Microinjection of anti-p21 antibodies induces senescent Hs68 human fibroblasts to synthesize DNA but not to divide

Replicative senescence is characterized by irreversible growth arrest and h as been defined by four genetic complementation groups. One of these groups is associated with the predominance of underphosphorylated growth-suppress ive retinoblastoma tumor suppressor protein (pRb), Although certain members of the cyclin-dependent kinase (cdk)/cyclin family, some of which phosphor ylate pRb, are underexpressed in senescent cells, others are expressed but inactive. This lack of cdk activity and arrest in the G(1) phase of the cel l cycle is likely attributable to the induction upon senescence of the G(1) -S cdk/cyclin inhibitors p21 (WAF1/CIP1/Sdi) and p16INK4, In fact, in early presenescent normal diploid fibroblasts in which p21 is inactivated, senes cence is bypassed or postponed. Moreover, in senescent cells in which p53 f unction was inhibited, DNA synthesis was reinitiated, an effect likely attr ibutable, in part, to the dependence of p21 expression on p53. We report he re that the apparent inactivation of p21 in senescent human fibroblasts thr ough the introduction of inhibitory alpha-p21 antibodies causes these cells to reenter the S-phase of the cell cycle. The disruption of p21 activity a ffects the p21-Rb-E2F pathway in that the expression of genes transcription ally regulated by E2F, such as cyclin A and cdc2 were found to be up-regula ted in injected cells. No evidence of cell division was observed. This sugg ests that p21 plays an important role in the maintenance of senescence and in the inhibition of S-phase progression, but inhibition of p21 activity is insufficient to permit cells to complete the cell cycle.