The v-ski oncogene cooperates with the v-sea oncogene in erythroid transformation by blocking erythroid differentiation.

The avian retrovirus oncogene v-ski was analysed for its ability to alter the differentiation program of erythroid cells and to cooperate with tyrosine kinase oncogenes in leukemogenesis. For this, a retrovirus combining v-ski with a temperature-sensitive version of the v-sea oncogene was constructed. In transformed erythroblasts, v-ski disturbed the concerted expression of several erythrocyte genes, leading to an abnormal erythroblast phenotype. Expression levels of hemoglobin and erythrocyte anion transporter (band 3) were elevated, while expression of the erythroid-specific histone H5 was strongly suppressed. v-ski could also be shown to repress or severely retard the temperature-induced erythroid differentiation of v-ski/ts-v-sea-transformed cells. The undifferentiated cells had an abnormal erythroblast or early reticulocyte phenotype characterized by unusually low levels of histone H5. In chicks, the v-ski/ts-v-sea virus displayed enhanced leukemogenicity compared with viruses containing just the single oncogenes. Thus, v-ski cooperates with tyrosine kinase oncogenes in a similar fashion to the v-erbA oncogene, however the pattern of genes affected by these two oncogenes is different.     

The v-erbA oncogene requires cooperation with tyrosine kinases to arrest erythroid differentiation induced by ligand-activated endogenous c-erbA and retinoic acid receptor.

The v-erbA oncogene, a mutated version of the thyroid hormone receptor alpha (c-erbA/TR-alpha), cooperates with tyrosine kinase oncogenes in erythroblast transformation. Here we show that the ligand-activated, endogenous retinoic acid receptor (RAR-alpha), in cooperation with c-erbA/TR-alpha, efficiently reverses the transforming effect of kinase oncogenes, overcoming oncogene-induced self-renewal by triggering terminal differentiation of the transformed cells into healthy erythrocytes. This differentiation induction was accompanied by up-regulation of erythrocyte gene expression. Similarly, RAR-alpha and over-expressed exogenous c-erbA/TR-alpha efficiently abolished the differentiation arrest caused by v-erbA, while the low levels of endogenous TR-alpha had no effect. In contrast, transformation by v-erbA plus a kinase oncogene was not affected at all by ligand-activated endogenous or over-expressed exogenous TR-alpha and RAR-alpha. These results suggest that oncogene cooperation is required to protect leukemic erythroblasts from differentiation induction via endogenous, nuclear hormone receptors. Endogenous c-erbA/TR-alpha and RAR-alpha apparently cooperated in abolishing erythroblast self-renewal and inducing differentiation, since the respective ligands acted in a synergistic fashion, and overexpressed, non-ligand-bound c-erbA/TR-alpha suppressed endogenous RAR-alpha function in differentiation induction. Genetic evidence is presented that this functional cooperation requires the receptor dimerization domain, suggesting that TR-alpha/RAR-alpha heterodimers play a role in regulation of erythroid differentiation.     

Localization and modulation of the DNA-binding activity of the human c-ets-1 protooncogene

The avian acute leukemia virus (E26) induces a mixed erythroid-myeloid leukemia in chickens and carries two distinct oncogenes, v-myb and v-ets. The viral protein responsible for transformation is a gag-myb-ets fusion protein that is located in the nucleus of the transformed cells. The cellular homologue of v-ets (c-ets-1) is highly expressed in lymphoid cells and differs from the v-ets gene at its carboxy terminal region. Here, we show that both the c-ets-1 and v-ets gene products are DNA-binding proteins and their DNA-binding activity is located in the carboxy terminal (46 amino acid residues) region. It appears that this DNA-binding activity is modulated by the extreme carboxy terminal region. The amino acid sequences of the putative ets DNA-binding domain at its carboxy terminal region showed a helix-turn-helix secondary structure. Exchanging the nonhomologous extreme carboxy terminal regions of c-ets-1 with v-ets gene sequences showed differences in DNA-binding affinity, indicating that these differences may be partly responsible for the activation of the ets oncogene.     

Leukemic transformation of normal murine erythroid progenitors: v- and c-ErbB act through signaling pathways activated by the EpoR and c-Kit in stress erythropoiesis.

Primary erythroid progenitors can be expanded by the synergistic action of erythropoietin (Epo), stem cell factor (SCF) and glucocorticoids. While Epo is required for erythropoiesis in general, glucocorticoids and SCF mainly contribute to stress erythropoiesis in hypoxic mice. This ability of normal erythroid progenitors to undergo expansion under stress conditions is targeted by the avian erythroblastosis virus (AEV), harboring the oncogenes v-ErbB and v-ErbA. We investigated the signaling pathways required for progenitor expansion under stress conditions and in leukemic transformation. Immortal strains of erythroid progenitors, able to undergo normal, terminal differentiation under appropriate conditions, were established from fetal livers of p53-/- mice. Expression and activation of the EGF-receptor (HER-1/c-ErbB) or its mutated oncogenic version (v-ErbB) in these cells abrogated the requirement for Epo and SCF in expansion of these progenitors and blocked terminal differentiation. Upon inhibition of ErbB function, differentiation into erythrocytes occurred. Signal transducing molecules important for renewal induction, i.e. Stat5- and phosphoinositide 3-kinase (PI3K), are utilized by both EpoR/c-Kit and v/c-ErbB. However, while v-ErbB transformed cells and normal progenitors depended on PI3K signaling for renewal, c-ErbB also induces progenitor expansion by PI3K-independent mechanisms.     

Transformation of early erythroid precursor cells (BFU-E) by a recombinant murine retrovirus containing v-erb-B

Avian erythroblastosis virus (AEV) is a replication-defective retrovirus that transforms erythroid and fibroblast cells in vitro and in vivo. The transforming ability of AEV is due primarily to the oncogene v-erb-B. A recombinant murine retrovirus has been constructed by inserting a chimeric gag-v-erb-B gene into a Moloney murine leukemia virus based vector. This retrovirus was used to examine v-erb-B-induced transformation of murine hematopoietic cells. Infection of murine primary fetal liver, adult bone marrow or adult spleen cells with the recombinant virus generated large hemoglobinized erythroid colonies in the absence of exogenous growth factors. Generation of such colonies usually requires the presence of erythropoietin (Epo) and interleukin-3 (IL-3). These growth-factor independent colonies were shown to be derived from early (BFU-E) and not late (CFU-E) erythroid progenitor cells, and the effect was not attributable to growth factors elicited by the virus-producing cell lines. In order to confirm that the recombinant virus was responsible for this transformation of BFU-E to growth factor independence, bone marrow cells from post 5-fluorouracil treated mice were infected and used to repopulate lethally-irradiated mice. Growth factor-independent BFU-E were obtained in up to 30% of day-13 spleen colonies and it was shown by DNA analysis that cells from these colonies contained integrated provirus. Our results indicate that v-erb-B transforms early erythroid progenitors to growth factor independent growth and subsequent differentiation to erythrocytes -a process that normally requires Epo plus either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF).     

v-erb A and v-erb B do not cooperate in quail myoblasts

We have previously shown that v-erb A stimulates quail myoblast differentiation in a T3 independent, cell-specific manner. In this work, we have studied the influence of v-erb B (the second oncogene carried in the AEV genome) upon quail myoblast proliferation and differentiation. v-erb B expression,moderately stimulates myoblast proliferation, and inhibits differentiation. Moreover, this oncoprotein fully inhibits the v-erb A myogenic influence. These data provide evidence that these two oncogenes do not cooperate in avian myoblasts. Consequently, in contrast to results obtained in other cell-types, coexpression of both oncogenes does not transform quail myoblasts.     

Cooperative effect of v-myc and v-erbA in the chick embryo.

We show that a construct designated as MAHEVA, which encodes oncogenes v-myc from MH2 virus and v-erbA from AEV under the control of the LTR of MH2, induces rapidly growing heart rhabdomyosarcomas, when it is injected in E3 but not E5 chick embryos. A similar pathology has previously been observed with MC29, within the same limited time frame. The tumors, which expressed P61-63myc, P75gag-erbA and Pr76gag proteins were detectable from E14 onwards. Compared with MC29, MAHEVA induced a secondary anomaly, not detectable prior to E17. This is the appearance of cartilage nodules within the heart rhabdomyosarcomas. The constant location of these nodules inside the rhabdomyosarcomas and their delayed appearance suggests that the chondrocytes originate from myoblasts prevented from differentiating by the expression of the v-myc product. This interpretation is supported by the appearance of chondrocytes in E3 heart muscle cells infected in vitro with MAHEVA.     

The v-erbA oncogene blocks expression of alpha2/beta1 integrin a normal inhibitor of erythroid progenitor proliferation

T2EC are chicken erythrocytic progenitors that balance between self-renewal and differentiation as a function of response to specific growth factors. Their transformation by the v-erbA oncogene locks them into the self-renewal program. We show here that the expression of the VLA-2 integrin alpha2 subunit mRNA is downregulated by v-erbA and that VLA-2 engagement and clustering, brought about by treatment with an alpha2-specific antibody or by culture on the VLA-2 ligand collagen I, inhibits T2EC proliferation. From competition studies using antibodies, VLA-2 was shown to be involved in the collagen-induced response. While engagement of VLA-2 inhibited proliferation, it was not sufficient to induce differentiation. The transformation of T2EC by v-erbA decreased their interaction with collagen I and the VLA-2 brake on cell proliferation, which may account for the increased proliferation potential of transformed erythrocytic progenitors and for their shedding into the blood of infected chickens. Our data suggest that the interaction between erythroid progenitors and collagen, mediated by VLA-2, play a major role in the control of erythropoiesis in vitro and that this pathway is a target of the v-erbA oncogene.     

Cell surface proteins of chicken hematopoietic progenitors, thrombocytes and eosinophils detected by novel monoclonal antibodies.

E26 is an acute avian leukemia virus that contains two nuclear oncogenes, v-myb and v-ets, and that is capable of transforming early cells of the erythroid and myeloid lineages. In another study, we have found that TPA (phorbol 12,13-dibutyrate) treatment of E26-transformants displaying an 'early erythroid' phenotype results in the production of cells with either myeloid or eosinophil characteristics. To analyze this induction in greater detail we have produced a panel of four monoclonal antibodies against E26-transformants before and after TPA-induced differentiation. Two antibodies, MEP21 and MEP26, reacted with proteins of 150 and 47-60 kDa, respectively, which are expressed on the surface of E26 progenitor cells but whose expression is extinguished following TPA-induced differentiation. A third antibody, EOS47, recognizes a 100 kDa molecule that is expressed on the surface of TPA-induced peroxidase positive cells (an enzyme that in avian species is restricted to cells of the eosinophilic lineage). MEP21, MEP26, and EOS47 do not react with lymphoid, myeloid, or more mature erythroid lineage cell lines. The fourth antibody, MEP17, recognizes a heterodimer of 140 and 150 kDa chains which is expressed at high levels by E26-transformed progenitor cells and at lower levels by TPA-induced cells. Further biochemical characterization of the MEP17 antigen revealed a structure similar to that of the leukocyte adhesion molecule VLA-4; a member of the integrin family of adhesion proteins. All four antibodies react with subpopulations of cells in the bone marrow and spleens of 1-day-old chickens. Although the MEP21 and MEP26 antibodies do not appear to react with mature cells of most hematopoietic lineages they are expressed at high levels by mature thrombocytes. In addition, MEP17 is expressed at high levels by the majority of bursal B-cells, thrombocytes, and more weakly by thymocytes. The reagents described should be useful as markers for the study of development, migration, and differentiation of normal avian hematopoietic progenitor cells and eosinophilic precursors, and for the study of retrovirus-induced neoplasia.     

Induction of in vitro differentiation of mouse erythroleukemia cells by genistein, an inhibitor of tyrosine protein kinases

We have found that genistein, a specific inhibitor of tyrosine protein kinases, induces in vitro erythroid differentiation of mouse erythroleukemia cells. Characterization of the induction process indicated that the genistein-induced differentiation is different from that induced by conventional inducing agents such as dimethyl sulfoxide or hexamethylene-bisacetamide. This conclusion was based upon the earlier appearance of differentiated cells, insensitivity to a specific inhibitor (dexamethasone), and responsiveness of some of the differentiation-resistant cells to genistein in the genistein-induced erythroid differentiation. Possible biological significance of this finding is discussed with respect to the involvement of protein phosphorylation (or dephosphorylation) in mouse erythroleukemia cell differentiation.     

Ets and retroviruses - transduction and activation of members of the Ets oncogene family in viral oncogenesis

Studies of retroviral-induced oncogenesis in animal systems led to the initial discovery of viral oncogenes and their cellular homologs, and provided critical insights into their role in the neoplastic process. V-ets, the founding member of the ETS oncogene family, was originally identified as part of the fusion oncogene encoded by the avian acute leukemia virus E26 and subsequent analysis of virus induced leukemias led to the initial isolation of two other members of the ETS gene family. PU.1 was identified as a target of insertional activation in the majority of tumors induced by the murine Spleen Focus Forming virus (SFFV), while fli-1 proved to be the target of Friend murine leukemia virus (F-MuLV) in F-MuLV induced erythroleukemia, as well as that of the 10A1 and Graffi viruses. The common features of the erythroid and myeloid diseases induced by these viruses provided the initial demonstration that these and other members of the ETS family play important roles in hematopoietic development as well as disease. This review provides an overview of the role of ETS genes in retrovirally induced neoplasia, their possible mechanisms of action, and how these viral studies relate to current knowledge of the functions of these genes in hematopoiesis.     

Apparent Epo-independence of erythroid cells infected with the polycythemia-inducing strain of Friend spleen focus-forming virus is not due to Epo production or change in number or affinity of Epo receptors

The polycythemia-inducing strain of the Friend spleen focus-forming virus (SFFVP) induces an acute erythroleukemia in mice. Erythroid cells from these mice differ from normal erythroid cells in that they can proliferate and differentiate in the apparent absence of the erythroid hormone erythropoietin (Epo). Although it was recently shown that the unique envelope protein encoded by SFFV is responsible for altering the hormonal requirements of erythroid cells for growth and differentiation, the mechanisms by which this occurs is not known. Since the SFFV envelope protein appears to interact with a target present only in erythroid cells and since Epo is specific for these cells, it is possible that the virus is exerting its effect through this hormone. In an effort to ascertain if this is the case, we examined cells from SFFVP-infected mice to determine (a) if they produce Epo or other erythroid growth factors that stimulate erythroid cells to grow in an autocrine-like manner and (b) if they express elevated numbers of Epo receptors that may result in a reduced requirement for the level of Epo needed for growth and differentiation. Our results indicate that SFFVP-infected cells do not secrete Epo or any other erythroid growth factors that could account for the reduced hormonal requirements of these cells. Also, our studies using iodinated Epo in cell binding assays and cross-linking studies indicate that SFFVP-infected cells are not significantly different from normal erythroid cells in the number, affinity, or size of their Epo receptors.     

Monoclonal antibodies to novel erythroid differentiation antigens reveal specific effects of oncogenes on the leukaemic cell phenotype

A panel of new monoclonal antibodies to antigens on the surface of chick erythroid progenitor cells is described. These are characterised with respect to their binding to different classes of normal haemopoietic cells of both the erythroid and myeloid lineages. Using these antibodies, we have examined the phenotype of avian leukaemic cells transformed by retroviruses carrying defined oncogenes. Our data show that these cells, although similar to the normal haematopoietic precursor from which they are derived, aberrantly express certain markers in an oncogene specific manner.