ORIGINAL ARTICLE Genotoxic exposure: novel cause of selection for a functional DN-p53 isoform JPM Melis 1 , EM Hoogervorst 1 , CTM van Oostrom 1 , E Zwart 1 , TM Breit 2 , JLA Pennings 1 , A de Vries 1 and H van Steeg 1 1 Laboratory for Health Protection Research, National Institute of Public Health and the Environment, Bilthoven, Utrecht, The Netherlands and 2 MicroArray Department, Faculty of Science, University of Amsterdam, Amsterdam, The Netherlands The p53 gene is frequently mutated in cancers and it is vital for cell cycle control, homeostasis and carcinogen- esis. We describe a novel p53 mutational spectrum, different to those generally observed in human and murine tumors. Our study shows a high prevalence of nonsense mutations in the p53 N terminus of 2-acetylaminofluorene (2-AAF)-induced urinary bladder tumors. These nonsense mutations forced downstream translation initiation at codon 41 of Trp53, resulting in the aberrant expression of the p53 isoform DN-p53 (or p44). We propose a novel mechanism for the origination and the selection for this isoform. We show that chemical exposure can act as a novel cause of selection for this truncated protein. In addition, our data suggest that the occurrence of DN-p53 accounts, at least in mice, for a cancer phenotype. We also show that gene expression profiles of embryonic stem (ES) cells carrying the DN-p53 isoform in a p53-null back- ground are divergent from p53 knockout ES cells, and therefore postulate that DN-p53 itself has functional transcriptional properties. Oncogene (2011) 30, 1764–1772; doi:10.1038/onc.2010.552; published online 13 December 2010 Keywords: DNA damage; gene expression; N-terminal p53 isoform (DN-p53, p44, p47); nonsense mutations; mouse urinary bladder tumor Introduction The tumor suppressor p53 regulates significant cellular functions to prevent the initiation and prolongation of cancer and is intensively studied. Several isoforms of p53 have been identified (Courtois et al., 2002; Candeias et al., 2006; Grover et al., 2009; Marcel and Hainaut, 2009), and point mutations influencing the functionality of p53 were elucidated and mimicked in vivo (de Vries et al., 2002; Wijnhoven et al., 2005; Hoogervorst et al., 2005a; Iwakuma and Lozano, 2007; Heinlein et al., 2008). The p53 gene is the most frequently mutated gene in cancer and analysis of many different human tumor types have shown a high prevalence of missense mutations located primarily in the central DNA-binding domain (Levine, 1997). Frequently, these mutations result in the expression of mutant p53 proteins, which are often more stable than wild-type p53 (Hailfinger et al., 2007). A small variety of isoforms, which lack an N-terminal and/or C-terminal part of the protein, have been discovered (Courtois et al., 2002; Marcel and Hainaut, 2009), and can result in a dominant-negative inhibition of the wild-type p53 protein (de Vries et al., 2002). However, a gain-of-function phenotype of mutant p53, which enhances the oncogenic properties of p53, has also been proposed (Strano et al., 2007; Xu, 2008). To gain more functional insight into p53 and the effect of alterations in its gene, several transgenic mouse models have been generated (Lozano, 2007). For example, the Trp53 þ /À model appears to be more susceptible than wild-type mice to carcinogenesis, both spontaneous and chemically induced (Hoogervorst et al., 2004, 2005b). We have previously investigated the effect of p53 heterozygosity to the exposure of the carcinogen 2-acetylaminofluorene (2-AAF), a strong inducer of urinary bladder tumors (Hoogervorst et al., 2004). As stress signals like DNA damage can affect p53 function, we also investigated Trp53 þ /À in combination with a deficiency in the DNA repair gene Xpa. Carcinogenesis in both models is putatively caused by Trp53 mutations in the remaining wild-type allele, shown by the presence of mutated p53 protein in early atypical preneoplastic lesions and tumors (Hoogervorst et al., 2004). Here, we reveal a mutational spectrum in tumors divergent from that generally observed after carcinogen exposure (http://p53.free.fr/index.html). A high frequency of predominantly N-terminal non- sense mutations was found in the Trp53 gene, resulting in the selective expression of an N-terminally truncated p53-isoform (DN-p53). We further investigated the impact of this isoform by creating and analyzing embryonic stem (ES) cells harboring a nonsense muta- tion at codon 5, expressing the DN-p53 isoform. We showed that this isoform has functional transcriptional properties that differ from both wild-type and p53 knockout cells. Received 17 March 2010; revised 17 August 2010; accepted 26 October 2010; published online 13 December 2010 Correspondence: Professor H van Steeg, Laboratory for Health Protection Research, National Institute of Public Health and the Environment, PO Box 1, Bilthoven, Utrecht 3720 BA, The Netherlands. E-mail: Harry.van.Steeg@rivm.nl Oncogene (2011) 30, 1764–1772 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc