Oncogene (2002) 21, 4595 – 4600 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc ORIGINAL PAPERS Novel gain of function activity of p53 mutants: activation of the dUTPase gene expression leading to resistance to 5-fluorouracil Elena N Pugacheva1,4,5, Alexey V Ivanov2,6, Julia E Kravchenko1, Boris P Kopnin3, Arnold J Levine4,7 and Peter M Chumakov*,1,2,6 1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, 117984 Moscow, Russia; 2Department of Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois, IL 60607, USA; 3Institute of Cancerogenesis, Russian Cancer Research Center, Moscow, Kashirskoye shosse 24, 115478 Moscow, Russia; 4Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA Mutated forms of p53 are often expressed in a variety of Introduction human tumors. In addition to loss of function of the p53 tumor suppressor, mutant p53s contribute to malignant The p53 tumor suppressor gene plays a key role in process by acquisition of novel functions that enhance maintaining genetic stability and protection against transformed properties of cells and resistance to antic- cancer. Accumulation and activation of functional p53 ancer therapy in vitro, and increase tumorigenecity, protein occurs in response to a variety of stresses and invasiveness and metastatic ability in vivo. Searching for insults potentially leading to genetic alterations genes that change expression in response to p53 gain of (Levine, 1997; Pugacheva et al., 2000). Acting mainly function mutants may give a clue to the mechanisms (but not exclusively) as a sequence-specific transcrip- underlying their oncogenic effects. Recently by subtrac- tion factor, p53 is capable of activating a set of genes tion hybridization cloning we found that the dUTPase required for cell cycle arrest or apoptosis (for review gene is transcriptionally upregulated in p53-null mouse see el-Deiry (1998); Sionov et al. (1999)). In this fibroblasts expressing the exogenous human tumor- manner the p53 gene controls elimination of potential derived His175 p53 mutant. Here we show that progenitors of malignant tumor cells. p53 deficient cells conditional expression of His175 and Trp248 hot-spot show greatly increased genetic instability, and most of p53 mutants in p53-negative mouse 10(1) fibroblasts and the p53 knockout mice develop malignant diseases by 6 human SK-OV3 and H1299 tumor cells results in months of age (Donehower et al., 1992). Different increase in dUTPase gene transcription, an important types of abnormalities in the p53 signaling pathways marker predicting the efficacy of cancer therapy with are observed in a majority of human malignancies. The fluoropyrimidine drugs. Using tetracycline-regulated most frequent type of defect represents missense retroviral vectors for conditional expression of p53 mutations within the p53 gene itself. Unexpectedly a mutants, we found that transcription of the dUTPase small fraction of mutations in the tumors corresponds gene is increased within 24 h after tetracycline with- to nonsense or frame shift mutations and that drawal, and the cells acquire higher resistance to 5-FU. discriminates the p53 gene from other tumor-suppres- Additional inactivation of the N-terminal transcription sors, such as RB or APC (Levine et al., 1995). This fact activation domain of mutant p53 (substitutions in amino- suggests that the presence of a mutated protein in acid residues 22 and 23) results in abrogation of both addition to the loss of wild type p53 protein might induction of dUTPase transcripts and 5-FU resistance. confer selective growth advantage and promote clonal Oncogene (2002) 21, 4595 – 4600. doi:10.1038/sj.onc. expansion (Greenblatt et al., 1994). In contrast to a 1205704 classical tumor suppressor gene, a missense mutation in the p53 gene can lead not only to loss of function (both Keywords: mutant p53; drug resistance; gain of in cis by mutation itself, or in trans by a dominant- function negative mechanism), but also to a gain of function. In particular, novel activities of mutant p53 are capable of promoting tumorigenicity when introduced into p53- *Correspondence: PM Chumakov, Department of Molecular Biol- null cell types (Dittmer et al., 1993; Michalovitz et al., ogy, Lerner Research Institute, Cleveland Clinic Foundation, 9500 1991). Also, over-expression of mutant p53 proteins in Euclid Avenue, Cleveland, Ohio, OH 44195, USA; p53-null cells of several lineages enhances their E-mail: [email protected] Current addresses: 5Fox Chase Cancer Center, 7701 Burtholme transformed properties and plating efficiency in culture, Avenue, Philadelphia, Pennsylvania, PA 19111, USA; 6Department and increases tumorigenicity and tissue invasiveness in of Molecular Biology, Lerner Research Institute, Cleveland Clinic mice (Dittmer et al., 1993; Gloushankova et al., 1997; Foundation, 9500 Euclid Avenue, Cleveland, Ohio, OH 44195, USA; Hsiao et al., 1994; Kremenetskaya et al., 1997; Sun et 7Rockefeller University, 1230 York Avenue, New York, NY 10021, USA al., 1993; Wolf et al., 1984). Introduction into p53-null Received 2 January 2002; revised 15 May 2002; accepted 22 May mice of a mutant p53 allele changes the tumor 2002 spectrum and results in higher frequencies of metas- Mutant p53s increase resistance to 5-FU EN Pugacheva et al 4596 tases (Liu et al., 2000). Some of the p53 mutants of dUTPase expression in colon cancer showed its provide increased resistance to p53-independent apop- strong correlation with poor response to 5-FU therapy tosis induced by a variety of treatments, including and a worse prognosis (Ladner et al., 2000). certain chemotherapeutic drugs (Kremenetskaya et al., In this study we show that certain types of mutant 1997; Li et al., 1998; Lotem and Sachs, 1995; Peled et p53s are capable of inducing expression of the al., 1996). For example, in p53 negative lung carcinoma dUTPase gene and, as a result, increasing resistance cell line H1299 introduction of His175 and His179 p53 of cells to fluoropyrimidine drugs. This observation mutants significantly reduces the rate of etoposide- provides a possible mechanism for increased resistance induced apoptosis, although His273 and Trp248 of certain malignant tumors to 5-FU, and may have mutants provide a much milder effect, indicating an potential value for prediction of sensitivity to allele specific impact, which provides further evidence chemotherapy based on analysis of mutant p53 that mutant p53 protein confers this activity (Blandino expression. et al., 1999). The mechanisms underlying gain of function activ- ities of p53 mutants remain poorly understood. Some Results and discussion studies emphasize the importance of the N-terminal transcription-activation domain for expression of We tested whether expression of different forms of mutant p53 gain of function phenotype (Lanyi et al., tumor-derived p53 mutants introduced into p53 1998; Lin et al., 1995). Mutant p53s seem to be capable negative cells can affect dUTPase expression and of activating promoters of genes that are usually not sensitivity to 5-FU. Constitutive ectopic expression of activated by the wild type protein. Among these are His175 and Ala281 p53 mutants in p53-negative mouse promoters of MDR1 (Chin et al., 1992; Dittmer et al., cell line 10(3) results in significant increase in dUTPase 1993; Kopnin et al., 1995), PCNA (Deb et al., 1992), transcripts as seen by Northern blot hybridization HSP70 (Tsutsumi-Ishii et al., 1995), 15-lipoxygenase (Figure 1a). This result is consistent with the fact that (Kelavkar and Badr, 1999), BAG-1 (Yang et al., 1999) the dUTPase cDNA clone was present in a high and c-myc (Frazier et al., 1998) genes. It is not clear proportion of a subtraction library from 10(3)-His175 whether mutant p53 acts directly as a transcription vs 10(3) cell lines. factor with altered sequence specificity, or modifies Conditional expression vectors were then used to expression of the genes by interaction with factors study immediate effects of mutant p53s. Various forms responsible for positive or negative transcriptional of mutant p53 cDNAs were inserted into self- regulation. Taking into account how common missense inactivating retroviral vectors designed for tetracy- p53 mutants are in human cancer, it is clear that cline-regulated gene expression. Three different p53 understanding the mechanisms underlying this gain of deficient cell lines bearing a tetracycline dependent function as well as identification of genes that are transactivator protein, tTA, were infected with recom- regulated by p53 mutants may help in development of binant retroviral stocks obtained after transfection of rational strategies of cancer treatment. the DNA constructs into the packaging cell line In a recent study using subtraction hybridization Phoenix. Mass cultures obtained after selection with cloning we have isolated several cDNA clones G418 were induced to express p53 by withdrawal of corresponding to genes that are activated in p53- doxacycline from the medium. Twenty-four hours after negative mouse fibroblast cell line 10(3) following induction, strong signals corresponding to p53 were expression of exogenous tumor-derived human mutant seen on Western blots (Figure 1b,d). In the mouse p53 p53-His175 (Pugacheva et al., 2000). One of the most deficient fibroblast cell line 10(1) induction of His175 abundant cDNAs differentially expressed in the 10(3)- p53 mutant expression correlated with a significant p53His175 cell line corresponded