Transformation of single myeloid precursor cells by the malignant histiocytosis sarcoma virus (MHSV): generation of growth-factor-independent myeloid colonies and permanent cell lines

Direct single-cell assays for oncogenic transformation are available for fibroblasts but not for other cell types. Using malignant histiocytosis sarcoma virus (MHSV), a member of the ras family of retroviruses, in vivo-infected granulocyte/macrophage and macrophage precursor cells lost the requirement for externally added hematopoietic growth factors. Factor-independent growth was demonstrated by colony-transfer experiments. More than 25% of the independent colonies were established as permanent macrophage cell lines following a phase of adaptation to tissue culture conditions. Factor-independent colony growth was also obtained by in vitro infection of single cells. As many as 50% of all myeloid precursor cells were target cells for MHSV as measured by this assay. About 2 x 10(-3) of these colony-forming cells acquired growth factor independence and immortality after in vitro infection. Cell lines derived from these colonies did not require adaptation to tissue culture conditions.     

EGF receptor activity and mitogenic response of Balb/3T3 cells expressing Ras and Myc oncogenes. EGF receptor activity in oncogene transformed cells.

EGF receptors are found on the surface of most cells, usually with high and low binding affinities. To investigate functional relationships between EGF (EGF-like growth factors) and oncogenes we have characterized the expression of the epidermal growth factor receptor (EGFr) in H-Ras, v-Myc, and H-Ras-v-Myc transformed Balb/3T3 cells. H-Ras cells show a marked decrease in the number of EGFr molecules per cell compared to parental cells. v-Myc and H-Ras-v-Myc transformed cells express an intermediate level of receptors. The majority of the EGF receptors on the parental and oncogene transformed cells bind EGF with low affinity and this low affinity receptor is down-regulated by oncogene transformation. v-Myc expression, in the H-Ras-v-Myc transformed cells, abrogates the receptor down-regulation seen with H-Ras transformation. The mechanism of abrogation is not a result of a change in the p21-Ras concentration in the H-Ras-v-Myc transformed cells. In addition, the mitogenic response to EGF was examined. H-Ras and H-Ras-v-Myc transformed cells do not respond to EGF mitogenically. In contrast, EGF stimulates DNA synthesis in parental cells and v-Myc transfected cells; this result suggests that growth promoting signals from the EGF receptor may not be required in H-Ras transformed cells.     

Flat revertants derived from Kirsten murine sarcoma virus-transformed cells produce transforming growth factors

Two flat cellular revertant cell lines, F-2 and C-11, which were originally selected from the DT line of Kirsten murine sarcoma virus (Ki-MuSV)-transformed NIH/3T3 cells, were examined for the production of transforming growth factors (TGFs). The revertant cells fail to grow in semisolid medium as colonies and exhibit a markedly reduced level of tumorigenicity in nude mice, although they are known to express high levels of p21ras, the product of the Kirsten sarcoma virus oncogene, ras, and they contain a rescuable transforming virus. TGF activity associated with the transformed, revertant, and non-transformed cell lines was measured by the ability of concentrated conditioned medium (CM) from these cells to induce normal rat kidney (NRK) and NIH/3T3 cells to form colonies in semisolid agar suspension cultures and to inhibit the binding of 125I epidermal growth factor (EGF) to specific cell surface receptors. CM from the transformed DT cells and from both the F-2 and C-11 revertants contains TGF activity, in contrast to CM obtained from normal NIH/3T3 cells. Furthermore, unlike NIH/3T3 cells, neither the DT nor the revertant cells were able to bind 125I EGF. All four cell lines were able to proliferate in serum-free medium supplemented with transferrin, insulin, EGF, and Pedersen fetuin. However, in basal medium lacking these growth factors, only DT cells and, to a lesser extent, the revertant cells were able to grow. These results suggest that the F-2 and C-11 revertants fail to exhibit all of the properties associated with transformation because the series of events leading to the transformed phenotype is blocked at a point(s) distal both to the expression of the p21 ras gene product and also to the production of TGFs and that the production of TGFs may be necessary but not sufficient for maintaining the transformed state.     

Chicken hematopoietic cells transformed by seven strains of defective avian leukemia viruses display three distinct phenotypes of differentiation.

Chicken hematopoietic cells transformed in vitro and in vivo by seven strains of replication-defective avian leukemia viruses were assayed for the expression of six erythroid and five myeloid differentiation parameters, including differentiation-specific surface antigens as detected by newly developed antisera. The transformed cells were found to display three distinct phenotypes of differentiation. First, cells transformed by AEV resemble erythroblasts. They express heme, globin, carbonic anhydrase and erythrocyte cell surface antigen at low levels, and histone H5 and erythroblast cell surface antigen at high levels. Second, cells transformed by MC29, CMII, OK10 and MH2 viruses have macrophage-like properties. They strongly express Fc receptors, phagocytic capacity and macrophage cell surface antigen, but only weakly express myeloblast cell surface antigen and are negative for ATPase activity. Third, cells transformed by AMV and E26 viruses resemble myeloblasts in that they weakly express Fc receptors, phagocytic capacity and macrophage cell surface antigen but strongly express myeloblast cell surface antigen and ATPase activity. No difference was found between in vitro- and in vivo-transformed cells in the parameters tested. In light of recent genetic and biochemical evidence, we believe that these phenotypes reflect the action of three new types of viral-transforming genes, designated erb (erythroblast), mac (macrophage) and myb (myeloblast).     

Role of colony-stimulating factors in the biology of acute myelogenous leukemia

A high proportion of acute myeloid leukemias (AML) recently investigated for their capacity to synthesize biologically active bioregulatory molecules was found to accumulate messenger (m) RNA and to produce membrane-bound or -secreted forms of stimulating factors for granulocyte, macrophage and mixed granulocyte-macrophage colony growth. Blast cells have also been found to secrete interleukin 1, tumor necrosis factor-alpha, interleukin 6, and to express receptors for various growth factors as well. However, growth factors like interleukin 2 and interleukin 3 have not been identified as AML products, and several other factors including interleukin 4, interleukin 5, etc. need further evaluation. Responsiveness of clonogenic leukemic cells to exogenous growth-promoting factors in vitro suggests a possible role of these biomolecules in the course of these disorders. Important evidence for the crucial role of growth factors, at least in some subtypes of AML, has been provided by demonstrating constitutive growth factor production by leukemic cells and their autonomous in vitro growth which is dependent on autocrine secretion of a specific growth factor. The concert of mechanisms providing stimulatory and inhibitory signals for hematopoiesis, which is adapted to the various physiological requirements of the organism, may have multiple defects in AML. This leads to successive steps of malfunctioning of cells, which finally express a fully malignant phenotype. In addition, these derangements also lead to defects in accessory cells on the level of mediator communication. However, there is evidence for autonomous growth promotion of AML blast by constitutive production of growth factors active in an autocrine fashion (GM-CSF, G-CSF, interleukin 6) and by recruitment of accessory cells to increase CSF supply (GM-CSF, G-CSF) via molecules such as interleukin 1 and TNF-alpha in a paracrine fashion. Molecular analysis of transformed hematopoietic cells has revealed changes of the genome, e.g., insertion of viral genetic information or cytogenetic fractures at DNA sites controlling growth factor gene activation. These events appear to be crucial in the induction of uncontrolled growth factor expression promoting oncogenic transformation of hematopoietic progenitor cells.     

Role of TGF alpha and its receptor in proliferation of immortalized rat tracheal epithelial cells: studies with tyrphostin and TGF alpha antisera

We have shown in the present study and in studies reported previously that preneoplastic and neoplastic rat tracheal epithelial (RTE) cell lines express TGF alpha and do so regardless of the mechanism by which they were transformed. In order to determine whether TGF alpha is an autocrine growth regulator of immortalized RTE cells, we have examined the function of TGF alpha/EGF receptors and the growth requirements for TGF alpha in these cells. The level of immunoprecipitated TGF alpha/EGF receptor protein in immortalized RTE cells was similar to or less than levels in primary RTE cells, indicating that chemically induced transformation of RTE cells does not involve overexpression of TGF alpha/EGF receptors. Scatchard analysis of TGF alpha/EGF receptors in the neoplastic EGV5T cell line revealed the presence of high-affinity (Kd = 0.4 nM) and low-affinity (Kd = 9.8 nM) binding sites. A tyrphostin TGF alpha/EGF receptor tyrosine kinase inhibitor decreased in a dose-dependent manner the proliferation as well as EGF-induced autophosphorylation of the TGF alpha/EGF receptor of transformed RTE cells. The inhibitory effect of tyrphostin on proliferation and receptor kinase activity was attenuated in late log and plateau phase cultures. The phosphotyrosine content of several other EGF-dependent and independent phosphoproteins was also decreased by the tyrphostin. Proliferation of transformed RTE cells was also inhibited when TGF alpha antisera was added to the media of growing cells. These data are consistent with the hypothesis that proliferation of transformed RTE cells involves autocrine regulation by TGF alpha and its receptor.     

Serum-free growth of normal and transformed fibroblasts in milk: differential requirements for fibronectin

Bovine milk may be used as a supplement for the serum-free growth of certain fibroblastic cells in culture. The growth properties of three representative cell types in milk-supplemented medium were examined; fibroblastic cell strains, fibroblastic cell lines, and transformed fibroblasts. Transformed fibroblasts, which included RNA and DNA tumor virus-transformed cells and carcinogen-transformed cells, grew in milk. Instead of growing attached to the culture dishes, as they normally do in serum, transformed fibroblasts grew in milk as large clusters in suspension. In contrast, nontransformed fibroblastic cell strains and cell lines did not grow in milk-supplemented medium. Fibroblasts transformed by a temperature-sensitive transformation mutant of Rous sarcoma virus were temperature-sensitive for growth in milk. The failure of cells to adhere to the substratum in milk-supplemented medium suggested that milk might be deficient in attachment factors for fibroblasts. When the attachment of fibroblastic cells in milk- supplemented medium was facilitated by pretreating culture dishes with fibronectin, (a) transformed cells grew attached rather than in suspension, (b) normal cell lines attached and grew to confluence, and (c) normal cell strains adhered and survived but did not exhibit appreciable cell proliferation.     

Release of hemopoietic factors by normal human T cell lines with either suppressor or helper activity

We analyzed the release of activities capable of stimulating the in vitro growth of human hemopoietic progenitor cells by long-term cultured T cell growth factor (TCGF)-dependent human T lymphocytes. Seven cell lines tested produced colony-stimulating activity (CSA) as well as burst-promoting activity (BPA). The CSA stimulated primarily the growth of the cells forming colonies after 14 days of incubation. In addition the supernatants from these seven T-cell lines showed the ability to induce the in vitro growth of mixed granulocyte, erythroid, megakaryocyte, macrophage colonies (CFU-GEMM). The release of hemopoietic factors did not depend on the presence of accessory cells or phytohemagglutinin or serum during the incubation for factor production. In six of the T cell lines the majority of the cells were reactive to the OKT 8 monoclonal antibody (MoAb), whereas one cell line contained mostly OKT 4+ cells. Suppressor activity was detected in three tested OKT 8+ cell lines, while the one OKT 4+ displayed helper activity. All cell lines produced hemopoietic factors with equal efficiency. These results indicate that factors affecting human hematopoiesis are produced by normal T lymphocytes in long-term culture and this property is not related to the helper or suppressor activity of the cultured cells.     

Transformation by the v-fms oncogene product: an analog of the CSF-1 receptor

The product of the c-fms proto-oncogene is related to, and possibly identical with, the receptor for the macrophage colony-stimulating factor, M-CSF (CSF-1). Unlike the product of the v-erbB oncogene, which is a truncated version of the EGF receptor, the glycoprotein encoded by the v-fms oncogene retains an intact extracellular ligand-binding domain so that cells transformed by v-fms express CSF-1 receptors at their surface. Although fibroblasts susceptible to transformation by v-fms generally produce CSF-1, v-fms-mediated transformation does not depend on an exogenous source of the growth factor, and neutralizing antibodies to CSF-1 do not affect the transformed phenotype. An alteration of the v-fms gene product at its extreme carboxyl-terminus represents the major structural difference between it and the c-fms-coded glycoprotein and may affect the tyrosine kinase activity of the v-fms-coded receptor. Consistent with this interpretation, tyrosine phosphorylation of the v-fms products in membranes was observed in the absence of CSF-1 and was not enhanced by addition of the murine growth factor. Cells transformed by v-fms have a constitutively elevated specific activity of a guanine nucleotide-dependent, phosphatidylinositol-4,5-diphosphate-specific phospholipase C. We speculate that the tyrosine kinase activity of the v-fms/c-fms gene products may be coupled to this phospholipase C, possibly through a G regulatory protein, thereby increasing phosphatidylinositol turnover and generating the intracellular second messengers diacylglycerol and inositol triphosphate.     

Target cells for avian myeloblastosis virus in embryonic yolk sac and relationship of cell differentiation to cell transformation

The yolk sac of the 12-day chicken embryo retains the blast stage progenitors to cells of the myeloid lineages with a very low level of contamination by more mature myeloid cells which have begun to express the characteristic myeloid cell markers. Both in vivo and in vitro experiments have supported the hypothesis that target cells for the BAI-A strain of avian myeloblastosis virus are contained within the myeloid lineages. An assay system for avian myeloblastosis virus was developed which utilizes this yolk sac cell system and which appears to be more sensitive than previous published assays. In addition, the kinetics of a liquid culture transformation system is presented in which at least 4% of the yolk sac cell population was transformed in a relatively synchronous fashion at 2 days after infection. The morphological transformation preceded an increased rate of cell proliferation. Cell separation procedures provided a 10- to 20-fold enrichment of target cells and demonstrated that the target cell population copurifies with macrophage colony-forming cells which are the committed progenitors to the macrophage lineage. In combination with earlier work, this work demonstrated that cells committed to the macrophage lineage at all stages of differentiation may serve as target cells for infection by avian myeloblastosis virus.     

Transformation of mouse bone marrow cells by transfection with a human oncogene related to c-myc is associated with the endogenous production of macrophage colony stimulating factor 1

We recently derived a series of transformed cell lines by transfecting mouse bone marrow cells highly enriched for macrophage progenitors with a newly described human gene, R-myc, which has homology to the c-myc oncogene. In this report, we show that these lines share some features characteristic of cells of the mononuclear phagocyte lineage. Specifically, all cell lines had macrophage- or monocytelike morphology, contained nonspecific esterase, were phagocytic for latex beads, secreted lysozyme, bore the Mac-1 antigen, and contained a minority of cells with Fc receptors. However, only a single monocytelike clone had appreciable numbers of cells which bore complement receptor 1, and none were phagocytic for antibody or complement-coated particles, or constitutively secreted Interleukin-1. All these cell lines secreted a growth factor capable of supporting the in vitro proliferation of bone marrow macrophages. Radioimmunoassay and receptor binding studies indicate that this factor is colony stimulating factor 1.     

Cell density-dependent regulation of cell surface expression of two types of human tumor necrosis factor receptors and its effect on cellular response

Tumor necrosis factor (TNF) is a multipotential cytokine known to regulate the growth of a wide variety of normal and tumor cells. It has been shown that the density of cells in culture can modulate the growth regulatory activities of TNF, the mechanism of which, however, is not understood. In this report, we investigated the effect of cell density on the expression of TNF receptors. The receptors were examined on epithelial cells (e.g., HeLa), which primarily express the p60 form, and on myeloid cells (e.g., HL-60) known to express mainly the p80 form. We observed that binding of TNF to both cell lines decreased with increase in cell density. Scatchard analysis of binding on HeLa and HL-60 cells revealed a 4- to 5-fold reduction in the number of TNF receptors without any significant change in receptor affinity in both cell types at high density. The decrease in TNF receptor numbers at high cell density was also observed in several other epithelial and myeloid cell lines. The downmodulation at high cell density was unique to TNF receptors, since minimum change in other cell surface proteins was observed as revealed by fluorescent activated cell sorter analysis. Neutralization of binding with antibodies specific to each type of the receptors revealed that both the p60 and p80 forms of the TNF receptor were equally downmodulated. A decrease in leucine incorporation into proteins was observed with increase in cell density, suggesting a reduction in protein synthesis. Since inhibition of protein synthesis by cycloheximide also leads to a decrease in TNF receptors, it is possible that the density-dependent reduction in TNF receptor number is due to an overall decrease in protein synthesis. The density-dependent decrease in TNF receptors was accompanied by a decrease in intracellular reduced glutathione levels. A reduction in the number of receptors on TNF sensitive tumor cells induced by cell-density correlated with increase in resistance to the cytokine.     

bcr-abl, the hallmark of chronic myeloid leukaemia in man, induces multiple haemopoietic neoplasms in mice.

The chromosome translocation forming the hybrid bcr-abl gene is thought to be the initiating event in chronic myeloid leukaemia (CML) and some cases of acute lymphoblastic leukaemia. To assess the impact of bcr-abl upon haemopoiesis, lethally irradiated mice were reconstituted with bone marrow cells enriched for cycling stem cells and infected with a bcr-abl bearing retrovirus. The mice developed several fatal diseases with abnormal accumulations of macrophage, erythroid, mast and lymphoid cells, and marked strain differences in disease distribution and kinetics. Some mice exhibited more than one neoplastic cell type and, in some instances, these were clonally related, indicating that a progenitor or stem cell had been transformed. While classical CML was not observed, the macrophage tumours were accompanied by a mild CML-like syndrome, probably due to myeloid growth factor production by tumour cells. The erythroid and mast cell diseases were rarely transplantable, in contrast to the macrophage tumours and lymphomas, but all disease types displayed limited clonality. These results establish that bcr-abl confers a proliferative advantage on diverse haemopoietic cells but complete transformation probably involves additional genetic changes.     

Transformation of mouse peritoneal macrophages and bone marrow cells by simian virus 40

A fraction of cultured mouse peritoneal macrophages and bone marrow cells acquired the ability to divide after infection by simian virus 40 (SV40). Two types of transformed lines were obtained. Most transformants isolated 400-60 days after infection did not display macrophage specific properties such as ingestion of opsonized red blood cells, possession of Fc receptors and complement receptors, and acid phosphatase activity throughout the whole culture phase. Cells of the transformed lines isolated by trypsin selection 2--6 months after infection displayed these properties when the cell density became high and cell growth was arrested. In the cells of the latter type of transformed lines, SV40 T-antigen was intensely demonstrated by immunofluorescence in the growing phase, but weakly or not at all in the stationary phase. It is suggested that the reversion to the phenotype of the progenitor (expression of macrophage specific functions) depends on the physiological state of the culture; however, it is uncertain whether the development of the macrophage functions is directly related to the SV40 T-antigen.     

NIH 3T3 cells transfected with a yeast H+-ATPase have altered sensitivity to insulin,insulin growth factor-I,and platelet-derived growth factor-AA

The role of intracellular pH (pHⁱⁿ) in the regulation of cell growth in both normal and transformed cells is a topic of considerable controversy. In an effort to study this relationship NIH 3T3 cells were stably transfected with the gene for the yeast H⁺-ATPase, constitutively elevating their pHⁱⁿ. The resulting cell line, RN1a, has a transformed phenotype: The cells are serum independent for growth, clone in soft agar, and form tumors in nude mice. In the present study, we further characterize this system in order to understand how transfection with this proton pump leads to serum-independent growth, using defined media to investigate the effects of specific growth factors on the transfected and parental NIH 3T3 cells. While both cell lines show similar growth increases in response to platelet-derived growth factor (PDGF)-BB and epidermal growth factor (EGF), they respond differently to insulin, insulin-like growth factor-I (IGF-I) and PDGF-AA. RN1a cells exhibit increased growth at nanomolar concentrations of insulin but the parental cells had only a relatively minor response to insulin at 10 μM. Both cell lines showed some response to IGF-I in the nanomolar range but the response of RN1a cells was much larger. Differences in insulin and IGF-I receptor number alone could not explain these results. The two cell lines also respond differently to PDGF-AA. RN1a cells are relatively insensitive to stimulation by PDGF-AA and express fewer PDGF α receptors as shown by Northern blots and receptor-binding studies. We propose a unifying hypothesis in which the H⁺-ATPase activates a downstream element in the PDGF-AA signal transduction pathway that complements insulin and IGF-I signals, while leading to downregulation of the PDGF α receptor. © 1994 wiley-Liss, Inc.