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.     

Splenic erythroblasts in anemia-inducing Friend disease: a source of cells for studies of erythropoietin-mediated differentiation

Splenic erythroblasts obtained from mice during the acute disease caused by either the polycythemia-inducing (FVP) or anemia-inducing (FVA) strain of Friend virus were examined for their degree of terminal differentiation. Morphology, benzidine staining, and heme synthesis kinetics showed that many erythroblasts from FVP-infected mice were undergoing terminal differentiation, while few erythroblasts from FVA-infected mice showed evidence of terminal differentiation. When cultured in methylcellulose medium, splenic erythroblasts from FVP-infected mice completed differentiation without the addition of erythropoietin (EP) to the medium. However, splenic erythroblasts from FVA-infected mice underwent terminal differentiation in vitro only when EP was added to the medium. From spleens of FVA-infected mice, a population of large, immature-appearing erythroblasts was obtained by separation with velocity sedimentation at unit gravity. Serial studies of the separated erythroblasts which were cultured with EP showed that despite some heterogeneity in their proliferative capacity, they were relatively homogeneous in their degree of differentiation in that they had not begun to synthesize heme or globin. Morphological changes and syntheses of heme and globins were monitored during terminal differentiation induced in vitro by EP. The accumulation of immature erythroblasts in vivo, their responsiveness in vitro to EP, and availability of large numbers of cells (10(8) or more) make the splenic erythroblasts of FVA-infected mice an ideal population of cells with which to study EP-mediated terminal differentiation. This erythroblast population should permit the biochemical and molecular studies in erythroid differentiation which heretofore had to be done with chemically induced erythroid differentiation in continuous cell lines.     

The thyroid hormone receptor functions as a ligand-operated developmental switch between proliferation and differentiation of erythroid progenitors.

The avian erythroblastosis virus (AEV) oncoprotein v-ErbA represents a mutated, oncogenic thyroid hormone receptor alpha (c-ErbA/ TRalpha). v-ErbA cooperates with the stem cell factor-activated, endogenous receptor tyrosine kinase c-Kit to induce self-renewal and to arrest differentiation of primary avian erythroblasts, the AEV transformation target cells. In this cooperation, v-ErbA substitutes for endogenous steroid hormone receptor function required for sustained proliferation of non-transformed erythroid progenitors. In this paper, we propose a novel concept of how v-ErbA transforms erythroblasts. Using culture media strictly depleted from thyroid hormone (T3) and retinoids, the ligands for c-ErbA/TRalpha and its co-receptor RXR, we show that overexpressed, unliganded c-ErbA/ TRalpha closely resembles v-ErbA in its activity on primary erythroblasts. In cooperation with ligand-activated c-Kit, c-ErbA/ TRalpha causes steroid-independent, long-term proliferation and tightly blocks differentiation. Activation of c-ErbA/ TRalpha by physiological T3 levels causes the loss of self-renewal capacity and induces synchronous, terminal differentiation under otherwise identical conditions. This T3-induced switch in erythroid progenitor development is correlated with a decrease of c-ErbA-associated histone deacetylase activity. Our results suggest that the crucial role of the mutations activating v-erbA as an oncogene is to 'freeze' c-ErbA/ TRalpha in its non-liganded, repressive conformation and to facilitate its overexpression.     

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.     

Sequence-specific DNA binding by the v-erbA oncogene protein of avian erythroblastosis virus.

The v-erbA oncogene, a transduced copy of a thyroid hormone receptor, plays an important role in establishment of the transformed cell phenotype induced by avian erythroblastosis virus. The ability of thyroid hormone receptors to bind to specific sites on chromatin and to thereby modify the expression of adjacent target genes is a crucial element in their mechanism of action in the normal cell. The v-erbA protein also bound at high affinity to a set of DNA fragments recognized by the rat thyroid hormone receptor, but the relative affinity of the v-erbA protein for the different binding sites was distinct from that previously reported for the thyroid hormone receptors.     

Effects of inhibitors of glycoprotein processing on the synthesis and biological activity of the erb B oncogene.

Three glycoprotein-processing inhibitors were used to resolve whether correct glycosylation was required for the oncogenic activity of erb B. The two glucosidase-I inhibitors, 1-deoxynojirimycin and 2,5-dihydroxymethyl 3,4-dihydroxypyrrolidine, arrested processing of v-erb B at the immature 68-kd form whereas, in the presence of the alpha-mannosidase-II inhibitor (swainsonine), cells synthesised an abnormally processed 70-kd form of v-erb B. Transport of incorrectly processed v-erb B to the cell surface was, however, unaffected, suggesting that correct processing is not a prerequisite for intracellular routing of v-erb B. Two systems were used to assess whether incorrectly processed erb B could maintain the transformed state. The first asked whether inhibitor treatment would release temperature-sensitive avian erythroblastosis virus (AEV) transformed erythroblasts kept at the viral permissive temperature from the erb B-induced block in differentiation, as seen when cells are normally shifted to the non-permissive temperature. The second tested the ability of AEV-transformed fibroblasts to grow in soft agar. In both systems, all three processing inhibitors did not alter the transformed phenotype suggesting that correct carbohydrate processing is not required for the transforming activity of erb B. In addition, none of the three processing inhibitors were found to have any effect on the normal maturation of bone marrow CFU-E or induced differentiation of temperature-sensitive AEV-transformed erythroblasts.     

Changes in erythroid membrane proteins during erythropoietin-mediated terminal differentiation

Membrane and membrane skeleton proteins were examined in erythroid progenitor cells during terminal differentiation. The employed model system of erythroid differentiation was that in which proerythroblasts from mice infected with the anemia-inducing strain of Friend virus differentiate in vitro in response to erythropoietin (EP). With this system, developmentally homogeneous populations of cells can be examined morphologically and biochemically as they progress from proerythroblasts through enucleated reticulocytes. alpha and beta spectrins, the major proteins of the erythrocyte membrane skeleton, are synthesized in the erythroblasts both before and after EP exposure. At all times large portions of the newly synthesized spectrins exist in and are turned over in the cytoplasm. The remaining newly synthesized spectrin is found in a cellular fraction containing total membranes. Pulse-chase experiments show that little of the cytoplasmic spectrins become membrane associated, but that the proportion of newly synthesized spectrin which is membrane associated increases as maturation proceeds. A membrane fraction enriched in plasma membranes has significant differences in the stoichiometry of spectrin accumulation as compared to total cellular membranes. Synthesis of band 3 protein, the anion transporter, is induced only after EP addition to the erythroblasts. All of the newly synthesized band 3 is membrane associated. A two-dimensional gel survey was conducted of newly synthesized proteins in the plasma membrane enriched fraction of the erythroblasts as differentiation proceeded. A majority of the newly synthesized proteins remain in the same proportion to each other during maturation; however, a few newly synthesized proteins greatly increase following EP induction while others decrease markedly. Of the radiolabeled proteins observed in two dimensional gels, only the spectrins, band 3 and actin become major proteins of the mature erythrocyte membrane. Examination of total proteins of the plasma membrane enriched fractions of EP-treated erythroblasts using silver staining and 32P autoradiography show that many proteins and phosphoproteins are selectively eliminated from this fraction late in the course of differentiation during the reticulocyte stage. The selective removal of many proteins at the reticulocyte stage of development combined with previous selective synthesis and accumulation of some specific proteins such as alpha and beta spectrin and band 3 in the differentiating erythroblasts lead to the final mammalian erythrocyte membrane structure.     

Changes in the Expression of Membrane Antigens During the Differentiation of Chicken Erythroblasts

Chicken erythroblasts can be transformed by the avian retrovirus, avian erythroblastosis virus (AEV). Earlier studies have shown that the mechanism of transformation appears to involve a “block” in differentiation, in that when erythroblasts are transformed by a temperature-sensitive mutant of ts34 AEV and incubated at the nonpermissive temperature, the cells start to differentiate and produce hemoglobin. We have decided to use this system to isolate pure populations of chicken erythroblasts and raise monoclonal antibodies against their cell surface proteins. Three monoclonal antibodies were isolated and tested for their ability to bind to various hematopoietic cell types; two were shown to be erythroid-specific, whereas the other antibody bound to proliferating cells but not to erythrocytes or granulocytes. Of the erythroid-specific antibodies, one precipitated a 94,000 molecular weight protein, whereas the other precipitated a 11,000 molecular weight protein that was tentatively identified as hemoglobin. The use of this system and approach to identify and evaluate changes that occur during the differentiation is discussed.     

Control of erythroid differentiation: asynchronous expression of the anion transporter and the peripheral components of the membrane skeleton in AEV- and S13-transformed cells

Chicken erythroblasts transformed with avian erythroblastosis virus or S13 virus provide suitable model systems with which to analyze the maturation of immature erythroblasts into erythrocytes. The transformed cells are blocked in differentiation at around the colony-forming unit-erythroid stage of development but can be induced to differentiate in vitro. Analysis of the expression and assembly of components of the membrane skeleton indicates that these cells simultaneously synthesize alpha-spectrin, beta-spectrin, ankyrin, and protein 4.1 at levels that are comparable to those of mature erythroblasts. However, they do not express any detectable amounts of anion transporter. The peripheral membrane skeleton components assemble transiently and are subsequently rapidly catabolized, resulting in 20-40-fold lower steady-state levels than are found in maturing erythrocytes. Upon spontaneous or chemically induced terminal differentiation of these cells expression of the anion transporter is initiated with a concommitant increase in the steady-state levels of the peripheral membrane-skeletal components. These results suggest that during erythropoiesis, expression of the peripheral components of the membrane skeleton is initiated earlier than that of the anion transporter. Furthermore, they point a key role for the anion transporter in conferring long-term stability to the assembled erythroid membrane skeleton during terminal differentiation.     

ISG15 modulates development of the erythroid lineage

Activation of erythropoietin receptor allows erythroblasts to generate erythrocytes. In a search for genes that are up-regulated during this differentiation process, we have identified ISG15 as being induced during late erythroid differentiation. ISG15 belongs to the ubiquitin-like protein family and is covalently linked to target proteins by the enzymes of the ISGylation machinery. Using both in vivo and in vitro differentiating erythroblasts, we show that expression of ISG15 as well as the ISGylation process related enzymes Ube1L, UbcM8 and Herc6 are induced during erythroid differentiation. Loss of ISG15 in mice results in decreased number of BFU-E/CFU-E in bone marrow, concomitant with an increased number of these cells in the spleen of these animals. ISG15(-/-) bone marrow and spleen-derived erythroblasts show a less differentiated phenotype both in vivo and in vitro, and over-expression of ISG15 in erythroblasts is found to facilitate erythroid differentiation. Furthermore, we have shown that important players of erythroid development, such as STAT5, Globin, PLC γ and ERK2 are ISGylated in erythroid cells. This establishes a new role for ISG15, besides its well-characterized anti-viral functions, during erythroid differentiation.     

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.     

Immunoelectron microscopic detection of band 3 protein during erythroid cell differentiation by a monoclonal antibody

We created a monoclonal antibody, designated EB1 (IgM, kappa), that reacts with erythroblasts by fusion of P3-X63-Ag8.653 with splenocytes of rats immunized with erythroblastic islands isolated from mice spleens. Western blotting revealed that EB1 reacted with the band 3 protein of the erythrocytic membrane. It stained erythrocytes and erythroblasts, forming clusters in the bone marrow, splenic red pulp, and fetal liver, but did not stain other tissues in the cryostat sections. The EB1 antigen was detected during dimethyl sulfoxide-induced differentiation of murine erythroleukemia cells. Immunoelectron microscopy revealed that the EB1 antigen was expressed from the basophilic erythroblasts during normal erythroid differentiation. Preferential segregation of the EB1 antigen on the cell membrane of the nucleating erythroblasts was not observed. These results suggest that EB1 is specific for erythrocyte band 3 protein and may be useful for studying erythroid cell differentiation.