WAF1 RETARDS S-PHASE PROGRESSION PRIMARILY BY INHIBITION OF CYCLIN-DEPENDENT KINASES

The p(2WAF1/CIP1/sdi1) gene product (WAF1) inhibits DNA replication in vitro (J. Chen, P. Jackson, M. Kirschner, and A. Dutta, Nature 374:38 6-388, 1995; S. Waga, G. Hannon, D. Beach, and B. Stillman, Nature 369 :574-578, 1994), but in vivo studies on the antiproliferative activity of WAF1 have not resolved G(1)-phase arrest from potential inhibition of S-phase progression. Here, we demonstrate that elevated WAF1 expre ssion can retard replicative DNA synthesis in vivo. The WAF1-mediated inhibitory effect could be antagonized by cyclin A, cyclin E, or the s imian virus 40 small-t antigen with no decrease in the levels of WAF1 protein in transfected cells, Proliferating-cell nuclear antigen (PCNA ) overexpression was neither necessary nor sufficient to antagonize WA F1 action, Expression of the N-terminal domain of WAF1, responsible fo r cyclin-dependent kinase (CDK) interaction, had the same effect as fu ll-length WAF1, while the PCNA binding C terminus exhibited modest act ivity. We conclude that S-phase progression in mammalian cells is depe ndent on continuing cyclin and CDK activity and that WAF1 affects S ph ase primarily through cyclin- and CDK-dependent pathways.