A proteomic analysis of protein variations during differentiation of v-myb-transformed monoblasts

v-myb oncogene of avian myeloblastosis virus (AMV) transforms myelomonocytic cells in vitro and induces acute monoblastic leukemia in vivo. The transforming effect of the v-myb can be suppressed using phorbol ester (TPA) or histone deacetylase inhibitor trichostatin A (TSA), the inducers of cell differentiation that are in clinical trials. In this study, we used proteomics-based approach to identify proteins with variable expression in differentiated BM2 cells. Proteome variations induced by TPA and TSA were compared to examine the mechanism of differentiation-promoting effects of these drugs. We found that expression of several proteins participating in cell cytoskeleton rearrangement, heat shock response, proteosynthesis and cell signaling was altered in TPA- or TSA-treated cells. We present here the first comparative proteome analysis of v-myb-transformed monoblasts BM2 focused on identification of proteins involved in their terminal differentiation.     

The nuclear oncogenes v-erbA and v-ets cooperate in the induction of avian erythroleukemia.

The nuclear oncogenes v-erbA and v-ets are known to cooperate with other viral oncogenes in the induction of avian erythroleukemia. Thus, in the case of avian erythroblastosis virus (AEV), v-erbA enhances the effect of the tyrosine kinase-encoding v-erbB oncogene by blocking the terminal differentiation of erythroid cells. In the case of E26 virus a fusion of the product from v-ets to that of the nuclear oncogene v-myb is a prerequisite for leukemogenicity. Here we show that an artificial virus carrying both v-erbA and v-ets induces a rapid, acute erythroleukemia phenotypically similar to that induced by AEV. In contrast, virus constructs containing either v-erbA or v-ets alone are non-leukemogenic, although they are capable of transforming erythroid cells in vitro. Analysis of in vitro-transformed cells showed that v-erbA induces a block of differentiation without abrogating dependence on anemic serum, while v-ets predominantly causes anemic serum independence. As expected, cells transformed by both oncogenes exhibit an increased proliferative potential, are blocked in differentiation and are anemic serum independent. These data demonstrate that two separately expressed nuclear oncoproteins can complement each other in vitro and in vivo. They also show that the v-Ets protein on its own can contribute to leukemogenesis.     

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.     

Transforming potential of truncated v-myb and stimulation of replication by gag-myb fusion products.

We have previously reported that truncated forms of the v-myb oncogene of avian myeloblastosis virus (AMV) are expressed in transformed chicken embryo fibroblasts (CEF). In this paper, we show that deletion mutants encoding v-myb products altered in either the DNA-binding or the negative regulatory domains are able to induce CEF transformation. In addition, we report that recombinant plasmids expressing gag-myb fusion proteins are maintained as extrachromosomal forms in transfected cells. This observation provides an important clue for a possible role of myb in the DNA replication processes.     

Identification of genes differentially expressed in two types of v-myb-transformed avian myelomonocytic cells.

In an earlier study we found that different forms of the v-myb oncogene transform myeloid cells which resemble either monoblasts [when v-myb of avian myeloblastosis virus (AMV) was used] or promyelocytes [when a point mutant in v-myb of AMV was used; Introna, M., Golay, J., Frampton J., Nakano, T., Ness, S.A. & Graf, T. (1990). Cell, 63, 1287-1297]. In the present study we have searched for genes expressed in AMV mutant-transformed promyelocytes that are not expressed in AMV-transformed monoblasts using a differential screening approach. Eight different genes were identified among more than 500 differentially expressed clones. The most abundant of these was the previously identified myb-regulated mim-1 gene. The others were found to encode a small calcium-binding (MRP-like) protein; the p20K protein; goose-type lysozyme; a ribonuclease A/angiogenin-related protein; and three non-identified proteins. Although these genes appear to be rather lineage restricted, their expression varied in different subtypes of transformed myelomonocytic cells, and only two of them (goose lysozyme and ribonuclease) showed a similar expression pattern in normal promyelocytes and macrophages, suggesting an aberrant gene regulation in the transformed cells. Co-transfection experiments of a reporter construct containing the promoter of the ribonuclease A-related gene indicated that this promoter is regulated by the v-Myb oncoprotein without the involvement of Myb-specific binding sequences.     

Mapping of a small phosphopeptide at the carboxyterminus of the viral myb protein by monoclonal antibodies

Several myb-specific monoclonal antibodies were produced and their antigen recognition sites characterized using a series of bacterially expressed truncated myb proteins. The monoclonal antibodies were used for analysing the in vivo phosphorylation site of the oncogene protein from avian myeloblastosis virus (AMV), p48v-myb. The p48v-myb protein labeled metabolically with [32P]orthophosphate was isolated from the AMV-transformed chicken myeloblast cell line BM-2 by immunoaffinity chromatography. Phosphoamino acid analysis indicated that it was phosphorylated mainly on serine and to a lesser extent (less than 5%) on threonine residues. Indirect immunoprecipitation of phosphopeptides from trypsin-digested [32P]-labeled purified p48v-myb protein by use of the myb-specific monoclonal antibodies allowed the mapping of a small phosphopeptide at the carboxyterminus of p48v-myb.     

A newly isolated avian sarcoma virus, ASV-1, carries the crk oncogene

Avian sarcoma virus 1 (ASV-1) was isolated from a spontaneous sarcoma of an adult chicken. It is a replication-defective virus that induces fibrosarcomas in young chickens and oncogenic transformation in cultured chick embryo fibroblasts. The ASV-1 genome has been cloned in the lambda gt WES.lambda B vector from closed circular viral DNA extracted from infected cells. The nucleotide sequence of the oncogene insert in the ASV-1 genome has been determined. It is virtually identical to the sequence of the oncogene crk recently discovered in the avian sarcoma virus CT10. ASV-1 and CT10 are two independent retrovirus isolates carrying the crk oncogene.     

Proto-oncogene expression in avian hematopoietic tissues

Previous findings from this laboratory (Kim & Baluda, 1988) have shown that the proto-oncogenes ETS, FPS, MHT (RAF), MYC and REL are expressed in avian myeloblastosis virus (AMV)-transformed cells, whereas the MYB gene is repressed. In this study five different chicken hematopoietic tissues which contained varying concentrations of target cells for AMV transformation were analyzed to determine whether the expression of these proto-oncogenes resulted from, or was altered by, v-myb-induced leukemogenesis. Poly-A+ RNA from hematopoietic cells of 11-13 day yolk sac, 16 day embryonic spleen, 1 day post-hatch bursa of Fabricius, bone marrow and thymus, as well as from chicken embryonic fibroblasts (CEF) was examined by Northern blot analysis. All five proto-oncogenes were found to be expressed in the normal hematopoietic tissues. The ETS, MHT (RAF), MYC, and REL genes, but not FPS, were expressed in CEF. The expression of these five proto-oncogenes was not quantitatively or qualitatively altered in AMV-transformed myeloid cells as compared with their normal counterparts. While their expression is part of the hematopoietic phenotype of the target cells and as such is necessary for susceptibility to AMV transformation, it is not sufficient because thymocytes with a high level of expression are not transformed. This is in contrast to MYB expression, which is totally repressed in leukemic cells but probably not as a result of v-myb expression.     

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).     

Transformation of murine pre-B-lymphocytes by v-erb-B: progression to growth factor independence and tumorigenicity.

v-erb-B is the principal transforming gene of avian erythroblastosis virus, a replication defective retrovirus that transforms erythroid and fibroblast cells in vitro and causes erythroleukemia and fibrosarcoma in vivo. We have used a recombinant murine retrovirus, based on the truncated genome of Moloney murine leukemia virus and containing a chimeric gag-v-erb-B gene, to determine the murine hematopoietic cells transformed by v-erb-B. Infection of day 16.5 murine embryonal cells in liquid culture with this virus resulted in the outgrowth of foci of loosely to non-adherent hematopoietic cells which grew in close association with an adherent monolayer. After several weeks in culture a clonal population of transformed pre-B-lymphocytes emerged from this transformation initiated, though still growth factor dependent, population. These transformed cells were growth factor independent, and were tumorigenic in syngeneic mice. The results indicate that although v-erb-B can initiate transformation of murine hematopoietic cells, additional events are required for establishment of the fully transformed growth factor independent, tumorigenic phenotype. This v-erb-B induced progression from growth factor dependence to independence provides an in vitro model system for analysing events involved in the initiation and maintenance of leukemia.     

Histone H5 in the immature blood cells of chickens with leukosis induced by avian leukosis virus strain E26

Whole histone was isolated from the immature cells of the peripheral blood of White Leghorn chickens, line 151, with leukosis experimentally induced by avian leukosis virus, strain E26 (ALV-E26). Histone H5 was demonstrated in all samples of these cells and was characterized by electrophoresis in polyacrylamide gel, extraction with perchloric acid, amino acid analysis, and immunodiffusion in agarose gel. Histone H5 was not detected in the myeloblasts of chickens with myeloblastosis caused by the avian myeloblastosis virus. It was concluded that the immature cells (IBC-E26) obtained from the peripheral blood of chickens responding positively to ALV-E26 belonged to the erythroid blood cell series and that ALV-E26 induced in vivo erythroblastosis but not myeloblastosis in chickens. The relative amount of histone H5 in IBC-E26 was two times higher than that in the erythroblasts obtained from the peripheral blood of chickens with erythroblastosis caused by avian erythroblastosis virus, strain R of Engelbreth-Holm. Thus the erythroblasts in the circulating blood of chickens with leukosis induced by ALV-E26 seemed to be more differentiated.     

Differential-effects of viral and cellular oncogenes on the growth factor-dependency of hematopoietic-cells

The effects of different viral and cellular oncogenes on the cytokine-dependency of murine hematopoietic cell lines were compared. The myeloid FDC-P1 cell line was sensitive to abrogation of growth factor-dependency by the constitutive expression of viral oncogenes (v-abl, v-src, v-Ha-ras, and v-fms) and the activated cellular oncogene BCR-ABL and Delta Nraf. The Delta Nraf encoded serine-threonine kinase was approximately 100-fold less efficient in relieving the factor-dependency of FDC-P1 cells than the other oncogenes examined. The synthesis of autocrine cytokines was not detected in the factor-independent FDC-P1 lines, indicating that the oncogene-mediated transformation occurred by a non-autocrine mechanism. A low frequency of cells were isolated after infection with the chronic retrovirus, murine leukemia virus and approximately 40% of these clones synthesized the autocrine lymphokine, granulocyte-macrophage colony stimulating factor. In contrast, only the v-abl and BCR-ABL oncogenes relieved the cytokine-dependency of the lymphoid FL5.12 cell line. In all the transformed cell lines, the rate of glucose transport was elevated above the basal level seen in uninfected cells indicating that this pivotal growth-regulated protein was associated with malignant transformation. In summary, these cell lines varied with respect to abrogation of growth factor-dependency as the myeloid FDC-P1 line was sensitive to transformation by all oncogenes examined whereas only the abl-family members would relieve the cytokine-requirement of lymphoid FL5.12 cells.     

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.     

Oncogene activation in human myeloid leukemia

We have studied by means of DNA-mediated gene transfer the activation of protooncogenes in human myeloid leukemias that represent various stages of myeloid differentiation. DNA from three cell lines, HL-60 (promyelocytic leukemia), Rc2a (myelomonocytic leukemia), and KG-1 (acute myeloblastic leukemia), was capable of transforming NIH/3T3 cells. Hybridization analysis indicated that, in all three tumor cell lines, the N-ras oncogene was activated. The cell lines U-937 ("histiocytic lymphoma") and K-562 (erythroblastic leukemia) yielded no transforming DNA. Fresh leukemia cells derived from an acute myelomonocytic leukemia patient and from a juvenile chronic myelogenous leukemia patient contained an activated N-ras and c-Ki-ras oncogene, respectively. DNA from some other myelogenous leukemia patients was not able to transform NIH/3T3 cells. Our results indicate that hematopoietic tumors of the myeloid lineage may contain oncogenes active in NIH/3T3 cell transformation and that, in particular, the N-ras oncogene may be activated in tumors representing various stages of maturation.     

Avian erythroblastosis virus isolated from chick erythroblastosis induced by lymphatic leukemia virus subgroup A

A new strain of avian erythroblastosis virus (AEV), designated "AEV-H," was established by serial passages of a field isolate of avian lymphatic leukemia virus subgroup A [LLV(A)] in chicks from a White Leghorn flock of line 151 chicks. One stock of AEV-H contained 10(4) focus-forming units/ml virus and 10(9) tissue culture infective dose/ml LLV(A). All of the chicks that received ip inoculations of 0.2 ml AEV-H developed erythroblastosis complicated by fibrosarcoma 16-24 days (approximately equal to 17.7 days) after inoculation. Pathologic changes of erythroblastosis were macroscopically observed, mainly in such visceral organs as the liver, spleen, and bone marrow. In addition, microscopic changes were observed in the lung, kidney, ovary, and heart. Pathologic changes of fibrosarcoma were so conspicuous that they were recognizable by the naked eye in the pancreas and in the serous membrane of the intestine.