Induction of p21(WAF1/CIP1) and inhibition of Cdk2 mediated by the tumor suppressor p16(INK4a)

The tumor suppressor p16(INK4 alpha) inhibits cyclin-dependent kinases 4 an d 6. This activates the retinoblastoma protein (pRB) and, through incomplet ely understood events, arrests the cell division cycle. To permit biochemic al analysis of the arrest, we generated U2-OS osteogenic sarcoma cell clone s 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(WAF 1/CIP1). Concomitantly, the total cellular level of p21 increased severalfo ld via a posttranscriptional mechanism. Most cyclin E-cdk2 complexes associ ated with p21 and became inactive, expression of cyclin A was curtailed, an d DNA synthesis was strongly inhibited. Induction of p21 alone, in a siblin g clone, to the level observed during p16 induction substantially reproduce d these effects. Overexpression of either cyclin E or A prevented p16 from mediating arrest. We then extended these studies to HCT 116 colorectal carc inoma cells and a p21-null clone derived by homologous recombination. In th e parental cells, p16 expression also augmented total cellular and cdk2-bou nd p21. Moreover, p16 strongly inhibited DNA synthesis in the parental cell s but not in the p21-null derivative. These findings indicate that p21-medi ated 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)/p 53 tumor suppressor pathways.