Oncogene (1998) 17, 1691 ± 1703 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Cell cycle arrest and DNA endoreduplication following p21Waf1/Cip1 expression Stewart Bates, Kevin M Ryan, Andrew C Phillips and Karen H Vousden ABL Basic Research Program, NCI-FCRDC, Building 560, Room 22-96, West 7th Street, Frederick, Maryland 21702-1201, USA p21Waf1/Cip1 is a major transcriptional target of p53 and there are an increasing number of regulatory mechan- has been shown to be one of the principal mediators of isms that serve to inhibit cdk activity. Primary amongst the p53 induced G1 cell cycle arrest. We show that in these is the expression of cdk inhibitors (CDKIs) that addition to the G1 block, p21Waf1/Cip1 can also contribute can bind to and inhibit cyclin/cdk complexes (Sherr to a delay in G2 and expression of p21Waf1/Cip1 gives rise and Roberts, 1996). These fall into two classes, the to cell cycle pro®les essentially indistinguishable from INK family which bind to cdk4 and cdk6 and inhibit those obtained following p53 expression. Arrest of cells complex formation with D-cyclins, and the p21Waf1/Cip1 in G2 likely re¯ects an inability to induce cyclin B1/cdc2 family that bind to cyclin/cdk complexes and inhibit kinase activity in the presence of p21Waf1/Cip1, although the kinase activity. inecient association of p21Waf1/Cip1 and cyclin B1 The p21Waf1/Cip1 family of CDKIs include p21Waf1/Cip1, suggests that the mechanism of inhibition is indirect. p27 and p57 and are broad range inhibitors of cyclin/ Cells released from an S-phase block were not retarded cdk complexes (Gu et al., 1993; Harper et al., 1993; in their ability to progress through S-phase by the Xiong et al., 1993; Firpo et al., 1994; Polyak et al., presence of p21Waf1/Cip1, despite ecient inhibition of 1994; Toyoshima and Hunter, 1994; Lee et al., 1995; cyclin E, A and B1 dependent kinase activity, suggesting Matsuoka et al., 1995). The crystal structure of p27/ that p21Waf1/Cip1 is inecient at inhibiting replicative DNA cyclin A/cdk2 indicated that this family of proteins synthesis in vivo. Interestingly, signi®cant numbers of make speci®c interactions with both cyclin and cdk cells released from the p21Waf1/Cip1 activated G2 block (Russo et al., 1996), and this is re¯ected by primary undergo endoreduplication, passing through another S- sequence conservation within the p21Waf1/Cip1 family phase before undergoing mitosis. This supports a members (Harper et al., 1993; Xiong et al., 1993; El- function of the mitotic kinases in both entry into mitosis, Deiry et al., 1994; Noda et al., 1994; Polyak et al., and also in preventing re-replication of DNA following 1994; Toyoshima and Hunter, 1994; Lee et al., 1995; S-phase and suggests a role for p21Waf1/Cip1 in coupling Matsuoka et al., 1995). This is supported by both in DNA synthesis and mitosis. Unlike p53, which induces vitro and in vivo evidence for complex formation apoptosis in these cells, extended expression of between p21Waf1/Cip1 and cyclin A/cdk2, cyclin E/cdk2 p21Waf1/Cip1 resulted in the expression of a senescent-like and cyclin D/cdk4/6 complexes (Gu et al., 1993; phenotype in these p53 null, pRB null tumor cells. Harper et al., 1993; Xiong et al., 1993; Hall et al., 1995). By contrast, the major mitotic cyclin B1/cdc2 Keywords: p21Waf1/Cip1; cell cycle arrest; cyclin dependent complex associates less eciently with p21Waf1/Cip1, and kinase; endoreduplication; senescence the kinase activity associated with this complex remains relatively insensitive to even high levels of p21Waf1/Cip1 in vitro (Harper et al., 1993; Xiong et al., 1993; Hall et al., 1995; Dulic et al., 1998). Interestingly, Introduction all of these inhibitors also serve as adapter proteins that allow assembly of the cyclins and cdks and target Cell cycle control is a tightly regulated process during their nuclear localization. Therefore, at low levels which alternating rounds of DNA synthesis (S-phase) p21Waf1/Cip1 contributes to the formation of active and mitosis (M-phase) are coordinated by a series of cyclin/cdk complexes, which are then inhibited as checkpoints. Progression through the cell cycle is p21Waf1/Cip1 levels increase (Zhang et al., 1994; LaBaer driven by a family of cyclin-dependent kinases (cdks) et al., 1997). whose activity is regulated through binding of cyclin A common theme in the cellular functioning of the regulatory molecules (Sherr, 1993; Hunter and Pines, CDKIs is their induced expression in response to a 1994). Cyclin D/cdk4 and cyclin D/cdk6 complexes broad spectrum of cellular stresses, thus allowing the regulate early events in the G1 phase of the cell cycle, cell to regulate cell cycle progression in response to while cdk2 activity regulated by cyclins A and E extracellular and intracellular cues (Sherr and Roberts, appears to be critical for the initiation of DNA 1996). One of the most signi®cant cellular stresses is synthesis. Transit through mitosis is regulated by the response to DNA damage, where p53-mediated cdc2 (cdk1) in combination with cyclin B1, although p21Waf1/Cip1 induction has been shown to play a critical cyclin A may also play some role. In addition to the role (Brugarolas et al., 1995; Deng et al., 1995; positive regulation of cdk activity provided by cyclins, Waldman et al., 1995; Polyak et al., 1996). p53 levels are elevated in response to DNA damage, associated with an increase in the half-life of the protein, and through its sequence-speci®c DNA binding function, Correspondence: KH Vousden p53 is able to enhance the transcription of a number of Received 16 March 1998; revised 5 May 1998; accepted 5 May 1998 genes, including p21Waf1/Cip1 (Bates and Vousden, 1996). Functions of p21waf1/Cip1 S Bates et al 1692 The p53-mediated response to genotoxic stress is also suggested a role for p21Waf1/Cip1 in both the p53- characterized by the inhibition of cell cycle progres- mediated G2 arrest response and the coupling of sion at both G1 and G2 checkpoints, and in some cells DNA synthesis and mitosis (Polyak et al., 1996; apoptotic cell death. The importance of p53's role in Waldman et al., 1996). the DNA damage response is underlined by its In addition to the ability to bind and inhibit cyclin/ mutation in many human tumours, allowing both the cdk complexes, p21Waf1/Cip1 also has the ability to interact survival of abnormal cells and replication of damaged with PCNA (Xiong et al., 1992; Chen et al., 1995; Luo DNA, which in turn can lead to genomic instability et al., 1995), to inhibit stress activated protein kinases and the acquisition of further oncogenic abnormalities. (Shim et al., 1996) and interact with Gadd45 (Kearsey et The generation of mice homozygously null for al., 1995). In vitro studies showed that the interaction of p21Waf1/Cip1 has shown that p21Waf1/Cip1 is not essential p21Waf1/Cip1 blocks the ability of PCNA to activate DNA for development and these animals fail to show the polymerase d, potentially by disrupting PCNA/Fen1 increased rate of tumorigenesis associated with p53 complexes (Chen et al., 1996; Warbrick et al., 1997). loss. Fibroblasts from these animals, however, are However, the subsequent inhibition of DNA synthesis impaired in their ability to arrest in Gl following was shown to be limited to replicative but not repair DNA damage (Brugarolas et al., 1995; Deng et al., synthesis (Li et al., 1994; Shivji et al., 1994), leading to 1995), and similar results have been reported for both an attractive model in which DNA damage induced human diploid ®broblasts and human tumour lines p21Waf1/Cip1 expression, through p53, arrests DNA deleted for p21Waf1/Cip1 (Waldman et al., 1995; Polyak et replication but allows repair of the damage to al., 1996; Brown et al., 1997). The latter cells have proceed. Indeed, functional p21Waf1/Cip1 appears to be Figure 1 p53 induced cell cycle arrest. (a) Cell cycle analysis of Saos-2 cells stably expressing the human temperature sensitive p53143A (Saos-ts p53) mutant either at the restrictive temperature (378C) or 24 h following shift to the permissive temperature (308C) and U20S cells either untreated or 24 h following treatment with 5 nM actinomycin D. (b) CD20 co-transfection was utilised to analyse the cell cycle pro®les of Saos-2 cells transiently transfected with 20 mg of the indicated plasmids and CD20 positive cells were identi®ed using a FITC conjugated anti-CD20 antibody. Percentage of live cells (excluding cells with less that 2N DNA content) in G1 and G2/M phases are indicated Functions of p21waf1/Cip1 S Bates et al 1693 required for ecient repair of DNA damage change in morphology and the induction of senescence (McDonald et al., 1996). associated b-Galactosidase activity, all characteristics of A role for both p53 and p21Waf1/Cip1 has also been senescent cells (Sugrue et al., 1997). It seems clear, implicated in cellular aging (Harvey et al., 1993; Noda however, that many other factors are also important in et al., 1994; Atadja et al., 1995; Bond et al., 1995). p53 the induction and maintenance of the senescent levels accumulate as diploid ®broblasts age, with a phenotype. commensurate increase in the levels of p21Waf1/Cip1 and In this study we show that p21Waf1/Cip1 can induce this coincides with a gradual slowing of cellular growth both a G1 arrest and G2 delay, but fails to signi®cantly and inhibition of cyclin/cdk activity. Many studies have delay progress through S-phase in the p53/pRB null shown that ®broblasts homozygously deleted for either osteosarcoma cell line, Saos-2.