Inhibition of proteasome-dependent degradation of Wee1 in G2-arrested Hep3B cells by TGF beta 1.
Transforming growth factor beta1 (TGF beta 1)-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 TGF beta1 (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 TGF beta 1 treatment. Moreover, a Wee1 inhibitor, PD0166285, abrogated the TGF beta 1-induced G2 arrest in Hep3B. These findings suggest that TGF beta 1 induced G2 arrest in Hep3B at least in part through stabilization of Wee1 and subsequent increase in Tyr-15 phosphorylation and inhibition of CDC2.
Hashimoto
Osamu
O
Liver Cancer Research Division, Research Center for Innovative Cancer Therapy, Kurume University School of Medicine, Kurume, Japan.
Ueno
Takato
T
Kimura
Rina
R
Ohtsubo
Motoaki
M
Nakamura
Toru
T
Koga
Hironori
H
Torimura
Takuji
T
Uchida
Sanae
S
Yamashita
Katsumi
K
Sata
Michio
M
D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects; D003546:Cysteine Endopeptidases; D017931:DNA Primers; D016195:G2 Phase / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors*; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D014407:Tumor Cells, Cultured
D018797:Cell Cycle Proteins; D017931:DNA Primers; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex