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Eftekhar Z, Aghaei M, Saki N. DNA damage repair in megakaryopoiesis: molecular and clinical aspects. Expert Rev Hematol 2024; 17:705-712. [PMID: 39117495 DOI: 10.1080/17474086.2024.2391102] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2024] [Revised: 07/09/2024] [Accepted: 08/07/2024] [Indexed: 08/10/2024]
Abstract
INTRODUCTION Endogenous DNA damage is a significant factor in the damage of hematopoietic cells. Megakaryopoiesis is one of the pathways of hematopoiesis that ends with the production of platelets and plays the most crucial role in hemostasis. Despite the presence of efficient DNA repair mechanisms, some endogenous lesions can lead to mutagenic alterations, disruption of pathways of hematopoiesis including megakaryopoiesis and potentially result in human diseases. AREAS COVERED The complex regulation of DNA repair mechanisms plays a central role in maintaining genomic integrity during megakaryopoiesis and influences platelet production efficiency and quality. Moreover, anomalies in DNA repair processes are involved in several diseases associated with megakaryopoiesis, including myeloproliferative disorders and thrombocytopenia. EXPERT OPINION In the era of personalized medicine, diagnosing diseases related to megakaryopoiesis can only be made with a complete assessment of their molecular aspects to provide physicians with critical molecular data for patient management and to identify the subset of patients who could benefit from targeted therapy.
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Affiliation(s)
- Zeinab Eftekhar
- Student Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
- Thalassemia & Hemoglobinopathy Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Mojtaba Aghaei
- Student Research Committee, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
- Thalassemia & Hemoglobinopathy Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Najmaldin Saki
- Thalassemia & Hemoglobinopathy Research Center, Health Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
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Thompson W, Papoutsakis ET. Similar but distinct: The impact of biomechanical forces and culture age on the production, cargo loading, and biological efficacy of human megakaryocytic extracellular vesicles for applications in cell and gene therapies. Bioeng Transl Med 2023; 8:e10563. [PMID: 37693047 PMCID: PMC10486331 DOI: 10.1002/btm2.10563] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2023] [Revised: 05/18/2023] [Accepted: 06/01/2023] [Indexed: 09/12/2023] Open
Abstract
Megakaryocytic extracellular vesicles (MkEVs) promote the growth and megakaryopoiesis of hematopoietic stem and progenitor cells (HSPCs) largely through endogenous miR-486-5p and miR-22-3p cargo. Here, we examine the impact of biomechanical force and culture age/differentiation on the formation, properties, and biological efficacy of MkEVs. We applied biomechanical force to Mks using two methods: shake flask cultures and a syringe pump system. Force increased MkEV production in a magnitude-dependent manner, with similar trends emerging regardless of whether flow cytometry or nanoparticle tracking analysis was used for MkEV counting. Both methods produced MkEVs that were relatively depleted of miR-486-5p and miR-22-3p cargo. However, while the shake flask-derived MkEVs were correspondingly less effective in promoting megakaryocytic differentiation of HSPCs, the syringe pump-derived MkEVs were more effective in doing so, suggesting the presence of unique, unidentified miRNA cargo components. Higher numbers of MkEVs were also produced by "older" Mk cultures, though miRNA cargo levels and MkEV bioactivity were unaffected by culture age. A reduction in MkEV production by Mks derived from late-differentiating HSPCs was also noted. Taken together, our results demonstrate that biomechanical force has an underappreciated and deeply influential role in MkEV biology, though that role may vary significantly depending on the nature of the force. Given the ubiquity of biomechanical force in vivo and in biomanufacturing, this phenomenon must be grappled with before MkEVs can attain clinical relevance.
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Affiliation(s)
- Will Thompson
- Department of Chemical and Biomolecular EngineeringUniversity of DelawareNewarkDelawareUSA
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Dressel N, Natusch L, Munz CM, Costas Ramon S, Morcos MNF, Loff A, Hiller B, Haase C, Schulze L, Müller P, Lesche M, Dahl A, Luksch H, Rösen-Wolff A, Roers A, Behrendt R, Gerbaulet A. Activation of the cGAS/STING Axis in Genome-Damaged Hematopoietic Cells Does Not Impact Blood Cell Formation or Leukemogenesis. Cancer Res 2023; 83:2858-2872. [PMID: 37335136 DOI: 10.1158/0008-5472.can-22-3860] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2022] [Revised: 05/04/2023] [Accepted: 06/14/2023] [Indexed: 06/21/2023]
Abstract
Genome damage is a main driver of malignant transformation, but it also induces aberrant inflammation via the cGAS/STING DNA-sensing pathway. Activation of cGAS/STING can trigger cell death and senescence, thereby potentially eliminating genome-damaged cells and preventing against malignant transformation. Here, we report that defective ribonucleotide excision repair (RER) in the hematopoietic system caused genome instability with concomitant activation of the cGAS/STING axis and compromised hematopoietic stem cell function, ultimately resulting in leukemogenesis. Additional inactivation of cGAS, STING, or type I IFN signaling, however, had no detectable effect on blood cell generation and leukemia development in RER-deficient hematopoietic cells. In wild-type mice, hematopoiesis under steady-state conditions and in response to genome damage was not affected by loss of cGAS. Together, these data challenge a role of the cGAS/STING pathway in protecting the hematopoietic system against DNA damage and leukemic transformation. SIGNIFICANCE Loss of cGAS/STING signaling does not impact DNA damage-driven leukemogenesis or alter steady-state, perturbed or malignant hematopoiesis, indicating that the cGAS/STING axis is not a crucial antioncogenic mechanism in the hematopoietic system. See related commentary by Zierhut, p. 2807.
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Affiliation(s)
- Nicole Dressel
- Institute for Immunology, Faculty of Medicine, TU Dresden, Dresden, Germany
| | - Loreen Natusch
- Institute for Immunology, Faculty of Medicine, TU Dresden, Dresden, Germany
| | - Clara M Munz
- Institute for Immunology, Faculty of Medicine, TU Dresden, Dresden, Germany
| | | | - Mina N F Morcos
- Institute for Immunology, Faculty of Medicine, TU Dresden, Dresden, Germany
| | - Anja Loff
- Institute for Immunology, Faculty of Medicine, TU Dresden, Dresden, Germany
| | - Björn Hiller
- Institute for Immunology, Faculty of Medicine, TU Dresden, Dresden, Germany
| | - Christa Haase
- Institute for Immunology, Faculty of Medicine, TU Dresden, Dresden, Germany
| | - Livia Schulze
- Institute for Immunology, Faculty of Medicine, TU Dresden, Dresden, Germany
| | - Patrick Müller
- Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
| | - Mathias Lesche
- DRESDEN-concept Genome Center, Center for Molecular and Cellular Bioengineering, TU Dresden, Dresden, Germany
| | - Andreas Dahl
- DRESDEN-concept Genome Center, Center for Molecular and Cellular Bioengineering, TU Dresden, Dresden, Germany
| | - Hella Luksch
- Department of Pediatrics, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany
| | - Angela Rösen-Wolff
- Department of Pediatrics, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany
| | - Axel Roers
- Institute for Immunology, Faculty of Medicine, TU Dresden, Dresden, Germany
- Institute for Immunology, Heidelberg University Hospital, Heidelberg, Germany
| | - Rayk Behrendt
- Institute for Immunology, Faculty of Medicine, TU Dresden, Dresden, Germany
- Institute for Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany
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Zhou XL, Wei Y, Chen P, Yang X, Lu C, Pan MH. A novel transcription factor, BmZFP67, regulates endomitosis switch by controlling the expression of cyclin B in silk glands. Int J Biol Macromol 2023:124931. [PMID: 37263320 DOI: 10.1016/j.ijbiomac.2023.124931] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2022] [Revised: 04/25/2023] [Accepted: 05/06/2023] [Indexed: 06/03/2023]
Abstract
Endomitosis is involved in developmental processes associated with an increase in metabolic cell activity, which is characterized by repeated rounds of DNA replication without cytokinesis. Endomitosis cells are widespread in protozoa, plants, animals and humans. Endomitosis cell cycle is currently viewed as a variation of the canonical cell cycle and transformed from mitotic cell cycle. However, the meaningful question about how endomitosis transformed from mitosis is still unclear. Herein, we identified a novel transcription factor in silk glands, ZFP67, which is gradually reduced in silk glands during the transition of mitosis to endomitosis. In addition, over-expressed ZFP67 in silk glands led to the transition delayed. And, knock-out of ZFP67 led to abnormal chromatin division and unsuccessful cell division. These data reveled that ZFP67 played an important role in transition of mitosis to endomitosis. Furthermore, ZFP67 can regulate the transcription of cyclin B, a key cyclin related to cell division and G2/M phase, which is demonstrated by chromatin immunoprecipitation and dual luciferase reporter system in this article. In conclusion, it can be speculated that the decreasing expression of ZFP67 in silk glands during the transition stage of mitosis-to-endomitosis resulted in the lack of cyclin B, which further led to unsuccessful cytokinesis and then promoted the transition from mitosis to endomitosis of silk gland cells.
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Affiliation(s)
- Xiao-Lin Zhou
- State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China; Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, Zhejiang 325001, China
| | - Yi Wei
- State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China; Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, Zhejiang 325001, China
| | - Peng Chen
- State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China; Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing 400716, China
| | - Xi Yang
- State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China
| | - Cheng Lu
- State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China; Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing 400716, China.
| | - Min-Hui Pan
- State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China; Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural Ministry, Southwest University, Chongqing 400716, China.
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Dahariya S, Raghuwanshi S, Thamodaran V, Velayudhan SR, Gutti RK. Role of Long Non-Coding RNAs in Human-Induced Pluripotent Stem Cells Derived Megakaryocytes: A p53, HOX Antisense Intergenic RNA Myeloid 1, and miR-125b Interaction Study. J Pharmacol Exp Ther 2023; 384:92-101. [PMID: 36243404 DOI: 10.1124/jpet.121.001095] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2021] [Revised: 09/21/2022] [Accepted: 09/22/2022] [Indexed: 12/27/2022] Open
Abstract
Megakaryocytes (MKs) are rare polyploid cells found in the bone marrow and produce platelets. Platelets are small cell fragments that are essential during wound healing and vascular hemostasis. In vitro differentiation of MKs from human-induced pluripotent stem cell-derived CD34+ hematopoietic stem cells (hiPSC-HSCs) could provide an alternative treatment option for thrombocytopenic patients as a platelet source. In this approach, we developed a method to produce functional MKs from hiPSC-HSCs using a xeno-free and feeder-free condition and minimize the variation and risk from animal-derived products in cell culture. We have also investigated the genome-wide expression as well as functional significance of long noncoding RNAs (lncRNAs) in hiPSC-HSC-derived MKs to get insight into MK biology. We have performed lncRNAs expression profiling by using the Human LncProfilers qPCR Array Kit and identified 26 differentially regulated lncRNAs in hiPSC-HSC-derived MKs as compared with those in hiPSC-HSCs. HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) was the most highly upregulated lncRNA in hiPSC-HSC-derived MKs and phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic-differentiating K562 cells. Furthermore, we have studied the potential mechanism of HOTAIRM1 based on the interactions between HOTAIRM1, p53, and miR-125b in PMA-induced K562 cells. Our results demonstrated that during MK maturation, HOTAIRM1 might be associated with the transcriptional regulation of p53 via acting as a decoy for miR-125b. Thus, the interaction between HOTAIRM1, p53, and miR-125b is likely involved in controlling cell cycling (cyclin D1), reactive oxygen species production, and apoptosis to support terminal maturation of MKs. SIGNIFICANCE STATEMENT: In vitro generation of megakaryocytes (MKs) from human-induced pluripotent stem cell-derived hematopoietic stem cells (hiPSC-HSCs) could provide an alternative source of platelets for treating thrombocytopenic patients. This study has investigated the functional significance of long non-coding RNAs in hiPSC-HSC-derived MKs, which remains unclear. This study's findings suggest that the regulatory role of HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) in p53-mediated regulation of cyclin D1 during megakaryocytopoiesis is to promote MK maturation by decoying miR-125b.
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Affiliation(s)
- Swati Dahariya
- Stem Cell Research Laboratory, Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India (S.D., S.R., R.K.G.) and Centre for Stem Cell Research, Christian Medical College, Vellore, India (V.T., S.R.V.)
| | - Sanjeev Raghuwanshi
- Stem Cell Research Laboratory, Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India (S.D., S.R., R.K.G.) and Centre for Stem Cell Research, Christian Medical College, Vellore, India (V.T., S.R.V.)
| | - Vasanth Thamodaran
- Stem Cell Research Laboratory, Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India (S.D., S.R., R.K.G.) and Centre for Stem Cell Research, Christian Medical College, Vellore, India (V.T., S.R.V.)
| | - Shaji R Velayudhan
- Stem Cell Research Laboratory, Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India (S.D., S.R., R.K.G.) and Centre for Stem Cell Research, Christian Medical College, Vellore, India (V.T., S.R.V.)
| | - Ravi Kumar Gutti
- Stem Cell Research Laboratory, Department of Biochemistry, School of Life Sciences, University of Hyderabad, Hyderabad, India (S.D., S.R., R.K.G.) and Centre for Stem Cell Research, Christian Medical College, Vellore, India (V.T., S.R.V.)
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Belliveau J, Papoutsakis ET. Extracellular Vesicles Facilitate Large-Scale Dynamic Exchange of Proteins and RNA Among Cultured Chinese Hamster Ovary (CHO) and Human Cells. Biotechnol Bioeng 2022; 119:1222-1238. [PMID: 35120270 DOI: 10.1002/bit.28053] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2022] [Accepted: 01/31/2022] [Indexed: 11/11/2022]
Abstract
Cells in culture are viewed as unique individuals in a large population communicating through extracellular molecules and, more recently extracellular vesicles (EVs). Our data here paint a different picture: large-scale exchange of cellular material through EVs. To visualize the dynamic production and cellular uptake of EVs, we used correlative confocal microscopy and scanning electron microscopy, as well as flow cytometry to interrogate labeled cells. Using cells expressing fluorescent proteins (GFP, miRFP703) and cells tagged with protein and RNA dyes, we show that Chinese Hamster Ovary (CHO) cells dynamically produce and uptake EVs to exchange proteins and RNAs at a large scale. Applying a simple model to our data, we estimate, for the first time, the per cell specific rates of EV production (68 and 203 microparticles and exosomes, respectively, per day). This EV-mediated massive exchange of cellular material observed in CHO cultures was also observed in cultured human CHRF-288-11 and primary hematopoietic stem and progenitor cells. This study demonstrates an underappreciated massive protein and RNA exchange between cells mediated by EVs spanning cell type, suggesting that the proximity of cells in normal and tumor tissues may also result in prolific exchange of cellular material. This exchange would be expected to homogenize the cell-population cytosol and dynamically regulate cell proliferation and the cellular state. This article is protected by copyright. All rights reserved.
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Affiliation(s)
- Jessica Belliveau
- Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, 19711.,Delaware Biotechnology Institute,, University of Delaware, Newark, DE, 19711
| | - Eleftherios T Papoutsakis
- Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, 19711.,Delaware Biotechnology Institute,, University of Delaware, Newark, DE, 19711.,Department of Biological Sciences, University of Delaware, Newark, DE, 19711
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Sun S, Jin C, Si J, Lei Y, Chen K, Cui Y, Liu Z, Liu J, Zhao M, Zhang X, Tang F, Rondina MT, Li Y, Wang QF. Single-cell analysis of ploidy and the transcriptome reveals functional and spatial divergency in murine megakaryopoiesis. Blood 2021; 138:1211-1224. [PMID: 34115843 PMCID: PMC8499048 DOI: 10.1182/blood.2021010697] [Citation(s) in RCA: 84] [Impact Index Per Article: 21.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2021] [Accepted: 05/26/2021] [Indexed: 11/20/2022] Open
Abstract
Megakaryocytes (MKs), the platelet progenitor cells, play important roles in hematopoietic stem cell (HSC) maintenance and immunity. However, it is not known whether these diverse programs are executed by a single population or by distinct subsets of cells. Here, we manually isolated primary CD41+ MKs from the bone marrow (BM) of mice and human donors based on ploidy (2N-32N) and performed single-cell RNA sequencing analysis. We found that cellular heterogeneity existed within 3 distinct subpopulations that possess gene signatures related to platelet generation, HSC niche interaction, and inflammatory responses. In situ immunostaining of mouse BM demonstrated that platelet generation and the HSC niche-related MKs were in close physical proximity to blood vessels and HSCs, respectively. Proplatelets, which could give rise to platelets under blood shear forces, were predominantly formed on a platelet generation subset. Remarkably, the inflammatory responses subpopulation, consisting generally of low-ploidy LSP1+ and CD53+ MKs (≤8N), represented ∼5% of total MKs in the BM. These MKs could specifically respond to pathogenic infections in mice. Rapid expansion of this population was accompanied by strong upregulation of a preexisting PU.1- and IRF-8-associated monocytic-like transcriptional program involved in pathogen recognition and clearance as well as antigen presentation. Consistently, isolated primary CD53+ cells were capable of engulfing and digesting bacteria and stimulating T cells in vitro. Together, our findings uncover new molecular, spatial, and functional heterogeneity within MKs in vivo and demonstrate the existence of a specialized MK subpopulation that may act as a new type of immune cell.
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Affiliation(s)
- Shu Sun
- Chinese Academy of Sciences (CAS) Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, CAS, Beijing, China
- China National Center for Bioinformation, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Chen Jin
- Chinese Academy of Sciences (CAS) Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, CAS, Beijing, China
- China National Center for Bioinformation, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Jia Si
- Chinese Academy of Sciences (CAS) Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, CAS, Beijing, China
- China National Center for Bioinformation, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Ying Lei
- Chinese Academy of Sciences (CAS) Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, CAS, Beijing, China
- China National Center for Bioinformation, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Kunying Chen
- Chinese Academy of Sciences (CAS) Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, CAS, Beijing, China
- China National Center for Bioinformation, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Yueli Cui
- Beijing Advanced Innovation Center for Genomics, College of Life Sciences, Peking University, Beijing, China
- Biomedical Institute for Pioneering Investigation via Convergence, Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China
- Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China
| | - Zhenbo Liu
- Chinese Academy of Sciences (CAS) Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, CAS, Beijing, China
- China National Center for Bioinformation, Beijing, China
| | - Jiang Liu
- Chinese Academy of Sciences (CAS) Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, CAS, Beijing, China
- China National Center for Bioinformation, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Meng Zhao
- Key Laboratory of Stem Cells and Tissue Engineering (Sun Yat-Sen University), Ministry of Education, Guangzhou, China
| | - Xiaohui Zhang
- Peking University People's Hospital, Peking University Institute of Hematology, Beijing, China
- National Clinical Research Center for Hematologic Disease, Beijing, China
- Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Beijing, China
- Collaborative Innovation Center of Hematology, Peking University, Beijing, China
| | - Fuchou Tang
- Beijing Advanced Innovation Center for Genomics, College of Life Sciences, Peking University, Beijing, China
- Biomedical Institute for Pioneering Investigation via Convergence, Ministry of Education Key Laboratory of Cell Proliferation and Differentiation, Beijing, China
- Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China
| | - Matthew T Rondina
- Department of Internal Medicine and Pathology, and the Molecular Medicine Program, University of Utah, Salt Lake City, UT; and
- Geriatric Research Education and Clinical Center, George E. Wahlen Veterans Affairs Medical Center, Salt Lake City, UT
| | - Yueying Li
- Chinese Academy of Sciences (CAS) Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, CAS, Beijing, China
- China National Center for Bioinformation, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Qian-Fei Wang
- Chinese Academy of Sciences (CAS) Key Laboratory of Genomic and Precision Medicine, Collaborative Innovation Center of Genetics and Development, Beijing Institute of Genomics, CAS, Beijing, China
- China National Center for Bioinformation, Beijing, China
- University of Chinese Academy of Sciences, Beijing, China
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Targeting the actin/tropomyosin cytoskeleton in epithelial ovarian cancer reveals multiple mechanisms of synergy with anti-microtubule agents. Br J Cancer 2021; 125:265-276. [PMID: 33981016 PMCID: PMC8292367 DOI: 10.1038/s41416-021-01420-y] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2020] [Revised: 04/12/2021] [Accepted: 04/22/2021] [Indexed: 02/03/2023] Open
Abstract
BACKGROUND Anti-microtubule agents are widely used to treat ovarian cancers, but the efficacy is often compromised by drug resistance. We investigated co-targeting the actin/tropomyosin cytoskeleton and microtubules to increase treatment efficacy in ovarian cancers and potentially overcome resistance. METHODS The presence of tropomyosin-3.1 (Tpm3.1) was examined in clinical specimens from ovarian cancer patients using immunohistochemistry. Combinatorial effects of an anti-Tpm3.1 compound, ATM-3507, with vinorelbine and paclitaxel were evaluated in ovarian cancer cells via MTS and apoptosis assays. The mechanisms of action were established using live- and fixed-cell imaging and protein analysis. RESULTS Tpm3.1 is overexpressed in 97% of tumour tissues (558 of 577) representing all histotypes of epithelial ovarian cancer. ATM-3507 displayed synergy with both anti-microtubule agents to reduce cell viability. Only vinorelbine synergised with ATM-3507 in causing apoptosis. ATM-3507 significantly prolonged vinorelbine-induced mitotic arrest with elevated activity of the spindle assembly checkpoint and mitotic cell death; however, ATM-3507 showed minor impact on paclitaxel-induced mitotic defects. Both combinations substantially increased post-mitotic G1 arrest with cyclin D1 and E1 downregulation and an increase of p21Cip and p27Kip. CONCLUSION Combined targeting of Tpm3.1/actin and microtubules is a promising treatment strategy for ovarian cancer that should be further tested in clinical settings.
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Human megakaryocytic microparticles induce de novo platelet biogenesis in a wild-type murine model. Blood Adv 2021; 4:804-814. [PMID: 32119736 DOI: 10.1182/bloodadvances.2019000753] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2019] [Accepted: 02/03/2020] [Indexed: 12/16/2022] Open
Abstract
Platelet transfusions are used to treat idiopathic or drug-induced thrombocytopenia. Platelets are an expensive product in limited supply, with limited storage and distribution capabilities because they cannot be frozen. We have demonstrated that, in vitro, human megakaryocytic microparticles (huMkMPs) target human CD34+ hematopoietic stem and progenitor cells (huHSPCs) and induce their Mk differentiation and platelet biogenesis in the absence of thrombopoietin. In this study, we showed that, in vitro, huMkMPs can also target murine HSPCs (muHSPCs) to induce them to differentiate into megakaryocytes in the absence of thrombopoietin. Based on that, using wild-type BALB/c mice, we demonstrated that intravenously administering 2 × 106 huMkMPs triggered de novo murine platelet biogenesis to increase platelet levels up to 49% 16 hours after administration. huMkMPs also largely rescued low platelet levels in mice with induced thrombocytopenia 16 hours after administration by increasing platelet counts by 51%, compared with platelet counts in thrombocytopenic mice. Normalized on a tissue-mass basis, biodistribution experiments show that MkMPs localized largely to the bone marrow, lungs, and liver 24 hours after huMkMP administration. Beyond the bone marrow, CD41+ (megakaryocytes and Mk-progenitor) cells were frequent in lungs, spleen, and especially, liver. In the liver, infused huMKMPs colocalized with Mk progenitors and muHSPCs, thus suggesting that huMkMPs interact with muHSPCs in vivo to induce platelet biogenesis. Our data demonstrate the potential of huMkMPs, which can be stored frozen, to treat thrombocytopenias and serve as effective carriers for in vivo, target-specific cargo delivery to HSPCs.
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Martinez-Høyer S, Karsan A. Mechanisms of lenalidomide sensitivity and resistance. Exp Hematol 2020; 91:22-31. [PMID: 32976949 DOI: 10.1016/j.exphem.2020.09.196] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2020] [Revised: 09/17/2020] [Accepted: 09/21/2020] [Indexed: 12/13/2022]
Abstract
The discovery that the immunomodulatory imide drugs (IMiDs) possess antitumor properties revolutionized the treatment of specific types of hematological cancers. Since then, much progress has been made in understanding why the IMiDs are so efficient in targeting the malignant clones in difficult-to-treat diseases. Despite their efficacy, IMiD resistance arises eventually. Herein we summarize the mechanisms of sensitivity and resistance to lenalidomide in del(5q) myelodysplastic syndrome and multiple myeloma, two diseases in which these drugs are at the therapeutic frontline. Understanding the molecular and cellular mechanisms underlying IMiD efficacy and resistance may allow development of specific strategies to eliminate the malignant clone in otherwise incurable diseases.
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Affiliation(s)
- Sergio Martinez-Høyer
- Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden.
| | - Aly Karsan
- Michael Smith Genome Sciences Centre, BC Cancer Research Institute, Vancouver, BC, Canada; Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.
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11
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Yang M, Liu Q, Niu T, Kuang J, Zhang X, Jiang L, Li S, He X, Wang L, Li J. Trp53 regulates platelets in bone marrow via the PI3K pathway. Exp Ther Med 2020; 20:1253-1260. [PMID: 32765666 PMCID: PMC7388439 DOI: 10.3892/etm.2020.8850] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2019] [Accepted: 11/07/2019] [Indexed: 12/23/2022] Open
Abstract
The p53 gene is well known as a key tumor suppressor gene; it is vital for hematopoietic stem cell differentiation and growth. In the present study, the change of platelets (PLTs) in p53 knockout mice (p53-/- mice) was investigated. The peripheral blood cell subsets and PLT parameters in p53-/-mice were compared with those in age-matched p53+/+ mice. Bleeding time as well as the alteration of PLT levels, were analyzed with the PLT marker CD41 antibody using flow cytometry. The results revealed that the number of PLTs in p53-/- mice was significantly lower than that in p53+/+ mice. Bleeding time was prolonged in the peripheral blood of p53-/- mice compared with that of p53+/+ mice. Furthermore, the related gene expression of the PI3K signaling pathway in the bone marrow of p53-/- mice was shown to be associated with plateletogenesis. PI3K inhibitor (LY294002) was also used to treat p53-/- mice, and the results demonstrated that LY294002 revert the change of PLTs in these mice. In summary, PLTs were altered in p53-/- mice, and the PI3K signaling pathway was involved in that process, suggesting that the p53-dependent PI3K signaling pathway is involved in thrombocytopenia or PLT diseases. PLT number is reduced in p53 deficiency; however, this reduction could be reverted by inhibiting the PI3K pathway.
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Affiliation(s)
- Mingming Yang
- Vascular Biology Research Institute, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, P.R. China
| | - Qing Liu
- Vascular Biology Research Institute, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, P.R. China
| | - Ting Niu
- Vascular Biology Research Institute, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, P.R. China
| | - Jianbiao Kuang
- Vascular Biology Research Institute, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, P.R. China
| | - Xiaohan Zhang
- Department of Pathology, Zhuhai Branch of Traditional Chinese Medicine Hospital of Guangdong Province, Zhuhai, Guangdong 519015, P.R. China
| | - Lingbi Jiang
- Vascular Biology Research Institute, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, P.R. China
| | - Siqi Li
- Vascular Biology Research Institute, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, P.R. China
| | - Xiaodong He
- Vascular Biology Research Institute, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, P.R. China
| | - Lijing Wang
- Vascular Biology Research Institute, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, P.R. China
| | - Jiangchao Li
- Vascular Biology Research Institute, School of Life Sciences and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, P.R. China
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12
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Lei XH, Yang YQ, Ma CY, Duan EK. Induction of differentiation of human stem cells ex vivo: Toward large-scale platelet production. World J Stem Cells 2019; 11:666-676. [PMID: 31616542 PMCID: PMC6789181 DOI: 10.4252/wjsc.v11.i9.666] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/14/2019] [Revised: 05/12/2019] [Accepted: 08/27/2019] [Indexed: 02/06/2023] Open
Abstract
Platelet transfusion is one of the most reliable strategies to cure patients suffering from thrombocytopenia or platelet dysfunction. With the increasing demand for transfusion, however, there is an undersupply of donors to provide the platelet source. Thus, scientists have sought to design methods for deriving clinical-scale platelets ex vivo. Although there has been considerable success ex vivo in the generation of transformative platelets produced by human stem cells (SCs), the platelet yields achieved using these strategies have not been adequate for clinical application. In this review, we provide an overview of the developmental process of megakaryocytes and the production of platelets in vivo and ex vivo, recapitulate the key advances in the production of SC-derived platelets using several SC sources, and discuss some strategies that apply three-dimensional bioreactor devices and biochemical factors synergistically to improve the generation of large-scale platelets for use in future biomedical and clinical settings.
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Affiliation(s)
- Xiao-Hua Lei
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Yi-Qing Yang
- Faculty of Laboratory Medical Science, Hebei North University, Zhangjiakou 075000, Hebei Province, China
| | - Chi-Yuan Ma
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - En-Kui Duan
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
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13
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Lei XH, Yang YQ, Ma CY, Duan EK. Induction of differentiation of human stem cellsex vivo: Toward large-scale platelet production. World J Stem Cells 2019. [DOI: dx.doi.org/10.4252/wjsc.v11.i9.666] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
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14
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Asnafi AA, Mohammadi MB, Rezaeeyan H, Davari N, Saki N. Prognostic significance of mutated genes in megakaryocytic disorders. Oncol Rev 2019; 13:408. [PMID: 31410247 PMCID: PMC6661530 DOI: 10.4081/oncol.2019.408] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2018] [Accepted: 06/28/2019] [Indexed: 01/19/2023] Open
Abstract
Megakaryopoiesis is a process during which platelets that play a major role in hemostasis are produced due to differentiation and maturation of megakaryocytic precursors. Several genes, including oncogenes and tumor suppressor genes, play a role in the regulation of this process. This study was conducted to investigate the oncogenes and tumor suppressor genes as well as their mutations during the megakaryopoiesis process, which can lead to megakaryocytic disorders. Relevant literature was identified by a PubMed search (1998-2019) of English language papers using the terms ‘Megakaryopoiesis’, ‘Mutation’, ‘oncogenes’, and ‘Tumor Suppressor’. According to investigations, several mutations occur in the genes implicated in megakaryopoiesis, which abnormally induce or inhibit megakaryocyte production, differentiation, and maturation, leading to platelet disorders. GATA-1 is one of the important genes in megakaryopoiesis and its mutations can be considered among the factors involved in the incidence of these disorders. Considering the essential role of these genes (such as GATA- 1) in megakaryopoiesis and the involvement of their mutations in platelet disorders, study and examination of these changes can be a positive step in the diagnosis and prognosis of these diseases.
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Affiliation(s)
- Ali Amin Asnafi
- Thalassemia and Hemoglobinopathy Research Center, Research Institute of Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Mohammad Bagher Mohammadi
- Thalassemia and Hemoglobinopathy Research Center, Research Institute of Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Hadi Rezaeeyan
- Thalassemia and Hemoglobinopathy Research Center, Research Institute of Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Nader Davari
- Thalassemia and Hemoglobinopathy Research Center, Research Institute of Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Najmaldin Saki
- Thalassemia and Hemoglobinopathy Research Center, Research Institute of Health, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
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15
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Megakaryocytes in Bone Metastasis: Protection or Progression? Cells 2019; 8:cells8020134. [PMID: 30744029 PMCID: PMC6406759 DOI: 10.3390/cells8020134] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2018] [Revised: 02/04/2019] [Accepted: 02/05/2019] [Indexed: 01/07/2023] Open
Abstract
Bone is the primary site where some cancers develop secondary growth, particularly those derived from breast and prostate tissue. The spread of metastasis to distant sites relies on complex mechanisms by which only cells endowed with certain characteristics are able to reach secondary growth sites. Platelets play a pivotal role in tumour growth, by conferring resistance to shear stress to the circulating tumour cells and protection against natural killer cell attack. Mature polyploid megakaryocytes (MKs) reside in close proximity to the vascular sinusoids of bone marrow, where their primary function is to produce platelets. Emerging evidence has demonstrated that MKs are essential for skeletal homeostasis, due to the expression and production of the bone-related proteins osteocalcin, osteonectin, bone morphogenetic protein, osteopontin, bone sialoprotein, and osteoprotegerin. Debate surrounds the role that MKs play in the development of bone metastasis, which is the topic of this mini-review.
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16
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Luff SA, Kao CY, Papoutsakis ET. Role of p53 and transcription-independent p53-induced apoptosis in shear-stimulated megakaryocytic maturation, particle generation, and platelet biogenesis. PLoS One 2018; 13:e0203991. [PMID: 30231080 PMCID: PMC6145578 DOI: 10.1371/journal.pone.0203991] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/21/2018] [Accepted: 09/02/2018] [Indexed: 12/18/2022] Open
Abstract
Megakaryocytes (Mks) derive from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and develop into large, polyploid cells that eventually give rise to platelets. As Mks mature, they migrate from the bone marrow niche into the vasculature, where they are exposed to shear forces from blood flow, releasing Mk particles (platelet-like particles (PLPs), pro/preplatelets (PPTs), and Mk microparticles (MkMPs)) into circulation. We have previously shown that transcription factor p53 is important in Mk maturation, and that physiological levels of shear promote Mk particle generation and platelet biogenesis. Here we examine the role of p53 in the Mk shear-stress response. We show that p53 is acetylated in response to shear in both immature and mature Mks, and that decreased expression of deacetylase HDAC1, and increased expression of the acetyltransferases p300 and PCAF might be responsible for these changes. We also examined the hypothesis that p53 might be involved in the shear-induced Caspase 3 activation, phosphatidylserine (PS) externalization, and increased biogenesis of PLPs, PPTs, and MkMPs. We show that p53 is involved in all these shear-induced processes. We show that in response to shear, acetyl-p53 binds Bax, cytochrome c is released from mitochondria, and Caspase 9 is activated. We also show that shear-stimulated Caspase 9 activation and Mk particle biogenesis depend on transcription-independent p53-induced apoptosis (TIPA), but PS externalization is not. This is the first report to show that shear flow stimulates TIPA and that Caspase 9 activation and Mk-particle biogenesis are directly modulated by TIPA.
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Affiliation(s)
- Stephanie A. Luff
- Department of Biological Sciences, University of Delaware, Newark, Delaware, United States of America
- Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, United States of America
| | - Chen-Yuan Kao
- Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, United States of America
- Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, United States of America
| | - Eleftherios T. Papoutsakis
- Department of Biological Sciences, University of Delaware, Newark, Delaware, United States of America
- Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, United States of America
- Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, Delaware, United States of America
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17
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Ding S, Wang M, Fang S, Xu H, Fan H, Tian Y, Zhai Y, Lu S, Qi X, Wei F, Sun G, Sun X. D-dencichine Regulates Thrombopoiesis by Promoting Megakaryocyte Adhesion, Migration and Proplatelet Formation. Front Pharmacol 2018; 9:297. [PMID: 29666579 PMCID: PMC5891617 DOI: 10.3389/fphar.2018.00297] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2017] [Accepted: 03/15/2018] [Indexed: 01/09/2023] Open
Abstract
Life-threatening chemotherapy-induced thrombocytopenia can increase the risk of bleeding due to a dramatic low platelet count, which may limit or delay treatment schedules in cancer patients. The pressing need for the rapid alleviation of the symptoms of thrombocytopenia has prompted us to search for novel highly effective and safe thrombopoietic agents. Pharmacological investigations have indicated that dencichine can prevent and treat blood loss and increase the number of platelets. On the basis of the neurotoxicity of dencichine, D-dencichine is artificially synthesized in the laboratory. Our initial results showed that D-dencichine had potential to elevate peripheral platelet levels in mice with carboplatin-induced thrombocytopenia. However, the mechanisms of D-dencichine on thrombopoiesis have been poorly understood. In this study, we found that sequential administration of D-dencichine had a distinct ability to elevate numbers of reticulated platelets, and did not alter their clearance. Moreover, we demonstrated that D-dencichine was able to modulate the return of hematopoietic factors to normal levels, including thrombopoietin and IL-6. However, subsequent analysis revealed that D-dencichine treatment had no direct effects on megakaryocytes proliferation, differentiation, and polyploidization. Further in vitro studies, we demonstrated for the first time that D-dencichine significantly stimulated megakaryocyte adhesion, migration, and proplatelet formation in a dose-dependent manner through extracellular regulated protein kinases1/2 (ERK1/2) and v-akt murine thymoma viral oncogene homolog (AKT) signaling pathways. This study sufficiently characterized the role of the effects of D-dencichine treatment on the regulation of thrombopoiesis and provided a promising avenue for CIT treating.
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Affiliation(s)
- Shilan Ding
- Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China
- Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing, China
- Zhongguancun Open Laboratory of the Research and Development of Natural Medicine and Health Products, Beijing, China
- Key Laboratory of Efficacy Evaluation of Chinese Medicine Against Glycolipid Metabolic Disorders, State Administration of Traditional Chinese Medicine, Beijing, China
| | - Min Wang
- Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China
- Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing, China
- Zhongguancun Open Laboratory of the Research and Development of Natural Medicine and Health Products, Beijing, China
- Key Laboratory of Efficacy Evaluation of Chinese Medicine Against Glycolipid Metabolic Disorders, State Administration of Traditional Chinese Medicine, Beijing, China
| | - Song Fang
- Kunming Shenghuo Pharmaceutical Group Co., Ltd., Kunming, China
| | - Huibo Xu
- Academy of Chinese Medical Sciences of Jilin Province, Jilin, China
| | - Huiting Fan
- Department of Oncology, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China
| | - Yu Tian
- Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China
- Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing, China
- Zhongguancun Open Laboratory of the Research and Development of Natural Medicine and Health Products, Beijing, China
- Key Laboratory of Efficacy Evaluation of Chinese Medicine Against Glycolipid Metabolic Disorders, State Administration of Traditional Chinese Medicine, Beijing, China
| | - Yadong Zhai
- Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China
- Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing, China
- Zhongguancun Open Laboratory of the Research and Development of Natural Medicine and Health Products, Beijing, China
- Key Laboratory of Efficacy Evaluation of Chinese Medicine Against Glycolipid Metabolic Disorders, State Administration of Traditional Chinese Medicine, Beijing, China
| | - Shan Lu
- Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China
- Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing, China
- Zhongguancun Open Laboratory of the Research and Development of Natural Medicine and Health Products, Beijing, China
- Key Laboratory of Efficacy Evaluation of Chinese Medicine Against Glycolipid Metabolic Disorders, State Administration of Traditional Chinese Medicine, Beijing, China
| | - Xin Qi
- Department of Oncology, Guang’anmen Hospital, China Academy of Chinese Medical Sciences, Beijing, China
| | - Fei Wei
- Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China
- Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing, China
- Zhongguancun Open Laboratory of the Research and Development of Natural Medicine and Health Products, Beijing, China
- Key Laboratory of Efficacy Evaluation of Chinese Medicine Against Glycolipid Metabolic Disorders, State Administration of Traditional Chinese Medicine, Beijing, China
| | - Guibo Sun
- Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China
- Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing, China
- Zhongguancun Open Laboratory of the Research and Development of Natural Medicine and Health Products, Beijing, China
- Key Laboratory of Efficacy Evaluation of Chinese Medicine Against Glycolipid Metabolic Disorders, State Administration of Traditional Chinese Medicine, Beijing, China
| | - Xiaobo Sun
- Beijing Key Laboratory of Innovative Drug Discovery of Traditional Chinese Medicine (Natural Medicine) and Translational Medicine, Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China
- Key Laboratory of Bioactive Substances and Resource Utilization of Chinese Herbal Medicine, Ministry of Education, Beijing, China
- Zhongguancun Open Laboratory of the Research and Development of Natural Medicine and Health Products, Beijing, China
- Key Laboratory of Efficacy Evaluation of Chinese Medicine Against Glycolipid Metabolic Disorders, State Administration of Traditional Chinese Medicine, Beijing, China
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18
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Yang J, Ma J, Xiong Y, Wang Y, Jin K, Xia W, Chen Q, Huang J, Zhang J, Jiang N, Jiang S, Ma D. Epigenetic regulation of megakaryocytic and erythroid differentiation by PHF2 histone demethylase. J Cell Physiol 2018; 233:6841-6852. [PMID: 29336484 DOI: 10.1002/jcp.26438] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2017] [Accepted: 01/05/2018] [Indexed: 12/16/2022]
Abstract
Plant homeodomain finger 2 (PHF2) is a JmjC family histone demethylase that demethylates H3K9me2, a repressive gene marker. PHF2 was found to play a role in the differentiation of several tissue types such as osteoblast and adipocyte differentiation. We report here that PHF2 plays a role in the epigenetic regulation of megakaryocytic (MK) and erythroid differentiation. We investigated PHF2 expression during MK and erythroid differentiation in K562 and human CD34+ progenitor (hCD34+ ) cells. Our data demonstrate that PHF2 expression is down-regulated during megakaryopoiesis and erythropoiesis. PHF2 has a negative role in MK and erythroid differentiation of K562 cells; knockdown of PHF2 promotes MK and erythroid differentiation of hCD34+ cells. Similarly, we found that p53 expression is also down-regulated during MK and erythroid differentiation, which parallels PHF2 expression. PHF2 binds to the p53 promoter and regulates the expression of p53 by demethylating H3K9me2 in the promoter region of p53. Taken together, our data show that PHF2 is a negative epigenetic regulator of MK and erythroid differentiation, and that one of the pathways through which PHF2 affects MK and erythroid differentiation is via regulation of p53 expression.
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Affiliation(s)
- Jichun Yang
- Institutes of Biomedical Sciences, Fudan University, Shanghai, China
| | - Jing Ma
- Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, Institute of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Yu Xiong
- Obstetrics and Gynecology Hospital, Fudan University, Shanghai, China
| | - Yanlin Wang
- International Peace Maternity & Child Health Hospital of China Welfare Institute, Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Kaiyue Jin
- Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, Institute of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Wenjun Xia
- Institutes of Biomedical Sciences, Fudan University, Shanghai, China
| | - Qing Chen
- Institutes of Biomedical Sciences, Fudan University, Shanghai, China
| | - Jianbo Huang
- Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, Institute of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Jin Zhang
- Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, Institute of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Nan Jiang
- Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, Institute of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai, China
| | - Shayi Jiang
- Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China
| | - Duan Ma
- Institutes of Biomedical Sciences, Fudan University, Shanghai, China.,Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, Institute of Biomedical Sciences, School of Basic Medical Sciences, Fudan University, Shanghai, China
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19
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Roy A, Lordier L, Pioche-Durieu C, Souquere S, Roy L, Rameau P, Lapierre V, Le Cam E, Plo I, Debili N, Raslova H, Vainchenker W. Uncoupling of the Hippo and Rho pathways allows megakaryocytes to escape the tetraploid checkpoint. Haematologica 2016; 101:1469-1478. [PMID: 27515249 DOI: 10.3324/haematol.2016.149914] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2016] [Accepted: 08/08/2016] [Indexed: 01/03/2023] Open
Abstract
Megakaryocytes are naturally polyploid cells that increase their ploidy by endomitosis. However, very little is known regarding the mechanism by which they escape the tetraploid checkpoint to become polyploid. Recently, it has been shown that the tetraploid checkpoint was regulated by the Hippo-p53 pathway in response to a downregulation of Rho activity. We therefore analyzed the role of Hippo-p53 pathway in the regulation of human megakaryocyte polyploidy. Our results revealed that Hippo-p53 signaling pathway proteins are present and are functional in megakaryocytes. Although this pathway responds to the genotoxic stress agent etoposide, it is not activated in tetraploid or polyploid megakaryocytes. Furthermore, Hippo pathway was observed to be uncoupled from Rho activity. Additionally, polyploid megakaryocytes showed increased expression of YAP target genes when compared to diploid and tetraploid megakaryocytes. Although p53 knockdown increased both modal ploidy and proplatelet formation in megakaryocytes, YAP knockdown caused no significant change in ploidy while moderately affecting proplatelet formation. Interestingly, YAP knockdown reduced the mitochondrial mass in polyploid megakaryocytes and decreased expression of PGC1α, an important mitochondrial biogenesis regulator. Thus, the Hippo pathway is functional in megakaryocytes, but is not induced by tetraploidy. Additionally, YAP regulates the mitochondrial mass in polyploid megakaryocytes.
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Affiliation(s)
- Anita Roy
- Institut National de la Santé et la Recherche Médicale (INSERM) UMR1170, Equipe Labellisée par la Ligue Nationale Contre le Cancer, Villejuif, France.,Université Paris-Saclay, Villejuif, France.,Gustave Roussy, Villejuif, France
| | - Larissa Lordier
- Institut National de la Santé et la Recherche Médicale (INSERM) UMR1170, Equipe Labellisée par la Ligue Nationale Contre le Cancer, Villejuif, France.,Université Paris-Saclay, Villejuif, France.,Gustave Roussy, Villejuif, France
| | - Catherine Pioche-Durieu
- Université Paris-Saclay, Villejuif, France.,Gustave Roussy, Villejuif, France.,Centre Nationale de la Recherche Scientifique (CNRS), UMR 8126, Gustave Roussy, Villejuif, France
| | - Sylvie Souquere
- Université Paris-Saclay, Villejuif, France.,Gustave Roussy, Villejuif, France.,CNRS UMR 8122, Gustave Roussy, Villejuif, France
| | - Lydia Roy
- Institut National de la Santé et la Recherche Médicale (INSERM) UMR1170, Equipe Labellisée par la Ligue Nationale Contre le Cancer, Villejuif, France.,Assistance Publique des Hôpitaux de Paris (AP-HP), Service d'Hématologie Clinique, Hôpital Henri Mondor, Créteil, France
| | | | | | - Eric Le Cam
- Université Paris-Saclay, Villejuif, France.,Gustave Roussy, Villejuif, France.,Centre Nationale de la Recherche Scientifique (CNRS), UMR 8126, Gustave Roussy, Villejuif, France
| | - Isabelle Plo
- Institut National de la Santé et la Recherche Médicale (INSERM) UMR1170, Equipe Labellisée par la Ligue Nationale Contre le Cancer, Villejuif, France.,Université Paris-Saclay, Villejuif, France.,Gustave Roussy, Villejuif, France
| | - Najet Debili
- Institut National de la Santé et la Recherche Médicale (INSERM) UMR1170, Equipe Labellisée par la Ligue Nationale Contre le Cancer, Villejuif, France.,Université Paris-Saclay, Villejuif, France.,Gustave Roussy, Villejuif, France
| | - Hana Raslova
- Institut National de la Santé et la Recherche Médicale (INSERM) UMR1170, Equipe Labellisée par la Ligue Nationale Contre le Cancer, Villejuif, France.,Université Paris-Saclay, Villejuif, France.,Gustave Roussy, Villejuif, France
| | - William Vainchenker
- Institut National de la Santé et la Recherche Médicale (INSERM) UMR1170, Equipe Labellisée par la Ligue Nationale Contre le Cancer, Villejuif, France .,Université Paris-Saclay, Villejuif, France.,Gustave Roussy, Villejuif, France
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20
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Malherbe JAJ, Fuller KA, Mirzai B, Kavanagh S, So CC, Ip HW, Guo BB, Forsyth C, Howman R, Erber WN. Dysregulation of the intrinsic apoptotic pathway mediates megakaryocytic hyperplasia in myeloproliferative neoplasms. J Clin Pathol 2016; 69:jclinpath-2016-203625. [PMID: 27060176 PMCID: PMC5136711 DOI: 10.1136/jclinpath-2016-203625] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2016] [Revised: 03/08/2016] [Accepted: 03/10/2016] [Indexed: 12/24/2022]
Abstract
AIMS Megakaryocyte expansion in myeloproliferative neoplasms (MPNs) is due to uncontrolled proliferation accompanied by dysregulation of proapoptotic and antiapoptotic mechanisms. Here we have investigated the intrinsic and extrinsic apoptotic pathways of megakaryocytes in human MPNs to further define the mechanisms involved. METHODS The megakaryocytic expression of proapoptotic caspase-8, caspase-9, Diablo, p53 and antiapoptotic survivin proteins was investigated in bone marrow specimens of the MPNs (n=145) and controls (n=15) using immunohistochemistry. The megakaryocyte percentage positivity was assessed by light microscopy and correlated with the MPN entity, JAK2V617F/CALR mutation status and platelet count. RESULTS The proportion of megakaryocytes in the MPNs expressing caspase-8, caspase-9, Diablo, survivin and p53 was significantly greater than controls. A greater proportion of myeloproliferative megakaryocytes expressed survivin relative to its reciprocal inhibitor, Diablo. Differences were seen between myelofibrosis, polycythaemia vera and essential thrombocythaemia for caspase-9 and p53. CALR-mutated cases had greater megakaryocyte p53 positivity compared to those with the JAK2V617F mutation. Proapoptotic caspase-9 expression showed a positive correlation with platelet count, which was most marked in myelofibrosis and CALR-mutated cases. CONCLUSIONS Disruptions targeting the intrinsic apoptotic cascade promote megakaryocyte hyperplasia and thrombocytosis in the MPNs. There is progressive dysfunction of apoptosis as evidenced by the marked reduction in proapoptotic caspase-9 and accumulation of p53 in myelofibrosis. The dysfunction of caspase-9, which is necessary for proplatelet formation, may be the mechanism for the excess thrombocytosis associated with CALR mutations. Survivin seems to be the key protein mediating the megakaryocyte survival signature in the MPNs and is a potential therapeutic target.
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Affiliation(s)
- Jacques A J Malherbe
- Schoolof Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia, Australia
| | - Kathryn A Fuller
- Schoolof Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia, Australia
- PathWest Laboratory Medicine, Nedlands, Western Australia, Australia
| | - Bob Mirzai
- Schoolof Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia, Australia
- PathWest Laboratory Medicine, Nedlands, Western Australia, Australia
| | - Simon Kavanagh
- Schoolof Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia, Australia
- PathWest Laboratory Medicine, Nedlands, Western Australia, Australia
| | - Chi-Chiu So
- Department of Pathology, Faculty of Medicine, University of Hong Kong, Hong Kong, Hong Kong
| | - Ho-Wan Ip
- Department of Pathology & Clinical Biochemistry, Queen Mary Hospital, Hong Kong, Hong Kong
| | - Belinda B Guo
- Schoolof Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia, Australia
| | - Cecily Forsyth
- Jarrett Street Specialist Centre, North Gosford, New South Wales, Australia
| | - Rebecca Howman
- PathWest Laboratory Medicine, Nedlands, Western Australia, Australia
| | - Wendy N Erber
- Schoolof Pathology and Laboratory Medicine, University of Western Australia, Crawley, Western Australia, Australia
- PathWest Laboratory Medicine, Nedlands, Western Australia, Australia
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21
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Duncan MT, Shin S, Wu JJ, Mays Z, Weng S, Bagheri N, Miller WM, Shea LD. Dynamic transcription factor activity profiles reveal key regulatory interactions during megakaryocytic and erythroid differentiation. Biotechnol Bioeng 2014; 111:2082-94. [PMID: 24853077 DOI: 10.1002/bit.25262] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2013] [Revised: 02/23/2014] [Accepted: 03/31/2014] [Indexed: 01/19/2023]
Abstract
The directed differentiation toward erythroid (E) or megakaryocytic (MK) lineages by the MK-E progenitor (MEP) could enhance the ex vivo generation of red blood cells and platelets for therapeutic transfusions. The lineage choice at the MEP bifurcation is controlled in large part by activity within the intracellular signal transduction network, the output of which determines the activity of transcription factors (TFs) and ultimately gene expression. Although many TFs have been implicated, E or MK differentiation is a complex process requiring multiple days, and the dynamics of TF activities during commitment and terminal maturation are relatively unexplored. Herein, we applied a living cell array for the large-scale, dynamic quantification of TF activities during MEP bifurcation. A panel of hematopoietic TFs (GATA-1, GATA-2, SCL/TAL1, FLI-1, NF-E2, PU.1, c-Myb) was characterized during E and MK differentiation of bipotent K562 cells. Dynamic TF activity profiles associated with differentiation towards each lineage were identified, and validated with previous reports. From these activity profiles, we show that GATA-1 is an important hub during early hemin- and PMA-induced differentiation, and reveal several characteristic TF interactions for E and MK differentiation that confirm regulatory mechanisms documented in the literature. Additionally, we highlight several novel TF interactions at various stages of E and MK differentiation. Furthermore, we investigated the mechanism by which nicotinamide (NIC) promoted terminal MK maturation using an MK-committed cell line, CHRF-288-11 (CHRF). Concomitant with its enhancement of ploidy, NIC strongly enhanced the activity of three TFs with known involvement in terminal MK maturation: FLI-1, NF-E2, and p53. Dynamic profiling of TF activity represents a novel tool to complement traditional assays focused on mRNA and protein expression levels to understand progenitor cell differentiation.
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Affiliation(s)
- Mark T Duncan
- Department of Chemical and Biological Engineering, Northwestern University, 2145 Sheridan Road, Evanston, Illinois, 60208
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22
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Shear enhances thrombopoiesis and formation of microparticles that induce megakaryocytic differentiation of stem cells. Blood 2014; 124:2094-103. [PMID: 24948658 DOI: 10.1182/blood-2014-01-547927] [Citation(s) in RCA: 63] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
In vivo visualization of thrombopoiesis suggests an important role for shear flow in platelet biogenesis. In vitro, shear stress was shown to accelerate proplatelet formation from mature megakaryocytes (Mks). Yet, the role of biomechanical forces on Mk biology and platelet biogenesis remains largely unexplored. In this study, we investigated the impact of shear stress on Mk maturation and formation of platelet-like particles (PLPs), pro/preplatelets (PPTs), and Mk microparticles (MkMPs), and furthermore, we explored a physiological role for MkMPs. We found that shear accelerated DNA synthesis of immature Mks in an exposure time- and shear stress level-dependent manner. Both phosphatidylserine exposure and caspase-3 activation were enhanced by shear stress. Exposure to physiological shear dramatically increased generation of PLPs/PPTs and MkMPs by up to 10.8 and 47-fold, respectively. Caspase-3 inhibition reduced shear-induced PLP/PPT and MkMP formation. PLPs generated under shear flow displayed improved functionality as assessed by CD62P exposure and fibrinogen binding. Significantly, coculture of MkMPs with hematopoietic stem and progenitor cells promoted hematopoietic stem and progenitor cell differentiation to mature Mks synthesizing α- and dense-granules, and forming PPTs without exogenous thrombopoietin, thus identifying a novel and unexplored potential physiological role for MkMPs.
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23
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Activation of p53 by the MDM2 inhibitor RG7112 impairs thrombopoiesis. Exp Hematol 2013; 42:137-45.e5. [PMID: 24309210 DOI: 10.1016/j.exphem.2013.11.012] [Citation(s) in RCA: 69] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2013] [Accepted: 11/10/2013] [Indexed: 12/18/2022]
Abstract
The tumor suppressor p53 is thought to play a role in megakaryocyte (MK) development. To assess the influence of the p53 regulatory pathway further, we studied the effect of RG7112, a small molecule MDM2 antagonist that activates p53 by preventing its interaction with MDM2, on normal megakaryocytopoiesis and platelet production. This drug has been previously been evaluated in clinical trials of cancer patients where thrombocytopenia was one of the major dose-limiting toxicities. In this study, we demonstrated that administration of RG7112 in vivo in rats and monkeys results in thrombocytopenia. In addition, we identified two distinct mechanisms by which RG7112-mediated activation of p53 affected human megakaryocytopoiesis and platelet production in vitro. RG7112 promoted apoptosis of MK progenitor cells, resulting in a reduction of their numbers and RG7112 affected mature MK by blocking DNA synthesis during endomitosis and impairing platelet production. Together, the disruption of these events provides an explanation for RG7112-induced thrombocytopenia and insight into the role of the p53-MDM2 auto-regulatory loop in normal megakaryocytopoiesis.
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24
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Chen PK, Chang HH, Lin GL, Wang TP, Lai YL, Lin TK, Hsieh MC, Kau JH, Huang HH, Hsu HL, Liao CY, Sun DS. Suppressive effects of anthrax lethal toxin on megakaryopoiesis. PLoS One 2013; 8:e59512. [PMID: 23555687 PMCID: PMC3605335 DOI: 10.1371/journal.pone.0059512] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2012] [Accepted: 02/15/2013] [Indexed: 01/14/2023] Open
Abstract
Anthrax lethal toxin (LT) is a major virulence factor of Bacillus anthracis. LT challenge suppresses platelet counts and platelet function in mice, however, the mechanism responsible for thrombocytopenia remains unclear. LT inhibits cellular mitogen-activated protein kinases (MAPKs), which are vital pathways responsible for cell survival, differentiation, and maturation. One of the MAPKs, the MEK1/2-extracellular signal-regulated kinase pathway, is particularly important in megakaryopoiesis. This study evaluates the hypothesis that LT may suppress the progenitor cells of platelets, thereby inducing thrombocytopenic responses. Using cord blood-derived CD34(+) cells and mouse bone marrow mononuclear cells to perform in vitro differentiation, this work shows that LT suppresses megakaryopoiesis by reducing the survival of megakaryocytes. Thrombopoietin treatments can reduce thrombocytopenia, megakaryocytic suppression, and the quick onset of lethality in LT-challenged mice. These results suggest that megakaryocytic suppression is one of the mechanisms by which LT induces thrombocytopenia. These findings may provide new insights for developing feasible approaches against anthrax.
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Affiliation(s)
- Po-Kong Chen
- Institute of Medical Science, Tzu-Chi University, Hualien, Taiwan
| | - Hsin-Hou Chang
- Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien, Taiwan
- Institute of Medical Science, Tzu-Chi University, Hualien, Taiwan
| | - Guan-Ling Lin
- Institute of Medical Science, Tzu-Chi University, Hualien, Taiwan
| | - Tsung-Pao Wang
- Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien, Taiwan
| | - Yi-Ling Lai
- Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien, Taiwan
| | - Ting-Kai Lin
- Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien, Taiwan
| | - Ming-Chun Hsieh
- Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien, Taiwan
| | - Jyh-Hwa Kau
- Department of Microbiology and Immunology, National Defense Medical Center, Taipei, Taiwan
| | - Hsin-Hsien Huang
- Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan
| | - Hui-Ling Hsu
- Institute of Preventive Medicine, National Defense Medical Center, Taipei, Taiwan
| | - Chi-Yuan Liao
- Department of Obstetrics and Gynecology, Mennonite Christian Hospital, Hualien, Taiwan
| | - Der-Shan Sun
- Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien, Taiwan
- Institute of Medical Science, Tzu-Chi University, Hualien, Taiwan
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25
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Apostolidis PA, Lindsey S, Miller WM, Papoutsakis ET. Proposed megakaryocytic regulon of p53: the genes engaged to control cell cycle and apoptosis during megakaryocytic differentiation. Physiol Genomics 2012; 44:638-50. [PMID: 22548738 DOI: 10.1152/physiolgenomics.00028.2012] [Citation(s) in RCA: 24] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
During endomitosis, megakaryocytes undergo several rounds of DNA synthesis without division leading to polyploidization. In primary megakaryocytes and in the megakaryocytic cell line CHRF, loss or knock-down of p53 enhances cell cycling and inhibits apoptosis, leading to increased polyploidization. To support the hypothesis that p53 suppresses megakaryocytic polyploidization, we show that stable expression of wild-type p53 in K562 cells (a p53-null cell line) attenuates the cells' ability to undergo polyploidization during megakaryocytic differentiation due to diminished DNA synthesis and greater apoptosis. This suggested that p53's effects during megakaryopoiesis are mediated through cell cycle- and apoptosis-related target genes, possibly by arresting DNA synthesis and promoting apoptosis. To identify candidate genes through which p53 mediates these effects, gene expression was compared between p53 knock-down (p53-KD) and control CHRF cells induced to undergo terminal megakaryocytic differentiation using microarray analysis. Among substantially downregulated p53 targets in p53-KD megakaryocytes were cell cycle regulators CDKN1A (p21) and PLK2, proapoptotic FAS, TNFRSF10B, CASP8, NOTCH1, TP53INP1, TP53I3, DRAM1, ZMAT3 and PHLDA3, DNA-damage-related RRM2B and SESN1, and actin component ACTA2, while antiapoptotic CKS1B, BCL2, GTSE1, and p53 family member TP63 were upregulated in p53-KD cells. Additionally, a number of cell cycle-related, proapoptotic, and cytoskeleton-related genes with known functions in megakaryocytes but not known to carry p53-responsive elements were differentially expressed between p53-KD and control CHRF cells. Our data support a model whereby p53 expression during megakaryopoiesis serves to control polyploidization and the transition from endomitosis to apoptosis by impeding cell cycling and promoting apoptosis. Furthermore, we identify a putative p53 regulon that is proposed to orchestrate these effects.
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Affiliation(s)
- Pani A Apostolidis
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, USA.
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26
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Calcium- and integrin-binding protein 1 regulates megakaryocyte ploidy, adhesion, and migration. Blood 2011; 119:838-46. [PMID: 22128142 DOI: 10.1182/blood-2011-04-346098] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Megakaryocytes are large, polyploid cells that produce platelets. We have previously reported that calcium- and integrin-binding protein 1 (CIB1) regulates endomitosis in Dami cells. To further characterize the role of CIB1 in megakaryopoiesis, we used a Cib1(-/-) mouse model. Cib1(-/-) mice have more platelets and BM megakaryocytes than wild-type (WT) controls (P < .05). Furthermore, subsequent analysis of megakaryocyte-CFU production revealed an increase with Cib1 deletion compared with WT (P < .05). In addition, BM from Cib1(-/-) mice, cultured with thrombopoietin (TPO) for 24 hours, produced more highly polyploid megakaryocytes than WT BM (P < .05). Subsequent analysis of TPO signaling revealed enhanced Akt and ERK1/2 phosphorylation, whereas FAK(Y925) phosphorylation was reduced in Cib1(-/-) megakaryocytes treated with TPO. Conversely, platelet recovery in Cib1(-/-) mice after platelet depletion was attenuated compared with WT (P < .05). This could be the result of impaired adhesion and migration, as adhesion to fibrinogen and fibronectin and migration toward an SDF-1α gradient were reduced in Cib1(-/-) megakaryocytes compared with WT (P < .05). In addition, Cib1(-/-) megakaryocytes formed fewer proplatelets compared with WT (P < .05), when plated on fibrinogen. These data suggest that CIB1 plays a dual role in megakaryopoiesis, initially by negatively regulating TPO signaling and later by augmenting proplatelet production.
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27
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Role of tumor suppressor p53 in megakaryopoiesis and platelet function. Exp Hematol 2011; 40:131-42.e4. [PMID: 22024107 DOI: 10.1016/j.exphem.2011.10.006] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2011] [Revised: 10/09/2011] [Accepted: 10/11/2011] [Indexed: 11/23/2022]
Abstract
The pathobiological role of p53 has been widely studied, however, its role in normophysiology is relatively unexplored. We previously showed that p53 knock-down increased ploidy in megakaryocytic cultures. This study aims to examine the effect of p53 loss on in vivo megakaryopoiesis, platelet production, and function, and to investigate the basis for greater ploidy in p53(-/-) megakaryocytic cultures. Here, we used flow cytometry to analyze ploidy, DNA synthesis, and apoptosis in murine cultured and bone marrow megakaryocytes following thrombopoietin administration and to analyze fibrinogen binding to platelets in vitro. Culture of p53(-/-) marrow cells for 6 days with thrombopoietin gave rise to 1.7-fold more megakaryocytes, 26.1% ± 3.6% of which reached ploidy classes ≥64 N compared to 8.2% ± 0.9% of p53(+/+) megakaryocytes. This was due to 30% greater DNA synthesis in p53(-/-) megakaryocytes and 31% greater apoptosis in p53(+/+) megakaryocytes by day 4 of culture. Although the bone marrow and spleen steady-state megakaryocytic content and ploidy were similar in p53(+/+) and p53(-/-) mice, thrombopoietin administration resulted in increased megakaryocytic polyploidization in p53(-/-) mice. Although their platelet counts were normal, p53(-/-) mice exhibited significantly longer bleeding times and p53(-/-) platelets were less sensitive than p53(+/+) platelets to agonist-induced fibrinogen binding and P-selectin secretion. In summary, our in vivo and ex vivo studies indicate that p53 loss leads to increased polyploidization during megakaryopoiesis. Our findings also suggest for the first time a direct link between p53 loss and the development of fully functional platelets resulting in hemostatic deficiencies.
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28
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Vatsyayan R, Singhal J, Nagaprashantha LD, Awasthi S, Singhal SS. Nutlin-3 enhances sorafenib efficacy in renal cell carcinoma. Mol Carcinog 2011; 52:39-48. [PMID: 22006587 DOI: 10.1002/mc.20875] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2011] [Revised: 09/20/2011] [Accepted: 09/21/2011] [Indexed: 12/19/2022]
Abstract
The renal cell carcinoma (RCC) is one of the top 10 cancers in USA. The renal tumors are highly angiogenic and are resistant to conventional interventions, particularly radiotherapy. The advent of multi-specific tyrosine kinase inhibitor sorafenib has improved the progression-free survival in RCC, but overall survival in recurrent and metastatic RCC is still a concern that has lead to characterization of combinatorial regimens. Hence, we studied the effect of combination of nutlin-3, an MDM2 inhibitor, which increases p53 levels, and sorafenib in RCC. Sorafenib along with nutlin-3 synergistically inhibited the cell survival and enhanced caspase-3 cleavage leading to apoptosis in RCC. Nutlin-3 and sorafenib were more effective in reducing the migration of RCC, in combination than as single agents. Sorafenib and nutlin-3 decreased the phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) and ERK along with inducing p53 activity. The sorafenib and nutlin-3 co-treatment lead to enhanced levels of p53, p-p53, and increase in the levels of p53 pro-apoptotic effector PUMA, Bax, and decrease in the anti-apoptotic Bcl-2 levels. Importantly, our studies revealed that sorafenib alone can activate p53 in a concentration dependent manner. Thus, co-treatment of nutlin-3 with sorafenib leads to increased half-life of p53, which in turn can be activated by sorafenib, to induce downstream pro-apoptotic and anti-proliferative effects. This is the first report showing the synergistic effect of sorafenib and nutlin-3 while providing a strong clinical-translational rationale for further testing of sorafenib and nutlin-3 combinatorial regimen in human RCC.
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Affiliation(s)
- Rit Vatsyayan
- Department of Molecular Biology and Immunology, University of North Texas Health Science Center, Fort Worth, Texas, USA
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29
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Lindsey S, Papoutsakis ET. The aryl hydrocarbon receptor (AHR) transcription factor regulates megakaryocytic polyploidization. Br J Haematol 2011; 152:469-84. [PMID: 21226706 PMCID: PMC3408620 DOI: 10.1111/j.1365-2141.2010.08548.x] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
We propose that the aryl hydrocarbon receptor (AHR) is a novel transcriptional regulator of megakaryopoietic polyploidization. Functional evidence was obtained that AHR impacts in vivo megakaryocytic differentiation and maturation; compared to wild-type mice, AHR-null mice had lower platelet counts, fewer numbers of newly synthesized platelets, increased bleeding times and lower-ploidy megakaryocytes (Mks). AHR mRNA increased 3·6-fold during ex vivo megakaryocytic differentiation, but reduced or remained constant during parallel isogenic granulocytic or erythroid differentiation. We interrogated the role of AHR in megakaryopoiesis using a validated Mk model of megakaryopoiesis, the human megakaryoblastic leukaemia CHRF cell line. Upon CHRF Mk differentiation, AHR mRNA and protein levels increased, AHR protein shifted from the cytoplasm to the nucleus and AHR binding to its consensus DNA binding sequence increased. Protein and mRNA levels of the AHR transcriptional target HES1 also increased. Mk differentiation of CHRF cells where AHR or HES1 was knocked-down using RNAi resulted in lower ploidy distributions and cells that were incapable of reaching ploidy classes ≥16n. AHR knockdown also resulted in increased DNA synthesis of lower ploidy cells, without impacting apoptosis. Together, these data support a role for AHR in Mk polyploidization and in vivo platelet function, and warrant further detailed investigations.
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Affiliation(s)
- Stephan Lindsey
- Department of Chemical Engineering and Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA.
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30
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Kostyak JC, Naik UP. Calcium- and integrin-binding protein 1 regulates endomitosis and its interaction with Polo-like kinase 3 is enhanced in endomitotic Dami cells. PLoS One 2011; 6:e14513. [PMID: 21264284 PMCID: PMC3021501 DOI: 10.1371/journal.pone.0014513] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2010] [Accepted: 12/15/2010] [Indexed: 12/24/2022] Open
Abstract
Endomitosis is a form of mitosis in which both karyokinesis and cytokinesis are interrupted and is a hallmark of megakaryocyte differentiation. Very little is known about how such a dramatic alteration of the cell cycle in a physiological setting is achieved. Thrombopoietin-induced signaling is essential for induction of endomitosis. Here we show that calcium- and integrin-binding protein 1 (CIB1), a known regulator of platelet integrin αIIbβ3 outside-in signaling, regulates endomitosis. We observed that CIB1 expression is increased in primary mouse megakaryocytes compared to mononuclear bone marrow cells as determined by Western blot analysis. Following PMA treatment of Dami cells, a megakaryoblastic cell line, we found that CIB1 protein expression increased concomitant with cell ploidy. Overexpression of CIB1 in Dami cells resulted in multilobated nuclei and led to increased time for a cell to complete cytokinesis as well as increased incidence of furrow regression as observed by time-lapse microscopy. Additionally, we found that surface expression of integrin αIIbβ3, an important megakaryocyte marker, was enhanced in CIB1 overexpressing cells as determined by flow cytometry. Furthermore, PMA treatment of CIB1 overexpressing cells led to increased ploidy compared to PMA treated control cells. Interestingly, expression of Polo-like kinase 3 (Plk3), an established CIB1-interacting protein and a key regulator of the mitotic process, decreased upon PMA treatment of Dami cells. Furthermore, PMA treatment augmented the interaction between CIB1 and Plk3, which depended on the duration of treatment. These data suggest that CIB1 is involved in regulating endomitosis, perhaps through its interaction with Plk3.
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Affiliation(s)
- John C Kostyak
- Department of Biological Sciences, University of Delaware, Newark, Delaware, United States of America
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31
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Zhao Y, Zhang Y, Wang S, Hua Z, Zhang J. The clock gene Per2 is required for normal platelet formation and function. Thromb Res 2010; 127:122-30. [PMID: 21186050 DOI: 10.1016/j.thromres.2010.11.025] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2010] [Revised: 11/15/2010] [Accepted: 11/26/2010] [Indexed: 01/01/2023]
Abstract
INTRODUCTION Apoptotic cell death is a highly regulated genetic program, which has been observed in mature megakaryocytes fragmenting into platelets. The clock gene Per2, a key component of core clock oscillator, was involved in affecting both cell cycle control and apoptosis. Thus, loss of Per2 function may be considered potential influence of platelet formation and function. METHODS Per2-null mice and C57BL/6 mice were used in the study. Bleeding time, platelet count, megakaryocyte count, megakaryocyte ploidy, megakaryocyte apoptosis, rate of proplatelet formation, clot retraction, platelet aggregation and secretion were performed to evaluate thrombopoiesis and hemostasis. Quantitative RT-PCR was employed to analyze genes expression in liver, bone marrow and enriched megakaryocytes. RESULTS The Per2-null mice had nearly 50% platelet counts in peripheral blood. Per2-null platelets were compromised in their ability to aggregate and secretion, consistent with a marked reduction in the number of dense and a-granules. Megakaryocytes from Per2-null mice showed no significant variation in number but increased in ploidy. Ultrastructural examination of Per2-null megakaryocytes revealed many vacuoles in demarcation membranes and reduction in platelet granules. Megakaryocytes from Per2-null bone marrow decreased the rate of proplatelet formation and impaired apoptosis. Per2-null mice showed increased both in Tpo in livers and its receptors C-mpl in bone marrow, and the megakaryocytes from these mice decreased P53 expression, consequently increased Bcl-xl and Bcl-2 level. CONCLUSIONS The clock gene Per2 modulating the apoptosis of megakaryocytes was required for platelet formation and function.
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Affiliation(s)
- Yue Zhao
- Center for Molecular Metabolism, Nanjing University of Science & Technology, Nanjing, China
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32
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Molchadsky A, Rivlin N, Brosh R, Rotter V, Sarig R. p53 is balancing development, differentiation and de-differentiation to assure cancer prevention. Carcinogenesis 2010; 31:1501-8. [DOI: 10.1093/carcin/bgq101] [Citation(s) in RCA: 116] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
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33
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Eidelman O, Jozwik C, Huang W, Srivastava M, Rothwell SW, Jacobowitz DM, Ji X, Zhang X, Guggino W, Wright J, Kiefer J, Olsen C, Adimi N, Mueller GP, Pollard HB. Gender dependence for a subset of the low-abundance signaling proteome in human platelets. HUMAN GENOMICS AND PROTEOMICS : HGP 2010; 2010:164906. [PMID: 20981232 PMCID: PMC2958630 DOI: 10.4061/2010/164906] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/03/2009] [Accepted: 01/05/2010] [Indexed: 11/23/2022]
Abstract
The incidence of cardiovascular diseases is ten-times higher in males than females, although the biological basis for this gender disparity is not known. However, based on the fact that antiplatelet drugs are the mainstay for prevention and therapy, we hypothesized that the signaling proteomes in platelets from normal male donors might be more activated than platelets from normal female donors. We report here that platelets from male donors express significantly higher levels of signaling cascade proteins than platelets from female donors. In silico connectivity analysis shows that the 24 major hubs in platelets from male donors focus on pathways associated with megakaryocytic expansion and platelet activation. By contrast, the 11 major hubs in platelets from female donors were found to be either negative or neutral for platelet-relevant processes. The difference may suggest a biological mechanism for gender discrimination in cardiovascular disease.
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Affiliation(s)
- Ofer Eidelman
- Department of Anatomy, Physiology and Genetics, USU Center for Medical Proteomics, Uniformed Services University, School of Medicine, USUHS, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
| | - Catherine Jozwik
- Department of Anatomy, Physiology and Genetics, USU Center for Medical Proteomics, Uniformed Services University, School of Medicine, USUHS, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
| | - Wei Huang
- Department of Anatomy, Physiology and Genetics, USU Center for Medical Proteomics, Uniformed Services University, School of Medicine, USUHS, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
| | - Meera Srivastava
- Department of Anatomy, Physiology and Genetics, USU Center for Medical Proteomics, Uniformed Services University, School of Medicine, USUHS, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
| | - Stephen W. Rothwell
- Department of Anatomy, Physiology and Genetics, USU Center for Medical Proteomics, Uniformed Services University, School of Medicine, USUHS, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
| | - David M. Jacobowitz
- National Institute for Mental Health, NIH, 9500 Rockville Pike, Bethesda, MD 20892, USA
| | - Xiaoduo Ji
- Department of Anatomy, Physiology and Genetics, USU Center for Medical Proteomics, Uniformed Services University, School of Medicine, USUHS, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
| | - Xiuying Zhang
- Department of Anatomy, Physiology and Genetics, USU Center for Medical Proteomics, Uniformed Services University, School of Medicine, USUHS, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
| | - William Guggino
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
| | - Jerry Wright
- Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
| | - Jeffrey Kiefer
- Translational Genomics Research Institute, Phoenix, AZ 85004, USA
| | - Cara Olsen
- Department of Preventive Medicine and Biometrics, Uniformed Services University School of Medicine, USUHS, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
| | - Nima Adimi
- Department of Anatomy, Physiology and Genetics, USU Center for Medical Proteomics, Uniformed Services University, School of Medicine, USUHS, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
| | - Gregory P. Mueller
- Department of Anatomy, Physiology and Genetics, USU Center for Medical Proteomics, Uniformed Services University, School of Medicine, USUHS, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
| | - Harvey B. Pollard
- Department of Anatomy, Physiology and Genetics, USU Center for Medical Proteomics, Uniformed Services University, School of Medicine, USUHS, 4301 Jones Bridge Road, Bethesda, MD 20814, USA
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Dou J, Li X, Cai Y, Chen H, Zhu S, Wang Q, Zou X, Mei Y, Yang Q, Li W, Han Y. Human cytomegalovirus induces caspase-dependent apoptosis of megakaryocytic CHRF-288-11 cells by activating the JNK pathway. Int J Hematol 2010; 91:620-9. [PMID: 20376580 DOI: 10.1007/s12185-010-0560-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2010] [Revised: 03/14/2010] [Accepted: 03/16/2010] [Indexed: 11/30/2022]
Abstract
Human cytomegalovirus (HCMV) infection is usually implicated in thrombocytopenia occurring in newborns and immunocompromised patients. However, the underlying mechanisms remain elusive. This study was conducted to investigate the effects of HCMV infection on the viability of megakaryocytic CHRF-288-11 cells and the underlying mechanisms involved. RT-PCR for determining mRNA expression of HCMV immediate early gene 1 and Western blot for measuring protein expression of late HCMV gene pp65 showed that CHRF-288-11 cells were susceptible to HCMV infection. HCMV infection reduced the viability of CHRF-288-11 cells via apoptosis in a dose- and time-dependent manner. Both caspase 3 and c-Jun terminal kinase (JNK) signaling pathway were activated in the HCMV-treated CHRF-288-11 cells. z-DEVD-fmk (a caspase inhibitor) and SP600125 (a JNK inhibitor) significantly prevented the death of CHRF-288-11 cells induced by HCMV, respectively. Furthermore, inhibition of JNK activity could reduce the formation of active caspase 3 induced by HCMV. Interestingly, the co-application of antivirus drug ganciclovir and SP600125 synergistically prevented the death of CHRF-288-11 cells induced by HCMV. Collectively, these findings suggest that HCMV infection may induce the caspase-dependent apoptosis of megakaryocytic CHRF-288-11 cells by the activation of JNK signaling pathway.
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Affiliation(s)
- Juan Dou
- Department of Pediatrics, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
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Giammona LM, Panuganti S, Kemper JM, Apostolidis PA, Lindsey S, Papoutsakis ET, Miller WM. Mechanistic studies on the effects of nicotinamide on megakaryocytic polyploidization and the roles of NAD+ levels and SIRT inhibition. Exp Hematol 2009; 37:1340-1352.e3. [PMID: 19715739 PMCID: PMC2763937 DOI: 10.1016/j.exphem.2009.08.004] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2009] [Revised: 08/20/2009] [Accepted: 08/20/2009] [Indexed: 12/24/2022]
Abstract
OBJECTIVE Megakaryocytic cells (Mks) undergo endomitosis and become polyploid. Mk ploidy correlates with platelet production. We previously showed that nicotinamide (NIC) greatly increases Mk ploidy in cultures of human mobilized peripheral blood CD34(+) cells. This study aims to examine the generality of NIC effects, NIC's impact on Mk ultrastructure, and potential mechanisms for the increased ploidy. MATERIALS AND METHODS We used electron microscopy to examine Mk ultrastructure and flow cytometry to evaluate NIC effects on Mk differentiation and ploidy in mobilized peripheral blood CD34(+) cell cultures under diverse megakaryopoietic conditions. Mk ploidy and NAD(H) content were evaluated for NIC and other NAD(+) precursors. We tested additional inhibitors of the sirtuin (or SIRT) 1 and SIRT2 histone/protein deacetylases and, after treatment with NIC, evaluated changes in the acetylation of SIRT1/2 targets. RESULTS NIC increased ploidy under diverse culture conditions and did not alter Mk ultrastructure; 6.25 mM NIC increased NAD(+) levels fivefold. Quinolinic acid increased NAD(+) similar to that for 1 mM NIC, but yielded a much smaller ploidy increase. Similar increases in Mk ploidy were obtained using NIC or the SIRT1/2 inhibitor cambinol, while the SIRT2 inhibitor AGK2 moderately increased ploidy. SIRT1/2 inhibition in cells treated with NIC was evidenced by increased acetylation of nucleosomes and p53. Greater p53 acetylation with NIC was associated with increased binding of p53 to its consensus DNA binding sequence. CONCLUSION NIC greatly increases Mk ploidy under a wide range of conditions without altering Mk morphology. Inhibition of SIRT1 and/or SIRT2 is primarily responsible for NIC effects on Mk maturation.
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Affiliation(s)
- Lisa M. Giammona
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, 60208
| | - Swapna Panuganti
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, 60208
| | - Jan M. Kemper
- Master of Biotechnology Program, Northwestern University, Evanston, Illinois, 60208
| | - Pani A. Apostolidis
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, 60208
- Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, 19711
| | - Stephan Lindsey
- Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, 19711
- Department of Chemical Engineering, University of Delaware, Newark, Delaware, 19711
| | - Eleftherios T. Papoutsakis
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, 60208
- Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, 19711
- Department of Chemical Engineering, University of Delaware, Newark, Delaware, 19711
| | - William M. Miller
- Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, 60208
- Master of Biotechnology Program, Northwestern University, Evanston, Illinois, 60208
- The Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, Illinois, 60611
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Bluteau D, Lordier L, Di Stefano A, Chang Y, Raslova H, Debili N, Vainchenker W. Regulation of megakaryocyte maturation and platelet formation. J Thromb Haemost 2009; 7 Suppl 1:227-34. [PMID: 19630806 DOI: 10.1111/j.1538-7836.2009.03398.x] [Citation(s) in RCA: 80] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Each day in every human, approximately 1 x 10(11) platelets are produced by the cytoplasmic fragmentation of megakaryocytes (MK), their marrow precursor cells. Platelets are the predominating factor in the process of hemostasis and thrombosis. Recent studies have shown that platelets also play a hitherto unsuspected role in several other processes such as inflammation, innate immunity, neoangiogenesis and tumor metastasis. The late phases of MK differentiation identified by polyploidization, maturation and organized fragmentation of the cytoplasm leading to the release of platelets in the blood stream represent a unique model of differentiation. The molecular and cellular mechanisms regulating platelet biogenesis are better understood and may explain several platelet disorders. This review focuses on MK polyploidization, and platelet formation, and discusses their alteration in some platelet disorders.
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Affiliation(s)
- D Bluteau
- INSERM, U790, 39 rue Camille Desmoulins, Villejuif, France
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Abstract
Polyploidy, an increased number of chromosome sets, is a surprisingly common phenomenon in nature, particularly in plants and fungi. In humans, polyploidy often occurs in specific tissues as part of terminal differentiation. Changes in ploidy can also result from pathophysiological events that are caused by viral-induced cell fusion or erroneous cell division. Tetraploidization can initiate chromosomal instability (CIN), probably owing to supernumerary centrosomes and the doubled chromosome mass. CIN, in turn, might persist or soon give way to a stably propagating but aneuploid karyotype. Both CIN and stable aneuploidy are commonly observed in cancers. Recently, it has been proposed that an increased number of chromosome sets can promote cell transformation and give rise to an aneuploid tumor. Here, we review how tetraploidy can occur and describe the cellular responses to increased ploidy. Furthermore, we discuss how the specific physiological changes that are triggered by polyploidization might be used as novel targets for cancer therapy.
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Affiliation(s)
- Zuzana Storchova
- Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
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Zhang H, Wang H, Zhang J, Qian G, Niu B, Fan X, Lu J, Hoffman AR, Hu JF, Ge S. Enhanced therapeutic efficacy by simultaneously targeting two genetic defects in tumors. Mol Ther 2009; 17:57-64. [PMID: 19018252 PMCID: PMC2834989 DOI: 10.1038/mt.2008.236] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2008] [Accepted: 09/25/2008] [Indexed: 01/11/2023] Open
Abstract
Targeting tumor-specific gene abnormalities has become an attractive approach in developing therapeutics to treat cancer. Overexpression of Bcl2 and mutations of p53 represent two of the most common molecular defects in tumors. In the nucleus, p53 induces cell cycle arrest, while it interacts with Bcl2 outside of the nucleus to regulate signal pathways involved in apoptosis. To potentiate antitumor activity, we tested a "double target" approach to antitumor therapy by combining H101, a recombinant oncolytic adenovirus that targets the inactive p53 in tumors, with a small interfering RNA (siBCL2) that targets Bcl2. In cell culture, the combined treatment significantly enhanced apoptosis and cytotoxicity as compared with treatment with either H101 or siBCL2 alone. In animals carrying tumor xenographs, combined H101 and siBCL2 treatment significantly inhibited tumor growth and prolonged survival. At the end of the study, all animals in the combined therapy group survived and two of the five animals showed complete eradication of their tumors. Interestingly, siBCL2 treatment increased H101 viral replication in both treated cells and tumor tissues. Simultaneously targeting two tumor-specific gene abnormalities using an oncolytic adenovirus and siRNA potentiates total antitumor activity.
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Affiliation(s)
- He Zhang
- Department of Biochemistry and Molecular Biology, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China
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