78–95 Nucleic Acids Research, 2009, Vol. 37, No. 1 Published online 16 November 2008 doi:10.1093/nar/gkn910 Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage Peter M. Wilson1, William Fazzone1, Melissa J. LaBonte1, Heinz-Josef Lenz2 and Robert D. Ladner1,* 1Department of Pathology and 2Division of Medical Oncology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA Received May 19, 2008; Revised October 28, 2008; Accepted October 29, 2008 ABSTRACT INTRODUCTION Deoxyuridine triphosphate nucleotidohydrolase Deoxyuridine triphosphate nucleotidohydrolase (dUT (dUTPase) catalyzes the hydrolysis of dUTP to Pase) is the sole enzyme responsible for the hydrolysis of dUMP and PPi. Although dUTP is a normal intermedi- dUTP to dUMP and pyrophosphate simultaneously pro- ate in DNA synthesis, its accumulation and misincor- viding substrate for thymidylate synthase (TS) and elim- poration into DNA is lethal. Importantly, uracil inating dUTP from the DNA biosynthetic pathway. Although dUTP is a normal intermediate in DNA synth- misincorporation is a mechanism of cytotoxicity esis, its extensive accumulation and misincorporation induced by fluoropyrimidine chemotherapeutic into DNA is lethal in both prokaryotic and eukaryotic agents including 5-fluorouracil (5-FU) and elevated organisms as evidenced from knockout models (1,2). expression of dUTPase is negatively correlated Importantly, uracil misincorporation also represents with clinical response to 5-FU-therapy. In this study a major mechanism of cytotoxicity induced by the we performed the first functional characterization of TS-inhibitor class of chemotherapeutic agents including the dUTPase promoter and demonstrate a role for the fluoropyrimidines 5-fluorouracil (5-FU), fluorodeox- E2F-1 and Sp1 in driving dUTPase expression. yuridine (FUdR) and capecitabine which are broadly We establish a direct role for both mutant and used in the treatment of cancers of the gastrointestinal wild-type forms of p53 in modulating dUTPase pro- tract, breast and head and neck (3). Inhibition of TS moter activity. Treatment of HCT116 p53+/+ cells induces a metabolic blockade, depleting thymidylate with the DNA-damaging agent oxaliplatin induced pools and in some instances promoting the accumulation a p53-dependent transcriptional downregulation of intracellular dUTP pools and subsequent misincorpora- of dUTPase not observed in the isogenic null cell tion of uracil into DNA resulting in DNA damage and cell line. Oxaliplatin treatment induced enrichment of death (4,5). Expression of dUTPase is reported to be an p53 at the dUTPase promoter with a concomitant important mediator of resistance to therapeutic agents reduction in Sp1. The suppression of dUTPase by that target TS both in vitro and in vivo. We previously demonstrated that diminished dUTPase expression greatly oxaliplatin promoted increased levels of dUTP that enhanced dUTP pool expansion following TS-inhibition, was enhanced by subsequent addition of fluoropy- sensitizing yeast cells to the effects of uracil misincorpora- rimidines. The novel observation that oxaliplatin tion while cells overexpressing dUTPase were significantly downregulates dUTPase expression may provide protected (6). In colon cancer cells, depletion of dUTPase a mechanistic basis contributing to the synergy by siRNA resulted in dUTP accumulation and growth observed between 5-FU and oxaliplatin in the clinic. arrest (7). Overexpression of dUTPase was also demon- Furthermore, these studies provide the first evi- strated to confer resistance to FUdR (8), while we pre- dence of a direct transcriptional link between the viously reported that depletion of dUTPase by siRNA essential enzyme dUTPase and the tumor suppres- sensitized both breast and colon cell lines to FUdR sor p53. through misincorporation of dUTP and enhanced DNA *To whom correspondence should be addressed. Tel: +1 323 865 3116; Fax: +1 323 865 0522; Email: [email protected] ß 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2009, Vol. 37, No. 1 79 damage (9). Moreover, we also reported the results of In MCF-7 (p53 wild-type) breast cancer cells, microarray a retrospective clinical study negatively correlating analysis also identified dUTPase mRNA within an exten- elevated nuclear expression of dUTPase with response to sive panel of genes repressed following 5-FU treatment 5-FU-therapy in colorectal cancer patients (10). These (21). However, the precise mechanism of the downregula- studies bolster the concept that dUTPase represents an tion of dUTPase has not been determined and it is attractive drug target and we recently reported the identi- unknown as to whether this phenomenon is the result of fication of novel small molecules with dUTPase inhibitory indirect downstream events induced by p53 itself or its activity (11). transactivation and repressive gene targets. Furthermore, While detailed analyses of dUTPase structure and dUTPase was one of a number of genes identified as catalytic activity have been described, the mechanisms upregulated in p53-null mouse embryonic fibroblasts fol- that govern human dUTPase gene regulation and expres- lowing introduction of the human tumor-derived p53 sion remain largely unknown (12–14). Previous R175H by subtraction hybridization (24). studies have reported that expression of the nuclear iso- As dUTPase is an essential enzyme in maintaining geno- form of dUTPase (DUT-N) is primarily cell cycle and mic stability, and demonstrates both aberrant intratu- proliferation-dependent, whereas the mitochondrial iso- moral expression and an association with resistance to form (DUT-M) is constitutively expressed (15). Immuno- 5-FU, we sought to perform the first functional character- histochemical staining of normal tissues demonstrated ization of the promoter and elucidate the mechanisms that high DUT-N expression is exclusively observed in involved in regulating dUTPase expression. In addition, replicating cell types whereas cytoplasmic expression is p53 mutations are widely observed in many cancers and as observed in mitochondria-rich tissues (10). However, the fluoropyrimidines remain the mainstay chemothera- both colon cancer cell lines (16) and tumor specimens peutics in gastrointestinal cancer treatment, characterizing demonstrate dramatic variation, both in magnitude of a role for p53 in regulating dUTPase gene expression in dUTPase expression and intracellular localization. tumor cells may be of major clinical significance and may Furthermore, the correlation between replication status lead to more targeted therapeutic options. In this study, and DUT-N expression was not observed in colon adeno- we demonstrate direct roles for Sp1 and E2F-1 in driving carcinomas (17). Importantly, dysregulation of dUTPase basal DUT-N expression and report a direct role for wild expression is observed in tumor types which are frequently type and mutant p53 in repressing and inducing dUTPase treated with agents that target TS, therefore elucidating promoter activity respectively. Furthermore, we demon- the molecular basis driving dUTPase expression is impor- strate the ability of oxaliplatin, an important chemother- tant from both a basic science and clinical standpoint apeutic agent primarily used in combination with 5-FU in (10,16,17). Functional analyses of various S-phase-specific the treatment of colorectal cancer, to acutely suppress the genes including TS and thymidine kinase (TK) identified dUTPase promoter, mRNA and protein expression and several transcriptional elements that are commonly pre- enzymatic activity in a p53-dependent manner. We also sent in their promoter regions including E2F and Sp1 present evidence that p53 is associated with the endogen- consensus sites. These genes are also characterized by a ous dUTPase promoter in vivo, and propose that inter- lack of TATA or CAAT box initiation sites, but are rich in ference with transcription machinery at the dUTPase GC boxes. Promoter sequence analysis of the DUT gene promoter by wild-type p53 represents a plausible mecha- reveals putative regulatory motifs including potential nism for this transcriptional repression. Finally, suppres- binding sites for NF-kB, E2F and Sp1 transcription fac- sion of dUTPase by oxaliplatin resulted in the expansion tors (15). Recently, a genome-wide ChIP-on-chip identi- of intracellular dUTP pools and subsequently enhanced fied dUTPase in a subset of 127 genes bound by E2F the downstream effects of TS-inhibition. This interaction family members (18). Despite the presence of these puta- may contribute to the clinical synergy observed between tive S-phase-specific binding sites in the DUT-N promoter 5-FU and oxaliplatin in the treatment of colorectal cancer. region, functional analysis of this gene has not been pre- viously reported. Several studies have also reported downregulation of MATERIALS AND METHODS dUTPase during apoptosis (19,20) and that dUTPase Compounds and reagents expression may be modulated by the tumor suppressor gene p53 (21,22). In response to stress stimuli such as 5-FU, fluorodeoxyuridine (FUdR), paclitaxel, doxycycline DNA damage, p53 can initiate