Gadd45a and Gadd45b Protect Haematopoetic Cells from Ultraviolet Radiation Induced Apoptosis Via Distinct Signaling Pathways.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1258-1258
Author(s):  
Mamta Gupta ◽  
Shiv K. Gupta ◽  
Barbara Hoffman ◽  
Dan A. Liebermann
Keyword(s):  
Bone Marrow ◽  
Ultraviolet Radiation ◽  
Cell Survival ◽  
Signaling Pathways ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Genotoxic Stress ◽  
Induced Apoptosis ◽  
Deficient Mice ◽  
Marrow Cells

Abstract Gadd45 expression, which is stress inducible, has been associated with growth arrest, but the exact role of gadd45 family genes in apoptosis still remains unclear. We have found that myeloid progenitor cells from gadd45a and gadd45b-deficient mice are more sensitive to ultra-violet radiation, VP-16 or daunorubicin induced apoptosis. indicating that gadd45a or gadd45b protect haematopoetic cells from DNA damaging agents. To determine, how gadd45a or gadd45b proteins exert their anti-apoptotic function, bone marrow cells from wild-type and gadd45a or gadd45b deficient mice were exposed to ultraviolet radiation (UV) and analyzed for expression of stress responsive kinases, including JNK and p38. It was observed that P38 and JNK were activated in wt bone marrow cells in response to UV but not in bone marrow cells defecient in gadd45a. Also, the transcription factor NF-kB was activated in wt bone marrow cells, but not in gadd45a−/− cells. The pharmacological inhibitor SB203580 specific for p38, increased apoptosis in reponse to UV, indicating that p38 is implicated in signaling myeloid cell survival. SB203580 was observed also to inhibit the expression of certain NF-kB target genes, including cIAP-1, c-IAP-2, bcl-2 and bcl-xl, in gadd45a+/+ cells but not in gadd45a deficient bone marrow cells. Taken together this data provides first evidence for the role gadd45a plays in the control of hematopoietic cell survival in response to UV, via modulation of P38 MAPK and NF-kB signaling pathways. Unlike in gadd45a−/− bone marrow cells, p38 activation appeared not to be impaired in gadd45b−/− cells, indicating that gadd45b is not involved in p38 activation in myeloid cells. However, UV induced JNK activation was sustained in gadd45b−/− myeloid cells compared to wt cells, indicating that gadd45b is a negative modulator of UV induced JNK signaling in myeloid cells. UV induced activation of MKK4 an upstream regulator of JNK also was impaired in gadd45b−/−. NF-kB was also found activated in wt cells, but not in gadd45b−/− cells. This data indicates that in bone marrow cells exposed to UV, NF-kB induced expression of Gadd45b plays a protective role against UV induced apoptosis via inhibition of MKK4 kinase which in turn results in suppression of JNK activity. Taken together this data provides evidence that Gadd45a and Gadd45b protect haematopoetic cells from genotoxic-stress induced apoptosis via distinct signaling pathways.

Myeloid Progenitor Cells from Gadd45a and Gadd45b-Deficient Mice Are Sensitized to Genotoxic-Stress Induced Apoptosis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4239-4239
Author(s):  
Mamta Gupta ◽  
Shiv K. Gupta ◽  
Arthur G. Balliet ◽  
Barbara Hoffman ◽  
Dan A. Lieberman
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Genotoxic Stress ◽  
Wild Type ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Induced Apoptosis ◽  
Deficient Mice ◽  
Marrow Cells

Abstract GADD45 (Growth arrest and DNA damge) regulates cell growth following exposure to diverse stimuli. It has been shown that, mice lacking the gadd45a gene exhibit genomic instability and increased carcinogenesis, but the exact role of the gadd45 family genes still remains unclear. In this study we have aimed at determining the effect of gadd45a or gadd45b deficiency on the response of bone marrow derived myeloid cells to genotoxic stress agents by using gadd45a or gadd45b null mice. We have found that myeloid progenitor cells from gadd45a or gadd45b-null mice are more sensitive to ultraviolet-radiation (UV), VP-16 or daunorubicin induced apoptosis. Introduction of wild-type gadd45 into gadd45-deficient bone marrow cells restored the wild-type apoptotic phenotype. In-vitro colony formation following stress responses has shown that bone marrow cells from gadd45a or gadd45b-deficient mice have a decreased ability to form haematopoetic colonies. Gadd45a or gadd45b-deficient bone marrow cells also displayed defective G2/M cell cycle checkpoint following exposure to either UV and V-16 but were still able to undergo G2/M arrest following exposure to daunorubicin, indicating the existence of different G2/M checkpoints in response to these anticancer agents. Taken together these findings identify gadd45a or gadd45b as anti-apoptotic gene(s), and suggests that the absence of gadd45a or gadd45b results in higher susceptibility of haematopoetic cells to UV radiation and certain anticancer drugs.

Hematopoietic Cells from Gadd45a, Gadd45b Deficent Mice Exhibit Altered Myelopoiesis and Higher Apoptosis after Cytokine Treatment.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1697-1697
Author(s):  
Shiv K. Gupta ◽  
Mamta Gupta ◽  
Barbara Hoffman ◽  
Dan A. Liebermann
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Methyl Cellulose ◽  
Myeloid Cell ◽  
Cell Maturation ◽  
Wild Type ◽  
Wild Type Littermate ◽  
Deficient Mice ◽  
Marrow Cells

Abstract Growth arrest and DNA damage, Gadd45 gene family members are rapidly induced by genotoxic agents as well as by apoptosis and differentiation inducing cytokines. Their role in hemetopoiesis, wherein proliferation, differentiation and apoptosis integrate to maintain cellular homeostasis, is not clear. Using bone marrow cells from gadd45a or gadd45b deficient and wild type littermate mice we have investigated the role of Gadd45 proteins in cytokine induced myeloid cell differentiation in vitro. Bone marrow cells obtained from either gadd45a or gadd45b deficient mice displayed compromised cytokines (IL3, GM-CSF, M-CSF or G-CSF) induced myelopoiesis, resulting in a quantitatively decreased population of mature myeloid cells. Immuno-phenotyping with antibodies to cell surface molecules associated with myeloid cell maturation confirmed impaired myeloid cell maturation in Gadd45a or b deficient bone marrow cells treated with the above cytokines. Analysis of apoptosis by annexin-V and PI staining followed by FACS analysis showed a substantially higher apoptosis in Gadd45a−/− as well as gadd45b−/− cells compared to wild type cells after treatment with M-CSF or G-CSF. Gadd45a−/− as well as gadd45b−/− bone marrow cells were found to be less clonogenic in methylcellulose medium. Morphologically compact and round colonies consisting of immature myeloid cells prevailed over dispersed- colonies consisting of mature myeloid cells in gadd45- deficient cells cultured in methyl cellulose containing IL-3. Furthermore, colony re-plating assay showed better self-renewal abilities in gadd45a−/− as well as gadd45b−/− progenitors, compared to wild type progenitor cells. Altered myelopoiesis in gadd45 a or b deficient mice was further confirmed in vivo by intra-peritoneal administration of sodium casienate - a known inducer of inflammatory response and myelopoiesis in mice bone marrow. Sodium casienate failed to enhance myelopoiesis in gadd45a or gadd45b deficient mice bone marrow, while wild type littermate mice showed a rapid induction of myelopoiesis. Simultaneously peritoneal exudates collected from gadd45 deficient mice consisted of 2–3 fold less myeloid cells compared to age matched wild type control mice after sodium casienate treatment. Gadd45a−/− or gadd45b−/− mice showed a slow recovery after myelo-suppressive effect of antimetabolite 5-Fluorouracil, which further confirmed that gadd45 deficiency leads to delayed myelopoiesis in mouse. Mechanistic aspects of Gadd45 deficiency, which results in impaired myelopoiesis are under investigation.

VEGF and AMD3100-Induced Rapid Mobilization of C-Kit/Sca-1 Positive Murine Bone Marrow Cells and of Gr-1+/CD-11b+ Myeloid Cells Is Impaired in Urokinase (uPA) and Urokinase Receptor (uPAR) Deficient Mice.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1384-1384
Author(s):  
Mohammad Dehghani ◽  
Damla Olcaydu ◽  
Pavel Uhrin ◽  
Bernd Binder ◽  
Johannes Breuss
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Myeloid Cells ◽  
Urokinase Receptor ◽  
Facs Analysis ◽  
Β1 Integrins ◽  
Deficient Mice ◽  
Marrow Cells

Abstract Hematopoietic Progenitor Cells (HPC) can be mobilized from bone marrow into the circulation in response to a number of stimuli including G-CSF, AMD3100 (antagonist of CXCR4-DSF-1 axis) and vascular endothelial growth factor (VEGF). The main mechanism for mobilization of HPCs upon stimulation by classical “mobilizers”as G-CSF is thought to be through extracellular matrix proteolysis in the marrow. Urokinase is a serine protease present in the marrow and contributes to mobilization of stem cells upon binding to its receptor (uPAR) and activating plasminogen that leads to matrix degradation. Our previous data show that the effect of VEGF on endothelial cell migration is exerted through activation of the uPA/uPAR system and through co-internalization of β 1 integrins. Upon internalization of these receptors, cells detach from their underlying extra-cellular matrix (ECM) as well as from stromal cells. We hypothesize that the contribution of VEGF to HPC mobilization occurs through a similar mechanism. We also want to analyze the influence of uPA/uPAR deficiencies on mobilization of Gr-1+/CD-11b+ myeloid and c-kit +/Sca-1+ (SK)cells by VEGF and AMD3100 and compare it with G-CSF as a classical “mobilizer”. Wild type, uPA knockout and uPAR knockout mice in C57BL6 background were used for in vivo experiments. We collected peripheral blood before and 2 hours after i.p. injection of VEGF-E and AMD3100 and assessed the number of SK cells and myeloid cells by FACS analysis. We also administered G-CSF for 5 days and compared blood samples before and after the experiment. To evaluate the effect of VEGF on HPC integrin expression, femurs of the respective animals were incubated with VEGF in an ex vivo experimental model and β1 expression was assessed by FACS analysis. In vivo data demonstrated a significantly reduced responsiveness of uPA−/− mice to VEGF-E in the first 2 hours after the injection. This decreased responsiveness to VEGFis observed in uPAR−/− mice but to a lesser degree than in uPA−/− mice..(40 +/−16 % and21 +/− 20% respectively vs 65 +/− 24 % in wt, means and SD). Injection of urokinase together with VEGF to uPA−/− mice rescues the lack of mobilization of SK cells. Ex vivo stimulation of uPAR knockout femoral bone marrow cells with VEGF for 20 minutes provides evidence that the internalization of β1 integrins upon VEGF stimulation is uPAR dependent. VEGF can also increase in vivo the number of Gr-1+/CD-11b+ myeloid cells after 2 hours in wt mice (96 +/− 45%) but not in urokinase deficient or urokinase receptor deficient mice (7 +/− 11% and 21+/−33%, respectively). AMD3100 has a strong effect on mobilization of SK cells in wt animals within 2 hours (increase of 2.8+/−0.78 times) but cannot mobilize these cells in uPA and uPAR deficient mice to the same extend (0.8+/−0.65 times and 0.1+/−0.07 respectively). G-CSF injection for 5 days mobilizes Gr-1+/CD-11b+and SK cells in wt and knock out mice to a similar extent, indicating that the capacity to release these cells from the bone marrow is not affected by uPA or uPAR gene deficiency. Our results demonstrate a reduced mobilization of uPA−/− and uPAR−/− HPCs and myeloid cells in response to VEGF compared to wt mice. VEGF leads to internalization of the expression of β1 integrins on the surface of SK cells in wt but not in uPAR−/− mice. In addition, we could show that the uPA/uPAR system plays a role in AMD3100-dependent mobilization of these cells. These data indicate that the uPA – uPAR system plays a pivotal role in short-term but not long-term bone marrow HPC and PMN leukocyte mobilization.

scholarly journals Radioprotective effects of fucoidan on bone marrow cells: improvement of the cell survival and immunoreactivity

2008 ◽  
Vol 9 (4) ◽  
pp. 359 ◽  
Author(s):  
Yun-Young Byon ◽  
Mi-Hyoung Kim ◽  
Eun-Sook Yoo ◽  
Kyu-Kye Hwang ◽  
Youngheun Jee ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Survival ◽  
Bone Marrow Cells ◽  
Marrow Cells

scholarly journals THE DISTRIBUTION OF HISTOCHEMICALLY DEMONSTRABLE GLYCOGEN IN HUMAN BLOOD AND BONE MARROW CELLS

Blood ◽  
1949 ◽  
Vol 4 (1) ◽  
pp. 54-59 ◽  
Author(s):  
MAX WACHSTEIN
Keyword(s):  
Bone Marrow ◽  
Human Blood ◽  
Acid Treatment ◽  
Bone Marrow Cells ◽  
Periodic Acid ◽  
Myeloid Cells ◽  
Staining Reaction ◽  
Cytoplasmic Staining ◽  
Chemical Association ◽  
Marrow Cells

Abstract By applying Schiff’s reagent after periodic acid treatment to blood and bone marrow films, a cytoplasmic staining reaction is seen in some cells of the myeloid series, as well as in megakaryocytes and platelets. The intensity of the staining reaction in the myeloid cells increases with their maturation. The staining reaction can be prevented altogether in alcohol-fixed films by salivary digestion, but only incompletely in air-dried films. The staining reaction is due to the presence of glycogen in some chemical association, possibly with protein.

Therapeutic angiogenesis in diabetic apolipoprotein E-deficient mice using bone marrow cells, functional hemangioblasts and metabolic intervention

2010 ◽  
Vol 209 (2) ◽  
pp. 403-414 ◽  
Author(s):  
Maria Luisa Balestrieri ◽  
Shi-Jiang Lu ◽  
Filomena de Nigris ◽  
Alfonso Giovane ◽  
Sharon Williams-Ignarro ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Apolipoprotein E ◽  
Bone Marrow Cells ◽  
Therapeutic Angiogenesis ◽  
Deficient Mice ◽  
Marrow Cells

Mutant CXCR4 Identified in Neutropenic Patients with Myelokathexis Impairs Survival of Human Myeloid Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3071-3071
Author(s):  
Vahagn Makaryan ◽  
David C. Dale ◽  
Andrew A. Aprikyan
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Survival ◽  
Mutational Analysis ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Receptor Internalization ◽  
Morphological Examination ◽  
Exon 2 ◽  
Whim Syndrome

Abstract Myelokathexis (WHIM syndrome) is a very rare hematopoietic congenital disorder that is characterized by extremely low level of circulating neutrophils in peripheral blood. It is inherited as an autosomal dominant disease and is diagnosed in early childhood. These patients may have hypogammaglobulinemia and suffer from recurrent infections associated with warts. The hallmark of myelokathexis is a hyperplastic bone marrow and hypersegmented neutrophils with nuclear lobs connected with thin filaments. Myelokathexis is due to a characteristic retention of mature neutrophils in bone marrow, which are not being released to peripheral circulation. We and others reported abnormal cell survival characteristics and impaired bcl-x expression in bone marrow myeloid cells of myelokathexis patients that was partially restored by G-CSF treatment. Recently, it has also been reported that heterozygous truncation mutations in the carboxyterminal domain of the CXCR4 gene, a sole receptor for SDF-1 chemokine, were observed in most, but not all of the families with WHIM syndrome. Subsequently, an impaired receptor internalization and increased chemotaxis towards SDF-1 have been observed in cells expressing truncated CXCR4. Nevertheless, the mechanism of mutant CXCR4 induced myelokathexis remains largely unknown. We performed mutational analysis of the CXCR4 gene in 3 unrelated families with myelokathexis and identified a previously reported R334ter truncation mutation in exon 2 in two of the families. In addition, two silent polymorphisms have been identified in exon 2 of the CXCR4 gene in one of these patients. The third family with afflicted mother and son had a new mutation in the CXCR4 carboxyterminal domain, which resulted in deletion of the last 16 amino acids and subsequent frame shift. None of these mutations were observed in healthy volunteers examined. Since the morphological examination by electron microscopy and flow cytometry analysis of bone marrow cells from some of these patients revealed characteristic apoptotic features, we examined the effect of mutant CXCR4 gene expression on survival of human promyelocytic HL-60 cells. Preliminary data demonstrated that human promyelocytic cells transfected with truncated CXCR4 exhibited impaired cell survival characteristics compared with control HL-60 cells transfected with intact CXCR4. The truncated, but not wild type CXCR4 also increased apoptosis in HL-60 cells induced to differentiate along the granulocytic pathway as determined by flow cytometry of annexin V labeled cells. Thus, these data link together the abnormal survival of proliferating and differentiating myeloid cells in WHIM syndrome with mutant CXCR4 expression. Current studies are focused on elucidation of specific signaling pathways mediating mutant CXCR4-triggered myelokathexis.

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.

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