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Signal transduction from membrane-bound macrophage colony-stimulating factor
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Abstracts/Experimental Hematology 28 (2000) 31–131
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Cytokines, Growth Factors and Receptors
SOLUBLE MACROPHAGE COLONY STIMULATING FACTOR RECEPTOR IN HUMAN SERUM AND UREA Q. RAO*, J.S. HAN*, Y.Q. GENG*, G.G. ZHENG*, K.F. WU National Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, P.R. China Soluble receptors are produced by proteolytic cleavage of membrane-bound receptors to release soluble forms, or by alternative mRNA splicing resulting in dedicated transcript encoding soluble receptors. It has been reported that M-CSF receptor could be cleaved from the cell surface by a protease induced by activation of protein kinase C. We have developed an enzyme-linked immunosorbent assay (ELISA) to quantify the concentration of soluble M-CSFR (Rao Q. et al. Exp Hematol 27:105, 1999). Now via M-CSFR specific ELISA, the soluble M-CSFR level in serum and urea from normal donors and patients with hematological diseases is reported. The average level of soluble M-CSFR in normal human individuals (n⫽102) was 0.457⫾0.303ng/ml. There was no statistical difference in soluble M-CSFR levels between males and females groups or between the different age groups. The average levels of soluble M-CSFR in the serum from ALL and AML patients were 0.184⫾0.368 ng/ml (n⫽36) and 0.124ng/ml⫾0.223ng/ ml (n⫽60), which were much lower than normal human (p⫽ 0.0002 and p⬍0.0001, respectively). For the patients with benign hematological disease, the serum levels of M-CSFR in IDA (n⫽ 25) and ITP patients (n⫽34) were slightly higher than normal individuals (0.662⫾0.468ng/ml and 0.828⫾0.960ng/ml respectively), but no statistical difference (p⫽0.05 and p⫽0.06) was found. Using our ELISA, the soluble M-CSFR in the urea of normal human (n⫽109) and hematological patients was also investigated. The results showed that the urea M-CSFR level of patients with malignant hematological disease (n⫽35) was significantly lower than that of normal donor (0.291⫾0.502ng/ml versus 0.894⫾0.715ng/ml, p⫽0.0007). In contrast, the urea M-CSFR level of patients with benign hematological disease (n⫽33) was higher than that of normal adult (6.98⫾13.14ng/ml versus 0.894⫾0.715ng/ml, p⫽0.01). Result of the urea M-CSFR levels is consistent with that of serum. It has been reported that detectable levels of some soluble receptors can be measured in normal individuals and in some diseases. Our results showed that soluble M-CSFR exited in normal human serum and urea. The function of soluble M-CSFR and whether the abnormalities of soluble M-CSFR level in serum and urea may contribute to leukemia or other hematological diseases should further be verified. 4
Sunday, July 9, 2000 (14:15–16:00) Session II-3: Signal Transduction
SIGNAL TRANSDUCTION FROM MEMBRANE-BOUND MACROPHAGE COLONY-STIMULATING FACTOR G.G. ZHENG*, K.F. WU, Y.Q. GENG*, AND Q. RAO* National Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, P.R. China Macrophage colony-stimulating factor (M-CSF) exists in different forms, which bind the same membrane receptor (M-CSFR),
due to alternative splicing and post-translational modifications. Membrane bound M-CSF (mM-CSF) was highly expressed in some malignancies including J6-1, J6-2 (leukemia), SMMC7721, BEL7402 (hepatoma) cell lines and specimens from patients with leukemia, Hodgkin’s Disease (HD), hepatoma, etc. (Zheng GG et al. Hematologica 1999, 84:951). Furthermore, we demonstrated that mM-CSF and its receptor played adhesion molecule-like roles in J6-1 cells and the activation of mM-CSF by recombinant soluble receptor (M-CSFsR) resulted in the decrease of cytoplasmic pH, mobilization of cytosolic calcium, tyrosine-phosphorylation on cytoplasmic protiens, etc. (Zheng GG et al. Leukemia Res 2000, in press). In the present study, we used cytochalasin B (CB) to study whether the integrity of cytoskeleton is necessary for mMCSF signaling. The results showed that one hour’s treatment of J6-1 cells by CB before exposed to M-CSFsR blocked the tyrosine-phosphorylation on cytoplasmic proteins with MW of 45 and 55-90kD, while decreased the sensitivity of cytoplasmic pH changes, but showed no obvious influence on cytosolic calcium mobilization. The initial activation process of most membrane proteins by their ligands involves dimerization or cluster of the membrane protein and cross-linking of the membrane protein by specific antibody can mimic the effects of their natural ligands, while dimerization or cluster is not indispensable for the signaling of some membrane proteins. We used a monoclonal antibody (McAb) B5, which was developed in our lab belonging to IgM subtype and recognizes N-terminal part of mM-CSF, to cross-link mM-CSF on J6-1 cell surface before underwent phospho-tyrosine Western blot analysis. While F(ab’)2 fragment of B5 caused no obvious tyrosine phosphorylation when compared with BSA control, 10 minutes’ treatment by B5 dose-dependently caused tyrosine phosphorylation on cytoplasmic proteins with MW of approximately 45 and 50-90kD, which mimicked the effects of M-CSFsR. The above results suggested that the initiation of mM-CSF signaling need dimerization/cluster of them on cell surface and cytoskeleton take part in some, but not all, of the downstream signal events. 5
Monday, July 10, 2000 (12:45–14:15) Session IV-2: Chronic Myelogenous Leukemia II
CML PATIENTS WITH ADVANCED PHASE DISEASE RETAIN CLONOGENIC PROGENITOR CELLS WHICH ARE RESPONSIVE TO INTERFERON ALFA AND STI571 IN VITRO MY Gordon, SB Marley, RJ Davidson, DX Nguyen, JL Lewis, JM Goldman LRF Centre, Department of Haematology, Imperial College School of Medicine, Hammersmith Hospital, London, UK We investigated if progenitor cells from CML patients with accelerating/transformed (AP) disease differ from chronic phase (CP) cells in their sensitivities to interferon(IFN)-␣ and STI571. We used a colony replating assay which measures the self-renewal capacity of clonogenic cells (CFU-GM). The results are expressed as the area-under-the-curve (AUC) of the cumulative distribution obtained by plotting the numbers of secondary colonies produced by replating individual primary colonies. The AUC for CP-CML CFU-GM is significantly greater than that for normal marrow CFU-GM (79.6–5.0 (mean–sem), n⫽90 vs 63–4.4, n⫽63; p⫽0.029, t test). CP CFU-GM exposed to pharmacological concentrations of IFN-␣ (50U/ml) or STI571 (0.1M) exhibit a reduced AUC (respectively 63–5% and 52–7% of control level; n⫽33 and 30) but
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Abstracts/Experimental Hematology 28 (2000) 31–131
Sunday, July 9, 2000 (18:30–19:30) Poster Session I: Cytokines, Growth Factors and Receptors
SOLUBLE MACROPHAGE COLONY STIMULATING FACTOR RECEPTOR IN HUMAN SERUM AND UREA Q. RAO*, J.S. HAN*, Y.Q. GENG*, G.G. ZHENG*, K.F. WU National Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, P.R. China Soluble receptors are produced by proteolytic cleavage of membrane-bound receptors to release soluble forms, or by alternative mRNA splicing resulting in dedicated transcript encoding soluble receptors. It has been reported that M-CSF receptor could be cleaved from the cell surface by a protease induced by activation of protein kinase C. We have developed an enzyme-linked immunosorbent assay (ELISA) to quantify the concentration of soluble M-CSFR (Rao Q. et al. Exp Hematol 27:105, 1999). Now via M-CSFR specific ELISA, the soluble M-CSFR level in serum and urea from normal donors and patients with hematological diseases is reported. The average level of soluble M-CSFR in normal human individuals (n⫽102) was 0.457⫾0.303ng/ml. There was no statistical difference in soluble M-CSFR levels between males and females groups or between the different age groups. The average levels of soluble M-CSFR in the serum from ALL and AML patients were 0.184⫾0.368 ng/ml (n⫽36) and 0.124ng/ml⫾0.223ng/ ml (n⫽60), which were much lower than normal human (p⫽ 0.0002 and p⬍0.0001, respectively). For the patients with benign hematological disease, the serum levels of M-CSFR in IDA (n⫽ 25) and ITP patients (n⫽34) were slightly higher than normal individuals (0.662⫾0.468ng/ml and 0.828⫾0.960ng/ml respectively), but no statistical difference (p⫽0.05 and p⫽0.06) was found. Using our ELISA, the soluble M-CSFR in the urea of normal human (n⫽109) and hematological patients was also investigated. The results showed that the urea M-CSFR level of patients with malignant hematological disease (n⫽35) was significantly lower than that of normal donor (0.291⫾0.502ng/ml versus 0.894⫾0.715ng/ml, p⫽0.0007). In contrast, the urea M-CSFR level of patients with benign hematological disease (n⫽33) was higher than that of normal adult (6.98⫾13.14ng/ml versus 0.894⫾0.715ng/ml, p⫽0.01). Result of the urea M-CSFR levels is consistent with that of serum. It has been reported that detectable levels of some soluble receptors can be measured in normal individuals and in some diseases. Our results showed that soluble M-CSFR exited in normal human serum and urea. The function of soluble M-CSFR and whether the abnormalities of soluble M-CSFR level in serum and urea may contribute to leukemia or other hematological diseases should further be verified. 4
Sunday, July 9, 2000 (14:15–16:00) Session II-3: Signal Transduction
SIGNAL TRANSDUCTION FROM MEMBRANE-BOUND MACROPHAGE COLONY-STIMULATING FACTOR G.G. ZHENG*, K.F. WU, Y.Q. GENG*, AND Q. RAO* National Laboratory of Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, P.R. China Macrophage colony-stimulating factor (M-CSF) exists in different forms, which bind the same membrane receptor (M-CSFR),
due to alternative splicing and post-translational modifications. Membrane bound M-CSF (mM-CSF) was highly expressed in some malignancies including J6-1, J6-2 (leukemia), SMMC7721, BEL7402 (hepatoma) cell lines and specimens from patients with leukemia, Hodgkin’s Disease (HD), hepatoma, etc. (Zheng GG et al. Hematologica 1999, 84:951). Furthermore, we demonstrated that mM-CSF and its receptor played adhesion molecule-like roles in J6-1 cells and the activation of mM-CSF by recombinant soluble receptor (M-CSFsR) resulted in the decrease of cytoplasmic pH, mobilization of cytosolic calcium, tyrosine-phosphorylation on cytoplasmic protiens, etc. (Zheng GG et al. Leukemia Res 2000, in press). In the present study, we used cytochalasin B (CB) to study whether the integrity of cytoskeleton is necessary for mMCSF signaling. The results showed that one hour’s treatment of J6-1 cells by CB before exposed to M-CSFsR blocked the tyrosine-phosphorylation on cytoplasmic proteins with MW of 45 and 55-90kD, while decreased the sensitivity of cytoplasmic pH changes, but showed no obvious influence on cytosolic calcium mobilization. The initial activation process of most membrane proteins by their ligands involves dimerization or cluster of the membrane protein and cross-linking of the membrane protein by specific antibody can mimic the effects of their natural ligands, while dimerization or cluster is not indispensable for the signaling of some membrane proteins. We used a monoclonal antibody (McAb) B5, which was developed in our lab belonging to IgM subtype and recognizes N-terminal part of mM-CSF, to cross-link mM-CSF on J6-1 cell surface before underwent phospho-tyrosine Western blot analysis. While F(ab’)2 fragment of B5 caused no obvious tyrosine phosphorylation when compared with BSA control, 10 minutes’ treatment by B5 dose-dependently caused tyrosine phosphorylation on cytoplasmic proteins with MW of approximately 45 and 50-90kD, which mimicked the effects of M-CSFsR. The above results suggested that the initiation of mM-CSF signaling need dimerization/cluster of them on cell surface and cytoskeleton take part in some, but not all, of the downstream signal events. 5
Monday, July 10, 2000 (12:45–14:15) Session IV-2: Chronic Myelogenous Leukemia II
CML PATIENTS WITH ADVANCED PHASE DISEASE RETAIN CLONOGENIC PROGENITOR CELLS WHICH ARE RESPONSIVE TO INTERFERON ALFA AND STI571 IN VITRO MY Gordon, SB Marley, RJ Davidson, DX Nguyen, JL Lewis, JM Goldman LRF Centre, Department of Haematology, Imperial College School of Medicine, Hammersmith Hospital, London, UK We investigated if progenitor cells from CML patients with accelerating/transformed (AP) disease differ from chronic phase (CP) cells in their sensitivities to interferon(IFN)-␣ and STI571. We used a colony replating assay which measures the self-renewal capacity of clonogenic cells (CFU-GM). The results are expressed as the area-under-the-curve (AUC) of the cumulative distribution obtained by plotting the numbers of secondary colonies produced by replating individual primary colonies. The AUC for CP-CML CFU-GM is significantly greater than that for normal marrow CFU-GM (79.6–5.0 (mean–sem), n⫽90 vs 63–4.4, n⫽63; p⫽0.029, t test). CP CFU-GM exposed to pharmacological concentrations of IFN-␣ (50U/ml) or STI571 (0.1M) exhibit a reduced AUC (respectively 63–5% and 52–7% of control level; n⫽33 and 30) but