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

Abstract

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.

ACKNOWLEDGMENTS

We are grateful to Youngsup Byun, who contributed to this project during undergraduate studies at Harvard University, and to Ed Harlow, in whose laboratory this project was initiated. We thank Jim Koh for antibodies and Liang Zhu for sharing data prior to publication.

W.S.E.-D. is an Assistant Investigator of the Howard Hughes Medical Institute. This work was supported in part by the following grants to G.H.E.: a pilot project grant from the University of Pennsylvania Comprehensive Cancer Center, an American Cancer Society institutional research grant administered through this Center, and grant 7-K08-CA61412 from the National Cancer Institute (NCI). We also acknowledge use of facilities of the Penn Digestive Disease Center, supported by National Institutes of Health grant P30 DK50306, and the Comprehensive Cancer Center, supported by grants from the NCI and the Markey Charitable Trust.