Inhibition of proteasome-dependent degradation of Wee1 in G2-arrested Hep3B cells by TGFβ1

Transforming growth factor beta1 (TGFbeta1)-induced G(2) arrest was observed when a proliferation inhibitory function of the retinoblastoma protein (Rb) was compromised, but the mechanism underlying the G(2) arrest was poorly characterized compared with that of G(1) arrest. In the present study, we characterized G(2) arrest induced by TGFbeta1 (1 ng/mL) in the Rb-negative hepatoma cell line (Hep3B) and compared with G, arrest in the Rb-positive hepatoma cell line (Huh7). Activities of cyclin-dependent kinases (CDK) 2 and cell division cycle (CDC) 2 were markedly decreased at 24 h, the time when cell-cycle arrest became apparent in both cell lines. However, considerable amounts of inactive CDC2-cyclinB1 complexes were present in the nucleus of G(2)-arrested Hep3B but were not present in G(1)-arrested Huh7. The inhibitory phosphorylation of CDC2 on Tyr-15 was significantly elevated at 12-24 h, and its levels gradually declined during G(2) arrest in Hep3B. In particular, augmentation of CDK inhibitors p21(cip1) and p27(kip1) and Weel kinase and diminution of CDC25C phosphatase coincided with induced Tyr-15 phosphorylation and inhibition of CDC2. Weel in Hep3B was unstable and was degraded in a proteasome-dependent manner, but it became substantially stabilized within 6 h of TGFbeta1 treatment. Moreover, a Weel inhibitor, PD0166285, abrogated the TGFbeta1-induced G(2) arrest in Hep3B. These findings suggest that TGFbeta1 induced G(2) arrest in Hep3B at least in part through stabilization of Weel and subsequent increase in Tyr-15 phosphorylation and inhibition of CDC2. (C) 2003 Wiley-Liss, Inc.