | - Gulcan Sarila
- Epigenetics in Human Health and Disease Program, Baker Heart and Diabetes Institute, 75 Commercial Road, Prahran, VIC 3004, Australia
| | - Samuel T Keating
- Epigenetics in Human Health and Disease Program, Baker Heart and Diabetes Institute, 75 Commercial Road, Prahran, VIC 3004, Australia
| | - Laura J Foong
- Epigenomic Medicine Laboratory at prospED Training, Carlton, VIC 3053, Australia
| | - Stefanie Fabris
- Epigenomic Medicine Laboratory at prospED Training, Carlton, VIC 3053, Australia
| | - Christina Ngo-Nguyen
- Epigenomic Medicine Laboratory at prospED Training, Carlton, VIC 3053, Australia
| | - Neha Malik
- Epigenomic Medicine Laboratory at prospED Training, Carlton, VIC 3053, Australia
| | - Jun Okabe
- Epigenetics in Human Health and Disease Program, Baker Heart and Diabetes Institute, 75 Commercial Road, Prahran, VIC 3004, Australia
| | - Andrew Hung
- School of Science, STEM College, RMIT University, VIC 3001, Australia
| | - Theo Mantamadiotis
- Department of Microbiology and Immunology, The University of Melbourne, Parkville, VIC 3010, Australia; Department of Surgery (RMH), The University of Melbourne, Parkville, VIC 3010, Australia
| | - Assam El-Osta
- Epigenetics in Human Health and Disease Program, Baker Heart and Diabetes Institute, 75 Commercial Road, Prahran, VIC 3004, Australia; Department of Diabetes, Central Clinical School, Monash University, Melbourne, VIC 3004, Australia; Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Sha Tin, Hong Kong; Hong Kong Institute of Diabetes and Obesity, Prince of Wales Hospital, The Chinese University of Hong Kong, 3/F Lui Che Woo Clinical Sciences Building, 30-32 Ngan Shing Street, Sha Tin, Hong Kong; Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Sha Tin, Hong Kong; Biomedical Laboratory Science, Department of Technology, Faculty of Health, University College Copenhagen, Copenhagen, Denmark
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Gökbuget D, Lenshoek K, Boileau RM, Bayerl J, Huang H, Wiita AP, Laird DJ, Blelloch R. Transcriptional repression upon S phase entry protects genome integrity in pluripotent cells. Nat Struct Mol Biol 2023; 30:1561-1570. [PMID: 37696959 DOI: 10.1038/s41594-023-01092-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2022] [Accepted: 08/07/2023] [Indexed: 09/13/2023]
Abstract
Coincident transcription and DNA replication causes replication stress and genome instability. Rapidly dividing mouse pluripotent stem cells are highly transcriptionally active and experience elevated replication stress, yet paradoxically maintain genome integrity. Here, we study FOXD3, a transcriptional repressor enriched in pluripotent stem cells, and show that its repression of transcription upon S phase entry is critical to minimizing replication stress and preserving genome integrity. Acutely deleting Foxd3 leads to immediate replication stress, G2/M phase arrest, genome instability and p53-dependent apoptosis. FOXD3 binds near highly transcribed genes during S phase entry, and its loss increases the expression of these genes. Transient inhibition of RNA polymerase II in S phase reduces observed replication stress and cell cycle defects. Loss of FOXD3-interacting histone deacetylases induces replication stress, while transient inhibition of histone acetylation opposes it. These results show how a transcriptional repressor can play a central role in maintaining genome integrity through the transient inhibition of transcription during S phase, enabling faithful DNA replication.
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Affiliation(s)
- Deniz Gökbuget
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA, USA
- Department of Urology, University of California, San Francisco, San Francisco, CA, USA
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA
| | - Kayla Lenshoek
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA, USA
- Department of Urology, University of California, San Francisco, San Francisco, CA, USA
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA
| | - Ryan M Boileau
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA, USA
- Department of Urology, University of California, San Francisco, San Francisco, CA, USA
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA
| | - Jonathan Bayerl
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA, USA
- Department of Obstetrics, Gynecology and Reproductive Science, University of California, San Francisco, San Francisco, CA, USA
| | - Hector Huang
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA
- Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA
| | - Arun P Wiita
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA
- Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA
| | - Diana J Laird
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA, USA
- Department of Obstetrics, Gynecology and Reproductive Science, University of California, San Francisco, San Francisco, CA, USA
| | - Robert Blelloch
- The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences, University of California, San Francisco, San Francisco, CA, USA.
- Department of Urology, University of California, San Francisco, San Francisco, CA, USA.
- Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA.
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3
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She R, Fair T, Schaefer NK, Saunders RA, Pavlovic BJ, Weissman JS, Pollen AA. Comparative landscape of genetic dependencies in human and chimpanzee stem cells. Cell 2023; 186:2977-2994.e23. [PMID: 37343560 PMCID: PMC10461406 DOI: 10.1016/j.cell.2023.05.043] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Revised: 03/14/2023] [Accepted: 05/26/2023] [Indexed: 06/23/2023]
Abstract
Comparative studies of great apes provide a window into our evolutionary past, but the extent and identity of cellular differences that emerged during hominin evolution remain largely unexplored. We established a comparative loss-of-function approach to evaluate whether human cells exhibit distinct genetic dependencies. By performing genome-wide CRISPR interference screens in human and chimpanzee pluripotent stem cells, we identified 75 genes with species-specific effects on cellular proliferation. These genes comprised coherent processes, including cell-cycle progression and lysosomal signaling, which we determined to be human-derived by comparison with orangutan cells. Human-specific robustness to CDK2 and CCNE1 depletion persisted in neural progenitor cells and cerebral organoids, supporting the G1-phase length hypothesis as a potential evolutionary mechanism in human brain expansion. Our findings demonstrate that evolutionary changes in human cells reshaped the landscape of essential genes and establish a platform for systematically uncovering latent cellular and molecular differences between species.
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Affiliation(s)
- Richard She
- Whitehead Institute for Biomedical Research, Cambridge, MA, USA
| | - Tyler Fair
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA
| | - Nathan K Schaefer
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA, USA
| | - Reuben A Saunders
- Whitehead Institute for Biomedical Research, Cambridge, MA, USA; Department of Cellular and Molecular Pharmacology, University of California at San Francisco, San Francisco, CA, USA
| | - Bryan J Pavlovic
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA, USA
| | - Jonathan S Weissman
- Whitehead Institute for Biomedical Research, Cambridge, MA, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA; David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute Technology, Cambridge, MA 02142, USA.
| | - Alex A Pollen
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA; Department of Neurology, University of California, San Francisco, San Francisco, CA, USA.
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4
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Zhao Q, Liu K, Zhang L, Li Z, Wang L, Cao J, Xu Y, Zheng A, Chen Q, Zhao T. BNIP3-dependent mitophagy safeguards ESC genomic integrity via preventing oxidative stress-induced DNA damage and protecting homologous recombination. Cell Death Dis 2022; 13:976. [PMID: 36402748 PMCID: PMC9675825 DOI: 10.1038/s41419-022-05413-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2022] [Revised: 11/03/2022] [Accepted: 11/07/2022] [Indexed: 11/21/2022]
Abstract
Embryonic stem cells (ESCs) have a significantly lower mutation load compared to somatic cells, but the mechanisms that guard genomic integrity in ESCs remain largely unknown. Here we show that BNIP3-dependent mitophagy protects genomic integrity in mouse ESCs. Deletion of Bnip3 increases cellular reactive oxygen species (ROS) and decreases ATP generation. Increased ROS in Bnip3-/- ESCs compromised self-renewal and were partially rescued by either NAC treatment or p53 depletion. The decreased cellular ATP in Bnip3-/- ESCs induced AMPK activation and deteriorated homologous recombination, leading to elevated mutation load during long-term propagation. Whereas activation of AMPK in X-ray-treated Bnip3+/+ ESCs dramatically ascended mutation rates, inactivation of AMPK in Bnip3-/- ESCs under X-ray stress remarkably decreased the mutation load. In addition, enhancement of BNIP3-dependent mitophagy during reprogramming markedly decreased mutation accumulation in established iPSCs. In conclusion, we demonstrated a novel pathway in which BNIP3-dependent mitophagy safeguards ESC genomic stability, and that could potentially be targeted to improve pluripotent stem cell genomic integrity for regenerative medicine.
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Affiliation(s)
- Qian Zhao
- grid.9227.e0000000119573309State Key Laboratory of Stem Cell and Reproductive Biology, Institute for Stem Cell and Regeneration, Institute of Zoology, Chinese Academy of Sciences Beijing, Beijing, 100101 China ,grid.512959.3Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101 China
| | - Kun Liu
- grid.9227.e0000000119573309State Key Laboratory of Stem Cell and Reproductive Biology, Institute for Stem Cell and Regeneration, Institute of Zoology, Chinese Academy of Sciences Beijing, Beijing, 100101 China ,grid.512959.3Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101 China
| | - Lin Zhang
- grid.9227.e0000000119573309State Key Laboratory of Stem Cell and Reproductive Biology, Institute for Stem Cell and Regeneration, Institute of Zoology, Chinese Academy of Sciences Beijing, Beijing, 100101 China ,grid.410726.60000 0004 1797 8419University of Chinese Academy of Sciences, Beijing, 100049 China
| | - Zheng Li
- grid.24696.3f0000 0004 0369 153XDepartment of Gastroenterology, Beijing Tiantan Hospital, Capital Medical University, Beijing, 100070 China
| | - Liang Wang
- grid.9227.e0000000119573309State Key Laboratory of Stem Cell and Reproductive Biology, Institute for Stem Cell and Regeneration, Institute of Zoology, Chinese Academy of Sciences Beijing, Beijing, 100101 China ,grid.410726.60000 0004 1797 8419University of Chinese Academy of Sciences, Beijing, 100049 China
| | - Jiani Cao
- grid.9227.e0000000119573309State Key Laboratory of Stem Cell and Reproductive Biology, Institute for Stem Cell and Regeneration, Institute of Zoology, Chinese Academy of Sciences Beijing, Beijing, 100101 China ,grid.512959.3Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101 China
| | - Youqing Xu
- grid.24696.3f0000 0004 0369 153XDepartment of Gastroenterology, Beijing Tiantan Hospital, Capital Medical University, Beijing, 100070 China
| | - Aihua Zheng
- grid.9227.e0000000119573309State Key Laboratory of Stem Cell and Reproductive Biology, Institute for Stem Cell and Regeneration, Institute of Zoology, Chinese Academy of Sciences Beijing, Beijing, 100101 China ,grid.410726.60000 0004 1797 8419University of Chinese Academy of Sciences, Beijing, 100049 China
| | - Quan Chen
- grid.216938.70000 0000 9878 7032College of Life Sciences, Nankai University, Tianjin, 300073 China
| | - Tongbiao Zhao
- grid.9227.e0000000119573309State Key Laboratory of Stem Cell and Reproductive Biology, Institute for Stem Cell and Regeneration, Institute of Zoology, Chinese Academy of Sciences Beijing, Beijing, 100101 China ,grid.512959.3Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, 100101 China ,grid.410726.60000 0004 1797 8419University of Chinese Academy of Sciences, Beijing, 100049 China
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5
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Liang T, Bai J, Zhou W, Lin H, Ma S, Zhu X, Tao Q, Xi Q. HMCES modulates the transcriptional regulation of nodal/activin and BMP signaling in mESCs. Cell Rep 2022; 40:111038. [PMID: 35830803 DOI: 10.1016/j.celrep.2022.111038] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2021] [Revised: 03/20/2022] [Accepted: 06/11/2022] [Indexed: 12/01/2022] Open
Abstract
Despite the fundamental roles of TGF-β family signaling in cell fate determination in all metazoans, the mechanism by which these signals are spatially and temporally interpreted remains elusive. The cell-context-dependent function of TGF-β signaling largely relies on transcriptional regulation by SMAD proteins. Here, we discover that the DNA repair-related protein, HMCES, contributes to early development by maintaining nodal/activin- or BMP-signaling-regulated transcriptional network. HMCES binds with R-SMAD proteins, co-localizing at active histone marks. However, HMCES chromatin occupancy is independent on nodal/activin or BMP signaling. Mechanistically, HMCES competitively binds chromatin to limit binding by R-SMAD proteins, thereby forcing their dissociation and resulting in repression of their regulatory effects. In Xenopus laevis embryo, hmces KD causes dramatic development defects with abnormal left-right axis asymmetry along with increasing expression of lefty1. These findings reveal HMCES transcriptional regulatory function in the context of TGF-β family signaling.
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Affiliation(s)
- Tao Liang
- School of Life Sciences, Tsinghua University, Beijing 100084, China
| | - Jianbo Bai
- School of Life Sciences, Tsinghua University, Beijing 100084, China; Joint Graduate Program of Peking-Tsinghua-NIBS, Tsinghua University, Beijing 100084, China
| | - Wei Zhou
- School of Life Sciences, Tsinghua University, Beijing 100084, China
| | - Hao Lin
- School of Life Sciences, Tsinghua University, Beijing 100084, China; MOE Key Laboratory of Protein Sciences, Tsinghua University, Beijing 100084, China
| | - Shixin Ma
- School of Life Sciences, Tsinghua University, Beijing 100084, China; Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing 100084, China
| | - Xuechen Zhu
- Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing 100084, China; Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China
| | - Qinghua Tao
- School of Life Sciences, Tsinghua University, Beijing 100084, China; MOE Key Laboratory of Protein Sciences, Tsinghua University, Beijing 100084, China
| | - Qiaoran Xi
- School of Life Sciences, Tsinghua University, Beijing 100084, China; MOE Key Laboratory of Protein Sciences, Tsinghua University, Beijing 100084, China.
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Ren W, Gao L, Mou Y, Deng W, Hua J, Yang F. DUX: One Transcription Factor Controls 2-Cell-like Fate. Int J Mol Sci 2022; 23:ijms23042067. [PMID: 35216182 PMCID: PMC8877164 DOI: 10.3390/ijms23042067] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2021] [Revised: 02/10/2022] [Accepted: 02/11/2022] [Indexed: 02/06/2023] Open
Abstract
The double homeobox (Dux) gene, encoding a double homeobox transcription factor, is one of the key drivers of totipotency in mice. Recent studies showed Dux was temporally expressed at the 2-cell stage and acted as a transcriptional activator during zygotic genome activation (ZGA) in embryos. A similar activation occurs in mouse embryonic stem cells, giving rise to 2-cell-like cells (2CLCs). Though the molecular mechanism underlying this expanded 2CLC potency caused by Dux activation has been partially revealed, the regulation mechanisms controlling Dux expression remain elusive. Here, we discuss the latest advancements in the multiple levels of regulation of Dux expression, as well as Dux function in 2CLCs transition, aiming to provide a theoretical framework for understanding the mechanisms that regulate totipotency.
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Affiliation(s)
- Wei Ren
- College of Veterinary Medicine, Northwest A & F University, Xianyang 712100, China; (W.R.); (L.G.); (Y.M.); (J.H.)
- Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Xianyang 712100, China
- College of Innovation and Experiment, Northwest A & F University, Xianyang 712100, China
| | - Leilei Gao
- College of Veterinary Medicine, Northwest A & F University, Xianyang 712100, China; (W.R.); (L.G.); (Y.M.); (J.H.)
- Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Xianyang 712100, China
| | - Yaling Mou
- College of Veterinary Medicine, Northwest A & F University, Xianyang 712100, China; (W.R.); (L.G.); (Y.M.); (J.H.)
- Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Xianyang 712100, China
| | - Wen Deng
- College of Veterinary Medicine, Northwest A & F University, Xianyang 712100, China; (W.R.); (L.G.); (Y.M.); (J.H.)
- Correspondence: (W.D.); (F.Y.)
| | - Jinlian Hua
- College of Veterinary Medicine, Northwest A & F University, Xianyang 712100, China; (W.R.); (L.G.); (Y.M.); (J.H.)
- Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Xianyang 712100, China
| | - Fan Yang
- College of Veterinary Medicine, Northwest A & F University, Xianyang 712100, China; (W.R.); (L.G.); (Y.M.); (J.H.)
- Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A & F University, Xianyang 712100, China
- Correspondence: (W.D.); (F.Y.)
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7
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Xin Y, Wang J, Wu Y, Li Q, Dong M, Liu C, He Q, Wang R, Wang D, Jiang S, Xiao W, Tian Y, Zhang W. Identification of Nanog as a novel inhibitor of Rad51. Cell Death Dis 2022; 13:193. [PMID: 35220392 PMCID: PMC8882189 DOI: 10.1038/s41419-022-04644-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2021] [Revised: 01/12/2022] [Accepted: 02/01/2022] [Indexed: 11/09/2022]
Abstract
AbstractTo develop inhibitors targeting DNA damage repair pathways is important to improve the effectiveness of chemo- and radiotherapy for cancer patients. Rad51 mediates homologous recombination (HR) repair of DNA damages. It is widely overexpressed in human cancers and overwhelms chemo- and radiotherapy-generated DNA damages through enhancing HR repair signaling, preventing damage-caused cancer cell death. Therefore, to identify inhibitors of Rad51 is important to achieve effective treatment of cancers. Transcription factor Nanog is a core regulator of embryonic stem (ES) cells for its indispensable role in stemness maintenance. In this study, we identified Nanog as a novel inhibitor of Rad51. It interacts with Rad51 and inhibits Rad51-mediated HR repair of DNA damage through its C/CD2 domain. Moreover, Rad51 inhibition can be achieved by nanoscale material- or cell-penetrating peptide (CPP)-mediated direct delivery of Nanog-C/CD2 peptides into somatic cancer cells. Furthermore, we revealed that Nanog suppresses the binding of Rad51 to single-stranded DNAs to stall the HR repair signaling. This study provides explanation for the high γH2AX level in unperturbed ES cells and early embryos, and suggests Nanog-C/CD2 as a promising drug candidate applied to Rad51-related basic research and therapeutic application studies.
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Merighi A, Gionchiglia N, Granato A, Lossi L. The Phosphorylated Form of the Histone H2AX (γH2AX) in the Brain from Embryonic Life to Old Age. Molecules 2021; 26:7198. [PMID: 34885784 PMCID: PMC8659122 DOI: 10.3390/molecules26237198] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2021] [Revised: 11/25/2021] [Accepted: 11/25/2021] [Indexed: 12/12/2022] Open
Abstract
The γ phosphorylated form of the histone H2AX (γH2AX) was described more than 40 years ago and it was demonstrated that phosphorylation of H2AX was one of the first cellular responses to DNA damage. Since then, γH2AX has been implicated in diverse cellular functions in normal and pathological cells. In the first part of this review, we will briefly describe the intervention of H2AX in the DNA damage response (DDR) and its role in some pivotal cellular events, such as regulation of cell cycle checkpoints, genomic instability, cell growth, mitosis, embryogenesis, and apoptosis. Then, in the main part of this contribution, we will discuss the involvement of γH2AX in the normal and pathological central nervous system, with particular attention to the differences in the DDR between immature and mature neurons, and to the significance of H2AX phosphorylation in neurogenesis and neuronal cell death. The emerging picture is that H2AX is a pleiotropic molecule with an array of yet not fully understood functions in the brain, from embryonic life to old age.
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Affiliation(s)
| | | | | | - Laura Lossi
- Department of Veterinary Sciences, University of Turin, I-10095 Grugliasco, Italy; (A.M.); (N.G.); (A.G.)
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9
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Novo CL. A Tale of Two States: Pluripotency Regulation of Telomeres. Front Cell Dev Biol 2021; 9:703466. [PMID: 34307383 PMCID: PMC8300013 DOI: 10.3389/fcell.2021.703466] [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: 04/30/2021] [Accepted: 06/08/2021] [Indexed: 01/01/2023] Open
Abstract
Inside the nucleus, chromatin is functionally organized and maintained as a complex three-dimensional network of structures with different accessibility such as compartments, lamina associated domains, and membraneless bodies. Chromatin is epigenetically and transcriptionally regulated by an intricate and dynamic interplay of molecular processes to ensure genome stability. Phase separation, a process that involves the spontaneous organization of a solution into separate phases, has been proposed as a mechanism for the timely coordination of several cellular processes, including replication, transcription and DNA repair. Telomeres, the repetitive structures at the end of chromosomes, are epigenetically maintained in a repressed heterochromatic state that prevents their recognition as double-strand breaks (DSB), avoiding DNA damage repair and ensuring cell proliferation. In pluripotent embryonic stem cells, telomeres adopt a non-canonical, relaxed epigenetic state, which is characterized by a low density of histone methylation and expression of telomere non-coding transcripts (TERRA). Intriguingly, this telomere non-canonical conformation is usually associated with chromosome instability and aneuploidy in somatic cells, raising the question of how genome stability is maintained in a pluripotent background. In this review, we will explore how emerging technological and conceptual developments in 3D genome architecture can provide novel mechanistic perspectives for the pluripotent epigenetic paradox at telomeres. In particular, as RNA drives the formation of LLPS, we will consider how pluripotency-associated high levels of TERRA could drive and coordinate phase separation of several nuclear processes to ensure genome stability. These conceptual advances will provide a better understanding of telomere regulation and genome stability within the highly dynamic pluripotent background.
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Affiliation(s)
- Clara Lopes Novo
- The Francis Crick Institute, London, United Kingdom
- Imperial College London, London, United Kingdom
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10
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Orlando L, Tanasijevic B, Nakanishi M, Reid JC, García-Rodríguez JL, Chauhan KD, Porras DP, Aslostovar L, Lu JD, Shapovalova Z, Mitchell RR, Boyd AL, Bhatia M. Phosphorylation state of the histone variant H2A.X controls human stem and progenitor cell fate decisions. Cell Rep 2021; 34:108818. [PMID: 33691101 DOI: 10.1016/j.celrep.2021.108818] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2017] [Revised: 10/28/2020] [Accepted: 02/11/2021] [Indexed: 02/07/2023] Open
Abstract
Histone variants (HVs) are a subfamily of epigenetic regulators implicated in embryonic development, but their role in human stem cell fate remains unclear. Here, we reveal that the phosphorylation state of the HV H2A.X (γH2A.X) regulates self-renewal and differentiation of human pluripotent stem cells (hPSCs) and leukemic progenitors. As demonstrated by CRISPR-Cas deletion, H2A.X is essential in maintaining normal hPSC behavior. However, reduced levels of γH2A.X enhances hPSC differentiation toward the hematopoietic lineage with concomitant inhibition of neural development. In contrast, activation and sustained levels of phosphorylated H2A.X enhance hPSC neural fate while suppressing hematopoiesis. This controlled lineage bias correlates to occupancy of γH2A.X at genomic loci associated with ectoderm versus mesoderm specification. Finally, drug modulation of H2A.X phosphorylation overcomes differentiation block of patient-derived leukemic progenitors. Our study demonstrates HVs may serve to regulate pluripotent cell fate and that this biology could be extended to somatic cancer stem cell control.
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Affiliation(s)
- Luca Orlando
- McMaster University, Michael G. DeGroote School of Medicine, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada
| | - Borko Tanasijevic
- McMaster University, Michael G. DeGroote School of Medicine, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada
| | - Mio Nakanishi
- McMaster University, Michael G. DeGroote School of Medicine, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada
| | - Jennifer C Reid
- McMaster University, Michael G. DeGroote School of Medicine, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada
| | - Juan L García-Rodríguez
- McMaster University, Michael G. DeGroote School of Medicine, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada
| | - Kapil Dev Chauhan
- McMaster University, Michael G. DeGroote School of Medicine, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada
| | - Deanna P Porras
- McMaster University, Michael G. DeGroote School of Medicine, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada
| | - Lili Aslostovar
- McMaster University, Michael G. DeGroote School of Medicine, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada
| | - Justin D Lu
- McMaster University, Michael G. DeGroote School of Medicine, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada
| | - Zoya Shapovalova
- McMaster University, Michael G. DeGroote School of Medicine, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada
| | - Ryan R Mitchell
- McMaster University, Michael G. DeGroote School of Medicine, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada
| | - Allison L Boyd
- McMaster University, Michael G. DeGroote School of Medicine, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada
| | - Mickie Bhatia
- McMaster University, Michael G. DeGroote School of Medicine, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON L8N 3Z5, Canada.
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11
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Bloom JC, Schimenti JC. Sexually dimorphic DNA damage responses and mutation avoidance in the mouse germline. Genes Dev 2020; 34:1637-1649. [PMID: 33184219 PMCID: PMC7706705 DOI: 10.1101/gad.341602.120] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2020] [Accepted: 10/07/2020] [Indexed: 12/20/2022]
Abstract
In this study, Bloom and Schimenti examine the response of primordial germ cells to DNA damage. Using both environmental and genetic stresses, the authors reveal the importance of the G1 checkpoint in preventing accumulation of complex mutations in the germline, and the differentiation of the DNA damage response during germ cell development. Germ cells specified during fetal development form the foundation of the mammalian germline. These primordial germ cells (PGCs) undergo rapid proliferation, yet the germline is highly refractory to mutation accumulation compared with somatic cells. Importantly, while the presence of endogenous or exogenous DNA damage has the potential to impact PGCs, there is little known about how these cells respond to stressors. To better understand the DNA damage response (DDR) in these cells, we exposed pregnant mice to ionizing radiation (IR) at specific gestational time points and assessed the DDR in PGCs. Our results show that PGCs prior to sex determination lack a G1 cell cycle checkpoint. Additionally, the response to IR-induced DNA damage differs between female and male PGCs post-sex determination. IR of female PGCs caused uncoupling of germ cell differentiation and meiotic initiation, while male PGCs exhibited repression of piRNA metabolism and transposon derepression. We also used whole-genome single-cell DNA sequencing to reveal that genetic rescue of DNA repair-deficient germ cells (Fancm−/−) leads to increased mutation incidence and biases. Importantly, our work uncovers novel insights into how PGCs exposed to DNA damage can become developmentally defective, leaving only those genetically fit cells to establish the adult germline.
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Affiliation(s)
- Jordana C Bloom
- Department of Biomedical Sciences,, Cornell University, Ithaca, New York 14853, USA.,Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA
| | - John C Schimenti
- Department of Biomedical Sciences,, Cornell University, Ithaca, New York 14853, USA.,Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA
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12
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Wang X, Ellenbecker M, Hickey B, Day NJ, Osterli E, Terzo M, Voronina E. Antagonistic control of Caenorhabditis elegans germline stem cell proliferation and differentiation by PUF proteins FBF-1 and FBF-2. eLife 2020; 9:52788. [PMID: 32804074 PMCID: PMC7467723 DOI: 10.7554/elife.52788] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2019] [Accepted: 08/14/2020] [Indexed: 02/07/2023] Open
Abstract
Stem cells support tissue maintenance, but the mechanisms that coordinate the rate of stem cell self-renewal with differentiation at a population level remain uncharacterized. We find that two PUF family RNA-binding proteins FBF-1 and FBF-2 have opposite effects on Caenorhabditis elegans germline stem cell dynamics: FBF-1 restricts the rate of meiotic entry, while FBF-2 promotes both cell division and meiotic entry rates. Antagonistic effects of FBFs are mediated by their distinct activities toward the shared set of target mRNAs, where FBF-1-mediated post-transcriptional control requires the activity of CCR4-NOT deadenylase, while FBF-2 is deadenylase-independent and might protect the targets from deadenylation. These regulatory differences depend on protein sequences outside of the conserved PUF family RNA-binding domain. We propose that the opposing FBF-1 and FBF-2 activities serve to modulate stem cell division rate simultaneously with the rate of meiotic entry.
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Affiliation(s)
- Xiaobo Wang
- Division of Biological Sciences, University of Montana, Missoula, United States
| | - Mary Ellenbecker
- Division of Biological Sciences, University of Montana, Missoula, United States
| | - Benjamin Hickey
- Division of Biological Sciences, University of Montana, Missoula, United States
| | - Nicholas J Day
- Division of Biological Sciences, University of Montana, Missoula, United States
| | - Emily Osterli
- Division of Biological Sciences, University of Montana, Missoula, United States
| | - Mikaya Terzo
- Division of Biological Sciences, University of Montana, Missoula, United States
| | - Ekaterina Voronina
- Division of Biological Sciences, University of Montana, Missoula, United States
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13
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Radiation Response of Murine Embryonic Stem Cells. Cells 2020; 9:cells9071650. [PMID: 32660081 PMCID: PMC7408589 DOI: 10.3390/cells9071650] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Revised: 06/18/2020] [Accepted: 07/01/2020] [Indexed: 12/13/2022] Open
Abstract
To understand the mechanisms of disturbed differentiation and development by radiation, murine CGR8 embryonic stem cells (mESCs) were exposed to ionizing radiation and differentiated by forming embryoid bodies (EBs). The colony forming ability test was applied for survival and the MTT test for viability determination after X-irradiation. Cell cycle progression was determined by flow cytometry of propidium iodide-stained cells, and DNA double strand break (DSB) induction and repair by γH2AX immunofluorescence. The radiosensitivity of mESCs was slightly higher compared to the murine osteoblast cell line OCT-1. The viability 72 h after X-irradiation decreased dose-dependently and was higher in the presence of leukemia inhibitory factor (LIF). Cells exposed to 2 or 7 Gy underwent a transient G2 arrest. X-irradiation induced γH2AX foci and they disappeared within 72 h. After 72 h of X-ray exposure, RNA was isolated and analyzed using genome-wide microarrays. The gene expression analysis revealed amongst others a regulation of developmental genes (Ada, Baz1a, Calcoco2, Htra1, Nefh, S100a6 and Rassf6), downregulation of genes involved in glycolysis and pyruvate metabolism whereas upregulation of genes related to the p53 signaling pathway. X-irradiated mESCs formed EBs and differentiated toward cardiomyocytes but their beating frequencies were lower compared to EBs from unirradiated cells. These results suggest that X-irradiation of mESCs deregulate genes related to the developmental process. The most significant biological processes found to be altered by X-irradiation in mESCs were the development of cardiovascular, nervous, circulatory and renal system. These results may explain the X-irradiation induced-embryonic lethality and malformations observed in animal studies.
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14
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Padgett J, Santos SDM. From clocks to dominoes: lessons on cell cycle remodelling from embryonic stem cells. FEBS Lett 2020; 594:2031-2045. [PMID: 32535913 DOI: 10.1002/1873-3468.13862] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2019] [Revised: 05/01/2020] [Accepted: 05/27/2020] [Indexed: 12/21/2022]
Abstract
Cell division is a fundamental cellular process and the evolutionarily conserved networks that control cell division cycles adapt during development, tissue regeneration, cell de-differentiation and reprogramming, and a variety of pathological conditions. Embryonic development is a prime example of such versatility: fast, clock-like divisions hallmarking embryonic cells at early developmental stages become slower and controlled during cellular differentiation and lineage specification. In this review, we compare and contrast the unique cell cycle of mouse and human embryonic stem cells with that of early embryonic cells and of differentiated cells. We propose that embryonic stem cells provide an extraordinarily useful model system to understand cell cycle remodelling during embryonic-to-somatic transitions. We discuss how cell cycle networks help sustain embryonic stem cell pluripotency and self-renewal and how they safeguard cell identity and proper cell number in differentiated cells. Finally, we highlight the incredible diversity in cell cycle regulation within mammals and discuss the implications of studying cell cycle remodelling for understanding healthy and disease states.
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Affiliation(s)
- Joe Padgett
- Quantitative Cell Biology Lab, The Francis Crick Institute, London, UK
| | - Silvia D M Santos
- Quantitative Cell Biology Lab, The Francis Crick Institute, London, UK
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15
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Prunellae Spica Extract Suppresses Teratoma Formation of Pluripotent Stem Cells through p53-Mediated Apoptosis. Nutrients 2020; 12:nu12030721. [PMID: 32182802 PMCID: PMC7146640 DOI: 10.3390/nu12030721] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2020] [Revised: 03/04/2020] [Accepted: 03/05/2020] [Indexed: 12/31/2022] Open
Abstract
Induced pluripotent stem cells (iPSCs) have similar properties to embryonic stem cells in terms of indefinite self-renewal and differentiation capacity. After in vitro differentiation of iPSCs, undifferentiated iPSCs (USCs) may exist in cell therapy material and can form teratomas after in vivo transplantation. Selective elimination of residual USCs is, therefore, very important. Prunellae Spica (PS) is a traditional medicinal plant that has been shown to exert anti-cancer, antioxidant, and anti-inflammatory activities; however, its effects on iPSCs have not been previously characterized. In this study, we find that ethanol extract of PS (EPS) effectively induces apoptotic cell death of USCs through G2/M cell cycle arrest, generation of intracellular reactive oxygen species, alteration of mitochondrial membrane potentials, and caspase activation of USCs. In addition, EPS increases p53 accumulation and expression of its downstream targets. In p53 knockout (KO) iPSCs, the EPS did not induce apoptosis, indicating that EPS-mediated apoptosis of USCs was p53-dependent. In addition, EPS was not genotoxic towards iPSCs-derived differentiated cells. EPS treatment before injection efficiently prevented in ovo teratoma formation of p53 wild-type (WT) iPSCs but not p53KO iPSCs. Collectively, these results indicate that EPS has potent anti-teratoma activity and no genotoxicity to differentiated cells. It can, therefore, be used in the development of safe and efficient iPSC-based cell therapies.
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16
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TRIM66 reads unmodified H3R2K4 and H3K56ac to respond to DNA damage in embryonic stem cells. Nat Commun 2019; 10:4273. [PMID: 31537782 PMCID: PMC6753139 DOI: 10.1038/s41467-019-12126-4] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2018] [Accepted: 08/20/2019] [Indexed: 12/19/2022] Open
Abstract
Recognition of specific chromatin modifications by distinct structural domains within “reader” proteins plays a critical role in the maintenance of genomic stability. However, the specific mechanisms involved in this process remain unclear. Here we report that the PHD-Bromo tandem domain of tripartite motif-containing 66 (TRIM66) recognizes the unmodified H3R2-H3K4 and acetylated H3K56. The aberrant deletion of Trim66 results in severe DNA damage and genomic instability in embryonic stem cells (ESCs). Moreover, we find that the recognition of histone modification by TRIM66 is critical for DNA damage repair (DDR) in ESCs. TRIM66 recruits Sirt6 to deacetylate H3K56ac, negatively regulating the level of H3K56ac and facilitating the initiation of DDR. Importantly, Trim66-deficient blastocysts also exhibit higher levels of H3K56ac and DNA damage. Collectively, the present findings indicate the vital role of TRIM66 in DDR in ESCs, establishing the relationship between histone readers and maintenance of genomic stability. TRIM66 protein has an N-terminal tripartite motif and a C-terminal PHD Bromodomain. Here the authors show the specific histone modification recognition of TRIM66-PHD-Bromodomain through crystallography and biochemistry assay, and further reveal that TRIM66 recognition of certain histone modification is important for DNA damage repair in ESCs.
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17
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Vallabhaneni H, Lynch PJ, Chen G, Park K, Liu Y, Goehe R, Mallon BS, Boehm M, Hursh DA. High Basal Levels of γH2AX in Human Induced Pluripotent Stem Cells Are Linked to Replication-Associated DNA Damage and Repair. Stem Cells 2018; 36:1501-1513. [PMID: 29873142 DOI: 10.1002/stem.2861] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2018] [Revised: 05/02/2018] [Accepted: 05/19/2018] [Indexed: 01/07/2023]
Abstract
Human induced pluripotent stem cells (iPSCs) have great potential as source cells for therapeutic uses. However, reports indicate that iPSCs carry genetic abnormalities, which may impede their medical use. Little is known about mechanisms contributing to intrinsic DNA damage in iPSCs that could lead to genomic instability. In this report, we investigated the level of DNA damage in human iPSC lines compared with their founder fibroblast line and derived mesenchymal stromal cell (MSC) lines using the phosphorylated histone variant, γH2AX, as a marker of DNA damage. We show that human iPSCs have elevated basal levels of γH2AX, which correlate with markers of DNA replication: 5-ethynyl-2'-deoxyuridine and the single-stranded binding protein, replication protein A. γH2AX foci in iPSCs also colocalize to BRCA1 and RAD51, proteins in the homologous repair pathway, implying γH2AX in iPSCs marks sites of double strand breaks. Our study demonstrates an association between increased basal levels of γH2AX and the rapid replication of iPSCs. Stem Cells 2018;36:1501-1513.
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Affiliation(s)
- Haritha Vallabhaneni
- Division of Cellular and Gene Therapy, Office of Tissue and Advanced Therapies, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, USA
| | - Patrick J Lynch
- Division of Biotechnology Review and Research II, Office of Pharmaceutical Quality, Center for Drugs Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, USA
| | - Guibin Chen
- Laboratory of Cardiovascular Regenerative Medicine, National Heart, Lung and Blood Institute, Bethesda, Maryland, USA
| | - Kyeyoon Park
- Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA
| | - Yangtengyu Liu
- Laboratory of Cardiovascular Regenerative Medicine, National Heart, Lung and Blood Institute, Bethesda, Maryland, USA
| | - Rachel Goehe
- Division of Cellular and Gene Therapy, Office of Tissue and Advanced Therapies, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, USA
| | - Barbara S Mallon
- Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland, USA
| | - Manfred Boehm
- Laboratory of Cardiovascular Regenerative Medicine, National Heart, Lung and Blood Institute, Bethesda, Maryland, USA
| | - Deborah A Hursh
- Division of Cellular and Gene Therapy, Office of Tissue and Advanced Therapies, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland, USA
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18
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Grigorash BB, Suvorova II, Pospelov VA. AICAR-Dependent Activation of AMPK Kinase Is Not Accompanied by G1/S Block in Mouse Embryonic Stem Cells. Mol Biol 2018. [DOI: 10.1134/s0026893318030056] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
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19
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Furuta T, Joo HJ, Trimmer KA, Chen SY, Arur S. GSK-3 promotes S-phase entry and progression in C. elegans germline stem cells to maintain tissue output. Development 2018; 145:dev.161042. [PMID: 29695611 DOI: 10.1242/dev.161042] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2017] [Accepted: 04/17/2018] [Indexed: 12/26/2022]
Abstract
Adult C. elegans germline stem cells (GSCs) and mouse embryonic stem cells (mESCs) exhibit a non-canonical cell cycle structure with an abbreviated G1 phase and phase-independent expression of Cdk2 and cyclin E. Mechanisms that promote the abbreviated cell cycle remain unknown, as do the consequences of not maintaining an abbreviated cell cycle in these tissues. In GSCs, we discovered that loss of gsk-3 results in reduced GSC proliferation without changes in differentiation or responsiveness to GLP-1/Notch signaling. We find that DPL-1 transcriptional activity inhibits CDK-2 mRNA accumulation in GSCs, which leads to slower S-phase entry and progression. Inhibition of dpl-1 or transgenic expression of CDK-2 via a heterologous germline promoter rescues the S-phase entry and progression defects of the gsk-3 mutants, demonstrating that transcriptional regulation rather than post-translational control of CDK-2 establishes the abbreviated cell cycle structure in GSCs. This highlights an inhibitory cascade wherein GSK-3 inhibits DPL-1 and DPL-1 inhibits cdk-2 transcription. Constitutive GSK-3 activity through this cascade maintains an abbreviated cell cycle structure to permit the efficient proliferation of GSCs necessary for continuous tissue output.
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Affiliation(s)
- Tokiko Furuta
- Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Hyoe-Jin Joo
- Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Kenneth A Trimmer
- Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.,Genes and Development Graduate Program, MD Anderson Cancer Center UT Health Graduate School of Biomedical Sciences, Houston, TX 77030, USA
| | - Shin-Yu Chen
- Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Swathi Arur
- Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA .,Genes and Development Graduate Program, MD Anderson Cancer Center UT Health Graduate School of Biomedical Sciences, Houston, TX 77030, USA
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20
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PARI Regulates Stalled Replication Fork Processing To Maintain Genome Stability upon Replication Stress in Mice. Mol Cell Biol 2017; 37:MCB.00117-17. [PMID: 28894029 DOI: 10.1128/mcb.00117-17] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2017] [Accepted: 09/07/2017] [Indexed: 12/14/2022] Open
Abstract
DNA replication is frequently perturbed by intrinsic, as well as extrinsic, genotoxic stress. At damaged forks, DNA replication and repair activities require proper coordination to maintain genome integrity. We show here that PARI antirecombinase plays an essential role in modulating the initial response to replication stress in mice. PARI is functionally dormant at replisomes during normal replication, but upon replication stress, it enhances nascent-strand shortening that is regulated by RAD51 and MRE11. PARI then promotes double-strand break induction, followed by new origin firing instead of replication restart. Such PARI function is apparently obstructive to replication but is nonetheless physiologically required for chromosome stability in vivo and ex vivo Of note, Pari-deficient embryonic stem cells exhibit spontaneous chromosome instability, which is attenuated by differentiation induction, suggesting that pluripotent stem cells have a preferential requirement for PARI that acts against endogenous replication stress. PARI is a latent modulator of stalled fork processing, which is required for stable genome inheritance under both endogenous and exogenous replication stress in mice.
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21
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Kozhukharova I, Zemelko V, Kovaleva Z, Alekseenko L, Lyublinskaya O, Nikolsky N. Therapeutic doses of doxorubicin induce premature senescence of human mesenchymal stem cells derived from menstrual blood, bone marrow and adipose tissue. Int J Hematol 2017; 107:286-296. [PMID: 29022209 DOI: 10.1007/s12185-017-2346-6] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2017] [Revised: 09/29/2017] [Accepted: 10/06/2017] [Indexed: 01/09/2023]
Abstract
Doxorubicin (Dox) is an effective anticancer drug with known activity against a wide spectrum of malignancies, hematologic malignancies in particular. Despite extensive clinical use, the mechanisms of its side effects and negative action on normal cells remain under study. The aim of this study was to investigate the effect of Dox on cultured human mesenchymal stem cells (MSCs) derived from menstrual blood (eMSCs), bone marrow (BMSCs) and adipose tissue (AMSCs). Dox treatment in high doses decreased the survival of MSCs in a dose-dependent manner. Clinically relevant low doses of Dox induced premature senescence of eMSCs, BMSCs and AMSCs, but did not kill the cells. Dox caused cell cycle arrest and formation of γ-H2AX foci, and increased the number of SA-β-gal-positive cells. BMSCs entered premature senescence earlier than other MSCs. It has been reported that neural-like cells differentiated from MSCs of various origins are more sensitive to Dox than their parent cells. Dox-treated differentiated MSCs exhibited lower viability and earlier generation of γ-H2AX foci. Dox administration inhibited secretory activity in neural-like cells. These findings suggest that a clinically relevant Dox dose damages cultured MSCs, inducing their premature senescence. MSCs are more resistant to this damage than differentiated cells.
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Affiliation(s)
- Irina Kozhukharova
- Department of Intracellular Signaling and Transport, Institute of Cytology Russian Academy of Sciences, Tikhoretskiy Prospect 4, Saint-Petersburg, 194064, Russia.
| | - Victoria Zemelko
- Department of Intracellular Signaling and Transport, Institute of Cytology Russian Academy of Sciences, Tikhoretskiy Prospect 4, Saint-Petersburg, 194064, Russia
| | - Zoya Kovaleva
- Department of Intracellular Signaling and Transport, Institute of Cytology Russian Academy of Sciences, Tikhoretskiy Prospect 4, Saint-Petersburg, 194064, Russia
| | - Larisa Alekseenko
- Department of Intracellular Signaling and Transport, Institute of Cytology Russian Academy of Sciences, Tikhoretskiy Prospect 4, Saint-Petersburg, 194064, Russia
| | - Olga Lyublinskaya
- Department of Intracellular Signaling and Transport, Institute of Cytology Russian Academy of Sciences, Tikhoretskiy Prospect 4, Saint-Petersburg, 194064, Russia
| | - Nikolay Nikolsky
- Department of Intracellular Signaling and Transport, Institute of Cytology Russian Academy of Sciences, Tikhoretskiy Prospect 4, Saint-Petersburg, 194064, Russia
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22
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Protein Kinases in Pluripotency—Beyond the Usual Suspects. J Mol Biol 2017; 429:1504-1520. [DOI: 10.1016/j.jmb.2017.04.013] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2016] [Revised: 04/21/2017] [Accepted: 04/21/2017] [Indexed: 12/14/2022]
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23
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Molchadsky A, Rotter V. p53 and its mutants on the slippery road from stemness to carcinogenesis. Carcinogenesis 2017; 38:347-358. [PMID: 28334334 DOI: 10.1093/carcin/bgw092] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/07/2016] [Accepted: 08/25/2016] [Indexed: 12/18/2022] Open
Abstract
Normal development, tissue homeostasis and regeneration following injury rely on the proper functions of wide repertoire of stem cells (SCs) persisting during embryonic period and throughout the adult life. Therefore, SCs employ robust mechanisms to preserve their genomic integrity and avoid heritage of mutations to their daughter cells. Importantly, propagation of SCs with faulty DNA as well as dedifferentiation of genomically altered somatic cells may result in derivation of cancer SCs, which are considered to be the driving force of the tumorigenic process. Multiple experimental evidence suggest that p53, the central tumor suppressor gene, plays a critical regulatory role in determination of SCs destiny, thereby eliminating damaged SCs from the general SC population. Notably, mutant p53 proteins do not only lose the tumor suppressive function, but rather gain new oncogenic function that markedly promotes various aspects of carcinogenesis. In this review, we elaborate on the role of wild type and mutant p53 proteins in the various SCs types that appear under homeostatic conditions as well as in cancer. It is plausible that the growing understanding of the mechanisms underlying cancer SC phenotype and p53 malfunction will allow future optimization of cancer therapeutics in the context of precision medicine.
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Affiliation(s)
- Alina Molchadsky
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel
| | - Varda Rotter
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel
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24
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Suchorska WM, Augustyniak E, Łukjanow M. Comparison of the early response of human embryonic stem cells and human induced pluripotent stem cells to ionizing radiation. Mol Med Rep 2017; 15:1952-1962. [PMID: 28259963 PMCID: PMC5364988 DOI: 10.3892/mmr.2017.6270] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2016] [Accepted: 01/09/2017] [Indexed: 12/14/2022] Open
Abstract
Despite the well-demonstrated efficacy of stem cell (SC) therapy, this approach has a number of key drawbacks. One important concern is the response of pluripotent SCs to treatment with ionizing radiation (IR), given that SCs used in regenerative medicine will eventually be exposed to IR for diagnostic or treatment-associated purposes. Therefore, the aim of the present study was to examine and compare early IR-induced responses of pluripotent SCs to assess their radioresistance and radiosensitivity. In the present study, 3 cell lines; human embryonic SCs (hESCs), human induced pluripotent SCs (hiPSCs) and primary human dermal fibroblasts (PHDFs); were exposed to IR at doses ranging from 0 to 15 gray (Gy). Double strand breaks (DSBs), and the gene expression of the following DNA repair genes were analyzed: P53; RAD51; BRCA2; PRKDC; and XRCC4. hiPSCs demonstrated greater radioresistance, as fewer DSBs were identified, compared with hESCs. Both pluripotent SC lines exhibited distinct gene expression profiles in the most common DNA repair genes that are involved in homologous recombination, non-homologous end-joining and enhanced DNA damage response following IR exposure. Although hESCs and hiPSCs are equivalent in terms of capacity for pluripotency and differentiation into 3 germ layers, the results of the present study indicate that these 2 types of SCs differ in gene expression following exposure to IR. Consequently, further research is required to determine whether hiPSCs and hESCs are equally safe for application in clinical practice. The present study contributes to a greater understanding of DNA damage response (DDR) mechanisms activated in pluripotent SCs and may aid in the future development of safe SC-based clinical protocols.
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Affiliation(s)
| | - Ewelina Augustyniak
- Radiobiology Laboratory, Greater Poland Cancer Centre, 61‑866 Poznan, Poland
| | - Magdalena Łukjanow
- Radiobiology Laboratory, Greater Poland Cancer Centre, 61‑866 Poznan, Poland
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25
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Fu X, Cui K, Yi Q, Yu L, Xu Y. DNA repair mechanisms in embryonic stem cells. Cell Mol Life Sci 2017; 74:487-493. [PMID: 27614628 PMCID: PMC11107665 DOI: 10.1007/s00018-016-2358-z] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2016] [Revised: 08/28/2016] [Accepted: 09/05/2016] [Indexed: 10/21/2022]
Abstract
Embryonic stem cells (ESCs) can undergo unlimited self-renewal and retain the pluripotency to differentiate into all cell types in the body. Therefore, as a renewable source of various functional cells in the human body, ESCs hold great promise for human cell therapy. During the rapid proliferation of ESCs in culture, DNA damage, such as DNA double-stranded breaks, will occur in ESCs. Therefore, to realize the potential of ESCs in human cell therapy, it is critical to understand the mechanisms how ESCs activate DNA damage response and DNA repair to maintain genomic stability, which is a prerequisite for their use in human therapy. In this context, it has been shown that ESCs harbor much fewer spontaneous mutations than somatic cells. Consistent with the finding that ESCs are genetically more stable than somatic cells, recent studies have indicated that ESCs can mount more robust DNA damage responses and DNA repair than somatic cells to ensure their genomic integrity.
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Affiliation(s)
- Xuemei Fu
- Shenzhen Children's Hospital, 7019 Yitian Road, Shenzhen, 518026, China.
| | - Ke Cui
- Center for Regenerative and Translational Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China
| | - Qiuxiang Yi
- Center for Regenerative and Translational Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China
| | - Lili Yu
- Guangdong Provincial Key Laboratory of Cancer Immunotherapy, Cancer Research Institute, Southern Medical University, Guangzhou, Guangdong, China
| | - Yang Xu
- Center for Regenerative and Translational Medicine, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China.
- Guangdong Provincial Key Laboratory of Cancer Immunotherapy, Cancer Research Institute, Southern Medical University, Guangzhou, Guangdong, China.
- Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA.
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26
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Suvorova II, Grigorash BB, Chuykin IA, Pospelova TV, Pospelov VA. G1 checkpoint is compromised in mouse ESCs due to functional uncoupling of p53-p21Waf1 signaling. Cell Cycle 2016; 15:52-63. [PMID: 26636245 DOI: 10.1080/15384101.2015.1120927] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Mouse embryonic stem cells (mESCs) lack of G1 checkpoint despite that irradiation (IR) activates ATM/ATR-mediated DDR signaling pathway. The IR-induced p53 localizes in the nuclei and up-regulates p21/Waf1 transcription but that does not lead to accumulation of p21/Waf1 protein. The negative control of the p21Waf1 expression appears to occur at 2 levels of regulation. First, both p21/Waf1 gene transcription and the p21/Waf1 protein content increase in mESCs treated with histone-deacetylase inhibitors, implying its epigenetic regulation. Second, proteasome inhibitors cause the p21/Waf1 accumulation, indicating that the protein is a subject of proteasome-dependent degradation in ESСs. Then, the dynamics of IR-induced p21Waf1 protein show its accumulation at long-term time points (3 and 5 days) that coincides with an increase in the proportion of G1-phase cells, down-regulation of Oct4 and Nanog pluripotent gene transcription and activation of endoderm-specific genes sox17 and afp. In addition, nutlin-dependent stabilization of p53 in mESC was also accompanied by the accumulation of p21/Waf1 as well as restoration of G1 checkpoint and an onset of differentiation. Thus, the lack of functional p21/Waf1 is indispensable for maintaining self-renewal and pluripotency of mESCs.
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Affiliation(s)
- Irina I Suvorova
- a Institute of Cytology , Russian Academy of Sciences , St-Petersburg , Russia.,b Saint-Petersburg State University, Saint-Petersburg State University , St-Petersburg , Russia
| | - Bogdan B Grigorash
- a Institute of Cytology , Russian Academy of Sciences , St-Petersburg , Russia.,b Saint-Petersburg State University, Saint-Petersburg State University , St-Petersburg , Russia
| | | | - Tatiana V Pospelova
- a Institute of Cytology , Russian Academy of Sciences , St-Petersburg , Russia.,b Saint-Petersburg State University, Saint-Petersburg State University , St-Petersburg , Russia
| | - Valery A Pospelov
- a Institute of Cytology , Russian Academy of Sciences , St-Petersburg , Russia.,b Saint-Petersburg State University, Saint-Petersburg State University , St-Petersburg , Russia
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27
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Czerwinska AM, Nowacka J, Aszer M, Gawrzak S, Archacka K, Fogtman A, Iwanicka-Nowicka R, Jańczyk-Ilach K, Koblowska M, Ciemerych MA, Grabowska I. Cell cycle regulation of embryonic stem cells and mouse embryonic fibroblasts lacking functional Pax7. Cell Cycle 2016; 15:2931-2942. [PMID: 27610933 PMCID: PMC5105925 DOI: 10.1080/15384101.2016.1231260] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
The transcription factor Pax7 plays a key role during embryonic myogenesis and in adult organisms in that it sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Recently we have shown that lack of Pax7 does not prevent the myogenic differentiation of pluripotent stem cells. In the current work we show that the absence of functional Pax7 in differentiating embryonic stem cells modulates cell cycle facilitating their proliferation. Surprisingly, deregulation of Pax7 function also positively impacts at the proliferation of mouse embryonic fibroblasts. Such phenotypes seem to be executed by modulating the expression of positive cell cycle regulators, such as cyclin E.
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Affiliation(s)
- Areta M Czerwinska
- a Department of Cytology , Institute of Zoology, Faculty of Biology, University of Warsaw , Warsaw , Poland
| | - Joanna Nowacka
- a Department of Cytology , Institute of Zoology, Faculty of Biology, University of Warsaw , Warsaw , Poland
| | - Magdalena Aszer
- a Department of Cytology , Institute of Zoology, Faculty of Biology, University of Warsaw , Warsaw , Poland
| | - Sylwia Gawrzak
- a Department of Cytology , Institute of Zoology, Faculty of Biology, University of Warsaw , Warsaw , Poland
| | - Karolina Archacka
- a Department of Cytology , Institute of Zoology, Faculty of Biology, University of Warsaw , Warsaw , Poland
| | - Anna Fogtman
- b Laboratory of Microarray Analysis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences , Warsaw , Poland
| | - Roksana Iwanicka-Nowicka
- b Laboratory of Microarray Analysis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences , Warsaw , Poland.,c Department of Systems Biology , Faculty of Biology, University of Warsaw , Warsaw , Poland
| | - Katarzyna Jańczyk-Ilach
- a Department of Cytology , Institute of Zoology, Faculty of Biology, University of Warsaw , Warsaw , Poland
| | - Marta Koblowska
- b Laboratory of Microarray Analysis, Institute of Biochemistry and Biophysics, Polish Academy of Sciences , Warsaw , Poland.,c Department of Systems Biology , Faculty of Biology, University of Warsaw , Warsaw , Poland
| | - Maria A Ciemerych
- a Department of Cytology , Institute of Zoology, Faculty of Biology, University of Warsaw , Warsaw , Poland
| | - Iwona Grabowska
- a Department of Cytology , Institute of Zoology, Faculty of Biology, University of Warsaw , Warsaw , Poland
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28
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Enhanced cartilage repair in 'healer' mice-New leads in the search for better clinical options for cartilage repair. Semin Cell Dev Biol 2016; 62:78-85. [PMID: 27130635 DOI: 10.1016/j.semcdb.2016.04.018] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2016] [Accepted: 04/25/2016] [Indexed: 12/13/2022]
Abstract
Adult articular cartilage has a poor capacity to undergo intrinsic repair. Current strategies for the repair of large cartilage defects are generally unsatisfactory because the restored cartilage does not have the same resistance to biomechanical loading as authentic articular cartilage and degrades over time. Recently, an exciting new research direction, focused on intrinsic cartilage regeneration rather than fibrous repair by external means, has emerged. This review explores the new findings in this rapidly moving field as they relate to the clinical goal of restoration of structurally robust, stable and non-fibrous articular cartilage following injury.
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29
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Turinetto V, Giachino C. Histone variants as emerging regulators of embryonic stem cell identity. Epigenetics 2016; 10:563-73. [PMID: 26114724 DOI: 10.1080/15592294.2015.1053682] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Dynamic regulation of chromatin structure is an important mechanism for balancing the pluripotency and cell fate decision in embryonic stem cells (ESCs). Indeed ESCs are characterized by unusual chromatin packaging, and a wide variety of chromatin regulators have been implicated in control of pluripotency and differentiation. Genome-wide maps of epigenetic factors have revealed a unique epigenetic signature in pluripotent ESCs and have contributed models to explain their plasticity. In addition to the well known epigenetic regulation through DNA methylation, histone posttranslational modifications, chromatin remodeling, and non-coding RNA, histone variants are emerging as important regulators of ESC identity. In this review, we summarize and discuss the recent progress that has highlighted the central role of histone variants in ESC pluripotency and ESC fate, focusing, in particular, on H1 variants, H2A variants H2A.X, H2A.Z and macroH2A and H3 variant H3.3.
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Affiliation(s)
- Valentina Turinetto
- a Department of Clinical and Biological Sciences; University of Turin ; Orbassano , Turin , Italy
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30
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Jacobs KM, Misri S, Meyer B, Raj S, Zobel CL, Sleckman BP, Hallahan DE, Sharma GG. Unique epigenetic influence of H2AX phosphorylation and H3K56 acetylation on normal stem cell radioresponses. Mol Biol Cell 2016; 27:1332-45. [PMID: 26941327 PMCID: PMC4831886 DOI: 10.1091/mbc.e16-01-0017] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2016] [Accepted: 02/22/2016] [Indexed: 01/08/2023] Open
Abstract
Normal stem cells from tissues often exhibiting radiation injury are highly radiosensitive and exhibit a muted DNA damage response, in contrast to differentiated progeny. These radioresponses can be attributed to unique epigenetic regulation in stem cells, identifying potential therapeutic targets for radioprotection. Normal tissue injury resulting from cancer radiotherapy is often associated with diminished regenerative capacity. We examined the relative radiosensitivity of normal stem cell populations compared with non–stem cells within several radiosensitive tissue niches and culture models. We found that these stem cells are highly radiosensitive, in contrast to their isogenic differentiated progeny. Of interest, they also exhibited a uniquely attenuated DNA damage response (DDR) and muted DNA repair. Whereas stem cells exhibit reduced ATM activation and ionizing radiation–induced foci, they display apoptotic pannuclear H2AX-S139 phosphorylation (γH2AX), indicating unique radioresponses. We also observed persistent phosphorylation of H2AX-Y142 along the DNA breaks in stem cells, which promotes apoptosis while inhibiting DDR signaling. In addition, down-regulation of constitutively elevated histone-3 lysine-56 acetylation (H3K56ac) in stem cells significantly decreased their radiosensitivity, restored DDR function, and increased survival, signifying its role as a key contributor to stem cell radiosensitivity. These results establish that unique epigenetic landscapes affect cellular heterogeneity in radiosensitivity and demonstrate the nonubiquitous nature of radiation responses. We thus elucidate novel epigenetic rheostats that promote ionizing radiation hypersensitivity in various normal stem cell populations, identifying potential molecular targets for pharmacological radioprotection of stem cells and hopefully improving the efficacy of future cancer treatment.
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Affiliation(s)
- Keith M Jacobs
- Department of Radiation Oncology, Cancer Biology Division, Washington University School of Medicine, St. Louis, MO 63108
| | - Sandeep Misri
- Department of Radiation Oncology, Cancer Biology Division, Washington University School of Medicine, St. Louis, MO 63108
| | - Barbara Meyer
- Department of Radiation Oncology, Cancer Biology Division, Washington University School of Medicine, St. Louis, MO 63108
| | - Suyash Raj
- Department of Radiation Oncology, Cancer Biology Division, Washington University School of Medicine, St. Louis, MO 63108
| | - Cheri L Zobel
- Department of Radiation Oncology, Cancer Biology Division, Washington University School of Medicine, St. Louis, MO 63108
| | - Barry P Sleckman
- Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO 63108 Department of Pathology, Laboratory and Genomic Medicine, Washington University School of Medicine, St. Louis, MO 63108
| | - Dennis E Hallahan
- Department of Radiation Oncology, Cancer Biology Division, Washington University School of Medicine, St. Louis, MO 63108 Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO 63108
| | - Girdhar G Sharma
- Department of Radiation Oncology, Cancer Biology Division, Washington University School of Medicine, St. Louis, MO 63108 Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO 63108
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31
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Ahuja AK, Jodkowska K, Teloni F, Bizard AH, Zellweger R, Herrador R, Ortega S, Hickson ID, Altmeyer M, Mendez J, Lopes M. A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells. Nat Commun 2016; 7:10660. [PMID: 26876348 PMCID: PMC4756311 DOI: 10.1038/ncomms10660] [Citation(s) in RCA: 136] [Impact Index Per Article: 15.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2015] [Accepted: 01/08/2016] [Indexed: 12/15/2022] Open
Abstract
Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity. In fast proliferating embryonic stem cells (ESC) the DNA damage response is activated by mechanisms that are as yet elusive. Here, Ahuja et al. link the DNA damage response to replication stress in mouse ESCs, caused by a short G1 phase, and propose fork remodelling as maintaining genome stability in embryos.
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Affiliation(s)
- Akshay K Ahuja
- Institute of Molecular Cancer Research, University of Zurich, Zurich CH-8057, Switzerland
| | - Karolina Jodkowska
- DNA Replication Group, Molecular Oncology Programme, CNIO, Madrid E-28029, Spain
| | - Federico Teloni
- Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Zurich CH-8057, Switzerland
| | - Anna H Bizard
- Department of Cellular and Molecular Medicine, Center for Chromosome Stability and Center for Healthy Aging, University of Copenhagen, Panum Institute, Copenhagen N DK-2200, Denmark
| | - Ralph Zellweger
- Institute of Molecular Cancer Research, University of Zurich, Zurich CH-8057, Switzerland
| | - Raquel Herrador
- Institute of Molecular Cancer Research, University of Zurich, Zurich CH-8057, Switzerland
| | - Sagrario Ortega
- Transgenic Mice Core Unit, Biotechnology Programme, CNIO, Madrid E-28029, Spain
| | - Ian D Hickson
- Department of Cellular and Molecular Medicine, Center for Chromosome Stability and Center for Healthy Aging, University of Copenhagen, Panum Institute, Copenhagen N DK-2200, Denmark
| | - Matthias Altmeyer
- Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Zurich CH-8057, Switzerland
| | - Juan Mendez
- DNA Replication Group, Molecular Oncology Programme, CNIO, Madrid E-28029, Spain
| | - Massimo Lopes
- Institute of Molecular Cancer Research, University of Zurich, Zurich CH-8057, Switzerland
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32
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Antonelli F, Campa A, Esposito G, Giardullo P, Belli M, Dini V, Meschini S, Simone G, Sorrentino E, Gerardi S, Cirrone GAP, Tabocchini MA. Induction and Repair of DNA DSB as Revealed by H2AX Phosphorylation Foci in Human Fibroblasts Exposed to Low- and High-LET Radiation: Relationship with Early and Delayed Reproductive Cell Death. Radiat Res 2015; 183:417-31. [PMID: 25844944 DOI: 10.1667/rr13855.1] [Citation(s) in RCA: 73] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2022]
Abstract
The spatial distribution of radiation-induced DNA breaks within the cell nucleus depends on radiation quality in terms of energy deposition pattern. It is generally assumed that the higher the radiation linear energy transfer (LET), the greater the DNA damage complexity. Using a combined experimental and theoretical approach, we examined the phosphorylation-dephosphorylation kinetics of radiation-induced γ-H2AX foci, size distribution and 3D focus morphology, and the relationship between DNA damage and cellular end points (i.e., cell killing and lethal mutations) after exposure to gamma rays, protons, carbon ions and alpha particles. Our results showed that the maximum number of foci are reached 30 min postirradiation for all radiation types. However, the number of foci after 0.5 Gy of each radiation type was different with gamma rays, protons, carbon ions and alpha particles inducing 12.64 ± 0.25, 10.11 ± 0.40, 8.84 ± 0.56 and 4.80 ± 0.35 foci, respectively, which indicated a clear influence of the track structure and fluence on the numbers of foci induced after a dose of 0.5 Gy for each radiation type. The γ-H2AX foci persistence was also dependent on radiation quality, i.e., the higher the LET, the longer the foci persisted in the cell nucleus. The γ-H2AX time course was compared with cell killing and lethal mutation and the results highlighted a correlation between cellular end points and the duration of γ-H2AX foci persistence. A model was developed to evaluate the probability that multiple DSBs reside in the same gamma-ray focus and such probability was found to be negligible for doses lower than 1 Gy. Our model provides evidence that the DSBs inside complex foci, such as those induced by alpha particles, are not processed independently or with the same time constant. The combination of experimental, theoretical and simulation data supports the hypothesis of an interdependent processing of closely associated DSBs, possibly associated with a diminished correct repair capability, which affects cell killing and lethal mutation.
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Affiliation(s)
- F Antonelli
- a Health and Technology Department, Istituto Superiore di Sanità, Roma, Italy
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Turinetto V, Giachino C. Multiple facets of histone variant H2AX: a DNA double-strand-break marker with several biological functions. Nucleic Acids Res 2015; 43:2489-98. [PMID: 25712102 PMCID: PMC4357700 DOI: 10.1093/nar/gkv061] [Citation(s) in RCA: 277] [Impact Index Per Article: 27.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
In the last decade, many papers highlighted that the histone variant H2AX and its phosphorylation on Ser 139 (γH2AX) cannot be simply considered a specific DNA double-strand-break (DSB) marker with a role restricted to the DNA damage response, but rather as a ‘protagonist’ in different scenarios. This review will present and discuss an up-to-date view regarding the ‘non-canonical’ H2AX roles, focusing in particular on possible functional and structural parts in contexts different from the canonical DNA DSB response. We will present aspects concerning sex chromosome inactivation in male germ cells, X inactivation in female somatic cells and mitosis, but will also focus on the more recent studies regarding embryonic and neural stem cell development, asymmetric sister chromosome segregation in stem cells and cellular senescence maintenance. We will discuss whether in these new contexts there might be a relation with the canonical DNA DSB signalling function that could justify γH2AX formation. The authors will emphasize that, just as H2AX phosphorylation signals chromatin alteration and serves the canonical function of recruiting DSB repair factors, so the modification of H2AX in contexts other than the DNA damage response may contribute towards creating a specific chromatin structure frame allowing ‘non-canonical’ functions to be carried out in different cell types.
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Affiliation(s)
- Valentina Turinetto
- Department of Clinical and Biological Sciences, University of Turin, Orbassano, Turin, Italy
| | - Claudia Giachino
- Department of Clinical and Biological Sciences, University of Turin, Orbassano, Turin, Italy
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Minieri V, Saviozzi S, Gambarotta G, Lo Iacono M, Accomasso L, Cibrario Rocchietti E, Gallina C, Turinetto V, Giachino C. Persistent DNA damage-induced premature senescence alters the functional features of human bone marrow mesenchymal stem cells. J Cell Mol Med 2015; 19:734-43. [PMID: 25619736 PMCID: PMC4395188 DOI: 10.1111/jcmm.12387] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2014] [Accepted: 06/24/2014] [Indexed: 12/26/2022] Open
Abstract
Human mesenchymal stem cells (hMSCs) are adult multipotent stem cells located in various tissues, including the bone marrow. In contrast to terminally differentiated somatic cells, adult stem cells must persist and function throughout life to ensure tissue homeostasis and repair. For this reason, they must be equipped with DNA damage responses able to maintain genomic integrity while ensuring their lifelong persistence. Evaluation of hMSC response to genotoxic insults is of great interest considering both their therapeutic potential and their physiological functions. This study aimed to investigate the response of human bone marrow MSCs to the genotoxic agent Actinomycin D (ActD), a well-known anti-tumour drug. We report that hMSCs react by undergoing premature senescence driven by a persistent DNA damage response activation, as hallmarked by inhibition of DNA synthesis, p21 and p16 protein expression, marked Senescent Associated β-galactosidase activity and enlarged γH2AX foci co-localizing with 53BP1 protein. Senescent hMSCs overexpress several senescence-associated secretory phenotype (SASP) genes and promote motility of lung tumour and osteosarcoma cell lines in vitro. Our findings disclose a multifaceted consequence of ActD treatment on hMSCs that on the one hand helps to preserve this stem cell pool and prevents damaged cells from undergoing neoplastic transformation, and on the other hand alters their functional effects on the surrounding tissue microenvironment in a way that might worsen their tumour-promoting behaviour.
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Affiliation(s)
- Valentina Minieri
- Department of Clinical and Biological Sciences, University of Turin, Orbassano, Turin, Italy
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35
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Liu Q, Wang G, Chen Y, Li G, Yang D, Kang J. A miR-590/Acvr2a/Rad51b axis regulates DNA damage repair during mESC proliferation. Stem Cell Reports 2014; 3:1103-17. [PMID: 25458897 PMCID: PMC4264031 DOI: 10.1016/j.stemcr.2014.10.006] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2014] [Revised: 10/16/2014] [Accepted: 10/16/2014] [Indexed: 11/23/2022] Open
Abstract
Embryonic stem cells (ESCs) enable rapid proliferation that also causes DNA damage. To maintain genomic stabilization during rapid proliferation, ESCs must have an efficient system to repress genotoxic stress. Here, we show that withdrawal of leukemia inhibitory factor (LIF), which maintains the self-renewal capability of mouse ESCs (mESCs), significantly inhibits the cell proliferation and DNA damage of mESCs and upregulates the expression of miR-590. miR-590 promotes single-strand break (SSB) and double-strand break (DSB) damage repair, thus slowing proliferation of mESCs without influencing stemness. miR-590 directly targets Activin receptor type 2a (Acvr2a) to mediate Activin signaling. We identified the homologous recombination-mediated repair (HRR) gene, Rad51b, as a downstream molecule of the miR-590/Acvr2a pathway regulating the SSB and DSB damage repair and cell cycle. Our study shows that a miR-590/Acvr2a/Rad51b signaling axis ensures the stabilization of mESCs by balancing DNA damage repair and rapid proliferation during self-renewal.
miR-590 promotes DNA damage repair and slows proliferation by targeting Acvr2a miR-590/Acvr2a/Rad51b axis balances SSB and DSB damage repair in mESCs
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Affiliation(s)
- Qidong Liu
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Health Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, People's Republic of China
| | - Guiying Wang
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Health Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, People's Republic of China
| | - Yafang Chen
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Health Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, People's Republic of China
| | - Guoping Li
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Health Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, People's Republic of China
| | - Dandan Yang
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Health Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, People's Republic of China
| | - Jiuhong Kang
- Clinical and Translational Research Center of Shanghai First Maternity and Infant Health Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Science and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, People's Republic of China.
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36
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Abramova MV, Svetlikova SB, Kukushkin AN, Aksenov ND, Pospelova TV, Pospelov VA. HDAC inhibitor sodium butyrate sensitizes E1A+Ras-transformed cells to DNA damaging agents by facilitating formation and persistence of γH2AX foci. Cancer Biol Ther 2014; 12:1069-77. [DOI: 10.4161/cbt.12.12.18365] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
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Heo SH, Cha Y, Park KS. Hydroxyurea induces a hypersensitive apoptotic response in mouse embryonic stem cells through p38-dependent acetylation of p53. Stem Cells Dev 2014; 23:2435-42. [PMID: 24836177 DOI: 10.1089/scd.2013.0608] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
While hydroxyurea (HU) is well known to deplete dNTP pools and lead to replication fork arrest in the cell, the mechanisms by which it exerts a cell response are poorly understood. Here, our results suggest that mouse embryonic stem cells (mESCs), unlike terminally differentiated cells such as mouse embryonic fibroblasts (MEFs), rapidly respond to low concentrations of HU by p53 acetylation, leading to activation of the caspase-dependent apoptotic pathway. We show that HU treatment induces the production of nitric oxide (NO), which plays a central role in the rapid induction of apoptosis in mESCs. By contrast, reactive oxygen species, which are expressed at significantly higher levels in mESCs compared with MEFs, are not related to the HU response. Furthermore, on exposure to HU, the p38 signaling pathway becomes activated in a dose-dependent manner, and chemical inhibition of the p38 pathway attenuates HU-dependent apoptosis in mESCs. Our data reveal that acetylation of p53 as a result of HU-dependent NO production plays a key role in the induction of the apoptotic response in mESCs. Finally, p38 signaling appears to be the main pathway underlying the activation of apoptosis in mESCs in response to HU exposure.
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Affiliation(s)
- Sun-Hee Heo
- 1 Department of Biomedical Science, College of Life Science, CHA University , Seoul, Korea
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Non-integrating gamma-retroviral vectors as a versatile tool for transient zinc-finger nuclease delivery. Sci Rep 2014; 4:4656. [PMID: 24722320 PMCID: PMC3983605 DOI: 10.1038/srep04656] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2013] [Accepted: 03/14/2014] [Indexed: 12/17/2022] Open
Abstract
Designer nucleases, like zinc-finger nucleases (ZFNs), represent valuable tools for targeted genome editing. Here, we took advantage of the gamma-retroviral life cycle and produced vectors to transfer ZFNs in the form of protein, mRNA and episomal DNA. Transfer efficacy and ZFN activity were assessed in quantitative proof-of-concept experiments in a human cell line and in mouse embryonic stem cells. We demonstrate that retrovirus-mediated protein transfer (RPT), retrovirus-mediated mRNA transfer (RMT), and retrovirus-mediated episome transfer (RET) represent powerful methodologies for transient protein delivery or protein expression. Furthermore, we describe complementary strategies to augment ZFN activity after gamma-retroviral transduction, including serial transduction, proteasome inhibition, and hypothermia. Depending on vector dose and target cell type, gene disruption frequencies of up to 15% were achieved with RPT and RMT, and >50% gene knockout after RET. In summary, non-integrating gamma-retroviral vectors represent a versatile tool to transiently deliver ZFNs to human and mouse cells.
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Pouliliou S, Koukourakis MI. Gamma histone 2AX (γ-H2AX)as a predictive tool in radiation oncology. Biomarkers 2014; 19:167-80. [DOI: 10.3109/1354750x.2014.898099] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Affiliation(s)
- Stamatia Pouliliou
- Department of Radiotherapy/Oncology, Radiobiology and Radiopathology Unit, Democritus University of Thrace
AlexandroupolisGreece
| | - Michael I. Koukourakis
- Department of Radiotherapy/Oncology, Radiobiology and Radiopathology Unit, Democritus University of Thrace
AlexandroupolisGreece
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Rivlin N, Koifman G, Rotter V. p53 orchestrates between normal differentiation and cancer. Semin Cancer Biol 2014; 32:10-7. [PMID: 24406212 DOI: 10.1016/j.semcancer.2013.12.006] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2013] [Revised: 12/29/2013] [Accepted: 12/30/2013] [Indexed: 12/18/2022]
Abstract
During recent years, it is becoming more and more evident that there is a tight connection between abnormal differentiation processes and cancer. While cancer and stem cells are very different, especially in terms of maintaining genomic integrity, these cell types also share many similar properties. In this review, we aim to provide an over-view of the roles of the key tumor suppressor, p53, in regulating normal differentiation and function of both stem cells and adult cells. When these functions are disrupted, undifferentiated cells may become transformed. Understanding the function of p53 in stem cells and its role in maintaining the balance between differentiation and malignant transformation can help shed light on cancer initiation and propagation, and hopefully also on cancer prevention and therapy.
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Affiliation(s)
- Noa Rivlin
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.
| | - Gabriela Koifman
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel
| | - Varda Rotter
- Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel
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Heber-Katz E, Zhang Y, Bedelbaeva K, Song F, Chen X, Stocum DL. Cell cycle regulation and regeneration. Curr Top Microbiol Immunol 2013; 367:253-76. [PMID: 23263201 DOI: 10.1007/82_2012_294] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Regeneration of ear punch holes in the MRL mouse and amputated limbs of the axolotl show a number of similarities. A large proportion of the fibroblasts of the uninjured MRL mouse ear are arrested in G2 of the cell cycle, and enter nerve-dependent mitosis after injury to form a ring-shaped blastema that regenerates the ear tissue. Multiple cell types contribute to the establishment of the regeneration blastema of the urodele limb by dedifferentiation, and there is substantial reason to believe that the cells of this early blastema are also arrested in G2, and enter mitosis under the influence of nerve-dependent factors supplied by the apical epidermal cap. Molecular analysis reveals other parallels, such as; (1) the upregulation of Evi5, a centrosomal protein that prevents mitosis by stabilizing Emi1, a protein that inhibits the degradation of cyclins by the anaphase promoting complex and (2) the expression of sodium channels by the epidermis. A central feature in the entry into the cell cycle by MRL ear fibroblasts is a natural downregulation of p21, and knockout of p21 in wild-type mice confers regenerative capacity on non-regenerating ear tissue. Whether the same is true for entry into the cell cycle in regenerating urodele limbs is presently unknown.
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Shirai H, Fujimori H, Gunji A, Maeda D, Hirai T, Poetsch AR, Harada H, Yoshida T, Sasai K, Okayasu R, Masutani M. Parg deficiency confers radio-sensitization through enhanced cell death in mouse ES cells exposed to various forms of ionizing radiation. Biochem Biophys Res Commun 2013; 435:100-6. [PMID: 23624507 DOI: 10.1016/j.bbrc.2013.04.048] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2013] [Accepted: 04/06/2013] [Indexed: 11/18/2022]
Abstract
Poly(ADP-ribose) glycohydrolase (Parg) is the main enzyme involved in poly(ADP-ribose) degradation. Here, the effects of Parg deficiency on sensitivity to low and high linear-energy-transfer (LET) radiation were investigated in mouse embryonic stem (ES) cells. Mouse Parg(-/-) and poly(ADP-ribose) polymerase-1 deficient (Parp-1(-/-)) ES cells were used and responses to low and high LET radiation were assessed by clonogenic survival and biochemical and biological analysis methods. Parg(-/-) cells were more sensitive to γ-irradiation than Parp-1(-/-) cells. Transient accumulation of poly(ADP-ribose) was enhanced in Parg(-/-) cells. Augmented levels of phosphorylated H2AX (γ-H2AX) from early phase were observed in Parg(-/-) ES cells. The induction level of p53 phophorylation at ser18 was similar in wild-type and Parp-1(-/-) cells and apoptotic cell death process was mainly observed in the both genotypes. These results suggested that the enhanced sensitivity of Parg(-/-) ES cells to γ-irradiation involved defective repair of DNA double strand breaks. The effects of Parg and Parp-1 deficiency on the ES cell response to carbon-ion irradiation (LET13 and 70 keV/μm) and Fe-ion irradiation (200 keV/μm) were also examined. Parg(-/-) cells were more sensitive to LET 70 keV/μm carbon-ion irradiation than Parp-1(-/-) cells. Enhanced apoptotic cell death also accompanied augmented levels of γ-H2AX in a biphasic manner peaked at 1 and 24h. The induction level of p53 phophorylation at ser18 was not different between wild-type and Parg(-/-) cells. The augmented level of poly(ADP-ribose) accumulation was noted after carbon-ion irradiation compared to γ-irradiation even in the wild-type cells. An enhanced poly(ADP-ribose) accumulation was further observed in Parg(-/-) cells. Both Parg(-/-) cells and Parp-1(-/-) cells did not show sensitization to Fe-ion irradiation. Parg deficiency sensitizes mouse ES cells to a wide therapeutic range of LET radiation through the effects on DNA double strand break repair responses and enhanced cell death.
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Affiliation(s)
- Hidenori Shirai
- Division of Genome Stability Research, National Cancer Center Research Institute, 5-1-1 Tsukiji, Tokyo 104-0045, Japan
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Khromov T, Dressel R, Siamishi I, Nolte J, Opitz L, Engel W, Pantakani DVK. Apoptosis-related gene expression profiles of mouse ESCs and maGSCs: role of Fgf4 and Mnda in pluripotent cell responses to genotoxicity. PLoS One 2012; 7:e48869. [PMID: 23145002 PMCID: PMC3492253 DOI: 10.1371/journal.pone.0048869] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2012] [Accepted: 10/02/2012] [Indexed: 01/27/2023] Open
Abstract
Stem cells in the developing embryo proliferate and differentiate while maintaining genomic integrity, failure of which may lead to accumulation of mutations and subsequent damage to the embryo. Embryonic stem cells (ESCs), the in vitro counterpart of embryo stem cells are highly sensitive to genotoxic stress. Defective ESCs undergo either efficient DNA damage repair or apoptosis, thus maintaining genomic integrity. However, the genotoxicity- and apoptosis-related processes in germ-line derived pluripotent cells, multipotent adult germ-line stem cells (maGSCs), are currently unknown. Here, we analyzed the expression of apoptosis-related genes using OligoGEArray in undifferentiated maGSCs and ESCs and identified a similar set of genes expressed in both cell types. We detected the expression of intrinsic, but not extrinsic, apoptotic pathway genes in both cell types. Further, we found that apoptosis-related gene expression patterns of differentiated ESCs and maGSCs are identical to each other. Comparative analysis revealed that several pro- and anti-apoptotic genes are expressed specifically in pluripotent cells, but markedly downregulated in the differentiated counterparts of these cells. Activation of the intrinsic apoptotic pathway cause approximately ∼35% of both ESCs and maGSCs to adopt an early-apoptotic phenotype. Moreover, we performed transcriptome studies using early-apoptotic cells to identify novel pluripotency- and apoptosis-related genes. From these transcriptome studies, we selected Fgf4 (Fibroblast growth factor 4) and Mnda (Myeloid cell nuclear differentiating antigen), which are highly downregulated in early-apoptotic cells, as novel candidates and analyzed their roles in apoptosis and genotoxicity responses in ESCs. Collectively, our results show the existence of common molecular mechanisms for maintaining the pristine stem cell pool of both ESCs and maGSCs.
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Affiliation(s)
- Tatjana Khromov
- Institute of Human Genetics, University of Goettingen, Goettingen, Germany
| | - Ralf Dressel
- Department of Cellular and Molecular Immunology, University of Goettingen, Goettingen, Germany
| | - Iliana Siamishi
- Institute of Human Genetics, University of Goettingen, Goettingen, Germany
| | - Jessica Nolte
- Institute of Human Genetics, University of Goettingen, Goettingen, Germany
| | - Lennart Opitz
- DNA Microarray Facility, University of Goettingen, Goettingen, Germany
| | - Wolfgang Engel
- Institute of Human Genetics, University of Goettingen, Goettingen, Germany
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Nagaria P, Robert C, Rassool FV. DNA double-strand break response in stem cells: mechanisms to maintain genomic integrity. Biochim Biophys Acta Gen Subj 2012; 1830:2345-53. [PMID: 22995214 DOI: 10.1016/j.bbagen.2012.09.001] [Citation(s) in RCA: 55] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2012] [Revised: 08/09/2012] [Accepted: 09/05/2012] [Indexed: 12/19/2022]
Abstract
BACKGROUND Embryonic stem cells (ESCs) represent the point of origin of all cells in a given organism and must protect their genomes from both endogenous and exogenous genotoxic stress. DNA double-strand breaks (DSBs) are one of the most lethal forms of damage, and failure to adequately repair DSBs would not only compromise the ability of SCs to self-renew and differentiate, but will also lead to genomic instability and disease. SCOPE OF REVIEW Herein, we describe the mechanisms by which ESCs respond to DSB-inducing agents such as reactive oxygen species (ROS) and ionizing radiation, compared to somatic cells. We will also discuss whether the DSB response is fully reprogrammed in induced pluripotent stem cells (iPSCs) and the role of the DNA damage response (DDR) in the reprogramming of these cells. MAJOR CONCLUSIONS ESCs have distinct mechanisms to protect themselves against DSBs and oxidative stress compared to somatic cells. The response to damage and stress is crucial for the maintenance of self-renewal and differentiation capacity in SCs. iPSCs appear to reprogram some of the responses to genotoxic stress. However, it remains to be determined if iPSCs also retain some DDR characteristics of the somatic cells of origin. GENERAL SIGNIFICANCE The mechanisms regulating the genomic integrity in ESCs and iPSCs are critical for its safe use in regenerative medicine and may shed light on the pathways and factors that maintain genomic stability, preventing diseases such as cancer. This article is part of a Special Issue entitled Biochemistry of Stem Cells.
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Affiliation(s)
- Pratik Nagaria
- Department of Radiation Oncology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
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Alekseenko LL, Zemelko VI, Zenin VV, Pugovkina NA, Kozhukharova IV, Kovaleva ZV, Grinchuk TM, Fridlyanskaya II, Nikolsky NN. Heat shock induces apoptosis in human embryonic stem cells but a premature senescence phenotype in their differentiated progeny. Cell Cycle 2012; 11:3260-9. [PMID: 22895173 DOI: 10.4161/cc.21595] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Embryonic stem cells (ESC) are able to self-renew and to differentiate into any cell type. To escape error transmission to future cell progeny, ESC require robust mechanisms to ensure genomic stability. It was stated that stress defense of mouse and human ESC against oxidative stress and irradiation is superior compared with differentiated cells. Here, we investigated heat shock response of human ESC (hESC) and their differentiated progeny. Fibroblast-like cells were generated by spontaneous hESC differentiation via embryoid bodies. Like normal human diploid fibroblasts, these cells have a finite lifespan in culture, undergo replicative senescence and die. We found that sublethal heat shock affected survival of both cell types, but in hESC it induced apoptosis, whereas in differentiated cells it produced cell cycle arrest and premature senescence phenotype. Heat shock survived hESC and differentiated cells restored the properties of initial cells. Heated hESC progeny exhibited pluripotent markers and the capacity to differentiate into the cells of three germ layers. Fibroblast-like cells resisted heat shock, proliferated for a limited number of passages and entered replicative senescence as unheated parental cells. Taken together, these results show for the first time that both hESC and their differentiated derivatives are sensitive to heat shock, but the mechanisms of their stress response are different: hESC undergo apoptosis, whereas differentiated cells under the same conditions exhibit stress-induced premature senescence (SIPS) phenotype. Both cell types that survived sublethal heat shock sustain parental cell properties.
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46
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Gordeeva OF. Antiproliferative and cytotoxic effects of different type cytostatics on mouse pluripotent stem and teratocarcinoma cells. Russ J Dev Biol 2012. [DOI: 10.1134/s1062360412040030] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
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47
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Thompson LH. Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: the molecular choreography. Mutat Res 2012; 751:158-246. [PMID: 22743550 DOI: 10.1016/j.mrrev.2012.06.002] [Citation(s) in RCA: 272] [Impact Index Per Article: 20.9] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2011] [Revised: 06/09/2012] [Accepted: 06/16/2012] [Indexed: 12/15/2022]
Abstract
The faithful maintenance of chromosome continuity in human cells during DNA replication and repair is critical for preventing the conversion of normal diploid cells to an oncogenic state. The evolution of higher eukaryotic cells endowed them with a large genetic investment in the molecular machinery that ensures chromosome stability. In mammalian and other vertebrate cells, the elimination of double-strand breaks with minimal nucleotide sequence change involves the spatiotemporal orchestration of a seemingly endless number of proteins ranging in their action from the nucleotide level to nucleosome organization and chromosome architecture. DNA DSBs trigger a myriad of post-translational modifications that alter catalytic activities and the specificity of protein interactions: phosphorylation, acetylation, methylation, ubiquitylation, and SUMOylation, followed by the reversal of these changes as repair is completed. "Superfluous" protein recruitment to damage sites, functional redundancy, and alternative pathways ensure that DSB repair is extremely efficient, both quantitatively and qualitatively. This review strives to integrate the information about the molecular mechanisms of DSB repair that has emerged over the last two decades with a focus on DSBs produced by the prototype agent ionizing radiation (IR). The exponential growth of molecular studies, heavily driven by RNA knockdown technology, now reveals an outline of how many key protein players in genome stability and cancer biology perform their interwoven tasks, e.g. ATM, ATR, DNA-PK, Chk1, Chk2, PARP1/2/3, 53BP1, BRCA1, BRCA2, BLM, RAD51, and the MRE11-RAD50-NBS1 complex. Thus, the nature of the intricate coordination of repair processes with cell cycle progression is becoming apparent. This review also links molecular abnormalities to cellular pathology as much a possible and provides a framework of temporal relationships.
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Affiliation(s)
- Larry H Thompson
- Biology & Biotechnology Division, L452, Lawrence Livermore National Laboratory, P.O. Box 808, Livermore, CA 94551-0808, United States.
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Turinetto V, Orlando L, Sanchez-Ripoll Y, Kumpfmueller B, Storm MP, Porcedda P, Minieri V, Saviozzi S, Accomasso L, Cibrario Rocchietti E, Moorwood K, Circosta P, Cignetti A, Welham MJ, Giachino C. High Basal γH2AX Levels Sustain Self-Renewal of Mouse Embryonic and Induced Pluripotent Stem Cells. Stem Cells 2012; 30:1414-23. [DOI: 10.1002/stem.1133] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
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49
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Wang R, Guo YL. Transient inhibition of cell proliferation does not compromise self-renewal of mouse embryonic stem cells. Exp Cell Res 2012; 318:2094-104. [PMID: 22705123 DOI: 10.1016/j.yexcr.2012.05.017] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2012] [Revised: 05/17/2012] [Accepted: 05/21/2012] [Indexed: 01/01/2023]
Abstract
Embryonic stem cells (ESCs) have unlimited capacity for self-renewal and can differentiate into various cell types when induced. They also have an unusual cell cycle control mechanism driven by constitutively active cyclin dependent kinases (Cdks). In mouse ESCs (mESCs). It is proposed that the rapid cell proliferation could be a necessary part of mechanisms that maintain mESC self-renewal and pluripotency, but this hypothesis is not in line with the finding in human ESCs (hESCs) that the length of the cell cycle is similar to differentiated cells. Therefore, whether rapid cell proliferation is essential for the maintenance of mESC state remains unclear. We provide insight into this uncertainty through chemical intervention of mESC cell cycle. We report here that inhibition of Cdks with olomoucine II can dramatically slow down cell proliferation of mESCs with concurrent down-regulation of cyclin A, B and E, and the activation of the Rb pathway. However, mESCs display can recover upon the removal of olomoucine II and are able to resume normal cell proliferation without losing self-renewal and pluripotency, as demonstrated by the expression of ESC markers, colony formation, embryoid body formation, and induced differentiation. We provide a mechanistic explanation for these observations by demonstrating that Oct4 and Nanog, two major transcription factors that play critical roles in the maintenance of ESC properties, are up-regulated via de novo protein synthesis when the cells are exposed to olomoucine II. Together, our data suggest that short-term inhibition of cell proliferation does not compromise the basic properties of mESCs.
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Affiliation(s)
- Ruoxing Wang
- Department of Biological Sciences, The University of Southern Mississippi, 118 College Drive # 5018, Hattiesburg, MS 39406, USA
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50
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Rebuzzini P, Pignalosa D, Mazzini G, Di Liberto R, Coppola A, Terranova N, Magni P, Redi CA, Zuccotti M, Garagna S. Mouse embryonic stem cells that survive γ-rays exposure maintain pluripotent differentiation potential and genome stability. J Cell Physiol 2012; 227:1242-9. [PMID: 21732352 DOI: 10.1002/jcp.22908] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
This study aimed to investigate the cell cycle, apoptosis, cytogenetics and differentiation capacity of mouse embryonic stem cells (mESCs) that survived a single dose of 2 or 5 Gy γ-rays during a period of up to 96 h of culture. After 2 Gy irradiation and 24 h culture, compared to control, a significant majority of cells was blocked at the G2/M phase and a massive apoptosis was recorded. Between 48 and 72 h post-irradiation, the parameters used to describe the cell cycle and apoptosis returned similar to those of control samples. When mESCs were irradiated with 5 Gy, a small fraction of cells, even after 96 h of culture, still presented clear evidences of a G2/M block and apoptosis. The cytogenetic analysis performed at 96 h showed that the structural stability of the aberrations did not change significantly when comparing control and 2 or 5 Gy-treated populations. However, the chromosomal damage observed in the progeny of the survived cells after 5 Gy exposure is significantly higher than that observed in control samples, although it is mostly of the stable and transmissible type. Ninety-six hours after irradiation, the survived mESCs maintained their undifferentiated status and capability to differentiate into the three germ layers. Overall, these results indicate a commitment of mESCs to maintain pluripotency and genome stability.
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Affiliation(s)
- Paola Rebuzzini
- Laboratorio di Biologia dello Sviluppo, Dipartimento di Biologia Animale, Università degli Studi di Pavia, Pavia, Italy
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