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

(CDKN1A). Inhibition of cyclin-dependentkinase (CDK)2 and CDK4/6 by p21 leads to depho-sphorylation and activation of Rb. We now show thatectopic p21 expression in human HT1080 fibrosarcomacells causes not only dephosphorylation but also depletionofRb; this effect was p53-independent and susceptible toaproteasome inhibitor. CDK inhibitor p27 (CDKN1B) alsocaused Rb dephosphorylation and depletion, but anotherCDK inhibitor p16 (CDKN2A) induced only depho-sphorylation but not depletion of Rb. Rb depletion wasobserved in both HT1080 and HCT116 colon carcinomacells, where p21 was induced by DNA-damaging agents.Rb depletion after DNA damage did not occur in theabsenceofp21,anditwasreducedwhenp21inductionwasinhibited by p21-targeting short hairpin RNA or by atransdominant inhibitor of p53. These results indicate thatp21 both activates Rb through dephosphorylation andinactivates it through degradation, suggesting negativefeedback regulation of damage-induced cell-cycle check-point arrest.Oncogene advance online publication, 7 May 2007;doi:10.1038/sj.onc.1210516Keywords: p21; Rb; p27; damage responsep53-inducible cyclin-dependent kinase (CDK) inhibitorp21 (CDKN1A) is the key mediator of damage-inducedcell-cyclearrest.p21interactswithdifferentcyclin/CDKcomplexes and other regulators of transcription andsignal transduction, exerting broad effects on cellsurvival, gene expression and morphology (Roninson,2002).p21effectsarepartiallymediatedbyRb,whichisinactivated in proliferating cells through phosphoryla-tion by CDK2 and CDK4/6, both of which are inhibi-ted by p21. As a result, p21 induction leads to Rbdephosphorylation and activation, with ensuing G1arrest.Whereas p21 activates Rb by dephosphorylation,several oncoproteins inactivate Rb by degradation viathe proteasome. Proteasome-mediated Rb degradationis promoted by Mdm2 (Sdek et al., 2005) and gankyrin(Higashitsuji et al., 2000), E7 of papilloma virus (Boyeret al., 1996) and Tax of HTLV1 (Kehn et al., 2005).Oncoprotein-induced proteasomal degradation of Rb isone of the mechanisms for Rb inactivation in carcino-genesis (Ying and Xiao, 2006), but Rb degradation hasnot been described in DNA damage response.Changes in Rb phosphorylation are most commonlydetected by immunoblotting through changes in theprotein’s electrophoretic mobility. Examination ofnumerous Rb immunoblots published by differentgroups showed that in many (but not all) cases Rbdephosphorylation, which results from drug treatment,cell senescence or ectopic p21 expression, is associatedwithareduction intheRb protein signal. In thepresentstudy, we have asked (i) whether a decrease in the Rbsignal in response to p21 reflects protein degradation ormerely altered immunoreactivity of dephosphorylatedRb, (ii) if p53 plays a p21-independent role in the de-crease in Rb, (iii) whether such decrease can be inducedby other CDK inhibitors that induce Rb dephosphory-lation and (iv) if the decrease in Rb in drug-damagedcells is dependent on p21 induction.We have described previously a subline of HT1080cells, p21-9, which expresses p21 from a promoterinducible by b-galactoside isopropyl-b-thio-galactoside(IPTG) (Chang et al., 1999). p21 induction in HT1080p21-9 leads to Rb dephosphorylation and depletionwithout any changes in Rb mRNA levels (Chang et al.,2000). As shown in Figure 1a (left lanes), Rb wasdephosphorylated and its signal intensity decreased 24–48h after IPTG addition. To determine if the reducedRb signal indicates a decrease in protein levels ratherthan altered antibody reactivity, we have used both amouse monoclonal and a rabbit polyclonal antibodyagainst Rb. Both antibodies produced the same result(Figure1a,leftlanes),indicatingthatthedecreaseinRbsignal intensity was indeed due to a decrease in theprotein level.The decrease in Rb protein was not accompanied bythe appearance of Rb fragments that arise after caspasecleavage of Rb (Tan et al., 1997). Since the best-knownmechanism of Rb degradation is via the proteasomepathway (Ying and Xiao, 2006), we have tested theeffect of a proteasome inhibitor ALLN (N-acetyl-leucinyl-leucinyl-norleucinal) on p21-induced Rb deple-