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A-myb is a member of the myb family of transcription factors, which regulates proliferation, differentiation, and apoptosis of hematopoietic cells. A-Myb expression is normally restricted to the proliferating B-cell centroblasts and transgenic mice overexpressing A-myb displayed enhanced hyperplasia of the lymph nodes. Because A-Myb is highly expressed in several subtypes of human B-cell neoplasias, we sought to determine whether the A-myb gene promoted proliferation and survival of B lymphocytes, using the WEHI 231 and CH33 murine B-cell lymphomas as models. Here, we show that ectopic expression of A-mybrescues WEHI 231 and CH33 cells from growth arrest and apoptosis induced by anti-IgM treatment. Previously, we demonstrated an essential role of the c-myc gene in promoting cell survival of WEHI 231 cells in response to a variety of apoptotic stimuli. Furthermore, we and others have shown that the c-myc gene is potently transactivated by A-Myb in several cell types. Thus, we sought to determine whether c-Myc would mediate the A-Myb antiapoptotic effect in B cells. Here we show that ectopic expression of A-myb leads to maintenance of c-myc expression, and that expression of antisense c-myc RNA ablates A-Myb–mediated survival signals. Thus, these findings strongly implicate the A-myb gene in the regulation of B-cell survival and confirm the c-myc gene as one of the downstream targets of A-myb in these cells. Overall, our observation suggests that A-mybexpression may be relevant to the pathology of human B-cell neoplasias.

ABSTRACTNF-?B/Rel factors have been implicated in the regulation of liver cell death during development, after partial hepatectomy, and in hepatocytes in culture. Rat liver epithelial cells (RLEs) display many biochemical and ultrastructural characteristics of oval cells, which are multipotent cells that can differentiate into mature hepatocytes. While untransformed RLEs undergo growth arrest and apoptosis in response to transforming growth factor ß1 (TGF-ß1) treatment, oncogenic Ras- or Raf-transformed RLEs are insensitive to TGF-ß1-mediated growth arrest. Here we have tested the hypothesis that Ras- or Raf-transformed RLEs have altered NF-?B regulation, leading to this resistance to TGF-ß1. We show that classical NF-?B is aberrantly activated in Ras- or Raf-transformed RLEs, due to increased phosphorylation and degradation of I?B-a protein. Inhibition of NF-?B activity with a dominant negative form of I?B-a restored TGF-ß1-mediated cell killing of transformed RLEs. IKK activity mediates this hyperphosphorylation of I?B-a protein. As judged by kinase assays and transfection of dominant negative IKK-1 and IKK-2 expression vectors, NF-?B activation by Ras appeared to be mediated by both IKK-1 and IKK-2, while Raf-induced NF-?B activation was mediated by IKK-2. NF-?B activation in the Ras-transformed cells was mediated by both the Raf and phosphatidylinositol 3-kinase pathways, while in the Raf-transformed cells, NF-?B induction was mediated by the mitogen-activated protein kinase cascade. Last, inhibition of either IKK-1 or IKK-2 reduced focus-forming activity in Ras-transformed RLEs. Overall, these studies elucidate a mechanism that contributes to the process of transformation of liver cells by oncogene Ras and Raf through the I?B kinase complex leading to constitutive activation of NF-?B.

NF-κB/Rel factors have been implicated in the regulation of liver cell death during development, after partial hepatectomy, and in hepatocytes in culture. Rat liver epithelial cells (RLEs) display many biochemical and ultrastructural characteristics of oval cells, which are multipotent cells that can differentiate into mature hepatocytes. While untransformed RLEs undergo growth arrest and apoptosis in response to transforming growth factor β1 (TGF-β1) treatment, oncogenic Ras- or Raf-transformed RLEs are insensitive to TGF-β1-mediated growth arrest. Here we have tested the hypothesis that Ras- or Raf-transformed RLEs have altered NF-κB regulation, leading to this resistance to TGF-β1. We show that classical NF-κB is aberrantly activated in Ras- or Raf-transformed RLEs, due to increased phosphorylation and degradation of IκB-α protein. Inhibition of NF-κB activity with a dominant negative form of IκB-α restored TGF-β1-mediated cell killing of transformed RLEs. IKK activity mediates this hyperphosphorylation of IκB-α protein. As judged by kinase assays and transfection of dominant negative IKK-1 and IKK-2 expression vectors, NF-κB activation by Ras appeared to be mediated by both IKK-1 and IKK-2, while Raf-induced NF-κB activation was mediated by IKK-2. NF-κB activation in the Ras-transformed cells was mediated by both the Raf and phosphatidylinositol 3-kinase pathways, while in the Raf-transformed cells, NF-κB induction was mediated by the mitogen-activated protein kinase cascade. Last, inhibition of either IKK-1 or IKK-2 reduced focus-forming activity in Ras-transformed RLEs. Overall, these studies elucidate a mechanism that contributes to the process of transformation of liver cells by oncogene Ras and Raf through the IκB kinase complex leading to constitutive activation of NF-κB.

Expression studies as well as the use of transgenic animals have demonstrated that the A-MYB transcription factor plays central and specific role in the regulation of mature B cell proliferation and/or differentiation. Furthermore, it is highly expressed in Burkitt's lymphoma cells and may participate in the pathogenesis of this disease. We have therefore investigated the transcriptional activity of A-MYB and its regulation in several human lymphoid cell lines using co-transfection assays and show that A-MYB is transcriptionally active in all the B cell lines studied, but not in T cells. In particular the best responder cell line was the Burkitt's cell line Namalwa. The activity of A-MYB in B and not T cells was observed when either an artificial construct or the c-MYCpromoter was used as a reporter. Furthermore, the functional domains responsible for DNA binding, transactivation, and negative regulation, previously characterized in a fibroblast context, were found to have similar activity in B cells. The region of A-MYB responsible for the B cell specific activity was defined to be the N-terminal 218 amino acids containing the DNA binding domain. Finally, a 110-kDa protein has been identified in the nuclei of all the B, but not T, cell lines that specifically binds to this A-MYB N-terminal domain. We hypothesize that this 110-kDa protein may be a functionally important B cell-specific co-activator of A-MYB.

Direct and indirect evidence strongly indicates that the proto-oncogene c-mybplays an important role in the regulation of both the proliferation and differentiation of hematopoietic cells. In addition, recent data suggest that the structurally related B-mybgene is also necessary for the proliferation of these cells. To help understand the relationship between these two related gene products during proliferation and differentiation of myeloid cells, we have studied in parallel the regulated expression of c-myband B-myb RNAs and proteins in human myeloid cells that were either growth-arrested or induced to differentiate along different pathways. For this purpose, we have produced a polyclonal antibody directed against a fragment of the recombinant B-mybprotein. We have thus been able to detect the B-mybprotein in human cell lines and have found it to be a 93-kD protein localized in the nucleus. We have chosen two models to study the expression of both c-myband B-mybmRIMAs and proteins during myeloid proliferation and differentiation. One of the models was the HL-60 cell line, which can be induced to differentiate towards the monocytic pathway with either phorbol ester (phorbol myristate acetate) or vitamin D3 and towards the granulocytic pathway with either dimethyl sulfoxide or retinoic acid. In addition, we have studied another recently established human leukemic cell line, called GF-D8, which is strictly dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF) for proliferation. The results show that the expression of B-mybRNA and protein closely correlates with proliferation in all experimental setups studied, whereas the c-mybprotein levels do not always do so. We observed that the c-mybprotein levels decreased well before the decrease of B-mybprotein and of proliferation itself during differentiation toward monocytes. Such a difference was not present during granulocytic differentiation, in which c-myblevels decreased, if anything, later than those of B-myb and proliferation. Most striking was the finding that high levels of c-mybRNA and protein, but not of B-myb, were present in the GF-D8 cell line, even after growth arrest by GM-CSF deprivation. These data suggest that B-mybmay function solely in the regulation of cellular proliferation, whereas c-mybhas additional functions, for example, in the maintenance of an undifferentiated state.

The Myb family of transcription factors is defined by homology within the DNA binding domain and includes c-Myb, A-Myb, and B-Myb. The protein products of the mybgenes all bind the Myb-binding site (MBS) [YG(A/G)C(A/C/G)GTT(G/A)]. A-mybhas been found to display a limited pattern of expression. Here we report that bovine aortic smooth muscle cells (SMCs) express A-myb. Sequence analysis of isolated bovine A-myb cDNA clones spanning the entire coding region indicated extensive homology with the human gene, including the putative transactivation domain. Expression of A-mybwas cell cycle dependent; levels of A-mybRNA increased in the late G1-to-S phase transition following serum stimulation of serum-deprived quiescent SMC cultures and peaked in S phase. Nuclear run-on analysis revealed that an increased rate of transcription can account for most of the increase in A-mybRNA levels. Treatment of SMC cultures with 5,6-dichlorobenzimidazole riboside, a selective inhibitor of RNA polymerase II, indicated an approximate 4-h half-life for A-mybmRNA during the S phase of the cell cycle. Expression of A-mybby SMCs was stimulated by basic fibroblast growth factor, in a cell density-dependent fashion. Cotransfection of a human A-mybexpression vector activated a multimerized MBS element-driven reporter construct approximately 30-fold in SMCs. The activity of c-myband c-mycpromoters, which both contain multiple MBS elements, were similarly transactivated, approximately 30- and 50-fold, respectively, upon cotransfection with human A-myb. Lastly, A-mybRNA levels could be increased by a combination of phorbol ester plus insulin-like growth factor 1. To test the role of mybfamily members in progression through the cell cycle, we comicroinjected c-mycand mybexpression vectors into serum-deprived quiescent SMCs. The combination of c-mycand either A-mybor c-mybbut not B-mybsynergistically led to entry into S phase, whereas microinjection of any vector alone had little effect on S phase entry. Thus, these results suggest that A-mybis a potent transactivator in bovine SMCs and that its expression induces progression into S phase of the cell cycle.