Activation of cMGF expression is a critical step in avian myeloid leukemogenesis.

A non-leukemogenic version of the v-myb oncogene causes in vitro transformation of avian myeloblasts, which are dependent on chicken myelomonocytic growth factor (cMGF). We have shown that this version of v-myb, when combined with the erythroleukemia-inducing v-erbB oncogene, is capable of causing a mixed myeloid and erythroid leukemia. Myeloid leukemic cells transformed by this construct produce cMGF. To test whether autocrine growth stimulation via cMGF is the essential contribution of the tyrosine kinase oncogene v-erbB in avian myeloid leukemogenesis we constructed another retrovirus containing both the non-leukemogenic v-myb and the cMGF cDNA. This virus induced myeloid leukemia at high efficiency. In a third construct we combined v-myb with the human EGF-receptor gene. Myeloid cells transformed by this construct could be stimulated to grow by the addition of cMGF or EGF. Growth stimulation with EGF was blocked by a cMGF antiserum indicating that activation of a normal tyrosine kinase-type receptor induces cMGF expression but does not bypass the cMGF requirement. We conclude that cMGF plays a key role in the growth regulation of normal and transformed avian myeloid cells.     

Protective Effect of Avian Myelomonocytic Growth Factor in Infection with Marek’s Disease Virus

Marek's disease virus (MDV) is a herpesvirus that induces T lymphomas in chickens. The aim of this study was to assess the role of the macrophage activator chicken myelomonocytic growth factor (cMGF) in controlling MDV infection. B13/B13 chickens, which are highly susceptible to MD, were either treated with cMGF delivered via a live fowlpox virus (fp/cMGF) or treated with the parent vector (fp/M3) or were left as untreated controls. Seven days later, when challenged with the very virulent RB-1B strain of MDV, the spleens of chickens treated with fp/cMGF showed increased expression of the inducible nitric oxide synthase (iNOS) gene compared to those of control chickens and fp/M3-treated chickens. Increased iNOS gene expression was also accompanied by greater induction of gamma interferon and macrophage inflammatory protein (K203) gene expression, both possible activators of iNOS. fp/cMGF treatment also increased the number of monocytes and systemic NO production in contrast to fp/M3 treatment. Even though cMGF treatment was unable to prevent death for the chickens, it did prolong their survival time, and viremia and tumor incidence were greatly reduced. In addition, cMGF treatment improved the partial protection induced by vaccination with HVT (herpesvirus isolated from turkeys) against RB-1B, preventing 100% mortality (versus 66% with vaccination alone) and greatly reducing tumor development. Treatment with fp/M3 did not have such effects. These results suggest that cMGF may play multiple roles in protection against MD. First, it may enhance the innate immune response by increasing the number and activity of monocytes and macrophages, resulting in increased NO production. Second, it may enhance the acquired immune response, indicated by its ability to enhance vaccine efficacy.     

Molecular cloning of the chicken myelomonocytic growth factor (cMGF) reveals relationship to interleukin 6 and granulocyte colony stimulating factor.

Normal as well as retrovirally transformed avian myeloid precursor cells require the colony stimulating factor cMGF for their survival, proliferation and colony formation in vitro. cMGF has been shown to be a glycoprotein which is active in the picomolar concentration range. Co-expression of kinase type oncogenes in v-myb or v-myc transformed myeloid cells induces cMGF expression and confers factor independence via an autocrine mechanism. Here we describe the molecular cloning of cMGF from a myeloblast cDNA library and show that it is a 201 amino acid residue secretory protein which is modified by signal peptide cleavage and glycosylation during translocation into the lumen of membrane vesicles. A bacterially expressed trpE-cMGF fusion protein induces proliferation of E26 transformed myeloblasts in a cMGF bioassay suggesting that glycosylation is not absolutely necessary for biological activity. Sequence comparison reveals that cMGF is distantly related to G-CSF and IL-6.     

Temperature-sensitive mutants of MH2 avian leukemia virus that map in the v-mil and the v-myc oncogene respectively.

MH2 is an avian retrovirus that contains the v-mil and v-myc oncogenes. In vitro it transforms chick macrophages that are capable of proliferation in the absence of growth factor. Earlier work showed that v-myc induces macrophage transformation and that v-mil induces the production of chicken myelomonocytic growth factor (cMGF), thus generating an autocrine system. We describe the isolation of temperature-sensitive (ts) mutants of MH2 virus. As suggested by marker rescue experiments, one mutant bears a ts lesion in v-mil, whereas the other carries a mutation in v-myc. Ts v-mil MH2-transformed macrophages become factor-dependent at the non-permissive temperature (42 degrees C), while ts-v-myc MH2-transformed macrophages cease growing and acquire a more normal macrophage phenotype at 42 degrees C irrespective of the presence of cMGF. Both phenotypes can be reversed by backshift to the permissive temperature. These results suggest that the gene products of v-mil and v-myc function independently of each other and that v-mil is necessary for the maintenance of autocrine growth, whereas v-myc is required to maintain the transformed phenotype.     

Hematopoietic growth factor glycosylation. Multiple forms of chicken myelomonocytic growth factor

The production of chicken myelomonocytic growth factor (cMGF) can be rapidly induced by bacterial lipopolysaccharide from the macrophage cell line HD11. Immunoprecipitation analysis of lipopolysaccharide-induced HD11 cells labeled with various radioactive precursors showed the secretion of a variety of cMGF forms. The precursor-product relationships of the different cMGF forms were studied by pulse-chase experiments, by long-term metabolic labeling in the presence or absence of glycosylation- and oligosaccharide-processing inhibitors, as well as by glycosidase treatment of immunoprecipitates. Our results show that the half-time for intracellular processing/secretion is less than 10 min, making cMGF one of the most rapidly processed proteins. The different forms of the factor are generated from a 24-kDa polypeptide precursor by co- and post-translational acquisition of one or two N-linked oligosaccharides and by O-linked glycosylation. In addition, a fraction of cMGF is modified by long chain, chondroitinase-sensitive, sulfated glycans. This modification is tunicamycin-sensitive, suggesting that the sulfated glycans are attached to N-linked rather than to O-linked oligosaccharides.     

v-mil induces autocrine growth and enhanced tumorigenicity in v-myc-transformed avian macrophages

MH2, an avian retrovirus containing the v-myc and v-mil oncogenes, rapidly transforms chick hematopoietic cells in vitro. The transformed cells belong to the macrophage lineage and proliferate in the absence of exogenous growth factors. Here we analyze a series of MH2 deletion mutants and show that these two oncogenes together establish an autocrine growth system in which v-myc stimulates cell proliferation, while v-mil induces the production of chicken myelomonocytic growth factor (cMGF). We also demonstrate that these two oncogenes cooperate in vivo. MH2 efficiently induces monocytic leukemias and liver tumors, while deletion mutants lacking either a functional v-mil or v-myc do not.     

B cell growth modulating and differentiating activity of recombinant human 26-kd protein (BSF-2, HuIFN-beta 2, HPGF).

The human "26-kd protein' is a secreted glycoprotein expressed, for example, in (blood) leukocytes, in epithelial cells treated with various inducers, but most strongly in interleukin-1 (IL-1)-treated fibroblasts. After finding it has antiviral and 2-5A synthetase-inducing activity, one group of authors called this protein IFN-beta 2. However, recently the full-length 26-kd cDNA sequence was shown to be identical with that of a B-cell-differentiating lymphokine called BSF-2, and another report suggested that the 26-kd protein could support the growth of some transformed murine B cell lines. To define its biological activities, we expressed the recombinant 26-kd protein by translating in Xenopus laevis oocytes a pure, synthetic chimeric mRNA containing the 26-kd protein coding region surrounded by Xenopus laevis beta-globin untranslated regions. A similar construction, but containing the HuIFN-beta cDNA coding region, was used to produce HuIFN-beta by the same procedure. Both recombinant glycoproteins were secreted, glycosylated, and their amounts were measured by [35S]methionine incorporation by the oocyte. Here we show that the recombinant 26-kd protein exhibits a high growth factor activity when assayed on an IL-HP1-dependent murine B cell hybridoma (sp. act. approximately 2 X 10(8) U/mg) as well as a potent differentiating activity on human CESS cells (sp. act. approximately 5 X 10(7) U/mg). While rHuIFN-beta was inactive in the latter two assays, it had the expected antiviral activity of 1-5 X 10(8) U/mg. The parallel recombinant 26-kd protein preparations had no detectable antiviral activity (i.e. a maximal specific activity of 1-3 X 10(2) U/mg, if any). The 26-kd protein is thus clearly an interleukin, and considering the confusing nomenclature now in use, this factor may better be renamed "interleukin 6'.     

Isolation of human type X collagen and immunolocalization in fetal human cartilage

Type X collagen was extracted with 1 M NaCl and 10 mM dithiothreitol at neutral pH from fetal human growth plate cartilage and purified to homogeneity by gel filtration and anion-exchange chromatography. The purified protein migrates in SDS/polyacrylamide gels with an apparent Mr of 66,000 under reducing conditions, and as a high-Mr oligomer under non-reducing conditions. Purified collagenase digests most of the molecule; pepsin digestion at 4 degrees C decreases the Mr of the monomer to 53,000. A rabbit antiserum was raised against purified human type X collagen; the IgG fraction was specific for this collagen by criteria of ELISA and immunoblotting after absorption with collagen types I, II, VI, IX and XI. Immunohistological studies localized type X collagen exclusively in the zone of hypertrophic and calcifying cartilage.     

Reconstitution into liposomes of membrane proteins involved in ribosome binding on rough endoplasmic reticulum. Ribosome-binding capacity.

Platelet-derived growth factor was purified from fresh platelets by a large-scale procedure not involving the use of SDS (sodium dodecyl sulphate). The product, 0.5 mg of platelet-derived growth factor, obtained from about 3 x 10(13) platelets migrated as a single component in analytical gel electrophoresis in the presence of SDS and showed no inhomogeneity on sedimentation-equilibrium analysis in the ultracentrifuge. It had a high specific activity, 2 ng of platelet-derived growth factor/ml (70pM) being equivalent to 1% (v/v) human serum in an assay for multiplication-stimulating activity. Amino acid analysis revealed that platelet-derived growth factor contains all the common amino acids, except tryptophan, but no hexosamine. The molecular weight of platelet-derived growth factor, as determined by sedimentation-equilibrium analysis, was about 33 000. A similar value was obtained by gel electrophoresis in SDS under non-reducing conditions. In the presence of reducing agents the factor molecule was converted into two distinct components of lower molecular weight (17 000 and 14 000 respectively), as demonstrated by protein staining. The molecular model implicated by these findings is that platelet-derived growth factor consists of two different polypeptides chains, linked by disulphide bridges.     

Purification of a novel growth inhibitory factor for partially differentiated myeloid leukemic cells

A novel factor termed growth inhibitory (GI) factor, which specifically inhibits the growth of mouse monocytic leukemia cells including monocytic cell lines (Mm-A and J774.1) and other partially differentiated myeloid leukemic cells, has been purified from conditioned medium of some clones of mouse myeloblastic leukemia M1 cells. The procedure for purification of the GI factor included ammonium sulfate precipitation, CM-Sepharose CL-6B and Sephadex G-200 chromatographies, reverse-phase high-performance liquid chromatography on a C18 hydrophobic support, and high-performance liquid chromatography on a gel filtration column. The purified factor gave a single band of protein with a molecular weight of 25,000 on sodium dodecyl sulfate-polyacrylamide gel. A concentration of 8 X 10(-10) M GI factor was required for 50% inhibition of growth of Mm-A cells. On chromatofocusing, the GI activity was eluted with Polybuffer 96/acetic acid at pH 8.2-8.4. The purified GI factor markedly inhibited growth of mouse bone marrow cells stimulated by macrophage colony-stimulating factor. The GI factor appeared to be a unique cytokine unrelated to known cytokines such as the tumor necrosis factor, interferons, and oncostatin M.     

Purification of melanoma growth stimulatory activity

The Hs0294 human malignant melanoma cell line produces a monolayer mitogen that stimulates the serum free growth of low-density cultures of Hs0294 cells. This report describes the purification of that mitogen, termed MGSA for melanoma growth stimulatory activity, from serum-free conditioned medium from the Hs0294 cultures. MGSA has been purified from acetic acid extracts of lyophilized conditioned medium by gel filtration, reverse-phase high-pressure liquid chromatography (RP-HPLC), and preparative electrophoresis, resulting in a greater than 400,000-fold purification. MGSA bioactivity resides in acid- and heat-stable polypeptides of high and low molecular weight (24-28 kd and less than 14-16 kd). However, the majority of the activity is reproducibly associated with the approximately 16-kd moiety eluting from RP-HPLC at approximately 35% acetonitrile. Reduction with dithiothreitol or B-mercaptoethanol results in a loss of biological activity but does not convert the 24-28-kd moieties to the less than 14-16-kd forms of MGSA. 125I-MGSA that has been purified by preparative electrophoresis (16 kd) specifically binds to Hs0294 melanoma cells and retains 100% of the growth-stimulatory activity. The 16-kd MGSA stimulates the proliferation of Hs0294 cells at concentrations of 0.3-30 pM. The electrophoretic mobility of MGSA is also unaltered by the preparative electrophoresis procedure, further demonstrating that this procedure does not alter the biochemical integrity of the growth factor. Purified MGSA does not enable anchorage-independent growth of normal rat kidney (NRK) cells and is therefore different from the previously described transforming growth factors. The amino acid composition of MGSA differs from that of other previously described growth factors. These data demonstrate that MGSA represents a separate class of growth factors with biological and biochemical properties different from other growth factors.     

Purification and characterization of natural and recombinant human plasminogen activator inhibitor-1 (PAI-1)

Human plasminogen activator inhibitor-1 (PAI-1) was purified from the conditioned medium of endotoxin-stimulated umbilical vein endothelial cell cultures by combinations of zinc-chelate-Sepharose chromatography, gel filtration on Sephacryl S-300 and immunoadsorption on an insolubilized murine monoclonal antibody (MA-7D4). The final product was obtained with a recovery of approximately 20% from conditioned medium containing about 3 micrograms/ml PAI-1. The yield of PAI-1 was 15-100 micrograms/umbilical cord, depending on the culture and harvest conditions. SDS gel electrophoresis revealed a main band with Mr = 46,000 both under reducing and non-reducing conditions. On gel filtration on Sephacryl S-300, however, the material was separated in two fractions, one eluting at the void volume, which contains active PAI-1, and one with Mr = 46,000 containing inactive material that could be reactivated with 12 M urea. SDS gel electrophoresis of the isolated high-Mr fraction revealed several bands including a main 46,000-Mr component, which reacted with anti-(PAI-1) antibodies on immunoblotting and neutralized tissue-type plasminogen activator (t-PA). The active high-Mr fraction and the reactivated low-Mr fraction of PAI-1 inhibited t-PA very rapidly with an apparent second-order rate constant of (1.5-4) x 10(7) M-1 s-1. The cDNA of endothelial cell PAI-1 was cloned and expressed in Chinese hamster ovary cells. The translation product, purified from conditioned medium of transfected cells, also revealed a high-Mr and a low-Mr fraction on gel filtration, which were indistinguishable from the natural proteins by physicochemical, immunochemical and functional analysis. On reduced SDS gel electrophoresis, the high-Mr fraction was separated into the Mr-46,000 low-Mr PAI-1 and two other components with Mr 65,000 and one barely entering the gel. When reactivated low-Mr PAI-1 was added to plasma, PAI activity and PAI-1 antigen eluted with an apparent Mr greater than or equal to 300,000 on gel filtration, indicating that active PAI-1 complexes with one or more binding proteins in plasma.     

Ionic responses rapidly elicited by porcine platelet-derived growth factor in Swiss 3T3 cells.

Addition of porcine platelet-derived growth factor (PDGF) to quiescent cultures of Swiss 3T3 cells caused a marked, dose-dependent stimulation of Na+ influx and Na-K pump-mediated 86Rb+ uptake. Porcine PDGF (a single component in SDS polyacrylamide gels) stimulated ion fluxes to the same maximal extent as partially purified preparations, and exhibited half-maximal effect at 6 ng/ml (2 X 10(-10) M). Maximal effect was achieved at 30 ng/ml (10(-9) M). In the presence of insulin, PDGF elicited mitogenesis at comparable concentrations. PDGF stimulated ion uptake in a time-dependent fashion; maximal effect was obtained after 5 min of exposure to the growth factor. PDGF stimulates Na+ influx via an amiloride-sensitive pathway, suggesting that PDGF enhances the activity of a Na+/H+ antiport system. In accordance with this possibility, the mitogen caused an increase of intracellular pH by 0.15 pH units, as judged by the steady-state distribution of labelled 5,5-dimethyloxazolidine-2,4-dione (DMO). Porcine PDGF stimulated E-type prostaglandin synthesis and cAMP accumulation but these events could be dissociated from the stimulation of the ionic fluxes, which was detected within minutes and was not blocked by indomethacin. It is suggested that PDGF elicits multiple signals to stimulate cell proliferation in 3T3 cells.     

Replacement of lys 622 in the ATP binding domain of P100gag-mil abolishes the in vitro autophosphorylation of the protein and the biological properties of the v-mil oncogene of MH2 virus.

Lysine 622 in the ATP-binding domain of P100gag-mil, the translation product of the v-mil oncogene of MH2, has been replaced with methionine using oligonucleotide site-directed mutagenesis. This substitution results in the inactivation of the serine/threonine-specific autophosphorylation of P100gag-mil in vitro, indicating that this activity is an intrinsic property of the viral protein. This substitution also suppresses two of the biological properties of MH2 which have previously been shown to be dependant upon the expression of v-mil, namely, the production of chicken myelomonocytic growth factor (cMGF) by v-myc-transformed chicken macrophages and the sustained proliferation of chicken neuroretina cells. These data strongly suggest that the biological properties of v-mil are mediated by the phosphorylation at serine/threonine residues of key cellular substrates. In contrast to the in vitro situation, both the mutant and wild-type proteins appear to be phosphorylated at the same sites and to the same extent in either transformed fibroblasts or macrophages. This, together with the fact that the sites phosphorylated in vivo and in vitro are essentially different indicate that most of the phosphate associated with P100gag-mil in transformed cells does not result from an obligate autophosphorylation event but from the phosphorylation by as yet uncharacterized cellular kinase(s).     

Diamine and polyamine oxidase activities in normal and transformed cells

1. The activity of diamine oxidase (EC 1.4.3.6) in normal rat kidney cells and in normal rat kidney cells transformed by avian sarcoma virus (B77 strain) growing in tissue culture varies with the stage of growth. There is an initial stimulation of activity by 24h after seeding, followed by a steep decline during exponential growth (48-72h). Enzyme activity decreases even further as the cells reach saturation density (confluence) after 4 days in culture when the activity in normal rat kidney cells is twice as high as that in transformed cells. 2. Differences of about the same order of magnitude are observed between transformed human cells HeLa, HEp2 (a human epithelioid carcinoma) and normal human fibroblasts, in chicken cells between normal myeloblasts and leukaemic myeloblasts, and in rats between biopsy material from normal mammary tissue and 9,10-dimethylbenz[a]anthracene-induced mammary tumours. 3. Polyamine oxidase activity also varies with the growth of transformed rat kidney cells, but shows no significant variation with the growth of normal rat kidney cells between 24 and 96h after seeding. The activity in cells at confluence is from 3- to 5-fold lower in the transformed than in the normal rat kidney cells. 4. A similar 5-10-fold decrease in activity has been found in 9,10-dimethylbenz[a]anthracene-induced mammary tumours in rats and in human oesophageal tumours. 5. Possible reasons for these observations and the contribution of these two enzymes to cellular putrescine concentrations are discussed.     

巨噬细胞源性神经营养因子的纯化和鉴定

Macrophage-derived neurotrophic factor (M phi DNF) is purified from macrophage conditioned medium by a procedure consisting of column chromatography with Sephacryl S-100-HR, high-performance liquid chromatography (HPLC), and a final step using reverse-phase HPLC. The product shows a single protein band in sodium dodecyl sulfate-polyacrylamide gel. It has a molecular weight of 60.5 kD and an isoelectric point of pI 5.1 and contains more leucine, lysine, glutamine and aspartic acids in its amino acid composition. Purified M phi DNF can promote the survival, activity, and neurite outgrowth of cultured cerebellar cortical neurons and that this effect reaches maximal levels with concentrations of the M phi DNF ranging from 500-1000 ng/ml.     

Blood coagulation induced by Bothrops atrox venom: identification and properties of a factor X activator

The coagulating activity of Bothrops atrox venom was investigated in vitro with purified fibrinogen, with normal plasma and with plasma deficient in various factors of the coagulation cascade. This study indicated that Bothrops atrox venom possesses a thrombin-like action caused by one or several serine proteases sensitive to DFP treatment, but that its clotting action is in fact mainly due to components insensitive to DFP which activate prothrombin and factor X (Stuart factor). We partially purified the DFP insensitive activator of factor X from Bothrops atrox venom and found it to be a protein of molecular weight 77,000. Analysis of the purified fraction by electrophoresis on polyacrylamide gel in the presence of SDS showed that it is mainly composed of one heavy polypeptide chain (65,000) and one light doublet (12 - 13,000). This activator is calcium-dependent and catalyzes in vitro the conversion of purified factor X into factor X alpha. By its molecular properties, it resembles the factor X activator from Vipera russellii venom rather than physiological activators.