Multilineage hematopoietic disorders induced by transplantation of bone marrow cells expressing the v-fms oncogene

Mouse bone marrow cells infected with a helper-free retrovirus containing v-fms were engrafted into lethally irradiated mice. Dominant provirus-positive clones emerged in the spleens of some recipients within 1 month. When spleen cells were transplanted into lethally irradiated secondary recipients, clonal erythroleukemias or B cell lymphomas expressing the v-fms-coded glycoprotein developed. Other secondary recipients repopulated by “unmarked” progenitor cells or by cryptic provirus-positive precursors present in the spleens of the same donor mice did not develop disease; thus cells expressing v-fms did not invariably have a proliferative advantage after transplantation. Several primary engrafted recipients developed myeloproliferative disorders that were provirus-positive without evidence of clonality. Although expression of the c-fms product (CSF-1 receptor) is normally restricted to cells of the mononuclear phagocyte series, the v-fms-coded glycoprotein can contribute to proliferative abnormalities of multiple hematopoietic lineages.     

Morphological analysis of osteoclastogenesis induced by RANKL in mouse bone marrow cell cultures

Under the influence of RANKL, in the presence of M-CSF, monocyte/macrophage precursor cells entered the osteoclast lineage and expressed the osteoclast marker tartrate-resistant acid phosphatase (TRAP). These cells were motile and began to differentiate by contacting and fusing together, initially forming cells with several nuclei. All sizes of cells continued to fuse, forming larger cells with more than 6 and as many as 50 nuclei. The degree of osteoclastogenesis was related to the concentration of RANKL. High cell density changed osteoclast morphology from a more rounded form with cytoplasm extended all round the cell to a form with cytoplasm concentrated around the nuclei and more restricted multiple cytoplasmic extensions. At optimal cell density and RANKL concentrations the large numbers of rounded cells fused into large cytoplasmic masses. On reaching a critical size, osteoclasts assumed a spread morphology with a peripheral ring structure. Most of the nuclei were associated with the peripheral ring. When cytoplasmic masses were present, rings also formed within the mass, often with no contact with the cell periphery. All forms of RANKL-induced osteoclastogenesis were blocked by the endogenous decoy receptor osteoprotegerin and were also strongly reduced by calcitonin, with the later arriving morphological categories being the first to disappear.     

Human long-term bone marrow cultures in aplastic anemia

Long-term bone marrow cultures (LTMC) were initiated with marrow from five normal subjects and eight patients with aplastic anemia (AA). Near confluent to confluent adherent layers developed in all cultures from normal subjects and AA patients. When present, the 'cobblestone' areas in LTMC from AA subjects were smaller than those observed in the LTMC from normal subjects. The decline in total and viable cell numbers in the LTMC was similar for both normal subjects and AA patients. Granulocyte-macrophage colony-forming units (CFU-gm) were present in nonadherent cells (NAC) from normal LTMC for a mean of 5.2 weeks. CFU-gm were present in the NAC of only two of the eight AA cultures for one week. The absent or small 'cobblestone' areas and the absence of CFU-gm production in AA-LTMC suggest a decrease in the reproductive potential of adherent hematopoietic stem cells, which may be the result of either an abnormal hematopoietic stem cell or an abnormal stromal microenvironment or both.     

Transmembrane orientation of glycoproteins encoded by the v-fms oncogene

The retroviral oncogene v-fms encodes a glycoprotein whose transport to the plasma membrane is required for transformation. Tryptic digestion of microsomes from transformed cells yielded membrane-protected amino-terminal fragments 40 kd smaller than intact molecules. These fragments were glycosylated, and they included v-fms-coded epitopes expressed at the cell surface. Deletion of the predicted membrane-spanning peptide generated polypeptides that were completely sequestered within microsomes. The mutant glycoproteins acquired more asparagine-linked oligosaccharide chains than did wild-type molecules, lacked kinase activity in vitro, were not transported to the cell surface, and had no transforming activity. Thus, the membrane-spanning segment in the middle of the glycoprotein interrupts translocation of nascent chains into the endoplasmic reticulum, ultimately orienting the amino-terminal domain outside the cell and the carboxy-terminal kinase domain in the cytoplasm. These topological features are similar to those of several growth factor receptors, suggesting that v-fms transforms cells through modified receptor-mediated signals.     

Residual radiation injury exhibited in long-term bone marrow cultures

Residual radiation injury was demonstrated in long-term primary cultures of mouse bone marrow. Control cultures underwent three phases of hematopoietic activity as distinguished by initial establishment, steady high (plateau) production of granulocytes, and gradual decline. Irradiation with 50, 300, or 550 rads, given at the end of the initial phase, did not prevent any culture flasks from entering the plateau phase. However, actual production levels and the time they were maintained varied inversely with the radiation dose so that the accumulated postradiation cell production corresponded to an exponential dose-response relationship at any time after treatment. The accumulated cell productions were found to be similar in all groups when expressed by the number of stem cell doublings necessary to produce them. The findings cannot be explained by reproductive cell death and are consistent with the notion of a limited division capacity in hematopoietic stem cells.     

Fibroblast transformation parameters induced by the avian v-mil oncogene.

An avian retrovirus containing only the v-mil oncogene (PA200-MH2) was analyzed for its ability to induce a transformed phenotype in chicken embryo fibroblasts. Infected cultures exhibited an altered morphology, disarranged actin cable filaments, and a decrease in the amount of cell surface fibronectin. In addition, these cells showed a high level of plasminogen activator protease activity and were also capable of growth in low serum concentrations. In contrast, PA200-MH2 was very inefficient at inducing foci under agar and colonies in semisolid medium relative to the Mill Hill 2 and Rous sarcoma viruses. This inefficiency was further reflected in vivo by the total inability of PA200-MH2 to induce wing tumors in young birds. However, 40% of the birds inoculated in the wing web with PA200-MH2-infected cells did develop slow-growing tumors at the site of injection, with no evidence of hematopoietic involvement. Our results indicate that the v-mil oncogene is transforming both in vitro and in vivo and that each of the oncogenes in the Mill Hill 2 virus, v-mil and v-myc, can independently transform fibroblasts. These data suggest that v-mil is functionally related to its homologous murine counterpart, v-raf, which also transforms fibroblasts.     

Selective transformation of primitive lymphoid cells by the BCR/ABL oncogene expressed in long-term lymphoid or myeloid cultures.

The BCR/ABL gene, formed by the Philadelphia chromosome translocation (Ph1) of human chronic myelogenous leukemia, encodes an altered ABL gene product, P210. P210 is strongly implicated in the malignant process of chronic myelogenous leukemia, but it precise role is unknown. Infection of long-term bone marrow cultures enriched for B-lymphoid cell types with a Moloney murine leukemia virus retroviral vector containing the BCR/ABL cDNA resulted in clonal outgrowths of immature B-lymphoid cells which expressed abundant P210 kinase activity. Surprisingly, infection of long-term myeloid lineage-enriched cultures also resulted in clonal outgrowths of immature B-lymphoid cells. The P210-expressing lymphoid cell lines resulting from either type of culture were resistant to the lethal effects of corticosteroids. These findings indicate that high levels of P210 expressed from a Moloney murine leukemia virus long terminal repeat preferentially stimulate the growth of immature B-lineage cells, and this effect is apparent even in myeloid lineage-enriched cultures, in which few if any lymphoid cells can be detected prior to infection.     

Subcellular localization of glycoproteins encoded by the viral oncogene v-fms

The McDonough strain of feline sarcoma virus encodes a polyprotein that is cotranslationally glycosylated and proteolytically cleaved to yield transforming glycoproteins specified by the viral oncogene v-fms. The major form of the glycoprotein (gp120fms) contains endoglycosidase H-sensitive, N-linked oligosaccharide chains lacking fucose and sialic acid, characteristic of glycoproteins in the endoplasmic reticulum. Kinetic and steady-state measurements showed that most gp120fms molecules were not converted to mature forms containing complex carbohydrate moieties. Fixed-cell immunofluorescence confirmed that the majority of v-fms-coded antigens were internally sequestered in transformed cells. Dual-antibody fluorescence performed with antibodies to intermediate filaments (IFs) showed that the IFs of transformed cells were rearranged, and their distribution coincided with that of v-fms-coded antigens. No specific disruption of actin cables was observed. The v-fms gene products cofractionated with IFs isolated from virus-transformed cells and reassociated with IFs self-assembled in vitro. A minor population of v-fms-coded molecules (gp140fms) acquired endoglycosidase H-resistant, N-linked oligosaccharide chains containing fucose and sialic acid residues, characteristic of molecules processed in the Golgi complex. Some gp140fms molecules were detected at the plasma membrane and were radiolabeled by lactoperoxidase-catalyzed iodination of live transformed cells. We suggest that v-fms-coded molecules are translated as integral transmembrane glycoproteins, most of which are inhibited in transport through the Golgi complex to the plasma membrane.     

Production of colony stimulating factor in long-term bone marrow cultures

Previous studies have shown no detectable colony-stimulating factor (CSF) in media harvested from long-term bone marrow cultures. In the present experiments supernatants from long-term cultures established in three laboratories were assayed for CSF by colony assay and by radioimmunoassay (RIA). Most samples were devoid of biologic activity but all contained CSF as judged by RIA. Biologic activity was found in the majority of samples after diafiltration to remove low molecular weight inhibitors or 5-fold concentration by ultrafiltration. Samples that remained inactive in the colony assay were subjected to gel filtration on Sephadex G-150 to remove potential high molecular weight inhibitors. Biologic activity remained lower than that by RIA in two of three samples tested. Thus, most long-term cultures appear to contain biologically active CSF but this activity is masked by various types of inhibitors. In addition some media appear to contain material that is only detected by RIA.     

Monoclonal antibodies to the transformation-specific glycoprotein encoded by the feline retroviral oncogene v-fms.

Monoclonal antibodies prepared to epitopes encoded by the transforming gene (v-fms) of the McDonough strain of feline sarcoma virus were used to study v-fms-coded antigens in feline sarcoma virus-transformed rat and mink cells. These antibodies reacted with three different polypeptides (gP180gag-fms, gp140fms, and gp120fms), all of which were shown to be glycosylated. Protein blotting with [125I]-labeled monoclonal immunoglobulin G's was used to determine the relative steady-state levels of these glycoproteins in transformed cells and showed that gp120 and gp140 were the predominant products. Immunofluorescence assays and subcellular fractionation experiments localized these molecules to the cytoplasm of transformed cells in quantitative association with sedimentable organelles. Thus, v-fms-coded glycoproteins differ both chemically and topologically from the partially characterized products of other known oncogenes and presumably transform cells by a different mechanism.     

Renewal of hematopoietic stem cell clones in long-term bone marrow cultures

In long-term cultures of murine bone marrow, clonal succession of hemopoietic cells was observed as measured by karyologic analysis. There were high oscillations in self-renewal of CFUs in the cultures. A close correlation between the CFUmix karyotype and mitotic non-adherent cells in culture (but not between these cell types and CFUs) was revealed.     

In vitro studies on lymphocyte differentiation. II. Generation of terminal deoxynucleotidyl transferase-positive cells in long-term cultures of mouse bone marrow.

The enzyme TdT is the earliest known marker of lymphocytic differentiation in rodents. Cells containing this enzyme were demonstrated in suspension cultures of mouse bone marrow cells that had been maintained in vitro for periods of 7 to 45 days. The cells were detected by immunofluorescence using purified antibodies to homogeneous TdT. Between 0.04 and 2.0% of cultured bone marrow cells from a variety of mouse strains were positive. More than 40% of the TdT-positive cells incorporated 3H-thymidine during a 20-min pulse. Surface Ig and Thy-1 antigens were not detected on the TdT-positive cells. The prevalence of TdT-positive cells was decreased 10-fold in cultures that had been treated with 10(-6) M hydrocortisone 24 hr before harvesting. The results indicate that lymphoid progenitor cells can be generated in vitro.     

The kinetics of granulopoiesis in long-term mouse bone marrow culture. Part I

The spontaneous stratification in long-term bone marrow cultures was illustrated and quantified. The cultures were separated into three hematopoietic layers: nonadherent cells in the supernatant medium, lightly adherent cells on top of the stromal layer, and remaining cells buried within the stromal layer. The cells of each layer were subcultured for 10 days in plastic tubes that inhibit the formation of a stromal layer. Daily samplings with absolute and differential cell counts were obtained. We identified three families of cell disappearance curves and cell types: CFU-s, hemocytoblasts, myeloblasts, and promyelocytes (G1, 2); myelocytes (G3); and postmitotic granulocytes (G4). Also, the numbers of mitotic and necrotic cells were determined. The longest half-time of CFU-s was 2.5 days. Lacking stromal support, CFU-s disappeared faster than other differentiated cells. Generally, these cells maintained their numbers for the first week of subcultures, which was attributable to a temporarily maintained balance of cell death and fresh cell production. After more than 7 days, there was a rapid decline of all differentiated cell types.     

The kinetics of granulopoiesis in long-term mouse bone marrow culture. Part II

A mathematical model of mouse granulopoiesis in long-term bone marrow culture was constructed, based on established in vivo cell kinetic parameters. We applied the model to the cell kinetic experiment presented in Part I. Comparing model-predicted cell kinetics with the experimental data led to iterative testing of several hypotheses. In the final model, the cell kinetics of intact tissue culture flasks were reconstructed, using the experimental data from 10 days of tube culture. Among other things, our analysis suggests that the parameters of normal in vivo granulopoiesis apply to bone marrow culture.     

Tyrosine phosphorylations in vivo associated with v-fms transformation.

The role of tyrosine-specific phosphorylation in v-fms-mediated transformation was examined by immunoblotting techniques together with a high-affinity antibody that is specific for phosphotyrosine. This antiphosphotyrosine antibody detected phosphorylated tyrosine residues on the gp140v-fms molecule, but not gP180v-fms or gp120v-fms, in v-fms-transformed cells. This antibody also identified a number of cellular proteins that were either newly phosphorylated on tyrosine residues or showed enhanced phosphorylation on tyrosine residues as a result of v-fms transformation. However, the substrates of the v-fms-induced tyrosine kinase activity were not the characterized pp60v-src substrates. The phosphorylation of some of these cellular proteins and of the gp140fms molecule was found to correlate with the ability of v-fms/c-fms hybrids to transform cells. In addition, immunoblotting with the phosphotyrosine antibody allowed a comparison to be made of the substrates phosphorylated on tyrosine residues in various transformed cell lines. This study indicates that the pattern of tyrosine phosphorylation in v-fms-transformed cells is strikingly similar to that in v-sis-transformed cells.     

The v-fms oncogene induces factor-independent growth and transformation of the interleukin-3-dependent myeloid cell line FDC-P1.

The normal cellular counterpart of the v-fms oncogene product is a receptor for the mononuclear phagocyte colony-stimulating factor, CSF-1. An interleukin-3 (IL-3)-dependent mouse myeloid cell line, FDC-P1, was infected with a murine retrovirus vector containing v-fms linked to a gene encoding resistance to neomycin (neo). Infected cells selected for resistance to the aminoglycoside G418 contained few proviral DNA copies per haploid genome, expressed low levels of the v-fms-coded glycoprotein, remained IL-3 dependent for growth, and were nontumorigenic in nude mice. In contrast, infected cells selected for their ability to grow in the absence of IL-3 contained an increased number of proviral insertions, expressed high levels of the v-fms-coded glycoprotein, and were tumorigenic in nude mice. The IL-3-independent cells expressed IL-3 receptors of comparable number and affinity to those detected in uninfected FDC-P1 cells and did not produce a growth factor able to support replication of the parental cells. Thus, the synthesis of high levels of the v-fms gene product in FDC-P1 cells abrogated their requirement for IL-3 and rendered the cells tumorigenic by a nonautocrine mechanism. The data suggest that v-fms encodes a promiscuous tyrosine kinase able to transform cells of the myeloid lineage that do not normally express CSF-1 receptors.     

Enhanced lymphoid and decreased myeloid reconstituting ability of stem cells from long-term cultures of mouse bone marrow

Mature, functional lymphocytes rapidly disappear from long-term cultures of mouse bone marrow cells and never reappear. One reason for the loss of B lymphocytes is that the optimal culture conditions for maintenance of myeloid stem cells are suboptimal for lymphocyte survival. However, despite the absence of functional lymphocytes, stem cells from such cultures retain the ability to reconstitute irradiated mice with mitogen-responsive B and T lymphocytes. In fact, in vitro grown stem cells repopulate the lymphoid system better than the myeloid system; the defective myeloid potential does not result from the absence in the cultures of Thy–1 bearing regulatory cells (TSRC). Although the cultures lack mature lymphocytes, they contain putative T cell precursors detectable with an in vitro colony-forming assay (CFU-T). In vitro maintenance of CFU-T requires an appropriate adherent monolayer. Monolyaters from congenitally anemic mice of genotype S1/S1^d fail to support either myeloid precursors or CFU-T.     

Characterization of early B lymphocyte precursors present in long-term bone marrow cultures

Lymphoid precursor cells are present in long-term bone marrow cultures (LTBMC), but their differentiation into mature lymphocytes is blocked. A quantitative assay for B cell precursors in LTBMC, which gives a linear relationship between the number of grafted LTBMC cells and the frequency of B cell colony forming units (CFU-B) in the spleen and bone marrow of immunodeficient CBA/N mice 19 days after reconstitution, is described. Characterization of the B cell precursor indicates that this assay is detecting a very early precursor and not a B lymphocyte or a late pre-B cell. This conclusion is based on the observations that a) pre-B cells transformable by Abelson murine leukemia virus are not present in LTBMC by 3 days postrecharge and CFU-B are absent by 6 days postrecharge; b) late B cell progenitors capable of rapid repopulation of irradiated CBA/N mice are not present in LTBMC, since a lag in the kinetics of B cell reconstitution in animals grafted with LTBMC cells is observed compared with fresh bone marrow cells; c) the B cell precursors in LTBMC have high proliferative potential, since they can stably repopulate recipient mice for at least 8 wk postreconstitution and through two serial passages in irradiated CBA/N recipients; and d) the B cell precursors are large, rapidly sedimenting cells as determined by velocity sedimentation. The serial transplantation experiment further shows that a split is often observed between lymphoid and myeloid reconstituting ability of LTBMC cells. The LTBMC B cell precursor may be a pluripotent stem cell or a lymphoid stem cell, although its differentiative potential remains to be determined.     

Regulation of haemopoiesis in long-term bone marrow cultures. IV. Glycosaminoglycan synthesis and the stimulation of haemopoiesis by beta- D-xylosides

Sulfated glycosaminoglycans (GAGs) are distributed in consistent and distinctive patterns between the cell surface and the growth medium of haemopoietically active long-term bone marrow cultures. Heparan sulfate is the main cell surface component and chondroitin sulfate is the major sulfated species in the medium. When the cultures are supplemented with beta-D-xylosides a significant increase in chondroitin sulfate synthesis is observed but no stimulation of heparan sulfate synthesis occurs. The chondroitin sulfate accumulates in the culture medium in beta-D-xyloside-treated cultures but the composition of sulfated GAGs in cell-surface derived material is unaffected. beta-D-xylosides also stimulate the production of haemopoietic cells without any apparent alteration in the adherent stromal cells of the marrow cultures. Equivalent increases are obtained in cells at all stages of development so that a fivefold increase in pluripotent stem cells (CFU-S) is matched by fivefold increase in the granulocyte-macrophage progenitors (GM-CFC) and in mature granulocytes. The stimulation persists for many weeks in beta-D-xyloside-treated cultures. These results indicate that the sulfated GAGs may play an important role in the regulation of haemopoiesis.     

Cellular interactions in erythroblastic islands in long-term bone marrow cultures, as studied by time-lapse video

Long-term bone marrow cultures (LTBMC) are readily converted from the usual granulopoietic to erythropoietic production by the addition of anemic mouse serum (AMS). The "statics" of proliferation and maturation, previously shown by ultrastructural methods to closely mirror the in vivo situation, were studied dynamically using a time-lapse video system. Several cell pedigrees were followed, but the most complete series showed three successive divisions and subsequent enucleations in the progeny of three synchronously mitotic cells observed in the culture; this is indicative of a five division sequence in the erythron. As in erythroblastic islets observed in marrow in vivo, the striking synchrony of maturation was maintained in vitro. Furthermore, when some of the erythroid progeny became displaced to other macrophages, the synchrony, which was maintained by the original erythroid group on the original erythroblastic islet macrophage, was lost. Time-lapse video, which is inexpensive to run and can be maintained in continuous recording for many weeks, is an ideal technique for recording both erythroid cell pedigrees, and the initial events leading to the formation of an erythroblastic islet in vitro after stimulation with AMS.