p21(Waf1/Cip1/Sdi1) mediates retinoblastoma protein degradation.

Damage-induced G1 checkpoint in mammalian cells involves upregulation of p53, which activates transcription of p21(Waf1) (CDKN1A). Inhibition of cyclin-dependent kinase (CDK)2 and CDK4/6 by p21 leads to dephosphorylation and activation of Rb. We now show that ectopic p21 expression in human HT1080 fibrosarcoma cells causes not only dephosphorylation but also depletion of Rb; this effect was p53-independent and susceptible to a proteasome inhibitor. CDK inhibitor p27 (CDKN1B) also caused Rb dephosphorylation and depletion, but another CDK inhibitor p16 (CDKN2A) induced only dephosphorylation but not depletion of Rb. Rb depletion was observed in both HT1080 and HCT116 colon carcinoma cells, where p21 was induced by DNA-damaging agents. Rb depletion after DNA damage did not occur in the absence of p21, and it was reduced when p21 induction was inhibited by p21-targeting short hairpin RNA or by a transdominant inhibitor of p53. These results indicate that p21 both activates Rb through dephosphorylation and inactivates it through degradation, suggesting negative feedback regulation of damage-induced cell-cycle checkpoint arrest.

Mechanism of G1-like arrest by low concentrations of paclitaxel: next cell cycle p53-dependent arrest with sub G1 DNA content mediated by prolonged mitosis.

Paclitaxel (PTX) and other microtubule inhibitors cause mitotic arrest. However, low concentrations of PTX (low PTX) paradoxically cause G1 arrest (without mitotic arrest). Here, we demonstrated that unexpectedly, low PTX did not cause G1 arrest in the first cell cycle and did not prevent cells from passing through S phase and entering mitosis. Mitosis was prolonged but cells still divided, producing either two or three cells (tripolar mitosis), thus explaining a sub G1 peak caused by low PTX. Importantly, sub G1 cells were viable and non-apoptotic. Some cells fused back and then progressed to mitosis, frequently producing three cells again before becoming arrested in the next cell-cycle interphase. Thus, low PTX caused postmitotic arrest in second and even the third cell cycles. By increasing concentration of PTX, tripolar mitosis was transformed to mitotic slippage, thus eliminating a sub G1 peak. Time-lapse microscopy revealed that prolonged mitosis ensured a p53-dependent postmitotic arrest. We conclude that PTX directly affects cells only in mitosis and the duration of mitosis determines cell fate, including p53-dependent G1-like arrest.