Human P14arf-Mediated Cell Cycle Arrest Strictly Depends on Intact P53 Signaling Pathways

Oncogene (2002) 21, 3207 ± 3212 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Human p14ARF-mediated cell cycle arrest strictly depends on intact p53 signaling pathways H Oliver Weber1,2, Temesgen Samuel1,3, Pia Rauch1 and Jens Oliver Funk*,1 1Laboratory of Molecular Tumor Biology, Department of Dermatology, University of Erlangen-Nuremberg, 91052 Erlangen, Germany; 2Regulation of Cell Growth Laboratory, National Cancer Institute, Frederick, Maryland, MD 21702-1201, USA The tumor suppressor ARF is transcribed from the INK4a/ 4 and 6 activities, thus leading to decreased phosphor- ARF locus in partly overlapping reading frames with the ylation of RB and to G1 arrest. Cells that are de®cient CDK inhibitor p16Ink4a. ARF is able to antagonize the for RB are resistant to p16Ink4a-mediated cell cycle MDM2-mediated ubiquitination and degradation of p53, arrest (Sherr and Roberts, 1995; Weinberg, 1995). leading to either cell cycle arrest or apoptosis, depending on ARF is also a cell cyle inhibitor. It does not directly the cellular context. However, recent data point to inhibit CDKs but interferes with the function of additional p53-independent functions of mouse p19ARF. MDM2 to destabilize p53. ARF may be activated by Little is known about the dependency of human p14ARF aberrant activation of oncoproteins such as Ras function on p53 and its downstream genes. Therefore, we (Palmero et al., 1998), c-myc (Zindy et al., 1998), analysed the mechanism of p14ARF-induced cell cycle arrest E1A (de Stanchina et al., 1998), Abl (Radfar et al., in several human cell types. Wild-type HCT116 colon 1998) and E2F-1 (Bates et al., 1998). In these scenarios, carcinoma cells (p53+/+p21CIP1+/+ 14-3-3s+/+), but not ARF is believed to counteract the oncogenic hyper- 7/7 p53 counterparts, underwent G1 and G2 cell cycle arrest proliferative eect, predominantly by induction of both ARF CIP17/7 following infection with a p14 -adenovirus. In p21 G1 and G2 arrest (Quelle et al., 1995). ARF 7/ ARF cells, p14 did not induce G1 or G2 arrest, while 14-3-3s Human p14 is only 49% homologous to murine 7 ARF counterparts were mainly arrested in G1, pointing to p19 and 5 kDa smaller. Despite such high size and CIP1 ARF ARF essential roles of p21 in G1 and G2 arrest and cooperative sequence divergence, p19 and p14 share similar ARF ARF roles of p21 and 14-3-3s in ARF-mediated G2 arrest. Our properties. Both p19 and p14 can directly data demonstrate a strict p53 and p21CIP1 dependency of interact with MDM2 (Pomerantz et al., 1998; Zhang p14ARF-induced cell cycle arrest in human cells. et al., 1998). MDM2 binds to p53 leading to the Oncogene (2002) 21, 3207 ± 3212. DOI: 10.1038/sj/ ubiquitin-mediated degradation of p53. Since p53 onc/1205429 transactivates MDM2, this constitutes a negative feedback loop between MDM2 and p53 (Sherr, Keywords: INK4a/ARF; p53; p21; 14-3-3s; DNA 1998). The interaction of ARF with MDM2 prevents tumor virus the MDM2-mediated p53 degradation and subse- quently leads to the stabilization and activation of p53, which then induces either cell cycle arrest or Introduction apoptosis, depending on the cellular context. The INK4a/ARF gene locus is one of the most The INK4a/ARF gene locus encodes for two cell cycle frequently targeted genes in human carcinogenesis. regulatory proteins, p16Ink4a and ARF (alternative p16Ink4a is a bona ®de tumor suppressor, showing reading frame; human p14ARF, murine p19ARF), in frequent deletions, point mutations and hypermethyla- dierent, overlapping reading frames. The two tran- tions exclusively targeting p16Ink4a in many human scripts have separate ®rst exons but share exons 2 and cancers (Chin et al., 1998a; Sharpless and DePinho, 3. Exon 2 is transcribed in distinct readings frames 1999). In contrast, the role of ARF in human such that there is no amino acid homology between the carcinogenesis is less clear. Many deletions target not two proteins (Chin et al., 1998b; Sharpless and only p16Ink4a, but also p14ARF. However, there are few DePinho, 1999). point mutations or deletions known so far to p16Ink4a belongs to the INK4 family of cyclin- exclusively target p14ARF (Chin et al., 1998b), and dependent kinase (CDK) inhibitors and inhibits CDK most point mutations in exon 2 render p16INK4A and not p14ARF unfunctional in in vitro assays (Quelle et al., 1997). On the other hand, the role of ARF as a tumor suppressor has been supported by a mouse *Correspondence; JO Funk; model in which exon 1b has exclusively been disrupted E-mail: [email protected] (Kamijo et al., 1997). The resulting p19ARF null mice 3Current address: The Burnham Institute, La Jolla, CA 92037, USA Received 11 December 2001; revised 23 January 2002; accepted 19 developed lymphomas and sarcomas at an early age, a February 2002 phenotype very similar to that of INK4a/ARF exon 2 ARF-mediated cell cycle arrest HO Weber et al 3208 knockout mice, which lack both p19ARF and p16Ink4a (Serrano et al., 1996). It is believed that the function of murine and human ARF to induce arrest mainly depends on the stabilization and activation of p53. However, the analysis of p19ARF in mouse embryo ®broblasts (MEFs) has pointed to additional p53-independent mechanisms of p19ARF function (Carnero et al., 2000; Weber et al., 2000). Furthermore, little is known about the downstream eectors once p53 is activated by ARF. In general, it is believed that the p53 function to arrest the cell cycle, i.e. following DNA damage, depends primarily on the transactivation of speci®c p53-responsive genes, such as p21CIP1, 14-3-3s,and others. (Levine, 1997; Vogelstein et al., 2000; Vousden, 2000). However, mouse p19ARF may inhibit the cell cycle through both p21CIP1-dependent and p21CIP1 - independent pathways (Modestou et al., 2001). Results We set out to analyse the dependency of p14ARF- induced cell cycle arrest on p53 and the p53 target CIP1 ARF genes, p21 and 14-3-3s, in the human colorectal Figure 1 p14 -induced G1 and G2 arrest depends on p53. An +/+ adenovirus encoding for p14ARF (ad-ARF) was used to express cancer cell line, HCT116. This cell line (p53 ARF 7/7 CIP1+/+ +/+ p14 in wild-type and p53 HCT116 cells. As a negative p21 14-3-3s ) and various isogenic knockout control an adenovirus encoding for GFP (ad-GFP) was used. cell lines, derived from the parental HCT116 cells by Treatment with adriamycin (ADR) to induce DNA damage homologous recombination (Waldman et al., 1995), served as a control for the activation of p53. At 24 h post- have been successfully used in a number of cell cycle treatment either BrdU-labeled cells were harvested for ¯ow cytometry analysis and whole-cell extracts were made for analysis studies (Bunz et al., 1998; Chan et al., 1999; McShea et of the relevant cell cycle regulators by immunoblotting or al., 2000; Samuel et al., 2001; Waldman et al., 1996, determination of CDK activities 1997). Their phenotype, mostly in the context of a DNA damage response, has been extensively docu- mented. In HCT cells, the endogenous p14ARF gene is silenced by mutation of one and methylation of the of CDK2 activity and the subsequent hypophosphor- other allele, respectively (Burri et al., 2001). Therefore, ylation of RB, indicative of G1 arrest. The CDK2 the introduction of p14ARF into these cells substitutes inhibition was mediated by p21CIP1, since p21CIP1 was missing p14ARF expression instead of increasing already detected in anti-CDK2 immunoprecipitates both in the existing p14ARF expression. ad-ARF-infected and ADR-treated cells (data not The adenoviral pAdEasy system (He et al., 1998) shown). Consequently, the percentage of S phase cells, was used to express p14ARF in the cells. The newly as evaluated by BrdU incorporation and ¯ow generated adenovirus (ad-ARF) encoded for p14ARF cytometry analysis, was decreased (Figure 1). In and the GFP gene under the control of two separated comparison, ADR-treated HCT cells showed a pro- cytomegalovirus promotors. Therefore, the infected found p53 induction and subsequent induction of CIP1 cells could be traced throughout the experiment. As a p21 and 14-3-3s with concomitant G1 and G2 cell control, an adenovirus encoding for GFP only (ad- cycle arrest. GFP) was used. GFP ¯uorescence indicated that 100% CDK1/Cyclin B activity, the key regulatory CDK of the target cells were infected (data not shown). To complex governing G2/M, was also markedly reduced compare the activation of p53 in response to ARF with both in ad-ARF- and ADR-treated wild-type cells, its activation by DNA damage, all cells were treated indicative of G2 arrest. CDK1 activity is primarily with adriamycin (ADR). We analysed the onset of regulated by activating and inhibitory phosphoryla- ARF-mediated eects in a time frame of up to 48 h tions. Therefore, the phosphorylation status of CDK1 following infection. was analysed by immunoblotting. Interestingly, CDK1 Following infection with ad-ARF, the HCT cells showed a hyperphosphorylated pattern, indicative of showed a high level of ARF expression as early as 12 h inhibitory tyrosine 14/threonine 15 phosphorylation post-infection, which was stable for at least another (Herzinger et al., 1995), only in ADR-treated cells but 36 h (data not shown). In the wild-type HCT cells, ad- not in ad-ARF-infected cells (Figure 1). ARF infection led to the stabilization of p53 and The current model of ARF function involves the induction of the p53 targets MDM2, p21CIP1 and 14-3- direct association of ARF and MDM2 leading to the 3s (Figure 1). This coincided with the downregulation inhibition of MDM2-mediated ubiquitination of p53 Oncogene ARF-mediated cell cycle arrest HO Weber et al 3209 (Kamijo et al., 1998; Pomerantz et al., 1998; Sherr, associated kinase activity was only marginally altered, 1998; Stott et al., 1998; Zhang et al., 1998); however, suggesting that 14-3-3s is dispensable for p14ARF- the precise requirement of MDM2 relocation for p53 induced G1 but important for G2 arrest.

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