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

• Published in: Molecular carcinogenesis Vol. 36; no. 4; pp. 171 - 182
• Main Authors: Hashimoto, Osamu, Ueno, Takato, Kimura, Rina, Ohtsubo, Motoaki, Nakamura, Toru, Koga, Hironori, Torimura, Takuji, Uchida, Sanae, Yamashita, Katsumi, Sata, Michio
• Format: Journal Article
• Language: English
• Published: New York Wiley Subscription Services, Inc., A Wiley Company 01.04.2003
• Subjects: CDC2; cell cycle; cyclin; MG132; p21cip1; PD0166285
• ISSN: 0899-1987; 1098-2744
• DOI: 10.1002/mc.10111

Transforming growth factor β1 (TGFβ1)‐induced G2 arrest was observed when a proliferation inhibitory function of the retinoblastoma protein (Rb) was compromised, but the mechanism underlying the G2 arrest was poorly characterized compared with that of G1 arrest. In the present study, we characterized G2 arrest induced by TGFβ1 (1 ng/mL) in the Rb‐negative hepatoma cell line (Hep3B) and compared with G1 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 G2‐arrested Hep3B but were not present in G1‐arrested Huh7. The inhibitory phosphorylation of CDC2 on Tyr‐15 was significantly elevated at 12–24 h, and its levels gradually declined during G2 arrest in Hep3B. In particular, augmentation of CDK inhibitors p21cip1 and p27kip1 and Wee1 kinase and diminution of CDC25C phosphatase coincided with induced Tyr‐15 phosphorylation and inhibition of CDC2. Wee1 in Hep3B was unstable and was degraded in a proteasome‐dependent manner, but it became substantially stabilized within 6 h of TGFβ1 treatment. Moreover, a Wee1 inhibitor, PD0166285, abrogated the TGFβ1‐induced G2 arrest in Hep3B. These findings suggest that TGFβ1 induced G2 arrest in Hep3B at least in part through stabilization of Wee1 and subsequent increase in Tyr‐15 phosphorylation and inhibition of CDC2. © 2003 Wiley‐Liss, Inc.