Kang S H, Bang Y J, Jong H S, Seo J Y, Kim N K, Kim S J
Cancer Research Center, Seoul National University College of Medicine, Korea.
Br J Cancer. 1999 Jun;80(8):1144-9. doi: 10.1038/sj.bjc.6690478.
Transforming growth factor-beta (TGF-beta) is a multifunctional polypeptide that inhibits cellular proliferation in most epithelial cells. cdk4 and several cyclin-dependent kinase (cdk) inhibitors (p15INK4B, p21WAF1/Cip1 and p27Kip1) have been implicated in the TGF-beta-induced cell cycle arrest. More recently, down-regulation of Cdc25A, a cdk activator, was additionally suggested as a mechanism underlying growth inhibition by TGF-beta. The existence of diverse cellular mediators of TGF-beta, however, raises the question of whether their involvement might occur in a redundant manner or coordinately in a certain cell type. Using two TGF-beta-sensitive gastric carcinoma cell lines (SNU-16 and -620), we addressed the contributory roles of several cdk inhibitors, and of cdk4 and Cdc25A, in TGF-beta-induced cell cycle arrest by comparing their temporal expression pattern in response to TGF-beta. Among the cdk inhibitors examined, p21 mRNA was most rapidly (in less than 1 h) and prominently induced by TGF-beta. In contrast, p15 mRNA was more slowly induced than p21 in SNU-620 cells, and not expressed in SNU-16 cells harbouring homozygous deletion of p15. Western blotting results confirmed the rapid increase of p21, while opposite patterns of p27 expression were observed in the two cell lines. The down-regulation of Cdc25A mRNA occurred, but was more delayed than that of p15 or p21. Until G1 arrest was established, changes in the protein levels of both Cdc25A and cdk4 were marginal. Co-immunoprecipitation with anti-cdk4 antibody showed that induced p21 associates with cdk4 and that its kinase activity is reduced by TGF-beta, which kinetically correlates closely with G1 arrest following TGF-beta treatment of both cell lines. These results suggest that in certain human epithelial cells, p21 may play an early role in TGF-beta-induced cell cycle arrest, and its cooperation with other cdk inhibitors is different depending on cell type. Delayed down-regulation of Cdc25A and cdk4 may contribute to cell adaptation to the quiescent state in the two gastric carcinoma cell lines studied.
转化生长因子-β（TGF-β）是一种多功能多肽，可抑制大多数上皮细胞的细胞增殖。细胞周期蛋白依赖性激酶4（cdk4）和几种细胞周期蛋白依赖性激酶（cdk）抑制剂（p15INK4B、p21WAF1/Cip1和p27Kip1）与TGF-β诱导的细胞周期停滞有关。最近，细胞周期蛋白依赖性激酶激活剂Cdc25A的下调也被认为是TGF-β抑制生长的一种机制。然而，TGF-β存在多种细胞介质，这就提出了一个问题，即它们的参与是可能以冗余的方式发生，还是在某种细胞类型中协同发生。我们使用两种对TGF-β敏感的胃癌细胞系（SNU-16和-620），通过比较它们对TGF-β反应的时间表达模式，研究了几种cdk抑制剂以及cdk4和Cdc25A在TGF-β诱导的细胞周期停滞中的作用。在所检测的cdk抑制剂中，p21 mRNA是最迅速（在1小时内）且显著地被TGF-β诱导。相比之下，在SNU-620细胞中，p15 mRNA的诱导比p21慢，并且在携带p15纯合缺失的SNU-16细胞中不表达。蛋白质印迹结果证实了p21的快速增加，而在这两种细胞系中观察到p27表达的相反模式。Cdc25A mRNA发生了下调，但比p15或p21的下调更延迟。在建立G1期停滞之前，Cdc25A和cdk4的蛋白质水平变化很小。用抗cdk4抗体进行的免疫共沉淀显示，诱导产生的p21与cdk4结合，并且其激酶活性被TGF-β降低，这在动力学上与用TGF-β处理这两种细胞系后的G1期停滞密切相关。这些结果表明，在某些人类上皮细胞中，p21可能在TGF-β诱导的细胞周期停滞中起早期作用，并且其与其他cdk抑制剂的协同作用因细胞类型而异。Cdc25A和cdk4的延迟下调可能有助于所研究的两种胃癌细胞系适应静止状态。
