Tel-PDGFβR Specifically Induces Interferon-Stimulated Genes.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1213-1213
Author(s):  
Hani Kim ◽  
Dwayne L. Barber
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Bone Marrow ◽  
Fusion Protein ◽  
Signaling Pathways ◽  
Bone Marrow Cells ◽  
Chromosomal Translocations ◽  
Time Points ◽  
Interferon Stimulated Genes ◽  
Marrow Cells

Abstract Chromosomal translocations involving tyrosine kinases play a significant role in human leukemia. Chronic myeloid leukemia (CML) is associated with the recurrent chromosomal translocation, BCR-ABL (t(9;22)(q34;q11)). Chronic myelomonocytic leukemia (CMML) is linked to TEL-PDGF-β Receptor (PDGFβR) (t(5;12)(q33;p13)) fusion. Another TEL fusion, TEL-JAK2 (t(9;12)(p24;p13) has been observed in CMML and Acute Lymphoid Leukemia. All three fusion proteins induce leukemia-like diseases in animal models, and this is attributed to the constitutive tyrosine kinase activity, which leads to dysregulation of their respective downstream signaling pathways. The downstream targets include STAT transcription factors, MAP kinases, and PI3 kinase. On the other hand, little is known about the gene transcription regulated by these fusions. The objective of our study is to determine whether BCR-ABL, TEL-PDGFβR and TEL-JAK2 induce distinct gene expression patterns when expressed in cell lines and retrovirally transduced bone marrow cells. Each fusion was expressed in an IL3-dependent murine myeloid cell line, Ba/F3. The specific inhibitor, Imatinib mesylate, was utilized to control the activation/inhibition of BCR-ABL and TEL-PDGFβR, and an inducible system was utilized for TEL-JAK2. Upon activation of the fusion protein, cells were collected at various time-points for cell cycle and microarray analysis (Affymetrix MOE430A). We utilized 8 hr, 12 hr, 24 hr and 1 wk time points. Our rationale was to monitor gene expression changes through the first cell cycle and then to examine the fingerprint at a steady state point. Analysis of the 1 wk data reveals that a subset of genes are co-regulated (2-fold, p<0.05) by BCR-ABL, TEL-PDGFβR and TEL-JAK2 (Pim1, Id1b, Podxl, Cxcr4, Gp49b and Scin). Interestingly, analysis of the TEL-PDGFβR induced genes (10-fold, p<0.05) revealed a significant overlap with Interferon-Stimulated Gene (ISG) dataset including Cxcl-10, Gbp1, Gbp2, Isg20, Ccl-5, Stat1, Irf7, Serpine-1 and Mx1. Genes identified in this microarray study have been confirmed by Q-PCR in Ba/F3 cells and confirmatory experiments in primary bone marrow cells transduced with each fusion protein are underway. In addition, we will determine whether the transcription of these targets is dependent on STAT1 by utilizing bone marrow cells from STAT1−/− mice. In conclusion, our data reveals that oncogenic chromosomal translocations activate both distinct and co-regulated gene expression and reveal a novel and specific role of Interferon-Stimulated Genes in signaling pathways downstream of TEL-PDGFβR.

scholarly journals Signaling Pathways of Mutant IDH1 Independent of R-2-Hydroxyglutarate

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2617-2617
Author(s):  
Charu Gupta ◽  
Michelle Maria Araujo Cruz ◽  
Nidhi Jyotsana ◽  
Amit Sharma ◽  
Ramya Goparaju ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Signaling Pathways ◽  
Splice Variant ◽  
Research Funding ◽  
Bone Marrow Cells ◽  
Stat3 Phosphorylation ◽  
Mutant Idh1 ◽  
Marrow Cells

Abstract #Michael Heuser and Anuhar Chaturvedi share senior authorship Background: Isocitrate dehydrogenase-1 (IDH1) is mutated in about 6% of AML patients. Mutant IDH produces R-2-hydroxyglutarate (R-2HG), which induces histone and DNA hypermethylation through inhibition of epigenetic regulators, thereby linking metabolism to tumorigenesis. We recently reported that at comparable intracellular R-2HG levels, mice receiving transplants of IDH1 mutant cells died significantly earlier than R-2HG treated mice in the context of HOXA9 overexpression. This suggests oncogenic functions of mutant IDH1 beyond R-2HG production. We employed a splice variant of mutated IDH1 that does not produce R-2HG (IDH1mutantΔ7) to decipher R-2HG independent signaling pathways that may contribute towards leukemogenesis. Methods: Bone marrow cells from mice were immortalized with HoxA9, and IDH1wildtype (IDH1wt), IDH1mutant (IDH1mut), IDH1wildtypeΔ7 (IDH1wtΔ7) and IDH1mutΔ7, were constitutively expressed and the leukemogenic potential was evaluated in vivo. Intracellular R-2HG was measured by enantiomer-specific quantification. Deletion of exon 7 from IDH1mut leads to a frameshift that creates a premature stop codon in the 9th exon, finally producing a 119 amino acids truncated protein, IDH1mutΔ7. This splice variant does not produce increased levels of R-2HG. The signaling pathways were explored by immunoblotting and immunofluorescence. Results: Mice receiving cells with IDH1mutΔ7 had the same short latency to leukemia as mice receiving cells with full-length mutant IDH1, while IDH1wt and IDH1wtΔ7 cells died with significantly longer latency. The WBC count increased over time in IDH1mutΔ7 mice similar to IDH1mut mice, whereas WBC counts in IDH1wtΔ7 mice remained normal. IDH1mutΔ7 mice died from monocytic leukemia that was phenotypically and morphologically indistinguishable from IDH1mut mice. HoxA9 IDH1mutΔ7 cells were readily transplantable into secondary recipients. During in vivo cell cycle analysis, we observed that the proportion of cells in S/G2/M phases was significantly higher in bone marrow cells transduced with IDH1mut or IDH1mutΔ7 when compared to cells transduced with IDH1wt or CTL. These data suggest that mutant IDH1 enhances myeloproliferation even in the absence of R-2HG. To identify R-2HG independent signaling pathways mediated by the mutant IDH1 protein, we first analyzed the gene expression of important regulators of cell cycle, differentiation, cell signaling and transcription by quantitative RT-PCR. Several genes (Ccnd1, Slc2a, Hdac3, Tgif2,and c-myc) were upregulated in IDH1mut and IDH1mutΔ7 cells compared to IDH1wt cells. Interestingly, we found a specific up-regulation of Ctnnb1 and Nfkb genes in IDH1mutΔ7 cells over both IDH1mut and IDH1wt cells. We next validated our mRNA expression results by immunoblotting and found that NFKB and ERK signaling were upregulated in both IDH1mut and IDH1mutΔ7 compared to IDH1wt and IDH1wtΔ7 cells. Interestingly, the protein level of β-catenin, STAT3 and STAT5 were many fold higher in IDH1mutΔ7 compared to IDH1mut and IDH1wt cells. β-catenin is known to be transactivated via c-Src, which is phosphorylated by EGFR to promote β-catenin nuclear localization and signaling. We traced this pathway for its relevance in our cells and found that IDH1mutΔ7 cells indeed showed higher levels of both EGFR and c-Src phosphorylation compared to IDH1mut cells. We performed immunofluorescence and cellular fractionation for β-catenin and found it to be partially localized in the nucleus in IDH1mutΔ7 but not in IDH1mut cells. We also observed an up-regulated STAT3 phosphorylation in IDH1mutΔ7 cells over IDH1mut. Conclusions: In summary, mutant IDH1 activates ERK and NFKB signaling, which is attributed to both R-2HG dependent and independent mechanisms of leukemogenesis. Interestingly, IDH1mutΔ7 employs R-2HG independent EGFR/β-catenin and JAK/STAT signaling for oncogenesis. This R-2HG-independent leukemogenesis reveals a novel signaling dynamic of IDH1mut which should be evaluated for its therapeutic potential. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Heuser:Astellas: Research Funding; Karyopharm: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; StemLine Therapeutics: Consultancy; Bayer Pharma AG: Consultancy, Research Funding; Sunesis: Research Funding; BergenBio: Research Funding; Tetralogic: Research Funding; Daiichi Sankyo: Research Funding.

scholarly journals Antioxidant Fusion Protein SOD1-Tat Increases the Engraftment Efficiency of Total Bone Marrow Cells in Irradiated Mice

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3395
Author(s):  
Ting Bei ◽  
Xusong Cao ◽  
Yun Liu ◽  
Jinmei Li ◽  
Haihua Luo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Fusion Protein ◽  
Bone Marrow Cells ◽  
Standard Procedure ◽  
Severe Infection ◽  
Copper Zinc Superoxide Dismutase ◽  
Donor Cells ◽  
Total Bone Marrow ◽  
Marrow Cells

Total body irradiation is a standard procedure of bone marrow transplantation (BMT) which causes a rapid increase in reactive oxygen species (ROS) in the bone marrow microenvironment during BMT. The increase in ROS reduces the engraftment ability of donor cells, thereby affecting the bone marrow recovery of recipients after BMT. In the early weeks following transplantation, recipients are at high risk of severe infection due to weakened hematopoiesis. Thus, it is imperative to improve engraftment capacity and accelerate bone marrow recovery in BMT recipients. In this study, we constructed recombinant copper/zinc superoxide dismutase 1 (SOD1) fused with the cell-penetrating peptide (CPP), the trans-activator of transcription (Tat), and showed that this fusion protein has penetrating ability and antioxidant activity in both RAW264.7 cells and bone marrow cells in vitro. Furthermore, irradiated mice transplanted with SOD1-Tat-treated total bone marrow donor cells showed an increase in total bone marrow engraftment capacity two weeks after transplantation. This study explored an innovative method for enhancing engraftment efficiency and highlights the potential of CPP-SOD1 in ROS manipulation during BMT.

scholarly journals Simultaneous flow cytometric DNA and volume measurements of bone marrow cells as sensitive indicator of abnormal proliferation patterns in rat leukemias.

1979 ◽  
Vol 27 (1) ◽  
pp. 398-403 ◽  
Author(s):  
G Valet ◽  
B Fischer ◽  
A Sundergeld ◽  
G Hanser ◽  
V Kachel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Parameter Analysis ◽  
Alpha Cell ◽  
Brown Norway ◽  
Normal Limits ◽  
Flow Cytometric ◽  
Volume Measurements ◽  
Marrow Cells

Simultaneous flow cytometric DNA and volume analysis of normal rat bone marrow cells shows three populations of nucleated cells with different mean volume. Each of these populations proliferates in a distinct cell cycle (alpha, beta, gamma). Normally the alpha-cell cycle has the highest amplitude, the beta-cell cycle is intermediate, and the gamma-cell cycle is low. The alpha-cell cycle was very significantly depressed and the beta + gamma-cell cycle was increased in three different rat leukemias (L5222, Shay, BNML), growing on three different rat strains (BDIX, Holtzmann, Brown Norway). The two parameter analysis further revealed that cells of the beta + gamma-cell cycle were slightly hyperdiploid and hypertetraploid in leukemic animals. The decrease of the alpha-cell cycle and the hyperploidies were more sensitive indicators for the abnormal proliferation pattern than the analysis of one parameter DNA distributions which remained within normal limits in all three leukemias.

scholarly journals Expression of Shelterin ComponentPOT1Is Associated with Decreased Telomere Length and Immunity Condition in Humans with Severe Aplastic Anemia

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Ting Wang ◽  
Shu-chong Mei ◽  
Rong Fu ◽  
Hua-quan Wang ◽  
Zong-hong Shao
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Telomere Length ◽  
Bone Marrow Cells ◽  
Culture Media ◽  
Telomere Attrition ◽  
High Concentrations ◽  
Relationship Of ◽  
The Relationship ◽  
Marrow Cells

Abnormal telomere attrition has been found to be closely related to patients with SAA in recent years. To identify the incidence of telomere attrition in SAA patients and investigate the relationship of telomere length with clinical parameters, SAA patients(n=27)and healthy controls(n=15)were enrolled in this study. Telomere length of PWBCs was significantly shorter in SAA patients than in controls. Analysis of gene expression of Shelterin complex revealed markedly low levels ofPOT1expression in SAA groups relative to controls. No differences in the gene expression of the other Shelterin components—TRF1,TRF2,TIN2,TPP1, andRAP1—were identified. Addition of IFN-γto culture media induced a similar fall in POT1 expression in bone marrow cells to that observed in cells cultured in the presence of SAA serum, suggesting IFN-γis the agent responsible for this effect of SAA serum. Furthermore, ATR, phosphorylated ATR, and phosphorylated ATM/ATR substrate were all found similarly increased in bone marrow cells exposed to SAA serum, TNF-α, or IFN-γ. In summary, SAA patients have short telomeres and decreased POT1 expression. TNF-αand IFN-γare found at high concentrations in SAA patients and may be the effectors that trigger apoptosis through POT1 and ATR.

Gene Expression Changes in Bone Marrow Cells from Diamond-Blackfan Anemia Patients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 720-720 ◽  
Author(s):  
Hanna T. Gazda ◽  
Despina Sanoudou ◽  
Alvin T. Kho ◽  
Jan M. Zaucha ◽  
Colin A. Sieff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Ribosomal Protein ◽  
Cell Population ◽  
Bone Marrow Cells ◽  
Principal Component ◽  
Fold Change ◽  
Ribosomal Protein Genes ◽  
Diamond Blackfan Anemia ◽  
Marrow Cells

Abstract Diamond-Blackfan anemia is usually characterized by anemia, absence or insufficiency of erythroid precursors in bone marrow, growth retardation and diverse congenital anomalies that are present in approximately half of patients, indicating that DBA is a broad disorder of development. Mutations of RPS19 are found in approximately 25% of DBA patients. There is good evidence for a second DBA gene, located on chromosome 8, and further genetic heterogeneity of the disease is likely. The aim of this study is to determine the most disturbed molecular pathways in DBA patients, based on gene expression changes in bone marrow cells. Knowing these pathways will possibly enable us to decipher the pathogenic mechanisms of DBA and find other genes involved in the disease. Bone marrow cells from 6 normal individuals and 3 DBA patients with RPS19 mutations, currently in remission, were FACS separated into 3 populations: primitive (P), erythroid (E) and myeloid (M) containing CD34+CD71-CD45RA-, CD34+CD71hiCD45RA- and CD34+CD71lowCD45RA+ cells, respectively. The purity of each sorted population was >97%. As a control for cell sorting accuracy, methylcellulose assay demonstrated that the P populations were highly enriched in primitive BFU-E and CFU-GEMM colonies, the E populations gave rise to BFU-E and CFU-E colonies in more than 90% of the CFCs, while more than 99% colonies from M populations were CFU-G, CFU-M and CFU-GM. RNA targets from these three FACS sorted cellular subsets was hybridized to Affymetrix HG-U133A chips (>22,000 probe sets). The data from all 27 samples were analyzed by hierarchical clustering and Principal Component Analysis, and each cell population was also studied separately. All pairwise comparisons among 27 datasets showed correlations with r=0.86–0.99. Hierarchical clustering identified three major specimen clusters, perfectly overlapping with the three different cell populations under study. Principal Component 1 and 2 separated the three studied subgroups P, E, and M. In each cell population analysis, 3 patient samples were compared to 6 control samples using 1)Significance Analysis of Microarrays with fold change 2 or greater and false discovery rate 1%, 2)Geometric Fold Change analysis and 3)Filter on Fold Change GeneSpring application (arithmetic analysis). All fold change analyses revealed the most significantly changed transcripts in patients vs. control individuals in E (45 upregulated and 184 downregulated) and P populations. The most changed genes in E subgroup were apoptosis related genes, namely TNFRSF10B and TNFRSF6 (CD95/Fas), upregulated in patients 10 and 3 fold, respectively. Other most changed genes were cancer related and genes involved in developmental processes and nucleic acid binding. Additionally, several ribosomal protein genes, namely RPL10L, RPL28, RPL36, RPL13, RPL27a and RPL37a were significantly underexpressed in P and E populations of DBA patients. All three analyses showed that RPL10L, RPL28 and RPL36 are underexpressed in the M population. This finding indicates that ribosomal protein genes are closely co-regulated and that RPS19 protein abnormalities result in downregulation of the additional ribosomal protein genes in both erythroid and nonerythroid cells in DBA patients.

Overlapping but Distinct Role of p21WAF1/CDKN1 and Survivin in Hematopoietic Progenitor Cell Proliferation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1334-1334
Author(s):  
Seiji Fukuda ◽  
Mariko Abe ◽  
Seiji Yamaguchi ◽  
Louis M. Pelus
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Growth Factor ◽  
Bone Marrow Cells ◽  
Mouse Bone Marrow ◽  
Hematopoietic Progenitor ◽  
Primary Mouse ◽  
Marrow Cells

Abstract Survivin is a member of the inhibitor of apoptosis protein family that has been implicated in cell cycle control, anti-apoptosis and cell division. Our previous studies and others have shown that Survivin and the cyclin dependent kinase inhibitor p21WAF1/CDKN1 (p21) are functionally associated and are involved in cell cycle, anti-apoptosis and cytokinesis in cancer cells and in normal hematopoietic progenitor cells (HPC). P21 is highly expressed in quiescent hematopoietic stem cells (HSC) in steady state, but the proportion of quiescent HSCs in G0 phase is reduced in p21−/− mice. In contrast, p21 has been shown as positive regulator on cell cycle of normal HPC since p21 deficiency results in fewer total CFU in mouse bone marrow (BM) cells with fewer CFU in S-phase and retrovirus transduction of p21 in p21 deficient bone marrow cells restores total and cycling CFU. We have previously reported that Survivin increases the proliferation of mouse primary HPC and that this enhancing effect is on HPC proliferation is absent when p21 is functionally deleted, suggesting that p21 is required for Survivin to enhance HPC proliferation. In addition, ITD-Flt3 mutations that are normally expressed in patients with acute myeloid leukemia and associate poor prognosis increase expression of both Survivin and p21, implicating their involvement in aberrant proliferation of HPC expressing ITD-Flt3. Herein we have characterized the functional association between p21 and Survivin in normal and transformed cell proliferation. Antagonizing wild-type Survivin in mouse BaF3 cells by retrovirus transduction of a T34A dominant negative mutant Survivin or anti-sense increased p21 expression, even though Survivin requires p21 to enhance HPC proliferation. Ectopic p21 in Survivin+/+ primary mouse bone marrow cells increased the number of immunophenotypically defined c-kit+, lin− (KL) cells, which is consistent with a positive role of p21 in HPC proliferation, however; ectopic expression of p21 failed to increase HPC proliferation in Survivin deficient primary bone marrow cells, suggesting that p21 alone is not sufficient to substitute for Survivin’s enhancing function on normal HPC proliferation. Over-expression of ITD-Flt3 enhanced growth factor independent proliferation of primary mouse marrow c-kit+, Sca-1+, lin− (KSL) cell number; however, co-expression of p21 with ITD-Flt3 dramatically decreased the number of growth factor independent KSL cells (80±6% reduction: P<0.01). Furthermore, the inhibitory effect of p21 on KLS proliferation was further enhanced by Survivin knockout bone marrow cells (64±5% reduction compared with presence of Survivin: P<0.05). These findings indicate that Survivin and p21 have a overlapping but distinct roles in regulating normal HPC proliferation and that manipulating p21 and Survivin may represent a potential therapeutic target for acute leukemia cells expressing ITD-Flt3.

Gene Expression Profiling of Murine HOXB4-Transduced HSCs Shows Multiple Counter-Regulatory Signals That May Control HSC Pool Size

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2361-2361
Author(s):  
Hui Yu ◽  
Sheng Zhou ◽  
Geoffrey A. Neale ◽  
Brian P. Sorrentino
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Expression Array ◽  
Self Renewal ◽  
Time Points ◽  
Time Point

Abstract Abstract 2361 HOXB4 is a homeobox transcription factor that can induce hematopoietic stem cell (HSC) expansion both in vivo and in vitro. An interesting feature of HOXB4-induced HSC expansion is that HSC numbers do not exceed normal levels in vivo due to an unexplained physiological capping mechanism. To gain further insight into HOXB4 regulatory signals, we transplanted mice with bone marrow cells that had been transduced with a MSCV-HOXB4-ires-YFP vector and analyzed gene expression profiles in HSC-enriched populations 20 weeks after transplant, a time point at which HSC numbers have expanded to normal levels but no longer increasing beyond physiologic levels. We used Affymetrix arrays to analyze gene expression profiles in bone marrow cells sorted for a Lin−Sca-1+c-Kit+ (LSK), YFP+ phenotype. Using ANOVA, we identified1985 probe sets with >2 fold difference in expression (FDR<, 0.1) relative to a control vector-transduced LSK cells. A cohort of genes was identified that were known positive regulators of HSC self-renewal and proliferation. Hemgn, which we identified in a previous screen as a positive regulator of expansion and a direct transcriptional target of HOXB4, was 3.5 fold up-regulated in HOXB4 transduced LSKs. Other genes known to be important for HSCs survival, self-renewal and differentiation were upregulated to significant levels including N-myc, Meis1, Hoxa9, Hoxa10 and GATA2. Microarray data for selected genes was validated by quantitative real-time PCR on HOXB4 transduced CD34low LSK cells, a highly purified HSC population, obtained from another set of transplanted mice at the 20 week time point. In contrast, other gene expression changes were noted that would potentially limit or decrease stem cell numbers. PRDM16, a set domain transcription factor critical for HSC maintenance and associated with clonal hematopoietic expansions when inadvertently activated as a result of retroviral insertion, was dramatically down-regulated on the expression array and 7.6 fold decreased in the real time PCR assay of CD34low LSK cells. TFG-beta signaling is a well defined inhibitor HSC proliferation and utilize Smad proteins as downstream effectors. Expression of Smad1 and Smad7 were significantly upregulated on the LSK expression array and 8.1 and 3.5 fold up-regulated by qPCR in CD34low LSK cells. Another potential counter-regulatory signal was down regulation of Bcl3 mRNA, a potential anti-apoptotic effector in HSCs. We hypothesize that the HOXB4 expansion program involves activation of genes that lead to increased HSC numbers with later activation of counter-regulatory signals that limit expansion to physiologic numbers of HSCs in vivo. We are now examining how this program changes at various time points after transplantation and hypothesize the capping limits are set at relatively later time points during reconstitution. We also are studying the functional effects of these gene expression changes, and in particular, whether enforced expression of HOXB4 and PRMD16 will result in uncontrolled HSC proliferation and/or leukemia. Disclosures: No relevant conflicts of interest to declare.

Gene expression profiling related to the enhanced erythropoiesis in mouse bone marrow cells

2008 ◽  
Vol 104 (1) ◽  
pp. 295-303 ◽  
Author(s):  
Hee-Young Yang ◽  
Dong Kee Jeong ◽  
Seok-Ho Kim ◽  
Kyoung-Jin Chung ◽  
Eun-Jin Cho ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Bone Marrow Cells ◽  
Mouse Bone Marrow ◽  
Mouse Bone Marrow Cells ◽  
Marrow Cells
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