Induction of p21(WAF1/CIP1) and Inhibition of Cdk2 Mediated by the Tumor Suppressor p16(INK4a)

The tumor suppressor p16(INK4a) inhibits cyclin-dependent kinases 4 and 6. This activates the retinoblastoma protein (pRB) and, through incompletely understood events, arrests the cell division cycle. To permit biochemical analysis of the arrest, we generated U2-OS osteogenic sarcoma cell clones in which p16 transcription could be induced. In these clones, binding of p16 to cdk4 and cdk6 abrogated binding of cyclin D1, p27(KIP1), and p21(WAF1/CIP1). Concomitantly, the total cellular level of p21 increased severalfold via a posttranscriptional mechanism. Most cyclin E-cdk2 complexes associated with p21 and became inactive, expression of cyclin A was curtailed, and DNA synthesis was strongly inhibited. Induction of p21 alone, in a sibling clone, to the level observed during p16 induction substantially reproduced these effects. Overexpression of either cyclin E or A prevented p16 from mediating arrest. We then extended these studies to HCT 116 colorectal carcinoma cells and a p21-null clone derived by homologous recombination. In the parental cells, p16 expression also augmented total cellular and cdk2-bound p21. Moreover, p16 strongly inhibited DNA synthesis in the parental cells but not in the p21-null derivative. These findings indicate that p21-mediated inhibition of cdk2 contributes to the cell cycle arrest imposed by p16 and is a potential point of cooperation between the p16/pRB and p14(ARF)/p53 tumor suppressor pathways.

抑癌蛋白p16(INK4a)可抑制细胞周期蛋白依赖性激酶4和6（cyclin-dependent kinases 4 and 6），进而激活视网膜母细胞瘤蛋白（retinoblastoma protein, pRB），并通过尚未完全阐明的分子事件阻断细胞周期进程。为对该阻断效应开展生化分析，我们构建了可诱导p16转录的U2-OS骨肉瘤细胞克隆株。在该克隆株中，p16与细胞周期蛋白依赖性激酶4（cyclin-dependent kinase 4, CDK4）、细胞周期蛋白依赖性激酶6（cyclin-dependent kinase 6, CDK6）的结合会阻断其与细胞周期蛋白D1（cyclin D1）、p27(KIP1)及p21(WAF1/CIP1)的结合；与此同时，p21的总细胞水平通过转录后机制（posttranscriptional mechanism）提升数倍。多数与p21结合的细胞周期蛋白E（cyclin E）-细胞周期蛋白依赖性激酶2（cyclin-dependent kinase 2, CDK2）复合物会发生失活，细胞周期蛋白A（cyclin A）的表达受到抑制，DNA合成也被显著阻断。在同胞克隆株（sibling clone）中单独诱导p21至p16诱导时的表达水平，可基本重现上述效应。过表达细胞周期蛋白E或细胞周期蛋白A均可阻断p16介导的细胞周期阻断效应。随后我们将该研究拓展至HCT 116结肠癌细胞（colorectal carcinoma cells），以及通过同源重组（homologous recombination）构建的p21基因敲除克隆株。在亲本细胞（parental cells）中，p16的表达同样会提升细胞内总p21以及与CDK2结合的p21的水平。此外，p16可显著抑制亲本细胞的DNA合成，但对p21基因敲除衍生细胞株无此效应。上述结果表明，p21介导的CDK2抑制参与了p16引发的细胞周期阻断效应，同时也是p16/pRB与p14(ARF)/p53抑癌通路之间潜在的协同作用位点。