Identification of the soluble granulocyte-macrophage colony stimulating factor receptor protein in vivo

Blood ◽  
2000 ◽  
Vol 95 (2) ◽  
pp. 461-469 ◽  
Author(s):  
Farzana Sayani ◽  
Felix A. Montero-Julian ◽  
Valerie Ranchin ◽  
Jay M. Prevost ◽  
Sophie Flavetta ◽  
...  
Keyword(s):  
Cell Lines ◽  
State Level ◽  
Leukemic Cell ◽  
Protein Product ◽  
Receptor Protein ◽  
Soluble Form ◽  
Human Neutrophils ◽  
Gm Csf ◽  
Leukemic Cell Lines

On the basis of the finding of alternatively spliced mRNAs, the -subunit of the receptor for GM-CSF is thought to exist in both a membrane spanning (tmGMR) and a soluble form (solGMR). However, only limited data has been available to support that the solGMR protein product exists in vivo. We hypothesized that hematopoietic cells bearing tmGMR would have the potential to also produce solGMR. To test this hypothesis we examined media conditioned by candidate cells using functional, biochemical, and immunologic means. Three human leukemic cell lines that express tmGMR (HL60, U937, THP1) were shown to secrete GM-CSF binding activity and a solGMR-specific band by Western blot, whereas a tmGMR-negative cell line (K562) did not. By the same analyses, leukapheresis products collected for autologous and allogeneic stem cell transplants and media conditioned by freshly isolated human neutrophils also contained solGMR. The solGMR protein in vivo displayed the same dissociation constant (Kd = 2-5 nmol) as that of recombinant solGMR. A human solGMR ELISA was developed that confirmed the presence of solGMR in supernatant conditioned by the tmGMR-positive leukemic cell lines, hematopoietic progenitor cells, and neutrophils. Furthermore, the ELISA demonstrated a steady state level of solGMR in normal human plasma (36 ± 17 pmol) and provided data suggesting that plasma solGMR levels can be elevated in acute myeloid leukemias.

scholarly journals Akacid Medical Formulation Induces Apoptosis in Myeloid and Lymphatic Leukemic Cell Lines In Vitro and In Vivo

PLoS ONE ◽  
2015 ◽  
Vol 10 (2) ◽  
pp. e0117806 ◽  
Author(s):  
Hannes Neuwirt ◽  
Elisabeth Wabnig ◽  
Clemens Feistritzer ◽  
Iris E. Eder ◽  
Christina Salvador ◽  
...  
Keyword(s):  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cell Lines

Metabolic Capability to Induce the Pasteur Effect Mediates Sensitivity of Human Leukemic Cells to Metformin

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2601-2601
Author(s):  
Sarah Scotland ◽  
Estelle Saland ◽  
Lindsay Peyriga ◽  
Rémi Peyraud ◽  
Elizabeth Micklow ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Pasteur Effect ◽  
Cytotoxic Response ◽  
Leukemic Cell Lines

Abstract Abstract 2601 An emerging hallmark of cancer cells is the reprogramming of intermediary and energy metabolism these cells undergo. Several epidemiological studies have shown that metformin, widely used to treat patients with type 2 diabetes, may reduce their risk of cancer. Despite several reports of anti-neoplastic activity of metformin, the mechanisms responsible for this activity have not been fully elucidated in cancer or leukemic cells. We hypothesized that metformin elicits a metabolic reprogramming driven by alterations in mitochondrial function and signaling, which induces apoptosis in leukemic cells, and that metabolic flexibility determines the variation(s) of the cytotoxic response to metformin among different leukemic cell lines. We first demonstrated that metformin markedly decreased oxygen consumption of six leukemic cell lines in a concentration-dependent manner. We also observed that the cytotoxic effect of metformin varies between cell lines reflecting their energetic capacity to compensate for the mitochondrial inhibition induced by metformin (eg. to induce the Pasteur effect). Importantly, metformin-insensitive leukemic cells did not exhibit a Pasteur effect in response to metformin. All leukemic cells exhibited high basal conversion of glucose to lactate (eg. aerobic glycolysis) and specific expression of key metabolic genes as compared to normal mononuclear cells. Despite dependence on glucose catabolism, metformin sensitivity was associated with relative resistance to glucose starvation. Metformin effects in drug-resistant cells were potentiated by the addition of a glycolytic inhibitor, but not by inhibitors of the pentose phosphate pathway or glutaminolysis. Leukemic cells with broad metabolic capacities to utilize other energetic substrates in response to diverse nutrient starvation showed insensitivity to metformin. Metformin induced a significant decrease in metabolites of the upper segment of glycolysis and the oxidative branch of the pentose phosphate pathway as well as a clear increase of PRPP and IMP biosynthesis. Energy charge, the nucleotide phosphate pool and lactate/glucose ratio remained stable after metformin treatment. Furthermore, our results showed that basal glucose uptake/consumption and the activity of the lower segment of the glycolytic pathway are key determinants of a cytotoxic response to metformin. In addition, high glutathione, malate, IMP and orotate content were observed in metformin-insensitive leukemic cells. Moreover, the cytotoxic effect of metformin was independent of AMPK/LKB1 status of the leukemic cells while p53 expression abrogated this effect. The presence of wild-type p53 appears to partially protect tumor cells from glucose starvation and metformin cytotoxicity and prevents the induction of the Pasteur effect. Finally, we demonstrated that metformin increased the cytotoxicity of chemotherapy agent, cytarabine, on all leukemic cell lines in vitro and significantly reduced leukemic colony-forming units (CFU-L) from six primary AML patient samples in a concentration-dependent manner. Additional experiments on metabolic and signaling pathways as well as in vivo studies are in progress to better understand the cytotoxic response of metformin in both AML cell lines and primary AML patient specimens that impact the therapeutic potential of metformin in vivo. Disclosures: Carroll: Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.

IFNα-stimulated neutrophils and monocytes release a soluble form of TNF-related apoptosis-inducing ligand (TRAIL/Apo-2 ligand) displaying apoptotic activity on leukemic cells

Blood ◽  
2004 ◽  
Vol 103 (10) ◽  
pp. 3837-3844 ◽  
Author(s):  
Cristina Tecchio ◽  
Veronica Huber ◽  
Patrizia Scapini ◽  
Federica Calzetti ◽  
Daniela Margotto ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Soluble Form ◽  
Human Neutrophils ◽  
Interferon Α ◽  
Cytotoxic Activities ◽  
Peripheral Blood Mononuclear ◽  
Trail Receptors

Abstract Tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily exerting cytotoxic activities toward tumor cells. Herein, we demonstrate that therapeutic concentrations of interferon α (IFNα) stimulate the expression of high levels of TRAIL mRNA and the release of elevated amounts of a soluble bioactive form of TRAIL (sTRAIL) in both human neutrophils and monocytes. Supernatants harvested from IFNα-treated neutrophils/monocytes elicited, on TRAIL-sensitive leukemic cell lines, proapoptotic activities that were significantly reduced by either a combination of TRAIL-R1/Fc and TRAIL-R2/Fc chimeras or neutralizing anti-TRAIL, anti–TRAIL-R1, and anti–TRAIL-R2 antibodies, suggesting that they were mediated by released sTRAIL acting on both TRAIL receptors. Since diseases such as chronic myeloid leukemia (CML) and melanoma are effectively treated with IFNα,we also demonstrate that CML neutrophils and peripheral blood mononuclear cells (PBMCs) cultured with IFNα at therapeutic concentrations retain the capacity of releasing sTRAIL, suggesting that CML leukocytes, in vivo, might represent an important source of sTRAIL. In this regard, we show that sTRAIL serum levels as well as leukocyte-associated TRAIL significantly increase in melanoma patients following IFNα administration. Collectively, these findings indicate that sTRAIL released by IFNα-activated neutrophils and monocytes contributes not only to the immunoregulatory actions but also to the therapeutic activities of IFNα.

scholarly journals Interleukin-3 regulates the activity of the LYN protein-tyrosine kinase in myeloid-committed leukemic cell lines

Blood ◽  
1992 ◽  
Vol 80 (3) ◽  
pp. 617-624 ◽  
Author(s):  
T Torigoe ◽  
R O'Connor ◽  
D Santoli ◽  
JC Reed
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Leukemic Cell ◽  
Beta Subunit ◽  
Gm Csf ◽  
Leukemic Cell Lines ◽  
Lyn Kinase

The lymphokine interleukin-3 (IL-3) promotes the growth and survival of immature hematopoietic cells. Previous studies have shown that IL-3 induces rapid increases in protein-tyrosine kinase (PTK) activity in IL- 3--dependent cells. Unlike some other hematopoietic growth factor receptors (eg, c-fms and c-kit), however, the known subunits of the IL- 3 receptor (IL-3R) lack intrinsic kinase activity. Recently, it was reported that the IL-2R (whose p75 beta-subunit shares sequence homology with a known murine IL-3R subunit and a common beta-subunit of the human IL-3R and granulocyte-macrophage colony-stimulating factor [GM-CSF] receptors) can physically associate with and regulate the activity of the SRC-family PTK, p56-LCK. Because most IL-3--dependent cells contain p53/56-LYN, but not p56-LCK, we explored the effects of IL-3 on the activities of LYN and other SRC-like PTKs in two human leukemic cell lines, AML-193 and TALL-101, which are phenotypically myeloid, and whose in vitro growth is dependent on IL-3. These cells expressed four of the eight known SRC-family proto-oncogenes: lyn, fyn, yes, and hck. When these factor-dependent leukemic cell lines were deprived of lymphokine to achieve cellular quiescence and then restimulated with IL-3, rapid increases (detectable within 1 minute and maximal by 10 minutes) were observed in the activity of the p53/56-LYN kinase, as assessed by in vitro kinase assays. In contrast, no alteration in the activities of other SRC-family PTKs present in these cells was detected after restimulation with IL-3 under the same conditions. This effect of IL-3 reflected an increase in the specific activity of the LYN kinase, because levels of the 53-Kd and 56-Kd LYN proteins were unaltered by IL-3 stimulation, as assessed by immunoblotting. Furthermore, the magnitude of these inducible increases in LYN kinase activity was dependent on the concentration of IL-3, and correlated with IL-3--induced proliferation. The IL-3--induced upregulation of LYN kinase activity may be mediated by the 120-Kd common subunit of the human IL-3 and GM-CSF receptors, because GM-CSF also stimulated marked increases in the activity of the LYN kinase, whereas granulocyte-CSF (G-CSF) did not, despite inducing cellular proliferation. These observations provide the first example of an IL-3-- regulable PTK, and strongly suggest that the p53/56-LYN kinase participates in early IL-3--initiated signalling events, at least in some human leukemic cell lines.

scholarly journals Response of newly established mouse myeloid leukemic cell lines to MC3T3-G2/PA6 preadipocytes and hematopoietic factors

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 49-54
Author(s):  
H Kodama ◽  
M Iizuka ◽  
T Tomiyama ◽  
K Yoshida ◽  
M Seki ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines ◽  
Hematopoietic Factors ◽  
In Vitro Cell Lines

Some mouse myeloid leukemias induced by X-irradiation and serially transplanted into syngenic mice do not proliferate in vitro even in the presence of hematopoietic factors. To examine whether such leukemic cells can proliferate in response to stromal cells, we cocultured them with MC3T3-G2/PA6 (PA6) preadipocytes, cells that can support the growth of hematopoietic stem cells. All leukemias developed into in vitro cell lines, showing a dependence on contact with the PA6 cells. Two cell lines responded to none of the known hematopoietic factors including interleukin-3 (IL-3), IL-4, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and Epo. These results demonstrate that the mechanism of the action of PA6 cells is different from that of any of the known hematopoietic factors, and that, because these two leukemic cell lines retained the ability to grow in vivo, responsiveness to the known hematopoietic factors is not essential for the leukemic cell growth in vivo. Furthermore, all leukemic cell lines could respond also to the preadipocytes fixed with formalin, paraformaldehyde, or glutaraldehyde, suggesting that some molecule(s) associated with the surface of PA6 cells or with extracellular matrix secreted by the preadipocytes is responsible for the leukemic cell growth.

scholarly journals Interleukin-3 regulates the activity of the LYN protein-tyrosine kinase in myeloid-committed leukemic cell lines

Blood ◽  
1992 ◽  
Vol 80 (3) ◽  
pp. 617-624 ◽  
Author(s):  
T Torigoe ◽  
R O'Connor ◽  
D Santoli ◽  
JC Reed
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Leukemic Cell ◽  
Beta Subunit ◽  
Gm Csf ◽  
Leukemic Cell Lines ◽  
Lyn Kinase

Abstract The lymphokine interleukin-3 (IL-3) promotes the growth and survival of immature hematopoietic cells. Previous studies have shown that IL-3 induces rapid increases in protein-tyrosine kinase (PTK) activity in IL- 3--dependent cells. Unlike some other hematopoietic growth factor receptors (eg, c-fms and c-kit), however, the known subunits of the IL- 3 receptor (IL-3R) lack intrinsic kinase activity. Recently, it was reported that the IL-2R (whose p75 beta-subunit shares sequence homology with a known murine IL-3R subunit and a common beta-subunit of the human IL-3R and granulocyte-macrophage colony-stimulating factor [GM-CSF] receptors) can physically associate with and regulate the activity of the SRC-family PTK, p56-LCK. Because most IL-3--dependent cells contain p53/56-LYN, but not p56-LCK, we explored the effects of IL-3 on the activities of LYN and other SRC-like PTKs in two human leukemic cell lines, AML-193 and TALL-101, which are phenotypically myeloid, and whose in vitro growth is dependent on IL-3. These cells expressed four of the eight known SRC-family proto-oncogenes: lyn, fyn, yes, and hck. When these factor-dependent leukemic cell lines were deprived of lymphokine to achieve cellular quiescence and then restimulated with IL-3, rapid increases (detectable within 1 minute and maximal by 10 minutes) were observed in the activity of the p53/56-LYN kinase, as assessed by in vitro kinase assays. In contrast, no alteration in the activities of other SRC-family PTKs present in these cells was detected after restimulation with IL-3 under the same conditions. This effect of IL-3 reflected an increase in the specific activity of the LYN kinase, because levels of the 53-Kd and 56-Kd LYN proteins were unaltered by IL-3 stimulation, as assessed by immunoblotting. Furthermore, the magnitude of these inducible increases in LYN kinase activity was dependent on the concentration of IL-3, and correlated with IL-3--induced proliferation. The IL-3--induced upregulation of LYN kinase activity may be mediated by the 120-Kd common subunit of the human IL-3 and GM-CSF receptors, because GM-CSF also stimulated marked increases in the activity of the LYN kinase, whereas granulocyte-CSF (G-CSF) did not, despite inducing cellular proliferation. These observations provide the first example of an IL-3-- regulable PTK, and strongly suggest that the p53/56-LYN kinase participates in early IL-3--initiated signalling events, at least in some human leukemic cell lines.

scholarly journals Response of newly established mouse myeloid leukemic cell lines to MC3T3-G2/PA6 preadipocytes and hematopoietic factors

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 49-54 ◽  
Author(s):  
H Kodama ◽  
M Iizuka ◽  
T Tomiyama ◽  
K Yoshida ◽  
M Seki ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cell Lines ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Leukemic Cell Lines ◽  
Hematopoietic Factors ◽  
In Vitro Cell Lines

Abstract Some mouse myeloid leukemias induced by X-irradiation and serially transplanted into syngenic mice do not proliferate in vitro even in the presence of hematopoietic factors. To examine whether such leukemic cells can proliferate in response to stromal cells, we cocultured them with MC3T3-G2/PA6 (PA6) preadipocytes, cells that can support the growth of hematopoietic stem cells. All leukemias developed into in vitro cell lines, showing a dependence on contact with the PA6 cells. Two cell lines responded to none of the known hematopoietic factors including interleukin-3 (IL-3), IL-4, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and Epo. These results demonstrate that the mechanism of the action of PA6 cells is different from that of any of the known hematopoietic factors, and that, because these two leukemic cell lines retained the ability to grow in vivo, responsiveness to the known hematopoietic factors is not essential for the leukemic cell growth in vivo. Furthermore, all leukemic cell lines could respond also to the preadipocytes fixed with formalin, paraformaldehyde, or glutaraldehyde, suggesting that some molecule(s) associated with the surface of PA6 cells or with extracellular matrix secreted by the preadipocytes is responsible for the leukemic cell growth.

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