Oncostatin M is a differentiation factor for myeloid leukemia cells.

Oncostatin M (OSM) is a 28-kDa glycoprotein produced by stimulated macrophages and T lymphocytes that inhibits the proliferation of a number of different cell lines derived from solid tumors. Analysis of both amino acid sequence and gene structure has demonstrated that OSM is a member of a cytokine family that includes leukemia inhibitory factor (LIF), IL-6, and granulocyte colony-stimulating factor (G-CSF). We demonstrate that, like LIF, IL-6 and G-CSF, OSM can induce the differentiation of the myeloblastic M1 murine leukemia cells into macrophage-like cells. The morphologic and functional changes induced by OSM are more similar to those observed with LIF and IL-6 than those induced with G-CSF. OSM can also induce the differentiation of the histiocytic U937 human leukemia cells in the presence of granulocyte-macrophage CSF, a property shared with LIF and IL-6. In murine M1 cells, binding of labeled OSM is completely inhibited by excess LIF or OSM, reflecting the binding of OSM to the high affinity form of the murine LIF receptor. In contrast, the binding of labeled OSM to human U937 leukemia cells is inhibited by OSM, but the inhibition by LIF is significantly less. These results suggest that, in human leukemia cells, OSM may act through the LIF receptor and an OSM-specific receptor. The existence of an OSM-specific receptor was confirmed by both growth inhibition and competition binding assays on A375 human melanoma cells. The growth of human A375 cells was inhibited by OSM and IL-6 but not LIF or G-CSF. Neither LIF, G-CSF, nor IL-6 could compete with the binding of labeled OSM to A375 cells.     

Biological Properties of Melanoma and Endothelial Cells after Plasmid AMEP Gene Electrotransfer Depend on Integrin Quantity on Cells

The data on the biological responsiveness of melanoma and endothelial cells that are targeted by Antiangiogenic MEtargidin Peptide (AMEP) are limited; therefore, the antiproliferative, antimetastatic and antiangiogenic effects of AMEP were investigated in murine melanoma and human endothelial cells after plasmid AMEP gene electrotransfer into the cells in vitro. Plasmid AMEP, a plasmid coding for the disintegrin domain of metargidin targeting specific integrins, had cytotoxic and antiproliferative effects on murine melanoma and human endothelial cells. Among the metastatic properties of cells, migration, invasion and adhesion were investigated. Plasmid AMEP strongly affected the migration of murine melanoma and human endothelial cell lines and also affected the invasion of highly metastatic murine melanoma B16F10 and human endothelial cell lines. There was no effect on cell adhesion on Matrigel^TM or fibronectin in all cell lines. The antiangiogenic effect was shown with tube formation assay, where human microvascular endothelial cell line (HMEC-1) proved to be more sensitive to plasmid AMEP gene electrotransfer than the human umbilical vein endothelial cell line (HUVEC). The study indicates that antiproliferative and antimetastatic biological responses to gene electrotransfer of plasmid AMEP in murine melanoma cells were dependent on the integrin quantity on melanoma cells and not on the expression level of AMEP. The strong antiangiogenic effect expressed in human endothelial cell lines was only partly dependent on the quantity of integrins and seemed to be plasmid AMEP dose dependent.     

Long-term in vitro culture of murine mast cells. III. Discrimination of mast cells growth factor and granulocyte-CSF

Long-term in vitro growth of murine mast cells was dependent on the presence of a mast cell growth factor (MCGF) present in media conditioned by mitogen-activated splenic leukocytes or by various murine leukemic cell lines. MCGF shared a number of properties with granulocyte colony-stimulating factor (G-CSF). Both factors were present in media conditioned by the myelomonocytic leukemic WEHI-3 and the T cell lymphoma, LBRM-33 cell lines. They were relatively sensitive to trypsin treatment, and were resistant to boiling temperature. NZB mice that failed to respond to WEHI-3-derived G-CSF also failed to respond to MCGF. MCGF differed from G-CSF, however, in sensitivity to neuraminidase and lactoferrin, an inhibitor of macrophage CSF production, suppressed G-CSF production by WEHI-3 cells without affecting MCGF production. Furthermore, peritoneal cells produced G-CSF but not MCGF when stimulated with lipopolysaccharide. In vitro production of MCGF by normal spleen cells required the presence of T lymphocytes and is relatively macrophage-independent. The role of T cells in the maturation and growth of mast cells and the physiologic function of MCGF are discussed.     

Expression cloning of a receptor for murine granulocyte colony-stimulating factor

Two cDNAs encoding the receptor for murine granulocyte colony-stimulating factor (G-CSF) were isolated from a CDM8 expression library of mouse myeloid leukemia NFS-60 cells, and their nucleotide sequences were determined. Murine G-CSF receptor expressed in COS cells could bind G-CSF with an affinity and specificity similar to that of the native receptor expressed by mouse NFS-60 cells. The amino acid sequence encoded by the cDNAs has demonstrated that murine G-CSF receptor is an 812 amino acid polypeptide (Mr, 90,814) with a single transmembrane domain. The extracellular domain consists of 601 amino acids with a region of 220 amino acids that shows a remarkable similarity to rat prolactin receptor. The cytoplasmic domain of the G-CSF receptor shows a significant similarity with parts of the cytoplasmic domain of murine interleukin-4 receptor. A 3.7 kb mRNA coding for the G-CSF receptor could be detected in mouse myeloid leukemia NFS-60 and WEHI-3B D+ cells as well as in bone marrow cells.     

Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic differentiation from the murine cell line 32D

Erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor- (G-CSF) dependent cell lines have been derived from the murine hematopoietic cell line 32D with a selection strategy involving the culture of the cells in FBS-deprived medium supplemented only with pure recombinant Epo, GM-CSF, or G-CSF. The cells retain the diploid karyotype of the original 32D clone, do not grow in the absence of exogenous growth factor, and do not induce tumors when injected into syngeneic recipients. The morphology of the Epo-dependent cell lines (32D Epo1, -2, and -3) was heterogeneous and evolved with passage. The percent of differentiated cells also was a function of the cell line investigated. Benzidine-positive cells ranged from 1-2% (32D Epo3) to 50-60% (32D Epo1). These erythroid cells expressed carbonic anhydrase I and/or globin mRNA but not carbonic anhydrase II. The GM-CSF- and G-CSF-dependent cell lines had predominantly the morphology of undifferentiated myeloblasts or metamyelocytes, respectively. The GM-CSF-dependent cell lines were sensitive to either GM-CSF or interleukin-3 (IL-3) but did not respond to G-CSF. The G-CSF-dependent cell lines grew to a limited extent in IL-3 but did not respond to GM-CSF. These results indicate that the cell line 32D, originally described as predominantly a basophil/mast cell line, has retained the capacity to give rise to cells which proliferate and differentiate in response to Epo, GM-CSF, and/or G-CSF. These cells represent the first nontransformed cell lines which can be maintained in growth factors other than IL-3 and which differentiate in the presence of physiologic signals. As such, they may represent a model to study the molecular mechanisms underlying the process of hematopoietic differentiation, as well as sensitive targets for bioassays of specific growth factors.     

Dissection of murine lymphocyte-endothelial cell interaction mechanisms by SV-40-transformed mouse endothelial cell lines: novel mechanisms mediating basal binding, and alpha 4-integrin-dependent cytokine-induced adhesion.

Lymph node-derived endothelial cells were immortalized by infection with SV40 virus and subclones expressing the marker MECA 325 specific for high-endothelial venules (HEV) were selected. These transformed mouse endothelial (TME-) cell lines grow permanently without requirement for special growth factors. Staining of the selected clones with endothelium-specific antibodies and with anti-von Willebrand factor antiserum and uptake of acetylated low-density lipoprotein provide evidence for their endothelial origin. The vascular addressins identified by mAbs MECA 79 and MECA 367 on HEV are not detectable, indicating that the phenotype of the cells differs from that of HEV-type endothelium. The TME cells display a constitutive capacity to bind lymphocytes. An additional binding component is induced by treatment of the TME cells with TNF alpha. Antibodies against the homing receptor LECAM-1 (lectin-related leucocyte-endothelial cell adhesion molecule 1), alpha 4-integrins, vascular addressins, LFA-1, or ICAM-1 known to block lymphocyte interaction with particular types of HEV were unable to inhibit the basal adhesion to TME cells, indicating that a further binding mechanism in mice is displayed by this cell type. The adhesion component induced by TNF alpha is mediated by alpha 4-integrins since enhanced binding could be blocked by an antibody against mouse alpha 4 (lymphocyte-Peyer's patch adhesion molecule 1/2). TME cell lines therefore seem to be a useful model for the dissection and analysis of hitherto poorly characterized murine lymphocyte/endothelial cell interaction mechanisms.     

鼠粒细胞集落刺激因子受体的纯化及鉴定

粒细胞集落刺激因子受体(G-CSFR)在鼠NFS-60细胞中有较高的含量,通过对NFS-60细胞的大规模培养,用CHAPS及超速离心抽提G-CSFR, 经G-CSF亲和层析纯化获得G-CSFR, 采用ABC-ELISA进行鉴定.     

Differential role of Rho GTPases in endothelial barrier regulation dependent on endothelial cell origin

From studies using macrovascular endothelium, it was concluded that Rho A activation generally leads to endothelial barrier breakdown. Here, we characterized the role of Rho GTPases in endothelial barrier regulation in four different cell lines, both microvascular and macrovascular. Rho A activation by cytotoxic necrotizing factor y (CNFy) induced stress fiber formation in all cell lines. This was paralleled by gap formation and barrier breakdown in microvascular mesenteric endothelial cells (MesEnd), human dermal microvascular endothelial cells (HDMEC) as well as in macrovascular pulmonary artery endothelial cells (PAEC) but not in microvascular myocardial endothelial cells (MyEnd). In MyEnd cells, activation of Rac 1 and Cdc42 by CNF-1 strengthened barrier properties whereas in MesEnd, HDMEC and PAEC all three GTPases were activated which increased permeability in PAEC but not in MesEnd and HDMEC. In PAEC, CNF-1-induced decrease of barrier properties was blocked by the Rho kinase inhibitor Y27632 indicating that co-activation of Rho A dominated the barrier response. Inactivation of Rac 1 by toxin B or by lethal toxin (LT) compromised barrier properties in all cell lines. Taken together, Rac 1 requirement for endothelial barrier maintenance but not the destabilizing role of Rho A seems to be ubiquitous. Y. Baumer and S. Burger contributed equally.     

Granulocyte colony-stimulating factor and its receptor in normal hematopoietic cell development and myeloid disease

Hematopoiesis, the process of blood cell formation, is orchestrated by cytokines and growth factors that stimulate the expansion of different progenitor cell subsets and regulate their survival and differentiation into mature blood cells. Granulocyte colony-stimulating factor (G-CSF) is the major hematopoietic growth factor involved in the control of neutrophil development. G-CSF is now applied on a routine basis in the clinic for treatment of congenital and acquired neutropenias. G-CSF activates a receptor of the hematopoietin receptor superfamily, the G-CSF receptor (G-CSF-R), which subsequently triggers multiple signaling mechanisms. Here we review how these mechanisms contribute to the specific responses of hematopoietic cells to G-CSF and how perturbations in the function of the G-CSF-R are implicated in various types of myeloid disease.     

Thalidomide analogues demonstrate dual inhibition of both angiogenesis and prostate cancer

The identification of agents with antiproliferative activity against endothelial cells has significant value for the treatment of many angiogenesis-dependent pathologies. Herein, we describe the discovery of a series of thalidomide analogues possessing inhibitory effects against both endothelial and prostate cancer cells. More specifically, several analogues exhibited low micromolar to mid-nanomolar potency in the inhibition of human microvascular endothelial cell (HMEC) proliferation, both in the presence and absence of vascular endothelial growth factor (VEGF), with the tetrafluorophthalimido class of compounds demonstrating the greatest potency. Additionally, all the compounds were screened against two different androgen independent prostate cancer cell lines (PC-3 and DU-145). Again, the tetrafluorophthalimido analogues exhibited the greatest effect with GI(50) values in the low micromolar range. Thalidomide was found to demonstrate selective inhibition of androgen receptor positive LNCaP prostate cancer cells. Furthermore, we showed that, as an example, tetrafluorophthalimido analogue 19 was able to completely inhibit the prostate specific antigen (PSA) secretion by the LNCaP cell line, while thalidomide demonstrated a 70% inhibition. We have also demonstrated that a correlation exists between HMEC and prostate cancer cell proliferation for this structural class. Altogether, our study suggests that these analogues may serve as promising leads for the development of agents that target both androgen dependent and independent prostate cancer and blood vessel growth.     

Hepatoma-derived growth factor is a pulmonary endothelial cell-expressed angiogenic factor

Hepatoma-derived growth factor (HDGF) was previously identified as a developmentally regulated cardiovascular and renal gene that is mitogenic for vascular smooth muscle and aortic endothelial cells. As reciprocal interactions of smooth muscle and endothelial cells are necessary for vascular formation, we examined whether HDGF plays a role in angiogenesis. According to immunohistochemistry, HDGF was highly expressed in endothelial cells of nonmuscularized, forming blood vessels of the fetal lung. HDGF was also expressed in endothelial cells of small (20 microm) mature arteries and veins. By Western immunoblotting, HDGF was highly expressed by human pulmonary microvascular endothelial cells in vitro. Adenoviral overexpression of HDGF was mitogenic for human pulmonary microvascular endothelial cells in serum-free medium, stimulating a 1.75-fold increase in bromodeoxyuridine (BrdU) uptake and a twofold increase in cell migration. With the chick chorioallantoic membrane (CAM), a biologic assay for angiogenesis, exogenous recombinant HDGF significantly stimulated blood vessel formation and a dose-dependent reorganization of cells within the CAM into a more compact, linear alignment reminiscent of tube formation. According to double immunostaining for endothelial cells with a transforming growth factor-betaII receptor antibody and BrdU as a marker of cell proliferation, exogenous HDGF selectively stimulated endothelial cell BrdU uptake. HDGF also activated specific ERK1/2 signaling and did not overlap with VEGF SAPK/JNK, Akt-mediated pathways. We conclude that HDGF is a highly expressed vascular endothelial cell protein in vivo and is a potent endothelial mitogen and regulator of endothelial cell migration by mechanisms distinct from VEGF.     

Age-associated impairment in TNF-alpha cardioprotection from myocardial infarction

Age-associated dysfunction in cardiac microvascular endothelial cells with impaired induction of cardioprotective platelet-derived growth factor (PDGF)-dependent pathways suggests that alterations in critical vascular receptor(s) may contribute to the increased severity of cardiovascular pathology in older persons. In vivo murine phage-display peptide library biopanning revealed a senescent decrease in cardiac microvascular binding of phage epitopes homologous to tumor necrosis factor-alpha (TNF-alpha), suggesting that its receptor(s) may be downregulated in older cardiac endothelial cells. Immunostaining demonstrated that TNF-receptor 1 (TNF-R1) density was significantly lower in the subendocardial endothelium of the aging murine heart. Functional studies confirmed the senescent dysregulation of TNF-alpha receptor pathways, demonstrating that TNF-alpha induced PDGF-B expression in cardiac microvascular endothelial cells of 4-mo-old, but not 24-mo-old, rats. Moreover, TNF-alpha mediated cardioprotective pathways were impaired in the aging heart. In young rat hearts, injection of TNF-alpha significantly reduced the extent of myocardial injury after coronary ligation: TNF-alpha, 7.9 +/- 1.9% left ventricular injury (n = 4) versus PBS, 16.2 +/- 7.9% (n = 10; P < 0.05). The addition of PDGF-AB did not augment the cardioprotective action of TNF-alpha. In myocardial infarctions of older hearts, however, TNF-alpha induced significant postcoronary occlusion mortality (TNF-alpha 80% vs. PBS 0%; n = 10 each, P < 0.05) that was reversed by the coadministration of PDGF-AB. Overall, these studies demonstrate that aging-associated alterations in TNF-alpha receptor cardiac microvascular pathways may contribute to the increased cardiovasular pathology of the aging heart. Strategies targeted at restoring TNF-alpha receptor-mediated expression of PDGF-B may improve cardiac microvascular function and provide novel approaches for treatment and possible prevention of cardiovascular disease in older individuals.     

Binding of 125I-labeled granulocyte colony-stimulating factor to normal murine hemopoietic cells

The binding of granulocyte colony-stimulating factor (G-CSF) to murine bone marrow cells was investigated using a radioiodinated derivative of high specific radioactivity which retained full biological activity. The binding was time- and temperature-dependent, saturable and highly specific. The apparent dissociation constant for the reaction was 60-80 pM at 37 degrees C and 90-110 pM at 4 degrees C, similar to that found for the binding of G-CSF to murine leukemic cells (WEHI-3B D+) and significantly higher than the concentration of G-CSF required to stimulate colony formation in vitro. Autoradiographic analysis confirmed the specificity of binding since granulocytic cells were labeled but lymphocytes, erythroid cells and eosinophils were not. Blast cells and monocytic cells were partially labeled, the latter at low levels. In the neutrophilic granulocyte series, grain counts increased with cell maturity, polymorphs being the most heavily labeled but all cells showed considerable heterogeneity in the degree of labeling. Combination of Scatchard analysis of binding with autoradiographic data indicated that mature granulocytes from murine bone marrow exhibited 50-500 G-CSF receptors per cell.     

Molecular cloning and expression of the cDNA encoding feline granulocyte colony-stimulating factor.

Both genomic DNA and cDNA of the feline granulocyte colony-stimulating factor (G-CSF) gene were cloned from CRFK cells. Southern blot analysis showed that the haploid genome contains a single copy of the G-CSF gene. The RT-PCR analysis of several feline cell lines revealed expression of G-CSF mRNA in response to lipopolysaccharide stimulation. Sequence analysis of genomic and cDNA clones indicated that the intron-exon junction structure is conserved between the human and the feline G-CSF genes. The G-CSF coding region encodes a predicted protein of 195 amino acids including a signal sequence of 21 amino acids. The feline G-CSF amino acid sequence shares a high degree of identity with the canine (90.8%), human (87.4%), ovine (83.9%), bovine (82.8%), porcine (80.5%), murine (70.7%) and rat (66.8%) G-CSF. The feline G-CSF expressed in insect cells using recombinant baculovirus vector was biologically active as measured in a proliferation assay using NFS-60 cells and an induction assay of leukocytes in cats.     

Expression profiles of micro RNA in proliferating and differentiating 32D murine myeloid cells

32D cells are murine myeloid cells that grow indefinitely in Interleukin-3 (IL-3). In these cells, the type 1 insulin-like growth factor (IGF-I) and granulocytic-colony stimulating factor (G-CSF) induce differentiation to granulocytes. 32D cells do not express insulin receptor substrate-1 (IRS-1) or IRS-2, docking proteins of the IGF-I receptor. Ectopic expression of IRS-1 in these cells inhibits differentiation, the cells become IL-3 independent and IGF-1 dependent and can form tumors in mice. 32D and 32D-derived cells offer a good model in which to study the expression profiles of Micro Rna (miR) related to sustained proliferation or differentiation. We present here the data obtained with miR micro-arrays and identify the miR that are regulated by IGF-1 or G-CSF and are associated with either differentiation or indefinite cell proliferation of 32D murine myeloid cells.     

Functional domains of the granulocyte colony-stimulating factor receptor.

The granulocyte colony-stimulating factor (G-CSF) receptor has a composite structure consisting of an immunoglobulin(Ig)-like domain, a cytokine receptor-homologous (CRH) domain and three fibronectin type III (FNIII) domains in the extracellular region. Introduction of G-CSF receptor cDNA into IL-3-dependent murine myeloid cell line FDC-P1 and pro-B cell line BAF-B03, which normally do not respond to G-CSF, enabled them to proliferate in response to G-CSF. On the other hand, expression of the G-CSF receptor cDNA in the IL-2-dependent T cell line CTLL-2 did not enable it to grow in response to G-CSF, although G-CSF could transiently stimulate DNA synthesis. Mutational analyses of the G-CSF receptor in FDC-P1 cells indicated that the N-terminal half of the CRH domain was essential for the recognition of G-CSF, but the Ig-like, FNIII and cytoplasmic domains were not. The CRH domain and a portion of the cytoplasmic domain of about 100 amino acids in length were indispensable for transduction of the G-CSF-triggered growth signal.     

Human microvascular endothelial cells immortalized with human telomerase catalytic protein: a model for the study of in vitro angiogenesis

Human microvascular endothelial cell-1 (HMEC-1) generated by transfection with SV40 large T antigen has been the prevailing model for in vitro studies on endothelium. However, the transduction of SV40 may lead to unwanted cell behaviors which are absent in primary cells. Thus, establishing a new microvascular endothelial cell line, which is capable of maintaining inherent features of primary endothelial cells, appears to be extremely important. Here, we immortalized primary human microvascular endothelial cells (pHMECs) by engineering the human telomerase catalytic protein (hTERT) into the cells. Endothelial cell-specific markers were examined and the angiogenic responses were characterized in these cells (termed as HMVECs, for human microvascular endothelial cells). We found that VEGF receptor 2 (Flk-1/KDR), tie1, and tie2 expression is preserved in HMVEC, whereas Flk-1/KDR is absent in HMEC-1. In addition, HMVEC showed similar angiogenic responses to VEGF as HMEC-1. Furthermore, the HMVEC line was found to generate a prominent angiogenic response to periostin, a potent angiogenic factor identified recently. The data indicate that HMVEC may serve as a suitable in vitro endothelium model.     

Thrombospondin 2 inhibits microvascular endothelial cell proliferation by a caspase-independent mechanism

The matricellular protein thrombospondin 2 (TSP2) regulates a variety of cell-matrix interactions. A prominent feature of TSP2-null mice is increased microvascular density, particularly in connective tissues synthesized after injury. We investigated the cellular basis for the regulation of angiogenesis by TSP2 in cultures of murine and human fibroblasts and endothelial cells. Fibroblasts isolated from murine and human dermis synthesize TSP2 mRNA and secrete significant amounts of immunoreactive TSP2, whereas endothelial cells from mouse lung and human dermis did not synthesize TSP2 mRNA or protein. Recombinant mouse TSP2 inhibited growth of human microvascular endothelial cells (HMVECs) mediated by basic fibroblast growth factor, insulin-like growth factor-1, epidermal growth factor, and vascular endothelial growth factor (VEGF). HMVECs exposed to TSP2 in the presence of these growth factors had a decreased proportion of cells in S and G2/M phases. HMVECs cultured with a combination of basic fibroblast growth factor, insulin-like growth factor-1, and epidermal growth factor displayed an increased proportion of nonviable cells in the presence of TSP2, but the addition of VEGF blocked this TSP2-mediated impairment of cell viability. TSP2-mediated inhibition of DNA synthesis by HMVECs in the presence of VEGF was not affected by the broad-spectrum caspase inhibitor zVAD-fmk. Similar findings were obtained with TSP1. Taken together, these observations indicate that either TSP2 or TSP1 can inhibit HMVEC proliferation by inhibition of cell cycle progression and induction of cell death, but the mechanisms responsible for TSP2-mediated inhibition of cell cycle progression are independent from those leading to cell death.