ORIGINAL ARTICLE PRIMA-1 MET induces nucleolar accumulation of mutant p53 and PML nuclear body-associated proteins N Ro¨kaeus 1 , G Klein 2 , KG Wiman 1 , L Szekely 2 and K Mattsson 2 1 Department of Oncology-Pathology, Karolinska Institute, Cancer Center Karolinska (CCK), Karolinska University Hospital, Stockholm, Sweden and 2 Microbiology and Tumor Biology Center (MTC), Karolinska Institute, Stockholm, Sweden We have previously identified PRIMA-1, a low molecular weight compound that restores the transcriptional trans- activation function to mutant p53 and induction of apoptosis. To explore the molecular mechanism for PRIMA-1-induced mutant p53-dependent apoptosis, we examined the intracellular distribution of mutant p53 upon treatment with PRIMA-1 MET by immunofluorescence staining. We found that PRIMA-1 MET induced nucleolar translocation of mutant p53 and the promyelocytic leukemia (PML) nuclear body-associated proteins PML, CBP and Hsp70. Levels of Hsp70 were significantly enhanced by PRIMA-1 MET treatment. PRIMA-Dead, a compound structurally related to PRIMA-1 but unable to induce mutant p53-dependent apoptosis, failed to induce nucleolar translocation of mutant p53. Our results suggest that redistribution of mutant p53 to nucleoli plays a role in PRIMA-1-induced apoptosis. Oncogene advance online publication, 14 August 2006; doi:10.1038/sj.onc.1209858 Keywords: mutant p53; PRIMA-1; nucleolus; PML bodies; Hsp70 Introduction The tumor suppressor p53 induces cell-cycle arrest, apoptosisand/orsenescenceinresponsetocellularstress such as DNA damage, oncogene activation, hypoxia andtelomereerosion(forareview,seeVousdenandLu (2002)). p53 is a transcription factor that binds DNA andactivatestranscriptionoftargetgenesincludingp21, MDM2,GADD45,BaxandPUMA.Thecriticalroleof p53 as tumor suppressor is illustrated by the fact that p53 is mutated in more than 50% of human tumors, including most if not all tumor types (Olivier et al., 2002).Thegreatmajorityofp53mutationsaremissense mutationsintheDNA-bindingcoredomain,resultingin inactivation or impairment of p53 DNA binding and transcriptional activity. Point mutant p53 is often expressed at high levels in tumor cells, due to failure to transactivate the p53 antagonist MDM2 which targets p53 for proteasome-mediated degradation (reviewed by Vousden and Lu (2002)). Mutant p53- carrying tumors are usually more resistant to currently used anticancer therapy. Therefore, reconstitution of wild–type p53 function in human tumors is an important therapeutic goal. Several low molecular weight compounds that reactivate mutant p53 have been identified, for example, CP-31398 (Foster et al., 1999), PRIMA-1 (Bykov et al., 2002a,b) and MIRA-1 (Bykov et al., 2005a). PRIMA-1 restores wild-type conformationandDNAbindingtomutantp53proteins andinducesapoptosisinhumantumorcellsinamutant p53-dependent manner. PRIMA-1 MET , a methylated form of PRIMA-1, is even more potent in inducing mutant p53-dependent apoptosis than PRIMA-1 itself (Bykov et al., 2005b). The promyelocytic leukemia nuclear body (PML- NBs, also termed Kremer bodies, PML oncogenic domains, PODs, nuclear domain 10, ND10, NBs or nuclear dots, NDs) are dynamic macromolecular structures dependent on the tumor suppressor PML for its formation. PML was originally identified as the fusionpartnerwiththeretinoicacidreceptorRARalpha in leukemic blasts from patients suffering from acute promyelocytic leukemia (APL), leading to disrupted PML-NBs (Melnick and Licht, 1999). Accumulating evidence indicates that PML-NBs play regulatory roles in cellular processes such as cell growth (Fagioli et al., 1998; Cohen et al., 2001), chromatin remodeling (Amin etal.,2001),cellulardifferentiation(Drouin etal.,2001), protein degradation (Lallemand-Breitenbach et al., 2001), transcriptional regulation (Li et al., 2000; Zhong et al., 2000), antiviral response (Everett, 2001; Regad and Chelbi-Alix, 2001), cellular senescence (Ferbeyre et al., 2000; Pearson et al., 2000; Bischof et al., 2002; Langley et al., 2002), tumor suppression (reviewed by Salomoni and Pandolfi (2002)), DNA repair (Bischof et al., 2001; Carbone et al., 2002) and apoptosis (Wang etal.,1998;Fogal etal.,2000;Guo etal.,2000;D’Orazi et al., 2002; Hofmann et al., 2002). Todatemorethan40differentcellularproteinsanda number of viral proteins have been found to colocalize with PML in the PML-NBs. Several reports indicate Received 22 November 2005; revised 4 April 2006; accepted 3 July 2006 Correspondence: Professor KG Wiman, Department of Oncology- Pathology, Karolinska Institute, Cancer Center Karolinska (CCK), R8:04 Karolinska University Hospital, Stockholm, SE-171 76, Sweden. E-mail: Klas.Wiman@ki.se Oncogene (2006), 1–11 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc