Myeloblasts transformed by the avian acute leukemia virus E26 are hormone-dependent for growth and for the expression of a putative myb-containing protein, p135 E26.

Avian leukemia virus E26 contains the myb oncogene and transforms erythroid and myeloid hematopoietic cells in vivo and in vitro. E26-transformed nonproducer myeloblasts but not avian erythroleukemia virus (AEV)-transformed erythroblasts nor MC29-transformed macrophages were shown to be dependent for growth on factor(s) present in supernatants from Concanavalin A-stimulated chicken spleen cells. The same factor enhanced the synthesis of p135 E26, the candidate transforming protein of E26, but did not induce the synthesis of the transforming proteins of AEV and MC29 viruses nor that of helper virus-derived structural proteins. P135 E26 was shown to contain sequences related to the viral gag gene as well as sequences which may be related to the myb gene product. P135 E26 might constitute the first example of a viral onc protein whose synthesis is regulated directly or indirectly by an exogenous hematopoietic growth factor.     

Properties of a murine retroviral recombinant of avian acute leukemia virus E26: a murine fibroblast assay for v-ets function.

A replication-defective murine retroviral construct, termed pME26, was generated by inserting avian gag-myb-ets sequences derived from the cloned avian acute leukemia virus E26 into an Abelson murine leukemia virus-derived retroviral vector. ME26 virus can be rescued efficiently from transfected NIH 3T3 cells by replicating murine leukemia viruses. Either pME26-transfected nonproducers or ME26 virus-infected NIH 3T3 cells expressed a 135-kilodalton fusion protein (p135) which was detectable by immunoprecipitation with antiserum directed against avian leukemia virus p27gag, myb or ets oncogene protein, or murine leukemia virus p15gag and was principally localized in the nucleus. NIH 3T3 cells infected with ME26 exhibited morphological alterations and increased proliferation in reduced serum and formed small colonies in agar suspension. Discrete foci could be readily recognized in cells maintained in a defined medium containing 0.03 to 0.1% calf serum. In newborn NFS/N mice, ME26 induced a significantly higher mortality and incidence of erythroid and myeloid leukemias. Analysis of a series of mutants affecting the expression of various portions of p135 indicated that the v-ets gene acts to mitogenically stimulate the proliferation of NIH 3T3 fibroblasts and reduces or abolishes their serum dependence. These properties provide an assay system to study functions of the ets gene family.     

Quail embryo fibroblasts transformed by four v-myc-containing virus isolates show enhanced proliferation but are non tumorigenic.

Quail embryo fibroblasts infected with any of the four natural avian myc gene-containing virus strains (MC29, CMII, OK10 and MH2) or with the myb, ets-containing E26 acute leukemia virus, were examined for their expression of several transformation-associated parameters. All myc-containing viruses, but not E26 or Rous sarcoma virus (used as a control) induced a dramatic stimulation of cell proliferation. In addition, the myc virus-transformed cells exhibited prominent nucleoli, possibly as a consequence of their increased proliferation. Cells transformed by MC29, OK10, MH2 and E26 were capable of growing in semi-solid medium and showed a loss of actin cables and, in most cases, of an ordered fibronectin distribution. All of the myc virus-transformed fibroblasts, as well as the E26-transformed cells, were unable to form tumors in nude mice, indicating that the myc gene (and the myb/ets genes) are not sufficient for the induction of a fully malignant phenotype in avian fibroblasts.     

Inversion of a chicken ets-1 proto-oncogene segment in avian leukemia virus E26.

The segment of the avian leukemia virus E26 genome near the termination of the p135gag-myb-ets open reading frame contains an inversion of the chicken ets-1 sequence. The inversion contains at least 41 bp and may be as large as 46 bp. This results in the replacement of 13 amino acids of chicken ets-1, with 16 amino acids derived from reverse complement of the normal ets-1 coding strand or read-through into E26 env sequences. At least 13 of these codons are specified by the inverted ets sequences. This represents the first reported occurrence of inverted oncogene sequences in a natural retrovirus. The inverted ets sequences are immediately followed by sequences homologous to the Rous sarcoma virus Prague B env gene. Since the E26 env sequence is more closely related to subgroup B avian retroviruses than to avian retroviruses from subgroups A, C, D, or E, the progenitor of E26 was a virus belonging to avian retrovirus subgroup B.     

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).     

A single point mutation in the v-ets oncogene affects both erythroid and myelomonocytic cell differentiation

The v-myb, ets-containing avian leukemia virus E26 is unique in its capacity to transform both erythroblasts and myeloblasts. Previous studies showing that v-myb is sufficient for the transformation of myeloid cells failed to definitively establish the role of the v-ets gene. We have now isolated a mutant of E26, ts1.1, that is temperature-sensitive for erythroid cell transformation and that we found to contain a single mutation in the v-ets gene. Surprisingly, myeloid cells transformed by this mutant showed an altered phenotype relative to wild-type-transformed cells, in that they resemble promyelocytes. In addition, infection of mature macrophages with ts1.1 led to their transformation and conversion into promyelocyte-like cells. We conclude that the v-ets domain of the p135gag-myb-ets protein of E26 has an effect on both erythroid and myeloid cell differentiation, suggesting a possible role for the c-ets/c-myb genes in the commitment of hematopoietic cells towards specific lineages.     

DNA-binding activity is associated with purified myb proteins from AMV and E26 viruses and is temperature-sensitive for E26 ts mutants

Oncogene protein products from avian myeloblastosis virus, p48v-myb, and from avian leukemia virus E26, p135gag-myb-ets, are located predominantly in the nucleus of nonproducer bone marrow cell clones, as revealed by indirect immunofluorescence. Both oncogene proteins were purified by immunoaffinity chromatography using monoclonal antibodies against p19 and immunoglobulins specific for myb, which was expressed in bacteria for antibody production. The purified proteins bind to DNA in vitro. In contrast, purified p135gag-myb-ets proteins from several mutants of E26 virus, temperature-sensitive for myeloblast transformation, either lost their abilities to bind to DNA or exhibited highly thermolabile DNA-protein interactions in vitro. DNA binding of AMV and E26 oncogene proteins is inhibited by myb-specific immunoglobulins. Our results suggest that lesions in the myb oncogene affect transformation as well as DNA binding of myb proteins in vitro.     

Detection of avian hematopoietic cell surface antigens with monoclonal antibodies to myeloid cells : Their distribution on normal and leukemic cells of various lineages

The isolation and characterization of monoclonal antibodies reacting with cell surface antigenic determinants of normal and leukemic avian hematopoietic cells is described. The antibodies were produced by immunizing mice with normal macrophages, as well as with myeloid cells transformed with the avian acute leukemia viruses MC29, AMV and E26. Eleven antibodies were characterized for their reactivity with a variety of normal and leukemic cells of the myeloid, B- and T-lymphoid and of the erythroid cell lineage. Using several methods, they could be subdivided into five distinct types: I. Four antibodies were specific for the myeloid lineage, predominantly reacting with immature myeloid cells. II. One antibody reacted with mature and immature myeloid cells as well as with T-lymphoid cells. III. Four antibodies reacted with myeloid, erythroid and T-lymphoid cells. IV. One antibody reacted with myeloid as well as with T- and B-lymphoid cells. V. One antibody reacted with all kinds of chicken hematopoietic cells except erythrocytes. The first type of antibodies detected glycoproteins with MWs of 170 and 130 kD. The pattern of antigens precipitated varied with the different monoclonal antibodies of this group. The antibody of the fourth type precipitated a 30 kD polypeptide from extracts of myeloid and lymphoid cells. None of the other antibodies precipitated any detectable proteins.     

env-encoded residues are not required for transformation by p48v-myb.

The v-myb oncogene of avian myeloblastosis virus induces acute myeloblastic leukemia in chickens and transforms avian myeloid cells in vitro. The protein product of this oncogene, p48v-myb, is partially encoded by the retroviral gag and env genes. We demonstrated that the env-encoded carboxyl terminus of p48v-myb is not required for transformation. Our results showed, in addition, that a coding region of c-myb which is not essential for transformation was transduced by avian myeloblastosis virus.     

Characterization of the hematopoietic target cells of AEV, MC29 and AMV avian leukemia viruses

The hematopoietic target cells of the three prototype strains of replication defective avian leukemia viruses (DLVs) were studied, using a newly developed, quantitative in vitro transformation assay. Our results show that the target cells of avian erythroblastosis virus (AEV) belong to the erythroid lineage while those of myelocytomatosis virus 29 (MC29) and avian myeloblastosis virus (AMV) belong to the myeloid lineage. As judged from suicide experiments using BUdR incorporation and irradiation with visible light, a higher proportion of AEV- and AMV-target cells are in cycle than MC29-target cells. Using differentiation specific antisera directed against cell surface antigens, we could demonstrate that the target cells of AEV express erythroblast-specific antigen(s) and less intensively erythrocyte-specific antigen(s), while those of MC29 and AMV express myeloblast-specific antigen(s). In addition, MC29-target cells express macrophage-specific antigen(s). None of the AEV-target cells are adherent or phagocytic, while a small proportion of the AMV-target cells are adherent and about half of the MC29-target cells are both adherent and phagocytic. Our results support the concept that DLVs specifically transform certain types of committed erythroid and myeloid progenitor cells. The target cells of AEV and AMV appear to resemble the respective transformed cells in their state of differentiation, whereas those of MC29 appear to be more immature than the corresponding transformed cells.     

v-myb does not prevent the expression of c-myb in avian erythroblasts.

The v-myb oncogene of avian myeloblastosis virus transforms myeloid cells exclusively, both in vivo and in vitro. The c-myb proto-oncogene from which v-myb arose is expressed at relatively high levels in immature hematopoietic cells of the lymphoid, erythroid, and myeloid lineages but not in myeloblasts transformed by v-myb. This finding suggested that the nuclear v-myb gene product p48v-myb might act directly to inhibit the normal expression of the c-myb gene. I have therefore used a selectable avian retroviral vector to express p48v-myb in avian erythroblasts which normally express high levels of the c-myb gene product p75c-myb. The results demonstrate that p48v-myb and p75c-myb can be coexpressed in the nuclei of cloned cells. Therefore, p48v-myb does not invariably prevent the expression of p75c-myb.     

HLS7, a hemopoietic lineage switch gene homologous to the leukemia-inducing gene MLF1.

Hemopoietic lineage switching occurs when leukemic cells, apparently committed to one lineage, change and display the phenotype of another pathway. cDNA representational difference analysis was used to identify myeloid-specific genes that may be associated with an erythroid to myeloid lineage switch involving the murine J2E erythroleukemic cell line. One of the genes isolated (HLS7) is homologous to the novel human oncogene myeloid leukemia factor 1 (MLF1) involved in the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia. Enforced expression of HLS7 in J2E cells induced a monoblastoid phenotype, thereby recapitulating the spontaneous erythroid to myeloid lineage switch. HLS7 also inhibited erythropoietin- or chemically-induced differentiation of erythroleukemic cell lines and suppressed development of erythropoietin-responsive colonies in semi-solid culture. However, intracellular signaling activated by erythropoietin was not impeded by ectopic expression of HLS7. In contrast, HLS7 promoted maturation of M1 monoblastoid cells and increased myeloid colony formation in vitro. These data show that HLS7 can influence erythroid/myeloid lineage switching and the development of normal hemopoietic cells.     

Activation of cMGF expression is a critical step in avian myeloid leukemogenesis.

A non-leukemogenic version of the v-myb oncogene causes in vitro transformation of avian myeloblasts, which are dependent on chicken myelomonocytic growth factor (cMGF). We have shown that this version of v-myb, when combined with the erythroleukemia-inducing v-erbB oncogene, is capable of causing a mixed myeloid and erythroid leukemia. Myeloid leukemic cells transformed by this construct produce cMGF. To test whether autocrine growth stimulation via cMGF is the essential contribution of the tyrosine kinase oncogene v-erbB in avian myeloid leukemogenesis we constructed another retrovirus containing both the non-leukemogenic v-myb and the cMGF cDNA. This virus induced myeloid leukemia at high efficiency. In a third construct we combined v-myb with the human EGF-receptor gene. Myeloid cells transformed by this construct could be stimulated to grow by the addition of cMGF or EGF. Growth stimulation with EGF was blocked by a cMGF antiserum indicating that activation of a normal tyrosine kinase-type receptor induces cMGF expression but does not bypass the cMGF requirement. We conclude that cMGF plays a key role in the growth regulation of normal and transformed avian myeloid cells.     

Sequences responsible for erythroid and lymphoid leukemia in the long terminal repeats of Friend-mink cell focus-forming and Moloney murine leukemia viruses.

Despite the high degree of homology (91%) between the nucleotide sequences of the Friend-mink cell focus-forming (MCF) and the Moloney murine leukemia virus (MuLV) genomic long terminal repeats (LTRs), the pathogenicities determined by the LTR sequences of the two viruses are quite different. Friend-MCF MuLV is an erythroid leukemia virus, and Moloney MuLV is a lymphoid leukemia virus. To map the LTR sequences responsible for the different disease specificities, we constructed nine viruses with LTRs recombinant between the Friend-MCF and Moloney MuLVs. Analysis of the leukemia induced with the recombinant viruses showed that a 195-base-pair nucleotide sequence, including a 75-base-pair nucleotide Moloney enhancer, is responsible for the tissue-specific leukemogenicity of Moloney MuLV. However, not only the enhancer but also its downstream sequences appear to be necessary. The Moloney virus enhancer and its downstream sequence exerted a dominant effect over that of the Friend-MCF virus, but the enhancer sequence alone did not. The results that three of the nine recombinant viruses induced both erythroid and lymphoid leukemias supported the hypothesis that multiple viral genetic determinants control both the ability to cause leukemia and the type of leukemia induced.