Transforming growth factor β1 increases the stability of p21/WAF1/CIP1 protein and inhibits CDK2 kinase activity in human colon carcinoma FET cells

We examined transforming growth factor-β1 (TGF-β1) effects on cell cycle progression of human colon carcinoma FET cells. TGF-β1 inhibited DNA synthesis and cyclin-dependent kinase (CDK) activity after release from growth arrest in association with induction of the p21 CDK inhibitor, whereas cyclins, CDKs, and p27 protein levels remained relatively unchanged. The decrease in CDK2 kinase activity was the result of increased p21 association with cyclin A-CDK2 and cyclin E-CDK2. TGF-β1 treatment in late G₁ showed reduced induction of p21 protein levels in association with increased DNA synthesis. Consequently, p21 induction in early G₁ is critical for TGF-β1 inhibition of CDK2 kinase activity. Although TGF-β1 treatments in late G₁ failed to induce p21 protein, p21 mRNA induction was observed in late G₁ and in S phase. Further analysis showed that TGF-β1 treatment in early G₁ increases p21 protein stability throughout the G₁ and S phases of the cell cycle. Our results demonstrate that TGF-β1 stimulation of p21 is regulated at the posttranscriptional and transcriptional levels. This is a novel mechanism of TGF-β1 inhibition requiring early G₁ induction and stabilization of p21 protein, which binds to and inhibits cyclin E-CDK2 and cyclin A-CDK2 kinase activity rather than direct modulation of cyclin or CDK protein levels as seen in other systems.