scholarly journals Regulation of Tyrosine Kinase Signaling by Cbl in Hematopoietic Stem Cells

10.5772/31930 ◽  
2012 ◽  
Author(s):  
Mayumi Naramura
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Hematopoietic Stem Cells ◽  
Kinase Signaling ◽  
Hematopoietic Stem ◽  
Tyrosine Kinase Signaling

Inhibition of PDGFRβ by Imatinib Mesylate Suppresses Proliferation and Alters Differentiation of Human Mesenchymal Stem Cells In Vitro.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2563-2563
Author(s):  
Fernando Fierro ◽  
Thomas Illmer ◽  
Duhoui Jing ◽  
Philip Le Coutre ◽  
Gerhard Ehninger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Tyrosine Kinase ◽  
Hematopoietic Stem Cells ◽  
Imatinib Mesylate ◽  
Dependent Manner ◽  
Differentiation Potential ◽  
Hematopoietic Stem

Abstract Recent data show that the tyrosine kinase inhibitor Imatinib mesylate (IM) also affects normal hematopoietic stem cells (HSC), T lymphocyte activation and dendritic cell function not relying on the specific inhibition of bcr-abl activity. Mesenchymal stem cells (MSC) have been identified in the bone marrow (BM) as multipotent non-hematopoietic progenitor cells that differentiate into osteoblasts, adipocytes, chondrocytes, tenocytes, skeletal myocytes, and cells of visceral mesoderm. MSC interact with HSC, influencing their homing and differentiation through cell-cell contact and the production of factors including chemokines We evaluated possible effects of IM in vitro on human bone marrow-derived MSC. Screening the activity of fourty-two receptor tyrosine kinases by a phospho-receptor tyrosine kinase (RTK)-array revealed an exclusive inhibition of platelet-derived growth factor receptor (PDGFRβ) by IM which consequently affects downstream targets of PDGFRβ as Akt and Erk1/2 signalling pathways in a concentration and time dependent manner. Furthermore, perinuclear multivesicular bodies harbouring PDGFRβ were found within 18–20 hours culture of MSC in the presence of 5 μM IM. Cell proliferation and clonogenicity (evaluated as the capability to form colony forming units - fibroblasts (CFU-F)) of MSC were significantly inhibited by IM in a concentration dependent fashion. IM inhibits significantly the differentiation process of MSC into osteoblasts as evaluated by decreased alkaline phosphatase activity and reduced calcium phosphate precipitates. In contrary, differentiation of MSC into adipocytes was strongly favoured in presence of IM. All these functional deficits described, probably contribute to an observed 50% reduction in the support of clonogenic hematopoietic stem cells, as evaluated by a long term culture-initiating cells (LTC-IC)-based assay. In summary our experiments show that IM inhibits the capacity of human MSC to proliferate and to differentiate into the osteogenic lineage, favouring adipogenesis. This effect is mainly mediated by an inhibition of PDGFRβ autophosphorylation leading to a more pronounced inhibition of PI3K/Akt compared to Erk1/2 signalling. This work confirms the role of PDGFRβ recently described for the proliferation and differentiation potential of MSC and provides a first possible explanation for the altered bone metabolism found in certain patients treated with IM.

scholarly journals Predominant expression of a receptor tyrosine kinase, TIE, in hematopoietic stem cells and B cells

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 93-101 ◽  
Author(s):  
M Hashiyama ◽  
A Iwama ◽  
K Ohshiro ◽  
K Kurozumi ◽  
K Yasunaga ◽  
...  
Keyword(s):  
Stem Cells ◽  
B Cells ◽  
Tyrosine Kinase ◽  
Hematopoietic Stem Cells ◽  
Receptor Tyrosine Kinase ◽  
Hematopoietic Cells ◽  
Erythroid Differentiation ◽  
Northern Blotting ◽  
Hematopoietic Stem ◽  
Epidermal Growth

Abstract A receptor tyrosine kinase (RTK), TIE (tyrosine kinase that contains immunoglobulin-like loops and epidermal growth factor [EGF] homology domains), is expressed in vascular endothelial and hematopoietic cells. We generated monoclonal antibodies (MoAbs) against the extracellular domain of TIE and a polyclonal antibody against the TIE carboxyterminus and used them to analyze expression of TIE in hematopoietic cells. Western blotting detected two forms of TIE protein with a molecular mass of 135 and 130 kD in hematopoietic and endothelial cells. Northern blotting analysis revealed that TIE was expressed preferentially in undifferentiated cell lines, especially when megakaryocytic, but not erythroid differentiation was induced. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that TIE was predominantly expressed in the human hematopoietic progenitor fraction, CD34+ cells. Fluorescence- activated cell sorting (FACS) showed that 42% of CD34+ and 17% of KIT- positive (KIT+) cells were TIE-positive (TIE+). The majority (81%) of the primitive hematopoietic stem cells, CD34+CD38- cells, were TIE+. Assays of progenitor cells and long-term culture-initiating cells (LTC- IC) showed that the TIE+ fraction contained more primitive cells than the TIE- fraction. Some TIE+ cells were in the CD34- fraction, which were CD19+ and CD20+ (B cells). These findings indicate that TIE has a unique spectrum of expression in primitive hematopoietic stem cells and B cells. Although its ligand has not been identified, TIE and its ligand may establish a novel regulatory pathway not only in early hematopoiesis, but also in the differentiation and/or proliferation of B cells.

scholarly journals Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3160-3169 ◽  
Author(s):  
A Iwama ◽  
K Okano ◽  
T Sudo ◽  
Y Matsuda ◽  
T Suda
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Hematopoietic Stem Cells ◽  
Tyrosine Kinases ◽  
Growth Regulation ◽  
Chromosome 9 ◽  
Cdna Clones ◽  
Hematopoietic Stem ◽  
Degenerate Oligonucleotide

To identify the novel receptor tyrosine kinases (RTKs) critical to the proliferation of hematopoietic stem cells, we performed polymerase chain reaction-based cloning from highly purified murine hematopoietic stem cells. Lineage marker-negative, c-KIT-positive, and Ly6A/E- or Sca- 1-positive (Lin-c-KIT+Sca-1+) cells were sorted by a fluorescence- activated cell sorter. Two sets of degenerate oligonucleotide primers were directed to the conserved sequences of the catalytic domain, and were used to amplify cDNAs that encode protein tyrosine kinases (PTKs). One hundred cDNA clones were sequenced and 8 RTKs were identified, as well as 12 non-RTKs and 2 serine/threonine kinases. Sixteen cDNAs were identical to the known kinase genes (PKC beta, JAK-1, JAK-2, TYK-2, HCK, FGR, FYN, BLK, c-FES, FER, c-ABL, c-KIT, FLK-1, FLK-2, IGF1R, and ECK). Six novel cDNA sequences (stk series) were identified. However, three of them turned out to be BPK, RYK, and TEK. The remaining three showed high homology to S6 kinase II, JAK-2, and v-SEA/c-MET, respectively. Characterization of full-length cDNA sequence of the v- SEA/cMET-related gene showed that this was a novel RTK gene and we named this gene STK (stem cell-derived tyrosine kinase). We identified two distinct forms of STK cDNA; the short one encoded a putative truncated protein that lacked most of the extracellular domain. STK was expressed at various stages of hematopoietic cells, including stem cells, but we could not detect any apparent expression in other adult tissues. The expression of the truncated form of mRNA was more predominant than that of the complete form. STK was assigned by fluorescent in situ hybridization to the R-positive F1 band of chromosome 9, the same region to which hepatic growth factor-like protein has been assigned. Characterization of these PTKs, including STK, will be helpful to elucidate the molecular mechanism of the growth regulation of hematopoietic stem cells.

scholarly journals Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3160-3169 ◽  
Author(s):  
A Iwama ◽  
K Okano ◽  
T Sudo ◽  
Y Matsuda ◽  
T Suda
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Hematopoietic Stem Cells ◽  
Tyrosine Kinases ◽  
Growth Regulation ◽  
Chromosome 9 ◽  
Cdna Clones ◽  
Hematopoietic Stem ◽  
Degenerate Oligonucleotide

Abstract To identify the novel receptor tyrosine kinases (RTKs) critical to the proliferation of hematopoietic stem cells, we performed polymerase chain reaction-based cloning from highly purified murine hematopoietic stem cells. Lineage marker-negative, c-KIT-positive, and Ly6A/E- or Sca- 1-positive (Lin-c-KIT+Sca-1+) cells were sorted by a fluorescence- activated cell sorter. Two sets of degenerate oligonucleotide primers were directed to the conserved sequences of the catalytic domain, and were used to amplify cDNAs that encode protein tyrosine kinases (PTKs). One hundred cDNA clones were sequenced and 8 RTKs were identified, as well as 12 non-RTKs and 2 serine/threonine kinases. Sixteen cDNAs were identical to the known kinase genes (PKC beta, JAK-1, JAK-2, TYK-2, HCK, FGR, FYN, BLK, c-FES, FER, c-ABL, c-KIT, FLK-1, FLK-2, IGF1R, and ECK). Six novel cDNA sequences (stk series) were identified. However, three of them turned out to be BPK, RYK, and TEK. The remaining three showed high homology to S6 kinase II, JAK-2, and v-SEA/c-MET, respectively. Characterization of full-length cDNA sequence of the v- SEA/cMET-related gene showed that this was a novel RTK gene and we named this gene STK (stem cell-derived tyrosine kinase). We identified two distinct forms of STK cDNA; the short one encoded a putative truncated protein that lacked most of the extracellular domain. STK was expressed at various stages of hematopoietic cells, including stem cells, but we could not detect any apparent expression in other adult tissues. The expression of the truncated form of mRNA was more predominant than that of the complete form. STK was assigned by fluorescent in situ hybridization to the R-positive F1 band of chromosome 9, the same region to which hepatic growth factor-like protein has been assigned. Characterization of these PTKs, including STK, will be helpful to elucidate the molecular mechanism of the growth regulation of hematopoietic stem cells.

scholarly journals Predominant expression of a receptor tyrosine kinase, TIE, in hematopoietic stem cells and B cells

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 93-101 ◽  
Author(s):  
M Hashiyama ◽  
A Iwama ◽  
K Ohshiro ◽  
K Kurozumi ◽  
K Yasunaga ◽  
...  
Keyword(s):  
Stem Cells ◽  
B Cells ◽  
Tyrosine Kinase ◽  
Hematopoietic Stem Cells ◽  
Receptor Tyrosine Kinase ◽  
Hematopoietic Cells ◽  
Erythroid Differentiation ◽  
Northern Blotting ◽  
Hematopoietic Stem ◽  
Epidermal Growth

A receptor tyrosine kinase (RTK), TIE (tyrosine kinase that contains immunoglobulin-like loops and epidermal growth factor [EGF] homology domains), is expressed in vascular endothelial and hematopoietic cells. We generated monoclonal antibodies (MoAbs) against the extracellular domain of TIE and a polyclonal antibody against the TIE carboxyterminus and used them to analyze expression of TIE in hematopoietic cells. Western blotting detected two forms of TIE protein with a molecular mass of 135 and 130 kD in hematopoietic and endothelial cells. Northern blotting analysis revealed that TIE was expressed preferentially in undifferentiated cell lines, especially when megakaryocytic, but not erythroid differentiation was induced. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that TIE was predominantly expressed in the human hematopoietic progenitor fraction, CD34+ cells. Fluorescence- activated cell sorting (FACS) showed that 42% of CD34+ and 17% of KIT- positive (KIT+) cells were TIE-positive (TIE+). The majority (81%) of the primitive hematopoietic stem cells, CD34+CD38- cells, were TIE+. Assays of progenitor cells and long-term culture-initiating cells (LTC- IC) showed that the TIE+ fraction contained more primitive cells than the TIE- fraction. Some TIE+ cells were in the CD34- fraction, which were CD19+ and CD20+ (B cells). These findings indicate that TIE has a unique spectrum of expression in primitive hematopoietic stem cells and B cells. Although its ligand has not been identified, TIE and its ligand may establish a novel regulatory pathway not only in early hematopoiesis, but also in the differentiation and/or proliferation of B cells.

Hypoxia-Adapted CML Cells Are More Primitive Population and Are Eradicated by Glyoxalase-1 Inhibitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2166-2166 ◽  
Author(s):  
Miki Takeuchi ◽  
Shinya Kimura ◽  
Junya Kuroda ◽  
Eishi Ashihara ◽  
Makoto Kawatani ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Hematopoietic Stem Cells ◽  
Cell Lines ◽  
Lactate Production ◽  
Glucose Consumption ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Nog Mice

Abstract Abstract 2166 Poster Board II-143 Normal hematopoietic stem cells reside in the epiphyses of the bone marrow that is a low oxygenated area and are protected against ROS-induced DNA damages. Recent study showed that the hypoxic environment plays a crucial role not only in maintaining stem cells but also in tumorigenesis. Hypoxia induces dramatical changes in cell characteristics including cell cycle quiescence, self renewal potency, and shift in energy production from an aerobic to anaerobic pathway, and it induces resistance to a variety of cell death stimuli. Chronic myelogenous leukemia (CML) is a disorder of hematopoietic stem cells caused by the constitutive activation of the Bcr-Abl tyrosine kinase. Tyrosine kinase inhibitors (TKIs) have led to marked improvement in prognosis of CML patients. However, CML cells could not be eradicated completely by TKI alone because quiescent CML stem cells are less sensitive to such molecular target drugs. Therefore, we speculate that the adaptation of leukemic cells to hypoxia in the bone marrow niche alters their characteristics contributing to minimal residual disease. We first evaluated the oxygen levels of engrafted leukemic cells by pimonidazol (hypoxia specific marker) staining.We transplanted K562 cells to the bone marrow of NOD/SCID/gcnull (NOG) mice and found that those cells engrafted and survived in the epiphysis of the bone marrow where O2 concentrations are less than 1.3%. Then, we generated two hypoxia-adapted (HA) CML subclones from K562 and KCL22 by cultivating under 1.0% O2, and were denoted as K562/HA and KCL22/HA, respectively. Both cell lines survived and proliferated continuously for years under 1.0% O2 conditions, although their growth was slower than that of their parental counterparts under 20% O2 conditions. Interestingly, HA-CML cells exhibited several unique characteristics compared to their parental cells. First, these HA cells showed higher transplantation efficacy in NOG mice. The transplanted HA cells grow more rapidly in vivo than the parental cells and mice transplanted with HA cells died earlier. Next, the percentage of G0 fractions in K562 and K562/HA cells were 0.87 ± 0.58 % and 4.9 ± 2.1 %, respectively, indicating that K562/HA cells included more quiescent fractions than the parental K562. Hoechst staining analysis confirmed that HA cell lines include more SP (side population) fractions than their parental cells, indicating that HA cells contains more dormant cells. We next examined the signaling pathway of HA cell lines. Despite the unchanged levels of AKT, STAT, and ERK phosphrylation, BCR-ABL phsophrylation was suppressed in HA cells. Both of HA cell lines showed higher expression of b-catenin which is considered essential for survival and self-renewality of CML stem cells. Furthermore, HA cells were less sensitive to TKIs (imatinib, dasatinib, and bafetinib) and chemotherapeutic agents (daunorubicin and busulfan). Taken together, our HA cell lines have characteristics of more primitive CML cell populations resistant to cytotoxic agents. We next examined the energy metabolites such as adenosine triphosphate (ATP), glucose consumption, and lactate production in HA cells. The amounts of ATP in K562/HA and KCL22/HA cells decreased, whereas glucose consumption and lactate production increased compared with those in their parental cell lines. These findings indicate that ATP production of HA cells depends on glycolysis. Furthermore, we found higher expression and kinase activity of Glyoxalase-1 (Glo-I). Glo-I is an enzyme that detoxifies glycolysis-specific cytocidal byproducts in glycolytis system. Glo-I inhibitors such as S-p-bromobenzylglutathione cyclopentyl diester (BBGC), 2-crotonyloxymethyl-4,5,6-trihydroxycylohex-2-enone (COTC), and methyl-gerfelin were much more cytotoxic against HA-CML cells than their parental cells in vitro. Notably, when K562/HA-transplanted mice were treated with 100 mg/kg/day BBGC for 8 days, the treated mice survived longer than the untreated mice (Figure 1). These findings suggest that Glo-1 plays an important role in primitive CML cells survival under hypoxia. In conclusion, Glo-1 is a novel attractive target against hypoxia-adapted primitive CML cells in the bone marrow milieu. Investigation of hypoxia-specific pathways and roles on CML cells could develop novel therapeutic approach targeting TKIs resistance. Disclosures: No relevant conflicts of interest to declare.

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