P14arf Is a Component of the P53 Response Following Ionizing Irradiation of Normal Human ﬁbroblasts

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Oncogene (2004) 23, 6040–6046 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc p14ARF is a component of the p53 response following ionizing irradiation of normal human ﬁbroblasts

Shireen Khan1, Claudia Guevara1, Greg Fujii1 and David Parry*,1

1Department of Discovery Research, DNAX Research Incorporated, Palo Alto, CA 94304, USA

Ionizing radiation leads to rapid stabilization and activa- to prevent downregulation of p53 when activation of cell tion of the p53 tumor suppressor.Previous reports cycle arrest or apoptosis is appropriate. demonstrate that murine p19ARF cooperates with p53 in P53 is rapidly induced following exposure to ionizing the cellular response to gamma irradiation.Here, we show radiation (IR) in wild-type mouse embryonic fibroblasts that endogenous ARF sequentially interacts with p53 and (MEFs). However, p53 induction is neither acute nor MDM2 following irradiation of primary human and sustained in irradiated ARFÀ/À MEFs, suggesting that mouse embryonic fibroblasts.Shortly after irradiation, p19ARF cooperates with p53 in response to DNA p14ARF binds p53 independently of MDM2.As nuclear damage (Khan et al., 2000). Similar toATM À/À and pools of p53 decline, endogenous p14ARF co-immuno- Chk2À/À MEFs, cell cycle profiling reveals that the precipitates with MDM2 and is localized within the majority of ARFÀ/À MEFs continue cycling post-IR nucleolus.Interestingly, p14ARF nucleolar localization (Khanna and Lavin, 1993; Xu and Baltimore, 1996; during this response is abrogated in cells lacking Hirao et al., 2000). Further evidence linking ARF with functional p53.Taken together, our data suggest that DNA damage responses came from the in vivo observa- human and murine ARF contribute to the mammalian tion that irradiated ARF-null mice have an increased DNA damage response. tumor frequency compared to wild-type mice, suggest- Oncogene (2004) 23, 6040–6046. doi:10.1038/sj.onc.1207824 ing that ARF suppresses tumor growth upon genetic Published online 14 June 2004 insult (Kamijo et al., 1999a). Recent data have also suggested that ARF, p53 and Rad6 form a supramole- Keywords: ARF; DNA damage; ionizing radiation; cular complex in response to adriamycin or cisplatin MDM2; p53 treatment (Lyakhovich and Shekhar, 2003). In addition to DNA damage responses, data from several laboratories suggest that ARF promotes p53 stabilization and prevents cell cycle progression in response to oncogenic activation. Enforced expression Introduction of E1A, ras, myc or E2F-1 leads to an increase in the cellular levels of ARF and stabilization of p53 (Bates The p53 tumor suppressor is activated via diverse stress et al., 1998; de Stanchina et al., 1998; Palmero et al., signals including DNA damage, hypoxia and ribonu- 1998; Zindy et al., 1998; Groth et al., 2000). cleotide depletion (Kastan et al., 1991; Huang et al., Several models of ARF function have been proposed. 1996; Linke et al., 1996). p53 relies on upstream and ARF can bind to and directly ablate the function of downstream regulators for correct function (Giaccia and MDM2 (Kamijo et al., 1998; Pomerantz et al., 1998; Kastan, 1998). Because p53 promotes growth arrest or Stott et al., 1998; Zhang et al., 1998). ARF alsoblocks apoptosis, modulation of p53 activity is critical for the ubiquitin ligase activity of MDM2 toward p53 in survival. Thus, p53 function and stability are regulated vitro (Honda and Yasuda, 1999; Midgley et al., 2000). by MDM2 (HDM2 in humans), which inhibits p53- An alternate model proposes that ARF promotes p53 mediated transactivation and promotes p53 degradation stability by sequestration of MDM2 into nucleolar (Momand et al., 1992; Haupt et al., 1997; Kubbutat compartments (Weber et al., 1999, 2000). Yet, other et al., 1997; Thut et al., 1997). The major target of reports show that ARF promotes p53 stabilization and MDM2 is p53, as demonstrated by embryonic lethality cell cycle arrest without nucleolar accumulation with of MDM2 nullizygous mice, a phenotype rescued by MDM2 (Llanos et al., 2001; Korgaonkar et al., 2002). inactivating p53 (Jones et al., 1995; Montes de Oca Luna One report demonstrates that ARF function can be et al., 1995). Interestingly, p53 induces MDM2 expres- exerted via direct p53/ARF interaction, as murine sion, generating an autoregulatory feedback loop (Wu p19ARF binds p53/DNA complexes in an MDM2- et al., 1993). Hence, countermeasures must be in place independent manner (Kamijo et al., 1998). Current models suggest that ectopically expressed *Correspondence: D Parry; E-mail: [email protected] ARF binds p53 and MDM2, yet the exact timing of Received 7 January 2004; revised 9 April 2004; accepted 9 April 2004; these interactions, at physiological expression levels, published online 14 June 2004 during a stress response, has not been well characterized p14ARF and the DNA damage response S Khan et al 6041 (Kamijo et al., 1998; Pomerantz et al., 1998; Stott et al., 1998; Zhang et al., 1998). While murine ARF has been implicated in the cellular response to IR, it is not known whether ARF forms complexes with p53 or MDM2 during this response. Endogenous pools of p53 and MDM2 are rapidly induced and tightly regulated after irradiation, setting up a physiological system in which to analyse the biochemical interactions between ARF, p53 and MDM2 following DNA damage. We demonstrate here that human and murine ARF associate transiently with p53 and MDM2 in a temporally separable manner after irradiation.

Results p19ARF transiently associates with p53 and MDM2 Figure 1 IR induces complex formation between p19ARF, p53 following irradiation and Mdm2. Early passage (p4) wild-type MEFs were left untreated (T0) or irradiated at 7 Gy and harvested after 2 (T2), 4 (T4), 6 (T6) p19ARF is required to promote cell cycle arrest of and 8 h (T8). (a) Whole-cell lysates (100 mg) were separated on a irradiated murine fibroblasts (Khan et al., 2000). 4–20% polyacrylamide gradient gel. After Western transfer, blots were probed for p19ARF, p53 and Mdm2. To verify equal protein Current models predict that p19ARF must interact with loading, the blot was probed with actin antisera. (b) p19ARF p53 and/or MDM2 during this response in order to immunoprecipitations were performed using 1 mg lysate per time induce cell cycle arrest. Thus, early-passage, wild-type point and immune complexes were resolved on 4–20% gradient MEFs were irradiated at 7 Gy and harvested at several SDS–PAGE. Blots were probed with p53 and Mdm2 antibodies. (c) time points post-IR. WS1 fibroblasts were treated with 7 Gy IR and harvested after 2 (T2), 4 (T4) and 8 h (T8). Whole-cell extracts (100 mg) were resolved p53 and MDM2 levels were elevated in irradiated on 4–20% gradient SDS–PAGE, transferred and immunoblotted MEFs relative to untreated populations, whereas with p14ARF, p53 and HDM2 monoclonal antibodies. To p19ARF levels remained relatively constant over the demonstrate equal protein loading, blots were probed with actin duration of the time course (Figure 1a). Immunopreci- antibodies. (d) Anti-p14ARF immunoprecipitations were performed and complexes were immunoblotted for p53 and HDM2 pitation of p19ARF revealed an interaction between p53 following SDS–PAGE. T0 represents the time zero, unirradiated and p19ARF within 2 h of irradiation, whereas p53 is control undetectable in ARF immune complexes isolated from nonirradiated cells (Figure 1b). Formation of p19ARF/ p53 complexes is rapid and transient, as p53 is not association occurred within 2 h of irradiation in WS1, detected within ARF immune complexes at later time whereas p14ARF/HDM2 binding was maximal at 4 h points (Figure 1b). Whereas MDM2 induction appears (Figure 1d). Peak levels of ARF/p53 complexes con- topeak 4 h after IR, the peak p19ARF/MDM2 complex sistently preceded those of ARF/HDM2, following IR. formation was not detected until 6–8 h after irradiation Moreover, the accumulation of p14ARF/HDM2 com- (Figure 1b). plexes at 4 h coincided with a decrease in detectable p53. Similar results were obtained in normal human lung p14ARF transiently associates with p53 and HDM2 fibroblasts (data not shown). following IR ARF re-localizes during the IR response Toassess whether human p14ARF responds similarly to IR, WS1 human diploid fibroblasts were irradiated Several reports suggest that overexpressed pools of at 7 Gy and harvested at various time points following p19ARF accumulate in the nucleolus (Weber et al., treatment. Immunoblotting of whole-cell extracts with 1999; Zhang and Xiong, 1999; Llanos et al., 2001). p53 and HDM2 antisera showed that p53 levels are Endogenous p19ARF also accumulates in the nucleolus maximal within 2 h of irradiation, whereas HDM2 during cellular senescence and upon overexpression of induction shows delayed kinetics (Figure 1c). In oncogenes (Weber et al., 1999, 2000; Zhang and Xiong, common with murine p19ARF, human p14ARF levels 1999). remained relatively unchanged during the time course, Staining of untreated, early-passage MEFs with anti- as assessed by Western blotting (Figure 1c). p19ARF antibodies revealed diffuse staining, distri- Anti-p14ARF immunoprecipitations were performed buted throughout the nucleus (Figure 2A, panels a, b). on lysates prepared from irradiated or untreated WS1, At 2–4 h post-IR, the majority of the p19ARF signal is followed by Western blotting with p53 and HDM2 detectable in large subnuclear domains, reminiscent of antisera. IR-induced interactions between p14ARF, p53 nucleoli, although some signal is still detectable within and HDM2 in WS1 fibroblasts were readily detectable the nucleoplasm (Figure 2A, panels c–f). At 6–8 h post- (Figure 1d). Significantly, the peak of p14ARF/p53 IR, the majority of the p19ARF signal accumulates

Oncogene p14ARF and the DNA damage response S Khan et al 6042

Figure 2 ARF localizes to the nucleolus in response to IR. (A) Early-passage (p3) MEFs were seeded onto coverslips and irradiated at 7 Gy. They were harvested after 2, 4, 6 and 8 h. Coverslips were stained using polyclonal p19ARF antibodies followed by FITC-conjugated secondary rabbit antibodies. Cells were counterstained with DAPI to identify the nucleoplasmic Figure 3 p14ARF nucleolar translocation requires a functional region. (B) NARF2 cells were seeded onto coverslips and either left p53 pathway. (A) WS1E6 fibroblasts were seeded onto coverslips untreated or treated with IPTG to induce p14ARF expression. and irradiated at 7 Gy. Cells were harvested after 2, 4, 6, 8, 14, 16, Cells were stained with anti-p14ARF reagents (MAb13C5 in this 18, 20 and 22 h. Coverslips were stained for p14ARF and case) to demonstrate antibody specificity. (C) WS1 fibroblasts were counterstained with DAPI. (B) WS1E6 cells were electroporated seeded onto coverslips and irradiated at 7 Gy. Coverslips were with HDM2 and mutant HDM2 (1–440) expression vectors and harvested at several intervals post-IR and stained with p14ARF seeded onto coverslips. At 7 h prior to harvesting, coverslips were antisera, followed by DAPI staining. Arrows indicate nucleolar irradiated at 7 Gy. Cells were sequentially stained for p14ARF pools of ARF protein (Texas red signal) followed by HDM2 (FITC signal) and DAPI. Arrows indicate nucleolar pools of ARF or HDM2

within these large nuclear domains (Figure 2A, panels (Figure 2C, panels i, j). The intense nucleolar p14ARF g–j). Co-staining experiments with p19ARF and B23, a staining is maintained up to10 h after irradiation,after nucleolar marker, confirmed the nucleolar re-localiza- which there is a significant diminution of the signal, as tion of p19ARF after irradiation (data not shown). ARF redistributes intonucleoplasmic ‘speckles’ The specificity of Mab13C5, a monoclonal antibody (Figure 2C, panels k, l). Thus, accumulation of raised against p14ARF, was first confirmed in the detectable ARF/HDM2 complexes (Figure 1) correlates NARF2 system (Stott et al., 1998). Importantly, with the re-localization of p14ARF to the nucleolus Mab13C5 detected p14ARF only in IPTG-induced 4–6 h post-IR (Figure 2C). In multiple experiments, NARF2 populations (Figure 2B). Similar analyses were there was a consistent increase in the proportion of cells then performed using WS1 human fibroblasts. with re-localized pools of ARF (typically 470–80% Staining of unirradiated WS1 cells with Mab13C5 showed nucleolar staining), following exposure to IR. revealed multiple, weak-staining nucleoplasmic ‘speckles’ (Figure 2C, panels a, b) in the majority of cells. How- p14ARF nucleolar localization is dependent on p53 ever, p14ARF staining becomes stronger within 2 h function of irradiation (Figure 2C, panels c, d). Significantly, 4–6 h after irradiation, p14ARF accumulates in regions To examine whether nucleolar re-localization of of the nucleus that exclude DAPI stain and are p14ARF after IR requires an intact p53 pathway, we reminiscent of nucleoli (Figure 2C, panels e–h). Nucleo- analysed the localization of p14ARF in irradiated lar localization was confirmed by co-staining with anti- WS1E6 fibroblasts. These cells express HPVE6 and nucleolin (data not shown). At 8 h post-irradiation, the have undetectable levels of p53. Consequently, they majority of p14ARF localizes within the nucleoli donotundergocell cycle arrest after exposureto

Oncogene p14ARF and the DNA damage response S Khan et al 6043 g-radiation (Kastan et al., 1991; Huang et al., 1996; Linke et al., 1996). Irradiated WS1E6 fibroblasts have undetectable levels of p53 and HDM2 in p14ARF immune complexes, although ARF is detectable at levels comparable to parental WS1 cells (data not shown). WS1E6 fibroblasts were seeded onto coverslips, irradiated at 7 Gy and harvested at several time points. The intensity of p14ARF staining increases within 2 h of irradiation (Figure 3A, panels a–d). However, in contrast to WS1, p14ARF does not re-localize to the nucleolus after IR (Figure 3A, panels e–j). Instead, the signal remained generally diffuse, yet intense, throughout the nucleus (Figure 3A, panels c–j). Significantly, the ARF signal intensity was maintained 22 h after irradiation. Hence, E6 expression can prevent the nucleolar accumulation of p14ARF in response to IR.

Ectopically expressed HDM2 rescues nucleolar localization of p14ARF in WS1E6 Nucleolar localization signals (NoLS) on both ARF and HDM2 are required totarget the ARF/HDM2 complex to the nucleolus (Lohrum et al., 2000a, b; Weber et al., 2000). Since WS1E6 cells lack functional p53, they are Figure 4 p14ARF/p53 complex formation in the absence of unable to induce HDM2 expression following IR. To HDM2. (a) p53À/ÀMdm2À/À MEFs were infected with adeno- test the hypothesis that HDM2 is necessary for the viruses expressing empty vector (AdVec), p14ARF (Adp14ARF), nucleolar translocation of p14ARF following IR, p53 (Adp53) or both p14ARF and p53 (Adp14ARF þ Adp53). At irradiated WS1E6 cells were transfected with HDM2 24 h post-infection, cells were harvested and p14ARF immunopre- expression constructs. Significantly, p14ARF nucleolar cipitations were performed. Blots were probed sequentially with anti-p53 and anti-p14ARF reagents. (b) WS1 fibroblasts were accumulation was restored in WS1E6 cells expressing irradiated at 7 Gy and harvested 2 h later. Three successive anti- HDM2, whereas p14ARF remained nucleoplasmic in HDM2 immunoprecipitations were performed to immunodeplete mock-transfected cells (Figure 3B, panels a–f). In the endogenous HDM2 (aHDM2 #1, #2 and #3). Immunopreci- agreement with previous reports, cells expressing pitation of p14ARF was then performed on the HDM2-depleted extract (ap14ARF*). HDM2-depleted whole-cell extract was also HDM2 1–440 (an HDM2 mutant lacking the C-terminal loaded (WCE*). Immunoblotting was performed with monoclonal ring finger domain and cryptic NoLS) did not target antibodies against HDM2, p53 and p14ARF as indicated p14ARF tothe nucleolus(Figure 3B, panels g–i). Instead, these cells display diffuse nuclear staining for both ARF and HDM2 1–440. Hence, localization of ARF to the nucleolus may be dependent on nucleolar associated with p53 in HDM2-depleted extracts accumulation of MDM2. (Figure 4b), suggesting that ARF may exert a direct effect on p53 during the IR response. p14ARF binds p53 in the absence of HDM2 The rapid initial interaction between p53 and ARF following irradiation suggests that ARF may exert a Discussion direct effect on p53 function in an HDM2-independent manner. ARF and p53 function cooperatively during oncogenic p53À/Àmdm2À/À MEFs were infected with adeno- transformation and cellular senescence (Bates et al., viruses expressing human p14ARF and p53, singly and 1998; de Stanchina et al., 1998; Palmero et al., 1998; in combination. Significantly, p53 was readily detectable Zindy et al., 1998; Weber et al., 1999). Initial character- in p14ARF immune complexes isolated from these cells ization of ARF-null, murine fibroblasts suggested that (Figure 4a), indicating ARF and p53 can interact in the an intact response to IR was present in such cells absence of MDM2. To determine if similar HDM2- (Kamijo et al, 1997). However, following ionizing independent complexes are detectable in human primary irradiation, ARF-null fibroblasts do not undergo cell cells following IR, WS1 fibroblasts were harvested 2 h cycle arrest, suggestive of an attenuated p53 response post-IR. Lysates were prepared and subjected to three (Khan et al., 2000). Furthermore, as shown in Figures 1 successive anti-HDM2 immunodepletions, followed by and 4, ionizing irradiation of human and murine an anti-p14ARF immunoprecipitation. Subsequent primary fibroblasts induces transient ARF/p53 and immunoblotting revealed that HDM2 was efficiently ARF/MDM2 interactions. Hence, ARF may be a immunodepleted from extracts (Figure 4b). Impor- critical component of the DNA damage response in tantly, a significant fraction of p14ARF remains mammals. Further genetic evidence includes the

Oncogene p14ARF and the DNA damage response S Khan et al 6044 observation that null mutations in p19ARF complement elucidated, one possibility is that ARF functions as a the premature senescence phenotype displayed by ATM- cofactor for p53 transactivation. Indeed, electrophoretic null MEFs (Kamijo et al., 1999b). Previously, this rescue mobility shift assays demonstrate that p19ARF super- had only been observed in ATM-null MEFs lacking shifts p53–DNA complexes, suggesting that ARF is either p53 or p21 function (Wang et al., 1997; Xu et al., capable of assembling into complexes with p53 on DNA 1998). Likewise, p19ARF-null MEFs, expressing ade- (Kamijo et al., 1998). noviral E1A oncoprotein, are highly resistant to IR- The cellular response to IR and DNA damage relies induced apoptosis when compared to wild-type MEFs on several signaling pathways to modify p53. These (de Stanchina et al., 1998). Moreover, recent data induce downstream protein–protein interactions, ensur- suggest that the suppression of genetic instability arising ing nuclear retention of p53 complexes and transactiva- from telomeric attrition may also be a component of tion of target genes. Here, we demonstrate that ARF is a ARF function (Smogorzewska and De Lange, 2002). component of this signaling mechanism. Taken together, the genetic and biochemical data support the model that ARF contributes to the p53 response following DNA damage. Materials and methods The subcellular localizations of ARF and HDM2 have led toa modelwherein ARF sequesters HDM2 in Cell culture the nucleolus to promote p53 stabilization in the WS1 embryonic fibroblasts (ATCC) and NARF2 cells were nucleoplasm (Weber et al., 1999). Yet, p14ARF can cultured in DMEM containing 10% FBS, penicillin/strepto- also promote p53 stabilization or activation without mycin and non-essential amino acids. WS1E6 fibroblasts nucleolar sequestration of HDM2 (Llanos et al., 2001; expressing the HPV16 E6 oncoprotein were maintained as Korgaonkar et al., 2002). Both p14ARF and HDM2 described for WS1, supplemented with 200 mg/ml G418. co-localize to the nucleolus after IR in primary p14ARF expression was induced in NARF2 cells by the fibroblasts. Interestingly, ARF contains two putative addition of 1 mM IPTG. Cells were harvested 18 h post- nucleolar localization signals (Zhang and Xiong, 1999; induction. Human cells were maintained at subconfluency and Weber et al., 2000), yet nucleolar translocation fails to split in 1 : 4 ratioevery 3–4 days. Wild-type, p53 À/À and occur during the DNA damage response in WS1E6 cells. p53À/Àmdm2À/À MEFs were grown in DMEM containing This was rescued by ectopic expression of full-length 10% FBS, penicillin/streptomycin and non-essential amino acids. All experiments utilizing wild-type MEFs were com- HDM2. Significantly, the cryptic NoLS in the C- pleted prior to passage 4. Adenoviruses were infected at terminus of HDM2 was required to promote nucleolar multiplicities of infection of two, overnight in complete localization of ARF after irradiation (Figure 3). This DMEM media. The supernatants were then removed and the observation is in broad agreement with previous findings cells placed in fresh complete media for a further 24 h, prior to (Lohrum et al., 2000b; Weber et al., 2000). The data harvesting. suggest that nucleolar accumulation of ARF with HDM2 is one component of the DNA damage response. Antibody development However, the peak of detectable ARF/p53 complex formation reproducibly precedes maximal detectable Monoclonal antibodies (MAbs 13C5, 11D11 and 5C9) were raised against human p14ARF by immunizing BALB/c mice levels of ARF/HDM2 interaction in both human and with a His-tagged p14ARF fusion protein. Splenocytes were mouse fibroblasts. Moreover, the peak of ARF/HDM2 fused to the SP2 myeloma cell line. Reactive hybridomas were complex formation correlates with the appearance and identified by ELISA and screened for the ability to immuno- co-localization of both proteins within the nucleolus of precipitate in vitro-translated p14ARF. Specificity of the human cells. Hence, following IR, significant nucleolar monoclonal reagents was confirmed by IP, Western blot and accumulation of p14ARF and HDM2 is apparent immunofluorescence using the NARF2, inducible p14ARF during the later stages of the response, after cellular system. Polyclonal murine p19ARF antisera were raised p53 levels reach their peak and are actually in decline. against a synthetic peptide comprising amino acids 54–72 Thus, we hypothesize that increased pools of HDM2 (CRRGPHRNPGDDDGQRSRSS) of the murine protein may negatively regulate p14ARF/p53 complexes, per- (Research Genetics). This was coupled to keyhole limpet hemocyanin (Pierce), and injected into rabbits (Pocono Rabbit haps by promoting p14ARF nucleolar localization. In Farms). Peptide antisera were affinity purified against the this scenario, HDM2 inhibits the functions of both p53 cognate immunogen (Sulfolink, Pierce). and p14ARF. Therefore, nucleolar localization of p14ARF may be indicative of a p53 response to IR Protein analysis and immunoprecipitation undergoing downregulation rather than initiation. Current models of ARF function focus on interac- WS1 and WS1E6 embryonic fibroblasts were irradiated tions between ARF and Mdm2, yet ARF can bind p53 at 7 Gy using a Cesium 137 irradiator and harvested at directly, in the absence of Mdm2 (Kamijo et al., 1998; several intervals after exposure. Cells were lysed in ice-cold Zhang et al., 1998; Figure 4a). Accordingly, we show ARF lysis buffer (50 mM HEPES pH 7.5, 350 mM NaCl, 1 mM EDTA, 0.3% NP-40) containing a cocktail of protease that endogenous p14ARF can bind p53 independently inhibitors (Calbiochem). Protein determinations were per- of HDM2 (Figure 4b) following IR. Such complexes formed using Bradford assay reagents (Bio-Rad). For Western may represent an immediate-early response to DNA blotting, whole-cell extracts (100 mg) were separated on damage. While the exact function of ARF/p53 com- SDS–PAGE and subsequently transferred to Immobilon-P plexes during the DNA damage response remains to be membranes using a semi-dry transfer apparatus (Bio-Rad).

Oncogene p14ARF and the DNA damage response S Khan et al 6045 Immunoprecipitations were performed using B1 mg of whole- times in PBS/0.5% Tween 20 after each antibody incubation. cell extract. Samples were incubated overnight at 41C with Coverslips were stained with FITC-conjugated anti-mouse rocking and washed four times in ice-cold medium salt lysis secondary antibodies (Amersham) diluted 1 : 400 and perma- buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, nently mounted in medium containing DAPI (Vectashield). 0.3% NP-40). Immune complexes were separated by SDS– For p14ARF/nucleolin co-stains, cells were first stained PAGE. Following transfer, blots were blocked in TBS/5% for p14ARF, followed by an isotype-specific, anti-IgG2a- non-fat dried milk/0.05% Tween 20 and probed for p53 FITC conjugate (Pharmingen). Cells were then incubated in (Oncogene Ab6, 1 : 1000), MDM2 (Oncogene Ab1 and/or anti-nucleolin antibody (MBL) diluted 1 : 100, followed by clone 2A10, kindly provided by Dr Jamil Momand), actin anti-IgG1-biotin diluted 1 : 400 and Texas red-conjugated (A2066, Sigma, 1 : 100), or a cocktail of MAb13C5 (1 : 400), streptavidin (Amersham) diluted 1 : 400. Tostain murine 11D11 (1 : 800) and 5C9 (1 : 800) todetect p14ARF. All p19ARF, a polyclonal p19ARF antibody (Novus Biologicals) primary antibodies were diluted in blocking buffer prior to use. was diluted 1 : 20 in PBS/0.5% Tween 20 and cells were HRP-conjugated secondary antibodies (Amersham) were incubated overnight at 41C. FITC-conjugated, anti-rabbit diluted 1 : 2500 in blocking buffer, and blots were incubated secondary antibodies (Amersham) were diluted 1 : 400 and for 1 h at room temperature. After extensive washing, incubated for 1 h at room temperature. Co-stains of p19ARF chemiluminescent signals were detected using Pierce reagents. and B23 were performed sequentially as described above. For immunoprecipitation of p19ARF, polyclonal antibodies Signals were detected using a LEICA confocal imaging system. generated in-house were coupled to protein A–sepharose beads and 1 mg of extracts was incubated with 50 ml bead slurry overnight at 41C with rocking. Immune complexes were then Transient transfections washed and analysed as described above. Murine p53 was Expression plasmids encoding an empty vector (pcDNA3), detected using monoclonal antibody Ab1 (Oncogene, 1 : 10) full-length HDM2 or HDM2 1–440 lacking the C-terminal and p19ARF was detected using Novus Biologicals polyclonal ring finger domain were electroporated into 1 Â 107 WS1E6 antibody (diluted 1 : 100). fibroblasts and seeded onto coverslips in six-well dishes. At 7 h prior to harvesting, cells were irradiated at 7 Gy. At 24 h after Immunofluorescence analysis electroporation, cells were harvested for immunofluorescence analysis as described above. p14ARF/HDM2 co-staining was Fibroblasts were seeded onto coverslips, grown overnight at performed as described for p14ARF/nucleolin, except that anti- 1 37 C and irradiated the next morning at 7 Gy. Cells were IgG2b-biotin (1 : 400, Pharmingen) followed by streptavidin permeabilized prior to fixation as described previously (Rubbi –Texas red was used todetect HDM2. HDM2 was detected and Milner, 2000). Briefly, cells were washed once in TBS, once using Ab1 (Oncogene diluted 1 : 400). in TBS containing 25% glycerol/0.05 mM EGTA, followed by a 4-min incubation in TBS containing 25% glycerol/0.05 mM EGTA/0.05% Triton X-100. Coverslips were then fixed in 1 : 1 Acknowledgements acetone/methanol followed by hydration in PBS. Coverslips We thank Dr Geoffrey Wahl and Dr Gordon Peters for were incubated for 1 h at 251C in blocking solution (10% goat reagents and helpful discussion. We are grateful to Dr Karen serum, 0.5% Tween 20, PBS). Coverslips of NARF2 cells were Vousden for providing HDM2 expression vectors, Dr Jamil fixed in 1 : 1 acetone/methanol without permeabilization. For Momand for the 2A10 reagent, Dr Pui Chan for anti-B23 p14ARF single stains, coverslips were incubated for 1 h with antibodies and Dr Gigi Lozano for providing p53À/À MAb13C5 diluted 1 : 200 in PBS/0.5% Tween 20. Unless mdm2À/À MEFs. We alsothank Drs Emma Lees and Bruno otherwise stated, all primary and secondary antibody incuba- Amati for critical review of this manuscript. DNAX Research tions were performed at 251C and coverslips were washed four Incorporated is a subsidiary of Schering Plough Corporation.

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