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Gene information | Literature | Expression | lncRNA | Mutation | Homolog

Basic Information

Gene ID





v-myb avian myeloblastosis viral oncogene homolog;MYB;v-myb avian myeloblastosis viral oncogene homolog


c-myb protein (140 AA)|c-myb10A_CDS|c-myb13A_CDS|c-myb14A_CDS|c-myb8B_CDS|proto-oncogene c-Myb|transcriptional activator Myb



Gene type




Myb-interacting protein, ATBF1, represses transcriptional activity of Myb oncoprotein.

Using the yeast two-hybrid system, the transcription factor ATBF1 was identified as v-Myb- and c-Myb-binding protein. Deletion mutagenesis revealed amino acids 2484-2520 in human ATBF1 and 279-300 in v-Myb as regions required for in vitro binding of both proteins. Further experiments identified leucines Leu325 and Leu332 of the Myb leucine zipper motif as additional amino acid residues important for efficient ATBF1-Myb interaction in vitro. In co-transfection experiments, the full-length ATBF1 was found to form in vivo complexes with v-Myb and inhibit v-Myb transcriptional activity. Both ATBF1 2484-2520 and Myb 279-300 regions were required for the inhibitory effect. Finally, the chicken ATBF1 was identified, showing high degree of amino acid sequence homology with human and murine proteins. Our data reveal Myb proteins as the first ATBF1 partners detected so far and identify amino acids 279-300 in v-Myb as a novel protein-protein interaction interface through which Myb transcriptional activity can be regulated.

The v-Myb oncoprotein activates C/EBPbeta expression by stimulating an autoregulatory loop at the C/EBPbeta promoter.

Previous studies have implicated the CCAAT box/enhancer binding protein beta (C/EBPbeta) in the regulation of cell-type specific gene expression in myelomonocytic cells and in the activation of target genes by the transcription factor v-Myb. To better understand the role of C/EBPbeta in myelomonocytic cells we have cloned the chicken C/EBPbeta gene and studied its regulation. The chicken C/EBPbeta promoter contains a number of C/EBP binding sites and is activated by C/EBPbeta, suggesting that the C/EBPbeta gene is autoregulated by its own protein product. Interestingly, the C/EBPbeta promoter is not activated by C/EBPalpha, another C/EBP family member highly expressed in myelomonocytic cells, indicating that the autoregulation is specific for C/EBPbeta. Comparison of different C/EBP inducible promoters shows that the relative transactivation potential of C/EBPalpha and beta is extremely dependent on the promoter context. By using the promoters of the mim-1 and C/EBPbeta genes and by exchanging the DNA-binding domains between C/EBPalpha and beta we show that the observed promoter preferences of C/EBPalpha and beta are not due to differential DNA-binding but instead depend on the transactivation domains of these proteins. The C/EBPbeta promoter also contains several Myb binding motifs, suggesting that the C/EBPbeta gene is also myb-inducible. We show that the C/EBPbeta promoter is activated synergistically by v-Myb and C/EBPbeta and that transcription of the endogenous C/EBPbeta gene is increased by v-Myb. Thus, our results identify the C/EBPbeta gene as a novel v-Myb target gene. Taken together, our data suggest a model for the regulation of C/EBPbeta expression in which v-Myb stimulates the synthesis of C/EBPbeta by enhancing an autoregulatory loop acting on the C/EBPbeta promoter.

B-myb proto-oncogene products interact in vivo with each other via the carboxy-terminal conserved region.

Using the yeast two-hybrid assay and in vivo binding assay, we investigated whether B-myb oncogene products (B-myb) can associate with each other. Specificity tests of the yeast two-hybrid system showed a self-association of B-myb proteins in yeast. Cotransfection experiments demonstrated that B-myb proteins form a complex in vivo. Deletion analysis revealed that this binding was sufficiently mediated by the carboxy-terminal conserved region of B-myb. In addition, the B-myb self-association is directly dependent on the amount of expressed B-myb in cells and slightly increased by the dephosphorylation state. These results suggested that B-myb could form a complex and influence its transcriptional activity.

A novel myb oncogene homologue in Arabidopsis thaliana related to hypersensitive cell death.

A novel myb oncogene homologue (AtMYB30) has been isolated by differential screening of a cDNA library prepared from Xanthomonas campestris pv. campestris (X. campestris)-inoculated Arabidopsis thaliana cells cultured in the presence of cycloheximide. AtMYB30 is a single-copy gene, and the encoded protein contains a MYB domain highly homologous to other plant and animal MYB proteins. Analyses of transcript levels in A. thaliana plants, or in cultured A. thaliana cells infected with either virulent or avirulent strains of the pathogens X. campestris and Pseudomonas syringae pv. tomato, showed that maximal levels of transcription of this gene occurred during the hypersensitive response. Furthermore, in A. thaliana mutants affected in the control of cell death initiation (lsd3, lsd4 and lsd5), constitutive expression or expression in lesion-positive plants was observed, while in suppressors of the mutations lsd5 and lsd4, AtMYB30 transcripts did not accumulate. However, AtMYB30 expression could not be detected in the lsd1 mutant, which was hyper-responsive to cell death initiators and unable to limit the extent of cell death, whatever the environmental conditions. The results presented here suggest a strong correlation between AtMYB30 and genetically controlled cell death, with a role in the initiation of cell death rather than in the limitation of its extent. Our results further indicate that the lsd mutants constitute an appropriate genetic model for studying the role of this gene in hypersensitive cell death, and their relation to different steps of the pathway(s) leading to cell death.

Retinoid X receptor suppresses transformation by the v-myb oncogene.

The v-myb oncogene of avian myeloblastosis virus causes acute monoblastic leukemia in vivo and transforms myelomonocytic cells in culture. Retinoids are potent regulators of proliferation and differentiation in various cell types, and they can initiate differentiation in certain types of leukemic cells. However, the BM2 v-myb-transformed chicken monoblastic cell line is resistant to retinoic acid treatment. We found that overexpression of the retinoid X receptor confers sensitivity of BM2 cells to retinoic acid, resulting in induction of growth arrest and terminal differentiation. In contrast, the frequency of apoptosis was not affected by the retinoid X receptor in this cell type. We also demonstrated that suppression of transformation by v-Myb results from the negative effect of retinoid X receptor on v-Myb transactivation function, similar to that previously described for the retinoic acid receptor. The retinoid X receptor-induced inhibition of transactivation by v-Myb seems to be enhanced by a cell type-specific factor(s), which is not required by retinoic acid receptor.

Interaction of V-Myb oncoprotein with spread chromatin of avian haematopoietic cells.

Interactions of v-Myb oncoprotein with spread chromatin of avian LSCC-BM2 cells expressing v-myb oncogene were studied by means of immunoelectron microscopy. The application of this technique using anti-Myb polyclonal antibody combined with the Miller type spreading for visualisation of chromatin revealed the presence of Myb protein on stretched chromatin fibres. Intense labelling was apparent on the chromatin dispersed by hypotonic treatment, where the label was present frequently in clusters, although individual marks along the fibrillar molecules were also found. The combination of hypotonic and detergent treatment resulted in better dispersal of chromatin, more frequent detection of active transcription units, but also in removal of some proteins from chromatin fibres. The labelling of chromatin with anti-Myb antibody was substantially reduced in this case and was dependent on detergent concentration used. The marker was found less frequently on chromatin fibres usually present in clusters on remaining protein structures. Our findings confirmed direct interaction of v-Myb protein with chromatin structure. This interaction is apparently affected by detergent treatment.

Baculovirus expression system cells expressing v-myb oncogene: the distribution of RNA and DNA in specific nuclear compartments with respect to structures interacting with anti-v-Myb antibody.

The distribution of RNA, total DNA and newly synthesized DNA within nucleoli-like structures in insect cells overexpressing v-myb oncogene was investigated. Three types of these structures which revealed interaction with anti-v-Myb oncoprotein antibody were found at the ultrastructural level. Specific staining by toluidine blue at pH 5.2 showed the presence of RNA in these nucleoli-like structures. To detect total DNA, the in situ terminal deoxynucleotidyl transferase-immunogold technique was used. In addition to an expected labeling of host condensed chromatin, the labeling of the three types of nucleoli-like structures differed from each other. While the compact (type I) and ring-shaped (type II) nucleoli-like structures were labeled only on their periphery and in the proximity of baculovirus particles that interacted with them, the structures with an appearance of nucleolonemas (type III) were labeled strongly. Incorporation of 5-bromo-2-deoxyuridine, in spite of a poor labeling of newly synthesized DNA, confirmed these results. We suggest that the nucleoli-like structures of type I and II are of nucleolar origin. The type III more likely represents virogenic stroma or viral DNA storage site.

c-MYB oncogene-like genes encoding three MYB repeats occur in all major plant lineages.

Since the identification of the first plant MYB-like protein, the Zea mays factor C1, the number of MYB-related genes described has greatly increased. ALL of the more than 150 plant MYB-like proteins known so far contain either two or only one sequence-related helix-turn-helix motif in their DNA-binding domain. Animal c-MYB genes contain three such helix-turn-helix motif-encoding repeats (R1R2R3 class genes). It has therefore been concluded that R2R3-MYB genes are the plant equivalents of c-MYB and that there are significant differences in the basic structure of MYB genes of plants and animals. Here, we describe expressed R1R2R3-MYB genes from Physcomitrella patients++ and Arabidopsis thaliana, designated PpMYB3R-1 and AtMYB3R-1. The amino acid sequences of their DNA-binding domains show high similarity to those of animal MYB factors, and less similarity to R2R3-MYB proteins from plants. In addition, R1R2R3-MYB genes were identified in different plant evolutionary lineages including mosses, ferns and monocots. Our data show that a DNA-binding domain consisting of three MYB repeats existed before the divergence of the animal and plant lineages. R1R2R3-MYB genes may have a conserved function in eukaryotes, and R2R3-MYB genes may predominantly regulate plant-specific processes which evolved during plant speciation.

Ultrastructural analysis of v-myb oncogene product cooperation with components of avian cell nuclear matrix.

The cooperation of the v-Myb oncoprotein with extracted nuclear matrix of avian haematopoietic cells expressing the v-myb oncogene was studied by means of immunoelectron microscopy. The nuclear matrix was extracted by a gentle method of detergent treatment at moderate ionic strength and visualized either in ultrathin LR White sections, in unembedded resin-free sections, and in addition by the aqueous spreading technique. Using anti-Myb polyclonal antibody we have shown interaction of the v-Myb protein product with extracted nuclear matrix. This oncoprotein, however, was easily released from the structure by a detergent as well as by DNAase treatment and ammonium sulphate extraction. Prefixation of structures before detergent treatment prevented this extraction. The v-Myb protein marker was distributed in clusters or associated with fibrillar structures in most cases. Single markers decorating these fibrillar or less dense structures were also detected.

MYB oncogene amplification in hereditary BRCA1 breast cancer.

Comparative genomic hybridization analysis has demonstrated that breast tumors from BRCA1 and BRCA2 germ-line mutation carriers contain a large number of chromosomal copy number gains and losses. A high regional copy number gain at 6q22-q24 was observed in one BRCA1 tumor, and fluorescence in situ hybridization analysis indicated a strong amplification of the MYB oncogene (15 copies of MYB compared with 1 copy of chromosome 6 centromere). Fluorescence in situ hybridization analysis revealed amplification of MYB in 5 (29%) of 17 BRCA1 breast tumors, whereas none of 8 BRCA2 tumors and 13 breast cancer cell lines, and only 2 of 100 sporadic breast tumors exhibited altered MYB copy numbers. Gene amplification resulted in mRNA overexpression as determined by Northern blot and cDNA microarray analysis, and protein overexpression by immunohistochemical staining. We conclude that MYB amplification is infrequent in sporadic breast cancer but common in breast tumors from BRCA1 mutation carriers, suggesting a role of this cell cycle regulator and transcription factor in the progression of some BRCA1 tumors. However, we cannot rule out the significance of other genes in the 6q22-q24 amplicon.

Tax oncoprotein trans-represses endogenous B-myb promoter activity in human T cells.

The B-myb gene was identified on the basis of its homology with the protooncogene c-myb, homolog of the avian myeloblastosis virus (AMV) and avian leukemia virus (E26) transforming genes. Several studies using antisense constructs or antisense oligonucleotides as well as overexpression experiments suggest that B-Myb plays an important role in the transition from G(1) to S phase of the cell cycle and that B-Myb expression is cell cycle regulated. We have previously demonstrated that the human T cell lymphotropic virus type 1 (HTLV1) trans-activator Tax is able to repress transcription from c-myb promoter reporter constructs as well as from the endogenous c-myb promoter in human T cells and that this effect is mediated through inhibition of the c-Myb trans-activating functions. Here we report that both HTLV-1 as well as HTLV-2 Tax proteins inhibit c-Myb trans-activation in mouse embryo fibroblasts (MEFs). In addition to c-Myb, B-Myb expression is also markedly downregulated in HTLV-1-transformed cells at both RNA and protein levels. Furthermore, by using a Jurkat T cell line stably transfected with a tax gene driven by a cadmium-inducible promoter (JPX9), we were able to demonstrate that Tax directly represses the endogenous B-myb promoter in T cells. Because c-Myb and B-Myb have been involved in cell cycle progression, our results suggest that Tax, by repressing both c-Myb and B-Myb endogenous promoters, may bypass their requirement for cell cycle progression in HTLV-1-transformed T cells.

Functional evolution of the Myb oncogene family.

Three Myb-related genes (A-Myb, B-Myb, and c- Myb) have been found in ALL vertebrates examined thus far including mammals, birds, and amphibians. Two invertebrates, the sea urchin and the fruit fly, have only one Myb-related gene. Our laboratory has used Drosophila as a model system to explore the function of its sole Myb gene. We have also reintroduced the three different vertebrate Myb genes into Drosophila in order to begin to understand how their different functions may have arisen following gene duplication during evolution.

Intra- and intermolecular triplex DNA formation in the murine c-myb proto-oncogene promoter are inhibited by mithramycin.

Mithramycin inhibits transcription by binding to G/C-rich sequences, thereby preventing regulatory protein binding. However, it is also possible that mithramycin inhibits gene expression by preventing intramolecular triplex DNA assembly. We tested this hypothesis using the DNA triplex adopted by the murine c-myb proto-oncogene. The 5 -regulatory region of c-myb contains two polypurine:polypyrimidine tracts with imperfect mirror symmetry, which are highly conserved in the murine and human c-myb sequences. The DNA binding drugs mithramycin and distamycin bind to one of these regions as determined by DNase I protection assay. Gel mobility shift assays, nuclease and chemical hypersensitivity and 2D-gel topological analyses as well as triplex-specific antibody binding studies confirmed the formation of purine*purine:pyrimidine inter- and pyrimidine*purine:pyrimidine intra-molecular triplex structures in this sequence. Mithramycin binding within the triplex target site displaces the major groove-bound oligonucleotide, and also abrogates the supercoil-dependent H-DNA formation, whereas distamycin binding had no such effects. Molecular modeling studies further support these observations. Triplex-specific antibody staining of cells pretreated with mithramycin demonstrates a reversal of chromosomal triplex structures compared to the non-treated and distamycin-treated cells. These observations suggest that DNA minor groove-binding drugs interfere with gene expression by precluding intramolecular triplex formation, as well as by physically preventing regulatory protein binding.

The leucine zipper region of Myb oncoprotein regulates the commitment of hematopoietic progenitors.

The development of blood cells proceeds from pluripotent stem cells through multipotent progenitors into mature elements belonging to at least 8 different lineages. The lineage choice process during which stem cells and progenitors commit to a particular lineage is regulated by a coordinated action of extracellular signals and transcription factors. Molecular mechanisms controlling commitment are largely unknown. Here, the transcription factor v-Myb and its leucine zipper region (LZR) are identified as regulators of the commitment of a common myeloid progenitor and progenitors restricted to the myeloid lineage. It is demonstrated that wild-type v-Myb with the intact LZR directs development of progenitors into the macrophage lineage. mutations in this region compromise commitment toward myeloid cells and cause v-Myb to also support the development of erythroid cells, thrombocytes, and granulocytes, similar to the c-Myb protein. In agreement with that, the wild-type v-Myb induces high expression of myeloid factors C/EBP beta, PU.1, and Egr-1 in its target cells, whereas SCL, GATA-1, and c-Myb are more abundant in cells expressing the v-Myb LZR mutant. It is proposed that Myb LZR can function as a molecular switch, affecting expression of lineage-specifying transcription factors and directing the development of hematopoietic progenitors into either myeloid or erythroid lineages.

Activation of c-MYC and c-MYB proto-oncogenes is associated with decreased apoptosis in tumor colon progression.

BACKGROUND: An increasing amount of evidence suggests that progression from normal mucosa to colorectal cancer is accompanied by morphological and genetic alterations. Genetic abnormalities affect malignant transformation via a gradual imbalance of normal tissue homeostasis involving programmed cell death (PCD) or apoptosis. Therefore, it has been hypothesized that alterations in apoptosis may contribute to carcinogenesis. The aim of the present work was to investigate the relationship between frequency of spontaneous apoptosis during transition adenoma-to-carcinoma of the colorectal tract and the incidence of activation of c-myc and c-myb proto-oncogenes, involved both in colon tumorigenesis and apoptosis. MATERIALS AND METHODS: Ninety-five tissue specimens (60 polyps and 35 adenocarcinomas) were removed with autologous normal adjacent mucosa from colon cancer patients. Genomic DNA was extracted and analyzed for both apoptosis frequency (DNA fragmentation assay) and proto-oncogene activation (Southern blot analysis). On the same samples, Bcl-2 protein expression was evaluated by immunohistochemistry. RESULTS: Our results showed that: i) a significant relationship exists between apoptosis and genesis of colorectal cancer since, compared to adenomatous polyps and adjacent normal mucosa, cell death is markedly inhibited in tumors (p = 0.01); ii) during colon tumor progression, apoptosis and amplifications of c-myc/c-myb genes are inversely related; iii) Bcl-2 expression is retained in colon tumors even though at a significantly lower level with respect to adenomatous polyps. CONCLUSION: These results indicate that failure of the normal apoptotic process together with de-regulation of c-myc and c-myb proto-oncogenes might promote the development of colorectal tumors.

Overexpression of v-myb oncogene or c-myb proto-oncogene in insect cells: characterization of newly induced nucleolus-like structures accumulating Myb protein.

The oncoprotein v-Myb induces myeloid leukemia and its cellular counterpart c-Myb is involved in the regulation of hematopoiesis. Although intensively studied, their precise subcellular localization is not known. In order to expand our knowledge in this respect, we used an artificial system overexpressing these proteins. We investigated the subcellular localization of Myb proteins in cultured non-synchronized insect cells transfected with recombinant baculoviruses overexpressing either v-myb oncogene or c-myb proto-oncogene. The cell expressing Myb proteins underwent extensive nuclear changes and exhibited distinct nuclear structures resembling nucleoli. The bulk of v-Myb and c-Myb proteins accumulated in such nucleolus-like structures which, according to the nucleolar nomenclature, we classified to three types: compact of enlarged size (type I), large ring-shaped (type II) and with nucleolonemas (type III). We investigated these structures for the presence of important nucleolar macromolecules in order to establish whether they were compatible with the function in the production of ribosomes. Strikingly, our results indicated that the different forms of these structures did not represent genuine nucleoli. They rather reflected progressive changes, induced by the virus infection and high expression of v-myb genes, accompanied by the formation of these prominent nucleolus-like structures highly enriched in Myb protein. Gradual changes in number of individual nucleolus-like forms during infection, increasing amount of Myb protein and DNA localized in them together with decreasing amount of RNA and their different interaction with viral particles indicate that the nucleolus-like structure of type I is a precursor of the type II and finally of the type III.

Phosphorylation-dependent down-regulation of c-Myb DNA binding is abrogated by a point mutation in the v-myb oncogene.

The viral Myb (v-Myb) oncoprotein of the avian myeloblastosis virus (AMV) is an activated form of the cellular transcription factor c-Myb causing acute monoblastic leukemia in chicken. Oncogenic v-Myb alterations include N- and C-terminal deletions as well as point mutations. Whereas truncations in Myb cause loss of various protein modifications, none of the point mutations in v-Myb has been directly linked to protein modifications. Here we show that the DNA-binding domain of c-Myb can be phosphorylated on serine 116 by the catalytic subunit of protein kinase A. Phosphorylation of Ser(116) differentially destabilizes a subtype of c-Myb-DNA complexes. The V117D mutation of the AMV v-Myb oncoprotein abolishes phosphorylation of the adjacent Ser(116) residue. Modification of Ser(116) was also detected in live cells in c-Myb, but not in AMV v-Myb. Phosphorylation-mimicking mutants of c-Myb failed to activate the resident mim-1 gene. Our data imply that protein kinase A or a kinase with similar specificity negatively regulates c-Myb function, including collaboration with C/EBP, and that the leukemogenic AMV v-Myb version evades inactivation by a point mutation that abolishes a phosphoacceptor consensus site. This suggests a novel link between Myb, a signal transduction pathway, cooperativity with C/EBP, and a point mutation in the myb oncogene.

Trichostatin A suppresses transformation by the v-myb oncogene in BM2 cells.

BM2 cells are chicken monoblasts transformed by the v-myb oncogene of avian myeloblastosis virus. The constitutively high v-myb expression interferes with the terminal differentiation of BM2 cells, but these cells can be induced to differentiate into macrophage-like cells by phorbol esters. Histone acetylation plays an important role in regulation of transcription and is particularly relevant to the regulation and pathology of hematopoiesis. In the present study, we examined the contribution of elevated histone acetylation to the differentiation of BM2 cells. Inhibition of the activity of endogenous histone deacetylases by trichostatin A (TSA) resulted in histone hyperacetylation causing cell cycle arrest and differentiation of BM2 cells into macrophage polykaryons. TSA did not affect the level of v-Myb protein in BM2 cells, but it downregulated its transcription activation capability. This suggests that chromatin remodeling can be significantly engaged in regulation of proliferation and differentiation of leukemic cells.

Transcriptional control by myb oncogene product.

Structure and function of two domains of c-Myb were analyzed. We show that a leucine zipper structure is a component of the negative regulatory domain, because its disruption markedly increases both the transactivating and transforming capacities of c-Myb. Our results suggest that an inhibitor which suppresses transactivation binds to c-Myb through the leucine zipper, and that c-Myb can be oncogenically activated by mis-sense mutation. We also proposed a model, the "tryptophan cluster", for the structure of the Myb DNA-binding domain, in which the three tryptophans form a cluster in the hydrophobic core in each repeat. The results of NMR analysis of repeat 3 revealed that the conserved tryptophans play a key role to make the hydrophobic core.

The cellular oncogene c-myb can interact synergistically with the Epstein-Barr virus BZLF1 transactivator in lymphoid cells.

Regulation of replicative functions in the Epstein-Barr virus (EBV) genome is mediated through activation of a virally encoded transcription factor, Z (BZLF1). We have shown that the Z gene product, which binds to AP-1 sites as a homodimer and has sequence similarity to c-Fos, can efficiently activate the EBV early promoter, BMRF1, in certain cell types (i.e., HeLa cells) but not others (i.e., Jurkat cells). Here we demonstrate that the c-myb proto-oncogene product, which is itself a DNA-binding protein and transcriptional transactivator, can interact synergistically with Z in activating the BMRF1 promoter in Jurkat cells (a T-cell line) or Raji cells (an EBV-positive B-cell), whereas the c-myb gene product by itself has little effect. The simian virus 40 early promoter is also synergistically activated by the Z/c-myb combination. Synergistic transactivation of the BMRF1 promoter by the Z/c-myb combination appears to involve direct binding by the Z protein but not the c-myb protein. A 30-bp sequence in the BMRF1 promoter which contains a Z binding site (a consensus AP-1 site) is sufficient to transfer high-level lymphoid-specific responsiveness to the Z/c-myb combination to a heterologous promoter. That the c-myb oncogene product can interact synergistically with an EBV-encoded member of the leucine zipper protein family suggests c-myb is likely to engage in similar interactions with cellularly encoded transcription factors.

Transformation of chicken myelomonocytic cells by a retrovirus expressing the v-myb oncogene from the long terminal repeats of avian myeloblastosis virus but not Rous sarcoma virus.

To test the effect of long terminal repeat (LTR) regulatory sequences on the transforming capability of the v-myb oncogene from avian myeloblastosis virus (AMV), we have constructed replication-competent avian retroviral vectors with nearly identical structural genes that express v-myb from either AMV or Rous sarcoma virus (RSV) LTRs. After transfection into chicken embryo fibroblasts, virus-containing cell supernatants were used to infect chicken myelomonocytic target cells from preparations of 16-day-old embryonic spleen cells. Both wild-type AMV and the virus expressing v-myb from AMV LTRs (RCAMV-v-myb) were able to transform the splenocyte cultures into a population of immature myelomonocytic cells. The transformed cells expressed the p48v-Myb oncoprotein and formed compact foci when grown in soft agar. In contrast, the virus expressing v-myb from RSV LTRs (RCAS-v-myb) was repeatedly unable to transform the same splenocyte cells, despite being able to infect fibroblasts with high efficiency. This difference in the transforming activities of v-myb-expressing viruses with different LTRs most likely results from the presence of a factor (or factors) within the appropriate myelomonocytic target cell that promotes specific expression from the AMV but not from the RSV LTR.

Myb p75 oncoprotein is expressed in developing otic and epibranchial placodes.

The c-myb proto-oncogene encodes a transcriptional regulatory protein which is highly conserved throughout evolution. Myb has been considered to be normally restricted to hematopoietic tissues, but there are indications that this might not always be the case. The present work shows the expression of a p75 Myb oncoprotein in the otic vesicle and epibranchial placodes of the early chick embryo. expression was sequential and followed the same time course as the formation of placode-derived cranial ganglia. The results suggest a potential role for c-myb in regulation of placode development and neurogenesis.

Protein truncation is not required for c-myb proto-oncogene activity in neuroretina cells.

The v-myb oncogene of avian myeloblastosis virus (AMV) differs from its normal cellular counterpart by a truncation at both its amino and carboxyl termini and by a substitution of 11 amino acid residues. We had previously shown that v-myb-containing AMV, in the presence of basic fibroblast growth factor, transformed chicken neuroretina (CNR) cells. To understand the mechanism of c-myb activation, we have tested whether avian retroviruses that express the full-length c-Myb are also active on CNR cells. We have found that c-Myb, like v-Myb, strongly increases the basic fibroblast growth factor response of CNR cells and that these c-myb-expressing cells are able to grow in soft agar in the presence of the growth factor. We have also found that, in contrast to normal or v-myb-expressing AMV-transformed CNR cells, c-Myb-transformed cells express mim-1, a granulocyte-specific gene. However, normal v-Myb- and c-Myb-expressing CNR cells ALL express the pax-QNR gene, a newly described paired and homeobox-containing gene specifically expressed in the neuroretina. We conclude that, in contrast to what has been described for hematopoietic cells, overexpression of c-Myb is sufficient to activate gene expression and to induce an abnormal behavior of CNR cells.

The c-myb proto-oncogene product binds to but does not activate the promoter of the DNA polymerase alpha gene.

The myb proto oncogene product (c-Myb) is a transcriptional regulator and its expression and function are tightly linked to the cellular entry into S phase and DNA synthesis. It has been shown [Venturelli, D., Travali, S. & Calabretta, B. (1990). Proc. Natl. Acad. Sci. USA, 87, 5963-5967] that inhibition of T-cell proliferation by a myb antisense oligomer is accompanied by down-regulation of DNA polymerase alpha expression. To examine whether the transcription of the DNA polymerase alpha gene is directly regulated by c-Myb, we have identified and characterized the 5 regulatory region of the human DNA polymerase alpha gene. Two major and several minor transcription start sites were identified by nuclease S1 mapping. DNA sequence analysis showed that the promoter region contains no TATA box, one CCAAT box and putative Ap-1, AP-2 and E2F binding sites. In DNAase I footprinting, the bacterially expressed c-Myb protected six sites in the 5 flanking region of the human DNA polymerase alpha gene. However, c-Myb did not activate the DNA polymerase alpha gene promoter in a co-transfection assay. Our results suggest that an unknown factor(s) is required for the c-Myb-induced activation of the DNA polymerase alpha gene promoter, or c-Myb does not directly activate this promoter.

Cooperation between the H-ras oncogene and a truncated derivative of the v-myb oncogene in transformation of hamster embryo fibroblasts.

The ras oncogenes alone fully transform established (immortalized) rodent fibroblasts in a few days, but generally transform early-passage fibroblasts only partially, unless their action is complemented by that of a nuclear, immortalizing, oncogene. Here we show that transfection of second-passage Syrian hamster embryo fibroblasts (HEFs) by the EJ-H-ras oncogene coupled to the neo gene, followed by selection with G418, gives rise to apparently normal, or only slightly transformed, clonal colonies, only a few of which become established. The study of two established clonal lines showed that they acquired only after some weeks, and stepwise, the main characteristics of full neoplastic transformation, i.e. anchorage independence, reduced requirement for serum growth factors and tumorigenicity. Later both clonal lines became increasingly tumorigenic and completely independent of exogenous growth and attachment factors, without increase in the expression of the H-ras oncogene. Transfection of one of the clones, early after its isolation, with a truncated derivative of the nuclear v-myb oncogene devoid of its transcriptional negative regulatory domain and able to partially transform chicken embryo fibroblasts [(myb(KXANM)] gave rise to more transformed cells, expressing both EJ-H-ras and myb(KXANM), which became tumorigenic earlier than the controls and remained more tumorigenic later on. With more efficient transfection techniques, numerous foci of fully transformed cells were subsequently obtained, in a few days, in cultures transfected sequentially with EJ-H-ras(neo) and myb(KXANM) and in cultures co-transfected with the two oncogenes. Highly tumorigenic, serum-independent and immortalized clones expressing both oncogenes were obtained from these cultures. Hence, the truncated myb(KXANM) oncogene accelerate the stepwise transformation of unestablished HEFs by the EJ-HH-ras oncogene and, together with this oncogene, fully transforms these same cells in a single step. The two oncogenes acting in cooperation also induce cell immortalization, but myb(KXANM), by itself, is not an immortalizing oncogene. No cooperation was observed between EJ-H-ras(neo) and the unaltered v-myb oncogene.

Regulation of the gene encoding glutathione S-transferase M1 (GSTM1) by the Myb oncoprotein.

The identification of Myb target genes will not only aid in the understanding of how overexpression of Myb, or expression of activated forms of Myb, leads to cellular transformation but will also shed light on its role in normal cells. Using a combination of an estrogen-regulated Myb-transformed cell line (ERMYB) and PCR-based subtractive hybridization, we have identified the gene (GSTM1) encoding the detoxification enzyme glutathione S-transferase M1 as being transcriptionally upregulated by Myb. Functional analysis of the GSTM1 promoter using reporter assays indicated that both the DNA binding and transactivation domains of Myb were required for transcriptional activation. mutational ana-lysis of consensus Myb-binding sites (MBS) in the promoter and electrophoretic mobility gel shift analysis indicated that one of the three potential MBS can bind Myb protein, and is the primary site involved in the regulation of this promoter by Myb.

Oncogenic activation of c-Myb correlates with a loss of negative regulation by TIF1beta and Ski.

The c-myb proto-oncogene product (c-Myb) regulates proliferation of hematopoietic cells by inducing the transcription of a group of target genes. Removal or mutations of the negative regulatory domain (NRD) in the C-terminal half of c-Myb leads to increased transactivating capacity and oncogenic activation. Here we report that TIF1beta directly binds to the NRD and negatively regulates the c-Myb-dependent trans-activation. In addition, three corepressors (Ski, N-CoR, and mSin3A) bind to the DNA-binding domain of c-Myb together with TIF1beta and recruit the histone deacetylase complex to c-Myb. Furthermore, the Drosophila TIF1beta homolog, Bonus, negatively regulates Drosophila Myb activity. The Ski corepressor competes with the coactivator CBP for binding to c-Myb, indicating that the selection of coactivators and corepressors is a key event for c-Myb-dependent transcription. mutations or deletion of the NRD of c-Myb and the mutations found in the DNA-binding domain of v-Myb decrease the interaction with these corepressors and weaken the corepressor-induced negative regulation of Myb activity. These observations have conceptual implications for understanding how the nuclear oncogene is activated.

Expression of the c-myb proto-oncogene in bovine vascular smooth muscle cells.

Previously we have shown that bovine vascular smooth muscle cells (SMCs) express c-myb mRNA (Reilly, C. F., Kindy, M. S., Brown, K. E., Rosenberg, R. D., and Sonenshein, G. E. (1989) J. Biol. Chem. 264, 6990-6995). Here we have characterized changes in the low level of c-myb mRNA expressed in quiescent serum-deprived subconfluent SMCs upon entry into the cell cycle. After serum stimulation, levels of c-myb mRNA increased 3-4-fold during late G1 and remained at this level during S phase. A 1.5-kilobase partial c-myb cDNA clone, isolated from a bovine SMC library, was partially sequenced and found to be 89 and 85% homologous to the human and murine c-myb genes, respectively. Using bovine and murine c-myb clones, no change in the rate of c-myb gene transcription or mRNA stability was detected during the cell cycle. Thus, the regulation of changes in c-myb mRNA levels in SMCs appears distinct from mechanisms seen in hematopoietic or fibroblastic cells. Vectors containing myb binding sites linked to the thymidine kinase promoter and the chloramphenicol acetyltransferase reporter gene were transiently transfected into SMC cultures. KHK-CAT-dAX, which contains nine concatenated myb binding sites, exhibited 7-fold more activity than the parental dAX-TK-CAT vector in exponentially growing SMCs. The levels of chloramphenicol acetyltransferase activity in exponentially growing cells were approximately 2-fold higher than in cells that had been serum deprived for 24 h and were entering quiescence. Thus SMCs produce a functional c-myb protein that can activate transcription from a heterologous promoter. Furthermore, introduction of antisense c-myb oligonucleotides to quiescent serum-deprived SMC cultures severely inhibited entry of cells into S phase upon serum addition. Thus, expression of the c-myb oncogene plays an important role in cell cycle progression of SMCs.

Mechanisms for the transformation of myeloid and erythroid cells by the nuclear oncogene V-myb.

Spontaneously immortalized human mammary epithelial cells MCF-10A were transfected with an activated c-Ha-ras oncogene. Transfected cells (MCF-10T) acquire a malignant phenotype, as already reported. Studies of 125I-2 -deoxyuridine incorporation in cultures given graded doses of hydrocortisone (HC), cholera toxin (CT), epidermal growth factor (EGF), and transforming growth factor alpha (TGF-alpha) showed that though MCF-10T had become almost independent on exogenous EGF and TGF-alpha, they continued to respond to the synergistic effect of HC and CT plus EGF. Both lines were phenotypically characterized with an immunoradiometric assay in live cells. expression of MHC class-I molecules, human milk-fat-globule-I antigen, and EGF receptor was reduced in ras-transfected cells, although other differentiation markers were unchanged. Exogenous EGF down-regulated the expression of functional EGF-R, selectively in transformed cells. TGF-alpha failed to modulate EGF-R. In contrast, HC strongly stimulated the expression of EGF-R while depressing MHC class-I molecules. Thus, it appears that in vivo HC may co-operate with TGF-alpha and EGF in promoting the growth of transformed mammary cells. This hormone might also favor the escape from immune surveillance by reducing the expression of surface differentiation markers.

Oncogenic mutations cause dramatic, qualitative changes in the transcriptional activity of c-Myb.

The v-Myb oncoprotein encoded by Avian Myeloblastosis Virus is highly oncogenic, induces leukemias in chickens and mice and transforms immature hematopoietic cells in vitro. The v-Myb protein is a mutated and truncated version of c-Myb, a DNA-binding transcription factor expressed in many cell types that is essential for normal hematopoiesis. Previous studies suggested that two types of differences, DNA binding domain mutations and the deletion of a C-terminal negative regulatory domain were important for increasing the transforming activity of v-Myb. Here, we combined structure-function studies of the v-Myb and c-Myb proteins with unbiased microarray-based transcription assays to compare the transcriptional specificities of the two proteins. In human cells, the v-Myb and c-Myb proteins displayed strikingly different activities and regulated overlapping, but largely distinct sets of target genes. Each type of mutation that distinguished v-Myb from c-Myb, including the N- and C-terminal deletions, DNA binding domain changes and mutations in the transcriptional activation domain, affected different sets of target genes and contributed to the different activities of c-Myb and v-Myb. The results suggest that v-Myb is not just a de-repressed version of c-Myb. Instead, it is a distinct transcriptional regulator with a unique set of activities.

Large-scale molecular mapping of human c-myb locus: c-myb proto-oncogene is not involved in 6q- abnormalities of lymphoid tumors.

Chromosomal abnormalities in the 6q region have been observed frequently in several T-cell and myeloid leukemias. Interestingly, this region was found to contain three cellular oncogenes, c-myb, c-fyn and c-ros. Several of the tumors that exhibit 6q- abnormalities have also been found to express high levels of c-myb and, in some cases, amplification of the c-myb gene, leading to the suggestion that this gene could lie in proximity to the deletions observed in these tumors. To determine if c-myb gene activation is associated with 6q- abnormalities, we developed a megabase map of the human c-myb locus using pulsed-field gel electrophoresis. We then examined the occurrence of abnormalities near the c-myb gene in several hematopoietic tumor cell lines containing well-characterized 6q- abnormalities. Our results show that no rearrangements or deletions occur within a region of 1.0 Mbp of the c-myb locus in these cell lines. However, several of the cell lines exhibited differential and partial methylation patterns which seem to be prevalent amongst different cell lines.

DNA binding-independent transcriptional activation of the vascular endothelial growth factor gene (VEGF) by the Myb oncoprotein.

Myb is a key transcription factor that can regulate proliferation, differentiation, and apoptosis, predominantly in the haemopoietic system. Abnormal expression of Myb is associated with a number of cancers, both haemopoietic and non-haemopoietic. In order to better understand the role of Myb in normal and tumorigenic processes, we undertook a cDNA array screen to identify genes that are regulated by this factor. In this way, we identified the gene encoding vascular endothelial growth factor (VEGF) as being potentially regulated by the Myb oncoprotein in myeloid cells. To determine whether this was a direct effect on VEGF gene transcription, we examined the activity of the murine VEGF promoter in the presence of either wild-type (WT) or mutant forms of Myb. It was found that WT Myb was able to activate the VEGF promoter and that a minimal promoter region of 120 bp was sufficient to confer Myb responsiveness. Surprisingly, activation of the VEGF promoter was independent of DNA binding by Myb. This was shown by the use of DNA binding-defective Myb mutants and by mutagenesis of a potential Myb-binding site in the minimal promoter. mutation of Sp1 sites within this region abolished Myb-mediated regulation of a reporter construct, suggesting that Myb DNA binding-independent activation of VEGF expression occurs via these Sp1 binding elements. Regulation of VEGF production by Myb has implications for the potential role of Myb in myeloid leukaemias and in solid tumours where VEGF may be functioning as an autocrine growth factor.

Histological detection of c-myb and c-myc proto-oncogene expression in infiltrating cells in cutaneous lupus erythematosus-like lesions of MRL/l mice by in situ hybridization.

A relationship between lymphocytic activation and the overexpression of proto-oncogenes such as c-myb or c-myc has been demonstrated in human autoimmune disease. In autoimmune-prone MRL/l mice, which spontaneously develop lupus erythematosus (LE)-like lesions on the back, increased expression of myb RNA has been found in the lymphoid organs. We detected the overexpression of c-myb and c-myc proto-oncogenes in infiltrating cells in the cutaneous lesions of MRL/l mice by using in situ hybridization. No specific hybridization signals of either of the probes used were seen in the nonlesional skin of MRL/l mice or in the apparently normal skin of aged MRL/n and young MRL/l mice. These results suggest that the increased expression of myb and myc proto-oncogenes in the cutaneous LE-like lesions of MRL/l mice is related to a state of activation in the infiltrating cells and is involved in the development of these lesions.

Duplication of the MYB oncogene in T cell acute lymphoblastic leukemia.

We identified a duplication of the MYB oncogene in 8.4% of individuals with T cell acute lymphoblastic leukemia (T-ALL) and in five T-ALL cell lines. The duplication is associated with a threefold increase in MYB expression, and knockdown of MYB expression initiates T cell differentiation. Our results identify duplication of MYB as an oncogenic event and suggest that MYB could be a therapeutic target in human T-ALL.

Oncogenic point mutations in the Myb DNA-binding domain alter the DNA-binding properties of Myb at a physiological target gene.

The oncoprotein v-Myb of avian myeloblastosis virus (AMV) transforms myelomonocytic cells by deregulating specific target genes. Previous work has shown that the oncogenic potential of v-Myb was activated by truncation of N- and C-terminal sequences of c-Myb and was further increased by amino acid substitutions in the DNA-binding domain and other parts of the protein. We have analyzed the activation of the chicken lysozyme gene which is strongly activated by c-Myb but not by its oncogenic counterpart v-Myb. We report that Myb acts on two different cis-regulatory elements, the promoter and an enhancer located upstream of the gene. Interestingly, the activation of the enhancer was abolished by the oncogenic amino acid substitutions. We demonstrated that a single Myb-binding site is responsible for the activation of the lysozyme enhancer by Myb and showed that the v-Myb protein of AMV was unable to bind to this site. Our data demonstrate for the first time that oncogenic activation of Myb alters its DNA-binding specificity at a physiological Myb target gene.

Isolation and functional assessment of common, polymorphic variants of the B-MYB proto-oncogene associated with a reduced cancer risk.

The B-MYB proto-oncogene is a transcription factor belonging to the MYB family that is frequently overexpressed or amplified in different types of human malignancies. While it is suspected that B-MYB plays a role in human cancer, there is still no direct evidence of its causative role. Looking for mutations of the B-MYB gene in human cell lines and primary cancer samples, we frequently isolated two nonsynonymous B-MYB polymorphic variants (rs2070235 and rs11556379). Compared to the wild-type protein, the B-MYB isoforms display altered conformation, impaired regulation of target genes and decreased antiapoptotic activity, suggesting that they are hypomorphic variants of the major allele. Importantly, the B-MYB polymorphisms are common; rs2070235 and rs11556379 are found, depending on the ethnic background, in 10-50% of human subjects. We postulated that, if B-MYB activity is important for transformation, the presence of common, hypomorphic variants might modify cancer risk. Indeed, the B-MYB polymorphisms are underrepresented in 419 cancer patients compared to 230 controls (odds ratio 0.53; (95%) confidence interval 0.385-0.755; P=0.001). This data imply that a large fraction of the human population is carrier of B-MYB alleles that might be associated with a reduced risk of developing neoplastic disease.

Proposed structure for the DNA-binding domain of the Myb oncoprotein based on model building and mutational analysis.

Myb-related proteins from plants to humans are characterized by a DNA-binding domain which contains two to three imperfect repeats of approximately 50 amino acids each. Based on the evolutionary conservation of specific residues, secondary structural predictions suggest an arrangement of alpha helices homologous to that seen in the homeodomains, members of the helix-turn-helix family of DNA-binding proteins. We have used molecular modelling in conjunction with site-directed mutagenesis to test the feasibility of this structure. We propose that each Myb repeat consists of three alpha helices packed over a hydrophobic core which is built around the three highly conserved tryptophan residues. The C-terminal helix forms part of the helix-turn-helix motif and can be positioned into the major groove of B-form DNA, allowing prediction of residues critical for specificity of interaction. Modelling also allowed positioning of adjacent repeats around the major groove over an 8 bp binding site.

C-myb proto-oncogene: evidence for intermolecular recombination of coding sequences.

We have characterized a novel chicken c-myb exon whose sequences are specifically expressed in thymic cells. In situ hybridization experiments indicate that this thymus-specific coding exon is localized on a small chromosome, distinct from the large acrocentric chromosome 3 on which we recently mapped the bulk of 15 exons, common to the c-myb mRNA species expressed in hematopoietic cells of both B and T lineages. These observations indicate that intermolecular recombination is required for the tissue-specific expression of the c-myb proto-oncogene. We also show that these thymus-specific sequences are conserved in human DNA and lie on chromosome 17q25, whereas the human c-myb locus is localized on chromosome 6q22-23. Sequencing data obtained from genomic DNA and PCR analyses performed with c-myb mRNA species expressed in chicken thymic cells strongly suggest that a repeated decameric sequence plays a key role in the recombination process.

c-Myb oncoprotein is an essential target of the dleu2 tumor suppressor microRNA cluster.

The dleu2 tumor suppressor locus encodes two microRNAs, miR-15a and miR-16, which are thought to play an important role in B-cell neoplasms. However, relatively little is known about proteins that regulate or are regulated by this microRNA cluster. Here we demonstrate that the Pax5 oncoprotein downregulates the dleu2 gene and at the same time boosts expression of its own heterodimeric partner c-Myb. Interestingly, c-Myb upregulation occurs primarily at a post-transcriptional level, suggesting that it might be a target for microRNAs such as miR-15a/16. Indeed, miR-15a/16 have predicted binding sites in the c-Myb 3-UTR and through them diminish protein output in luciferase sensor assays. Moreover, forced overexpression of miR-15a/16 reduces endogenous c-Myb levels and compromises Pax5 function. Conversely, restoration of c-Myb levels partly alleviates tumors suppressive effects of miR-15a/16, suggesting that c-Myb is a key downstream target of this microRNA cluster.

The c-myb proto-oncogene and microRNA-15a comprise an active autoregulatory feedback loop in human hematopoietic cells.

The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor important for lineage commitment, proliferation, and differentiation. Given its critical functions, c-Myb regulatory factors are of great interest but remain incompletely defined. Herein we show that c-Myb expression is subject to posttranscriptional regulation by microRNA (miRNA)-15a. Using a luciferase reporter assay, we found that miR-15a directly binds the 3 -UTR of c-myb mRNA. By transfecting K562 myeloid leukemia cells with a miR-15a mimic, functionality of binding was shown. The mimic decreased c-Myb expression, and blocked the cells in the G(1) phase of cell cycle. Exogenous expression of c-myb mRNA lacking the 3 -UTR partially rescued the miR-15a induced cell-cycle block. Of interest, the miR-15a promoter contained several potential c-Myb protein binding sites. Occupancy of one canonical c-Myb binding site was demonstrated by chromatin immunoprecipitation analysis and shown to be required for miR-15a expression in K562 cells. Finally, in studies using normal human CD34(+) cells, we showed that c-Myb and miR-15a expression were inversely correlated in cells undergoing erythroid differentiation, and that overexpression of miR-15a blocked both erythroid and myeloid colony formation in vitro. In aggregate, these findings suggest the presence of a c-Myb-miR-15a autoregulatory feedback loop of potential importance in human hematopoiesis.

v-myb transformation of Xeroderma pigmentosum human fibroblasts: overexpression of the c-Ha-ras oncogene in the transformed cells.

Human Xeroderma pigmentosum "normal" fibroblasts AS16 (XP4 VI) were transformed after transfection with a recombinant v-myb clone. In this clone (pKXA 3457) derived from avian myeloblastosis virus (AMV), the expression of the oncogene sequences is driven by the AMV U-5 LTR promoter. The transformed cells (ASKXA), which have integrated a rearranged v-myb oncogene, grow in agar, are not tumorigenic in nude mice, and express a 45-kDa v-myb protein. The HMW DNA of these cells transform chicken embryo fibroblasts. The c-Ha-ras oncogene is overexpressed in the ASKXA cells but not in the parental "normal" AS16 cells and a revertant clone (ASKXA Cl 1.1 G). Our results lead to the conclusion that the XP fibroblasts are phenotypically transformed by the presence of the transfected v-myb oncogene, which is able to induce an overexpression of the c-Ha-ras gene.

Intermolecular recombination of human c-myb proto-oncogene coding sequences.

We have previously reported evidence suggesting that intermolecular recombination events are involved in the tissue specific expression of the c-myb proto-oncogene in chicken. We show in this paper that recombined c-myb mRNA species are also expressed in human thymic cells, therefore indicating that intermolecular recombination of coding sequences occurs in higher eucaryotes.

Myb-induced chromatin remodeling at a dual enhancer/promoter element involves non-coding rna transcription and is disrupted by oncogenic mutations of v-myb.

The oncogene v-myb of avian myeloblastosis virus (AMV) encodes a transcription factor (v-Myb) that transforms myelomonocytic cells by deregulating the expression of specific target genes. v-myb has acquired its oncogenic potential by truncation as well as by a number of point mutations of its cellular progenitor c-myb. As a result of these changes, the target gene spectrum v-Myb differs from that of c-Myb. We recently showed that the chicken mim-1 gene, a c-Myb target gene that is not activated by v-Myb harbors a powerful cell type-specific and Myb-inducible enhancer in addition to a Myb-responsive promoter. We now show that Myb-dependent activation of the mim-1 gene is accompanied by extensive remodeling of the nucleosomal architecture at the enhancer. We found that the mim-1 enhancer region also harbors a promoter whose activity is stimulated by Myb and which directs the transcription of an apparently non-coding RNA. Furthermore, our data show that the oncogenic mutations of AMV have disrupted the ability of v-Myb to induce remodeling of chromatin structure at the mim-1 enhancer. Together, our results demonstrate for the first time directly that Myb induces alterations of the nucleosomal organization at a relevant target site and provide new insight into the functional consequences of the oncogenic amino acid substitutions.

Presence of circulating anti-c-myb oncogene product antibodies in human sera.

Bacterially expressed mouse c-myb protein was purified from E. coli extracts and used as antigen to screen human sera for circulating anti-c-myb oncogene product antibodies. Using Western blotting, we have found that human sera contain IgG antibodies to the c-myb gene product. The percentage of positive sera in cancer patients appears to be dependent upon cancer type and is significantly higher in breast-cancer patients than in normal donors: 43% of sera from breast-cancer patients are positive, whereas in neuroblastoma cancer patients the production of IgG anti-c-myb appears to be rare. No significant correlation was observed between the presence of circulating anti-c-myb antibodies and the expression of the c-myb gene in breast tumors.

Mitochondrial Hep27 is a c-Myb target gene that inhibits Mdm2 and stabilizes p53.

The ever-expanding knowledge of the role of p53 in cellular metabolism, apoptosis, and cell cycle control has led to increasing interest in defining the stress response pathways that regulate Mdm2. In an effort to identify novel Mdm2 binding partners, we performed a large-scale immunoprecipitation of Mdm2 in the osteosarcoma U2OS cell line. One significant binding protein identified was Hep27, a member of the short-chain alcohol dehydrogenase/reductase (SDR) family of enzymes. Here, we demonstrate that the Hep27 preprotein contains an N-terminal mitochondrial targeting signal that is cleaved following mitochondrial import, resulting in mitochondrial matrix accumulation of mature Hep27. A fraction of the mitochondrial Hep27 translocates to the nucleus, where it binds to Mdm2 in the central domain, resulting in the attenuation of Mdm2-mediated p53 degradation. In addition, Hep27 is regulated at the transcriptional level by the proto-oncogene c-Myb and is required for c-Myb-induced p53 stabilization. Breast cancer gene expression analysis correlated estrogen receptor (ER) status with Hep27 expression and p53 function, providing a potential in vivo link between estrogen receptor signaling and p53 activity. Our data demonstrate a unique c-Myb-Hep27-Mdm2-p53 mitochondria-to-nucleus signaling pathway that may have functional significance for ER-positive breast cancers.

Ribosomal protein L4 positively regulates activity of a c-myb proto-oncogene product.

The c-myb proto-oncogene product (c-Myb) induces transcription of a group of target genes involved in the G1/S transition and in anti-apoptosis. The level of c-Myb is negatively regulated by the Wnt signal, but it remains obscure how c-Myb activity is positively regulated. We have found that ribosomal protein L4 (RPL4) binds to the DNA-binding domain of c-Myb. Co-immunoprecipitation experiments also indicated that RPL4 interacts with c-Myb. When c-Myb was overexpressed in CV-1 cells, significant amounts of RPL4 moved to the nucleoplasm from the nucleolus. RPL4 stimulated the c-Myb-dependent expression of a c-myc-luciferase reporter construct. Chromatin immunoprecipitation assays indicated that RPL4 binds to the 5 -regulatory region of the c-myc gene via c-Myb. Serum starvation and 2-deoxyglucose treatment of NIH3T3 cells induced the movement of RPL4 from the nucleoplasm to the nucleolus. Furthermore, c-myc mRNA levels were reduced by either serum starvation or 2-deoxyglucose treatment, and the degree of reduction in the c-myc mRNA level was correlated with the RPL4 level. These results suggest that growth factor and nutritional signals positively regulate c-Myb activity via its interaction with RPL4. Thus, RPL4 plays an important role in c-myc expression by interacting with c-Myb in response to growth factor and nutritional signals.

Expression of Slug is regulated by c-Myb and is required for invasion and bone marrow homing of cancer cells of different origin.

In metastatic cancer cells, the process of invasion is regulated by several transcription factors that induce changes required for migration and resistance to apoptosis. Slug (SNAI2, Snail2) is involved in epithelial mesenchymal transition in physiological and in pathological contexts. We show here that in embryonic kidney, colon carcinoma, chronic myeloid leukemia-blast crisis, and in neuroblastoma cells, expression of Slug is transcriptionally regulated by c-Myb via Myb binding sites in the 5 -flanking region and in the first intron of the slug gene. In embryonic kidney and neuroblastoma cells, c-Myb induced vimentin, fibronectin, and N-cadherin expression and membrane ruffling via actin polymerization consistent with the acquisition of a mesenchymal-like phenotype. Furthermore, down-regulation of endogenous c-Myb levels in colon carcinoma cells led to increased expression of E-cadherin and reduced levels of vimentin. Some of these changes are predominantly Slug-dependent as Slug silencing via RNA interference (RNAi) reverts the cells to a quasi-parental condition. Changes in gene expression and morphology induced by c-Myb-activated Slug correlated with increased ability to migrate (embryonic kidney) and to invade through a Matrigel membrane (embryonic kidney, colon carcinoma, neuroblastoma). c-Myb-dependent Slug expression was also essential for the homing of chronic myeloid leukemia K562 cells to the bone marrow. In summary, we show here that the proto-oncogene c-Myb controls Slug transcription in tumor cells of different origin. Such a regulatory pathway contributes to the acquisition of invasive properties that are important for the metastatic process.

New tricks from an old oncogene: gene fusion and copy number alterations of MYB in human cancer.

MYB is a leucine zipper transcription factor that is essential for hematopoesis and for renewal of colonic crypts. There is also ample evidence showing that MYB is leukemogenic in several animal species. However, it was not until recently that clear evidence was presented showing that MYB actually is an oncogene rearranged in human cancer. In a recent study, a novel mechanism of activation of MYB involving gene fusion was identified in carcinomas of the breast and head and neck. A t(6;9) translocation was shown to generate fusions between MYB and the transcription factor gene NFIB. The fusions consistently result in loss of the 3 -end of MYB, including several highly conserved target sites for microRNAs that negatively regulate MYB expression. Deletion of these target sites may disrupt the repression of MYB, leading to overexpression of MYB-NFIB transcripts and protein and to transcriptional activation of critical MYB target genes associated with apoptosis, cell cycle control, cell growth/angiogenesis and cell adhesion. This study, together with previous and recent data showing rearrangements and copy number alterations of the MYB locus in T-cell leukemia and certain solid tumors, will be the main focus of this review.

Protein truncation is required for the activation of the c-myb proto-oncogene.

The protein product of the v-myb oncogene of avian myeloblastosis virus, v-Myb, differs from its normal cellular counterpart, c-Myb, by (i) expression under the control of a strong viral long terminal repeat, (ii) truncation of both its amino and carboxyl termini, (iii) replacement of these termini by virally encoded residues, and (iv) substitution of 11 amino acid residues. We had previously shown that neither the virally encoded termini nor the amino acid substitutions are required for transformation by v-Myb. We have now constructed avian retroviruses that express full-length or singly truncated forms of c-Myb and have tested them for the transformation of chicken bone marrow cells. We conclude that truncation of either the amino or carboxyl terminus of c-Myb is sufficient for transformation. In contrast, the overexpression of full-length c-Myb does not result in transformation. We have also shown that the amino acid substitutions of v-Myb by themselves are not sufficient for the activation of c-Myb. Rather, the presence of either the normal amino or carboxyl terminus of c-Myb can suppress transformation when fused to v-Myb. Cells transformed by c-Myb proteins truncated at either their amino or carboxyl terminus appear to be granulated promyelocytes that express the Mim-1 protein. Cells transformed by a doubly truncated c-Myb protein are not granulated but do express the Mim-1 protein, in contrast to monoblasts transformed by v-Myb that neither contain granules nor express Mim-1. These results suggest that various alterations of c-Myb itself may determine the lineage of differentiating hematopoietic cells.

Potential tumor suppressor role for the c-Myb oncogene in luminal breast cancer.

BACKGROUND: The transcription factor c-Myb has been well characterized as an oncogene in several human tumor types, and its expression in the hematopoietic stem/progenitor cell population is essential for proper hematopoiesis. However, the role of c-Myb in mammopoeisis and breast tumorigenesis is poorly understood, despite its high expression in the majority of breast cancer cases (60-80%). METHODOLOGY/PRINCIPAL FINDINGS: We find that c-Myb high expression in human breast tumors correlates with the luminal/ER+ phenotype and a good prognosis. Stable RNAi knock-down of endogenous c-Myb in the MCF7 luminal breast tumor cell line increased tumorigenesis both in vitro and in vivo, suggesting a possible tumor suppressor role in luminal breast cancer. We created a mammary-derived c-Myb expression signature, comprised of both direct and indirect c-Myb target genes, and found it to be highly correlated with a published mature luminal mammary cell signature and least correlated with a mammary stem/progenitor lineage gene signature. CONCLUSIONS/SIGNIFICANCE: These data describe, for the first time, a possible tumor suppressor role for the c-Myb proto-oncogene in breast cancer that has implications for the understanding of luminal tumorigenesis and for guiding treatment.

Intermolecular recombination of the c-myb proto-oncogene coding sequences in chicken: clues for a mechanism.

The physical separation of c-myb coding exons on two different chromosomes, both in chicken and human, indicated that intermolecular recombination events might occur in higher eucaryotes. We present evidence in this paper suggesting that the expression of "antisens" mRNA species could be involved in the regulation of the intermolecular recombination process.

[Progress of research on Proto-oncogene c-myc, c-myb in platelet diseases].

The proto-oncogene c-myc and c-myb has been shown to be crucial in the development of the hematopoietic system. The changes in the expression of c-myc are concerned the cell proliferation and differentiation, the expression products of which play an important regulatory role in cell growth, differentiation or malignant transformation. The c-myb involves in transcription and affects cell proliferation, differentiation, apoptosis. More recently, the researches on proto-oncogene c-myc, c-myb in hematopoietic regulation have gradually increased along with development of molecular biology, molecular immunology and cell biology. Scientists point out that the directive differentiation of erythroid and megakaryocytic progenitors, and platelet abnormalities ALL relate to the level of their expressions. The most common thrombocytopathy includes thrombocytopenia, thrombocytosis and so on. The etiology and the mechanism of these diseases are unknown. This article reviews the structure, function and the expression of c-myc and c-myb in platelet diseases and their significance.

Activation of v-Myb avian myeloblastosis viral oncogene homolog-like2 (MYBL2)-LIN9 complex contributes to human hepatocarcinogenesis and identifies a subset of hepatocellular carcinoma with mutant p53.

Up-regulation of the v-Myb avian myeloblastosis viral oncogene homolog-like2 B-Myb (MYBL2) gene occurs in human hepatocellular carcinoma (HCC) and is associated with faster progression of rodent hepatocarcinogenesis. We evaluated, in distinct human HCC prognostic subtypes (as defined by patient survival length), activation of MYBL2 and MYBL2-related genes, and relationships of p53 status with MYBL2 activity. Highest total and phosphorylated protein levels of MYBL2, E2F1-DP1, inactivated retinoblastoma protein (pRB), and cyclin B1 occurred in HCC with poorer outcome (HCCP), compared to HCC with better outcome (HCCB). In HCCP, highest LIN9-MYBL2 complex (LINC) and lowest inactive LIN9-p130 complex levels occurred. MYBL2 positively correlated with HCC genomic instability, proliferation, and microvessel density, and negatively with apoptosis. Higher MYBL2/LINC activation in HCC with mutated p53 was in contrast with LINC inactivation in HCC harboring wildtype p53. Small interfering RNA (siRNA)-mediated MYBL2/LINC silencing reduced proliferation, induced apoptosis, and DNA damage at similar levels in HCC cell lines, irrespective of p53 status. However, association of MYBL2/LINC silencing with doxorubicin-induced DNA damage caused stronger growth restraint in p53(-/-) Huh7 and Hep3B cells than in p53(+/+) Huh6 and HepG2 cells. Doxorubicin triggered LIN9 dissociation from MYBL2 in p53(+/+) cell lines and increased MYBL2-LIN9 complexes in p53(-/-) cells. Doxorubicin-induced MYBL2 dissociation from LIN9 led to p21(WAF1) up-regulation in p53(+/+) but not in p53(-/-) cell lines. Suppression of p53 or p21(WAF1) genes abolished DNA damage response, enhanced apoptosis, and inhibited growth in doxorubicin-treated cells harboring p53(+/+) . CONCLUSION: We show that MYBL2 activation is crucial for human HCC progression. In particular, our data indicate that MYBL2-LIN9 complex integrity contributes to survival of DNA damaged p53(-/-) cells. Thus, MYBL2 inhibition could represent a valuable adjuvant for treatments against human HCC with mutated p53.

Binding of the c-myb proto-oncogene product to the simian virus 40 enhancer stimulates transcription.

The proto-oncogene c-myb encodes a nuclear protein which binds to DNA. Here we find that bacterially synthesized c-myb protein binds to one site of the simian virus 40 enhancer. The c-myb protein purified from the human T-cell line, Molt4, was also shown to recognize the same sequence. In co-transfection experiments with a c-myb expression plasmid, tandem repeats of a c-myb-binding sequence were shown to function as a c-myb-dependent enhancer. These results indicate the c-myb protein is a simian virus 40 enhancer-binding protein that can positively regulate transcription.

Establishment of myeloid progenitor lines from primary cultures of murine bone-marrow cells expressing a v-myb oncoprotein.

Infection of primary hematopoietic progenitors from post 5-fluorouracil-treated murine bone marrow with a novel replication-defective retroviral vector [murine stem cell virus-v-myb (MSCV-v-myb)] bearing a v-myb oncogene reproducibly gave rise to permanent myeloid cell lines that were dependent on either interleukin (LL)-3 or granulocyte-macrophage colony-stimulating factor (GM-CSF) for sustained growth in vitro. ALL of the v-Myb-transformed myeloid cells synthesized an apparently normal c-Myb protein in addition to the predicted v-Myb species, indicating that expression of c-Myb is not incompatible with transformation by v-Myb. Cell lines derived in the presence of GM-CSF proliferated maximally in response to this factor but also responded well to IL-3 and macrophage-CSF (M-CSF), and to a lesser extent to granulocyte-CSF (G-CSF) and Steel factor (SF). In contrast, v-Myb-transformed cell lines maintained in IL-3-supplemented medium were optimally stimulated by SF but, besides IL-3, did not respond to any of the other factors tested. Unlike the GM-CSF-dependent cell lines, these latter cell lines expressed the CD34 surface antigen that is present on a population of bone marrow cells with short- and long-term hematopoietic repopulating ability. To our knowledge, this is the first report documenting transformation of mammalian hematopoietic cells by v-Myb. The well-characterized murine hematopoietic system should prove valuable in further investigations aimed at elucidating the mechanisms by which ectopic v-Myb expression results in the immortalization of target cells belonging to varying stages of early myeloid development.

Myb DNA binding inhibited by phosphorylation at a site deleted during oncogenic activation.

The c-Myb nuclear oncoprotein is phosphorylated in vitro and in vivo at an N-terminal site near its DNA-binding domain by casein kinase II (CK-II) or a CK-II-like activity. This in vitro phosphorylation reversibly inhibits the sequence-specific binding of c-Myb to DNA. The site of this phosphorylation is deleted in nearly ALL oncogenically activated Myb proteins, resulting in DNA-binding that is independent of CK-II. Because CK-II activity is modulated by growth factors, loss of the site could uncouple c-Myb from its normal physiological regulator.

Formation and recognition of G-quadruplex in promoter of c-myb oncogene by electrospray ionization mass spectrometry.

In this study, electrospray ionization mass spectrometry (ESI-MS) is used to study the formation of G-quadruplex by d(GGAGGAGGAGGA) which locates at the promoter region of c-myb gene. In addition, a natural small molecule, dehydrocorydaline from a Chinese herb, is found to have the highest binding affinity with the G-quadruplex in nine natural small molecules studied, and the binding selectivity of this natural molecule toward the c-myb G-quadruplex with respect to corresponding duplex DNA is significantly higher than that of the broad-spectrum G-quadruplex-ligand TMPyP4. The result from ESI-MS indicates that the gas-phase kinetic stability of the G-quadruplex can be enhanced by binding of dehydrocorydaline. To further investigate the binding properties of dehydrocorydaline to the G-quadruplex, Autodock3 is used to calculate the docked sites and docked energies of small molecules binding to the G-quadruplex and the result shows that the docked energy of dehydrocorydaline is the biggest in the nine small molecules used, consistent with the result from ESI-MS.

The myb oncogene.

The distribution of the c-myc oncogene product p62 was examined by immunohistochemistry using the monoclonal antibody Mycl-9E10 in a series of 50 colorectal resections for carcinoma. The specimens were specially handled to ensure rapid fixation in formalin, and a significant improvement was shown in the quality and localization of staining compared with routinely handled specimens. Non-neoplastic mucosa showed the presence of nuclear staining of epithelial cells in 93 per cent of the samples, whilst ALL carcinomas showed cytoplasmic staining and infrequent nuclear staining. Adenomas showed an intermediate pattern, with significantly more frequent cytoplasmic distribution than non-neoplastic mucosa, but less than carcinomas. The results show that whilst fixation conditions are important in the immunolocalization of the c-myc protein product, there may be a consistent difference between non-neoplastic mucosa and carcinoma in the manner of association of p62 with the nucleus.

Animal-specific C-terminal domain links myeloblastosis oncoprotein (Myb) to an ancient repressor complex.

Members of the Myb oncoprotein and E2F-Rb tumor suppressor protein families are present within the same highly conserved multiprotein transcriptional repressor complex, named either as Myb and synthetic multivuval class B (Myb-MuvB) or as Drosophila Rb E2F and Myb-interacting proteins (dREAM). We now report that the animal-specific C terminus of Drosophila Myb but not the more highly conserved N-terminal DNA-binding domain is necessary and sufficient for (i) adult viability, (ii) proper localization to chromosomes in vivo, (iii) regulation of gene expression in vivo, and (iv) interaction with the highly conserved core of the MuvB/dREAM transcriptional repressor complex. In addition, we have identified a conserved peptide motif that is required for this interaction. Our results imply that an ancient function of Myb in regulating G2/M genes in both plants and animals appears to have been transferred from the DNA-binding domain to the animal-specific C-terminal domain. Increased expression of B-MYB/MYBL2, the human ortholog of Drosophila Myb, correlates with poor prognosis in human patients with breast cancer. Therefore, our results imply that the specific interaction of the C terminus of Myb with the MuvB/dREAM core complex may provide an attractive target for the development of cancer therapeutics.

Dynamic long-range chromatin interactions control Myb proto-oncogene transcription during erythroid development.

The key haematopoietic regulator Myb is essential for coordinating proliferation and differentiation. ChIP-Sequencing and Chromosome Conformation Capture (3C)-Sequencing were used to characterize the structural and protein-binding dynamics of the Myb locus during erythroid differentiation. In proliferating cells expressing Myb, enhancers within the Myb-Hbs1l intergenic region were shown to form an active chromatin hub (ACH) containing the Myb promoter and first intron. This first intron was found to harbour the transition site from transcription initiation to elongation, which takes place around a conserved CTCF site. Upon erythroid differentiation, Myb expression is downregulated and the ACH destabilized. We propose a model for Myb activation by distal enhancers dynamically bound by KLF1 and the GATA1/TAL1/LDB1 complex, which primarily function as a transcription elongation element through chromatin looping.

Telomestatin impairs glioma stem cell survival and growth through the disruption of telomeric G-quadruplex and inhibition of the proto-oncogene, c-Myb.

PURPOSE: Glioma stem cells (GSC) are a critical therapeutic target of glioblastoma multiforme (GBM). EXPERIMENTAL DESIGN: The effects of a G-quadruplex ligand, telomestatin, were evaluated using patient-derived GSCs, non-stem tumor cells (non-GSC), and normal fetal neural precursors in vitro and in vivo. The molecular targets of telomestatin were determined by immunofluorescence in situ hybridization (iFISH) and cDNA microarray. The data were then validated by in vitro and in vivo functional assays, as well as by immunohistochemistry against 90 clinical samples. RESULTS: Telomestatin impaired the maintenance of GSC stem cell state by inducing apoptosis in vitro and in vivo. The migration potential of GSCs was also impaired by telomestatin treatment. In contrast, both normal neural precursors and non-GSCs were relatively resistant to telomestatin. Treatment of GSC-derived mouse intracranial tumors reduced tumor sizes in vivo without a noticeable cell death in normal brains. iFISH revealed both telomeric and non-telomeric DNA damage by telomestatin in GSCs but not in non-GSCs. cDNA microarray identified a proto-oncogene, c-Myb, as a novel molecular target of telomestatin in GSCs, and pharmacodynamic analysis in telomestatin-treated tumor-bearing mouse brains showed a reduction of c-Myb in tumors in vivo. Knockdown of c-Myb phenocopied telomestatin-treated GSCs both in vitro and in vivo, and restoring c-Myb by overexpression partially rescued the phenotype. Finally, c-Myb expression was markedly elevated in surgical specimens of GBMs compared with normal tissues. CONCLUSIONS: These data indicate that telomestatin potently eradicates GSCs through telomere disruption and c-Myb inhibition, and this study suggests a novel GSC-directed therapeutic strategy for GBMs.

[Research progress of proto-oncogene c-myb in megakaryocyte-erythroid hematopoiesis].

The nuclear proto-oncogene c-myb is an essential regulator of hematopoiesis, it involves in the growth, survival, proliferation and differentiation of hematopoietic cells. More recently, different cell lines and transgenic mouse studies have suggested that c-myb plays a pivotal role in the megakaryocyte-erythroid progenitor cell lineage commitment. The deletion of the proto-oncogene c-myb would lead to profoundly impaired definitive erythropoiesis, but little influence in definitive megakaryopoiesis. Moreover, transient transfection and immunoprecipitation studies have demonstrated that c-myb exerts its physiological function in normal hematopoiesis by influencing a network of regulator molecules. Now therefore, insight into the structure, function and related molecular regulation mechanism of c-myb gene can help to further clarify its function in megakaryocyte-erythroid hematopoiesis and can provide new ideas for molecular target therapy of the platelet diseases and red blood cell diseases. In this article, c-myb structure, function and related effects involved in megakaryocyte-erythroid hematopoiesis as well as related molecular mechanisms are reviewed.

Mutations in v-myb alter the differentiation of myelomonocytic cells transformed by the oncogene.

Chick myelomonocytic cells transformed by the v-myb oncogene-containing viruses E26 and AMV differ in that the former resemble myeloblasts and express the v-myb-regulated granulocyte-specific mim-1 gene, while the latter resemble monoblasts and are mim-1 negative. We constructed a series of AMV-E26 chimeras and localized the critical differences between these viruses to three point mutations within the second repeat of the v-myb DNA binding domain. These three positions are altered in the v-myb protein of AMV relative to the proteins encoded by c-myb or E26 v-myb. Back mutating AMV v-myb at any of these three sites restored the oncogene s ability to activate the mim-1 gene. Surprisingly, two of these changes led to the transformation, in vitro and in vivo, of cells having a promyelocyte-like phenotype. These results indicate that different forms of v-myb impose alternate phenotypes of differentiation on transformed myeloid cells, probably by regulating unique sets of differentiation-specific genes.

The MYB oncogene can suppress apoptosis in acute myeloid leukemia cells by transcriptional repression of DRAK2 expression.

RNA interference-mediated suppression of MYB expression promoted apoptosis in the AML cell line U937, without affecting expression of the anti-apoptotic MYB target BCL2. This was accompanied by up-regulation of the pro-apoptotic gene DRAK2 and stimulation of caspase-9 activity. Moreover, RNA interference-mediated suppression of DRAK2 in U937 cells alleviated apoptosis induced by MYB down-regulation. Finally ChIP assays showed that in U937 cells MYB binds to a conserved element upstream of the DRAK2 transcription start site. Together, these findings identify a novel mechanism by which MYB suppresses apoptosis in an AML model cell line.

Long noncoding RNA MALAT1 controls cell cycle progression by regulating the expression of oncogenic transcription factor B-MYB.

The long noncoding MALAT1 RNA is upregulated in cancer tissues and its elevated expression is associated with hyper-proliferation, but the underlying mechanism is poorly understood. We demonstrate that MALAT1 levels are regulated during normal cell cycle progression. Genome-wide transcriptome analyses in normal human diploid fibroblasts reveal that MALAT1 modulates the expression of cell cycle genes and is required for G1/S and mitotic progression. Depletion of MALAT1 leads to activation of p53 and its target genes. The cell cycle defects observed in MALAT1-depleted cells are sensitive to p53 levels, indicating that p53 is a major downstream mediator of MALAT1 activity. Furthermore, MALAT1-depleted cells display reduced expression of B-MYB (Mybl2), an oncogenic transcription factor involved in G2/M progression, due to altered binding of splicing factors on B-MYB pre-mRNA and aberrant alternative splicing. In human cells, MALAT1 promotes cellular proliferation by modulating the expression and/or pre-mRNA processing of cell cycle-regulated transcription factors. These findings provide mechanistic insights on the role of MALAT1 in regulating cellular proliferation.

Genomic analysis of diffuse pediatric low-grade gliomas identifies recurrent oncogenic truncating rearrangements in the transcription factor MYBL1.

Pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children but, apart from BRAF kinase mutations or duplications in specific subclasses, few genetic driver events are known. Diffuse PLGGs comprise a set of uncommon subtypes that exhibit invasive growth and are therefore especially challenging clinically. We performed high-resolution copy-number analysis on 44 formalin-fixed, paraffin-embedded diffuse PLGGs to identify recurrent alterations. Diffuse PLGGs exhibited fewer such alterations than adult low-grade gliomas, but we identified several significantly recurrent events. The most significant event, 8q13.1 gain, was observed in 28% of diffuse astrocytoma grade IIs and resulted in partial duplication of the transcription factor MYBL1 with truncation of its C-terminal negative-regulatory domain. A similar recurrent deletion-truncation breakpoint was identified in two angiocentric gliomas in the related gene v-myb avian myeloblastosis viral oncogene homolog (MYB) on 6q23.3. Whole-genome sequencing of a MYBL1-rearranged diffuse astrocytoma grade II demonstrated MYBL1 tandem duplication and few other events. Truncated MYBL1 transcripts identified in this tumor induced anchorage-independent growth in 3T3 cells and tumor formation in nude mice. Truncated transcripts were also expressed in two additional tumors with MYBL1 partial duplication. Our results define clinically relevant molecular subclasses of diffuse PLGGs and highlight a potential role for the MYB family in the biology of low-grade gliomas.

Differential amplification and expression of c-myb oncogene in Zajdela ascitic hepatoma.

Organisation and expression of c-myb protooncogene have been studied in a heterogeneous tumour the Zajdela ascitic hepatoma (ZAH). The myb gene is selectively amplified in the more tumorigenic subpopulation of the tumour while the non-lethal subpopulation does not show any change. Analysis of transcripts of the myb gene in tumorigenic versus nontumorigenic cells shows that the level of amplification of the gene does not correspond to the level of its transcription. Results have been discussed in the light of existing evidence regarding the role of c-myb gene expression during cell cycle.

The MYB proto-oncogene suppresses monocytic differentiation of acute myeloid leukemia cells via transcriptional activation of its target gene GFI1.

The MYB gene is a master regulator of hematopoiesis and contributes to leukemogenesis in several species including humans. Although it is clear that MYB can promote proliferation, suppress apoptosis and block differentiation, the identities of the MYB target genes that mediate these effects have only been partially elucidated. Several studies, including our own, have collectively identified substantial numbers of MYB target genes, including candidates for each of these activities; however, functional validation, particularly in the case of differentiation suppression, has lagged well behind. Here we show that GFI1, which encodes an important regulator of hematopoietic stem cell (HSC) function and granulocytic differentiation, is a direct target of MYB in myeloid leukemia cells. Chromatin immunoprecipitation and reporter studies identified a functional MYB-binding site in the promoter region of GFI, whereas ectopic expression and small hairpin RNA-mediated knockdown of MYB resulted in concomitant increases and decreases, respectively, in GFI1 expression. We also demonstrate that GFI1, like MYB, can block the induced monocytic differentiation of a human acute myeloid leukemia cell line, and most importantly, that GFI1 is essential for MYB s ability to block monocytic differentiation. Thus, we have identified a target of MYB that is a likely mediator of its myeloid differentiation-blocking activity, and which may also be involved in MYB s activities in regulating normal HSC function and myeloid differentiation.

Transforming potential of the v-myb oncogene from avian myeloblastosis virus: alterations in the oncogene product may reveal a new target specificity.

Transfection of brown leghorn chicken embryo fibroblasts by DNA containing v-myb sequences cloned either in a complete AMV proviral DNA or in a retroviral derived vector has led to the isolation of two kinds of transformed cells. A characterization of the proviral sequences retained and expressed in these transformed cells revealed that they contained either new or altered v-myb-related RNA species. The experiments presented in this paper also show that both types of transformants expressed truncated myb-related polypeptides, suggesting that alterations of the v-myb product may result in a new target specificity, leading to the transformation of chicken embryo fibroblasts.

ESI mass spectrometric exploration of selective recognition of G-quadruplex in c-myb oncogene promoter using a novel flexible cyclic polyamide.

In this research, electrospray ionization mass spectrometry (ESI-MS) was used to probe the binding selectivity of a flexible cyclic polyamide (cbeta) to G-quadruplexes from the long G-rich sequences in the c-myb oncogene promoter. The results show that three G-rich sequences, including d[(GGA)3GGTCAC(GGA)4], d[(GGA)4GAA(GGA)4], and d[(GGA)3GGTCAC(GGA)4GAA(GGA)4] species in the c-myb promoter can form parallel G-quadruplexes, and cbeta selectively binds towards these G-quadruplexes over both several other G-quadruplexes and the duplex DNA. These properties of cbeta have profound implications on future studies of the regulation of c-myb oncogene expression.

The oncoprotein v-Myb activates transcription of Gremlin 2 during in vitro differentiation of the chicken neural crest to melanoblasts.

The neural crest (NC) is a transient dynamic structure of ectodermal origin, found in early vertebrate embryos. The multipotential NC cells migrate along well defined routes, differentiate to various cell types including melanocytes and participate in the formation of various permanent tissues. As there is only limited information about the molecular mechanisms controlling early events in melanocyte specification and development, we exploited the AMV v-Myb transcriptional regulator, which directs differentiation of in vitro chicken NC cells to the melanocyte lineage. This activity is strictly dependent on v-Myb specifically binding to the Myb recognition DNA element (MRE). The two tamoxifen-inducible v-Myb alleles were constructed one which recognizes the MRE and one which does not. These were activated in ex ovo NC cells, and the expression profiles of resulting cells were analyzed using Affymetrix microarrays and RT-PCR. These approaches revealed up-regulation of the BMP antagonist Gremlin 2 mRNA, and down-regulation of mRNAs encoding several epithelial genes including KRT19 as very early events following the activation of melanocyte differentiation by v-Myb. The enforced v-Myb expression in neural tubes of chicken embryos resulted in detectable presence of Gremlin 2 mRNA. However, expression of Gremlin 2 in NC cells did not promote formation of melanocytes suggesting that Gremlin 2 is not the master regulator of melanocytic differentiation.

Analysis of MYB oncogene in transformed adenoid cystic carcinomas reveals distinct pathways of tumor progression.

Adenoid cystic carcinomas can occasionally undergo dedifferentiation, a phenomenon also referred to as high-grade transformation. However, cases of adenoid cystic carcinomas have been described showing transformation to adenocarcinomas that are not poorly differentiated, indicating that high-grade transformation may not necessarily reflect a more advanced stage of tumor progression, but rather a transformation to another histological form, which may encompass a wide spectrum of carcinomas in terms of aggressiveness. The aim of this study was to gain more insight in the biology of this pathological phenomenon by means of genetic profiling of both histological components. Using microarray comparative genomic hybridization, we compared the genome-wide DNA copy-number changes of the conventional and transformed area of eight adenoid cystic carcinomas with high-grade transformation, comprising four with transformation into moderately differentiated adenocarcinomas and four into poorly differentiated carcinomas. In general, the poorly differentiated carcinoma cases showed a higher total number of copy-number changes than the moderately differentiated adenocarcinoma cases, and this correlated with a worse clinical course. Special attention was given to chromosomal translocation and protein expression of MYB, recently being considered to be an early and major oncogenic event in adenoid cystic carcinomas. Our data showed that the process of high-grade transformation is not always accompanied by an accumulation of genetic alterations; both conventional and transformed components harbored unique genetic alterations, which indicate a parallel progression. Our data further demonstrated that the MYB/NFIB translocation is not necessarily an early event or fundamental for the progression to adenoid cystic carcinoma with high-grade transformation.

Structure and biological activity of the transcriptional initiation sequences of the murine c-myb oncogene.

To study the control mechanism(s) that govern the transcription of c-myb, genomic clones corresponding to the 5 region of the murine c-myb gene have been isolated and characterized structurally and functionally. Primer extension and nuclease protection analysis have revealed the presence of multiple transcriptional initiation sites, that are utilized in several hemopoietic cell lines (WEHI3B(D+). FDC-P1 and RB22.2). Some of the sites are used in ALL cell lines but others are unique; ALL are located in a region of the c-myb gene that is G-C rich, contains a number of potential Sp1 binding sites and lacks classical promoter consensus sequences. Experiments in which well characterized promoters controlling expression of a reporter gene have been replaced by fragments of c-myb DNA (including the observed cap sites) were performed in an attempt to demonstrate promoter activity in various cell types. It was shown that a region of the c-myb gene (approximately 1.0 kbp upstream from the splice donor site of the first exon) contains a weak promoter that has a low level of transcriptional activity in hemopoietic as well as in fibroblastic cells. These results support the suggestion that c-myb expression is not regulated primarily at the level of initiation of transcription.

Molecular mapping of the oncogene MYB and rearrangements in malignant melanoma.

The human cellular oncogene MYB has been mapped to 6q22-q23. Deletions and translocations involving this region of the long arm of chromosome 6 occur frequently in human malignant melanoma, and there are anecdotal reports of MYB gene rearrangements in this cancer. In the current study, Southern blotting and pulsed field gel electrophoresis (PFGE) have been performed to determine whether MYB or its flanking regions are commonly altered in malignant melanoma. Southern blotting failed to document obvious rearrangement of the MYB gene in 15 cases studied. To extend analysis of the MYB region, a long-range restriction map was established by PFGE. This map was then linked to the known restriction map of frequent cutting enzymes. Based on the mapping data and analysis of the MYB region in melanomas, ClaI tissue-specific variation due to methylation was demonstrated. Also, two melanomas (containing alterations in band 6q13) also demonstrated by PFGE a unique restriction fragment for the MYB gene. These results extend significantly the physical map surrounding the MYB locus and provide further evidence for the rearrangement of chromosome 6 in malignant melanoma.

MicroRNA-193b-3p acts as a tumor suppressor by targeting the MYB oncogene in T-cell acute lymphoblastic leukemia.

The MYB oncogene is a leucine zipper transcription factor essential for normal and malignant hematopoiesis. In T-cell acute lymphoblastic leukemia (T-ALL), elevated MYB levels can arise directly through T-cell receptor-mediated MYB translocations, genomic MYB duplications or enhanced TAL1 complex binding at the MYB locus or indirectly through the TAL1/miR-223/FBXW7 regulatory axis. In this study, we used an unbiased MYB 3 untranslated region-microRNA (miRNA) library screen and identified 33 putative MYB-targeting miRNAs. Subsequently, transcriptome data from two independent T-ALL cohorts and different subsets of normal T-cells were used to select miRNAs with relevance in the context of normal and malignant T-cell transformation. Hereby, miR-193b-3p was identified as a novel bona fide tumor-suppressor miRNA that targets MYB during malignant T-cell transformation thereby offering an entry point for efficient MYB targeting-oriented therapies for human T-ALL.

Trans-activation by the c-myb proto-oncogene.

We present evidence that the mouse c-myb proto-oncogene encodes a transcriptional trans-activator. Trans-activation was assayed by cotransfection into CV1 monkey kidney cells of a c-myb cDNA expression plasmid together with a reporter plasmid carrying the chloramphenicol acetyltransferase (CAT) gene under the control of a test promoter and enhancer. Cotransfection with the c-myb cDNA plasmid caused a 20-fold stimulation of transcription from the promoter of the mouse alpha 2(I) collagen gene linked to tandem repeats of the simian virus 40 (SV40) enhancer element. Using different promoters in combination with varying numbers of repeats of the SV40 enhancer element, it was shown that tandem repeats of the SV40 enhancer mediated the c-myb-induced activation of transcription. These results show that the mouse c-myb gene product either is itself or induces, an activator of transcription that recognizes specific sequences in the SV40 enhancer.

Oncogene regulation. An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element.

In certain human cancers, the expression of critical oncogenes is driven from large regulatory elements, called super-enhancers, that recruit much of the cell s transcriptional apparatus and are defined by extensive acetylation of histone H3 lysine 27 (H3K27ac). In a subset of T-cell acute lymphoblastic leukemia (T-ALL) cases, we found that heterozygous somatic mutations are acquired that introduce binding motifs for the MYB transcription factor in a precise noncoding site, which creates a super-enhancer upstream of the TAL1 oncogene. MYB binds to this new site and recruits its H3K27 acetylase-binding partner CBP, as well as core components of a major leukemogenic transcriptional complex that contains RUNX1, GATA-3, and TAL1 itself. Additionally, most endogenous super-enhancers found in T-ALL cells are occupied by MYB and CBP, which suggests a general role for MYB in super-enhancer initiation. Thus, this study identifies a genetic mechanism responsible for the generation of oncogenic super-enhancers in malignant cells.

Transcription of the chicken myb proto-oncogene starts within a CpG island.

The nucleotide sequence of an 8.2-kb DNA fragment from the 5 proximal part of the chicken myb proto-oncogene spanning 1761 nucleotides upstream and 6436 nucleotides downstream from a presumed c-myb initiation codon was determined. A 3.3-kb G + C-rich region found in this sequence had also other features characterizing CpG islands, i.e. no CpG underrepresentation and lack of CpG methylation. In haematopoietic tissues c-myb mRNA synthesis starts in two major regions of the CpG island, namely 98 to 108 and 143 to 145 nucleotides upstream from the c-myb initiation codon. These two regions are in or close to the 124-bp evolutionarily conserved element located in the middle part of the CpG island. No alternative splicing of the 5 end of c-myb mRNA suggested earlier (1,2) was observed. The c-myb promoter contains neither TATA nor CAAT box-like structures at the usual positions. Instead, numerous potential Sp1 factor binding sites were found both upstream and downstream from the transcription initiation sites. Moreover, consensus v-myb protein DNA-binding sites were revealed in the promoter region and in sequences downstream from it.

Transcriptional trans-repression by the c-myb proto-oncogene product.

We report that the c-myb protein binds to another site, MBS-II, in the SV40 enhancer with low affinity. In co-transfection experiments with a c-myb expression plasmid, tandem repeats of the sequence containing the MBS-II site induced c-myb-dependent transcriptional repression. Results of mutational analyses of the sequence around the MBS-II site suggested that the c-myb protein represses transcription by competing with another trans-activator. These results indicate that c-myb protein can regulate transcription not only positively but also negatively.

Therapeutic DNA vaccination against colorectal cancer by targeting the MYB oncoprotein.

cancers can be addicted to continued and relatively high expression of nuclear oncoproteins. This is evident in colorectal cancer (CRC) where the oncoprotein and transcription factor MYB is over expressed and essential to continued proliferation and tumour cell survival. Historically, targeting transcription factors in the context of cancer has been very challenging. Nevertheless, we formulated a DNA vaccine to generate a MYB-specific immune response in the belief MYB peptides might be aberrantly presented on the cell surface of CRC cells. MYB, like many tumour antigens, is weakly immunogenic as it is a self antigen and is subject to tolerance. To break tolerance, a fusion vaccine was generated comprising a full-length MYB complementary DNA (cDNA) flanked by two potent CD4-epitopes derived from tetanus toxoid. Vaccination was achieved against tumours initiated by two distinct highly aggressive, syngeneic cancer cell lines (CT26 and MC38) that express MYB. This was done in BALB/c and C57BL/6 mouse strains respectively. We introduced multiple inactivating mutations into the oncogene sequence for safety and sub-cloned the cDNA into a Food and Drug Administration (FDA)-compliant vector. We used low dose cyclophosphamide (CY) to overcome T-regulatory cell immune suppression, and anti-program cell death receptor 1 (anti-PD-1) antibodies to block T-cell exhaustion. Anti-PD-1 administered alone slightly delayed tumour growth in MC38 and more effectively in CT26 bearing mice, while CY treatment alone did not. We found that therapeutic vaccination elicits protection when MC38 tumour burden is low, mounts tumour-specific cell killing and affords enhanced protection when MC38 and CT26 tumour burden is higher but only in combination with anti-PD-1 antibody or low dose CY, respectively.

Transcription factor and microRNA interactions in lung cells: an inhibitory link between NK2 homeobox 1, miR-200c and the developmental and oncogenic factors Nfib and Myb.

BACKGROUND: The transcription factor NK2 homeobox 1 (Nkx2-1) plays essential roles in epithelial cell proliferation and differentiation in mouse and human lung development and tumorigenesis. A better understanding of genes and pathways downstream of Nkx2-1 will clarify the multiple roles of this critical lung factor. Nkx2-1 regulates directly or indirectly numerous protein-coding genes; however, there is a paucity of information about Nkx2-1-regulated microRNAs (miRNAs). METHODS AND RESULTS: By miRNA array analyses of mouse epithelial cell lines in which endogenous Nkx2-1 was knocked-down, we revealed that 29 miRNAs were negatively regulated including miR-200c, and 39 miRNAs were positively regulated by Nkx2-1 including miR-1195. Mouse lungs lacking functional phosphorylated Nkx2-1 showed increased expression of miR-200c and alterations in the expression of other top regulated miRNAs. Moreover, chromatin immunoprecipitation assays showed binding of NKX2-1 protein to regulatory regions of these miRNAs. Promoter reporter assays indicated that 1kb of the miR-200c 5 flanking region was transcriptionally active but did not mediate Nkx2-1- repression of miR-200c expression. 3 UTR reporter assays support a direct regulation of the predicted targets Nfib and Myb by miR-200c. CONCLUSIONS: These studies suggest that Nkx2-1 controls the expression of specific miRNAs in lung epithelial cells. In particular, we identified a regulatory link between Nkx2-1, the known tumor suppressor miR-200c, and the developmental and oncogenic transcription factors Nfib and Myb, adding new players to the regulatory mechanisms driven by Nkx2-1 in lung epithelial cells that may have implications in lung development and tumorigenesis.

Expression of the c-myb oncogene in human small cell lung carcinoma.

We have found that the oncogene c-myb is differentially expressed in human lung cancer cell lines and that myb-homologous RNA can be detected only in small cell lung cancer (SCLC) cell lines. Polyadenylic acid-RNA from 13 established cell lines was examined by Northern blotting for its ability to hybridize to a radiolabeled v-myb probe. A 3.5-kilobase RNA transcript homologous to v-myb is present in four of four lines of classic SCLC and in three of four SCLC variant lines but not in five of five non-small cell lung cancer lines tested. This transcript is the same size as that found in the immature myeloid cell lines KG1, but the amount of RNA is only about 10% of that in the KG1 line. A second transcript hybridizing to v-myb, 2.4 kilobases in size, is also present in the variant SCLC lines and the COLO 320 line, ALL of which have amplification of the c-myc gene and markedly increased c-myc messenger RNA. The presence of myb transcripts in SCLC suggests that the myb gene may have a specific role in the initiation or maintenance of an important human epithelial tumor.

A single amino-acid substitution in the DNA-binding domain of the myb oncogene confers a thermolabile phenotype to E26-transformed myeloid cells.

A biologically active provirus of the ts 143 E26 mutant that is temperature-sensitive (ts) for myeloblast transformation was molecularly cloned. The predicted amino-acid sequence of the v-myb-encoded domain of the mutant P135gag-myb-ets protein displayed two single amino-acid changes, one of which was non-conservative when compared to the wild-type E26 v-myb sequence. This mutation, which substitutes a threonine residue (wild-type) for an arginine residue (mutant), is located within the amino-terminal part of v-myb in the DNA-binding domain at a position which is conserved between the c-myb genes of chicken, humans, mice and Drosophila. Introduction of this mutation into the genome of a wild-type E26 virus was sufficient to induce a ts phenotype similar to that obtained with the original ts 143 E26 virus.

Supercoil-induced Z-DNA formation within 5 -end of chicken myb proto-oncogene.

We have analyzed the recently sequenced and characterized 2.9 kb fragment derived from the 5 -end of chicken myb proto-oncogene with respect to structural perturbations induced by DNA supercoiling. Within the first intron a 50 bp sequence stretch was localized, starting approximately 450 nucleotides downstream from putative ATG initiation codon, which forms a non-B-DNA structure. Fine mapping with structural probes revealed the three adjacent regions with imperfect purine-pyrimidine alternation creating together relatively long Z-forming tract, parts of which may undergo a B-Z DNA transition at different superhelical densities.

Transcriptional activation by the v-myb oncogene and its cellular progenitor, c-myb.

The v-myb oncogene, like its cellular progenitor c-myb, encodes a short-lived nuclear protein involved in processes affecting growth and differentiation in a number of cell types. Fusion proteins, in which v-myb sequences are linked to the DNA binding domain of the yeast transcriptional activator GAL4, can activate transcription from a reporter gene linked in cis to a GAL4 binding site. The domain of v-myb responsible for transcriptional activation is located between residues 204 and 254, and is both necessary and sufficient for activation. Intact v-myb and c-myb proteins can also activate transcription, via a myb binding site linked in cis to a reporter gene. A v-myb protein bearing a deletion in the activator domain is no longer capable of stimulating transcription.

The v-myb oncogene product binds to and activates the promyelocyte-specific mim-1 gene.

The v-myb oncogene induces myeloid leukemias in chickens, transforms myeloid cells in vitro, and encodes a sequence-specific DNA binding protein. We used differential hybridization to screen for v-myb-regulated genes in cells transformed by a temperature-sensitive mutant of the oncogene and identified a new gene, mim-1, which encodes a specifically expressed, secretable protein contained in the granules of both normal and v-myb-transformed promyelocytes. The promoter of the mim-1 gene contains three closely spaced binding sites for v-myb protein and is strongly activated by v-myb in a cotransfection assay. Synthetic copies of the binding sites are both necessary and sufficient to confer v-myb protein-dependent activation to a heterologous promoter. We conclude that mim-1 is a cellular gene that is directly regulated by the product of the v-myb oncogene.

The highly conserved amino-terminal region of the protein encoded by the v-myb oncogene functions as a DNA-binding domain.

The retroviral oncogene v-myb encodes a 45,000 Mr nuclear protein (p45v-myb) that is predominantly associated with the chromatin of transformed cells. It has previously been shown that p45v-myb, when released from chromatin by salt-treatment, binds to DNA. To analyse the biochemical properties of p45v-myb in more detail we have expressed the v-myb coding region in Escherichia coli. Our results demonstrate that bacterially expressed myb protein has an intrinsic DNA-binding activity. Using two alternative strategies, (i) inhibition of DNA-binding by monoclonal antibodies and (ii) analysis of DNA-binding activities of partially deleted forms of the bacterial myb protein, we show that the DNA-binding domain is located in the amino-terminal region of the v-myb protein. This region has been highly conserved between myb genes of different species. Our results are therefore consistent with the hypothesis that DNA-binding is an important aspect of myb protein function.

Structural and functional domains of the myb oncogene: requirements for nuclear transport, myeloid transformation, and colony formation.

The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in vivo and transforms only myeloid cells in vitro. Its product, p48v-myb, is a nuclear protein of unknown function. To determine structure-function relationships for this protein, we constructed a series of deletion mutants of v-myb, expressed them in retroviral vectors, and studied their biochemical and biological properties. We used these mutants to identify two separate domains of p48v-myb which had distinct roles in its accumulation in the cell nucleus. We showed that the viral sequences which normally encode both termini of p48v-myb were dispensible for transformation. In contrast, both copies of the highly conserved v-myb amino-terminal repeat were required for transformation. We also identified a carboxyl-terminal domain of p48v-myb which was required for the growth of v-myb-transformed myeloblasts in soft agar but not for morphological transformation.

myb oncogene in human hematopoietic neoplasia with 6q- anomaly.

Molecular and cytogenetic analyses were performed on human T-cell leukemia cell lines (PEER and MOLT-4) with the 6q- anomaly. The PEER cells contained an interstitial deletion of the long arm of chromosome 6, that is, del(6)(q13q21), as well as other changes. The MOLT-4 cells showed a terminal deletion of the long arm of chromosome 6, that is, del(6)(q24). The 700-bp BamHI/XbaI-digested c-myb probe hybridized to a 4.3-kb fragment in EcoRI digested DNAs from these two cell lines, showing no deletion, rearrangement, or amplification. On the other hand, ML cells [ML-1, -2 and -3; human myeloid/T-cell biphenotypic leukemia cell lines with del(6)(q24)] showed an amplification of the c-myb gene and had a high level of the c-myb-related mRNA at 3.5 kb. Though no amplification of the c-myb at the DNA level was noted in the PEER or MOLT-4 cell lines, apparent high expression of the c-myb was detected in these human T-cell neoplastic lines. These results indicate that high c-myb expression is related to lineage of hematopoietic neoplasia rather than to the 6q- change.

DNA-binding activity associated with the v-myb oncogene product is not sufficient for transformation.

The product of the v-myb oncogene of avian myeloblastosis virus is a nuclear protein with an associated DNA-binding activity. We demonstrated that the highly conserved amino-terminal domain of p48v-myb is required for its associated DNA-binding activity. This activity is not required for the nuclear localization of p48v-myb. Furthermore, the associated DNA-binding activity and nuclear localization of p48v-myb together are not sufficient for transformation.

A point mutation in the DNA binding domain of the v-myb oncogene of E26 virus confers temperature sensitivity for transformation of myelomonocytic cells.

We have molecularly cloned a mutant of the v-myb, ets-containing E26 avian leukemia virus which is temperature sensitive for the transformation of myeloid cells. Cells infected with this mutant, ts21E26, are immature at 37 degrees C and can be induced to differentiate into resting, macrophage-like cells when shifted to 42 degrees C. The sequence of ts21E26 reveals a single relevant nucleotide alteration resulting in a threonine to arginine change in the highly conserved, putative DNA binding v-myb portion of the p135gag-myb-ets protein. Surprisingly, a ts21E26 viral construct in which the v-ets gene domain was deleted was only weakly temperature sensitive, although temperature sensitivity was largely restored in another v-ets deletion mutant whose 3 terminal sequences were replaced with those from the AMV v-myb gene. These results suggest that the temperature sensitive lesion in v-myb of ts21E26 alters the DNA binding capacity of p135 at 42 degrees C and that the primary structure of the C-terminus of this protein has an influence on the activity of sequences that are further upstream.

Nucleotide sequence of cDNA clones of the murine myb proto-oncogene.

We have isolated cDNA clones of murine c-myb mRNA which contain approximately 2.8 kb of the 3.9-kb mRNA sequence. Nucleotide sequencing has shown that these clones extend both 5 and 3 to sequences homologous to the v-myb oncogenes of avian myeloblastosis virus and avian leukemia virus E26. The sequence contains an open reading frame of 1944 nucleotides, and could encode a protein which is both highly homologous, and of similar size (71 kd), to the chicken c-myb protein. Examination of the deduced amino acid sequence of the murine c-myb protein revealed the presence of a 3-fold tandem repeat of 52 residues near the N terminus of the protein, and has enabled prediction of some of the likely structural features of the protein. These include a high alpha-helix content, a basic region toward the N terminus of the protein and an overall globular configuration. The arrangement of genomic c-myb sequences, detected using the cDNA clones as probes, was compared with the reported structure of rearranged c-myb in certain tumour cells. This comparison suggested that the rearranged c-myb gene may encode a protein which, like the v-myb protein, lacks the N-terminal region of c-myb.

A cryptic transcription promoter in the myb oncogene of avian myeloblastosis virus.

The potential regulatory signals contained in the v-myb oncogene of avian myeloblastosis virus have been inserted upstream to the herpes simplex type 1 thymidine kinase gene in order to test their promoter activity. The isolation of TK+ transformants after transfection of clone 1D(TK-) mouse cells with the resulting recombinant DNAs indicated that the expression of the TK gene was made possible by the myb-derived sequences. Analysis of the TK specific RNA expressed in different TK+ transformants revealed that the regulatory signals contained in v-myb correspond to a weak functional promoter.

Structural organization and nucleotide sequence of mouse c-myb oncogene: activation in ABPL tumors is due to viral integration in an intron which results in the deletion of the 5 coding sequences.

Bacteriophage libraries of mouse DNA were screened for sequences homologous to the v-myb oncogene and two overlapping clones containing the v-myb related region were isolated. Restriction enzyme mapping, heteroduplex analysis and nucleotide sequence analysis revealed the presence of nine exons. Six of these exons are homologous to the v-myb region while the other three exons are derived from the 5 region which is deleted in the viral oncogene. The sequences downstream to the sixth v-myb exon are not included in the 17 kbp of DNA sequences analyzed in this study. Comparison of the structure of the normal c-myb clone with its rearranged couterpart present in plasmacytoid lymphosarcomas revealed that the rearrangements occur in this locus as a result of viral integration. Present studies demonstrate that such a viral insertion interrupts the c-myb coding region at a region identical to that observed in the generation of the v-myb gene of avian myeloblastosis virus and results in the synthesis of mRNAs that lack the same 5 coding region.

Nuclear compartmentalization of the v-myb oncogene product.

Nuclei obtained from chicken leukemic myeloblasts transformed by avian myeloblastosis virus were fractionated into various subnuclear compartments, which were then analyzed by specific immunoprecipitation for the presence of the leukemogenic product, p48v-myb, of the viral oncogene. In cells labeled for 30 or 60 min with L-[35S]methionine and in unlabeled exponentially dividing leukemic cells analyzed by Western blotting, p48v-myb was detected within the nucleoplasm (29 +/- 9% [standard deviation] of the total), chromatin (7 +/- 4%), and lamina-nuclear matrix (64 +/- 9%). Also, in myeloblasts analyzed by immunofluorescence during mitosis, p48v-myb appeared to be dispersed through the cell like the lamina-nuclear matrix complex. Strong attachment to the nuclear matrix-lamina complex suggests that p48v-myb may be involved in DNA replication or transcription or both.

Subnuclear localization of proteins encoded by the oncogene v-myb and its cellular homolog c-myb.

The retroviral transforming gene v-myb encodes a 45,000-Mr nuclear transforming protein (p45v-myb). p45v-myb is a truncated and mutated version of a 75,000-Mr protein encoded by the chicken c-myb gene (p75c-myb). Like its viral counterpart, p75c-myb is located in the cell nucleus. As a first step in identifying nuclear targets involved in cellular transformation by v-myb and in c-myb function, we determined the subnuclear locations of p45v-myb and p75c-myb. Approximately 80 to 90% of the total p45v-myb and p75c-myb present in nuclei was released from nuclei at low salt concentrations, exhibited DNA-binding activity, and was attached to nucleoprotein particles when released from the nuclei after digestion with nuclease. A minor portion of approximately 10 to 20% of the total p45v-myb and p75c-myb remained tightly associated with the nuclei even in the presence of 2 M NaCl. These observations suggest that both proteins are associated with two nuclear substructures tentatively identified as the chromatin and the nuclear matrix. The function of myb proteins may therefore depend on interactions with several nuclear targets.

Structure of the protein encoded by the chicken proto-oncogene c-myb.

The retroviral oncogene v-myb arose by transduction of the chicken proto-oncogene c-myb. We isolated and sequenced cDNA that represents the entire coding domain of chicken c-myb. By transcribing the cDNA into mRNA in vitro and then translating the RNA, we were able to document the integrity of the cDNA and to identify the codon responsible for initiation of translation from c-myb. Two different alleles of v-myb are extant, one in the genome of avian myeloblastosis virus (AMV) and the other in the genome of erythroblastosis virus 26 (E26V). The proteins encoded by the AMV and E26V alleles of v-myb differ from the product of c-myb in three ways: at their amino termini, they lack 71 and 80 amino acids respectively; at their carboxy termini, they are deficient in 199 and 278 residues; and 11 substitutions of amino acids are scattered throughout the product of AMV allele, whereas the product of the E26V allele contains only a single substitution. The structural origins of tumorigenicity by v-myb and the biological functions of c-myb remain enigmatic. The findings and molecular clones described here should now permit a systematic exploration of these enigmas.

Two exons specific for the myb proto-oncogene found upstream from the avian myeloblastosis virus-transduced myb sequences.

A partial restriction map of cloned 5.42-kb chicken DNA (clone P542, Perbal et al. 1983), covering a portion of the c-myb locus, is presented. The 5 end of the v-myb gene (approximately 0.5 kb) is located at the 3 end of P542 DNA, the remainder are the cellular sequences not transduced by avian myeloblastosis virus. Two non-contiguous DNA segments were detected within these cellular sequences which code for the 5 end of c-myb mRNA. These two exons, designated e1 and e2, are separated by a approximately equal to 1.5-kb non-coding region. Both of them are transcribed into 0.4 kb located near the 5 end of c-myb mRNA. The second exon e2 (approximately equal to 0.2 kb) is flanked at its 3 end by a short non-coding region within which virus-cell recombination took place. The possible presence of a portion of this intron in the 2.1-kb v-myb mRNA is discussed.

Expression and function of the c-myb oncogene during hematopoietic differentiation.

The human ets-2 gene is a homolog of the v-ets oncogene of the E26 virus and codes for a 56-kilodalton nuclear protein. The ets-2 protein is phosphorylated and has a rapid turnover, with a half-life of 20 min. When human lymphocytic CEM cells were treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), the level of the ets-2 protein was quickly elevated 5- to 20-fold. This effect of TPA was mimicked by a synthetic diacylglycerol, 1-oleoyl-2-acetyl glycerol, and was blocked by the protein kinase C inhibitor H7, indicating that protein kinase C is involved in the induction. The increase in the ets-2 protein was due to stabilization of the protein, because the protein had a half-life of more than 2 h in the presence of TPA and the ets-2 mRNA level did not increase significantly upon TPA treatment. The protein synthesis inhibitor cycloheximide enhanced the effect of TPA on the ets-2 protein and could itself slow turnover of the protein. Properties of the ets-2 protein, such as nuclear localization, phosphorylation, rapid turnover, and response to protein kinase C, indicate that this protein belongs to a group of oncogene proteins which are generally thought to have regulatory functions in the nucleus (e.g., myc, fos, myb, and p53). Our results suggest that protein kinase C, either directly or indirectly, regulates the level of the ets-2 protein by posttranslational mechanisms.

Regulation of human T-lymphocyte gene expression by interleukin 2: immediate-response genes include the proto-oncogene c-myb.

Antigen-stimulated human T lymphocytes must bind the immunoregulatory hormone interleukin 2 (IL-2) if they are to transit from the G1 to the S phase of the cell cycle. Indirect methods, such as the measurement of thymidine uptake rates, were previously the only means available for exploring the mechanism of action of IL-2. Several cDNA clones have been isolated which are expressed subsequent to IL-2 binding, and the expression of two of these genes. Tact52 and Tact75, is regulated directly at the level of transcription; expression of the proto-oncogene c-myb is also regulated directly by IL-2 binding. These genes thus constitute a set which is coordinately regulated in the course of the transition from G1 to S phase of human T lymphocytes, and their expression depends on IL-2 binding.

Viral myb oncogene encodes a sequence-specific DNA-binding activity.

The retroviral oncogene v-myb and its cellular progenitor c-myb encode nuclear DNA-binding proteins. Myb genes have been identified in a broad range of species, including vertebrates, the fruit fly Drosophila melanogaster and the plant Zea mays. The localization of the DNA-binding domain of the v-MYB protein to the highly conserved amino-terminal region suggests that the MYB/DNA interaction is important for MYB function. We show here that v-MYB specifically recognizes the nucleotide sequence pyAACG/TG. So like other nuclear transforming proteins, v-MYB seems to be a member of the class of sequence-specific DNA-binding factors presumably involved in gene regulation.

Solid-phase synthesis of a nonadecapeptide coded for by the v-myb oncogene.

A nonadecapeptide comprising a predicted B-cell determinant from the v-myb oncoprotein was synthesized by Merrifield s solid-phase method. Hydrogen chloride in dichloromethane was used for protective t-butyloxycarbonyl group removal; the deprotection was monitored using a new qualitative deprotection test. The nonadecapeptide coupled to a carrier elicited a high titre of protein-reactive antipeptide antibodies.

Differential expression of the c-myb proto-oncogene marks the pre-B cell/B cell junction in murine B lymphoid tumors.

A series of murine B lymphoid tumor cell lines which are representative of the pre-B cell, immature and mature B cell, and plasma cell stages of B cell development have been examined for expression of c-myb proto-oncogene mRNA. The pre-B cell lymphoma cell lines express equivalent high steady state levels of c-myb mRNA. In contrast, the B cell lymphoma and plasmacytoma cell lines express steady state c-myb mRNA at levels which are 0.005 to 0.1 times that of the pre-B cell lymphoma lines. These results correlate high levels of c-myb mRNA expression with the pre-B cell stage of development. Subclones of the 1881 pre-B cell lymphoma which express K light chain and are surface IgM-positive as well as two types of hybrid B lymphoid cell lines have been used to demonstrate that surface immunoglobulin expression is not sufficient to result in the down-regulation of c-myb mRNA levels or changes in the expression N-myc mRNA, lambda 5 mRNA, or the BP-1 surface antigen which are markers of the pre-B cell stage of development. Thus, changes in the expression of genes which are independent of immunoglobulin expression are associated with transition from the pre-B cell to the immature B cell stage of development.

Mechanism of activation of the myb oncogene in myeloid leukemias.

Rat pheochromocytoma (PC12) cells differentiate to neuronal cells in response to nerve growth factor. It has been shown that microinjection of oncogenic but not proto-oncogenic p21 protein induces morphological differentiation in PC12 cells (D. Bar-Sagi and J. R. Feramisco, Cell 42:841-848, 1985). In this paper we describe a recombinant human proto-oncogenic Ha-ras protein which can effectively induce neurite extension of PC12 cells when microinjected as a complex with guanosine-5 -O-(3-thiotriphosphate). The protein was found to be less effective when complexed with GTP. On the other hand, an oncogenic ras protein coinjected with guanosine-5 -O-(2-thiodiphosphate) was entirely inactive. These results indicate that the binary p21-GTP complex, but not the p21-GDP complex, is effective in inducing differentiation in PC12 cells, irrespective of the oncogenic or the proto-oncogenic protein.

The myb oncogene.

The highly conserved, single copy c-myb gene has been independently transduced by two avian acute leukemia viruses, AMV and E26. This gene has also undergone insertional mutagenesis by non-acutely transforming murine leukemia viruses in a number of hematopoietic tumors. The common denominator of these retroviral activations of c-myb appears to be truncation of the normal coding region at either or both ends. The role of point mutations in myb-induced leukemogenesis is currently unknown. The products of the c-myb gene and its altered viral counterparts are nuclear proteins, a large fraction of which are associated with the nuclear matrix. In addition, the myb gene products have short half-lives and bind DNA in vitro. These features suggest that myb may act by regulating DNA replication or transcription. Consistent with this notion, the expression of c-myb is cell cycle dependent in several cell types. However, the abundant expression of c-myb in the thymus is not similarly regulated and may serve a different function. The expression of c-myb appears not to be limited to hematopoietic tissues as previously thought and the nature of the hematopoietic specificity of transformation by v-myb is not currently understood. Nevertheless, hematopoietic growth factors and their receptors appear to play an important role in such transformation. Two new experimental systems for studying myb have recently been described. First, the discovery of a myb-related gene in Drosophila should allow the application of powerful classical and molecular genetic approaches. The functional similarity of this distantly related gene to the much more closely related avian and mammalian myb genes is unknown. Second, recent studies of murine myb in normal and abnormal hematopoiesis offers several advantages relative to the avian system, such as in-bred animal strains, a wealth of specific cell-surface markers, and cloned hematopoietic growth factor and receptor genes. Isolation or construction of an acutely transforming murine myb retrovirus may thus be very useful. Several obvious goals for future research will be to define the function of myb proteins within the nucleus, to understand the regulation of myb expression during the cell cycle, to establish which molecular alterations are essential for converting c-myb into a transforming gene, and the determine the role of myb in human malignancies.

Structure and mechanism of activation of the myb oncogene.

The results summarized in this review show that the normal chicken myb gene codes for a protein of 77 kd which appears to play an important role in the control and/or differentiation of hematopoietic cells of myeloid and T lymphoid series. The activation of this gene has been observed in chicken and murine systems. In the avian system, this has been achieved by transduction of the myb oncogene into a retrovirus. Such a transduction resulted in the deletion of coding sequences from both the 5 and 3 ends of the gene. Initiation and terminator codons in helper viral sequences have been substituted for the analogous sequences in the proto-oncogene. Deletion of similar stretches of sequence in both the viruses suggested the possibility that these deletions may play an important role in the activation of this gene. The availability of the murine model system allowed us to examine this question further. In the ABPL tumor system, the activation of the myb locus occurred as a result of viral integration in a region immediately upstream to the v-myb related sequences. In NSF-60 cell line, the activation is due to the viral integration toward the 3 end of the gene. In both cases the viral integration results in the synthesis of aberrant mRNAs that have suffered deletions similar to those observed in the avian system. In ALL instances this results in the synthesis of truncated proteins which appear to mediate the transforming function. The availability of chicken and mouse c-myb cDNA clones makes it possible to test this hypothesis directly by construction of retroviruses containing various deletion mutations.

The regulatory c1 locus of Zea mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators.

The structure of the wild-type c1 locus of Zea mays was determined by sequence analysis of one genomic and two cDNA clones. The coding region is composed of three exons (150 bp, 129 bp and one, at least 720 bp) and two small introns (88 bp and 145 bp). Transcription of the mRNAs corresponding to the two cDNA clones cLC6 (1.1 kb) and cLC28 (2.1 kb) starts from the same promoter. Both cDNAs are identical except that cLC28 extends further at its 3 end. A putative protein, 273 amino acids in length was deduced from the sequence of both transcripts. It contains two domains, one basic and the other acidic and might function as a transcriptional activator. The basic domain of this c1-encoded protein shows 40% sequence homology to the protein products of animal myb proto-oncogenes.

Expression of the c-myb proto-oncogene during cellular proliferation.

In several cell types, messenger RNA levels of the nuclear proto-oncogene c-myb vary as a function of cellular proliferation; a transient increase in c-myb steady-state mRNA, mediated by post-transcriptional mechanisms, occurs during cell-cycle progression. In contrast, both quiescent and proliferating immature thymocytes contain exceptionally high levels of c-myb mRNA as a consequence of increased c-myb transcription.

Identification and characterization of the protein encoded by the human c-myb proto-oncogene.

We have identified the product of the human c-myb proto-oncogene as a 80,000-Mr protein, p80c-myb, by using polyclonal and monoclonal antibodies raised against a bacterially synthesized polypeptide from the amino terminus of the viral myb protein. p80c-myb shares at least two distinct antigenic sites with the amino terminal region of the v-myb protein. p80c-myb is found only in hematopoietic cells or in cells that contain amplified c-myb genes. Like the chicken myb proteins, p80c-myb is a nuclear DNA-binding protein that is predominantly associated with chromatin and exhibits a short half-life of approximately 1 hour.

Levels of fos, ets2, and myb proto-oncogene RNAs correlate with segregation of chromosome 11 of normal cells and with suppression of tumorigenicity in human cell hybrids.

The tumorigenicity in nude mice of human carcinoma-derived D98AH2 (D98) cells is suppressed when cell hybrids are made by fusing these cells with normal human diploid cells. Selection for hybrids that have segregated chromosomes results in the recovery of tumorigenic segregants. These segregants have ALL lost at least one copy of chromosome 11 of the diploid cell parent. Earlier we found that the parental D98 cells had detectable levels of mRNA specific for 13 of 21 proto-oncogenes examined. To determine if transregulation of proto-oncogenes by genes of the normal cell occurs in such hybrids, the steady-state levels of mRNA specific to 22 proto-oncogenes in the parental cells were compared with those of nontumorigenic D98 X human diploid hybrids as well as with those of their tumorigenic segregants and with the cells of the resulting tumors. The only chromosome consistently segregated in the latter was chromosome 11 of the diploid cell. fos and ets2 RNA levels and the amount of fos protein were consistently elevated in the segregants compared with amounts in the original hybrids. An unexpected finding was the inverse relationship for myb RNA that was barely detected in the parental D98 cells but was at least 10-fold elevated in hybrids that did not have segregated chromosomes compared with those that did. These patterns were evident in RNAs prepared from both subconfluent and confluent cell cultures. The findings suggest that genes of the normal cell parent can affect proto-oncogene expression. Whether the genes affecting fos, ets2, and myb RNA levels are on chromosome 11 and whether these alterations are causally related to the tumorigenic phenotype of the hybrid remain to be determined.

Subnuclear associations of the v-myb oncogene product and actin are dependent on ionic strength during nuclear isolation.

The method used to isolate nuclei has a direct effect on the subnuclear association of the v-myb product, p48v-myb, and nuclear actin. Analysis of nuclei subjected to various isolation procedures showed that disruption of native nuclear structure during hypotonic treatment resulted in dissociation of p48v-myb from the nuclear matrix.

Expression of c-Myc, c-Myb, c-Erb-B and c-H-Ras oncogene mRNAs in fibroblasts cultured from psoriatic patients.

The abnormally high rate of proliferation described in cultured psoriatic fibroblasts could result from inappropriate expression of cellular oncogenes (c-onc) associated to the control of cell division. Four c-onc (c-Myc, c-Myb, c-Erb-B, c-H-Ras) were studied in cultured fibroblasts from lesional and nonlesional psoriatic skin (n = 6) and compared with normal subjects (n = 3). RNA was analyzed by hybridization with nick-translated cloned human DNA probes after extraction by the guanidinium thiocyanate/LiCl procedure, electrophoresis and transfer on nitrocellulose. No difference in the level of c-Myc and c-Myb mRNA could be detected in psoriatic skin compared with controls. N-Ras did not give a detectable signal and c-Erb-B exhibited individual variations which were not linked to the disease. These results do not rule out subtle qualitative changes of these genes; moreover, an abnormal mRNA expression of other c-onc remains possible in psoriasis.

Nucleotide sequence of chicken c-myb complementary DNA and implications for myb oncogene activation.

Avian myeloblastosis virus (AMV), like other acute transforming viruses, arose by recombination between its helper virus and host cellular sequences. The latter sequences, termed v-myb, are responsible for the oncogenic properties of the virus. AMV causes acute myeloblastic leukaemia in chickens and transforms a specific class of haematopoietic cells in vitro, but does not induce morphological transformation of cultured fibroblasts, suggesting that only a restricted target-cell population is responsive to its transforming gene product. The normal cellular counterpart of v-myb, c-myb, is highly conserved and is present in ALL vertebrate and some invertebrate species examined. DNA rearrangements and altered expression of the myb oncogene have been reported in mouse lymphoid tumours and human myeloid and colon tumours. The mechanism of activation of the cellular proto-oncogenes is thought to involve the structural alteration of the coding regions that result in either the synthesis of an altered gene product or the enhanced expression of a proto-oncogene caused by alterations in its regulatory elements. To distinguish between these two mechanisms, we have cloned and sequenced the chicken c-myb complementary DNA and compared it with that of v-myb sequences. We demonstrate that during the transduction of the cellular sequences and/or viral passage a substantial portion of the coding region of the c-myb gene has been lost from both the 5 and 3 ends, resulting in the generation of a truncated gene product that mediates the transforming function of the virus.

Mapping of amplified c-myb oncogene, sister chromatid exchanges, and karyotypic analysis of the COLO 205 colon carcinoma cell line.

We have studied molecular and chromosomal details of cytogenetic status in a human tumor cell line COLO 205 that shows a stable, approximately tenfold amplification of the c-myb oncogene. The amplified copies of c-myb reside in two marker chromosomes that may have evolved from chromosome #6 by complex chromosomal rearrangements. No homogeneously staining regions can be discerned at the site of c-myb amplification. We suggest that c-myb was amplified in situ in a chromosomal segment (6q22-24) that became a part of the marker chromosome, possibly through isochromosome formation followed by duplication, and without the extrachromosomal intermediate form of double minute chromosomes. There is an enhanced frequency of sister chromatid exchanges at the site of amplified c-myb. These results are discussed in the context of models for gene amplification and oncogene activation.

[Existence of sequences homologous to the V-MYB oncogene in the genome of archaebacteria].

The presence of DNA sequences homologous to the v-myb oncogene in the genome of both halophilic and methanogenic archaebacteria was revealed after hybridization of restriction fragments with cloned probes. No myb-related sequences were detected in the DNA from S. acidocaldarius.

Isolation of the proto-oncogene c-myb from D. melanogaster.

We have isolated the proto-oncogene c-myb from Drosophila melanogaster. This gene is represented by a single locus at position 13E-F on the X chromosome, and is expressed in early embryos by transcription into two polyadenylated RNAs with lengths of approximately 3.0 and 3.8 kb. The gene may encode a protein with a molecular weight of at least 55,000 that shares a domain with c-myb (chicken) in which 91 of 125 (or 73%) of the amino acids are identical in the Drosophila and chicken genes. These findings represent the first rigorous identification of a Drosophila proto-oncogene that can encode what may be a nuclear protein, and they set the stage for a genetic analysis of how c-myb serves the normal organism.

Identical chromosome translocations involving the region of the c-myb oncogene in four metastases of a mediastinal teratocarcinoma.

A 39-year-old white male presented with a disseminated mediastinal teratocarcinoma. Karyotyping was performed on two mature residual metastatic lesions in the lungs immediately following chemotherapy, on a recurring lung lesion after 5 months, and on a metastasis in the right thigh 5 months after salvage chemotherapy. ALL four lesions were pseudoeuploid and showed identical chromosomal abnormalities: a translocation with the two chromosomes #6 and one chromosome #11 involved, resulting in 46, XY, t (6;6;11) (q21;q23;q13). The breakpoint in chromosome #6 is in the region to which the oncogene c-myb has been localized, and the breakpoint in chromosome #11 is at a known fragile and possibly oncogenic site, suggesting that the translocations in this case may have played a crucial role in the development of the malignancy.

Early decline in c-myb oncogene expression in the differentiation of human myeloblastic leukemia (ML-1) cells induced with 12-O-tetradecanoylphorbol-13-acetate.

The relationship of oncogene expression to proliferation and differentiation has been examined in a line of human myeloblastic leukemia (ML-1) cells. Proliferating leukemic cells were found to express a 4.3-kilobase cellular homologue (c-myb) of the transforming sequence of avian myeloblastosis virus. A rapid decline in the expression of this transcript was seen in cells induced to differentiate with 12-O-tetradecanoylphorbol-13-acetate. The level of c-myb RNA was decreased by greater than 50% as early as 3 hr after 12-O-tetradecanoylphorbol-13-acetate exposure, and at 8 to 72 hr the reduction was greater than or equal to 4-fold. Subsequent to the decrease in oncogene expression at 3 hr, DNA synthesis began to decline; by 24 hr, cell proliferation had ceased. At this time, monocyte- and macrophage-like cells were beginning to emerge. These findings demonstrate that c-myb is expressed during ML-1 cell proliferation and declines prior to the loss of DNA synthesis that accompanies the differentiation process.

Nucleotide sequence of the retroviral leukemia gene v-myb and its cellular progenitor c-myb: the architecture of a transduced oncogene.

The oncogene (v-myb) of avian myeloblastosis virus and a large portion of its cellular homolog (c-myb) have been molecularly cloned and sequenced. The portion of c-myb we analyzed contains seven interspersed segments (or exons). Fusion of these exons creates a continuous nucleotide sequence that is remarkably similar to the sequence of v-myb and that potentially encodes a protein very similar to that specified by v-myb. Comparisons between the sequences of v-myb and c-myb indicate that transduction of c-myb to form v-myb probably resulted from an initial DNA rearrangement and the subsequent use of a spliced RNA as an intermediate.

The product of the retroviral transforming gene v-myb is a truncated version of the protein encoded by the cellular oncogene c-myb.

Avian myeloblastosis virus (AMV) is an oncogenic retrovirus that rapidly causes myeloblastic leukemia in chickens and transforms myeloid cells in culture. AMV carries an oncogene, v-myb, that is derived from a cellular gene, c-myb, found in the genomes of vertebrate species. We constructed a plasmid vector that allows expression of a portion of the coding region for v-myb in a procaryotic host. We then used the myb-encoded protein produced in bacteria to immunize rabbits. The antisera obtained permitted identification of the proteins encoded by both v-myb and chicken c-myb. The molecular weights of the products of v-myb and c-myb (45,000 and 75,000 respectively) indicate that the v-myb protein is an appreciably truncated version of the c-myb protein.

Amplification of the c-myb oncogene in a case of human acute myelogenous leukemia.

Amplification is one of the mechanisms by which cellular oncogenes may be altered in their function, possibly leading to neoplastic transformation. The oncogenes c-myc, c- abl , and c-Ki-ras are amplified in several different human neoplasias. The oncogene c-myb, which is specifically expressed and regulated in hematopoietic cells, was found to be amplified in cell lines ML-1, ML-2, and ML-3, which were separately cultured from cells of a patient with acute myelogenous leukemia (AML). A five- to tenfold amplification was correlated with high levels of expression of normal size c-myb messenger RNA and with chromosomal abnormalities in the region 6q22 -24, where the c-myb locus is normally located. Amplification and cytogenetic abnormalities were detected in DNA s from primary and secondary cultures of ML cells, suggesting that they may have contributed to leukemogenesis. The similar AML cell lines HL-60 and ML s contain different amplified oncogenes: c-myc and c-myb, respectively. Alternative activation of structurally and possibly functionally similar oncogenes may distinguish--at the pathogenetic level--phenotypically similar tumors.

Aberrant expression of an amplified c-myb oncogene in two cell lines from a colon carcinoma.

Two cell lines (COLO 201 and COLO 205) derived independently from a single adenocarcinoma of the human colon each harbored an approximately 10-fold amplification of the cellular oncogene c-myb and a proportional abundance of the 4-kilobase mRNA derived from c-myb. By contrast, expression of c-myb could not be detected in cells from a variety of other solid tumors, including other colon carcinomas. Analysis of the amplified DNA with restriction endonucleases failed to reveal any topographical abnormalities within c-myb. Neither COLO 201 nor COLO 205 carry the double minute chromosomes and homogeneously staining regions of chromosomes that frequently serve as karyotypic signatures of amplified DNA. Instead, amplified c-myb is carried on what appear to be disomic or trisomic copies of the same anomalous marker chromosome that is characteristic of both COLO 201 and COLO 205. The karyological origin of this abnormal chromosome is not presently apparent. Our findings show c-myb expression by cells outside of the hemopoietic lineage, raise the possibility that amplification and/or ectopic expression of c-myb may have contributed to the genesis of the tumor from which the cells of COLO 201 and COLO 205 arose, and suggest that amplification of cellular oncogenes may be a more common factor in tumorigenesis than might have been suspected from available karyological data.

Differential expression of c-erbB, c-myc and c-myb oncogene loci in human lymphomas and leukemias.

Total cellular polyadenylated RNA from a variety of fresh human lymphoma and leukemia cells, characterized by histopathology and certain cell surface markers, was analyzed for the expression of three distinct cellular oncogenes (c-onc genes), c-erbB, c-myc and c-myb by dot-blot hybridization assays. Probes used were molecularly cloned DNA containing the respective oncogene sequence of avian erythroblastosis virus, myelocytomatosis virus (MC29) and myeloblastosis virus. ALL lymphoma-leukemia cells irrespective of B, T or non-B/non-T lymphocyte lineage expressed the c-erbB locus. This gene was also found to be active in normal peripheral blood lymphocytes and lymphocytes from lymph nodes showing reactive hyperplasia. This observation suggested that c-erbB might be normally involved in cell growth functions since it was not unique to hematopoietic malignancies. In contrast to c-erbB, elevated expressions of c-myc or c-myb were detected in certain neoplasms of B-lymphocytes and some other lymphoproliferative disorders as compared to the majority of the samples tested which showed either low or undetectable levels of these transcripts. An examination of B-cell lymphomas and leukemias in which the majority of the cellular populations expressed either Kappa or lambda surface lg light chain molecules revealed variations in the levels of c-onc transcripts within a morphologic and immunologic subtype. These findings support the notion that, in general, genetic heterogeneity exists in groups of hematopoietic proliferations defined by conventional histopathologic and immunologic criteria. Although with the majority of the specimens there was no obvious correlation between the morphologic cell type of lymphoma/leukemia and the c-onc RNA levels, interestingly two of the three samples diagnosed as chronic lymphocytic leukemia, B-cell type, showed considerably increased transcription of the c-myc gene relative to the other B-cell neoplasms. Thus a class of differentiated B-cell leukemia has been identified in which the molecular mechanisms which affect c-myc gene expression can now be investigated.

DNA rearrangement and altered RNA expression of the c-myb oncogene in mouse plasmacytoid lymphosarcomas.

Three types of tumors termed plasmacytomas (ABPC s), lymphosarcomas (ABLS s), and plasmacytoid lymphosarcomas (ABPL s) arise in BALB/c mice treated with pristane and Abelson murine leukemia virus (A-MuLV). While most ABPC s and BLS s contain integrated A-MuLV proviral genome and synthesize the v-abl RNA, most ABPL s do not. The ABPL tumors were examined for the expression of other oncogenes that may be associated with their transformed state, in the absence of transforming virus. These tumors expressed abundant c-myb RNA of unusually large size and showed DNA rearrangements of the c-myb locus.

Dissociation of transcriptional activation and oncogenic transformation by v-Myb.

The nuclear oncoprotein v-Myb is a transcriptional activator in both animal cells and the budding yeast Saccharomyces cerevisiae. Previous studies have suggested that an acidic domain of approximately 50 amino acids (amino acids 204-254 of v-Myb) is necessary and sufficient for transcriptional activation by v-Myb, c-Myb and GAL4-Myb fusion proteins. However, we find that first, none of the acidic residues within this region is essential for transcriptional activation in either animal cells or yeast. Second, transcriptional activation requires cooperation among multiple domains of v-Myb. In animal cells, transcriptional activation by v-Myb requires a central domain (amino acids 234-295), a C-terminal domain (amino acids 295-356), plus either of two more N-terminal domains (amino acids 163-197 or 198-232); in yeast, it requires the central domain plus either the C-terminal domain or a more N-terminal domain (amino acids 163-233). Third, although various subsets of these domains are sufficient for transcriptional activation by v-Myb, ALL of the domains must be present for transformation of primary hematopoietic cells. These results demonstrate that transcriptional activation by v-Myb is not sufficient for oncogenic transformation.

Proviral activation of the c-myb proto-oncogene is detectable in preleukemic mice infected neonatally with Moloney murine leukemia virus but not in resulting end stage T lymphomas.

Moloney murine leukemia virus induces myeloid leukemia when inoculated intravenously into pristane-primed adult BALB/c mice. One hundred percent of these tumors show insertional activation of the c-myb proto-oncogene, and reverse transcriptase PCR assays have shown that the c-myb activation could be detected soon after infection. We tested BALB/c and NIH Swiss mice that had been inoculated as newborns with Moloney murine leukemia virus, under which conditions they develop T lymphomas exclusively. Reverse transcriptase-PCR assays indicated that c-myb activations were detectable soon after neonatal infection. However, none of the resulting T lymphomas contained c-myb activations. The implications of these results to the timing of proto-oncogene activations in leukemogenesis and the specificity of proto-oncogene activations for different diseases are discussed.

Induction of B cell lymphomas by overexpression of a Myb oncogene truncated at either terminus.

The c-myb oncogene encodes a nuclear transcriptional transactivator that is often terminally truncated in hematopoietic tumors. To directly assess the tumorigenic activity of full length and terminally-truncated variants of c-myb, we have overexpressed several structurally-altered forms of myb within an avian retroviral vector and have shown that overexpression of truncated (but not full length) myb transforms both myeloid cells in vitro and mesenchymal cells in vivo. In vivo infection with these truncated myb viruses is now shown to induce metastatic B cell lymphomas in a significant minority of animals. Evaluation of the lymphomas revealed two distinct mechanisms of myb-induced tumorigenesis. In most of the lymphomas, proviral DNA inserted into the endogenous chicken c-myb gene and promoted the expression of a 5 -truncated myb transcript encoding an amino terminal truncated protein. In comparison, some animals infected with a virus encoding a carboxyl (C) terminal truncated myb (T-myb) developed non-insertional B cell lymphomas that directly expressed the provirally-encoded T-myb gene. The lymphomagenic T-myb protein lacks 214 C terminal amino acids including ALL of the myb transcription inhibition domain. This novel lymphomagenic activity for a C terminal truncated myb suggests that a loss of regulatory sequences at either end of c-myb is sufficient to create a B cell-specific transforming gene.

Rem-1, a putative direct target gene of the Myb-Ets fusion oncoprotein in haematopoietic progenitors, is a member of the recoverin family.

The Myb-Ets oncoprotein encoded by the E26 avian leukaemia virus represents a fusion of two transcription factors which cooperate in transforming multipotent haematopoietic progenitors (MEPs) in vitro and in vivo. Previous studies with a temperature sensitive mutant in ets (ts1.1 E26) have suggested that the Ets part of the Myb-Ets fusion protein blocks multilineage differentiation of transformed MEPs, by regulating specific target genes. Using this system in a differential screening approach we have now identified a new gene, called rem-1, as a target for the E26 virus. Following shift of ts1.1 mutant transformed cells to the nonpermissive temperature a decreased expression of rem-1 was observed which increased upon downshift. The finding that this reexpression did not require new protein synthesis suggests that the Ets component of the fusion protein directly regulates rem-1 transcription. Rem-1 is related to a family of EF-hand-containing calcium-binding proteins that are predominantly expressed in the brain and in retinal cells. This family includes recoverin and visinin, proteins that have been implicated in regulating photoreception. Rem-1 is likewise expressed in these tissues but in addition in haematopoietic cells and in the gut. Enforced expression of rem-1 in ts1.1-transformed MEP cells, using a retroviral vector, showed that this gene is not sufficient to block their differentiation, but that it may provide them with a growth advantage.

The myb oncogene family of transcription factors: potent regulators of hematopoietic cell proliferation and differentiation.

The myb family of genes includes the virally encoded v-myb oncogene, the c-myb protooncogene from which it is derived, and two structurally related genes, B-myb and A-myb. C-myb is most highly expressed in hematopoietic cells and its oncogenic activation leads to transformation, primarily of myeloid cells. Several lines of evidence suggest that c-myb functions in regulating both the proliferation and differentiation of hematopoietic cells of different lineages, including early progenitors. The mechanisms of action and the regulation of expression of c-myb and v-myb will be described. The possible role of the B-myb and A-myb gene products will also be discussed.

HPV16 E7 oncoprotein deregulates B-myb expression: correlation with targeting of p107/E2F complexes.

HPV16 is a human tumour virus encoding two principal oncoproteins, E6 and E7. expression of E7 can induce DNA synthesis in quiescent cells and this property coincides with its ability to bind to the cell proteins pRb and p107. As these cell proteins are regulators of the transcription factor E2F, we have investigated whether the interaction with E7 could result in induction of cell cycle regulated genes. We show that B-myb, whose induction at the G1/S boundary is regulated by release from E2F mediated transcriptional repression, is a target for transcriptional activation by E7 and is the first E7 responsive cell gene to be identified. E7 transactivation leads to both inappropriate transcription of B-myb during G1 and constitutive over-expression in cycling cells. B-Myb plays an essential role in cell cycle progression, and activation by E7 is likely to contribute to the mitogenic activity of the viral oncoprotein. Regulation of the B-myb promoter in NIH3T3 cells correlates with binding of distinct p107-containing complexes at the E2F binding site, and analysis of E7 mutants confirms that B-myb transcription in these cells is regulated through interactions with p107 rather than pRb. These results provide the first example of a potentially specific role for p107 in the regulation of the cell cycle.

The Myb oncogene product induces DNA-bending.

The nuclear oncogene v-myb and its cellular counterpart c-myb code for proteins that bind to DNA in a sequence specific manner and act as regulators of transcription. The Myb protein contains DNA binding and trans-regulatory domains which are important for its function. The DNA binding domain of Myb protein has been shown to contain three imperfectly conserved repeats of 50-52 amino acids that constitute the amino terminal end. In this communication, we show that Myb protein induces conformational change in DNA after protein-DNA complex formation. Circular permutation assays indicate that Myb protein induces DNA bending at the site of binding. Phasing analysis confirm the DNA bending and allowed the detection of relative orientation of bend. Myb proteins which comprise only DNA-binding domains either with three repeats or two repeats also bend DNA in the same orientation as the larger proteins with both DNA-binding and transactivating domains. However, the transactivating region seems to influence the magnitude of bend angle. We used molecular modeling to analyse the structure of Myb-DNA complex formation resulting in the bending of DNA. Data presented here show that Myb protein, like other transcriptional regulators, bends DNA upon binding allowing the interaction of regulatory elements.

Transformation of myelomonocytic cells by the avian myeloblastosis virus is determined by the v-myb oncogene, not by the unique long terminal repeats of the virus.

The avian myeloblastosis virus (AMV) induces acute monoblastic leukemia in chickens and transforms only myelomonocytic cells in vitro. The long terminal repeat (LTR) regulatory region of AMV is unique among the known classes of avian retrovirus LTRs. We demonstrate that the substitution of the AMV LTRs by Rous sarcoma virus LTRs did not alter the cell type specificity or the transforming ability of the virus.

Overexpression of C-terminally but not N-terminally truncated Myb induces fibrosarcomas: a novel nonhematopoietic target cell for the myb oncogene.

The myb oncogene encodes a DNA-binding transcriptional transactivator which can become a hematopoietic cell-transforming protein following the deletion of amino acid sequences from either its amino or carboxyl terminus. Although a number of hematopoietic tumors express terminally deleted variants of Myb, the involvement of truncated Myb in nonhematopoietic tumors has not been adequately investigated. To assess the full spectrum of Myb s oncogenic capability, a replication-competent retroviral vector (RCAMV) was used to express a full-length protein (C-Myb), an amino-terminally truncated protein (VCC- or delta N-Myb), a carboxyl-terminally truncated protein (T-Myb), or a doubly truncated protein (VCT-Myb) in vivo. These viruses were injected intravenously into 10-day chicken embryos, and the infected chicks were monitored for tumors. Approximately 4 to 8 weeks after hatching, the majority (30 of 39 [77%]) of animals infected with the T-Myb retrovirus (without 214 carboxyl-terminal residues) developed nodular muscle tumors which could be identified by both morphologic and immunohistochemical criteria as fibrosarcomas. Identically appearing tumors could also be found in the kidney of some T-Myb-infected animals. The T-Myb-induced fibrosarcomas expressed the appropriately sized T-Myb protein, contained an unaltered proviral T-myb gene, and showed clonal proviral integration sites. In comparison, no sarcomas were observed in any of the animals infected with the amino-terminally truncated (VCC- and delta N-Myb) or doubly truncated (VCT-Myb) viruses. A loss of carboxyl-terminal but not amino-terminal sequences can thus convert Myb into a potent in vivo transforming protein for nonhematopoietic mesenchymal cells. In comparison, a truncation of either or both ends of the protein can activate Myb into a hematopoietic cell-transforming protein.

Two functionally distinct half sites in the DNA-recognition sequence of the Myb oncoprotein.

The oncoprotein Myb is a sequence-specific DNA-binding protein with a pivotal function in the development and proliferation of hematopoietic precursor cells. A minimal DNA-binding domain composed of two tryptophan-rich repeats R2 and R3 is responsible for sequence recognition. Based on model building and mutational analysis, Myb was proposed to recognise its target through a double helix-turn-helix (HTH)-related motif using two recognition helices, one in R2 and one in R3. We found, by mutational analysis, that the DNA-binding site for c-Myb is functionally bipartite. While the first half site is dominant and absolutely required for binding, the second half site is only modulatory and mainly affects the half life of the complex. This bipartite nature of the binding site parallels the proposed bipartite structure of R2R3 with two HTH-related domains. Analysis of the DNA-binding site of R2R3 by missing-base interference-footprint analysis showed that the protein interacted with a 9-bp region. The same was found with a larger protein containing ALL three repeats. The effect of adding R1 was mainly to stabilise the complex. The borders of the complex, as revealed by exonuclease III footprinting, did not change due to the presence of R1. However, both borders became more refractory to the nuclease when R1 was present, but with a difference that suggested a specific orientation of the repeat domains relative to the DNA-binding site. We propose that the first half site is recognised by R3, while the second modulatory half site interacts with the R2 repeat.

DNA and redox state induced conformational changes in the DNA-binding domain of the Myb oncoprotein.

The DNA-binding domain of the oncoprotein Myb comprises three imperfect repeats, R1, R2 and R3. Only R2 and R3 are required for sequence-specific DNA-binding. Both are assumed to contain helix-turn-helix (HTH)-related motifs, but multidimensional heteronuclear NMR spectroscopy revealed a disordered structure in R2 where the second HTH helix was predicted [Jamin et al. (1993) Eur. J. Biochem., 216, 147-154]. We propose that the disordered region folds into a recognition helix and generates a full HTH-related motif upon binding to DNA. This would move Cys43 into the hydrophobic core of R2. We observed that Cys43 was accessible to N-ethylmaleimide alkylation in the free protein, but inaccessible in the DNA complex. Mutant proteins with charged (C43D) or polar (C43S) side chains in position 43 bound DNA with reduced affinity, while hydrophobic replacements (C43A, C43V and C43I) gave unaltered or improved DNA-binding. Specific DNA-binding enhanced protease resistance dramatically. Fluorescence emission spectra and quenching experiments supported a DNA-induced conformational change. Moreover, reversible oxidation of Cys43 had an effect similar to the inactivating C43D mutation. The highly oxidizable Cys43 could function as a molecular sensor for a redox regulatory mechanism turning specific DNA-binding on or off by controlling the DNA-induced conformational change in R2.

Oncogenic truncation of the first repeat of c-Myb decreases DNA binding in vitro and in vivo.

Oncogenic activation of c-Myb in both avian and murine systems often involves N-terminal truncation. In particular, the first of three DNA-binding repeats in c-Myb has been largely deleted during the genesis of the v-myb oncogenes of avian myeloblastosis virus and E26 avian leukemia virus. This finding suggests that the first DNA-binding repeat may have an important role in cell growth control. We demonstrate that truncation of the first DNA-binding repeat of c-Myb is sufficient for myeloid transformation in culture, but deletion of the N-terminal phosphorylation site and adjacent acidic region is not. Truncation of the first repeat decreases the ability of a Myb-VP16 fusion protein to trans activate the promoter of a Myb-inducible gene (mim-1) involved in differentiation. Moreover, truncation of the first repeat decreases the ability of the Myb protein to bind DNA both in vivo and in vitro. These results suggest that N-terminal mutants of c-Myb may transform by regulating only a subset of those genes normally regulated by c-Myb.

Identification of a second promoter in the human c-myb proto-oncogene.

We have previously described an alternatively spliced cDNA clone of the human c-myb proto-oncogene which has been shown to enhance the differentiation of Friend murine erythroleukemia cells. This clone, pMbm-2, contains unique 5 sequences which replace exon 1. The human c-myb intron 1 was sequenced to determine the exact position of this unique sequence and to further characterize the role of intron 1 in the regulation of the human c-myb gene. Here we report that intron 1 of c-myb is highly conserved between human and mouse throughout the intron, while only those sequences directly adjacent to exons 1 and 2 are conserved between human and chicken. The unique sequence of pMbm-2 was located directly adjacent to exon 2, suggesting that it arose as a product of alternative transcription initiation within intron 1. RNAase protection analysis was used to map a cluster of transcription start sites at the 5 end of exon 2. Levels of messages utilizing these start sites are proportional to those arising from the primary promoter. Functional characterization of this region revealed that this region can function as a promoter. Deletion studies have revealed the presence of negative and positive regulatory elements within this region which are utilized with different efficiencies in different cell lines. These studies suggest that cis or trans factors acting in this region may serve a dual function in both attenuation and transcription initiation.

Regulation of c-myb oncogene expression in immature and mature murine T cells.

The c-myb oncogene encodes a nuclear binding protein which may play a major role in differentiation during early T cell development. However, the functionally important transcription regions in the GC promoter site have not been defined and the significance of the regulation of this promoter site in T cell differentiation has not been determined. Therefore, the promoter strength was determined by measurement of the CAT activity in cell extracts of EL-4 cells that were transfected with a CAT expression vector that contained cloned segments of the 5 myb gene. Stepwise removal of DNA sequences between -2300 bp and -346 bp upstream from the ATG initiation codon resulted in a gradual loss of 50% of CAT activity, whereas deletion of DNA sequences from -346 to -295 and -232 to -155 bp upstream from the ATG initiation codon eliminated promoter activity. On analysis of the CAT activity after transfection of various cell lines with these same constructs, it was found that the same two promoter regions were required for high CAT activity in ALL the cell lines, including murine cell lines which express the alpha/beta TCR and high levels of c-myb (BW5147), the alpha/beta TCR and low levels of c-myb (Yac-1), or the gamma/delta TCR (KN 12.1 and KN 2.4 T), a murine fibroblast T cell line (NIH-3T3), and a human epithelial cell line (HeLa). However, the CAT activity did not correlate with steady state levels of expression of the c-myb gene in the murine cell lines. Our data indicate that the c-myb oncogene promoter is constitutively expressed is highly dependent on a limited region of the 5 myb gene, requires two DNA elements for optimal activity, and is functional in diverse T cell lines.

Stress-associated modulation of proto-oncogene expression in human peripheral blood leukocytes.

Changes in the cellular immune response associated with psychological stress were studied by using an academic stress model with medical students. The authors examined the expression of 2 proto-oncogenes, c-myc and c-myb, in peripheral blood leukocytes (PBLs) obtained from medical students at the time of examinations and at a baseline period approximately 1 month prior to the examinations. The level of messenger ribonucleic acid (mRNA) expression of both protooncogenes was significantly lower in PBLs obtained during examinations than in those from the baseline period. In addition, a significant decrease in the level of mRNA to the glucocorticoid receptor and gamma interferon was also found in the same preparations. The decrease in mRNA content of c-myc, c-myb, the glucocorticoid receptor, and gamma interferon in PBLs obtained from subjects during examinations is consistent with data from previous studies using the same model that have demonstrated a down-regulation of T-lymphocyte activation and proliferation in response to mitogens.

Thermal stability of the DNA-binding domain of the Myb oncoprotein.

The DNA-binding domain of the c-myb protooncogene product consists of three homologous tandem repeats of 51-52 amino acids (denoted as R1, R2, and R3 from the N-terminal side). In order to analyze conformational and thermodynamic characteristics of the homologous repeats, we have examined the DNA-binding domain by circular dichroism (CD) and differential scanning calorimetry (DSC). The CD spectra for the three individual repeats are significantly different in the fine profiles, indicating subtle differences in their conformations. The melting analyses for the fragments show that the thermal stability of each fragment is different from one another, with the following order of stability: R1(Tm = 61 degrees C) approximately greater than R3(57 degrees C) >> R2(43 degrees C), where R2 is much less stable than the other repeats. The denaturing process for the whole DNA-binding domain, measured by DSC, is characterized by a very broad transition ranging from 30 to 80 degrees C. The denaturation curve can be fit well by a three-state transition with one intermediate state. The transition temperature for the native-to-intermediate transition coincides with the melting temperature of R2, indicating that the intermediate state corresponds to the unfolding of unstable R2. The CD spectrum of the whole domain is almost identical to the sum of the individual spectra. Thus, these results suggest that the individual repeats in the whole DNA-binding domain behave independently in terms of conformation and stability. The addition of DNA to the DNA-binding fragment drastically changed the melting profile, in which the broad transition curve was replaced by a sharp peak at 58 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)FAU - Sarai, A

Modulation of c-myc, c-myb, c-fos, c-sis and c-fms proto-oncogene expression and of CSF-1 transcripts and protein by phorbol diester in human malignant histiocytosis DEL cell line with 5q 35 break point.

Following exposure to phorbol ester (TPA), DEL cell line, a human malignant histiocytosis (MH) cell line, is able to differentiate along a macrophage phenotype and thus it provides a suitable model for analyzing the sequential and differential gene expression associated with monocyte/macrophage differentiation. C-myc, c-myb, c-fos, c-sis and c-fms expression were determined by Northern analysis at various times following TPA treatment. The results showed that TPA down-modulated the constitutive expression of c-myc, c-myb, and c-fms, mRNA to low but still detectable levels. Conversely, TPA-induced differentiation resulted in transient appearance of c-fos, whereas no change in the level of c-sis and actin transcripts were observed. Thus, the c-fms and c-sis genes appear to be regulated in a specific manner in this malignant histiocytosis derived cell line. Furthermore, these investigations demonstrated a constitutive CSF-1 gene expression which transiently increased at mRNA and also at protein level as evaluated by a murine bone marrow CFU bioassay. Through this drug-induced modulation, the DEL cell line offers an additional model for studying some of the subtle interrelations existing between a growth factor (CSF-1) and its receptor (c-fms) in the monocyte/macrophage system.

Secondary structure of the DNA-binding domain of the c-Myb oncoprotein in solution. A multidimensional double and triple heteronuclear NMR study.

The DNA-binding domain of the c-Myb oncoprotein contains two repeats, R2 and R3, both of which have been proposed to be related to the helix-turn-helix (HTH) motif. As a first step towards determination of the three-dimensional structure of this domain and of the mode of interaction with the DNA, we have undertaken multidimensional heteronuclear NMR studies using uniformly 15N-labeled and 13C, 15N double-labeled R2R3 and, a selectively 15N-enriched sample on ALL lysine, histidine and leucine residues of R2R3. We present almost complete assignments of the backbone 1H, 15N and 13C" atoms and determine the secondary structure of R2R3 in solution. The R3 repeat is composed of three helices (residues 62-75, 78-85 and 91-100) while for the R2 repeat only two helices are found (residues 10-23 and 28-34). The remaining C-terminal part of the R2 repeat, predicted to be helical and part of the HTH motif, undergoes intermediate conformational exchange processes. Stabilization of this segment might occur upon binding to DNA.

The proto-oncogene c-myb mediates an intracellular calcium rise during the late G1 phase of the cell cycle.

The intracellular concentration of ionized calcium is involved in regulating mitosis. However, little is known about intracellular levels of calcium during G1. We have demonstrated in vascular smooth muscle cells a mid-G1 decrease in ionized calcium concentration followed by a 2-fold rise at the G1/S interface (44 nM +/- 0.6 nM versus 98 nM +/- 1.1 nM, p < 0.01). The elevation of intracellular calcium is preceded by an increase in c-myb mRNA levels and is abolished with antisense but not missense c-myb oligonucleotides. Furthermore, cells stably transfected with c-myb show a similar 2-fold augmentation in intracellular calcium concentrations, as compared with untransfected cells, which is also abolished by antisense c-myb oligonucleotides. The c-myb-induced rise in intracellular calcium is dependent upon the presence of extracellular calcium and is not suppressed by L type calcium channel blockers. We conclude that c-myb induces an elevation in intracellular calcium levels of vascular smooth muscle cells at the G1/S interface which provides a novel role for this proto-oncogene as well as a potentially important control point for cell cycle regulation.

The Gag-Myb-Ets fusion oncogene alters the apoptotic response and growth factor dependence of interleukin-3 dependent murine cells.

expression of the avian E26-derived Gag-Myb-Ets fusion oncogene in interleukin-3(IL3)-dependent murine hematopoietic cell lines results in a pattern of cell line dependent changes in growth factor-induced proliferation and apoptosis. A drug-selectable retrovirus expressing p135Gag-Myb-Ets induced an erythropoietin(Epo)-responsive phenotype in the cell lines FDC-P2, BaF3 and 32Dc123. Gag-Myb-Ets expression alone did not increase expression of GATA-1 or the Epo receptor(EpoR) in the presence of IL3, and infected cell lines express increased GATA-1 and EpoR only when IL3 was replaced by Epo in the culture media. Indicative of Epo-induced erythroid differentiation, these cells also began to express beta-globin after 3-5 days growth in Epo. Unlike control cells, infected FDC-P2 cells failed to undergo programmed cell death (apoptosis) when transferred from IL3- to Epo-containing media, although a fraction of the cells failed to proliferate following the media shift. Three other IL3-dependent cell lines showed no changes in growth behavior when induced to express the fusion oncogene. Our data shows that Gag-Myb-Ets can have different affects on growth factor pathways depending on the cell background, suggesting a model in which the p135gag-myb-ets fusion oncogene promotes these different responses through its affect on apoptosis.

Retroviral insertional mutagenesis in murine promonocytic leukemias: c-myb and Mml1.

Studies have focused on two genetic loci, c-myb and Mml1, whose activation by retroviral insertional mutagenesis contribute to promonocytic leukemia in our acute monocytic leukemia (AMoL) model. Multiple mechanisms of activation of c-myb by retroviral insertional mutagenesis implicate both transcriptional deregulation and protein truncation in conversion of this proto-oncogene to an oncogene. Because transformation by c-Myb can be viewed as a block to differentiation our studies moved into two in vitro systems to evaluate effects of truncated forms of c-Myb on cytokine induced maturation of myeloid progenitors to the granulocyte and macrophage lineages. Deregulated expression of truncated and full length c-Myb did not result in maintenance of the myelomonocytic progenitor state but rather a block in differentiation at intermediate to late steps in the maturation processes of myelomonocytic cells. Our results argue that inhibition of differentiation is due to c-Myb s ability to maintain the proliferative state of cells. Interestingly, the phenotype of continuously proliferating monocytic cells resembles that of the tumor cell phenotype. Recently we identified a new target of integration, Mml1, which is rearranged in ten promonocytic leukemias that do not have c-myb rearrangements. This locus which was mapped to chromosome 10 is presently being characterized.

Functional analysis of the c-myb proto-oncogene.

Targeted mutagenesis studies were initiated to determine the normal biological function of the c-myb proto-oncogene. While heterozygous mice are phenotypically indistinguishable from their wild-type littermates, homozygous mutant fetuses die at approximately 15.5 days of gestation apparently due to anemia, which results from an inability to switch from embryonic yolk sac to fetal liver erythropoiesis. Studies are currently being done to determine the extent of hematopoietic abnormalities in the homozygous mutant fetuses. In vitro assays for hematopoietic colony-forming cells have been used to determine the frequency of both erythroid and myeloid progenitors in the fetal livers of wild-type, heterozygous, and homozygous mutant c-myb fetuses. The reduced number of erythroid progenitors was not unexpected considering the mutant fetus s pale color and reduced hematocrit. The dramatically reduced number of colonies derived from myeloid progenitors in the mutant fetuses in comparison to the number detected in phenotypically normal littermates suggests that expression of the c-myb proto-oncogene is critical for the proliferation and/or differentiation of early hematopoietic progenitors and possibly hematopoietic stem cells. Other possible explanations would include a hematopoietic progenitor migration problem from the yolk sac to the fetal liver or a defect in the microenvironment of the liver. Whether the lymphoid lineage is also adversely affected by the lack of c-myb expression remains to be determined. RT-PCR and Northern blot analyses were used in an attempt to identify downstream genes which may be directly or indirectly regulated by the Myb gene product. While the levels of expression of several genes involved in erythropoiesis (GATA-1, NF-E2, SCL, and EpoR) were reduced in the livers of homozygous mutant fetuses in comparison to phenotypically normal littermates and one gene, Kit ligand (KL), was expressed at higher levels in the mutant livers, these results must be viewed with caution. The livers of the mutant fetuses have been shown to be hypocellular in comparison to those of phenotypically normal littermates (35). It is possible that the Myb gene product is directly or indirectly modulating the expression of these genes. Conversely, the alteration in expression may be due to the reduced number or absence of specific hematopoietic lineages in the livers of the mutant fetuses. Differential display has also been used to identify putative novel genes that are involved in hematopoiesis. Preliminary studies suggest that this may be a powerful methodology to compare the expression pattern of genes in the fetal liver of wild-type, heterozygous, and homozygous mutant littermates at 14.5 days of gestation. To date nearly 60% of the partial cDNAs subcloned analyzed have been shown to be differentially expressed. More importantly, 75% of the differentially expressed cDNAs that have been sequenced appear to encode novel genes. Whether any of these novel genes are involved in the c-myb transcriptional cascade remains to be determined. Overall, analysis of the c-myb mutant fetuses have provided valuable insight into the biological function of this interesting proto-oncogene. The continued analysis of this resource will undoubtedly provide additional information concerning the role of the c-myb gene in hematopoiesis.

Apoptotic response to oncogenic stimuli: cooperative and antagonistic interactions between c-myb and the growth suppressor p53.

c-myb, a protooncogene prevalently expressed in the hematopoietic tissue, is a transcription factor that contains a DNA-binding domain and an acidic domain and is able to transactivate specific viral and cellular genes. In this report, we show that c-myb can stimulate apoptosis in both the murine promyelocytic 32D and the human osteosarcoma SAOS2 cell lines when coexpressed with p53. Apoptosis is accompanied by increased transactivation of the cell death-associated BAX gene. This effect is c-myb specific, because B-myb is not able to cooperate with p53 in the induction of BAX transcription and apoptosis. Immunoprecipitation studies and gel shift analysis indicate that c-myb does not directly interact with the BAX promoter or the p53 protein but, rather, cooperates through an indirect mechanism. Consistent with the existence of a functional link between c-myb and p53, we also observed that c-myb represses p53-induced activation of the WAF-1 promoter and induces proliferation of SAOS2 cells growth arrested by p53. These results might contribute to the elucidation of the mechanisms underlying p53-dependent pathways of oncogene-induced apoptosis and provide a further example of DNA-binding independent myb activity.

The c-myb proto-oncogene: a novel target for human gene therapy.

OBJECTIVE: To determine, for canine mammary tumors, whether malignancy, with or without local invasion or regional metastasis, was associated with overexpression of the oncogene c-erbB-2. DESIGN: c-erbB-2 expression was measured in canine mammary tumor-derived cell lines and in mammary tumor tissues from clinical cases. Clinical samples were examined histologically to determine whether they were benign or malignant and, if malignant, whether they had evidence of local invasion or regional metastasis. Canine fibroblast cultures and normal canine mammary epithelial tissues were used as reference standards for cell lines and mammary tumors, respectively. SAMPLE POPULATIONS: 28 canine mammary tumor tissue samples obtained surgically from clinical cases and samples from 7 canine mammary tumor cell lines derived from primary canine mammary tumors. PROCEDURE: c-erbB-2 mRNA levels were determined by means of hybridization of total polysomal RNA with a 32P-labeled human c-erbB-2 probe on dot blots, and results were quantified by means of scanning densitometry. Overexpression of c-erbB-2 was defined as an autoradiographic density > or = 2 times the density of reference samples on the same blot. RESULTS: Overexpression of c-erbB-2 was detected in 17 of 23 malignant tumors, 0 of 5 benign tumors, and 2 of 7 mammary tumor cell lines. c-erbB-2 overexpression was correlated with a histopathologic diagnosis of malignancy (P = 0.005) but not with the presence of local invasion or regional metastatic disease (P = 0.621). CONCLUSIONS: Results suggest that overexpression of c-erbB-2 occurs prior to the development of metastatic disease in canine mammary tumors and plays a role in the development of malignancy.

Regulation of hematopoietic cell proliferation and differentiation by the myb oncogene family of transcription factors.

The myb family of genes include the virally encoded v-myb oncogene, its normal cellular equivalent c-myb and two related members called A-myb and B-myb. They are ALL transcription factors that recognize the same DNA sequence (PyAACG/TG) and are ALL involved in the regulation of proliferation and differentiation in different cell types, including hematopoietic cells. C-myb is most highly expressed in hematopoietic cells and its oncogenic activation leads to transformation of these cells. Several lines of evidence have demonstrated that c-myb regulates both the proliferation and differentiation of hematopoietic cells of different lineages. The mechanisms of action of c-myb and v-myb are becoming clearer, mostly through the study of the different genes that are regulated by these transcription factors and the cofactors with which c-myb and v-myb co-operate. More recently the biological and biochemical functions of the B-myb and A-myb gene products have been investigated. Evidence for the function of the different members of the myb family in relation to hematopoietic proliferation and differentiation is presented, and the different roles of the myb genes are discussed.

c-myb proto-oncogene is expressed by quiescent scleroderma fibroblasts and, unlike B-myb gene, does not correlate with proliferation.

Systemic sclerosis (scleroderma) is characterized by excessive deposition of extracellular matrix constituents. Although it has been proposed that tissue fibrosis is due to increased fibroblast synthesis of various collagen polypeptides, there is some experimental evidence that patients with systemic sclerosis have a defect in the control of fibroblast growth. The myb family of genes includes, among others, the c-myb proto-oncogene and the structurally related gene, B-myb, which are both implicated in the regulation of differentiation and/or proliferation of hematopoietic and nonhematopoietic cells. To elucidate the molecular basis responsible for scleroderma fibroblast proliferation, we therefore elected to investigate the expression of c-myb and B-myb genes in scleroderma and control cells. Using the reverse transcriptase polymerase chain reaction technique, we detected c-myb transcripts in scleroderma skin fibroblasts rendered quiescent by serum deprivation. Under the same experimental conditions, c-myb message was not found in normal skin fibroblasts, but, after serum stimulation, c-myb RNA was clearly evident from 3 to 72 h in both normal and pathologic cells. Treatment of these cells with c-myb antisense oligonucleotides caused downregulation of c-myb expression, and the inhibition of scleroderma fibroblast proliferation was 42%, whereas in normal fibroblasts the inhibition was weaker (22%). In contrast to c-myb, in normal and scleroderma fibroblasts the level of expression of B-myb correlated with cell proliferation assessed by cell count, and densitometric analysis showed that B-myb message was 1.5-5 times higher in most of pathologic cells studied. The antisense B-myb oligonucleotides had a weaker antiproliferative effect compared with antisense c-myb, inhibiting scleroderma and normal fibroblasts by 23% and 13%, respectively. These data suggest that the B-myb and c-myb genes may play a role in scleroderma fibroblast proliferation and function.

V-myb oncogene and c-myb proto-oncogene expression in avian cells: morphological changes of the cells and topographic localization of myb proteins.

Morphological changes of avian cells expressing the v-myb oncogene or c-myb proto-oncogene were studied by means of electron microscopy. expression of both genes lead to distinct morphological changes of these cells. The nucleus of LSCC-BM2 cells espressing v-myb gene was of normal size but usually of irregular shape. It contained large unravelled nucleoli with typical interstices in some cells. Small nucleolar structures were also localized in the periphery of nuclear membrane. Nuclear envelope revealed reduced perinuclear space between two membranes. LSCC-BK3 cells expressing the c-myb gene were characterized by distinctly enlarged nucleus, in most cases of irregular shape. It contained only one nucleolus markedly enlarged, often unravelled, with apparent interstitial area. Nucleoli with nucleolonemas were observed in some cells. Nuclear envelope formed by two obscure membranes showed reduced perinuclear space. Topographic localization of v-Myb and c-Myb protein products was not basically different, both being detected in the nucleus of avian cells. v-Myb and c-Myb markers were distributed mostly in clusters, usually associated with interchromatin granules, but some marker was associated also with the nuclear membrane. Both Myb products were never detected in nucleolar structures of avian cells. Morphological changes of avian cells expressing myb genes and topographic localization of Myb proteins in these cells were different from those found in the insect cells expressing myb genes. The observed differences are discussed.

Myb-Ets fusion oncoprotein inhibits thyroid hormone receptor/c-ErbA and retinoic acid receptor functions: a novel mechanism of action for leukemogenic transformation by E26 avian retrovirus.

The E26 and avian erythroblastosis virus (AEV) avian retroviruses induce acute leukemia in chickens. E26 can block both erythroid and myeloid differentiation at an early multipotent stage. Moreover, E26 can block erythroid differentiation at the erythroid burst-forming unit/erythroid CFU (BFU-E/CFU-E) stage, which also corresponds to the differentiation stage blocked by AEV. AEV carries two oncogenes, v-erbA and v-erbB, whereas E26 encodes a single 135-kDa Gag-Myb-Ets fusion oncoprotein. v-ErbA is responsible for the erythroid differentiation arrest through negative interferences with both the retinoic acid receptor (RAR) and the thyroid hormone receptor (T3R/c-ErbA). We investigated whether Myb-Ets could block erythroid differentiation in a manner similar to v-ErbA. We show here that Myb-Ets inhibits both RAR and c-ErbA activities on specific hormone response elements in transient-expression assays. Moreover, Myb-Ets abrogates the inactivation of transcription factor AP-1 by RAR and T3R, another feature shared with v-ErbA. Myb-Ets also antagonizes the biological response of erythrocytic progenitor cells to retinoic acid and T3. Analysis of a series of mutants of Myb-Ets reveals that the domains of the oncoprotein involved in these inhibitory activities are the same as those involved in oncogenic transformation of hematopoietic cells. These data demonstrate that the Myb-Ets oncoprotein shares properties with the v-ErbA oncoprotein and that inhibition of ligand-dependent RAR and c-ErbA functions by Myb-Ets is responsible for blocking the differentiation of hematopoietic progenitors.

Retroviral insertional activation of the c-myb proto-oncogene in a Marek s disease T-lymphoma cell line.

Marek s disease virus (MDV) is an avian herpesvirus that causes, in chickens, a lymphoproliferative disease characterized by malignant transformation of T lymphocytes. The rapid onset of polyclonal tumors indicates the existence of MDV-encoded oncogenic products. However, the molecular basis of MDV-induced lymphoproliferative disease and latency remains largely unclear. Several lines of evidence suggest that MDV and Rous-associated virus (RAV) might cooperate in the development of B-cell lymphomas induced by RAV. Our present results indicate for the first time that MDV and RAV might also act synergistically in the development of T-cell lymphomas. We report an example of an MDV-transformed T-lymphoblastoid cell line (T9) expressing high levels of a truncated C-MYB protein as a result of RAV integration within one c-myb allele. The chimeric RAV-c-myb mRNA species initiated in the 5 long terminal repeat of RAV are deprived of sequences corresponding to c-myb exons 1 to 3. The attenuation of MDV oncogenicity has been strongly related to structural changes in the MDV BamHI-D and BamHI-H DNA fragments. We have established that both DNA restriction fragments are rearranged in the T9 MDV-transformed cells. Our results suggest that retroviral insertional activation of the c-myb proto-oncogene is a critical factor involved in the maintenance of the transformed phenotype and the tumorigenic potential of this T-lymphoma cell line.

Optimizing the cell efficacy of synthetic ribozymes. Site selection and chemical modifications of ribozymes targeting the proto-oncogene c-myb.

expression of the proto-oncogene c-myb is necessary for proliferation of vascular smooth muscle cells. We have developed synthetic hammerhead ribozymes that recognize and cleave c-myb RNA, thereby inhibiting cell proliferation. Herein, we describe a method for the selection of hammerhead ribozyme cleavage sites and optimization of chemical modifications that maximize cell efficacy. In vitro assays were used to determine the relative accessibility of the ribozyme target sites for binding and cleavage. Several ribozymes thus identified showed efficacy in inhibiting smooth muscle cell proliferation relative to catalytically inactive controls. A combination of modifications including several phosphorothioate linkages at the 5 -end of the ribozyme and an extensively modified catalytic core resulted in substantially increased cell efficacy. A variety of different 2 -modifications at positions U4 and U7 that confer nuclease resistance gave comparable levels of cell efficacy. The lengths of the ribozyme binding arms were varied; optimal cell efficacy was observed with relatively short sequences (13-15 total nucleotides). These synthetic ribozymes have potential as therapeutics for hyperproliferative disorders such as restenosis and cancer. The chemical motifs that give optimal ribozyme activity in smooth muscle cell assays may be applicable to other cell types and other molecular targets.

The importance of the linker connecting the repeats of the c-Myb oncoprotein may be due to a positioning function.

The DNA-binding domain of the oncoprotein c-Myb consists of three imperfect tryptophan-rich repeats, R1, R2 and R3. Each repeat forms an independent mini-domain with a helix-turn-helix related motif and they are connected by linkers containing highly conserved residues. The location of the linker between two DNA-binding units suggests a function analogous to a dimerisation motif with a critical role in positioning the recognition helices of each mini-domain. mutational analysis of the minimal DNA-binding domain of chicken c-Myb (R2 and R3), revealed that besides the recognition helices of each repeat, the linker connecting them was of critical importance in maintaining specific DNA-binding. A comparison of several linker sequences from different Myb proteins revealed a highly conserved motif of four amino acids in the first half of the linker: LNPE (L138 to E141 in chicken c-Myb R2R3). Substitution of residues within this sequence led to reduced stability of protein-DNA complexes and even loss of DNA-binding. The two most affected mutants showed increased accessibility to proteases, and fluorescence emission spectra and quenching experiments revealed greater average exposure of tryptophans which suggests changes in conformation of the proteins. From the structure of R2R3 we propose that the LNPE motif provides two functions: anchorage to the first repeat (through L) and determination of the direction of the bridge to the next repeat (through P).

Isolation and characterization of a tobacco mosaic virus-inducible myb oncogene homolog from tobacco.

Salicylic acid (SA) plays an important role in signaling the activation of plant defense responses against pathogen attack including induction of pathogenesis-related (PR) proteins. To gain further insight into the SA-mediated signal transduction pathway, we have isolated and characterized a tobacco mosaic virus (TMV)-inducible myb oncogene homolog (myb1) from tobacco. The myb1 gene was induced upon TMV infection during both the hypersensitive response and development of systemic acquired resistance in the resistant tobacco cultivar following the rise of endogenous SA, but was not activated in the susceptible cultivar that fails to accumulate SA. The myb1 gene was also induced by incompatible bacterial pathogen Pseudomonas syringae pv. syringae during the hypersensitive response. Exogenous SA treatment rapidly (within 15 min) activated the expression of myb1 in both resistant and susceptible tobacco cultivars with the subsequent induction of PR genes occurring several hours later. Biologically active analogs of SA and 2,6-dichloroisonicotinic acid (a synthetic functional analog of SA), which induce PR genes and enhanced resistance, also activated the myb1 gene. In contrast, biologically inactive analogs were poor inducers of myb1 gene expression. Furthermore, the recombinant Myb1 protein was shown to specifically bind to a Myb-binding consensus sequence found in the promoter of the PR-1a gene. Taken together, these results suggest that the tobacco myb1 gene encodes a signaling component down-stream of SA that may participate in transcriptional activation of PR genes and plant disease resistance.

The proto-oncogene c-myb is expressed in sporadic bovine lymphoma, but not in enzootic bovine leukosis.

We examined bovine c-myb gene expression in six samples of sporadic bovine lymphomas (two calf, three thymic and one intermediate) and five of enzootic bovine leukosis. tumor cells of the sporadic bovine lymphomas were of immature cell lineage (one B lymphoma and five T lymphomas). The c-myb mRNA was expressed in almost ALL the sporadic bovine lymphomas (except for one thymic form) including a BoCD8 single positive T lymphoma. On the contrary, c-myb was not expressed in mature B lymphomas of enzootic bovine leukosis. The results suggest that c-myb expression is closely associated with tumor cell differentiation of bovine lymphomas.

Oncogenic activation of c-Myb by carboxyl-terminal truncation leads to decreased proteolysis by the ubiquitin-26S proteasome pathway.

c-myb activation by insertional mutagenesis in murine myeloid leukemias can lead to amino (NH2)-terminal or carboxyl (COOH)-terminal truncation of its protein product. We observed that in these leukemias, the steady state level of the protein truncated at the COOH terminus was remarkably higher than that of the protein truncated at the NH2-terminus or full length wild-type protein. To examine the rate of proteolysis of different forms of Myb in a uniform cellular background, the proteins were constitutively expressed in the myeloblast cell line M1, using the retrovirus vector LXSN. In pulse chase experiments, using metabolically 35S-labeled proteins, it was determined that COOH-terminal truncation of c-Myb by 248 aa (CT-c-Myb) substantially increases protein stability, resulting in a t1/2 of about 140 min, as compared to 50 min for full length c-Myb (FL-c-Myb). In an investigation of the mechanism involved in the in vivo degradation of this short lived transcription factor, inhibitors of the lysosomal (chloroquine), proteasomal (ALLM, ALLN, lactacystin) and calpains (EGTA, E-64d, BAPTA/AM) pathways were utilized. Results of this experiment identified the 26S proteasome as a major pathway responsible for rapid breakdown of the protein in hematopoietic cells. Further experiments carried out in vitro demonstrated that c-Myb can be ubiquitinated, suggesting that this process may be involved in the targeting of wild-type c-Myb to degradation by the 26S proteasome. In addition, it was demonstrated that CT-c-Myb was less efficiently ubiquitinated than wild-type protein indicating that defects in modification account for its escape from rapid turnover. We speculate that the increased half-life of c-Myb resulting from truncation could contribute to its transforming potential.

The Myb oncoprotein: regulating a regulator.

Hox proteins control genetic programs that orchestrate development, and a large subset of Hox proteins can bind DNA elements as heterodimers with the Pbx family of homeodomain proteins. A transcriptionally activated version of Pbx1, E2a-Pbx1, is an oncoprotein in human pre-B cell leukemia that strongly suppresses differentiation and retains its ability to heterodimerize with Hox proteins. Because monomeric Hox proteins bind very similar DNA motifs, it is unclear how they activate diverse developmental programs. Here we demonstrate that heterodimers containing different Hox proteins and a common Pbx1 or E2a-Pbx1 partner bind different DNA motifs. Structural models suggest that the specificity of the Hox protein is altered by a conformation change involving residues in the N-terminal arm of the Hox homeodomain. mutational analysis also supported the hypothesis that unique sequences in the N-terminal arm of the Hox homeodomain are at least partially responsible for mediating this specificity. In vivo, Hox proteins directed E2a-Pbx1-mediated transactivation with moderate specificity to cognate Hox-Pbx motifs. Thus, the development specificity of individual Hox proteins may be mediated, in part, by differential targeting of cellular genes by Pbx1-Hox complexes. Likewise, through its function as a common heterodimer partner, oncoprotein E2a-Pbx1 may be able to interfere with multiple programs of development that are induced by the sequential or simultaneous expression of Hox proteins during hematopoiesis.

Oncogenic point mutations induce altered conformation, redox sensitivity, and DNA binding in the minimal DNA binding domain of avian myeloblastosis virus v-Myb.

c-Myb is the founder member of a class of transcription factors with tryptophan-rich repeats responsible for DNA binding. Activated oncogenic forms of Myb are encoded by the avian retroviruses, avian myeloblastosis virus (AMV) and E26. AMV v-Myb encodes a truncated protein with 11 point mutations relative to c-Myb. The mutations in the DNA binding domain (DBD) were reported to impose distinct phenotypes of differentiation on transformed myeloid cells (Introna, M., Golay, J., Frampton, J., Nakano, T., Ness, S. A., and Graf, T. (1990) Cell 63, 1287-1297). The molecular mechanism operating has remained elusive since no change in sequence specificity has been found. We introduced AMV-specific point mutations in the minimal DBD of chicken c-Myb and studied their effect on structure and function of the purified protein. Fluorescence emission spectra and fluorescence quenching experiments showed that the AMV-specific point mutations had a significant effect on the conformation of the DBD, giving rise to a more compact structure, a change that was accompanied by a reduced sensitivity toward cysteine-specific alkylation and oxidation. The DNA binding properties were also altered by the AMV-specific point mutations, leading to protein-DNA complexes with highly reduced stability. This reduction in stability was, however, more severe with certain subtypes of binding sequences than with others. This differential behavior was also observed in an in vivo model system where DBD-VP16 fusions were coexpressed with various reporters. These findings imply that different subsets of Myb-responsive promoters may react differentially toward the AMV-specific mutations, a phenomenon that could contribute to the altered patterns of gene expression induced by the AMV v-Myb relative to wild type c-Myb.

Antisense oligonucleotide to proto-oncogene c-myb inhibits the formation of intimal hyperplasia in experimental vein grafts.

BACKGROUND: The development of intimal hyperplasia is a major cause of early vein graft failure. The study examines the effects of locally delivered antisense oligonucleotides to the proto-oncogene c-myb on the development of vein graft intimal hyperplasia. METHODS: Common carotid vein bypass grafting procedures were performed on 60 New Zealand White rabbits. Seventeen grafts were controls, 14 had grafts coated with a commercial gel, 17 had grafts coated with gel containing 200 micrograms of an antisense c-myb oligonucleotide, and 6 rabbits each had grafts coated with gel containing one of two control oligonucleotides. Grafts were harvested 28 days after surgery, and sections were taken for dimensional analysis, morphologic evaluation, and vasomotor function. Grafts were also harvested at 1 day for oligonucleotide uptake/localization analysis and at 3 days for c-myb mRNA analysis. RESULTS: Oligonucleotides were uniformly distributed within the media and adventitia by 1 day. A 38% reduction occurred in mean intimal thickness in the vein grafts coated with antisense to c-myb compared with the other groups. No difference in medial thickness was seen among groups. By scanning and transmission electron microscopy ALL vein grafts showed a confluent endothelium. In contrast to control vein grafts, which did not relax to acetylcholine, most of the gel and ALL of the gel/oligonucleotide-coated grafts relaxed by more than 40% of precontracted tension. Responses to a panel of contractile agents were unchanged in the treated groups compared with those in the control group. CONCLUSIONS: Locally delivered antisense oligonucleotides to proto-oncogene c-myb significantly reduces intimal hyperplasia with preservation of acetylcholine-mediated endothelium-dependent relaxation in experimental vein grafts. These findings suggest that targeting a common regulatory pathway of vascular smooth muscle mitogenesis can be successful in reducing the early development of intimal hyperplasia.

Cytotoxic effect of sodium nitroprusside on cancer cells: involvement of apoptosis and suppression of c-myc and c-myb proto-oncogene expression.

Nitric oxide (NO) is an unstable free radical gas known as an effector molecule of macrophage cytotoxicity against cancer cells. Although several mechanisms of NO-mediated cytotoxicity have been proposed, this phenomenon remains to be characterized in detail. To explore the mechanisms by which NO kills cancer cells, we made use of sodium nitroprusside (SNP), which releases NO in the culture medium. SNP showed a dose-dependent cytotoxic effect on NA cells, an epithelial cancer cell line. When NA cells were killed by SNP, high levels of NO2- (stable end product of NO) were detected in the culture medium. The cell death induced by SNP was mediated by apoptosis, as demonstrated by the presence of nuclear condensation and blebbing of the nuclear membrane, and internucleosomal DNA fragmentation quantified by a specific ELISA. Northern blot analysis revealed that c-myc mRNA expression of NA cells was rapidly reduced by treatment with SNP. RT-PCR analysis showed that c-myb mRNA was expressed in untreated NA cells, and c-myb mRNA level of NA cells was dose-dependently reduced by treatment with SNP. These results indicate that SNP exerts its cytotoxic effect on NA cells through spontaneous release of NO. Cytotoxicity induced by SNP is at least partially mediated via the process known as apoptosis. Our results also suggest that down-regulation of c-myc and c-myb proto-oncogenes might be involved in SNP-induced cytotoxicity.

Characterization of a high copy number amplification at 6q24 in pancreatic cancer identifies c-myb as a candidate oncogene.

In a recent study designed to identify chromosomal aberrations in pancreatic cancer tissues using comparative genomic hybridization, a high copy number amplification on 6q was detected. To identify the most likely candidate oncogene, the extension of the amplification in pancreatic cancer tissues and cell lines was determined by Southern blot analysis. Exon trapping was performed with DNA from a yeast artificial chromosome clone containing the complete minimally amplified region. Only fragments from two genes, namely, the c-myb oncogene and a novel gene, were shown to be amplified. The c-myb proto-oncogene was amplified in 10% of the pancreatic carcinoma tissues and in the pancreatic cancer cell line PC2. Interestingly, the c-myb oncogene was overexpressed not only in the amplified samples but also in the majority of the examined pancreatic cancer tissues and cell lines, suggesting that amplification is only one of the mechanisms leading to overexpression. In contrast, the novel gene, which was called human eRF3b (eukaryotic release factor 3b), seems to be only coamplified with c-myb. Genetic alterations of c-myb were mainly found in advanced tumors, indicating a possible correlation to tumor progression and aggressive tumor phenotypes.

Two regions in c-myb proto-oncogene product negatively regulating its DNA-binding activity.

The c-myb proto-oncogene product (c-Myb) is a transcriptional regulator that binds to the specific DNA sequence. Deletion of the negative regulatory domain (NRD) in the carboxyl-proximal region of c-Myb results in both increased trans-activating capacity and oncogenic activation. One possible mechanism to modulate c-Myb activity is a regulation of DNA-binding activity. However, it is not known whether any region in NRD affects the in vivo DNA-binding activity of c-Myb. Using the highly transfectable cell line 293T, we developed a system to precisely measure the DNA-binding activity of Myb expressed in mammalian cells. Using this system, two regions in NRD were shown to repress DNA-binding activity. These results suggest that DNA-binding activity of c-Myb is independently regulated by multiple mechanisms through these subdomains.

The homeobox gene GBX2, a target of the myb oncogene, mediates autocrine growth and monocyte differentiation.

The homeobox gene GBX2 was identified as a target gene of the v-Myb oncoprotein encoded by the avian myeloblastosis virus (AMV). GBX2 activation by c-Myb requires signal transduction emanating from the cell surface while the leukemogenic AMV v-Myb constitutively induces the GBX2 gene. mutations in the DNA binding domain of AMV-Myb render it independent of signaling events and concomitantly abrogate the collaboration between Myb and CCAAT Enhancer Binding Proteins (C/EBP), which are involved in granulocyte differentiation. Ectopic expression of GBX2 in growth factor-dependent myeloblasts induces monocytic features and independence from exogenous cytokines, reflecting distinct features of AMV-transformed cells. Our results suggest that Myb or factors it interacts with contribute to hematopoietic lineage choice and differentiation in a signal transduction-dependent fashion.

Regulation of fibroblast growth factor 2 expression in melanoma cells by the c-MYB proto-oncoprotein.

Dysregulated expression of basic fibroblast growth factor [fibroblast growth factor 2 (FGF-2)] mediates autocrine growth of melanoma cells. The presence of a consensus Myb binding site in the human FGF-2 promoter prompted us to investigate whether this transcription factor could regulate FGF-2 expression in melanomas. We report that c-MYB mRNA is overexpressed in melanoma cell lines compared to normal melanocytes and that ectopic expression of murine c-Myb in SK-MEL-2 human melanoma cells resulted in increased expression of FGF-2 mRNA and FGF-2 protein. Furthermore, murine c-Myb transactivated a reporter plasmid containing the human FGF-2 promoter region in contransfected SK-MEL-2 human melanoma cells. Although a functional DNA-binding domain was required for transactivation, responsiveness to c-Myb was independent of the putative Myb binding site and mapped to two regions of the FGF-2 promoter that did not bind c-Myb in vitro. We suggest that c-MYB contributes to FGF-2-mediated autocrine growth of melanomas by indirectly regulating the FGF-2 promoter.

Regulating ancient regulators: the function of the B-Myb oncoprotein.

Stimulation of c-Jun transcriptional activity via phosphorylation mediated by the stress-activated or c-Jun amino-terminal (SAPK/JNK) subgroup of mitogen-activated protein kinases (MAP kinases) is thought to depend on a kinase-docking site (the delta region) within the amino-terminal activation domain, which is deleted from the oncogenic derivative, v-Jun [1] [2] [3]. This mutation markedly enhances v-Jun oncogenicity [4] [5]; however, its transcriptional consequences have not been resolved. In part, this reflects uncertainty as to whether binding of SAPK/JNK inhibits c-Jun function directly [6] [7] or, alternatively, serves to facilitate and maintain the specificity of positive regulatory phosphorylation [8]. Using a two-hybrid approach, we show that SAPK/JNK stimulates c-Jun transactivation in yeast and that this depends on both catalytic activity and physical interaction between the kinase and its substrate. Furthermore, c-Jun is active when tethered to DNA via SAPK/JNK, demonstrating that kinase binding does not preclude transactivation. Taken together, these results suggest that SAPK/JNK acts primarily as a positive regulator of c-Jun transactivation in situ, and that loss of the docking site physically uncouples v-Jun from this control. This loss-of-function model accounts for the deficit of v-Jun regulatory phosphorylation and repression of TPA response element (TRE)-dependent transcription observed in v-Jun-transformed cells and predicts that an important property of the oncoprotein is to antagonise SAPK/JNK-dependent gene expression.

The c-Myb oncoprotein.

The c-myb gene is the cellular homologue of the v-myb oncogenes carried by the avian leukaemia viruses AMV and E26. It encodes a transcription factor (c-Myb), as does each of the viral oncogenes, which recognises the core DNA sequence C/T-A-A-C-G/T-G via a repeated helix-turn-helix-like motif. c-myb is expressed in immature haemopoietic cells, as well as immature cells of the gastro-intestinal epithelium and is down-regulated with differentiation. Enforced expression of activated or even normal forms of Myb can transform haemopoietic cells, most often of the myeloid lineage, in vitro and in vivo. Although many genes have been identified which are likely to be regulated by c-Myb, the critical target genes involved in Myb s transforming activity are not known. Together with data showing increased c-myb expression in colonic tumours, these observations raise the possibility that c-myb may play a role in human malignant disease.

Oncoprotein v-Myb and retinoic acid receptor alpha are mutual antagonists.

The process of hematopoiesis is critically dependent on correct interactions of multiple regulatory molecules and transcription factors. We have studied the interactions of the v-Myb and retinoic acid receptor proteins which have opposing effects on hematopoiesis. While v-myb acts as a transforming oncogene preventing differentiation of monoblasts to macrophages, RAR alpha functions as an anti-oncogene arresting the growth of v-myb-transformed cells and allowing their final myeloid differentiation steps to occur. We found that the retinoic acid receptor alpha inhibits v-Myb transformation by suppressing the expression of v-Myb target genes typified by the mim-1 gene. Conversely, v-Myb protein interferes with RAR alpha transactivation function as well as with retinoic acid-induced apoptosis of HL-60 cells. These results demonstrate that retinoic acid receptor and v-Myb proteins act in antagonistic ways and reciprocally modify each other s functions.

The human T-cell leukemia virus type 1 oncoprotein Tax inhibits the transcriptional activity of c-Myb through competition for the CREB binding protein.

Tax, the transforming protein of human T-cell leukemia virus type 1 (HTLV-1), is required for strong activation of HTLV-1 transcription. This activation is mediated through interaction with the KIX domain of the cellular coactivator CREB binding protein (CBP). In this study we examined the possibility that the Tax-KIX interaction may mediate effects on cellular gene transcription in vivo, as a growing number of cellular transcription factors have been shown to utilize CBP as a coactivator. We tested the ability of Tax to deregulate the activity of the cellular transcription factor, c-Myb, since both Tax and c-Myb interact with the KIX domain of CBP. Our results show that in vivo, Tax antagonizes the transcriptional activity of c-Myb and, reciprocally, c-Myb antagonizes the transcriptional activity of Tax. Furthermore, c-Myb competes for KIX binding to Tax in vitro, indicating that these two transcription factors bind CBP in a mutually exclusive manner. This novel mechanism of transcriptional interference by Tax may promote globally deregulated cellular gene expression in the HTLV-1-infected cell, possibly leading to leukemogenesis.

Posttranscriptional regulation of the c-myb proto-oncogene in estrogen receptor-positive breast cancer cells.

We have determined that expression of the c-myb proto-oncogene is associated with estrogen receptor (ER) status and not with tumor progression in human breast epithelial cells. Analysis of normal, immortalized, nontumorigenic, and tumorigenic mammary epithelial cells showed that only ER+ tumor cell lines expressed readily detectable levels of c-myb mRNA and a Mr 75,000 protein that was the same size as the c-myb transcripts and protein products present in hematopoietic cells. In this report we show that c-myb mRNA and protein levels are down-regulated during estrogen withdrawal. A 20-fold increase in c-myb mRNA and protein expression was observed upon addition of beta-estradiol to the culture medium. Nuclear run-on transcription analyses showed that c-myb was transcribed at the same rate in the presence and absence of estrogen, suggesting that c-myb mRNA accumulation was regulated at a posttranscriptional level. To provide additional evidence that c-myb mRNA was dependent on ER expression, we examined c-myb mRNA levels in MCF-7 cells selected for resistance to antineoplastic drugs. c-myb expression was decreased only in cell lines that showed concomitant loss of ER expression. Moreover, c-myb mRNA was expressed and modulated by estrogen in ER-, MDA-MB-231 cells stably transfected with a human ER gene. When considered together, these data indicate that c-myb mRNA levels are regulated by estrogens and further suggest that this proto-oncogene plays a role in the biology of ER+ breast tumor cells.