Hashimoto Osamu, Ueno Takato, Kimura Rina, Ohtsubo Motoaki, Nakamura Toru, Koga Hironori, Torimura Takuji, Uchida Sanae, Yamashita Katsumi, Sata Michio
Liver Cancer Research Division, Research Center for Innovative Cancer Therapy, Kurume University School of Medicine, Kurume, Japan.
Mol Carcinog. 2003 Apr;36(4):171-82. doi: 10.1002/mc.10111.
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.
当视网膜母细胞瘤蛋白（Rb）的增殖抑制功能受损时，观察到转化生长因子β1（TGFβ1）诱导的G2期阻滞，但与G1期阻滞相比，G2期阻滞的潜在机制尚未得到充分表征。在本研究中，我们对TGFβ1（1 ng/mL）在Rb阴性肝癌细胞系（Hep3B）中诱导的G2期阻滞进行了表征，并与Rb阳性肝癌细胞系（Huh7）中的G1期阻滞进行了比较。在24小时时，细胞周期蛋白依赖性激酶（CDK）2和细胞分裂周期（CDC）2的活性显著降低，此时两个细胞系中细胞周期阻滞均明显出现。然而，在G2期阻滞的Hep3B细胞核中存在大量无活性的CDC2-细胞周期蛋白B1复合物，而在G1期阻滞的Huh7中则不存在。CDC2在Tyr-15位点的抑制性磷酸化在12至24小时显著升高，其水平在Hep3B的G2期阻滞期间逐渐下降。特别是，CDK抑制剂p21cip1和p27kip1以及Wee1激酶的增加和CDC25C磷酸酶的减少与诱导的Tyr-15磷酸化和CDC2的抑制相一致。Hep3B中的Wee1不稳定，以蛋白酶体依赖性方式降解，但在TGFβ1处理6小时内其稳定性显著增强。此外，一种Wee1抑制剂PD0166285消除了TGFβ1诱导的Hep3B中的G2期阻滞。这些发现表明，TGFβ1至少部分通过稳定Wee1以及随后增加Tyr-15磷酸化和抑制CDC2来诱导Hep3B中的G2期阻滞。
