Temperature-sensitive mutants of avian erythroblastosis virus: surface expression of the erbB product correlates with transformation

The v-erbB gene of avian erythroblastosis virus (AEV) codes for an integral plasma membrane glycoprotein, gp74erbB. Expression of gp74erbB and its intracellular precursors, gp66erbB and gp68erbB, has been studied in cells transformed by two temperature-sensitive mutants of AEV. After shift to 42 degrees C, the processing of gp68erbB is blocked in tsAEV-transformed, but not in wtAEV-transformed, erythroblasts and fibroblasts. In addition, gp74erbB disappears from the surface of tsAEV cells within 12 hr after shift. Thus tsAEV mutants probably bear a lesion in v-erbB that affects the maturation and subcellular localization of gp74erbB. The tsAEV erythroblasts, when "committed" to differentiation by a pulse-shift to 42 degrees C, reexpress gp74erbB during terminal differentiation at 36 degrees C. This suggests that tsAEV erythroblasts become insensitive to the transforming functions of gp74erbB at a certain stage of differentiation.     

v-erbA cooperates with sarcoma oncogenes in leukemic cell transformation

The v-erbB, v-src, v-fps, v-sea, and v-Ha-ras oncogenes induce avian erythroid progenitor cells to self-renew in an erythropoietin-independent manner. These transformed erythroblasts retain both their capacity to differentiate into erythrocytes and their requirement for complex growth media. However, previous studies showed that erythroblasts transformed by v-erbB plus v-erbA (which by itself is not oncogenic) are blocked in differentiation and grow in standard media. Here we show that the introduction of v-erbA into erythroblasts transformed with v-src, v-fps, v-sea, or v-Ha-ras likewise induces a fully transformed phenotype. It also reduces the capacity of ts sea- and ts erbB-transformed erythroblasts to differentiate terminally in an erythropoietin-dependent manner after a temperature shift. Cooperativity involving v-erbA also occurs in vivo since chicks infected with a retroviral construct encoding v-erbA and v-src develop both acute erythroblastosis and sarcomas.     

Expression of the v-erbA oncogene in chicken embryo fibroblasts stimulates their proliferation in vitro and enhances tumor growth in vivo

In contrast to uninfected chicken embryo fibroblasts (CEFs), CEFs infected with a retroviral vector that carries the v-erbA gene of avian erythroblastosis virus displayed new properties. These included limited anchorage-independent growth in soft agar, growth without latency in serum-supplemented medium, ability to overcome quiescence induced by serum deprivation, growth at low cell density, and an extended life span in vitro. Furthermore, when explanted in vivo onto the chorioallantoic membrane of chicken embryo, the transformed CEFs expressing v-erbA in addition to v-erbB exhibited a high proliferative rate, giving rise to fibrosarcoma tumors that were ten times larger than those developed from transformed CEFs expressing v-erbB alone. All these data show that CEFs expressing the v-erbA oncogene display activated growth and suggest that the v-erbA product interferes with the mechanisms regulating the growth and/or differentiation of primary CEFs.     

Mitogenic action of factors secreted by avian erythroblastosis virus transformed cells

We describe here the capacity of erythroid LSCC HD3 cells, transformed with a ts mutant of avian erythroblastosis virus, to grow in a chemically defined medium without serum at 36 degrees C, but not at 41 degrees C. At this latter temperature the activity of v-erbB oncogene is suppressed. However, cell growth at 41 degrees C could take place either by addition of the medium derived from LSCC HD3 cells grown at 36 degrees C (conditioned medium), or by addition of fetal calf serum. These results show that LSCC HD3 cells, maintained under conditions in which the v-erbB oncogene is active, secrete growth factor(s) which exhibit a mitogenic effect similar to that observed with calf serum.     

A single point mutation in erbA restores the erythroid transforming potential of a mutant avian erythroblastosis virus (AEV) defective in both erbA and erbB oncogenes.

We have characterized the v-erbA and v-erbB oncogenes of td359, a transformation-defective mutant of avian erythroblastosis virus (AEV) unable to transform erythroblasts, and the revertant r12, obtained after in vivo passage of the mutant. Molecular cloning, sequencing, construction of chimeric viruses and testing of their oncogenic capacities revealed that both oncogenes of td359 are mutated and biologically defective. The r12 virus, although still containing a mutant v-erbB gene, recovered its erythroid transforming potential by acquiring a highly active gag-erbA gene. These results demonstrate that two co-operating oncogenes, an active v-erbA and a defective v-erbB, can transform a cell type not transformed by either oncogene alone. Furthermore, a single amino acid substitution inactivated the td359 v-erbA protein and we show that its reversion led to the reactivation of the protein. This lesion is located in the same region as several previously described inactivating mutations of glucocorticoid receptors, suggesting that the structure/function relationship of the virally transduced form of the c-erbA/thyroid hormone receptor is closely similar to that of steroid hormone receptors.     

Protein phosphorylation at tyrosine is induced by the v-erbB gene product in vivo and in vitro

The v-erbB gene product of avian erythroblastosis virus (AEV) has extensive homology with the receptor for epidermal growth factor (EGF). We report here that chicken embryo fibroblasts (CEF) transformed by AEV show enhanced tyrosine phosphorylation of a number of cellular polypeptides, including the 36 kd protein, which is phosphorylated in avian sarcoma virus-transformed fibroblasts, and the 42 kd protein, which is phosphorylated in mitogen-stimulated cells. CEF infected by AEV mutants with deletions in v-erbA showed enhanced tyrosine phosphorylation, whereas CEF infected by mutants with deletions in v-erbB did not. When membranes from AEV-transformed cells were incubated with gamma-32P-ATP, both the v-erbB gene product and the 36 kd cellular protein became phosphorylated at tyrosine. These results indicate that the v-erbB protein induces tyrosine phosphorylation in vivo and in vitro, and suggest that, like the EGF receptor, it possesses tyrosine-specific protein kinase activity.     

Antibodies against a synthetic peptide as a probe for the kinase activity of the avian EGF receptor and v-erbB protein

The transforming protein v-erbB of avian erythroblastosis virus (AEV) displays extensive sequence homology with the presumptive protein-tyrosine kinase domain of the human EGF receptor and with the src protein-tyrosine kinase family of oncogenes. However, no kinase activity has previously been demonstrated for the v-erbB protein. Here antibodies generated against a synthetic peptide from the C terminus of human EGF receptor are shown to immunoprecipitate the EGF receptor from human and avian cells, as well as the v-erbB proteins from AEV-transformed cells that become phosphorylated on tyrosine residues upon the addition of gamma-32P-ATP. The immunoprecipitates are also able to phosphorylate exogenous tyrosine-containing substrates. Hence, it is likely that both avian EGF receptor and v-erbB proteins are protein tyrosine-specific protein kinases. Since the kinase activity of v-erbB protein cannot be regulated by EGF, it is proposed that the tyrosine protein kinase function of v-erbB may be constitutively activated.     

Isolation and transformation of primary mesenchymal cells of the chick embryo.

Pure primary mesenchymal cells from definitive streak stage chick embryos have been prepared free of epiblast and hypoblast cells. These cells have the potential in culture to differentiate into erythroid cells, beating heart muscle tissue, chondrocytes and epithelial cells. Transformation in vitro of pure primary mesenchymal cells by avian erythroblastosis virus (wt-AEV) and a temperature-sensitive mutant (ts34-AEV) gave rise to rapidly growing cells which remained largely undifferentiated, could be cloned in semi-solid medium and could be maintained for up to 3 months in culture. The majority of mesenchymal cells transformed by wt-AEV (MAE cells) are benzidine-negative. Gel electrophoresis of radioactively labeled cell proteins, immunoprecipitated with specific antisera against chicken hemoglobin, showed that MAE cell clones synthesize the alpha D, pi (or pi') and some unidentified "globin" polypeptide chains. Treatment of MAE cell clones with 1.0 mM n-butyrate stops cell proliferation reversibly and causes an increased synthesis of alpha D and pi (or pi') globin polypeptide chains. In certain clones of mesenchymal cells transformed by a temperature-sensitive mutant of the virus, ts34-AEV (MAE-ts34 cells), benzidine-positive cells can be induced by a shift from 37 degrees to 41 degrees C. The ability of the clone to undergo an increase in benzidine-positivity by temperature shift is decreased with the age of the clone. Different clones show a variable proportion of cells which are positive by immunofluorescence for both globin and chicken-specific histone H5. The alpha A and alpha D globin chains are synthesized in MAE-ts34 clones, but the ratios and quantities of these chains vary for different clones. Temperature shift made little difference in the types and quantities of globin chains synthesized; the increase in benzidine positivity is probably due to an increase in heme biosynthesis.     

Chromosomal localisation of the human homologues to the oncogenes erbA and B.

Avian erythroblastosis virus (AEV) induces acute erythroleukemia and sarcomas in vivo and it transforms erythroblasts and fibroblasts in vitro. The virus has two host cell-derived genes, v-erbA and v-erbB. The latter encodes the oncogenic capacity of the virus, whereas v-erbA enhances the erythroblast transforming effects of v-erbB while being unable to induce neoplasms independently. Recently, human cellular homologues of these viral erb genes have been isolated. The chromosomal locations of two of these genes have been determined using EcoRI-digested DNA prepared from human-mouse somatic cell hybrids. The human c-erbA1 gene has been assigned to chromosome 17 and is located between 17p11 and 17q21. The human c-erbB sequence has been assigned to chromosome 7 and is located between 7pter and 7q22. Thus, in the human genome these genes are on two separate chromosomes. No evidence for the involvement of the human c-erb genes in neoplasia has been found.     

Structural domains of the avian erythroblastosis virus erbB protein required for fibroblast transformation: dissection by in-frame insertional mutagenesis.

Avian erythroblastosis virus (AEV) induces erythroblastosis and fibrosarcomas. The viral erbB protein is required for AEV-mediated oncogenesis. To explore the structural aspects of the v-erbB polypeptide necessary for its oncogenic function, we created a series of small in-frame insertions in different domains of the v-erbB oncogene. AEV genomes bearing lesions within the v-erbB kinase domain demonstrated a drastically decreased ability to transform avian fibroblasts, establishing a functional role for this structurally conserved oncogene domain. In contrast, mutations in the extracellular domain, between the transmembrane region and the kinase domain, or at the extreme C terminus of the v-erbB protein had no effect on AEV-mediated fibroblast transformation. One lesion within the v-erbB kinase domain, a 10-amino acid insertion, produced a temperature-sensitive mutant capable of fibroblast transformation at 36 degrees C but not at 41 degrees C, suggesting that small in-frame insertions have general utility for the in vitro creation of conditional mutants.     

Isolation of an MH2 retrovirus mutant temperature sensitive for macrophage but not fibroblast transformation.

A conditional mutant of the MH2 avian retrovirus, termed ts41MH2, was isolated. Unlike wtMH2, ts41MH2 permitted transformed macrophages to differentiate during a 5- to 7-day temperature shift from 37 to 42 degrees C. Mutant-infected cells incubated at 42 degrees C exhibited a flattened morphology and then fused to form giant multinucleated cells that closely resembled normal macrophage maturation in vitro. These differentiated cells reacted strongly with a myeloid-macrophage-specific monoclonal antibody. The process of differentiation was inhibited when ts41MH2-transformed nonproducer clones were superinfected before the temperature shift with the myc gene-containing MC29 or OK10 viruses. By contrast, no inhibition was observed in clones superinfected with the MH2-PA200 virus that contains only the mil gene. The mutant also demonstrated a reduced oncogenic potential relative to that of wtMH2 when it was inoculated intravenously into young birds. However, in contrast to the results obtained with hematopoietic cells, none of the five fibroblast transformation parameters tested for ts41MH2 were altered from those induced by wtMH2. These results suggest that the mutation in ts41MH2 is located in a region of myc required for macrophage transformation, but not required for fibroblast transformation.     

AEV-transformed chicken erythroid cells secrete autocrine factors which promote soft agar growth and block erythroleukemia cell differentiation

LSCC HD3 chicken erythroleukemia cells, transformed by a temperature-sensitive avian erythroblastosis virus (tsAEV), secreted into the medium several transforming factors which after separation by Bio-Cel P-60 chromatography, stimulated quiescent (G0) chicken embryo fibroblasts and NIH 3T3 mouse cells to replicate DNA in serum-free medium and to form colonies in soft agar. Most of these factors were also mitogenic for the LSCC HD3 cells themselves when they were rendered phenotypically untransformed by incubation at 42 degrees C to inactivate the ts AEV. The transformed LSCC HD3 cells also secreted a non-mitogenic 40 kDa factor which blocked the erythropoietin-induced differentiation of untransformed LSCC HD3 (at 42 degrees C) and the DMSO-induced differentiation of Friend murine erythroleukemia cells into hemoglobin-synthetizing erythroid cells.     

Hormone-dependent terminal differentiation in vitro of chicken erythroleukemia cells transformed by ts mutants of avian erythroblastosis virus

Chicken erythroblast cell strains and a cell line transformed by ts mutants of avian erythroblastosis virus (AEV) terminally differentiate when shifted to the nonpermissive temperature (42°C). The differentiated cells resemble mature erythrocytes with respect to morphology and ultrastructure, expression of differentiation-specific cell-surface antigens, pattern of protein synthesis and hemoglobin content. Terminal differentiation is dependent on conditions favoring the differentiation of normal erythroid progenitor cells, including an erythropoietin-like factor. Colonies of ts AEV cells grown at 42°C in semisolid medium resemble erythrocyte colonies derived from normal erythroid progenitor cells. The colonies obtained were comparable in size or slightly larger than the late erythroid precursor (CFU-E) colonies. These results suggest that AEV-transformed cells are blocked at a stage of differentiation that is more advanced than that of the uninfected target cells. ts AEV cells are irreversibly committed to terminal differentiation within 20 to 30 hr after shift to 42°C.     

Ligand-independent dimerization of oncogenic v-erbB products involves covalent interactions.

Mutant v-erbB products of avian c-erbB1 have previously been used to correlate structural domains of the receptor encoded by this proto-oncogene with tissue-specific transformation potential. In these studies, deletion of the ligand-binding domain of the receptor has been shown to be required for transformation of erythroblasts, fibroblasts, and endothelial cells. It has, therefore, been postulated that deletion of this domain results in an allosteric change in the receptor analogous to the ligand-bound state of the epidermal growth factor receptor; i.e., it induces a receptor conformation that is constitutively active with respect to mitogenic signaling. While oncogenic v-erbB products have been shown to be expressed on the cell surface of both fibroblasts and erythroblasts, no comprehensive analysis of the oligomeric potential of these products has been conducted. Since the first event known to follow epidermal growth factor binding to its receptor is oligomerization, and receptor dimerization has been correlated with mitogenic signaling, we have carefully analyzed the ability of several v-erbB products to oligomerize in the three target cell types transformed by these oncogenes. In this report, we demonstrate the v-erbB products can efficiently homodimerize in all three target tissues, that this dimerization is ligand independent and occurs at the cell surface, and that there is no apparent correlation between v-erbB dimerization and transformation of avian fibroblasts. Furthermore, both oncogenic and nononcogenic v-erbB products can heterodimerize with the native c-erbB1 product in chicken embryo fibroblasts, suggesting that heterodimerization between v-erB and native c-erbB1 is not sufficient to result in c-erbB1-mediated sarcomagenesis.