Rapid induction of p21(WAF1) but delayed down-regulation of Cdc25A in the TGF-beta-induced cell cycle arrest of gastric carcinoma cells

Transforming growth factor-beta (TGF-beta) is a multifunctional polypeptide that inhibits cellular proliferation in most epithelial cells. cdk4 and se veral cyclin-dependent kinase (cdk) inhibitors (p15(INK4B), p21(WAFI/Cip1) and p27(Kip1)) have been implicated in the TGF-beta-induced cell cycle arre st. More recently, down-regulation of Cdc25A, a cdk activator, was addition ally 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 c arcinoma 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 SN U-16 cells harbouring homozygous deletion of p15. Western blotting results confirmed the rapid increase of p21 while opposite patterns of p27 expressi on 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 ma rginal. Co-immunoprecipitation with anti-cdk4 antibody showed that induced p21 associates with cdk4, and that its kinase activity is reduced by TGF-be ta, which kinetically correlates closely with G1 arrest following TGF-beta treatment of both cell lines. These results suggest that in certain human e pithelial cells, p21 may play an early role in TGF-beta-induced cell cycle arrest, and its cooperation with other cdk inhibitors is different dependin g on cell type. Delayed down-regulation of Cdc25A and cdk4 may contribute t o cell adaptation to the quiescent state in the two gastric carcinoma cell lines studied.