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Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disease of transformed hematopoietic progenitor cells. It is now clear that the chimeric bcr/abl P210(bcr/abl) fusion protein, which is generated by the reciprocal translocation t (9; 22), inhibits apoptosis and increase proliferation. P210(bcr/abl) plays a central role in the pathophysiology of CML. The purpose of this study was to construct a cell line model that bcr/abl expression can be regulated by Tet-off inducing-expression-system. The full-length b3a2 bcr/abl cDNA was subcloned into the pTRE2hyg expression vector to construct the pT2-P210 plasmid. 293 cells were firstly transfected with Tet-off plasmid and the clone that the Tet-off system can work effectively after transfected with pTRE2hyg-LUC was selected by luciferase activity assay. The pT2-P210 plasmid was then transfected into the selected clone and cells were then selected for hygromycin B and G418 resistance. The results showed that individual subclones expressing bcr/abl after withdrawing doxycycline were 293pT2-P210 cell line. In conclusion, selected 293pT2-P210 cells are cells that bcr/abl expression can be regulated by Tet-off inducing-expression-system. They are suitable thoroughly to study the function of bcr/abl fusion gene and its signal regulation mechanism.

The aim of this research was to understand the influence of rhG-CSF on the sphingosine kinase (SphK) activity of monocytes. The peripheral blood monocytes were collected from 6 peripheral blood progenitor cell donors on the fifth day of mobilization with rhG-CSF and from 5 blood donors' buffy coats. The mRNA expressions of monocyte G-CSF receptor and SphK were tested with RT-PCR. The changes of SphK activity of monocytes were assayed after being treated with rhG-CSF. The results showed that the two kinds monocytes collected from both blood donors and peripheral blood progenitor cell donors mobilized with rhG-CSF expressed mRNA of G-CSF receptor and SphK. The SphK activity of monocytes collected from blood donors was not changed significantly after being treated with rhG-CSF (P0.05). The SphK activity of monocytes collected from peripheral blood progenitor cell donors transiently increased by (39.6 - 87.2)% after being treated by means of rhG-CSF (P0.05) without obviously dose-dependent effect. It is concluded that the SphK activity of monocytes collected from peripheral blood progenitor cell donors can be activated by rhG-CSF.

The study was to understand the impact on the proliferation and cytotoxicity of donor's T cells during mobilization with rhG-CSF. The peripheral blood mononuclear cells (PBMNC) were collected from 15 donors before mobilization and on fifth day of mobilization with rhG-CSF. After the PBMNC were activated with 500 ng/ml of CD3 monoclonal antibody and 500 microg/ml of rhIL-2 for 96 hours, the activated T cells were collected for testing proliferation, cytotoxicity, Fas expression, perforin and Fas ligand (FasL) mRNA expression, the IFN-gamma concentration in the culture medium of the activated T cells was determined by radioimmunoassay. The results showed that the proliferation activity of T lymphocytes and the cytotoxicity of T cells activated with CD3 monoclonal antibody and rhIL-2 were reduced markedly after mobilization with rhG-CSF (P0.05). The Fas molecule expression in the activated T cells was very high both before and after mobilization with rhG-CSF (P0.10). The activated T cells expressed perforin mRNA and didn't express FasL mRNA both before and after mobilization with rhG-CSF. The concentration of IFN-gamma in the culture medium of the activated T cells decreased significantly after mobilization with rhG-CSF (P0.01). It is concluded that activity of proliferation and cytotoxicity of donor's T cells is impaired after mobilization with rhG-CSF.

Sphingosine 1-phosphate (S1P), which can be impacted by different growth factors through sphingosine kinase (SphK), is a bioactive lipid produced by metabolism of sphingolipid with various biological responses. Recombinant human granulocyte-colony-stimulating factor (rhG-CSF) have been used widely in peripheral blood stem/progenitor cell mobilization. This study was aimed to investigate the effects of rhG-CSF on S1P concentration in plasma of donors. The peripheral blood mononuclear cells and blood plasma were collected from 8 peripheral blood progenitor cell donors before mobilization and on the fifth day of mobilization with rhG-CSF. The SphK mRNA expression of blood mononuclear cells were detected by RT-PCR. The changes of S1P concentration were assayed with SphK enzyme catalyzed reaction. The results showed that both kinds blood mononuclear cells collected before and after rhG-CSF mobilization expressed SphK mRNA. The S1P concentration is low in donor's plasma both before and after mobilization with rhG-CSF, and there was no markedly change of S1P concentration in plasma before and after mobilization (P0.05). In conclusion, mobilization with rhG-CSF does not impact S1P concentration in donors' plasma.

[reaction: see text] Highly efficient rhodium-catalyzed asymmetric addition of arylboronic acids to aldehydes has been realized by using chiral spiro monophosphite ligands, affording diarylmethanols in excellent yields and good enantiomeric excesses.

To explore the effect of alphastatin on angiogenesis activity of human umbilical vein endothelial cells and mechanism thereof.Human umbilical vein endothelial cells of the line ECV304 was cultured. Hepatic growth factor (HGF) and alphastatin of the concentrations of 10, 100, or 1000 nmol/L were use in migration test ECV304 cells at logarithmic stage were inoculated in a 96-well plate. Alphastatin of different concentrations was added, 24, 48, 72, and 96 hours later MTT was added to detect the A(490) nm values with and draw a growth curve. Phorbol 12-myristate 13-acetate (PMA) was used as positive group. The formation of capillary-like structure was examined with fluorescent microscopy. RT-PCR was used to examine the expression of sphingosine kinase (SPK) mRNA in the ECV304 cells. ECV304 cells were treated with alphastatin of 10 nmol/L, 100 nmol/L, or 1000 nmol/L respectively. SPK activity was examined by using [gamma-(32)P] ATP.The number of migrated cells treated with alphastatin of the concentrations of 10, 100, and 1000 nmol/L were (103 +/- 4), (75 +/- 3), and (13 +/- 1) respectively, all significantly lower than that of the HGF group (131 +/- 4, all P0.05). The values at different time points of the ECV304 cells treated with alphastatin of different concentrations were not significantly different from that of the HGF group. The area of capillary-like structure of the PMA group was 2996 +/- 31 microm(2)/field, and those of the groups of alphastatin of the concentrations of 100 and 1000 nmol/L were 1509 +/- 30 microm(2)/field and 1301 +/- 20 microm(2)/field respectively, all significantly less than that of the PMA group (2996 +/- 31 microm(2)/field, all P0.05). However, there was not a dose-dependent manner in the inhibitive activity of alphastatin on the formation of capillary-like structure. RT-PCR showed that quantity of SPK mRNA product was amplified from the ECV304 cells. The S1P activity of the ECV304 treated with alphastatin was significantly lower than that of the control group, especially when the concentration of alphastatin was 100 nmol/L and 12 hours after treatment.Alphastatin shows a potent antiangiogenic property, which may be closely associated with the downregulation of endothelial cells SPK activity.

To explore the impact of mobilization with recombinant human granulocyte colony stimulated factor (rhG-CSF) on the migration and adhesive function and their related signal mechanism mediated by the CXCR4 and lymphocyte function antigen-1 (LFA-1) molecules on the surfaces of CD4(+) T cells.Before and at day 5 on rhG-CSF mobilization, the expression rates of CXCR4 and LFA-1 (CD11a) on CD4(+) T cells in the peripheral blood were detected by tricolor fluorescence labeling, and the migration and adhesive activities of CD4(+) T cells to stroma cell-derived factor 1 alpha (SDF-1 alpha) and intercellular adhesion molecule-1 (ICAM-1) were also tested.The expression of CXCR4 on CD4(+) T lymphocytes was (84.58 +/- 20.31)% before mobilization and (81.23 +/- 22.46)% at day 5 on mobilization. The expression of LFA-1 on CD4(+) T lymphocytes before and at day 5 on mobilization was 100%. There was no significant difference in the expression CXCR-4 and LFA-1 on CD4(+) T lymphocytes whether mobilization (P0.05). SDF-1 alpha induced 4 hours' CD4(+) T cells migration didn't change markedly before and after mobilization \[(28.5 +/- 10.3)% vs (31.2 +/- 8.9)%\] (P0.05). The adhesive activity of CD4(+) T cells to ICAM-1 was decreased from (85.59 +/- 14.21)% to (61.45 +/- 15.07)% after mobilization (P0.05).The expression of CXCR4 and LFA-1 on CD4(+) T lymphocytes didn't change markedly during rhG-CSF mobilization, but the adhesive activity of CD4(+) T cells to ICAM-1 was frustrated after that.

Shp-2, a ubiquitously expressed protein tyrosine phosphatase containing two Src homology 2 domains, plays an important role in integrating signaling from the cell surface receptors to intracellular signaling mechanisms. Previous studies have demonstrated that the Shp-2 is involved in hepatocyte growth factor (HGF)-induced cell scattering. Here we report that Shp-2 is required for the HGF-induced activation of sphingosine kinase-1 (SPK1), a highly conserved lipid kinase that plays an important role in cell migration. Loss-of-function mutation of Shp-2 did not affect the expression of SPK1, but resulted in its inactivation and the blockage of HGF-induced migration in embryonic fibroblasts. Reintroduction of functional wild type (WT) Shp-2 into the mutant cells partially restored SPK1 activation, and overexpression of SPK1 in these mutant cells enhanced HGF-induced cell migration. Inhibition of expression or activity of SPK1 in WT cells markedly decreased intracellular S1P levels and HGF-induced cell migration. Furthermore, we found that Shp-2 co-immunoprecipitated with SPK1 and c-Met in embryonic fibroblasts. These studies suggest that Shp-2 is an SPK1-interacting protein and that it plays an indispensable role in HGF-induced SPK1 activation.

Hepatocyte growth factor (HGF) is one of major growth factors in the bone marrow microenvironments with which the proliferation, differentiation and migration of bone marrow-derived mesenchymal stem cells were closely contacted. However, its roles in the regulation of proliferation, differentiation and migration of bone marrow-derived mesenchymal stem cells remain unclear. This study was aimed to investigate the effect of HGF on biological characteristics of bone marrow-derived mesenchymal stem cells. Expression of c-Met, the receptor for HGF was detected by immunohistochemistry assay, cell proliferation was determined by MTT, activity of ALP was quantitatively assayed, cell migration and anoikis-induced MSC apoptosis were analyzed. The results showed that HGF not influenced the proliferation and osteogenic differentiation of bone marrow-derived mesenchymal stem cells. Treatment of bone marrow-derived mesenchymal stem cells with recombinant human hepatocyte growth factor resulted in inhibition of anoikis-induced apoptosis. HGF significantly stimulated the migration of bone marrow-derived mesenchymal stem cells. Both PI-3 kinase and MAPK kinase were proved to be involved in HGF-induced migration. It is concluded that HGF/c-Met signal regulates the apoptosis and migration of bone marrow-derived mesenchymal stem cells.

The Ru(II) complexes of SDP and DPEN combined with t-BuOK in 2-propanol formed a very effective catalyst for the hydrogenation of simple aromatic ketones with high activity and enantioselectivity. The racemic alpha-arylcycloalkanones can also be hydrogenated by this system, providing alpha-arylcycloalkanols in excellent cis/trans stereoselectivity (>99:1) and enantioselectivity (up to 99.9%) for the cis isomer. In the study of the effect of the alkali metal cation in the hydrogenation of acetophenone using RuCl(2)(Tol-SDP)(DPEN) and RuCl(2)(Xyl-SDP)(DPEN) catalysts, we found that t-BuONa provided a faster reaction than t-BuOK, which indicated that the sterically hindered diphosphine ligands preferred the base with the smaller metal cation.

The proliferation and apoptosis of multiple myeloma (MM) cells were regulated by bone marrow microenvironments in which Notch signal plays important role in mediating cell-cell communication. However, the regulatory effect of Notch signal on the proliferation and apoptosis of multiple myeloma cells remains unclear. In this study, regulatory effect of Notch signal on the apoptosis of MM cells induced by DMS (N, N-dimethylsphingosine) was investigated. RT-PCR was used to identify the expression of Notch receptor and related molecules such as Dll-1, Jagged-1, Deltex-1 in MM cell lines. The intracellular domain of Notch (ICN), active form of Notch, was transferred into MM cells by retrovirus. The apoptosis of MM cells was determined by trypan blue exclusion tests and TdT-mediated dUTP nick end labeling (TUNEL) assay. The results showed that multiple myeloma cells expressed the Notch-1 and its related molecules. Notch activated multiple myeloma cell lines were obtained. Activation of Notch protected the multiple myeloma cells from the apoptosis induced by DMS,which was determined by cell viability and TUNEL assay. In conclusion, Notch signal suppressed the apoptosis of multiple myeloma cells and would possibly be a novel therapeutic target.

Recombinant adenoviral vectors have been widely applied for the basic research and clinical trials of gene therapy. However, the inability of adenovirus to infect hematopoietic cells which lack the specific adenovirus receptors-coxsackie virus and adenovirus receptor (CAR) represents an important limitation in therapeutic applications. This limitation may be overcome by several approaches including modification of adenovirus vector and improvement of the susceptibility of hematopoietic cells. The current progresses in this field were summarized.

Mesenchymal stem cells could differentiate into cardiomyocytes in vitro and have been shown to reconstitute the impaired myocardium in vivo. Hepatocyte growth factor, a recognized angiogenic factor and endothelial cell chemoattractant, has been applied in the treatment of myocardial ischemia. In this study, we used a ligation model of proximal left anterior descending coronary artery of rats to evaluate the effect of mesenchymal stem cells overexpressing hepatocyte growth factor in the treatment of myocardial ischemia. Bone marrow-derived mesenchymal stem cells were isolated, expanded, characterized, and infected with adenovirus carrying human hepatocyte growth factor cDNA (Ad-HGF). Mesenchymal stem cells infected by Ad-HGF released soluble HGF protein at a high level, which was maintained at least for 2 weeks. Implantation of mesenchymal stem cells overexpressing hepatocyte growth factor into left anterior descending risk areas improved the functions of impaired myocardium, including diminishing the area of ischemia, increasing the number of capillaries, and reducing collagen content. By using the sry gene as a marker, we also demonstrated that the engrafted cells or their progeny incorporated into ischemic cardiac muscle. These results showed that treatment of myocardial ischemia with bone marrow-derived mesenchymal stem cells overexpressing hepatocyte growth factor could be a novel strategy that can both restore local blood flow and regenerate lost cardiomyocytes.

Hepatocyte growth factor (HGF)-induced migration of endothelial cells is critical for angiogenesis. Sphingosine kinase (SPK) is a key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through both intracellular and extracellular mechanisms. The aim of this study was to investigate whether activation of SPK is involved in the migration of endothelial cells induced by HGF. The biological functions of HGF are mediated through the activation of its high-affinity tyrosine kinase receptor, c-met protooncogene. In the present study, Treatment of ECV304 endothelial cells with HGF resulted in tyrosine phosphorylation of c-Met and activation of SPK in a concentration-dependent manner. Either Ly294002 or PD98059, specific inhibitor of the PI3K and ERK/MAPK pathways, respectively, blocked the HGF-induced activation of SPK. HGF stimulation significantly increased intracellular S1P level, but no detectable secretion of S1P into the cell culture medium was observed. Treatment of ECV304 cells with pertussis toxin (PTX) has no effect on the HGF-induced migration, indicating extracellular S1P is dispensable for this process. Overexpression of wild-type SPK gene in ECV 304 cells increased the intracellular S1P and enhanced the HGF-induced migration, whereas inhibition of cellular SPK activity by N,N-dimethylsphingosine (DMS), a potent inhibitor of SPK, or by expression of a dominant-negative SPK (DN-SK) blocked the HGF-induced migration of ECV 304 cells. It is suggested that PI3K and ERK/MAPK mediated the activation of SPK and would be involved in the HGF-induced migration of endothelial cells. These results elucidate a novel mechanism by which intracellularly generated S1P mediates signaling from HGF/c-Met to the endothelial cell migration.

Sphingosine 1-phosphate, a bioactive sphingolipid produced from the metabolism of sphingomyelin, play important roles in diverse biological process, including cell proliferation, survival, cytoskeleton changes, migration, angiogenesis, wound healing and embryonic development. Here we review the role of sphingosine 1-phosphate in cell biological function regulation and signal transduction.

A highly efficient nickel-catalyzed asymmetric reductive coupling of dienes and aldehydes has been realized by using bulky spirobiindane phosphoramidite ligands, affording bishomoallylic alcohols in high yields with excellent diastereoselectivities and enantioselectivities.