In vertebrate cells, the c-myb proto-oncogene product, Myb, plays a critical role in regulating transition through the G1/S interface of the cell division cycle. C-myb family transcription factors are found throughout the phyla, and recent studies have demonstrated that Drosophila myb (Dm-myb), as well as plant and yeast c-myb-like transcription factors, also play an important role in cell cycle passage. In apparent contrast to vertebrates however, these proteins seem to exert an effect on transit through the G2/M phase of cell cycle, as demonstrated by studies using the Drosophila eye imaginal disc model. The mechanism was thought to be Dm-myb’s ability to upregulate cyclin B expression as a result of binding to its promoter. Since we are unaware of studies that directly address the role of c-myb in regulating the G2/M transition in human cells, we addressed this possibility using phytohemagglutinin (PHA) stimulated human T- lymphocytes. We began by quantitating c-myb expression in human T cells by western and northern blotting, pre and post PHA stimulation. c-Myb expression began to gradually increase in the G1 phase of cell cycle, but unexpectedly, continued to increase after the peak of [3H] TdR incorporation, with the maximal protein level being found in G2/M phase, and concordant with cyclin B1 expression. Next, we engineered K562 cells to express a 4-hydroxy-tamoxifen (4-OHT) responsive, dominant negative, engrailed Myb (DN-Myb) (K562-MERT). After 72 hrs, 4-OHT treated K562-MERT cell proliferation was considerably slowed compared to untreated control cells (∼4x). Expression of cyclin A, B1, D1/2, and E, and cdk1/cdc2 was also examined in these cells. At 24-48 hrs post 4-OHT induction of DN-Myb, cyclin B1 expression clearly decreased (>8.0 times vs. control). In contrast, expression of cyclin A, D1/2, and E, and cdk1/cdc2 did not change. In Jurkat cells, a PI3-kinase inhibitor concordantly down-regulated c-Myb and cyclin B1. To determine if human c-Myb directly regulated cyclin B1 expression, we did CAT assay which showed that c-Myb protein overexpression increased cyclin B1 promoter activity ∼5X compared to K562 control cells. Examination of the cyclin B1 promoter showed eight potential c-Myb binding sites. Two were canonical (5’-pyrimidine AACG/TG-3’) and located upstream of 3 others which were [5’-AACNG-3’] in type. By use of simple deletion constructs, it was found that only the canonical sites bound c-Myb. Anti-c-Myb antibody specifically enriched cyclin B1 promoter DNA sequences in Chromatin immunoprecipitation assay, which strongly suggested that Myb bound cyclin B1 promoter in vivo. We conclude that in addition to its role in regulating G1/S cell cycle progression, mammalian Myb also plays a previously unappreciated role in the transition through G2/M.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]