|
1
|
Ye Y, Xie W, Wang X, Tan S, Yang L, Ma Z, Zhu Z, Chen X, Liu X, O'Neill E, Chang L, Zhang W. DNA-damage orchestrates self-renewal and differentiation via reciprocal p53 family and Hippo/Wnt/TGF-β pathway activation in embryonic stem cells. Cell Mol Life Sci 2025; 82:38. [PMID: 39762370 PMCID: PMC11704118 DOI: 10.1007/s00018-024-05561-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2024] [Revised: 11/18/2024] [Accepted: 12/22/2024] [Indexed: 01/30/2025]
Abstract
The mechanism by which DNA-damage affects self-renewal and pluripotency remains unclear. DNA damage and repair mechanisms have been largely elucidated in mutated cancer cells or simple eukaryotes, making valid interpretations on early development difficult. Here we show the impact of ionizing irradiation on the maintenance and early differentiation of mouse embryonic stem cells (ESCs). Our findings demonstrate that irradiation induces the upregulation of the p53 family genes, including p53, p63, and p73, resulting in elevated expression of the E3 ubiquitin ligases Itch and Trim32. Consequently, this impairs ESC maintenance by reducing the protein levels of key pluripotency transcription factors in both mouse ESCs and early embryos. Notably, our study reveals that irradiation-induced DNA damage leads to the recruitment of the BAF complex, causing it to dissociate from its binding sites on the target genes associated with the Yap, Wnt, and TGF-β pathways, thereby increasing signaling and promoting differentiation of ESCs into all three lineages. Importantly, pathway inhibition demonstrates that DNA damage accelerated ESC differentiation relies on Wnt and TGF-β, and is selectively dependent on p53 or p63/ p73 for mesoderm and endoderm respectively. Finally, our study reveals that p53 family proteins form complexes with effector proteins of key signaling pathways which actively contribute to ESC differentiation. In summary, this study uncovered a mechanism by which multiple differentiation signaling pathways converge on the p53 family genes to promote ESC differentiation and are impacted by exposure to ionizing radiation.
Collapse
Affiliation(s)
- Ying Ye
- Department of Clinical Pathobiology and Immunological Testing, School of Medical Laboratory, Qilu Medical University, Zibo, 255300, China
| | - Wenyan Xie
- Cam-Su Genomic Resource Center, Medical College of Soochow University, Suzhou, China
| | - Xuepeng Wang
- Cam-Su Genomic Resource Center, Medical College of Soochow University, Suzhou, China
| | - Shuping Tan
- Cam-Su Genomic Resource Center, Medical College of Soochow University, Suzhou, China
| | - Lingyue Yang
- Institute for Regenerative Medicine, Shanghai East Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, 200120, China
- Guangzhou National Laboratory, Guangzhou International Bio Island, Guangzhou, 510005, Guangdong, China
| | - Zhaoru Ma
- Cam-Su Genomic Resource Center, Medical College of Soochow University, Suzhou, China
| | - Zhexin Zhu
- Institute of Health and Medicine, Hefei Comprehensive National Science Center, 4090 Guanhai Road, Heifei, China
| | - Xi Chen
- Department of Biology, Southern University of Science and Technology, Shenzhen, China
| | - Xiaoyu Liu
- Institute for Regenerative Medicine, Shanghai East Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, 200120, China
| | - Eric O'Neill
- Department of Oncology, University of Oxford, Oxford, UK.
| | - Lei Chang
- State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Jiangsu Province International Joint Laboratory for Regeneration Medicine, Medical College of Soochow University, Suzhou, China.
| | - Wensheng Zhang
- Cam-Su Genomic Resource Center, Medical College of Soochow University, Suzhou, China.
- State Key Laboratory of New Targets Discovery and Drug Development for Major Diseases, Gannan Innovation and Translational Medicine Research Institute, Gannan Medical University, Ganzhou, China.
| |
Collapse
|
|
2
|
Shi G, Pang Q, Lin Z, Zhang X, Huang K. Repetitive Sequence Stability in Embryonic Stem Cells. Int J Mol Sci 2024; 25:8819. [PMID: 39201503 PMCID: PMC11354519 DOI: 10.3390/ijms25168819] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/10/2024] [Revised: 08/07/2024] [Accepted: 08/12/2024] [Indexed: 09/02/2024] Open
Abstract
Repetitive sequences play an indispensable role in gene expression, transcriptional regulation, and chromosome arrangements through trans and cis regulation. In this review, focusing on recent advances, we summarize the epigenetic regulatory mechanisms of repetitive sequences in embryonic stem cells. We aim to bridge the knowledge gap by discussing DNA damage repair pathway choices on repetitive sequences and summarizing the significance of chromatin organization on repetitive sequences in response to DNA damage. By consolidating these insights, we underscore the critical relationship between the stability of repetitive sequences and early embryonic development, seeking to provide a deeper understanding of repetitive sequence stability and setting the stage for further research and potential therapeutic strategies in developmental biology and regenerative medicine.
Collapse
Affiliation(s)
- Guang Shi
- MOE Key Laboratory of Gene Function and Regulation, Guangzhou Key Laboratory of Healthy Aging Research and SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China; (Q.P.); (Z.L.); (X.Z.)
| | - Qianwen Pang
- MOE Key Laboratory of Gene Function and Regulation, Guangzhou Key Laboratory of Healthy Aging Research and SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China; (Q.P.); (Z.L.); (X.Z.)
| | - Zhancheng Lin
- MOE Key Laboratory of Gene Function and Regulation, Guangzhou Key Laboratory of Healthy Aging Research and SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China; (Q.P.); (Z.L.); (X.Z.)
| | - Xinyi Zhang
- MOE Key Laboratory of Gene Function and Regulation, Guangzhou Key Laboratory of Healthy Aging Research and SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China; (Q.P.); (Z.L.); (X.Z.)
| | - Kaimeng Huang
- Division of Radiation and Genome Stability, Department of Radiation Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA;
- Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
| |
Collapse
|
|
3
|
Deng D, Zhang Y, Tang B, Zhang Z. Sources and applications of endothelial seed cells: a review. Stem Cell Res Ther 2024; 15:175. [PMID: 38886767 PMCID: PMC11184868 DOI: 10.1186/s13287-024-03773-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2024] [Accepted: 05/26/2024] [Indexed: 06/20/2024] Open
Abstract
Endothelial cells (ECs) are widely used as donor cells in tissue engineering, organoid vascularization, and in vitro microvascular model development. ECs are invaluable tools for disease modeling and drug screening in fundamental research. When treating ischemic diseases, EC engraftment facilitates the restoration of damaged blood vessels, enhancing therapeutic outcomes. This article presents a comprehensive overview of the current sources of ECs, which encompass stem/progenitor cells, primary ECs, cell lineage conversion, and ECs derived from other cellular sources, provides insights into their characteristics, potential applications, discusses challenges, and explores strategies to mitigate these issues. The primary aim is to serve as a reference for selecting suitable EC sources for preclinical research and promote the translation of basic research into clinical applications.
Collapse
Affiliation(s)
- Dan Deng
- Department of Cardiovascular Medicine, Center for Circadian Metabolism and Cardiovascular Disease, Southwest Hospital, Army Medical University, Chongqing, China
| | - Yu Zhang
- Department of Cardiovascular Medicine, Center for Circadian Metabolism and Cardiovascular Disease, Southwest Hospital, Army Medical University, Chongqing, China
| | - Bo Tang
- Chongqing International Institute for Immunology, Chongqing, China.
| | - Zhihui Zhang
- Department of Cardiovascular Medicine, Center for Circadian Metabolism and Cardiovascular Disease, Southwest Hospital, Army Medical University, Chongqing, China.
| |
Collapse
|
|
4
|
Yang Q, Zhou X, Fang J, Lin A, Zhang H, Cheng Q, Liu Z, Luo P, Zhang J. Development and validation of a radiosensitivity model to evaluate radiotherapy benefits in pan-cancer. Cancer Sci 2024; 115:1820-1833. [PMID: 38571294 PMCID: PMC11145160 DOI: 10.1111/cas.16168] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2023] [Revised: 03/14/2024] [Accepted: 03/19/2024] [Indexed: 04/05/2024] Open
Abstract
Radiotherapy, one of the most fundamental cancer treatments, is confronted with the dilemma of treatment failure due to radioresistance. To predict the radiosensitivity and improve tumor treatment efficiency in pan-cancer, we developed a model called Radiation Intrinsic Sensitivity Evaluation (RISE). The RISE model was built using cell line-based mRNA sequencing data from five tumor types with varying radiation sensitivity. Through four cell-derived datasets, two public tissue-derived cohorts, and one local cohort of 42 nasopharyngeal carcinoma patients, we demonstrated that RISE could effectively predict the level of radiation sensitivity (area under the ROC curve [AUC] from 0.666 to 1 across different datasets). After the verification by the colony formation assay and flow cytometric analysis of apoptosis, our four well-established radioresistant cell models successfully proved higher RISE values in radioresistant cells by RT-qPCR experiments. We also explored the prognostic value of RISE in five independent TCGA cohorts consisting of 1137 patients who received radiation therapy and found that RISE was an independent adverse prognostic factor (pooled multivariate Cox regression hazard ratio [HR]: 1.84, 95% CI 1.39-2.42; p < 0.01). RISE showed a promising ability to evaluate the radiotherapy benefit while predicting the prognosis of cancer patients, enabling clinicians to make individualized radiotherapy strategies in the future and improve the success rate of radiotherapy.
Collapse
Affiliation(s)
- Qi Yang
- Department of Oncology, Zhujiang HospitalSouthern Medical UniversityGuangzhouChina
| | - Xinyi Zhou
- Department of Oncology, Zhujiang HospitalSouthern Medical UniversityGuangzhouChina
| | - Jianbo Fang
- Department of Oncology, Zhujiang HospitalSouthern Medical UniversityGuangzhouChina
| | - Anqi Lin
- Department of Oncology, Zhujiang HospitalSouthern Medical UniversityGuangzhouChina
| | - Hongman Zhang
- Department of Oncology, Zhujiang HospitalSouthern Medical UniversityGuangzhouChina
| | - Quan Cheng
- Department of Neurosurgery, Xiangya HospitalCentral South UniversityChangshaHunanChina
| | - Zaoqu Liu
- Department of Interventional RadiologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouHenanChina
| | - Peng Luo
- Department of Oncology, Zhujiang HospitalSouthern Medical UniversityGuangzhouChina
| | - Jian Zhang
- Department of Oncology, Zhujiang HospitalSouthern Medical UniversityGuangzhouChina
| |
Collapse
|
|
5
|
Shah P, Hill R, Dion C, Clark SJ, Abakir A, Willems J, Arends MJ, Garaycoechea JI, Leitch HG, Reik W, Crossan GP. Primordial germ cell DNA demethylation and development require DNA translesion synthesis. Nat Commun 2024; 15:3734. [PMID: 38702312 PMCID: PMC11068800 DOI: 10.1038/s41467-024-47219-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2023] [Accepted: 03/25/2024] [Indexed: 05/06/2024] Open
Abstract
Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell development and meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1-/- or PcnaK164R/K164R) or extension (Rev7 -/-) result in a > 150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.
Collapse
Affiliation(s)
- Pranay Shah
- MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK.
| | - Ross Hill
- MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK
| | - Camille Dion
- MRC Laboratory of Medical Sciences, London, W12 0HS, UK
- Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, W12 0HS, UK
| | - Stephen J Clark
- Altos Labs, Cambridge, UK
- Epigenetics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK
| | - Abdulkadir Abakir
- Altos Labs, Cambridge, UK
- Epigenetics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK
| | - Jeroen Willems
- Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Utrecht, The Netherlands
| | | | - Juan I Garaycoechea
- Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Utrecht, The Netherlands
| | - Harry G Leitch
- MRC Laboratory of Medical Sciences, London, W12 0HS, UK
- Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, W12 0HS, UK
| | - Wolf Reik
- Altos Labs, Cambridge, UK
- Epigenetics Programme, The Babraham Institute, Cambridge, CB22 3AT, UK
| | - Gerry P Crossan
- MRC Laboratory of Molecular Biology, Cambridge, CB2 0QH, UK.
| |
Collapse
|
|
6
|
Asana Marican HT, Shen H. Dynamics of Chromosome Aberrations and Cell Death in Zebrafish Embryos Exposed to 137Cs Total-Body Irradiation. ENVIRONMENTAL SCIENCE & TECHNOLOGY 2024; 58:2204-2213. [PMID: 38269402 DOI: 10.1021/acs.est.3c05389] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/26/2024]
Abstract
Ionizing radiation exposure induces significant DNA damage and cell death in aquatic species. Accurate sensing and quantification play pivotal roles in environmental monitoring and surveillance. Zebrafish (Danio rerio) is a well-suited animal model for research into this aspect, especially with recent development of cytogenetic and transgenic tools. In this study, we present time-course studies of chromosome aberrations and cell death in zebrafish embryos exposed to 2 Gy 137Cs total-body irradiation. Using a cytogenetic approach, we quantified chromosome and chromatid aberrations in irradiated embryos at 6, 14, 20, and 24 h postirradiation. Metaphases with aberrations showed rapid declining kinetics, accompanied by incomplete karyotypes and irregular chromatin contents. Using an apoptosis-reporting transgenic zebrafish, we found increasing cell death along these time points, with the embryonic eyes and brain contributing the majority of the cell death volumes. We provide evidence that self-proliferating progenitor cells form the underlying linkage between the two kinetics and their positions define radiosensitive niches in zebrafish embryos. Our results provide detailed chromosome aberration and cell death dynamics in 137Cs-irradiated zebrafish embryos and unveil the appropriate timeline and tissue positions for accurate sensing and quantification of radiation-induced damages in zebrafish embryos.
Collapse
Affiliation(s)
- Halida Thanveer Asana Marican
- Singapore Nuclear Research and Safety Initiative, National University of Singapore, CREATE Tower, 1 Create Way, Singapore 138602, Singapore
| | - Hongyuan Shen
- Singapore Nuclear Research and Safety Initiative, National University of Singapore, CREATE Tower, 1 Create Way, Singapore 138602, Singapore
| |
Collapse
|
|
7
|
Tichy ED. Specialized Circuitry of Embryonic Stem Cells Promotes Genomic Integrity. Crit Rev Oncog 2023; 27:1-15. [PMID: 36734869 DOI: 10.1615/critrevoncog.2022042332] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Embryonic stem cells (ESCs) give rise to all cell types of the organism. Given the importance of these cells in this process, ESCs must employ robust mechanisms to protect genomic integrity or risk catastrophic propagation of mutations throughout the organism. Should such an event occur in daughter cells that will eventually contribute to the germline, the overall species health could dramatically decline. This review describes several key mechanisms employed by ESCs that are unique to these cells, in order to maintain their genomic integrity. Additionally, the contributions of cell cycle regulators in modulating ESC differentiation, after DNA damage exposure, are also examined. Where data are available, findings reported in ESCs are extended to include observations described in induced pluripotent stem cells (IPSCs).
Collapse
Affiliation(s)
- Elisia D Tichy
- Department of Orthopaedic Surgery, Perelman School of Medicine, The University of Pennsylvania, 371 Stemmler Hall, 3450 Hamilton Walk, Philadelphia, PA 19104-6081
| |
Collapse
|
|
8
|
p53 at the crossroad of DNA replication and ribosome biogenesis stress pathways. Cell Death Differ 2022; 29:972-982. [PMID: 35444234 PMCID: PMC9090812 DOI: 10.1038/s41418-022-00999-w] [Citation(s) in RCA: 67] [Impact Index Per Article: 22.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/22/2022] [Revised: 03/28/2022] [Accepted: 03/30/2022] [Indexed: 01/05/2023] Open
Abstract
Despite several decades of intense research focused on understanding function(s) and disease-associated malfunction of p53, there is no sign of any “mid-life crisis” in this rapidly advancing area of biomedicine. Firmly established as the hub of cellular stress responses and tumor suppressor targeted in most malignancies, p53’s many talents continue to surprise us, providing not only fresh insights into cell and organismal biology, but also new avenues to cancer treatment. Among the most fruitful lines of p53 research in recent years have been the discoveries revealing the multifaceted roles of p53-centered pathways in the fundamental processes of DNA replication and ribosome biogenesis (RiBi), along with cellular responses to replication and RiBi stresses, two intertwined areas of cell (patho)physiology that we discuss in this review. Here, we first provide concise introductory notes on the canonical roles of p53, the key interacting proteins, downstream targets and post-translational modifications involved in p53 regulation. We then highlight the emerging involvement of p53 as a key component of the DNA replication Fork Speed Regulatory Network and the mechanistic links of p53 with cellular checkpoint responses to replication stress (RS), the driving force of cancer-associated genomic instability. Next, the tantalizing, yet still rather foggy functional crosstalk between replication and RiBi (nucleolar) stresses is considered, followed by the more defined involvement of p53-mediated monitoring of the multistep process of RiBi, including the latest updates on the RPL5/RPL11/5 S rRNA-MDM2-p53-mediated Impaired Ribosome Biogenesis Checkpoint (IRBC) pathway and its involvement in tumorigenesis. The diverse defects of RiBi and IRBC that predispose and/or contribute to severe human pathologies including developmental syndromes and cancer are then outlined, along with examples of promising small-molecule-based strategies to therapeutically target the RS- and particularly RiBi- stress-tolerance mechanisms to which cancer cells are addicted due to their aberrant DNA replication, repair, and proteo-synthesis demands. ![]()
Collapse
|
|
9
|
Raj S, Jaiswal SK, DePamphilis ML. Cell Death and the p53 Enigma During Mammalian Embryonic Development. Stem Cells 2022; 40:227-238. [PMID: 35304609 PMCID: PMC9199838 DOI: 10.1093/stmcls/sxac003] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2021] [Accepted: 12/20/2021] [Indexed: 01/30/2023]
Abstract
Twelve forms of programmed cell death (PCD) have been described in mammalian cells, but which of them occurs during embryonic development and the role played by the p53 transcription factor and tumor suppressor remains enigmatic. Although p53 is not required for mouse embryonic development, some studies conclude that PCD in pluripotent embryonic stem cells from mice (mESCs) or humans (hESCs) is p53-dependent whereas others conclude that it is not. Given the importance of pluripotent stem cells as models of embryonic development and their applications in regenerative medicine, resolving this enigma is essential. This review reconciles contradictory results based on the facts that p53 cannot induce lethality in mice until gastrulation and that experimental conditions could account for differences in results with ESCs. Consequently, activation of the G2-checkpoint in mouse ESCs is p53-independent and generally, if not always, results in noncanonical apoptosis. Once initiated, PCD occurs at equivalent rates and to equivalent extents regardless of the presence or absence of p53. However, depending on experimental conditions, p53 can accelerate initiation of PCD in ESCs and late-stage blastocysts. In contrast, DNA damage following differentiation of ESCs in vitro or formation of embryonic fibroblasts in vivo induces p53-dependent cell cycle arrest and senescence.
Collapse
Affiliation(s)
- Sonam Raj
- National Cancer Institute, Bethesda, MD 20892, USA
| | - Sushil K Jaiswal
- National Institute of Child Health and Human Development, Bethesda, MD 20892, USA
| | - Melvin L DePamphilis
- National Institute of Child Health and Human Development, Bethesda, MD 20892, USA
| |
Collapse
|
|
10
|
Chen S, Chen D, Liu B, Haisma HJ. Modulating CRISPR/Cas9 genome-editing activity by small molecules. Drug Discov Today 2021; 27:951-966. [PMID: 34823004 DOI: 10.1016/j.drudis.2021.11.018] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2021] [Revised: 10/25/2021] [Accepted: 11/17/2021] [Indexed: 12/12/2022]
Abstract
Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated genome engineering has become a standard procedure for creating genetic and epigenetic changes of DNA molecules in basic biology, biotechnology, and medicine. However, its versatile applications have been hampered by its overall low precise gene modification efficiency and uncontrollable prolonged Cas9 activity. Therefore, overcoming these problems could broaden the therapeutic use of CRISPR/Cas9-based technologies. Here, we review small molecules with the clinical potential to precisely modulate CRISPR/Cas9-mediated genome-editing activity and discuss their mechanisms of action. Based on these data, we suggest that direct-acting small molecules for Cas9 are more suitable for precisely regulating Cas9 activity. These findings provide useful information for the identification of novel small-molecule enhancers and inhibitors of Cas9 and Cas9-associated endonucleases.
Collapse
Affiliation(s)
- Siwei Chen
- Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen 9713 AV, the Netherlands
| | - Deng Chen
- Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen 9713 AV, the Netherlands
| | - Bin Liu
- Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen 9713 AV, the Netherlands; RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA(1)
| | - Hidde J Haisma
- Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen, Groningen 9713 AV, the Netherlands.
| |
Collapse
|
|
11
|
German OL, Vallese-Maurizi H, Soto TB, Rotstein NP, Politi LE. Retina stem cells, hopes and obstacles. World J Stem Cells 2021; 13:1446-1479. [PMID: 34786153 PMCID: PMC8567457 DOI: 10.4252/wjsc.v13.i10.1446] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/28/2021] [Revised: 07/14/2021] [Accepted: 09/17/2021] [Indexed: 02/07/2023] Open
Abstract
Retinal degeneration is a major contributor to visual dysfunction worldwide. Although it comprises several eye diseases, loss of retinal pigment epithelial (RPE) and photoreceptor cells are the major contributors to their pathogenesis. Early therapies included diverse treatments, such as provision of anti-vascular endothelial growth factor and many survival and trophic factors that, in some cases, slow down the progression of the degeneration, but do not effectively prevent it. The finding of stem cells (SC) in the eye has led to the proposal of cell replacement strategies for retina degeneration. Therapies using different types of SC, such as retinal progenitor cells (RPCs), embryonic SC, pluripotent SCs (PSCs), induced PSCs (iPSCs), and mesenchymal stromal cells, capable of self-renewal and of differentiating into multiple cell types, have gained ample support. Numerous preclinical studies have assessed transplantation of SC in animal models, with encouraging results. The aim of this work is to revise the different preclinical and clinical approaches, analyzing the SC type used, their efficacy, safety, cell attachment and integration, absence of tumor formation and immunorejection, in order to establish which were the most relevant and successful. In addition, we examine the questions and concerns still open in the field. The data demonstrate the existence of two main approaches, aimed at replacing either RPE cells or photoreceptors. Emerging evidence suggests that RPCs and iPSC are the best candidates, presenting no ethical concerns and a low risk of immunorejection. Clinical trials have already supported the safety and efficacy of SC treatments. Serious concerns are pending, such as the risk of tumor formation, lack of attachment or integration of transplanted cells into host retinas, immunorejection, cell death, and also ethical. However, the amazing progress in the field in the last few years makes it possible to envisage safe and effective treatments to restore vision loss in a near future.
Collapse
Affiliation(s)
- Olga L German
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, Bahia blanca 8000, Buenos Aires, Argentina
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, and Neurobiology Department, Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB) Conicet, Bahía Blanca 8000, Buenos Aires, Argentina
| | - Harmonie Vallese-Maurizi
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, Bahia blanca 8000, Buenos Aires, Argentina
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, and Neurobiology Department, Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB) Conicet, Bahía Blanca 8000, Buenos Aires, Argentina
| | - Tamara B Soto
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, and Neurobiology Department, Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB) Conicet, Bahía Blanca 8000, Buenos Aires, Argentina
| | - Nora P Rotstein
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, Bahia blanca 8000, Buenos Aires, Argentina
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, and Neurobiology Department, Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB) Conicet, Bahía Blanca 8000, Buenos Aires, Argentina
| | - Luis Enrique Politi
- Department of Biology, Biochemistry and Pharmacy, Universidad Nacional del Sur, and Neurobiology Department, Instituto de Investigaciones Bioquímicas de Bahía Blanca (INIBIBB) Conicet, Bahía Blanca 8000, Buenos Aires, Argentina
| |
Collapse
|
|
12
|
Jaiswal SK, Raj S, DePamphilis ML. Developmental Acquisition of p53 Functions. Genes (Basel) 2021; 12:genes12111675. [PMID: 34828285 PMCID: PMC8622856 DOI: 10.3390/genes12111675] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2021] [Revised: 10/14/2021] [Accepted: 10/21/2021] [Indexed: 12/12/2022] Open
Abstract
Remarkably, the p53 transcription factor, referred to as “the guardian of the genome”, is not essential for mammalian development. Moreover, efforts to identify p53-dependent developmental events have produced contradictory conclusions. Given the importance of pluripotent stem cells as models of mammalian development, and their applications in regenerative medicine and disease, resolving these conflicts is essential. Here we attempt to reconcile disparate data into justifiable conclusions predicated on reports that p53-dependent transcription is first detected in late mouse blastocysts, that p53 activity first becomes potentially lethal during gastrulation, and that apoptosis does not depend on p53. Furthermore, p53 does not regulate expression of genes required for pluripotency in embryonic stem cells (ESCs); it contributes to ESC genomic stability and differentiation. Depending on conditions, p53 accelerates initiation of apoptosis in ESCs in response to DNA damage, but cell cycle arrest as well as the rate and extent of apoptosis in ESCs are p53-independent. In embryonic fibroblasts, p53 induces cell cycle arrest to allow repair of DNA damage, and cell senescence to prevent proliferation of cells with extensive damage.
Collapse
Affiliation(s)
- Sushil K. Jaiswal
- National Institute of Child Health and Human Development, Bethesda, MD 20892, USA;
- National Human Genome Research Institute, Bethesda, MD 20892, USA
| | - Sonam Raj
- National Cancer Institute, Bethesda, MD 20892, USA;
| | - Melvin L. DePamphilis
- National Institute of Child Health and Human Development, Bethesda, MD 20892, USA;
- Correspondence:
| |
Collapse
|
|
13
|
Munisha M, Schimenti JC. Genome maintenance during embryogenesis. DNA Repair (Amst) 2021; 106:103195. [PMID: 34358805 DOI: 10.1016/j.dnarep.2021.103195] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2021] [Revised: 07/26/2021] [Accepted: 07/27/2021] [Indexed: 12/25/2022]
Abstract
Genome maintenance during embryogenesis is critical, because defects during this period can be perpetuated and thus have a long-term impact on individual's health and longevity. Nevertheless, genome instability is normal during certain aspects of embryonic development, indicating that there is a balance between the exigencies of timely cell proliferation and mutation prevention. In particular, early embryos possess unique cellular and molecular features that underscore the challenge of having an appropriate balance. Here, we discuss genome instability during embryonic development, the mechanisms used in various cell compartments to manage genomic stress and address outstanding questions regarding the balance between genome maintenance mechanisms in key cell types that are important for adulthood and progeny.
Collapse
Affiliation(s)
- Mumingjiang Munisha
- Dept. of Biomedical Sciences, Cornell University College of Veterinary Medicine, Ithaca, NY 14853, United States
| | - John C Schimenti
- Dept. of Biomedical Sciences, Cornell University College of Veterinary Medicine, Ithaca, NY 14853, United States.
| |
Collapse
|
|
14
|
Riggs MJ, Sheridan SD, Rao RR. ARHGDIA Confers Selective Advantage to Dissociated Human Pluripotent Stem Cells. Stem Cells Dev 2021; 30:705-713. [PMID: 34036793 PMCID: PMC8309423 DOI: 10.1089/scd.2021.0079] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Human pluripotent stem cells (hPSCs) have generated significant interest in the scientific community based on their potential applications in regenerative medicine. However, numerous research groups have reported a propensity for genomic alterations during hPSC culture that poses concerns for basic research and clinical applications. Work from our laboratory and others has demonstrated that amplification of chromosomal regions is correlated with increased gene expression. To date, the phenotypic association of common genomic alterations remains unclear and is a cause for concern during clinical use. In this study, we focus on trisomy 17 and a list of candidate genes with increased gene expression to hypothesize that overexpressing 17q25 located ARHGDIA will confer selective advantage to hPSCs. HPSC lines overexpressing ARHGDIA exhibited culture dominance in co-cultures of overexpression lines with nonoverexpression lines. Furthermore, during low-density seeding, we demonstrate increased clonality of our ARHGDIA lines against matched controls. A striking observation is that we could reduce this selective advantage by varying the hPSC culture conditions with the addition of ROCK inhibitor (ROCKi). This work is unique in (1) demonstrating a novel gene that confers selective advantage to hPSCs when overexpressed and may help explain a common trisomy dominance, (2) providing a selection model for studying culture conditions that reduce the appearance of genomically altered hPSCs, and (3) aiding in elucidation of a mechanism that may act as a molecular switch during culture adaptation.
Collapse
Affiliation(s)
- Marion J Riggs
- Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, USA.,Center for Genomic Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA.,Department of Neurology, Harvard Medical School, Boston, Massachusetts, USA
| | - Steven D Sheridan
- Center for Quantitative Health, Center for Genomic Medicine and Department of Psychiatry, Massachusetts General Hospital, Boston, Massachusetts, USA.,Department of Psychiatry, Harvard Medical School, Boston, Massachusetts, USA
| | - Raj R Rao
- Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, USA.,Department of Biomedical Engineering, College of Engineering, University of Arkansas, Fayetteville, Arkansas, USA
| |
Collapse
|
|
15
|
Pinto MT, Cárcano FM, Vieira AGS, Cabral ERM, Lopes LF. Molecular Biology of Pediatric and Adult Male Germ Cell Tumors. Cancers (Basel) 2021; 13:cancers13102349. [PMID: 34068019 PMCID: PMC8152248 DOI: 10.3390/cancers13102349] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2021] [Revised: 03/31/2021] [Accepted: 04/12/2021] [Indexed: 12/13/2022] Open
Abstract
Simple Summary Although testicular germ cell tumors (TGCTs) are rare pediatric malignancies, they are the most common malignancies in young adult men. The similarities and differences between TGCTs in adults and children, taking into account the clinic presentation, biology, and molecular changes, are underexplored. In this paper, we aim to provide an overview of the molecular aspects of TGCTs, drawing a parallel between the findings in adult and pediatric groups. Abstract Cancer is a leading cause of death by disease in children and the second most prevalent of all causes in adults. Testicular germ cell tumors (TGCTs) make up 0.5% of pediatric malignancies, 14% of adolescent malignancies, and are the most common of malignancies in young adult men. Although the biology and clinical presentation of adult TGCTs share a significant overlap with those of the pediatric group, molecular evidence suggests that TGCTs in young children likely represent a distinct group compared to older adolescents and adults. The rarity of this cancer among pediatric ages is consistent with our current understanding, and few studies have analyzed and compared the molecular basis in childhood and adult cancers. Here, we review the major similarities and differences in cancer genetics, cytogenetics, epigenetics, and chemotherapy resistance between pediatric and adult TGCTs. Understanding the biological and molecular processes underlying TGCTs may help improve patient outcomes, and fuel further investigation and clinical research in childhood and adult TGCTs.
Collapse
Affiliation(s)
- Mariana Tomazini Pinto
- Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos 14784400, Brazil; (M.T.P.); (F.M.C.); (E.R.M.C.)
- Brazilian Childhood Germ Cell Tumor Study Group, The Brazilian Pediatric Oncology Society (SOBOPE), Barretos 14784400, Brazil;
| | - Flavio Mavignier Cárcano
- Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos 14784400, Brazil; (M.T.P.); (F.M.C.); (E.R.M.C.)
- Department of Clinical Oncology, Barretos Cancer Hospital, Barretos 14784400, Brazil
- Barretos School of Health Sciences Dr. Paulo Prata—FACISB, Barretos 14785002, Brazil
| | - Ana Glenda Santarosa Vieira
- Brazilian Childhood Germ Cell Tumor Study Group, The Brazilian Pediatric Oncology Society (SOBOPE), Barretos 14784400, Brazil;
- Barretos Children’s Cancer Hospital from Hospital de Amor, Barretos 14784400, Brazil
| | - Eduardo Ramos Martins Cabral
- Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos 14784400, Brazil; (M.T.P.); (F.M.C.); (E.R.M.C.)
| | - Luiz Fernando Lopes
- Brazilian Childhood Germ Cell Tumor Study Group, The Brazilian Pediatric Oncology Society (SOBOPE), Barretos 14784400, Brazil;
- Barretos Children’s Cancer Hospital from Hospital de Amor, Barretos 14784400, Brazil
- Correspondence: ; Tel.: +55-17-3321-6600
| |
Collapse
|
|
16
|
Abstract
PURPOSE OF REVIEW Breast milk (BM) is a peculiar fluid owing unique properties and resulting the ideal food during early neonatal period. As widely known, it can improve the outcome of both neonate and lactating mother, influencing their whole life. BM is characterized by several beneficial components; among these, a great role is played by BM own and specific microbiome, deeply investigated in many studies. Moreover, the use of metabolomics in BM analysis allowed a better characterization of its metabolic pathways that vary according to lactation stage and neonatal gestational age. The aim of this review is to describe growth factors, cytokines, immunity mediators, and stem cells (SCs) contained in BM and investigate their functions and effects on neonatal outcome, especially focusing on immuno- and neurodevelopment. RECENT FINDINGS We evaluated recent and updated literature on this field. The article that we analyzed to write this review have been found in MEDLINE using breast milk-derived stem cells, biofactors, growth factors, breastfeeding-related outcomes, neurodevelopment, and neonatal immunological system as keywords. Discovering and characterizing BM components could result very useful to clarify the pathophysiology of their influence on neonatal growth and even to improve artificial formulations' composition. Moreover, since SCs abilities and their involvement in the development of several diseases, they could help to discover specific targets for new therapies. It could be useful to characterize BM-derived SC markers, properties, and variations during lactation stages, to understand their potential role in therapeutic applications, since they could be noninvasively isolated from BM. More studies will help to describe more in detail the characteristics of mother-to-child communication through breastfeeding and its potential role in the next future.
Collapse
|
|
17
|
Liao X, Zou T, Chen M, Song Y, Yang C, Qiu B, Chen ZF, Tsang SY, Qi Z, Cai Z. Contamination profiles and health impact of benzothiazole and its derivatives in PM 2.5 in typical Chinese cities. THE SCIENCE OF THE TOTAL ENVIRONMENT 2021; 755:142617. [PMID: 33045602 DOI: 10.1016/j.scitotenv.2020.142617] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/02/2020] [Revised: 09/22/2020] [Accepted: 09/23/2020] [Indexed: 06/11/2023]
Abstract
Although benzothiazole and its derivatives (BTHs) are considered emerging contaminants in diverse environments and organisms, little information is available about their contamination profiles and health impact in ambient particles. In this study, an optimized method of ultrasound-assisted extraction coupled with the selected reaction monitoring (SRM) mode of GC-EI-MS/MS was applied to characterize and analyze PM2.5-bound BTHs from three cities of China (Guangzhou, Shanghai, and Taiyuan) during the winter of 2018. The total BTH concentration (ΣBTHs) in PM2.5 samples from the three cities decreased in the order of Guangzhou > Shanghai > Taiyuan, independently of the PM2.5 concentration. Despite the large variation in concentration of ΣBTHs in PM2.5, 2-hydroxybenzothiazole (OTH) was always the predominant compound among the PM2.5-bound BTHs and accounted for 50-80% of total BTHs in the three regions. Results from human exposure assessment and toxicity screening indicated that the outdoor exposure risk of PM2.5-bound BTHs in toddlers was much higher than in adults, especially for OTH. The developmental and reproduction toxicity of OTH was further explored in vivo and in vitro. Exposure of mouse embryonic stem cells (mESCs) to OTH for 48 h significantly increased the intracellular reactive oxygen species (ROS) and induced DNA damage and apoptosis via the functionally activating p53 expression. In addition, the growth and development of zebrafish embryos were found to be severely affected after OTH treatment. An overall metabolomics study was conducted on the exposed zebrafish larvae. The results indicated that exposure to OTH inhibited the phenylalanine hydroxylation reaction, which further increased the accumulation of toxic phenylpyruvate and acetylphenylalanine in zebrafish. These findings provide important insights into the contamination profiles of PM2.5-bound BTHs and emphasize the health risk of OTH.
Collapse
Affiliation(s)
- Xiaoliang Liao
- Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, China
| | - Ting Zou
- Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, China
| | - Min Chen
- Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, China
| | - Yuanyuan Song
- State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, China
| | - Chun Yang
- Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, China
| | - Bojun Qiu
- Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, China
| | - Zhi-Feng Chen
- Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, China
| | - Suk Ying Tsang
- School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China
| | - Zenghua Qi
- Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, China.
| | - Zongwei Cai
- Guangzhou Key Laboratory of Environmental Catalysis and Pollution Control, Guangdong Key Laboratory of Environmental Catalysis and Health Risk Control, School of Environmental Science and Engineering, Institute of Environmental Health and Pollution Control, Guangdong University of Technology, Guangzhou 510006, China; State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, China.
| |
Collapse
|
|
18
|
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.
Collapse
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
| |
Collapse
|
|
19
|
Chung CYT, Lo PHY, Lee KKH. Babam2 Regulates Cell Cycle Progression and Pluripotency in Mouse Embryonic Stem Cells as Revealed by Induced DNA Damage. Biomedicines 2020; 8:biomedicines8100397. [PMID: 33050379 PMCID: PMC7600899 DOI: 10.3390/biomedicines8100397] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2020] [Revised: 09/28/2020] [Accepted: 10/03/2020] [Indexed: 12/15/2022] Open
Abstract
BRISC and BRCA1-A complex member 2 (Babam2) plays an essential role in promoting cell cycle progression and preventing cellular senescence. Babam2-deficient fibroblasts show proliferation defect and premature senescence compared with their wild-type (WT) counterpart. Pluripotent mouse embryonic stem cells (mESCs) are known to have unlimited cell proliferation and self-renewal capability without entering cellular senescence. Therefore, studying the role of Babam2 in ESCs would enable us to understand the mechanism of Babam2 in cellular aging, cell cycle regulation, and pluripotency in ESCs. For this study, we generated Babam2 knockout (Babam2−/−) mESCs to investigate the function of Babam2 in mESCs. We demonstrated that the loss of Babam2 in mESCs leads to abnormal G1 phase retention in response to DNA damage induced by gamma irradiation or doxorubicin treatments. Key cell cycle regulators, CDC25A and CDK2, were found to be degraded in Babam2−/− mESCs following gamma irradiation. In addition, Babam2−/− mESCs expressed p53 strongly and significantly longer than in control mESCs, where p53 inhibited Nanog expression and G1/S cell cycle progression. The combined effects significantly reduced developmental pluripotency in Babam2−/− mESCs. In summary, Babam2 maintains cell cycle regulation and pluripotency in mESCs in response to induced DNA damage.
Collapse
Affiliation(s)
- Cheuk Yiu Tenny Chung
- MOE Key Laboratory for Regenerative Medicine, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong; (C.Y.T.C.); (P.H.Y.L.)
- Chinese University of Hong Kong-University of Southampton Joint Laboratory for Stem Cell and Regenerative Medicine, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong
| | - Paulisally Hau Yi Lo
- MOE Key Laboratory for Regenerative Medicine, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong; (C.Y.T.C.); (P.H.Y.L.)
- Chinese University of Hong Kong-University of Southampton Joint Laboratory for Stem Cell and Regenerative Medicine, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong
| | - Kenneth Ka Ho Lee
- MOE Key Laboratory for Regenerative Medicine, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong; (C.Y.T.C.); (P.H.Y.L.)
- Chinese University of Hong Kong-University of Southampton Joint Laboratory for Stem Cell and Regenerative Medicine, School of Biomedical Sciences, Chinese University of Hong Kong, Shatin, Hong Kong
- Correspondence:
| |
Collapse
|
|
20
|
Halliwell J, Barbaric I, Andrews PW. Acquired genetic changes in human pluripotent stem cells: origins and consequences. Nat Rev Mol Cell Biol 2020; 21:715-728. [DOI: 10.1038/s41580-020-00292-z] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/19/2020] [Indexed: 12/14/2022]
|
|
21
|
Maintenance of genome integrity and active homologous recombination in embryonic stem cells. Exp Mol Med 2020; 52:1220-1229. [PMID: 32770082 PMCID: PMC8080833 DOI: 10.1038/s12276-020-0481-2] [Citation(s) in RCA: 27] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2020] [Revised: 06/12/2020] [Accepted: 06/23/2020] [Indexed: 12/11/2022] Open
Abstract
Embryonic stem cells (ESCs) possess specific gene expression patterns that confer the ability to proliferate indefinitely and enable pluripotency, which allows ESCs to differentiate into diverse cell types in response to developmental signals. Compared to differentiated cells, ESCs harbor an elevated level of homologous recombination (HR)-related proteins and exhibit exceptional cell cycle control, characterized by a high proliferation rate and a prolonged S phase. HR is involved in several aspects of chromosome maintenance. For instance, HR repairs impaired chromosomes and prevents the collapse of DNA replication forks during cell proliferation. Thus, HR is essential for the maintenance of genomic integrity and prevents cellular dysregulation and lethal events. In addition, abundant HR proteins in the prolonged S phase can efficiently protect ESCs from external damages and protect against genomic instability caused by DNA breaks, facilitating rapid and accurate DNA break repair following chromosome duplication. The maintenance of genome integrity is key to preserving the functions of ESCs and reducing the risks of cancer development, cell cycle arrest, and abnormal replication. Here, we review the fundamental links between the stem cell-specific HR process and DNA damage response as well as the different strategies employed by ESCs to maintain genomic integrity. Embryonic stem cells (ESCs), which give rise to the many specialized cells of the body, have highly effective molecular processes of DNA maintenance and repair which protect their genetic information from damage. Keun Pil Kim and colleagues at Chung-Ang University, Seoul, South Korea, review the strategies found in ESCs to maintain the integrity of their DNA as they develop and multiply. A key feature is the process of homologous recombination (HR) in which one copy of a section of DNA acts as the template allowing a damaged version of the DNA to be repaired. HR also facilitates swapping of sections of DNA when sperm and egg cells form, promoting genetic diversity. HR appears to be especially significant in maintaining ESC DNA as ESCs possess higher levels of key proteins involved in its maintenance and regulation.
Collapse
|
|
22
|
SOX2 and p53 Expression Control Converges in PI3K/AKT Signaling with Versatile Implications for Stemness and Cancer. Int J Mol Sci 2020; 21:ijms21144902. [PMID: 32664542 PMCID: PMC7402325 DOI: 10.3390/ijms21144902] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2020] [Revised: 07/03/2020] [Accepted: 07/08/2020] [Indexed: 12/12/2022] Open
Abstract
Stemness and reprogramming involve transcriptional master regulators that suppress cell differentiation while promoting self-renewal. A distinguished example thereof is SOX2, a high mobility group (HMG)-box transcription factor (TF), whose subcellular localization and turnover regulation in embryonic, induced-pluripotent, and cancer stem cells (ESCs, iPSCs, and CSCs, respectively) is mediated by the PI3K/AKT/SOX2 axis, a stem cell-specific branch of the PI3K/AKT signaling pathway. Further effector functions associated with PI3K/AKT induction include cell cycle progression, cellular (mass) growth, and the suppression of apoptosis. Apoptosis, however, is a central element of DNA damage response (DDR), where it provides a default mechanism for cell clearance when DNA integrity cannot be maintained. A key player in DDR is tumor suppressor p53, which accumulates upon DNA-damage and is counter-balanced by PI3K/AKT enforced turnover. Accordingly, stemness sustaining SOX2 expression and p53-dependent DDR mechanisms show molecular–functional overlap in PI3K/AKT signaling. This constellation proves challenging for stem cells whose genomic integrity is a functional imperative for normative ontogenesis. Unresolved mutations in stem and early progenitor cells may in fact provoke transformation and cancer development. Such mechanisms are also particularly relevant for iPSCs, where genetic changes imposed through somatic cell reprogramming may promote DNA damage. The current review aims to summarize the latest advances in the understanding of PI3K/AKT/SOX2-driven stemness and its intertwined relations to p53-signaling in DDR under conditions of pluripotency, reprogramming, and transformation.
Collapse
|
|
23
|
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.
Collapse
|
|
24
|
Stage-Specific Effects of Ionizing Radiation during Early Development. Int J Mol Sci 2020; 21:ijms21113975. [PMID: 32492918 PMCID: PMC7312565 DOI: 10.3390/ijms21113975] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2020] [Revised: 05/28/2020] [Accepted: 05/30/2020] [Indexed: 02/07/2023] Open
Abstract
Early embryonic cells are sensitive to genotoxic stressors such as ionizing radiation. However, sensitivity to these stressors varies depending on the embryonic stage. Recently, the sensitivity and response to ionizing radiation were found to differ during the preimplantation period. The cellular and molecular mechanisms underlying the change during this period are beginning to be elucidated. In this review, we focus on the changes in radio-sensitivity and responses to ionizing radiation during the early developmental stages of the preimplantation (before gastrulation) period in mammals, Xenopus, and fish. Furthermore, we discuss the underlying cellular and molecular mechanisms and the similarities and differences between species.
Collapse
|
|
25
|
Rebuzzini P, Civello C, Nantia Akono E, Fassina L, Zuccotti M, Garagna S. Chronic cypermethrin exposure alters mouse embryonic stem cell growth kinetics, induces Phase II detoxification response and affects pluripotency and differentiation gene expression. Eur J Histochem 2020; 64. [PMID: 32214279 PMCID: PMC7036707 DOI: 10.4081/ejh.2020.3084] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2019] [Accepted: 02/05/2020] [Indexed: 12/21/2022] Open
Abstract
Worldwide uncontrolled use of synthetic pyrethroids contaminates water and soil leading to health hazards. Cypermethrin (CYP), the most used pyrethroid, induces detrimental effects on adults and embryos at different stages of development of several vertebrate species. In Mammals, CYP-induced alterations have been previously described in adult somatic cells and in post-implantation embryos. It remains unknown whether CYP has effects during pre-implantation development. Studies to access pre-implantation embryo toxicity are complicated by the restricted number of blastocysts that may be obtained, either in vivo or in vitro. Embryonic stem cells (ESCs) are an in vitro model study that overcomes these limitations, as millions of pluripotent cells are available to the analysis. Also, ESCs maintain the same pluripotency characteristics and differentiation capacity of the inner cell mass (ICM) present in the blastocyst, from which they derive. In this work, using mouse R1 ESCs, we studied CYP-induced cell death, ROS production, the activation of oxidative stress-related and detoxification responses and the population growth kinetics following 72 h exposure at the 0.3 mM LD50 dose. Also, the expression levels of pluripotency genes in exposed ESCs and of markers of the three germ layers after their differentiation into embryoid bodies (EBs) were determined. Two apoptotic waves were observed at 12-24 h and at 72 h. The increase of ROS production, at 24 h until the end of the culture period, was accompanied by the induction, at 48 h, of redox-related Cat, Sod1, Sod2, Gpx1 and Gpx4 genes. Up-regulation of Cyp1b1, but not of Cyp1a1, phase I gene was detected at 72 h and induction of Nqo1, Gsta1 and Ugt1a6 phase II genes began at 24 h exposure. The results show that exposed R1 ESCs activate oxidative stress-related and detoxification responses, although not sufficient, during the culture period tested, to warrant recovery of the growth rate observed in untreated cells. Also, CYP exposure altered the expression of Oct-4 and Nanog pluripotency genes in ESCs and, when differentiated into EBs, the expression of Fgf5, Brachyury and Foxa2, early markers of the ectoderm, mesoderm and endoderm germ layers, respectively. NIH/3T3 cells, a differentiated cell line of embryonic origin, were used for comparison.
Collapse
Affiliation(s)
- Paola Rebuzzini
- Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia.
| | | | | | | | | | | |
Collapse
|
|
26
|
Critical Role for P53 in Regulating the Cell Cycle of Ground State Embryonic Stem Cells. Stem Cell Reports 2020; 14:175-183. [PMID: 32004494 PMCID: PMC7013234 DOI: 10.1016/j.stemcr.2020.01.001] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2019] [Revised: 12/30/2019] [Accepted: 01/02/2020] [Indexed: 12/11/2022] Open
Abstract
Mouse embryonic stem cells (ESCs) grown in serum-supplemented conditions are characterized by an extremely short G1 phase due to the lack of G1-phase control. Concordantly, the G1-phase-specific P53-P21 pathway is compromised in serum ESCs. Here, we provide evidence that P53 is activated upon transition of serum ESCs to their pluripotent ground state using serum-free 2i conditions and that is required for the elongated G1 phase characteristic of ground state ESCs. RNA sequencing and chromatin immunoprecipitation sequencing analyses reveal that P53 directly regulates the expression of the retinoblastoma (RB) protein and that the hypo-phosphorylated, active RB protein plays a key role in G1-phase control. Our findings suggest that the P53-P21 pathway is active in ground state 2i ESCs and that its role in the G1-checkpoint is abolished in serum ESCs. Taken together, the data reveal a mechanism by which inactivation of P53 can lead to loss of RB and uncontrolled cell proliferation.
The P53-P21 pathway is activated upon adaptation of ESCs to their pluripotent ground state. P53 is required for the elongated G1-phase characteristic to 2i ESCs. P53 binds the promoter and activates Rb1 expression.
Collapse
|
|
27
|
Alrefaei GI, Alkarim SA, Abduljabbar HS. Impact of Mothers' Age on Telomere Length and Human Telomerase Reverse Transcriptase Expression in Human Fetal Membrane-Derived Mesenchymal Stem Cells. Stem Cells Dev 2019; 28:1632-1645. [PMID: 31650883 DOI: 10.1089/scd.2019.0144] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Age-related cellular changes and limited replicative capacity of adult mesenchymal stem cells (MSCs) are few of the challenges confronting stem cell research. MSCs from human fetal membranes (hFM-MSCs), including placental, umbilical cord, and amniotic membrane, are considered an alternative to adult MSCs. However, the effect of mothers' age on hFM-MSC cellular properties is still not clearly established. This study aimed to evaluate the effect of mothers' age on hFM-MSC telomere length, telomerase activity, and proliferation ability in three different age groups: GI (20-29 years), GII (30-39 years), and GIII (≥40 years). hFM samples were collected from pregnant women ≤37 weeks after obtaining consent. hFM-MSCs were isolated and cultured to characterize them by flow cytometry and assess proliferation by MTT assay and doubling time. Telomere length and expression levels of human telomerase reverse transcriptase were assessed by quantitative real-time polymerase chain reaction (qRT-RCR). hFM-MSCs in the three age groups were spindle-shaped, plastic-adherent, and exhibited high proliferation rates and strong expression of hMSC markers. GI showed the longest telomere length in hMSCs in various FM regions, whereas GIII showed the highest level of telomerase expression. There was no difference in telomere length between GII and GIII, and both groups showed the same hMSC characteristics. In conclusion, although the hFM-MSCs derived from different fetal membranes maintained the MSC characteristics in all study groups, the hFM-MSCs of older mothers had shorter telomeres and higher telomerase activity and proliferation rate than did those derived from younger mothers. Thus, the hFM-MSCs of older mothers could be unsuitable for expansion in vitro or stem cell therapy. Determination of telomere length and telomerase expression level of hFM might help characterizing and understanding the biological differences of hFM-MSCs in different age groups.
Collapse
Affiliation(s)
- Ghadeer I Alrefaei
- Biology Department, Faculty of Sciences, University of Jeddah, Jeddah, Saudi Arabia
| | - Saleh A Alkarim
- Biology Department, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia.,Embryonic and Cancer Stem Cell Research Group, King Fahad Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
| | - Hassan S Abduljabbar
- Obstetrics and Gynecology Department, King Abdulaziz University Hospital, Jeddah, Saudi Arabia.,Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
| |
Collapse
|
|
28
|
|
|
29
|
Nai S, Shi Y, Ru H, Ding Y, Geng Q, Li Z, Dong MQ, Xu X, Li J. Chk2-dependent phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) regulates centrosome maturation. Cell Cycle 2019; 18:2651-2659. [PMID: 31416392 PMCID: PMC6773232 DOI: 10.1080/15384101.2019.1654795] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2019] [Revised: 07/17/2019] [Accepted: 08/07/2019] [Indexed: 12/16/2022] Open
Abstract
Checkpoint kinase 2 (Chk2) is a pivotal effector kinase in the DNA damage response, with an emerging role in mitotic chromosome segregation. In this study, we show that Chk2 interacts with myosin phosphatase targeting subunit 1 (MYPT1), the targeting subunit of protein phosphatase 1cβ (PP1cβ). Previous studies have shown that MYPT1 is phosphorylated by CDK1 at S473 during mitosis, and subsequently docks to the polo-binding domain of PLK1 and dephosphorylates PLK1. Herein we present data that Chk2 phosphorylates MYPT1 at S507 in vitro and in vivo, which antagonizes pS473. Chk2 inhibition results in failure of γ-tubulin recruitment to the centrosomes, phenocopying Plk1 inhibition defects. These aberrancies were also observed in the MYPT1-S507A stable transfectants, suggesting that Chk2 exerts its effect on centrosomes via MYPT1. Collectively, we have identified a Chk2-MYPT1-PLK1 axis in regulating centrosome maturation. Abbreviations: Chk2: checkpoint kinase 2; MYPT1: myosin phosphatase targeting subunit 1; PP1cβ: protein phosphatase 1c β; Noc: nocodazole; IP: immunoprecipitation; IB: immunoblotting; LC-MS/MS: liquid chromatography-tandem mass spectrometry; Chk2: checkpoint kinase 2; KD: kinase domain; WT: wild type; Ub: ubiquitin; DAPI: 4',6-diamidino-2-phenylindole; IF: Immunofluorescence; IR: ionizing radiation; siCHK2: siRNA targeting CHK2.
Collapse
Affiliation(s)
- Shanshan Nai
- Beijing Key Laboratory of DNA damage Response, College of Life Sciences, Capital Normal University, Beijing, China
| | - Yingxin Shi
- Beijing Key Laboratory of DNA damage Response, College of Life Sciences, Capital Normal University, Beijing, China
| | - Huanwei Ru
- Beijing Key Laboratory of DNA damage Response, College of Life Sciences, Capital Normal University, Beijing, China
| | - Yuehe Ding
- National Institute of Biological Sciences, Beijing, China
| | - Qizhi Geng
- Beijing Key Laboratory of DNA damage Response, College of Life Sciences, Capital Normal University, Beijing, China
| | - Zhe Li
- Beijing Key Laboratory of DNA damage Response, College of Life Sciences, Capital Normal University, Beijing, China
| | - Meng-Qiu Dong
- National Institute of Biological Sciences, Beijing, China
| | - Xingzhi Xu
- Beijing Key Laboratory of DNA damage Response, College of Life Sciences, Capital Normal University, Beijing, China
- Guangdong Key Laboratory of Genome Stability & Disease Prevention, Shenzhen University School of Medicine, Shenzhen, China
| | - Jing Li
- Beijing Key Laboratory of DNA damage Response, College of Life Sciences, Capital Normal University, Beijing, China
| |
Collapse
|
|
30
|
Gonnot F, Langer D, Bourillot PY, Doerflinger N, Savatier P. Regulation of Cyclin E by transcription factors of the naïve pluripotency network in mouse embryonic stem cells. Cell Cycle 2019; 18:2697-2712. [PMID: 31462142 PMCID: PMC6773236 DOI: 10.1080/15384101.2019.1656475] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Continuous, non-cell cycle-dependent expression of cyclin E is a characteristic feature of mouse embryonic stem cells (mESCs). We studied the 5′ regulatory region of Cyclin E, also known as Ccne1, and identified binding sites for transcription factors of the naïve pluripotency network, including Esrrb, Klf4, and Tfcp2l1 within 1 kilobase upstream of the transcription start site. Luciferase assay and chromatin immunoprecipitation-quantitative polymerase chain reaction (ChiP–qPCR) study highlighted one binding site for Esrrb that is essential to transcriptional activity of the promoter region, and three binding sites for Klf4 and Tfcp2l1. Knockdown of Esrrb, Klf4, and Tfcp2l1 reduced Cyclin E expression whereas overexpression of Esrrb and Klf4 increased it, indicating a strong correlation between the expression level of these factors and that of cyclin E. We observed that cyclin E overexpression delays differentiation induced by Esrrb depletion, suggesting that cyclin E is an important target of Esrrb for differentiation blockade. We observed that mESCs express a low level of miR-15a and that transfection of a miR-15a mimic decreases Cyclin E mRNA level. These results lead to the conclusion that the high expression level of Cyclin E in mESCs can be attributed to transcriptional activation by Esrrb as well as to the absence of its negative regulator, miR-15a.
Collapse
Affiliation(s)
- Fabrice Gonnot
- Stem Cell and Brain Research Institute, Univ Lyon, Université Lyon 1, Inserm , Bron , France
| | - Diana Langer
- Stem Cell and Brain Research Institute, Univ Lyon, Université Lyon 1, Inserm , Bron , France
| | - Pierre-Yves Bourillot
- Stem Cell and Brain Research Institute, Univ Lyon, Université Lyon 1, Inserm , Bron , France
| | - Nathalie Doerflinger
- Stem Cell and Brain Research Institute, Univ Lyon, Université Lyon 1, Inserm , Bron , France
| | - Pierre Savatier
- Stem Cell and Brain Research Institute, Univ Lyon, Université Lyon 1, Inserm , Bron , France
| |
Collapse
|
|
31
|
Bloom JC, Loehr AR, Schimenti JC, Weiss RS. Germline genome protection: implications for gamete quality and germ cell tumorigenesis. Andrology 2019; 7:516-526. [PMID: 31119900 DOI: 10.1111/andr.12651] [Citation(s) in RCA: 20] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2019] [Revised: 04/25/2019] [Accepted: 04/26/2019] [Indexed: 12/19/2022]
Abstract
BACKGROUND Germ cells have a unique and critical role as the conduit for hereditary information and therefore employ multiple strategies to protect genomic integrity and avoid mutations. Unlike somatic cells, which often respond to DNA damage by arresting the cell cycle and conducting DNA repair, germ cells as well as long-lived pluripotent stem cells typically avoid the use of error-prone repair mechanisms and favor apoptosis, reducing the risk of genetic alterations. Testicular germ cell tumors, the most common cancers of young men, arise from pre-natal germ cells. OBJECTIVES To summarize the current understanding of DNA damage response mechanisms in pre-meiotic germ cells and to discuss how they impact both the origins of testicular germ cell tumors and their remarkable responsiveness to genotoxic chemotherapy. MATERIALS AND METHODS We conducted a review of literature gathered from PubMed regarding the DNA damage response properties of testicular germ cell tumors and the germ cells from which they arise, as well as the influence of these mechanisms on therapeutic responses by testicular germ cell tumors. RESULTS AND DISCUSSION This review provides a comprehensive evaluation of how the developmental origins of male germ cells and their inherent germ cell-like DNA damage response directly impact the development and therapeutic sensitivity of testicular germ cell tumors. CONCLUSIONS The DNA damage response of germ cells directly impacts the development and therapeutic sensitivity of testicular germ cell tumors. Recent advances in the study of primordial germ cells, post-natal mitotically dividing germ cells, and pluripotent stem cells will allow for new investigations into the initiation, progression, and treatment of testicular germ cell tumors.
Collapse
Affiliation(s)
- J C Bloom
- Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA
| | - A R Loehr
- Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA
| | - J C Schimenti
- Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA
| | - R S Weiss
- Department of Biomedical Sciences, Cornell University, Ithaca, NY, USA
| |
Collapse
|
|
32
|
Dubbury SJ, Boutz PL, Sharp PA. CDK12 regulates DNA repair genes by suppressing intronic polyadenylation. Nature 2018; 564:141-145. [PMID: 30487607 PMCID: PMC6328294 DOI: 10.1038/s41586-018-0758-y] [Citation(s) in RCA: 204] [Impact Index Per Article: 29.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2017] [Accepted: 10/11/2018] [Indexed: 12/28/2022]
Abstract
Mutations that attenuate homologous recombination (HR)-mediated repair promote tumorigenesis and sensitize cells to chemotherapeutics that cause replication fork collapse, a phenotype known as 'BRCAness'1. BRCAness tumours arise from loss-of-function mutations in 22 genes1. Of these genes, all but one (CDK12) function directly in the HR repair pathway1. CDK12 phosphorylates serine 2 of the RNA polymerase II C-terminal domain heptapeptide repeat2-7, a modification that regulates transcription elongation, splicing, and cleavage and polyadenylation8,9. Genome-wide expression studies suggest that depletion of CDK12 abrogates the expression of several HR genes relatively specifically, thereby blunting HR repair3-7,10,11. This observation suggests that the mutational status of CDK12 may predict sensitivity to targeted treatments against BRCAness, such as PARP1 inhibitors, and that CDK12 inhibitors may induce sensitization of HR-competent tumours to these treatments6,7,10,11. Despite growing clinical interest, the mechanism by which CDK12 regulates HR genes remains unknown. Here we show that CDK12 globally suppresses intronic polyadenylation events in mouse embryonic stem cells, enabling the production of full-length gene products. Many HR genes harbour more intronic polyadenylation sites than other expressed genes, and these sites are particularly sensitive to loss of CDK12. The cumulative effect of these sites accounts for the enhanced sensitivity of HR gene expression to CDK12 loss, and we find that this mechanism is conserved in human tumours that contain loss-of-function CDK12 mutations. This work clarifies the function of CDK12 and underscores its potential both as a chemotherapeutic target and as a tumour biomarker.
Collapse
Affiliation(s)
- Sara J Dubbury
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.,Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Paul L Boutz
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.,Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
| | - Phillip A Sharp
- Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. .,Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
| |
Collapse
|
|
33
|
Keratinocyte stem cells are more resistant to UVA radiation than their direct progeny. PLoS One 2018; 13:e0203863. [PMID: 30208100 PMCID: PMC6135485 DOI: 10.1371/journal.pone.0203863] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2017] [Accepted: 08/29/2018] [Indexed: 12/16/2022] Open
Abstract
The epidermis undergoes constant renewal during its lifetime. This is possible due to a special population of keratinocyte stem cells (KSCs) located at the basal layer. These cells are surrounded by their direct progeny, keratinocyte progenitors or transient amplifying cells (TAs), which arise from cell division. Skin is exposed every day to sun radiation; in particular, UVA radiation penetrates through the epidermis and induces damage to KSCs and TAs. Although keratinocytes in the basal layer are the most likely skin carcinomas and/or photoaging cells of origin, surprisingly few studies have addressed the specific responses of these cells to UV radiation. In this study, we showed for the first time that keratinocyte stem cells were more resistant to UVA irradiation than their direct progeny, transient amplifying cells. Using both the MTT assay and clonogenic assay, we found that KSCs were more photo-resistant compared to TAs after exposure to different doses of UVA (from 0 to 50 J/cm2). Moreover, KSCs had a greater ability to reconstruct human epidermis (RHE) after UVA exposure compared with TAs. Finally, investigations of DNA repair using the comet assay showed that DNA single-strand breaks and thymine dimers were repaired quicker and more efficiently in KSCs compared with TAs. In a previous work, we showed that the same stem cell population was more resistant to ionizing radiation, another carcinogenic agent. Collectively, our results combined with other observations demonstrate that keratinocyte stem cells, which are responsible for epidermal renewal throughout life, are equipped with an efficient arsenal against several genotoxic agents. Our future work will try to identify the factors or signaling pathways that are responsible for this differential photo-sensitivity and DNA repair capacity between KSCs and TAs.
Collapse
|
|
34
|
Liu Z, Zhang C, Skamagki M, Khodadadi-Jamayran A, Zhang W, Kong D, Chang CW, Feng J, Han X, Townes TM, Li H, Kim K, Zhao R. Elevated p53 Activities Restrict Differentiation Potential of MicroRNA-Deficient Pluripotent Stem Cells. Stem Cell Reports 2018; 9:1604-1617. [PMID: 29141234 PMCID: PMC5688240 DOI: 10.1016/j.stemcr.2017.10.006] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2017] [Revised: 10/09/2017] [Accepted: 10/10/2017] [Indexed: 12/21/2022] Open
Abstract
Pluripotent stem cells (PSCs) deficient for microRNAs (miRNAs), such as Dgcr8−/− or Dicer−/– embryonic stem cells (ESCs), contain no mature miRNA and cannot differentiate into somatic cells. How miRNA deficiency causes differentiation defects remains poorly understood. Here, we report that miR-302 is sufficient to enable neural differentiation of differentiation-incompetent Dgcr8−/− ESCs. Our data showed that miR-302 directly suppresses the tumor suppressor p53, which is modestly upregulated in Dgcr8−/− ESCs and serves as a barrier restricting neural differentiation. We demonstrated that direct inactivation of p53 by SV40 large T antigen, a short hairpin RNA against Trp53, or genetic ablation of Trp53 in Dgcr8−/− PSCs enables neural differentiation, while activation of p53 by the MDM2 inhibitor nutlin-3a in wild-type ESCs inhibits neural differentiation. Together, we demonstrate that a major function of miRNAs in neural differentiation is suppression of p53 and that modest activation of p53 blocks neural differentiation of miRNA-deficient PSCs.
miR-302 enables neural differentiation of differentiation-incompetent Dgcr8−/− ESCs miR-302 directly suppresses p53 expression p53 inhibits neural differentiation of Dgcr8−/− and wild-type PSCs p53 may eliminate genetically defective embryos to save maternal resources
Collapse
Affiliation(s)
- Zhong Liu
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Cheng Zhang
- Department of Molecular Pharmacology and Experimental Therapeutics, Center for Individualized Medicine, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
| | - Maria Skamagki
- Cancer Biology and Genetics Program, Center for Cell Engineering, Center for Stem Cell Biology, Sloan-Kettering Institute, Cell and Developmental Biology Program, Weill Medical College of Cornell University, New York, NY 10065, USA
| | - Alireza Khodadadi-Jamayran
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Wei Zhang
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Dexin Kong
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Chia-Wei Chang
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Jingyang Feng
- Cook County Health and Hospital System, John H. Stroger Hospital, Chicago, IL 60612, USA
| | - Xiaosi Han
- Department of Neurology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Tim M Townes
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Hu Li
- Department of Molecular Pharmacology and Experimental Therapeutics, Center for Individualized Medicine, Mayo Clinic College of Medicine, Rochester, MN 55905, USA
| | - Kitai Kim
- Cancer Biology and Genetics Program, Center for Cell Engineering, Center for Stem Cell Biology, Sloan-Kettering Institute, Cell and Developmental Biology Program, Weill Medical College of Cornell University, New York, NY 10065, USA.
| | - Rui Zhao
- Department of Biochemistry and Molecular Genetics, Stem Cell Institute, University of Alabama at Birmingham, Birmingham, AL 35294, USA; Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
| |
Collapse
|
|
35
|
Zaveri L, Dhawan J. Cycling to Meet Fate: Connecting Pluripotency to the Cell Cycle. Front Cell Dev Biol 2018; 6:57. [PMID: 29974052 PMCID: PMC6020794 DOI: 10.3389/fcell.2018.00057] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2018] [Accepted: 05/14/2018] [Indexed: 01/26/2023] Open
Abstract
Pluripotent stem cells are characterized by their high proliferative rates, their ability to self-renew and their potential to differentiate to all the three germ layers. This rapid proliferation is brought about by a highly modified cell cycle that allows the cells to quickly shuttle from DNA synthesis to cell division, by reducing the time spent in the intervening gap phases. Many key regulators that define the somatic cell cycle are either absent or exhibit altered behavior, allowing the pluripotent cell to bypass cell cycle checkpoints typical of somatic cells. Experimental analysis of this modified stem cell cycle has been challenging due to the strong link between rapid proliferation and pluripotency, since perturbations to the cell cycle or pluripotency factors result in differentiation. Despite these hurdles, our understanding of this unique cell cycle has greatly improved over the past decade, in part because of the availability of new technologies that permit the analysis of single cells in heterogeneous populations. This review aims to highlight some of the recent discoveries in this area with a special emphasis on different states of pluripotency. We also discuss the highly interlinked network that connects pluripotency factors and key cell cycle genes and review evidence for how this interdependency may promote the rapid cell cycle. This issue gains translational importance since disruptions in stem cell proliferation and differentiation can impact disorders at opposite ends of a spectrum, from cancer to degenerative disease.
Collapse
Affiliation(s)
- Lamuk Zaveri
- Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, India.,CSIR - Centre for Cellular and Molecular Biology, Hyderabad, India.,Manipal Academy of Higher Education, Manipal, India
| | - Jyotsna Dhawan
- Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, India.,CSIR - Centre for Cellular and Molecular Biology, Hyderabad, India
| |
Collapse
|
|
36
|
Low-dose irradiation of mouse embryos increases Smad-p21 pathway activity and preserves pluripotency. J Assist Reprod Genet 2018; 35:1061-1069. [PMID: 29546598 PMCID: PMC6030001 DOI: 10.1007/s10815-018-1156-y] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2017] [Accepted: 03/05/2018] [Indexed: 12/31/2022] Open
Abstract
Purpose To study the outcomes of mouse preimplantation embryos irradiated with low doses of X-rays (≤ 1 Gy) and investigate apoptosis and pluripotency of the irradiated embryos. Methods Mouse embryos at the 2-cell stage were collected for in vitro culture. After reaching the 8-cell stage, embryos were irradiated with various low doses of X-rays (0–1 Gy). Blastocysts with a normal appearance were transferred into a pseudopregnant uterus. The developmental rate to blastocysts and the survival rate following embryo transfer were examined. Expression levels of p21, Smad2, Foxo1, Cdx2, Oct4, and Nanog genes were measured by RT-PCR. Apoptotic cells in mouse blastocysts were examined immunofluorescently by staining for cleaved caspase-3. Results More than 90% of non-irradiated and low-dose X-ray-irradiated preimplantation embryos developed to morphologically normal blastocysts that could be implanted and survive in the uterus. However, embryos irradiated with X-rays had more apoptotic cells in a dose-dependent manner. Expression of p21, Smad2, and Foxo1 genes in X-ray-irradiated embryos was increased significantly, while expression of Cdx2, Oct4, and Nanog genes was maintained in comparison with non-irradiated embryos. Conclusions Although irradiated embryos contained apoptotic cells, the low doses of irradiation did not disturb development of 8-cell stage embryos to blastocysts or their survival in utero. The underlying mechanisms might involve anti-apoptotic systems, including the Smad-p21 pathway, and preservation of pluripotency. Electronic supplementary material The online version of this article (10.1007/s10815-018-1156-y) contains supplementary material, which is available to authorized users.
Collapse
|
|
37
|
Brown N, Song L, Kollu NR, Hirsch ML. Adeno-Associated Virus Vectors and Stem Cells: Friends or Foes? Hum Gene Ther 2018; 28:450-463. [PMID: 28490211 DOI: 10.1089/hum.2017.038] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
The infusion of healthy stem cells into a patient-termed "stem-cell therapy"-has shown great promise for the treatment of genetic and non-genetic diseases, including mucopolysaccharidosis type 1, Parkinson's disease, multiple sclerosis, numerous immunodeficiency disorders, and aplastic anemia. Stem cells for cell therapy can be collected from the patient (autologous) or collected from another "healthy" individual (allogeneic). The use of allogenic stem cells is accompanied with the potentially fatal risk that the transplanted donor T cells will reject the patient's cells-a process termed "graft-versus-host disease." Therefore, the use of autologous stem cells is preferred, at least from the immunological perspective. However, an obvious drawback is that inherently as "self," they contain the disease mutation. As such, autologous cells for use in cell therapies often require genetic "correction" (i.e., gene addition or editing) prior to cell infusion and therefore the requirement for some form of nucleic acid delivery, which sets the stage for the AAV controversy discussed herein. Despite being the most clinically applied gene delivery context to date, unlike other more concerning integrating and non-integrating vectors such as retroviruses and adenovirus, those based on adeno-associated virus (AAV) have not been employed in the clinic. Furthermore, published data regarding AAV vector transduction of stem cells are inconsistent in regards to vector transduction efficiency, while the pendulum swings far in the other direction with demonstrations of AAV vector-induced toxicity in undifferentiated cells. The variation present in the literature examining the transduction efficiency of AAV vectors in stem cells may be due to numerous factors, including inconsistencies in stem-cell collection, cell culture, vector preparation, and/or transduction conditions. This review summarizes the controversy surrounding AAV vector transduction of stem cells, hopefully setting the stage for future elucidation and eventual therapeutic applications.
Collapse
Affiliation(s)
- Nolan Brown
- 1 Gene Therapy Center, University of North Carolina at Chapel Hill , North Carolina.,2 Department of Ophthalmology, University of North Carolina at Chapel Hill , North Carolina
| | - Liujiang Song
- 1 Gene Therapy Center, University of North Carolina at Chapel Hill , North Carolina.,2 Department of Ophthalmology, University of North Carolina at Chapel Hill , North Carolina
| | - Nageswara R Kollu
- 1 Gene Therapy Center, University of North Carolina at Chapel Hill , North Carolina.,2 Department of Ophthalmology, University of North Carolina at Chapel Hill , North Carolina
| | - Matthew L Hirsch
- 1 Gene Therapy Center, University of North Carolina at Chapel Hill , North Carolina.,2 Department of Ophthalmology, University of North Carolina at Chapel Hill , North Carolina
| |
Collapse
|
|
38
|
BRE/BRCC45 regulates CDC25A stability by recruiting USP7 in response to DNA damage. Nat Commun 2018; 9:537. [PMID: 29416040 PMCID: PMC5803202 DOI: 10.1038/s41467-018-03020-6] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2017] [Accepted: 01/12/2018] [Indexed: 01/07/2023] Open
Abstract
BRCA2 is essential for maintaining genomic integrity. BRCA2-deficient primary cells are either not viable or exhibit severe proliferation defects. Yet, BRCA2 deficiency contributes to tumorigenesis. It is believed that mutations in genes such as TRP53 allow BRCA2 heterozygous cells to overcome growth arrest when they undergo loss of heterozygosity. Here, we report the use of an insertional mutagenesis screen to identify a role for BRE (Brain and Reproductive organ Expressed, also known as BRCC45), known to be a part of the BRCA1-DNA damage sensing complex, in the survival of BRCA2-deficient mouse ES cells. Cell viability by BRE overexpression is mediated by deregulation of CDC25A phosphatase, a key cell cycle regulator and an oncogene. We show that BRE facilitates deubiquitylation of CDC25A by recruiting ubiquitin-specific-processing protease 7 (USP7) in the presence of DNA damage. Additionally, we uncovered the role of CDC25A in BRCA-mediated tumorigenesis, which can have implications in cancer treatment. Loss of BRCA2 leads to cancer formation. Here, the authors use an insertional mutagenesis approach and identify a multiprotein complex consisting of BRE, USP7 and CDC25A that can support the survival of BRCA2-deficient cells.
Collapse
|
|
39
|
A p53-dependent response limits the viability of mammalian haploid cells. Proc Natl Acad Sci U S A 2017; 114:9367-9372. [PMID: 28808015 DOI: 10.1073/pnas.1705133114] [Citation(s) in RCA: 52] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
The recent development of haploid cell lines has facilitated forward genetic screenings in mammalian cells. These lines include near-haploid human cell lines isolated from a patient with chronic myelogenous leukemia (KBM7 and HAP1), as well as haploid embryonic stem cells derived from several organisms. In all cases, haploidy was shown to be an unstable state, so that cultures of mammalian haploid cells rapidly become enriched in diploids. Here we show that the observed diploidization is due to a proliferative disadvantage of haploid cells compared with diploid cells. Accordingly, single-cell-sorted haploid mammalian cells maintain the haploid state for prolonged periods, owing to the absence of competing diploids. Although the duration of interphase is similar in haploid and diploid cells, haploid cells spend longer in mitosis, indicative of problems in chromosome segregation. In agreement with this, a substantial proportion of the haploids die at or shortly after the last mitosis through activation of a p53-dependent cytotoxic response. Finally, we show that p53 deletion stabilizes haploidy in human HAP1 cells and haploid mouse embryonic stem cells. We propose that, similar to aneuploidy or tetraploidy, haploidy triggers a p53-dependent response that limits the fitness of mammalian cells.
Collapse
|
|
40
|
Jeong HC, Cho SJ, Lee MO, Cha HJ. Technical approaches to induce selective cell death of pluripotent stem cells. Cell Mol Life Sci 2017; 74:2601-2611. [PMID: 28246701 PMCID: PMC11107638 DOI: 10.1007/s00018-017-2486-0] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2016] [Revised: 01/24/2017] [Accepted: 02/06/2017] [Indexed: 01/24/2023]
Abstract
Despite the recent promising results of clinical trials using human pluripotent stem cell (hPSC)-based cell therapies for age-related macular degeneration (AMD), the risk of teratoma formation resulting from residual undifferentiated hPSCs remains a serious and critical hurdle for broader clinical implementation. To mitigate the tumorigenic risk of hPSC-based cell therapy, a variety of approaches have been examined to ablate the undifferentiated hPSCs based on the unique molecular properties of hPSCs. In the present review, we offer a brief overview of recent attempts at selective elimination of undifferentiated hPSCs to decrease the risk of teratoma formation in hPSC-based cell therapy.
Collapse
Affiliation(s)
- Ho-Chang Jeong
- Dept. of Life Sciences, College of Natural Sciences, Sogang University, #1 Sinsu-dong, Mapo-gu, Seoul,, 121-742, Republic of Korea
| | - Seung-Ju Cho
- Dept. of Life Sciences, College of Natural Sciences, Sogang University, #1 Sinsu-dong, Mapo-gu, Seoul,, 121-742, Republic of Korea
| | - Mi-Ok Lee
- Stem Cell Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon,, 305-806, Republic of Korea
| | - Hyuk-Jin Cha
- Dept. of Life Sciences, College of Natural Sciences, Sogang University, #1 Sinsu-dong, Mapo-gu, Seoul,, 121-742, Republic of Korea.
| |
Collapse
|
|
41
|
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.
Collapse
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.
| |
Collapse
|
|
42
|
Resistance for Genotoxic Damage in Mesenchymal Stromal Cells Is Increased by Hypoxia but Not Generally Dependent on p53-Regulated Cell Cycle Arrest. PLoS One 2017; 12:e0169921. [PMID: 28081228 PMCID: PMC5231334 DOI: 10.1371/journal.pone.0169921] [Citation(s) in RCA: 249] [Impact Index Per Article: 31.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2016] [Accepted: 12/23/2016] [Indexed: 11/19/2022] Open
Abstract
Adult stem cells including multipotent mesenchymal stromal cells (MSC) acquire a high amount of DNA-damage due to their prolonged lifespan. MSC may exert specific mechanisms of resistance to avoid loss of functional activity. We have previously shown that resistance of MSC is associated with an induction of p53 and proliferation arrest upon genotoxic damage. Hypoxia may also contribute to resistance in MSC due to the low oxygen tension in the niche. In this study we characterized the role of p53 and contribution of hypoxia in resistance of MSC to genotoxic damage. MSC exhibited increased resistance to cisplatin induced DNA-damage. This resistance was associated with a temporary G2/M cell cycle arrest, induction of p53- and p21-expression and reduced cyclin B / cdk1-levels upon subapoptotic damage. Resistance of MSC to cisplatin was increased at hypoxic conditions i. e. oxygen <0.5%. However, upon hypoxia the cisplatin-induced cell cycle arrest and expression of p53 and p21 were abrogated. MSC with shRNA-mediated p53 knock-down showed a reduced cell cycle arrest and increased cyclin B / cdk1 expression. However, this functional p53 knock down did not alter the resistance to cisplatin. In contrast to cisplatin, functional p53-knock-down increased the resistance of MSC to etoposide. We conclude that resistance of MSC to genotoxic damage is influenced by oxygen tension but is not generally dependent on p53. Thus, p53-dependent and p53-independent mechanisms of resistance are likely to contribute to the life-long functional activity of MSC in vivo. These findings indicate that hypoxia and different resistance pathways contribute to the phenotype that enables the prolonged lifespan of MSC.
Collapse
|
|
43
|
Meyer B, Fabbrizi MR, Raj S, Zobel CL, Hallahan DE, Sharma GG. Histone H3 Lysine 9 Acetylation Obstructs ATM Activation and Promotes Ionizing Radiation Sensitivity in Normal Stem Cells. Stem Cell Reports 2016; 7:1013-1022. [PMID: 27974220 PMCID: PMC5161741 DOI: 10.1016/j.stemcr.2016.11.004] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2016] [Revised: 11/07/2016] [Accepted: 11/08/2016] [Indexed: 01/09/2023] Open
Abstract
Dynamic spatiotemporal modification of chromatin around DNA damage is vital for efficient DNA repair. Normal stem cells exhibit an attenuated DNA damage response (DDR), inefficient DNA repair, and high radiosensitivity. The impact of unique chromatin characteristics of stem cells in DDR regulation is not yet recognized. We demonstrate that murine embryonic stem cells (ES) display constitutively elevated acetylation of histone H3 lysine 9 (H3K9ac) and low H3K9 tri-methylation (H3K9me3). DNA damage-induced local deacetylation of H3K9 was abrogated in ES along with the subsequent H3K9me3. Depletion of H3K9ac in ES by suppression of monocytic leukemia zinc finger protein (MOZ) acetyltransferase improved ATM activation, DNA repair, diminished irradiation-induced apoptosis, and enhanced clonogenic survival. Simultaneous suppression of the H3K9 methyltransferase Suv39h1 abrogated the radioprotective effect of MOZ inhibition, suggesting that high H3K9ac promoted by MOZ in ES cells obstructs local upregulation of H3K9me3 and contributes to muted DDR and increased radiosensitivity.
Collapse
Affiliation(s)
- Barbara Meyer
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park, Saint Louis, MO 63108, USA
| | - Maria Rita Fabbrizi
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park, Saint Louis, MO 63108, USA
| | - Suyash Raj
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park, Saint Louis, MO 63108, USA
| | - Cheri L Zobel
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park, Saint Louis, MO 63108, USA
| | - Dennis E Hallahan
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park, Saint Louis, MO 63108, USA; Siteman Cancer Center, Washington University School of Medicine, Saint Louis, MO 63108, USA
| | - Girdhar G Sharma
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 4511 Forest Park, Saint Louis, MO 63108, USA; Siteman Cancer Center, Washington University School of Medicine, Saint Louis, MO 63108, USA.
| |
Collapse
|
|
44
|
He H, Wang C, Dai Q, Li F, Bergholz J, Li Z, Li Q, Xiao ZX. p53 and p73 Regulate Apoptosis but Not Cell-Cycle Progression in Mouse Embryonic Stem Cells upon DNA Damage and Differentiation. Stem Cell Reports 2016; 7:1087-1098. [PMID: 27866875 PMCID: PMC5161534 DOI: 10.1016/j.stemcr.2016.10.008] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2016] [Revised: 10/21/2016] [Accepted: 10/24/2016] [Indexed: 01/23/2023] Open
Abstract
Embryonic stem cells (ESCs) are fast proliferating cells capable of differentiating into all somatic cell types. In somatic cells, it is well documented that p53 is rapidly activated upon DNA damage to arrest the cell cycle and induce apoptosis. In mouse ESCs, p53 can also be functionally activated, but the precise biological consequences are not well characterized. Here, we demonstrated that doxorubicin treatment initially led to cell-cycle arrest at G2/M in ESCs, followed by the occurrence of massive apoptosis. Neither p53 nor its target gene p73 was required for G2/M arrest. Instead, p53 and p73 were fully responsible for apoptosis. p53 and p73 were also required for differentiation-induced apoptosis in mouse ESCs. In addition, doxorubicin treatment induced the expression of retinoblastoma protein in a p53-dependent manner. Therefore, both p53 and p73 are critical in apoptosis induced by DNA damage and differentiation.
p53/p73 are key for DNA damage-induced apoptosis but not G2/M arrest in mESCs Both p53 and p73 are required for differentiation-induced apoptosis in mESCs Doxorubicin induces RB via p53-mediated suppression of miR-17-92 and miR-106a-363 p73 expression is induced upon differentiation in mESCs
Collapse
Affiliation(s)
- Hanbing He
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Cheng Wang
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Qian Dai
- Department of Pediatrics, West China Second University Hospital, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Sichuan University, Chengdu 610041, China
| | - Fengtian Li
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Johann Bergholz
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Zhonghan Li
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Qintong Li
- Department of Pediatrics, West China Second University Hospital, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Sichuan University, Chengdu 610041, China.
| | - Zhi-Xiong Xiao
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China.
| |
Collapse
|
|
45
|
Histone modifications and p53 binding poise the p21 promoter for activation in human embryonic stem cells. Sci Rep 2016; 6:28112. [PMID: 27346849 PMCID: PMC4921813 DOI: 10.1038/srep28112] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2016] [Accepted: 05/31/2016] [Indexed: 11/08/2022] Open
Abstract
The high proliferation rate of embryonic stem cells (ESCs) is thought to arise partly from very low expression of p21. However, how p21 is suppressed in ESCs has been unclear. We found that p53 binds to the p21 promoter in human ESCs (hESCs) as efficiently as in differentiated human mesenchymal stem cells, however it does not promote p21 transcription in hESCs. We observed an enrichment for both the repressive histone H3K27me3 and activating histone H3K4me3 chromatin marks at the p21 locus in hESCs, suggesting it is a suppressed, bivalent domain which overrides activation by p53. Reducing H3K27me3 methylation in hESCs rescued p21 expression, and ectopic expression of p21 in hESCs triggered their differentiation. Further, we uncovered a subset of bivalent promoters bound by p53 in hESCs that are similarly induced upon differentiation in a p53-dependent manner, whereas p53 promotes the transcription of other target genes which do not show an enrichment of H3K27me3 in ESCs. Our studies reveal a unique epigenetic strategy used by ESCs to poise undesired p53 target genes, thus balancing the maintenance of pluripotency in the undifferentiated state with a robust response to differentiation signals, while utilizing p53 activity to maintain genomic stability and homeostasis in ESCs.
Collapse
|
|
46
|
Liao YX, Zeng JM, Zhou JJ, Yang GH, Ding K, Zhang XJ. Silencing of RTKN2 by siRNA suppresses proliferation, and induces G1 arrest and apoptosis in human bladder cancer cells. Mol Med Rep 2016; 13:4872-8. [PMID: 27082503 DOI: 10.3892/mmr.2016.5127] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2015] [Accepted: 12/21/2015] [Indexed: 11/05/2022] Open
Abstract
Human bladder cancer is the most common urological malignancy in China. One of the causes of carcinogenesis in the cancer may be gene mutation. Therefore, the present study investigated the expression levels of Rhotekin 2 (RTKN2), a Rho effector protein, in human bladder cancer tissues and cell lines, and examined the effect of RTKN2 on the proliferation, cell cycle, apoptosis and invasion of human bladder cancer cell lines. The mRNA expression levels of RTKN2 in 30 human bladder cancer tissue samples were significantly higher, compared with those in 30 normal human bladder tissue samples. The protein expression levels of RTKN2 was markedly higher in T24 and 5637 cells, compared with those in four other human bladder cancer cell lines. The silencing of RTKN2 by small interfering (si)RNA inhibited cell proliferation and arrested cell cycle at the G1 phase, via reducing the expression levels of the MCM10, CDK2, CDC24A and CDC6 cell cycle‑associated proteins in the T24 and 5637 cells. Furthermore, RTKN2 knockdown in the cells led to cell apoptosis and the suppression of invasion. These results suggested that RTKN2 is involved in the carcinogenesis and progression of human bladder cancer, indicating that RTKN2 may be a molecular target in cancer therapy.
Collapse
Affiliation(s)
- Yi-Xiang Liao
- Department of Urology, Jingzhou Central Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jingzhou, Hubei 434020, P.R. China
| | - Jin-Min Zeng
- Department of Urology, Jingzhou Central Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jingzhou, Hubei 434020, P.R. China
| | - Jia-Jie Zhou
- Department of Urology, Jingzhou Central Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jingzhou, Hubei 434020, P.R. China
| | - Guang-Hua Yang
- Department of Urology, Jingzhou Central Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jingzhou, Hubei 434020, P.R. China
| | - Kun Ding
- Department of Urology, Jingzhou Central Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jingzhou, Hubei 434020, P.R. China
| | - Xian-Jue Zhang
- Department of Urology, Jingzhou Central Hospital, Tongji Medical College, Huazhong University of Science and Technology, Jingzhou, Hubei 434020, P.R. China
| |
Collapse
|
|
47
|
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.
Collapse
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
| |
Collapse
|
|
48
|
Helm A, Arrizabalaga O, Pignalosa D, Schroeder IS, Durante M, Ritter S. Ionizing Radiation Impacts on Cardiac Differentiation of Mouse Embryonic Stem Cells. Stem Cells Dev 2016; 25:178-88. [PMID: 26506910 PMCID: PMC4733326 DOI: 10.1089/scd.2015.0260] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2015] [Accepted: 10/27/2015] [Indexed: 12/14/2022] Open
Abstract
Little is known about the effects of ionizing radiation on the earliest stages of embryonic development although it is well recognized that ionizing radiation is a natural part of our environment and further exposure may occur due to medical applications. The current study addresses this issue using D3 mouse embryonic stem cells as a model system. Cells were irradiated with either X-rays or carbon ions representing sparsely and densely ionizing radiation and their effect on the differentiation of D3 cells into spontaneously contracting cardiomyocytes through embryoid body (EB) formation was measured. This study is the first to demonstrate that ionizing radiation impairs the formation of beating cardiomyocytes with carbon ions being more detrimental than X-rays. However, after prolonged culture time, the number of beating EBs derived from carbon ion irradiated cells almost reached control levels indicating that the surviving cells are still capable of developing along the cardiac lineage although with considerable delay. Reduced EB size, failure to downregulate pluripotency markers, and impaired expression of cardiac markers were identified as the cause of compromised cardiomyocyte formation. Dysregulation of cardiac differentiation was accompanied by alterations in the expression of endodermal and ectodermal markers that were more severe after carbon ion irradiation than after exposure to X-rays. In conclusion, our data show that carbon ion irradiation profoundly affects differentiation and thus may pose a higher risk to the early embryo than X-rays.
Collapse
Affiliation(s)
- Alexander Helm
- Biophysics Department, GSI Helmholtz Centre for Heavy Ion Research, Darmstadt, Germany
| | - Onetsine Arrizabalaga
- Biophysics Department, GSI Helmholtz Centre for Heavy Ion Research, Darmstadt, Germany
- IKERBASQUE, Basque Foundation for Science, Bilbao, Spain
| | - Diana Pignalosa
- Biophysics Department, GSI Helmholtz Centre for Heavy Ion Research, Darmstadt, Germany
| | - Insa S. Schroeder
- Biophysics Department, GSI Helmholtz Centre for Heavy Ion Research, Darmstadt, Germany
| | - Marco Durante
- Biophysics Department, GSI Helmholtz Centre for Heavy Ion Research, Darmstadt, Germany
- Physics Department, Institute for Condensed Matter Physics, Technical University Darmstadt, Darmstadt, Germany
| | - Sylvia Ritter
- Biophysics Department, GSI Helmholtz Centre for Heavy Ion Research, Darmstadt, Germany
| |
Collapse
|
|
49
|
Suchorska WM, Augustyniak E, Łukjanow M. Genetic stability of pluripotent stem cells during anti-cancer therapies. Exp Ther Med 2016; 11:695-702. [PMID: 26997981 PMCID: PMC4774348 DOI: 10.3892/etm.2016.2993] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2015] [Accepted: 12/10/2015] [Indexed: 12/12/2022] Open
Abstract
Regenerative medicine is a rapidly growing field that holds promise for the treatment of many currently unresponsive diseases. Stem cells (SCs) are undifferentiated cells with long-term self-renewal potential and the capacity to develop into specialized cells. SC-based therapies constitute a novel and promising concept in regenerative medicine. Radiotherapy is the most frequently used method in the adjuvant treatment of tumorous alterations. In the future, the usage of SCs in regenerative medicine will be affected by their regular and inevitable exposure to ionizing radiation (IR). This phenomenon will be observed during treatment as well as diagnosis. The issue of the genetic stability of SCs and cells differentiated from SCs is crucial in the context of the application of these cells in clinical practice. This review examines current knowledge concerning the DNA repair mechanisms (base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end-joining) of SCs in response to the harmful effects of genotoxic agents such as IR and chemotherapeutics.
Collapse
Affiliation(s)
- Wiktoria Maria Suchorska
- Radiobiology Laboratory, Greater Poland Cancer Centre, 61-866 Poznań, Poland; The Postgraduate School of Molecular Medicine, Medical University of Warsaw, 20-091 Warsaw, Poland; Department of Electroradiology, Poznań University of Medical Sciences, 61-866 Poznań, Poland
| | - Ewelina Augustyniak
- Radiobiology Laboratory, Greater Poland Cancer Centre, 61-866 Poznań, Poland; The Postgraduate School of Molecular Medicine, Medical University of Warsaw, 20-091 Warsaw, Poland
| | - Magdalena Łukjanow
- Radiobiology Laboratory, Greater Poland Cancer Centre, 61-866 Poznań, Poland
| |
Collapse
|
|
50
|
Moehrle BM, Nattamai K, Brown A, Florian MC, Ryan M, Vogel M, Bliederhaeuser C, Soller K, Prows DR, Abdollahi A, Schleimer D, Walter D, Milsom MD, Stambrook P, Porteus M, Geiger H. Stem Cell-Specific Mechanisms Ensure Genomic Fidelity within HSCs and upon Aging of HSCs. Cell Rep 2015; 13:2412-2424. [PMID: 26686632 DOI: 10.1016/j.celrep.2015.11.030] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2015] [Revised: 08/13/2015] [Accepted: 11/08/2015] [Indexed: 01/22/2023] Open
Abstract
Whether aged hematopoietic stem and progenitor cells (HSPCs) have impaired DNA damage repair is controversial. Using a combination of DNA mutation indicator assays, we observe a 2- to 3-fold increase in the number of DNA mutations in the hematopoietic system upon aging. Young and aged hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) do not show an increase in mutation upon irradiation-induced DNA damage repair, and young and aged HSPCs respond very similarly to DNA damage with respect to cell-cycle checkpoint activation and apoptosis. Both young and aged HSPCs show impaired activation of the DNA-damage-induced G1-S checkpoint. Induction of chronic DNA double-strand breaks by zinc-finger nucleases suggests that HSPCs undergo apoptosis rather than faulty repair. These data reveal a protective mechanism in both the young and aged hematopoietic system against accumulation of mutations in response to DNA damage.
Collapse
Affiliation(s)
- Bettina M Moehrle
- Institute of Molecular Medicine, University of Ulm, 89081 Ulm, Germany
| | - Kalpana Nattamai
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center and University of Cincinnati, Cincinnati, OH 45229, USA
| | - Andreas Brown
- Institute of Molecular Medicine, University of Ulm, 89081 Ulm, Germany
| | - Maria C Florian
- Institute of Molecular Medicine, University of Ulm, 89081 Ulm, Germany
| | - Marnie Ryan
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center and University of Cincinnati, Cincinnati, OH 45229, USA
| | - Mona Vogel
- Institute of Molecular Medicine, University of Ulm, 89081 Ulm, Germany
| | | | - Karin Soller
- Institute of Molecular Medicine, University of Ulm, 89081 Ulm, Germany
| | - Daniel R Prows
- Division of Human Genetics, Cincinnati Children's Hospital Medical Center and University of Cincinnati, Cincinnati, OH 45229, USA
| | - Amir Abdollahi
- German Cancer Consortium (DKTK), 69120 Heidelberg, Germany; Molecular and Translational Radiation Oncology, Heidelberg Ion Therapy Center (HIT), 69120 Heidelberg, Germany
| | - David Schleimer
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center and University of Cincinnati, Cincinnati, OH 45229, USA
| | - Dagmar Walter
- Heidelberg Institute for Stem Cell Technology and Experimental Medicine gGmbH (HI-STEM), 69120 Heidelberg, Germany
| | - Michael D Milsom
- Heidelberg Institute for Stem Cell Technology and Experimental Medicine gGmbH (HI-STEM), 69120 Heidelberg, Germany; Deutsches Krebsforschungszentrum (DKFZ), Division of Stem Cells and Cancer, Experimental Hematology Group, 69120 Heidelberg, Germany
| | - Peter Stambrook
- Department of Molecular Genetics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA
| | - Matthew Porteus
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Hartmut Geiger
- Institute of Molecular Medicine, University of Ulm, 89081 Ulm, Germany; Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center and University of Cincinnati, Cincinnati, OH 45229, USA.
| |
Collapse
|