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.     

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.     

Purification and characterization of cMGF, a novel chicken myelomonocytic growth factor.

We describe the purification of a novel hematopoietic growth factor from conditioned medium of a transformed macrophage cell line. The factor, termed chicken myelomonocytic growth factor (cMGF) stimulates the growth of chicken myeloblasts transformed by myb oncogene-containing retroviruses and induces the formation of macrophage colonies in uninfected chick bone marrow cultures. The biological activity of the factor is destroyed by trypsin and by reducing reagents but not by SDS. Analysis of crude conditioned medium on non-reducing SDS gels reveals two active species of cMGF with mol. wts. of 23 and 27 kd. Incubation of radioiodinated partially purified cMGF with myeloblasts demonstrates the specific binding of 23- and 27-kd components under non-reducing, and 25- and 29-kd components under reducing conditions. Glycosylation inhibition experiments indicate that the larger molecules represent glycosylated forms of a single protein moiety. The 27-kd species has been purified to homogeneity (80 000-fold enrichment) and exerts its half maximal activity at 2 X 10(-12) M and its maximal activity at 3 X 10(-11) M. Antibodies prepared to purified cMGF completely neutralize the growth-stimulating activity of the factor.     

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.     

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.     

Structural characterization of a murine myeloid leukaemia inhibitory factor

A leukaemia inhibitory factor (LIF) which induces macrophage differentiation in M1 murine myeloid leukaemia cells and suppresses their proliferation in vitro has been isolated in sufficient quantities (30 micrograms) from Krebs ascites tumour cell conditioned medium to permit its partial characterization by amino acid sequence analysis. The combination of sensitive microbore column (1.0 and 2.1 mm internal diameter) HPLC technology and microsequence analysis has enabled the positive identification of 125 of the total 179 amino acid residues (70%) in the molecule. The amino acid sequence data reported here permitted the isolation of a partial cDNA clone encoding LIF [Gearing et al. (1987) EMBO J. 6, 3995-4002]. A candidate C-terminus of the LIF molecule predicted from the amino acid sequence was confirmed by subsequent isolation of a cDNA clone corresponding to the C-terminus of the protein. No strong similarity was revealed when the amino acid sequence of LIF was compared with other haemopoietic growth factors, in particular granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor and tumour necrosis factor-alpha or interleukins. The protein sequence data reported here indicate three sites of post-translational modification (N-linked glycosylation).     

Downregulation of the upstream binding factor1 by glycogen synthase kinase3beta in myeloid cells induced to differentiate

The upstream binding factor 1 (UBF1), one of the proteins that regulate the activity of RNA polymerase I, is downregulated in 32D myeloid cells induced to differentiate into granulocytes, either by the type 1 insulin-like growth factor (IGF-1) or the granulocytic colony stimulating factor (G-CSF). Downregulation of UBF1 is largely due to protein degradation, while mRNA levels are not affected. Inhibition of UBF1 degradation by lithium chloride (LiCl)and lactacystin suggest a role of glycogen synthase kinase beta (GSK3beta) in a proteasome-dependent degradation of UBF. GSK3beta phosphorylates in vitro and in vivo the UBF protein, which has five putative motifs for phosphorylation by GSK3beta. Elimination and/or mutations of these motifs stabilize the UBF1 protein even in cells induced to differentiate. Conversely, a stably transfected, constitutively active GSK3beta accelerates the downregulation of UBF1. We show further that activation of the differentiating protein C/EPBalpha in 32D cells transformed by the oncogenic BCR/ABL protein causes downregulation of UBF1. Finally, inhibition of differentiation of myeloid cells by a dominant negative mutant of Stat3 stabilizes the UBF1 protein, while rapamycin-induced differentiation of myeloid cells downregulates UBF1 levels. Taken together, our results indicate that the induction of granulocytic differentiation in 32D murine myeloid cells causes the degradation of UBF1, via GSK3beta and the proteasome pathway.     

Macrophage cell lines transformed by the malignant histiocytosis sarcoma virus: increase of CSF receptors suggests a model for transformation

The malignant histiocytosis sarcoma virus (MHSV) contains Ha-v-ras-related oncogenic sequences and rapidly transforms myeloid cells in vivo and in vitro. Myeloid cell lines can be derived which do not require growth factor for continued proliferation. We initiated this work to define the process of transformation leading to autonomy of cell growth in transformed myeloid cells. Five established cell lines were examined. All express macrophage-specific cell-surface antigens and exhibit several other properties typical for mature macrophages. Growth properties, growth factor release, and growth factor receptor presentation were examined: Release of growth factors is not a consistent feature. All cell lines show cell-density-independent colony formation and do not release self-stimulating factors, thus excluding autocrine stimulation as a model leading to transformation. All cell lines express unusually high levels of granulocyte-macrophage (GM)- and multi-CSF receptors and, except for one, M-CSF receptors. The high increase in GM-CSF and other growth factor receptors may be causally related to the transformed state of the cells. MHSV can be used as a tool to easily derive cell lines of the macrophage pathway as a model to study myeloid transformation, differentiation, and macrophage function.     

Reassignment of the human macrophage colony stimulating factor gene to chromosome 1p13-21.

Macrophage colony stimulating factor (CSF-1) is a member of a family of glycoproteins that are necessary for the normal proliferation and differentiation of myeloid progenitor cells. The human CSF-1 gene has previously been assigned to chromosome 5 using somatic cell hybrids, and further localized to 5q33 by in situ hybridization with a 3H labelled cDNA probe. However, the murine macrophage colony stimulating factor gene (csfm) has been localized to a region on mouse chromosome 3 which was previously shown to be syntenic with the proximal region of 1p and not 5q. Using a human genomic DNA clone that contains the CSF-1 gene, we have localized CSF-1 to chromosome 1p13-21 by fluorescence in situ hybridization. The reassignment of the CSF-1 gene argues against its involvement in myeloid disorders with deletions of the long arm of chromosome 5.     

Primary structure and functional expression of rat and human stem cell factor DNAs

Partial cDNA and genomic clones of rat stem cell factor (SCF) have been isolated. Using probes based on the rat sequence, partial and full-length cDNA and genomic clones of human SCF have been isolated. Based on the primary structure of the 164 amino acid protein purified from BRL-3A cells, truncated forms of the rat and human proteins have been expressed in E. coli and mammalian cells and have been shown to possess biological activity. SCF is able to augment the proliferation of both myeloid and lymphoid hematopoietic progenitors in bone marrow cultures. SCF exhibits potent synergistic activities in conjunction with colony-stimulating factors, resulting in increased colony numbers and colony size.     

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.