Identification of both Myt-1 and Wee-1 as necessary mediators of the p21-independent inactivation of the Cdc-2/cyclin B1 complex and growth inhibition of TRAMP cancer cells by genistein

BACKGROUND. The G(2)/M cell-cycle arrest is one mechanism by which genistein exerts its anti-proliferative effects, and the proposed underlying causes encompass the transcriptional repression of cyclin B1 and the activation of p21. However, the involvement of upstream kinases Myt-1 and Wee-1 in this arrest remains to be elucidated. METHODS. Myt-1 and Wee-1 modulation by genistein was examined via Western blot analysis and the effect of their inhibition by siRNA on cyclin B1 levels /localization, cdc2 kinase activity, and cellular proliferation of genistein-treated TRAMP-C2 cells was determined. RESULTS. The sustained G2/M arrest by genistein in TRAMP-C2 cells is associated with increased phospho-cdc2(Tyr15), decreased cdc2 protein, and cytoplasmic retention of cyclinB1, resulting in decreased cdc2 kinase activity independently of p2l. Genistein treatment increased Myt-1 levels and decreased Wee-1 phosphorylation. Downregulation of Myt-1 and Wee-1 by siRNA restored cdc2 levels, its kinase activity, cyclinB1 nuclear localization, and partially restored cell proliferation of genistein-treated cells. CONCLUSIONS. Myt-1 and Wee-1 rather than p2l are necessary for genistein-induced G(2)/M arrest in TRAMP-C2 cells and their inhibition partially restores proliferation of TRAMP-C2 cells in the presence of genistein.