B-MYB is a ubiquitously expressed transcription factor involved in the regulation of cell survival, proliferation, and differentiation. In an attempt to isolate B-MYB-regulated genes that may explain the role of B-MYB in cellular processes, representational difference analysis was performed in neuroblastoma cell lines with different levels of B-MYB expression. One of the genes, the mRNA levels of which were enhanced in B-MYB expressing cells, was ApoJ/Clusterin(SGP-2/TRMP-2) (ApoJ/Clusterin), previously implicated in regulation of apoptosis and tumor progression. Here we show that the human ApoJ/Clusterin gene contains a Myb binding site in its 5' flanking region, which interacts with bacterially synthesized B-MYB protein and mediates B-MYB-dependent transactivation of the ApoJ/Clusterin promoter in transient transfection assays. Endogenous ApoJ/Clusterin expression is induced in mammalian cell lines following transient transfection of a B-MYB cDNA. Blockage of secreted clusterin by a monoclonal antibody results in increased apoptosis of neuroblastoma cells exposed to the chemotherapeutic drug doxorubicin. Thus, activation of ApoJ/Clusterin by B-MYB may be an important step in the regulation of apoptosis in normal and diseased cells.

Since its isolation exactly a decade ago, B-Myb has intrigued a growing number of scientists interested in understanding the mechanisms of cell proliferation. In many aspects the B-Myb story resembles that of a fashionable transcription factor involved in cell cycle control: E2F-1. Similar to E2F-1, B-Myb is a transcription factor whose expression is regulated at the G1/S border of the cell cycle. Given the ubiquitous expression of B-Myb within different cell types, its link with the cell cycle, and augmented expression in transformed cells, studies are in progress to define the potential role of B-Myb in human cancer. The purpose of this review is not to provide an extensive background to the B-Myb field but rather to describe the latest developments. A comprehensive outline of B-Myb structure and function can be found in the review by Saville and Watson (1998a, Adv. Cancer Res., 72:109-140).

B-MYB expression is associated with cell proliferation and recent studies have suggested that it promotes the S phase of mammalian cells. Based on its homology to the transcription factors c-MYB and A-MYB, B-MYB is thought to be involved in transcriptional regulation; however, its activity is not detectable in several cell lines. It was postulated that B-MYB function may depend on the presence of a cofactor, and recent studies suggested that B-MYB is phosphorylated specifically during S phase in murine fibroblasts. In this report we provide evidence that the product of the human B-myb gene can be activated in vivo by coexpression with cyclin A or cyclin E. Transfection studies showed that B-MYB was a weak transcriptional activator in SAOS-2 cells and was unable to promote their proliferation. In contrast, overexpression of both B-MYB and cyclin A or cyclin E caused a drastic increase in the number of SAOS-2 cells in S phase. Also, overexpression of cyclin A and cyclin E in SAOS-2 cells enhanced the ability of B-MYB, but not c-MYB, to transactivate various promoters, including the cdc2 promoter, the HIV-1-LTR, and the simian virus 40 minimal promoter. A direct role for cyclin-dependent activation of B-MYB was demonstrated using an in vitro transcription assay. These observations suggest that one mechanism by which cyclin A and E may promote the S phase is through modification and activation of B-MYB.

The retinoblastoma protein family has been implicated in growth control and modulation of the activity of genes involved in cell proliferation, such as B-myb. Recent evidence indicates that the product of the B-myb gene is necessary for the growth and survival of several human and murine cell lines. Upon overexpression, B-myb induces deregulated cell growth of certain cell lines. Here we show that B-myb overexpression is able to induce DNA synthesis in p107 growth-arrested human osteosarcoma cells (SAOS2). p107 might exert its growth-suppressive activity by regulating B-myb gene transcription. Indeed, p107 down-modulated B-myb promoter activity and drastically decreased E2F-mediated transactivation. Finally, B-myb was able to stimulate DNA synthesis of both stably and transiently transfected human glioblastoma cells (T98G). Altogether, these data provide definitive evidence that the human B-myb protein is involved in growth control of human cells, and that p107 has a significant role in regulating B-myb gene activity.

It was recently reported that B-MYB can overcome p107-induced growth arrest. Here we show that B-MYB autoregulation of its own transcription is specifically suppressed by p107 and transient transfection assays with p107 deletion constructs determined that the carboxyl terminus of the protein, containing the major pocket region, was associated with inhibition of B-MYB-dependent transactivation. Consistent with these results, co-immunoprecipitation studies showed that p107 interacted in vivo with B-MYB through its pocket and carboxyl terminus domain. Thus, B-MYB-dependent promotion of cell proliferation and gene transactivation might be specifically repressed by the growth suppressor p107 through direct interaction with B-MYB.

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 encodes a nuclear protein involved in the regulation of cell proliferation, differentiation, and development. Myb protein contains a DNA binding and a transactivating domain thought to mediate its biologic properties. The DNA binding domain consists of three repeats (R1, R2, and R3), each containing a highly conserved motif of tryptophan residues. A c-myb mutant (DR1-myb) lacking the last 46 amino acids of R1 and 23 amino terminal residues of R2, a region homologous to the ADA-2 yeast transcriptional adaptor, lost DNA binding ability, but remained able to transactivate the human heat-shock promoter. Transfection of murine 32D and murine erythroleukemia (MEL) cell lines with DR1-myb caused inhibition of cellular differentiation induced by granulocyte colony-stimulating factor (G-CSF) and dimethyl sulfoxide (DMSO), respectively. A second c-myb mutant (D-ADA2-myb) lacking the first 23 amino acids of R2, also lost DNA binding and transactivation activity, but did not inhibit DMSO-induced differentiation of MEL transfected cells. These findings suggest that deletion of R1 activates a DNA binding-independent mechanism of c-myb function, which may involve interaction of Myb with cellular factors.

The B-myb gene belongs to a family of transcription factors that also includes A-myb and c-myb. B-myb is expressed in many cell types including human neuroblastoma cells. Here we demonstrate that B-myb expression is down-regulated during retinoic acid-induced neural and glial differentiation of neuroblastoma cells. This modulation is an early event, is maintained at late times of induction, and is in part regulated at the transcriptional level. Constitutive expression of B-myb prevents retinoic acid-induced neural differentiation as reflected by morphological features and the expression of (or lack of) biochemical markers associated with the undifferentiated phenotype. Furthermore, the expression of antisense B-myb transcripts does not allow the rescue of viable cells, suggesting an important role for B-myb in the survival of neuroblastoma cells. These results indicate that B-myb plays a functional role in the differentiative potential of neuroblastoma cells, raising the possibility that this gene is one of the nuclear regulators in the cascade of events leading to cellular differentiation.

Overexpression of wild-type p53 protein has been shown to induce arrest in the G1 stage of the cell cycle and to transactivate expression of the gene that encodes the 21-kDa Waf1/Cip1 protein, a potent inhibitor of cyclin-dependent kinase activity. p53-dependent G1 arrest is accompanied by decreased expression of the B-myb gene, a relative of the c-myb cellular oncogene. In this study we show that B-myb expression is required for cells to progress from G1 into S phase and that high levels of ectopic B-myb expression uncoupled from cell cycle regulation rescues cells from p53-induced G1 arrest even in the presence of Waf1/Cip1 transactivation and inhibition of cyclin E/Cdk2 kinase activity. Cotransfection experiments with p53 expression plasmids and expression plasmids encoding in-frame deletion mutations in B-myb coding sequences indicate that the DNA-binding domain of the B-Myb protein is required for this activity. These results provide evidence of a bypass of p53-induced Waf1/Cip1-mediated cell cycle regulatory pathways by a member of the myb oncogene family.

The mammalian nuclear protein E2F-1 has recently been cloned based on its ability to bind the retinoblastoma protein. To determine whether E2F-1 plays a role in the control of the cell proliferation, we introduced an inducible construct expressing an E2F-1 antisense RNA into the human glioblastoma T98G cell line and assessed DNA synthesis during the cell cycle. Expression of the antisense transcripts during the G1-S transition resulted in a marked delay in the completion of DNA synthesis. Band-shift analysis of bacterially produced E2F-1 showed that this protein bound to the promoters of human DNA polymerase-alpha, cyclin D1, and c-myb but not to the cdc2 gene promoter. E2F-1 also transactivated the bound promoters in transient transfection assays. These results suggest a major role for E2F-1 in the control of cell cycle progression via transcriptional regulation of proliferation-associated genes.

The B-myb gene is expressed in many cell types at the G1/S transition of the cell cycle. Inhibition of B-myb expression in BALB/c 3T3 fibroblasts by introduction of a B-myb antisense construct greatly diminished cell proliferation, whereas constitutive expression of a human B-myb cDNA in these cells reduced their growth factor requirements and induced a transformed phenotype. Constitutive expression of B-myb cDNA was accompanied by activation of cyclin D1 and cdc2 expression but not of cyclin A and cyclin B. Transfection of BALB/B-myb cells (a cell line expressing high levels of exogenous human B-myb) with a cyclin D1 antisense construct drastically reduced cloning efficiency of these cells. These results suggest that the B-myb-encoded product regulates fibroblast proliferation by activating cdc2 and cyclin D1 gene expression and that abnormal expression of cyclin D1 might be a step in the process of transformation.