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 G2/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 p21. 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 p21 are necessary for genistein‐induced G2/M arrest in TRAMP‐C2 cells and their inhibition partially restores proliferation of TRAMP‐C2 cells in the presence of genistein. Prostate 66: 1542–1555, 2006. © 2006 Wiley‐Liss, Inc.