p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells

Oncogene (1998) 16, 311 ± 320  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-de®cient cells Corinne Cayrol, Martine Knibiehler and Bernard Ducommun Institut de Pharmacologie et de Biologie Structurale du CNRS Universite Paul Sabatier, 205 route de Narbonne, 31077 Toulouse cedex France A unique feature of p21 that distinguishes it from the other cyclin-dependent kinase (CDK) inhibitors is its ability to associate with the proliferating cell nuclear antigen (PCNA), an auxiliary factor for DNA polymerases d and e. While it is now well established that inhibition of cyclin/CDK complexes by p21 can result in G1 cell cycle arrest, the consequences of p21/PCNA interaction on cell cycle progression have not yet been determined. Here, we show, using a tetracyclineregulated system, that expression of wild-type p21 in p53-de®cient DLD1 human colon cancer cells inhibits DNA synthesis and causes G1 and G2 cell cycle arrest. Similar e€ects are observed in cells expressing p21CDK7, a mutant impaired in the interaction with CDKs, but not in cells expressing p21PCNA7, a mutant de®cient for the interaction with PCNA. Analysis of cells treated with a p21-derived PCNA-binding peptide provides additional evidence that the growth inhibitory e€ects of p21 and p21CDK7 result from their ability to bind to PCNA. Our results suggest that p21 might inhibit cell cycle progression by two independent mechanisms, inhibition of cyclin/CDK complexes, and inhibition of PCNA function resulting in both G1 and G2 arrest. Keywords: cell cycle; cyclin-dependent kinase; growth arrest; p21CIP1/WAF1; PCNA Introduction Over the last few years a new class of cell cycle regulators, the cyclin-dependent kinase inhibitors (CKIs), has been the focus of a great deal of attention. The major interest for these proteins stems mainly in the connections their discovery allowed to establish between the growth control pathways and the cell cycle machinery (Elledge et al., 1996). Although all the inhibitors function to block the activity of cyclindependent kinases (CDKs) ± the key protein kinases that regulate major cell cycle transitions ± they belong to two di€erent classes that have been de®ned on the basis of their primary sequences and their biochemical properties. Members of the CIP/KIP family (p21Cip1, p27Kip1 and p57Kip2) inhibit the activity of the CDKs through their binding to the enzymatic complexes formed by the association of CDK catalytic subunits with cyclin regulatory subunits. A conserved bipartite motif located in the amino-terminal part of these inhibitors is required for binding and inhibition Correspondence: B Ducommun Received 25 July 1997; revised 1 September 1997; accepted 2 September 1997 of cyclin-CDK complexes both in vitro and in vivo (Fotedar et al., 1996; Goubin and Ducommun, 1995; Nakanishi et al., 1995; Zakut and Givol, 1995). The INK4 family members (p16, p15, p18 and p19) function di€erently by forming speci®c binary complexes with CDK4 and CDK6 that prevent association of these CDKs with cyclin D and thus result in inhibition of pRb-dependent cell cycle progression in G1 (Fahraeus et al., 1996; Medema et al., 1995; Serrano et al., 1993). A number of studies have now clearly established links between CKI expression and developmental events or cancer predisposition (for review see Elledge et al., 1996). In all these situations, CKIs appear to regulate cell cycle progression through the modulation of cyclin/CDK complexes activity in response to various intracellular or extracellular signals. p21Cip1/Waf1 (hereafter referred to as p21) is a key mediator of the growth arrest induced by the tumour suppressor protein p53 in response to DNA damage (ElDeiry et al., 1993). Accordingly, homozygous deletion of the p21 gene in mouse embryonic ®broblasts or human colon cancer cells partially or completely abrogates the DNA-damage-induced G1 arrest mediated by p53 (Brugarolas et al., 1995; Deng et al., 1995; Waldman et al., 1995). A unique feature of p21 that distinguishes it from the other CKIs is its ability to associate with the proliferating cell nuclear antigen (PCNA) (Xiong et al., 1992; Zhang et al., 1993), an auxiliary factor for DNA polymerases d and e, that is essential for both DNA replication and DNA repair (Kelman, 1997). p21 contains a PCNA binding motif located in the carboxyterminal part of the protein between residues 144 ± 151 (Chen et al., 1995a; Goubin and Ducommun, 1995; Nakanishi et al., 1995b; Warbrick et al., 1995). Crystal structure of human PCNA complexed with a 22 residue PCNA-binding peptide (PBP) containing this motif has revealed that the p21 carboxy-terminal domain interacts with the interdomain connector loop of PCNA and is likely to prevent the interaction of PCNA with other components of polymerase assembly (Gulbis et al., 1996). Although it is still controversial, this C-terminal domain of p21 might inhibit cell cycle progression independently of the N-terminal CDK inhibitory domain and thus contribute to the antiproliferative activity of p21 (Luo et al., 1995; Nakanishi et al., 1995b). p21 has been shown to inhibit PCNA-dependent DNA replication and mismatch repair in vitro (FloresRozas et al., 1994; Li et al., 1994; Podust et al., 1995; Umar et al., 1996; Waga et al., 1994). PCNA is also required for nucleotide excision repair but p21 does not appear to block the function of PCNA in this process (Li et al., 1994, 1996; Shivji et al., 1994). Recently, p21Cip1 has been shown to impair loading of Fen1 by p21 binding to PCNA causes G1 and G2 growth arrest C Cayrol et al 312 PCNA on the replication fork (Chen et al., 1996b; Warbrick et al., 1997). Since Fen1 is an essential exonuclease involved in the processing of ARN-DNA junctions during Okazaki fragments replication, this impairment might contribute to p21-dependent inhibition of replication. In contrast, Fen1 does not appear to be required for nucleotide excision repair and inhibition of PCNA/Fen1 interaction by p21 is therefore unlikely to a€ect this later process. p21 induction after DNA damage may thus lead to inhibition of cell cycle progression and inactivation of PCNA-dependent DNA replication, while permitting active nucleotide excision repair. This will ensure that any errors caused by the damage are corrected before being propagated by DNA replication. To further characterize the molecular mechanisms underlying p21 growth suppressive function, we have generated p53-de®cient human colon cancer cell lines expressing wild-type or mutants p21 under the control of a tetracycline-regulated promoter. To dissociate the e€ects of p21 due to the interaction with CDKs from the e€ects due to the interaction with PCNA, we used p21 mutants de®cient for either one or the other interaction. Here we report the characterization of the e€ects of these mutations on cell proliferation and demonstrate that p21-binding to PCNA is sucient to block (...truncated)