Critical role of cyclin B1/Cdc2 up-regulation in the induction of mitotic prometaphase arrest in human breast cancer cells treated with 2-methoxyestradiol

Earlier studies showed that 2-methoxyestradiol (2ME2), an endogenous nonpolar metabolite of estradiol-17β, is a strong inducer of G2/M cell cycle arrest (based on analysis of cellular DNA content) in human cancer cell lines. The present study sought to investigate the molecular mechanism underlying 2ME2-induced cell cycle arrest. We found that 2ME2 can selectively induce mitotic prometaphase arrest, but not G2 phase arrest, in cultured MDA-MB-435s and MCF-7 human breast cancer cells. During the induction of prometaphase arrest, there is a time-dependent initial up-regulation of cyclin B1 and Cdc2 proteins, occurring around 12–24 h. The strong initial up-regulation of cyclin B1 and Cdc2 matches in timing the 2ME2-induced prometaphase arrest. The 2ME2-induced prometaphase arrest is abrogated by selective knockdown of cyclin B1 and Cdc2, or by pre-treatment of cells with roscovitine, an inhibitor of cyclin-dependent kinases, or by co-treatment of cells with cycloheximide, a protein synthesis inhibitor that was found to suppress the early up-regulation of cyclin B1 and Cdc2. In addition, we provided evidence showing that MAD2 and JNK1 are important upstream mediators of 2ME2-induced up-regulation of cyclin B1 and Cdc2 as well as the subsequent induction of mitotic prometaphase arrest. In conclusion, treatment of human cancer cells with 2ME2 causes up-regulation of cyclin B1 and Cdc2, which then mediate the induction of mitotic prometaphase arrest.

► 2ME2 induces prometaphase arrest, but not G2 arrest, in human breast cancer cells.
► Up-regulation of cyclin B1 and Cdc2 by 2ME2 mediates mitotic prometaphase arrest.
► MAD2 and JNK1 are key upstream mediators of 2ME2-induced cell cycle changes.