[CANCER RESEARCH 60, 3493–3503, July 1, 2000] dUTP Nucleotidohydrolase Isoform Expression in Normal and Neoplastic Tissues: Association with Survival and Response to 5-Fluorouracil in Colorectal Cancer Robert D. Ladner,1 Frank J. Lynch, Susan Groshen, Yi Ping Xiong, Andrew Sherrod, Salvatore J. Caradonna, Jan Stoehlmacher, and Heinz-Josef Lenz Department of Molecular Biology, at the University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Stratford, New Jersey 08084 [R. D. L., S. J. C.]; QualTek Molecular Laboratories, Inc., Santa Barbara, California 93111 [F. J. L.]; and Division of Medical Oncology, University of Southern California/Norris Comprehensive Cancer Center, University of Southern California School of Medicine, Los Angeles, California 90033 [S. G., Y. P. X., A. S., J. S., H-J. L.] ABSTRACT survival in this limited study. Nuclear dUTPase staining within these .(0.06 ؍ tumors was also associated with TS gene expression (P Aberrant dUTP metabolism plays a significant role in the underlying This study demonstrates that low intratumoral levels of nuclear molecular mechanisms of cell killing mediated by inhibitors of thymidy- dUTPase protein expression is associated with response to 5-FU-based late biosynthesis. dUTP nucleotidohydrolase (dUTPase) is the key regu- chemotherapy, greater time to progression, and greater overall survival in lator of dUTP pools, and significant evidence exists suggesting that the colorectal cancer. Conversely, high levels of nuclear dUTPase protein expression of this enzyme may be an important determinant of cytotox- expression predict for tumor resistance to chemotherapy, shorter time to icity mediated by inhibitors of thymidylate synthase (TS). In this study, we progression, and shorter overall survival. This report represents the first have determined the expression patterns of dUTPase in normal and clinical study implicating dUTPase overexpression as a mechanism of neoplastic tissues and examined the association between dUTPase expres- resistance to TS inhibitor-based chemotherapy. sion and response to 5-fluorouracil (5-FU)-based chemotherapy and over- all survival in colorectal cancer. Immunohistochemistry was performed on formalin-fixed, paraffin- INTRODUCTION embedded tissue sections using a monoclonal antibody (MAb), DUT415, Thymidylate metabolism has long been an important target for that cross-reacts with both nuclear and mitochondrial isoforms of human widely used chemotherapeutic agents (e.g., the fluoropyrimidines) dUTPase. Nuclear and cytoplasmic staining was observed in both normal and neoplastic tissues. In normal tissues, nuclear dUTPase staining was that provide benefit in the treatment of head and neck, breast, and observed exclusively in replicating cell types. This observation is in agree- gastrointestinal cancers (1, 2). The major mechanism of action of this ment with cell culture studies where expression of the nuclear isoform class of antineoplastic drugs is the inhibition of enzymes that mediate (DUT-N) is proliferation dependent. In contrast, cytoplasmic expression of critical steps in thymidylate metabolism. The de novo biosynthesis of dUTPase does not correlate with proliferation status and was observed in TMP occurs by the reductive methylation of dUMP by the enzyme tissues rich in mitochondria. Consistent with this observation, cell culture TS2 to yield TMP, which is then converted to TTP for DNA replica- studies reveal that the mitochondrial isoform (DUT-M) is expressed con- tion (Fig. 1). The methyl donor in this reaction, MTHF, is oxidized to stitutively, independent of cell cycle status. These data suggest that in dihydrofolate so that the TS reaction constitutes a significant drain on normal tissues, nuclear staining with the DUT415 antibody represents the cellular tetrahydrofolate pools. The levels of MTHF are maintained DUT-N isoform, whereas cytoplasmic staining represents the DUT-M during TMP synthesis by the combined actions of DHFR and serine isoform. hydroxymethyltransferase (Fig. 1A). Chemotherapeutic agents such as In colon cancer tumor specimens, expression of dUTPase was shown to be highly variable in both amount and intracellular localization. Patterns 5-FU and FUdR block TMP biosynthesis by inhibiting TS directly. of dUTPase protein expression observed included exclusive nuclear, ex- Inhibitors of DHFR (e.g., methotrexate) indirectly block TMP pro- clusive cytoplasmic, and combined nuclear and cytoplasmic staining. duction by limiting the availability of MTHF (Fig. 1B). Thus, immunohistochemical detection of dUTPase in colon cancers pro- Studies attempting to elucidate the molecular mechanisms of cell vides distinct intracellular phenotypes of expression that may be of sig- killing mediated by inhibitors of TS and DHFR suggest that cytotox- nificant prognostic value. icity results from a process termed “thymineless death.” Historically, To examine the association between dUTPase expression and response cell death initiated by thymineless conditions was presumed to be the to 5-FU-based chemotherapy and overall survival, we initiated a retro- result of DNA synthesis arrest and DNA degradation because of spective study including tumor specimens from 20 patients who had extreme TTP pool depletion (3). However, more recent investigations received protracted infusion of 5-FU and leucovorin for treatment of suggest that multiple factors contribute to the underlying mechanism metastatic colon cancer. Positive nuclear staining was found in 8 patients, whereas 12 lacked nuclear expression. Of the patients lacking nuclear of thymineless death, particularly imbalance of other deoxynucleotide dUTPase expression, 6 responded to 5-FU-based chemotherapy, 4 had triphosphate pools. It has now become largely accepted that elevated stable disease, and 2 had progressive disease. Of the patients presenting dUTP pools and misincorporation of uracil into DNA play a signifi- positive nuclear dUTPase expression, 0 responded to chemotherapy, 1 had cant role in initiating DNA damage and cell death in response to The median inhibition of de novo thymidylate metabolism (Ref. 4 and references .(0.005 ؍ stable disease, and 7 had progressive disease (P survival for patients with tumors lacking nuclear staining was 8.5 months therein). Because of these findings, there is growing interest in the and 6.9 months for patients with tumors demonstrating positive nuclear role of dUTP metabolism as a mediator of cytotoxicity and as a -Time to progression was significantly determinant of efficacy in the clinical use of antithymidylate chemo .(0.09 ؍ dUTPase expression (P ؍ longer for patients with tumors lacking nuclear staining (P 0.017). therapeutics. Variable cytoplasmic dUTPase expression was observed in these tumors; In virtually all known organisms, uracil is not a native component however, there was no apparent association with clinical response or of DNA. However, uracil can arise in DNA either by the spontaneous deamination of cytosine residues or through dUTP utilization by DNA Received 12/20/99; accepted 4/28/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 2 The abbreviations used are: TS, thymidylate synthase; MTHF, 5,10-methylenetetra- 18 U.S.C. Section 1734 solely to indicate this fact. hydrofolate; DHFR, dihydrofolate reductase; 5-FU, 5-fluorouracil; FUdR, fluorodeoxyuri- 1 To whom requests for reprints should be addressed, at Department of Molecular dine; UDG, uracil-DNA glycosylase; dUTPase, dUTP nucleotidohydrolase; MAb, Biology, University of Medicine and Dentistry of New Jersey, School of Osteopathic monoclonal antibody; PBL, peripheral blood lymphocyte; PHA, phytohemagglutinin; Medicine, 2 Medical Center Drive, Stratford, NJ 08084. Phone: (856) 566-6043; Fax: DAB, diaminobenzidine; LV, leucovorin; mtDNA, mitochondrial DNA; TP, thymidine (856) 566-6232; E-mail: [email protected]. phosphorylase. 3493 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2000 American Association for Cancer Research. dUTPase EXPRESSION IN NORMAL AND NEOPLASTIC TISSUES Although dUTP is a normal intermediate in thymidylate biosynthe- sis, its extensive accumulation and misincorporation into DNA is lethal in both prokaryotic and eukaryotic organisms (8, 9). The exact biochemical basis for uracil-DNA-mediated cell death has not been definitively proven; however, there is substantial evidence suggesting that UDG-initiated repair is a central component of this process. For example, inactivation of dUTPase in Escherichia coli results in the dramatic accumulation of dUTP pools leading to extensive uracil misincorporation during replication. Under conditions of elevated dUTP pools, the cell engages in repeated cycles of uracil misincor- poration and UDG-mediated repair. This iterative process results in increased recombination, DNA strand breaks, and ultimately cell death (8). A similar phenomenon is thought to occur during inhibition of de novo thymidylate metabolism by anticancer agents (10–15). Inhibi- tion of the TS reaction leads to the accumulation of cellular dUMP pools and, as a result of mono- and diphosphate kinases, induces a dramatic increase in dUTP pools. Once levels of dUTP accumulate beyond a threshold level, overwhelming cellular dUTPase activity, the dUTP:TTP ratio increases. Under these conditions, dUTP is misin- corporated into replicating DNA, resulting in uracil-DNA-mediated cytotoxicity (Fig. 1B). Implicit