scholarly journals Using a new HSPC senescence model in vitro to explore the mechanism of cellular memory in aging HSPCs

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yongpin Dong ◽  
Chunni Guo ◽  
Wuxiong Zhou ◽  
Wenfang Li ◽  
Lina Zhang
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Distribution ◽  
Human Populations ◽  
Myeloproliferative Disease ◽  
Cellular Memory ◽  
Cell Memory ◽  
Hematopoietic Stem ◽  
Memory Mechanism ◽  
Stem And Progenitor Cells

Abstract Background Age-associated changes attenuate human blood system functionality through the aging of hematopoietic stem and progenitor cells (HSPCs), manifested in human populations an increase in myeloproliferative disease and even leukemia; therefore, study on HSPC senescence bears great significance to treat hematopoietic-associated disease. Furthermore, the mechanism of HSPC aging is lacking, especially the cellular memory mechanism. Here, we not only reported a new HSPC senescence model in vitro, but also propose and verify the cellular memory mechanism of HSPC aging of the Polycomb/Trithorax system. Methods HSPCs (Lin−c-kit+ cells) were isolated and purified by magnetic cell sorting (MACS). The proportions and cell cycle distribution of cells were determined by flow cytometry; senescence-related β-galactosidase assay, transmission electron microscope (TEM), and colony-forming unit (CFU)-mix assay were detected for identification of the old HSPC model. Proteomic tests and RNA-seq were applied to analyze differential pathways and genes in the model cells. qPCR, Western blot (WB), and chromatin immunoprecipitation PCR (CHIP-PCR) were used to detect the gene expression of cell memory-related proteins. Knockdown of cell memory-related key genes was performed with shRNA interference. Results In the model old HSPCs, β-gal activity, cell cycle, colony-forming ability, aging-related cell morphology, and metabolic pathway were significantly changed compared to the young HSPCs. Furthermore, we found the model HSPCs have more obvious aging manifestations than those of natural mice, and IL3 is the major factor contributing to HSPC aging in the model. We also observed dramatic changes in the expression level of PRC/TrxG complexes. After further exploring the downstream molecules of PRC/TrxG complexes, we found that Uhrf1 and TopII played critical roles in HSPC aging based on the HSPC senescence model. Conclusions These findings proposed a new HSPC senescence model in vitro which we forecasted could be used to preliminary screen the drugs of the HSPC aging-related hemopathy and suggested cellular memory mechanism of HSPC aging.

scholarly journals Using a new HSPC aging model in vitro to explore the mechanism of cellular memory in aging HSPCs

2021 ◽  
Author(s):  
Yongpin Dong ◽  
Wenfang Li ◽  
Wuxiong Zhou ◽  
Lina Zhang
Keyword(s):  
Progenitor Cells ◽  
Critical Role ◽  
Human Populations ◽  
Myeloproliferative Disease ◽  
Cellular Memory ◽  
Hematopoietic Stem ◽  
Aging Model ◽  
Memory Mechanism ◽  
Stem And Progenitor Cells

Abstract Age-associated changes attenuate human blood system functionality through the aging of hematopoietic stem and progenitor cells (HSPCs). Hematopoietic aging is manifested in human populations in the form of an increase in myeloproliferative disease,therefore, study on hematopoietic stem and progenitor cells (HSPCs) senescence bears great significance to treat hematopoietic associated disease. However, the mechanism of HSPC aging is lacking, especially cellular memory mechanism. Here, we not only reported a new HSPC aging model in vitro, but also propose and verify the cellular memory mechanism of HSPC aging of Polycomb/Trithorax system. In this model cells, the senescence-related β-gal activity, cell cycle, colony-forming ability, aging-related cell morphology and metabolic pathway are significantly changed compare to the young group. Furthermore, we found the model HSPCs have more obvious aging manifestation than those of natural mice and IL3 is the major factor contributing to HSPC aging in the model. We also observed dramatically changes in the expression level of PRC/TrxG complexes. We further identified downstream molecules of PRC/TrxG complexes,Uhrf1 and TopII, which were found to play a critical role in HSPC aging based on the HSPC aging model. So, these findings proposed a new aging HSPC model in vitro which we forecasted could be used to preliminary screen the drugs of the HSPC aging related hemopathy and suggested cellular memory mechanism of HSPC aging.

Enforced Expression of GATA-2 Confers Quiescence on Human Hematopoietic Stem and Progenitor Cells In Vitro and In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 83-83
Author(s):  
Alex J. Tipping ◽  
Cristina Pina ◽  
Anders Castor ◽  
Ann Atzberger ◽  
Dengli Hong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Zinc Finger ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Colony Number

Abstract Hematopoietic stem cells (HSCs) in adults are largely quiescent, periodically entering and exiting cell cycle to replenish the progenitor pool or to self-renew, without exhausting their number. Expression profiling of quiescent HSCs in our and other laboratories suggests that high expression of the zinc finger transcription factor GATA-2 correlates with quiescence. We show here that TGFβ1-induced quiescence of wild-type human cord blood CD34+ cells in vitro correlated with induction of endogenous GATA-2 expression. To directly test if GATA-2 has a causative role in HSC quiescence we constitutively expressed GATA-2 in human cord blood stem and progenitor cells using lentiviral vectors, and assessed the functional output from these cells. In both CD34+ and CD34+ CD38− populations, enforced GATA-2 expression conferred increased quiescence as assessed by Hoechst/Pyronin Y staining. CD34+ cells with enforced GATA-2 expression showed reductions in both colony number and size when assessed in multipotential CFC assays. In CFC assays conducted with more primitive CD34+ CD38− cells, colony number and size were also reduced, with myeloid and mixed colony number more reduced than erythroid colonies. Reduced CFC activity was not due to increased apoptosis, as judged by Annexin V staining of GATA-2-transduced CD34+ or CD34+ CD38− cells. To the contrary, in vitro cultures from GATA-2-transduced CD34+ CD38− cells showed increased protection from apoptosis. In vitro, proliferation of CD34+ CD38− cells was severely impaired by constitutive expression of GATA-2. Real-time PCR analysis showed no upregulation of classic cell cycle inhibitors such as p21, p57 or p16INK4A. However GATA-2 expression did cause repression of cyclin D3, EGR2, E2F4, ANGPT1 and C/EBPα. In stem cell assays, CD34+ CD38− cells constitutively expressing GATA-2 showed little or no LTC-IC activity. In xenografted NOD/SCID mice, transduced CD34+ CD38−cells expressing high levels of GATA-2 did not contribute to hematopoiesis, although cells expressing lower levels of GATA-2 did. This threshold effect is presumably due to DNA binding by GATA-2, as a zinc-finger deletion variant of GATA-2 shows contribution to hematopoiesis from cells irrespective of expression level. These NOD/SCID data suggest that levels of GATA-2 may play a part in the in vivo control of stem and progenitor cell proliferation. Taken together, our data demonstrate that GATA-2 enforces a transcriptional program on stem and progenitor cells which suppresses their responses to proliferative stimuli with the result that they remain quiescent in vitro and in vivo.

TIMP-1 Deficiency Subverts Cell Cycle Dynamics in Long-Term HSCs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2514-2514
Author(s):  
Lara Rossi ◽  
Margaret Goodell
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Conflicts Of Interest ◽  
Biological Behavior ◽  
Cell Cycle Distribution ◽  
Matrix Remodeling ◽  
Hematopoietic Stem ◽  
Stem Cell Quiescence

Abstract Abstract 2514 Poster Board II-491 In addition to the consolidated role in extracellular matrix remodeling, the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) has been suggested to be involved in the regulation of numerous biological functions, including cell proliferation and survival. We therefore hypothesized that TIMP-1 might be involved in the homeostatic regulation of hematopoietic stem cells (HSCs), whose biological behavior is the synthesis of both microenvironmental and intrinsic cues. We found that TIMP-1−/− mice have decreased HSC numbers and, consistent with this finding, TIMP-1−/− HSCs display reduced capability of long-term repopulation. Interestingly, the cell cycle distribution of TIMP-1−/− LT-HSCs is profoundly distorted, with a consistent proportion of the stem cell pool arrested in the G1 phase, suggesting that TIMP-1 is intrinsically involved in the regulation of the HSC proliferation dynamics. Indeed, HSCs exhibit a higher proliferation rate, leading to an increased formation of CFU-C in vitro and spleen colonies (CFU-S) after transplant. Of note, TIMP-1−/− HSCs present decreased levels of CD44 glycoprotein, whose expression has been proven to be controlled by p53, the master regulator of the G1/S transition. Our findings establish TIMP-1 role in HSC function, suggesting a novel mechanism presiding over stem cell quiescence and potentially involved in the development of hematological diseases. Disclosures: No relevant conflicts of interest to declare.

Self-Renewal and Differentiation in Hematopoietic Stem and Progenitor Cells Is Controlled By the APC/C Coactivator Cdh1

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2370-2370
Author(s):  
Daniel Ewerth ◽  
Stefanie Kreutmair ◽  
Birgit Kügelgen ◽  
Dagmar Wider ◽  
Julia Felthaus ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Research Funding ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Abstract Introduction: Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously renew the hematopoietic system by differentiation into mature blood cells. The process of differentiation is predominantly initiated in G1 phase of the cell cycle when stem cells leave their quiescent state. During G1 the anaphase-promoting complex or cyclosome (APC/C) associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate proliferation. In addition, Cdh1 has been shown to control terminal differentiation in neurons, muscle cells or osteoblasts. Here we show that Cdh1 is also a critical regulator of human HSPC differentiation and self-renewal. Methods: Human CD34+ cells were collected from peripheral blood (PB) of G-CSF mobilized donors and cultured in the presence of different cytokine combinations. To analyze cell division and self-renewal versus differentiation, CFSE staining was used in combination with flow cytometric detection of CD34 expression. The knockdown and overexpression of Cdh1 was achieved by lentiviral delivery of suitable vectors into target cells. After cell sorting transduced (GFP+) CD34+ cells were used for in vitro differentiation in liquid culture or CFU assay. For in vivo experiments purified cells were transplanted into NSG mice. Results: G-CSF mobilized CD34+ cells showed effective differentiation into granulocytes (SCF, G-CSF), erythrocytes (SCF, EPO) or extended self-renewal (SCF, TPO, Flt3-L) when stimulated in vitro. The differentiation was characterized by a fast downregulation of Cdh1 on protein level, while Cdh1 remained expressed under self-renewal conditions. A detailed analysis of different subsets, both in vitro and in vivo, showed high Cdh1 level in CD34+ cells and low expression in myeloid cells. Analysis of proliferation revealed lowest division rates during self-renewal, accompanied by higher frequency of CD34+ cells. The fastest proliferation was found after induction of erythropoiesis. These experiments also showed a more rapid decrease of HSPCs' colony-forming ability and of CD34+ cells during granulopoiesis after 2-3 cell divisions in contrast to a moderate decline under self-renewal conditions. The depletion of Cdh1 (Cdh1-kd) had no effect on total cell numbers or proliferation detected by CFSE during differentiation and self-renewal, but showed an increase in S phase cells. These results were confirmed at the single cell level by measuring the cell cycle length of individual cells. Independent of cell cycle regulation, Cdh1-kd cells showed a significant maintenance of CD34+ cells under self-renewal conditions and during erythropoiesis with lower frequency of Glycophorin A+ cells. In CFU assays, the Cdh1-kd resulted in less primary colony formation, notably CFU-GM and BFU-E, but significantly more secondary colonies compared to control cells. These results suggest that the majority of cells reside in a more undifferentiated state due to Cdh1-kd. The overexpression of Cdh1 showed reversed results with less S phase cells and tendency to increased differentiation in liquid culture and CFU assays. To further validate our results in vivo, we have established a NSG xenotransplant mouse model. Human CD34+ cells depleted of Cdh1 engrafted to a much higher degree in the murine BM 8 and 12 weeks after injection as shown by higher frequencies of human CD45+ cells. Moreover, we also found an increased frequency of human CD19+ B cells after transplantation of CD34+ Cdh1-kd cells. These results suggest an enhanced in vivo repopulation capacity of human CD34+ HSCs in NSG mice when Cdh1 is depleted. Preliminary data in murine hematopoiesis support our hypothesis showing enhanced PB chimerism upon Cdh1-kd. Looking for a mediator of these effects, we found the Cdh1 target protein TRRAP, a cofactor of many HAT complexes, increased upon Cdh1-kd under self-renewal conditions. We use currently RT-qPCR to determine, if this is caused by a transcriptional or post-translational mechanism. Conclusions: Loss of the APC/C coactivator Cdh1 supports self-renewal of CD34+ cells, represses erythropoiesis in vitro and facilitates engraftment capacity and B cell development of human HSPCs in vivo. This work was supported by Josè Carreras Leukemia Foundation grant DCJLS R10/14 (to ME+RW) Disclosures Ewerth: Josè Carreras Leukemia Foundation: Research Funding. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

scholarly journals Cytokine Rescue and Targeting of Inflammation-Sensitive RUNX1 Deficient Human CD34+ Hematopoietic Stem and Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 14-15
Author(s):  
Amy Fan ◽  
Armon Azizi ◽  
Kevin Nuno ◽  
Yusuke Nakauchi ◽  
Feifei Zhao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Publicly Traded ◽  
Hematopoietic Stem ◽  
The Past ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Stem And Progenitor Cells

Introduction: Loss-of-function mutations in Runt-related transcription factor 1 (RUNX1) are commonly found in both germline and somatic hematopoietic malignancies and confer particularly poor prognosis in AML. However, it remains unclear how RUNX1 functions during hematopoietic and leukemic development, particularly because RUNX1 mutations alone are not sufficient to cause myeloid malignancy and some models show that RUNX1 mutations confer hematopoietic stem cell defects. Recently, mouse models have shown that RUNX1-deficient neutrophils upregulate NFκB activity, and hematopoietic stem and progenitor cells (HSPCs) with overactive inflammatory pathways gain competitive advantage under chronic inflammation. Thus, we hypothesized that while RUNX1 mutations impair normal HSPC function, inflammation may select for or rescue RUNX1 mutant HSPCs. Methods: To interrogate the effect of RUNX1 loss in human CD34+ HSPCs, we disrupted the RUNX1 locus using CRISPR/Cas9 and AAV6-mediated homology directed repair. Importantly, by using an AAV6 vector that carries arms of homology flanking a fluorescent reporter expression cassette, we are able to track and isolate cells edited at the RUNX1 locus for in vitro and in vivo functional analyses and for molecular characterization using RNA-seq and ATAC-seq. Results: First, we used this system to evaluate the functional consequences of RUNX1 knockout (KO) in human CD34+ HSPCs. Loss of RUNX1 caused early erythroid-megakaryocytic differentiation arrest and skewing toward monocytic differentiation. RUNX1 KO cells demonstrated decreased proliferation, cell cycle arrest, and reduction in serial replating potential in vitro. In competitive transplantation experiments in NSG mice, RUNX1 KO engraftment decreased over time in both primary and secondary transplant, revealing a competitive disadvantage. Second, ATAC-seq peak motif analysis showed that PU.1 and NFκB motifs are more accessible upon RUNX1 KO whereas GATA, TAL1, and RUNX motifs were less accessible. Similarly, gene set enrichment analysis of transcriptional data confirmed the broad upregulation of NFκB-mediated inflammatory programs; downregulation of GATA1-dependent heme metabolism and platelet development pathways; and downregulation of MYC- and E2F-dependent cell cycle programs. These observations imply that RUNX1 directs cell fate decisions by recruiting and activating lineage-specific hematopoietic transcription factors and augmenting stem cell proliferation programs. We next sought to determine which cytokines are sufficient to drive RUNX1 KO cell expansion. RUNX1 KO cells not only expanded preferentially in NSG mice expressing human SCF, GM-CSF, and IL-3 (NSGS mice), but also were no longer defective in competitive transplants in these mice. Further, treatment with IL-3 was sufficient to significantly expand RUNX1 KO cells in vitro. Flow cytometry revealed that the IL-3 receptor CD123 is upregulated in RUNX1 KO cells compared to control. Similarly, RUNX1-mutant AML patient samples express higher levels of CD123 than RUNX1-wildtype AML patient samples. Finally, evaluation of publicly available RUNX1 ChIP-seq of bone marrow CD34+ HSPCs revealed that RUNX1 directly binds the promoter of CD123. Ongoing efforts are aimed at determining whether targeting CD123 and IL-3 signaling may be a viable therapeutic approach for the prevention or treatment of RUNX1-mutant myeloid malignancies. Conclusion: In summary, we established a RUNX1-deficient human HSPC model not only to evaluate the role of RUNX1 in hematopoiesis, but also to characterize intrinsic and extrinsic factors involved in RUNX1-deficient clonal expansion and leukemic transformation. We show that RUNX1 KO causes monocytic skew at the expense of erythro-megakaryocytic potential and severely limits HSC engraftment and expansion in vivo. Molecular profiling reveals that these effects are associated with dysregulation of both transcription factor activity and cytokine signaling. However, exposure to IL-3 rescues RUNX1-deficient cell proliferative defects in vitro and competitive engraftment defects in vivo. This hypersensitivity to IL-3 signaling is mediated in part by increased expression of the IL-3 receptor CD123. These findings reveal how RUNX1 mutations may initially behave in a deleterious manner but can ultimately confer an advantage to HSPCs under certain environmental conditions. Disclosures Majeti: CD47 Inc.: Divested equity in a private or publicly-traded company in the past 24 months; Gilead Sciences: Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties; Kodikaz Therapeutic Solutions Inc: Membership on an entity's Board of Directors or advisory committees.

mTOR Regulates DNA Damage Response of Hematopoietic Stem and Progenitor Cells Through Modulation of Fanconi Anemia Core Complex

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 864-864 ◽  
Author(s):  
Fukun Guo ◽  
Jie Li ◽  
Wei Du ◽  
Shuangmin Zhang ◽  
Wei Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Dna Damage ◽  
Dna Damage Response ◽  
Fanconi Anemia ◽  
Hematopoietic Stem ◽  
Damage Response ◽  
Stem And Progenitor Cells

Abstract Abstract 864 The mammalian target of rapamycin (mTOR) integrates signals from nutrients, growth factors, and cellular energy status to control protein synthesis, cell growth, proliferation, survival and metabolism in various cancer cells, but its physiological function in the hematopoiesis process and signaling role in hematopoietic stem cell (HSC) regulation remain unknown. By using the inhibitor rapamycin, mTOR has previously been suggested to regulate megakaryocyte and dendritic cell proliferation and differentiation. Hyperactivation of mTOR by deletion of the negative regulators of mTOR, TSC1/TSC2 or PTEN, causes a loss of quiescence and long-term exhaustion of HSCs. Since conventional gene targeting of mTOR leads to early embryonic lethality, a conditional mTOR knockout mouse model has recently been generated. We have produced mTORflox/flox; Mx-Cre compound mice that allow interferon-induced mTOR deletion in bone marrow (BM) following a transplantation and polyI:C induction protocol. We found that depletion of mTOR drastically affected hematopoiesis: the mTORflox/flox;Mx-Cre BM recipient mice showed a marked reduction in total BM cellularity and in the numbers and frequency of myeloid and lymphoid cells, erythrocytes, and platelets in peripheral blood, bone marrow, and thymus, after induced mTOR deletion, resulting in bone marrow failure and lethality. Interestingly, the numbers of hematopoietic stem and progenitor cells (HSPCs; Lin−Sca-1+c-Kit+) and HSCs (CD150+ CD41−CD48− Lin−Sca-1+c-Kit+) in bone marrow increased transiently by approximately 5- and 8-fold, respectively, while the numbers of early progenitors (CMP, GMP, MEP, CLP) were mildly affected in the mutant mice 7–14 days after polyI:C treatment. While the more mature lineage committed mTOR−/− blood cells showed a cell cycle blockage and significantly increased apoptosis, mTOR−/− HSPCs and HSCs displayed a loss of quiescence and increased proliferation, as assessed by 5-bromodeoxyuridine incorporation assays, and a normal survival index. Transplantation of mTOR−/− BM cells into immunodeficient or syngeneic mice demonstrated that the mTOR−/− HSPCs failed to engraft and repopulate in the recipients. At the molecular level, mRNA microarray, quantitative real-time PCR and immunoblotting analyses of mTOR−/− HSPCs or Lin− cells revealed that the cell cycle inhibitor Rb was downregulated while the positive regulator of cell cycle E2F5 and pro-survival regulators MCL1 and BCL-xL were upregulated. mTOR deficiency abolished the activation of translational regulators S6K and 4E-BP but led to an increased activation of Akt. In addition, mTOR deficiency sensitized Lin− cells to DNA damage induced in vitro or in vivo by melphalan or mitomycin C (MMC), evidenced by a marked increase in γH2AX foci as well as DNA double-strand breaks (comet-tailed value of 30.2 ± 7.6 in mTOR−/− cells treated in vitro with melphalan and 37.6 ± 3.4 in mTOR−/− cells treated in vivo with MMC compared to 7.6 ± 2.1 in melphalan-treated WT cells and 17.3 ± 6.7 in MMC-treated WT cells, respectively). The increased DNA damage response can be attributed to an ∼300-fold reduction of the expression of FANCD2, a key component of the Fanconi DNA damage repair complex. Significantly, the effect of mTOR deficiency on Fanconi gene expression was specific to FANCD2, because the expression of other Fanconi proteins such as FANCA and FANCC was not affected in mTOR−/− Lin− cells. Intriguingly, the mTOR−/− Lin− cells phenocopied the DNA damage response of FANCD2−/− Lin− cells in vitro and in vivo. Similar effects of reduced FANCD2 expression and dampened DNA damage response were observed in human lymphoblasts treated with pp242, a mTOR kinase inhibitor. FANCD2-deficient human Fanconi anemia patient cells recapitulated the pp242-induced DNA damage phenotypes that could be rescued by FANCD2 reconstitution. Taken together, these results demonstrate that mTOR is a critical regulator of HSC quiescence and engraftment through the regulation of cell cycle machinery and is essential in multiple stages of hematopoiesis. Moreover, mTOR is required for maintaining genomic stability of HSPCs through modulation of the Fanconi anemia DNA damage response pathway. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hematopoietic stem/progenitor cell senescence is associated with altered expression profiles of cellular memory-involved gene

2018 ◽  
Vol 38 (1) ◽  
Author(s):  
Yongpin Dong ◽  
Xiaolan Lian ◽  
Yanwu Xu ◽  
Haiyan Hu ◽  
Cen Chang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mrna Expression ◽  
Progenitor Cell ◽  
Expression Profiles ◽  
Cell Cycle Distribution ◽  
Cellular Memory ◽  
Hematopoietic Stem ◽  
Altered Expression ◽  
Trithorax Group ◽  
Expression Of Genes

To evaluate the contributions of cellular memory mechanisms to hematopoietic stem/progenitor cell (HSPC) senescence. HSPCs (Lin−CD117+, hereafter referred to as HSPC) were separated from young (6-week-old) and aged (18-month-old) mice using Magnetic Activated Cell Sorting (MACS). Cell cycle distribution of HSPCs was determined using flow cytometry. The mixed colony forming unit (CFU-Mix) assay was used to study the HSPCs’ ability to proliferate. The mRNA expression levels of cellular memory-implicated PCG family (enhancer of zeste homolog 2 (Ezh2), B lymphoma mo-MLV insertion region 1 (Bmi-1), embryonic ectoderm development (Eed), melanoma nuclear protein 18 (Mel18), Mph1/polyhomeotic-like protein 1 (Rae-28)) and Trithorax group (TrxG) family (mixed lineage leukemia (Mll), thioredoxin (Trx)) were determined by quantitative real-time PCR. We obtained highly purified populations of mouse HSPCs (Lin−CD117+) (92.2 ± 4.5% CD117+). The percentage of HSPCs was significantly higher in older mice compared with younger control mice and the percentage of SA-β-galactosidase positive cells was significantly higher in HSPCs isolated from older mice (P<0.05). The percentage of HSPCs in G0/G1 was significantly higher in older mice compared with younger control mice (52.0 compared with 47.1%), indicating increased cell cycle arrest in senescent HSPCs. The amount of CFU-Mix was significantly decreased in aged group (13.8 compared with 40.0), indicating a diminished ability to proliferate in senescent HSPCs. Ezh1, Bmi-1, Eed, Rae-28 gene mRNA expression was significantly lower in HSPCs from older mice compared to younger controls, while Mel18 mRNA expression was significantly higher in HSPCs from older mice (P<0.05). The expression of genes associated with cellular memory is altered in senescent (Lin− CD117+) HSPCs, which may affect the potential plasticity of aged hematopoietic stem cells (HSCs) and thereby contribute to senescence-associated disease processes.

scholarly journals The role of the PZP domain of AF10 in acute leukemia driven by AF10 translocations

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Brianna J. Klein ◽  
Anagha Deshpande ◽  
Khan L. Cox ◽  
Fan Xuan ◽  
Mohamad Zandian ◽  
...  
Keyword(s):  
Critical Role ◽  
Cellular Transformation ◽  
Acute Leukemias ◽  
Hematopoietic Stem ◽  
Nucleosome Core ◽  
Stem And Progenitor Cells ◽  
Transforming Activity

AbstractChromosomal translocations of the AF10 (or MLLT10) gene are frequently found in acute leukemias. Here, we show that the PZP domain of AF10 (AF10PZP), which is consistently impaired or deleted in leukemogenic AF10 translocations, plays a critical role in blocking malignant transformation. Incorporation of functional AF10PZP into the leukemogenic CALM-AF10 fusion prevents the transforming activity of the fusion in bone marrow-derived hematopoietic stem and progenitor cells in vitro and in vivo and abrogates CALM-AF10-mediated leukemogenesis in vivo. Crystallographic, biochemical and mutagenesis studies reveal that AF10PZP binds to the nucleosome core particle through multivalent contacts with the histone H3 tail and DNA and associates with chromatin in cells, colocalizing with active methylation marks and discriminating against the repressive H3K27me3 mark. AF10PZP promotes nuclear localization of CALM-AF10 and is required for association with chromatin. Our data indicate that the disruption of AF10PZP function in the CALM-AF10 fusion directly leads to transformation, whereas the inclusion of AF10PZP downregulates Hoxa genes and reverses cellular transformation. Our findings highlight the molecular mechanism by which AF10 targets chromatin and suggest a model for the AF10PZP-dependent CALM-AF10-mediated leukemogenesis.

scholarly journals Mechanism of Action of Diallyl Sulphide in Ameliorating the Hematopoietic Radiation Injury

2021 ◽  
Author(s):  
Omika Katoch ◽  
Mrinalini Tiwari ◽  
Namita Kalra ◽  
Paban K. Agrawala
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation ◽  
Stem And Progenitor Cells ◽  
Radiation Induced ◽  
Medicinal Properties ◽  
Marrow Cellularity

AbstractDiallyl sulphide (DAS), the pungent component of garlic, is known to have several medicinal properties and has recently been shown to have radiomitigative properties. The present study was performed to better understand its mode of action in rendering radiomitigation. Evaluation of the colonogenic ability of hematopoietic progenitor cells (HPCs) on methocult media, proliferation and differentiation of hematopoietic stem cells (HSCs), and transplantation of stem cells were performed. The supporting tissue of HSCs was also evaluated by examining the histology of bone marrow and in vitro colony-forming unit–fibroblast (CFU-F) count. Alterations in the levels of IL-5, IL-6 and COX-2 were studied as a function of radiation or DAS treatment. It was observed that an increase in proliferation and differentiation of hematopoietic stem and progenitor cells occurred by postirradiation DAS administration. It also resulted in increased circulating and bone marrow homing of transplanted stem cells. Enhancement in bone marrow cellularity, CFU-F count, and cytokine IL-5 level were also evident. All those actions of DAS that could possibly add to its radiomitigative potential and can be attributed to its HDAC inhibitory properties, as was observed by the reversal radiation induced increase in histone acetylation.

A novel PLK1 inhibitor onvansertib effectively sensitizes MYC-driven medulloblastoma to radiotherapy

2021 ◽  
Author(s):  
Dong Wang ◽  
Bethany Veo ◽  
Angela Pierce ◽  
Susan Fosmire ◽  
Krishna Madhavan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Tumor Growth ◽  
Radiation Treatment ◽  
Tumor Regression ◽  
Tumor Growth Inhibition ◽  
Cell Cycle Distribution ◽  
Cell Renewal ◽  
Group 3

Abstract Background Group 3 medulloblastoma (MB) is often accompanied by MYC amplification. PLK1 is an oncogenic kinase that controls cell cycle and proliferation and has been preclinically validated as a cancer therapeutic target. Onvansertib (PCM-075) is a novel, orally available PLK1 inhibitor, which shows tumor growth inhibition in various types of cancer. We aim to explore the effect of onvansertib on MYC-driven medulloblastoma as a monotherapy or in combination with radiation. Methods Crisper-Cas9 screen was used to discover essential genes for MB tumor growth. Microarray and immunohistochemistry on pediatric patient samples were performed to examine the expression of PLK1. The effect of onvansertib in vitro was measure by cell viability, colony-forming assays, extreme limiting dilution assay and RNA-Seq. ALDH activity, cell-cycle distribution and apoptosis were analyzed by flow cytometry. DNA damage was assessed by immunofluorescence staining. Medulloblastoma xenografts were generated to explore the monotherapy or radio-sensitizing effect. Results PLK1 is overexpressed in Group 3 MB. The IC50 concentrations of onvansertib in Group 3 MB cell lines were in a low nanomolar range. Onvansertib reduced colony formation, cell proliferation, stem cell renewal and induced G2/M arrest in vitro. Moreover, onvansertib in combination with radiation increased DNA damage and apoptosis compare with radiation treatment alone. The combination radiotherapy resulted in marked tumor regression in xenografts. Conclusions These findings demonstrate the efficacy of a novel PLK1 inhibitor onvansertib in vitro and in xenografts of Group 3 MB, which suggests onvansertib is an effective strategy as monotherapy or in combination with radiotherapy in MB.

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