Dutp Nucleotidohydrolase Isoform Expression in Normal and Neoplastic Tissues: Association with Survival and Response to 5-Fluorouracil in Colorectal Cancer

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[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 overall 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

Although dUTP is a normal intermediate in thymidylate biosynthesis, 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 misincorporation 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 misincorporated into replicating DNA, resulting in uracil-DNA-mediated cytotoxicity (Fig. 1B). Implicit to this model of cell killing is the central role of dUTPase. As the main regulator of dUTP pools, the expression of dUTPase could have profound effects on the utility of chemotherapeutics that inhibit thymidylate biosynthesis. Normally, dUTPase mediates a protective role by limiting the expansion of dUTP pools and countering the cytotoxic effect of uracil misincorporation. According to this model, elevated levels of dUTPase would prevent the accumulation of dUTP required for cell killing. To test this hypothesis, Canman et al. (16) ectopically overexpressed the E. coli dUTPase in a FUdR-sensitive human colorectal tumor cell line (HT29) and measured the response to the TS inhibitor FUdR. The manipulated cell lines (containing dUTPase activity 4–5-fold higher than controls) were protected from FUdR-induced DNA strand breaks and showed an increased viability over control cells (16). This study provided the first evidence in human cells implicating dUTPase enzyme levels as an important factor in determining the efficacy of TS inhibition. Although the contribution of uracil-DNA-mediated cytotoxicity toward overall cell death is likely to vary between different cell lines (17), these data support the role of dUTP pool imbalance and uracil misincorporation as a contributing mechanism of TS inhibitor- based cytotoxicity. Cancer of the gastrointestinal system is one of the leading causes of Fig. 1. Partial schematic diagram of de novo thymidylate metabolism. A, this simplified diagram of TMP synthesis illustrates the role of thymidylate synthase, dUTPase, DHFR, cancer death, and the fluoropyrimidines remain the chemotherapeutic and serine hydroxymethyltransferase. Under normal cellular conditions, dUTP pools are agent of choice used to combat these diseases (18). The difficulty in eliminated by the action of dUTPase. See text for details. B, this simplified diagram successfully treating patients with this class of drugs is the high illustrates the mechanism of action of inhibitors of TS (5-FU and FUdR) and DHFR (methotrexate). Inhibition of the TS reaction leads to the accumulation of dUTP, which is incidence of resistance intrinsic to these cancers. Because resistance is misincorporated into DNA, leading to DNA strand breaks and ultimately cell death by the a common phenomenon, understanding and predicting response and action of uracil-DNA glycosylase. See text for details. overall patient outcome is of great importance for the clinical evaluation and management of the cancer patient. Considerable effort has polymerases during replication (5, 6). Because cytosine deamination been directed toward identifying useful molecular markers that predict can lead to G:C to A:T transition mutations, the cell has evolved for patient response to chemotherapy and overall survival in gastro- highly efficient mechanisms to facilitate the exclusion of uracil from intestinal cancers. For example, recent advances have focused on DNA (7). When uracil does occur in DNA, UDG initiates the base- measuring the intratumoral expression of molecular determinants of excision repair pathway to remove and correct the misincorporated drug action (i.e., TS and TP) and mechanisms of response to DNA nucleotide. To prevent dUTP utilization during DNA replication, the damage (e.g., p53; Refs. 19–23). However, there has been no clinical enzyme dUTPase hydrolyzes dUTP to yield dUMP and PPi. This evaluation of potential predictive markers involved in dUTP metab- reaction effectively eliminates dUTP from the DNA biosynthetic olism. Considering the central role of dUTPase in uracil-DNA-medi- pathway and also provides substrate (dUMP) for the de novo synthesis ated cytotoxicity, we conducted a retrospective study to evaluate the of thymidylate. Therefore, under normal cellular conditions, the main- ability of dUTPase isoform expression to predict for response to tenance of uracil-free DNA is achieved through the combined actions 5-FU-based chemotherapy and overall patient survival in colorectal of dUTPase and UDG. cancers. 3494

PATIENTS AND METHODS for 30 min (Vector Elite). After a final rinse in PBS, the tissues were incubated with DAB substrate (0.02% DAB, 0.005% hydrogen peroxidase) for 15 min to Antibodies, Immunoprecipitation, Western Blot Analysis, and Enzyme develop the colorimetric reaction, then counterstained in hematoxylin 100% Assays. dUTPase-specific MAbs (MAb DUT415) were generated and pre- (GILL 11 formula), and mounted with glass coverslips using Permount. pared as described previously (24). The DUT415 MAb is useful for immuno- Antibodies for Immunohistochemistry. All antibodies were individually precipitation experiments and immunohistochemistry but is not effective for optimized for working dilutions. All tissue samples were pretreated by steam- immunoblot analysis. For immunoblot analysis, dUTPase-specific polyclonal ing as described above for antigen recovery. The DUT415 MAb was generated antibodies were generated against recombinant DUT-N protein (expressed in a in our laboratory and used at 2 ␮g/ml. Ki67 (MIB-1 clone) was purchased from baculovirus system) as described previously (25). The dUTPase-specific poly- Immunotech and used at a dilution of 1:60. The mitochondrial marker 113-1 clonal antibodies were purified by immunoaffinity chromatography and used (Mito-M) was purchased from BioGenex and used at a concentration of 1:100. for immunoblot analysis at a dilution of 1:1000. Immunoprecipitation of Patients. Specimens from 20 patients with metastatic colon cancer were dUTPase was performed using DUT415. Total HeLa S3 cell extracts (1 mg) available for analysis of dUTPase expression. These are a subset of 36 patients were incubated for 2 h with 5 ␮g of DUT415. Protein A-Sepharose (Sigma) who had received protracted infusion of 5-FU with leucovorin for treatment of was added (150 ␮l of a 10% solution) to complex and pellet the antigen- the metastatic disease and who had consented to have biopsies for analysis of antibody complexes. The immunoprecipitations were washed five times in molecular markers. Tumor specimens from these patients had been analyzed RIPA buffer (150 mM NaCl, 1.0% NP40, 0.5% deoxycholic acid, 0.1% SDS, previously for TS gene expression by reverse transcription-PCR (28). and 50 mM Tris, pH 8.0) and subsequently prepared for SDS-PAGE analysis Patients who experienced a complete response or partial response (Ͼ50% as described previously (25). For Western/immunoblot analysis, protein was reduction of measurable tumor) were classified as responders. Survival was fractionated by 15% SDS-PAGE and transferred to nitrocellulose. The mem- calculated as the time from start of treatment until death of any cause or until brane was blocked in TBST [10 mM Tris (pH 8.0), 150 mM NaCl, and 0.05% last date known to be alive. Time to tumor progression (Ͼ25% increase in Tween 20] containing 5% powdered skim milk. Antibody incubations were tumor mass) was determined from patients that had stable disease or responded performed as described previously (25). Visualization of the protein bands was to chemotherapy. Response and survival information was available for all 20 performed using the ECL chemiluminescent Western blotting detection system patients included in this study. See Table 1 for a summary of the demographic (Amersham Corp). characteristics of these patients. Cell Culture, Peripheral Blood Lymphocyte Preparation, and Meta- Statistical Analysis. Contingency tables and Fisher’s exact test were used bolic Labeling. All cell lines were maintained in a humidified atmosphere of to summarize the association between dUTPase expression in the nucleus and

5.5% CO2 at 37°C. HeLa S3 cells (CCL 2.2) were purchased from American in the cytoplasm with other molecular variables and with tumor response to Type Culture Collection and maintained in DMEM supplemented with 5% chemotherapy. The t test after logarithm transformation was used to compare FCS purchased from Life Technologies, Inc. Human PBLs were prepared from TS levels according to dUTPase status. Kaplan-Meier plots (29) and the venous whole blood with LeucoPREP cell separation tubes (Becton Dickinson) log-rank test (30) were used to compare survival of patients according to using the manufacturer’s recommendations. PBLs were cultured in RPMI 1640 dUTPase expression. Median survival was calculated based on the Kaplan- supplemented with 10% fetal bovine serum. PBLs were stimulated by the Meier estimator. All Ps are two-sided. addition of PHA to a final concentration of 15 ␮g/ml (Sigma). The onset of 3 DNA replication was monitored in stimulated PBLs by [ H]thymidine incor- RESULTS poration. At 12-h intervals after PHA stimulation, cells were labeled for 30 min with 10 ␮lof[3H]thymidine (1 mCi/ml). The remainder of the procedures was Isoforms of Human dUTPase. The overall goal of this study is to performed as described previously (26). assess the utility of measuring intratumoral dUTPase isoform expres- Cytospins and Immunocytochemistry. Cultured cells were harvested at sion as predictive markers of tumor response to 5-FU-based chemo- the appropriate times, washed in PBS, counted, and resuspended in PBS at therapy and overall patient survival in colon cancer. To begin this 3 ϫ 105 cells/ml. Positively charged slides were placed in cytocarriers (adapter analysis, it is important to understand the molecular and biochemical 1024) for the IEC-Centra-8R centrifuge (International Equipment Co.). One-ml aliquots of the cell suspension were centrifuged at 400 ϫ g for 5 min. details of the dUTPase isoforms, the specificity of the monoclonal Cells were fixed in 4% paraformaldehyde in PBS (pH 7.2) for 30 min at room antibody used to detect these proteins in patient tumor specimens, and temperature while the slides were still attached to the slide carrier. Slides were how these proteins are expressed in normal tissues. removed from the carrier and washed five times for 2 min in PBS (pH 7.0). In previous studies, we identified and characterized distinct mito- Cells were then lightly digested with Proteinase K Dako cat# 53020 diluted chondrial and nuclear isoforms of dUTPase in humans (DUT-M and 1:500 with PBS for 15 min at room temperature. Cells were washed again five DUT-N, respectively) that have identical kinetic affinities for dUTP times in PBS, and immunocytochemistry was performed using the ABC- (25). Detailed mass spectrometry-based analysis of native dUTPase alkaline phosphatase protocols. dUTPase-specific MAb (DUT415) was used at proteins revealed that the isoforms are largely identical, differing only ␮ a concentration of 2 g/ml and incubated for 30 min at room temperature. in a short region of their NH termini (25, 31). Fig. 2A summarizes Immunohistochemistry. Formalin-fixed, paraffin-embedded human tissue 2 these data in schematic format. The protein sequences shown indicate specimens were sectioned at 4–5 ␮m and placed on Capillary Gap charged and precleaned glass microscope slides (Ventana Medical Systems, Inc.). The tissue sections were deparaffinized in xylene and rehydrated through graded Table 1 Basic demographic information alcohol solutions. Prior to immunohistochemical labeling, epitope recovery Metastatic colon cancer was performed by steam pretreatment (Ref. 27 and references therein). Cap- illary Gap slide pairs were placed in a slide holder in 10 mM sodium citrate (pH No. of patients 20 Males 12 (60%) 6.0). Water was brought to a boil in a steamer (Handy Steamer Plus; Black and Females 8 (40%) Decker), and the entire slide holder was placed in the upper chamber of the Median age (range) 61 (33–81)yr steamer with the lid on and steamed for 20 min. After steam pretreatment, the Ethnicity slides were cooled for 10 min, rinsed in PBS for 5 min, and incubated in a 3% Asian 4 (20%) Hispanic 6 (30%) hydrogen peroxide solution for 5 min to quench any endogenous peroxidase Black 1 (5%) activity. The tissues were rinsed again in PBS and placed in normal goat serum White 9 (45%) (5% in PBS) for 5 min to reduce nonspecific staining. The tissues were Response to chemotherapy subsequently incubated with primary antibody (e.g., DUT415) for 30 min at Number evaluable 20 Number of responses 6 (30%) room temperature. After incubation, the tissues were rinsed in PBS and Median time to progressiona 4.4 mo incubated in biotinylated goat-anti-mouse secondary antibody for 30 min Median survivala 8.3 mo (Vector Laboratories). The slides were washed again in PBS to reduce non- a Median survival and time to progression were calculated based on the Kaplan-Meier specific staining of secondary antibody and incubated with ABC-peroxidase estimator. 3495

Fig. 2. Protein structure and Western blot analysis of dUTPase isoforms. A, DUT-N and DUT-M proteins were characterized in detail by a combination of NH2-terminal sequencing and mass spectrometry (25, 31, 32). The protein sequences presented here illustrate the unique NH2 termini and overlapping regions of the DUT-N and DUT-M proteins. Large lettering indicates overlapping sequence. The remainder of the protein sequences not shown is identical between the two isoforms. The underlined region of DUT-N identifies a consensus target sequence for cyclin-dependent protein kinase phosphorylation. The bold face and italicized lettering indicates the in vivo site of DUT-N serine phosphorylation. B, to demonstrate the cross-reactivity of DUT415, dUTPase protein was immunoprecipitated from total HeLa cell extract and fractionated by 15% SDS- PAGE. dUTPase protein isoforms were detected by Western blot analysis using dUTPase-specific polyclonal antibodies. The identity of each dUTPase isoform is indicated, and molecular weight stand- ards are expressed in thousands.

native NH2-terminal sequence and illustrate where the isoforms over- PBLs were isolated from venous whole blood and either maintained lap in their primary sequences. The identity of the amino acid se- in culture in a resting state or were stimulated by the addition of PHA. quence at the protein level reflects the fact that the same nuclear gene Fig. 3A illustrates a time-course experiment where protein extracts encodes both isoforms, with each isoform arising through the use of from equivalent numbers of PBLs were fractionated by SDS-PAGE alternative 5Ј exons (32). Additionally, the DUT-N isoform is phos- and dUTPase was detected by Western blot analysis. Time points phorylated on Ser-11 in vivo (31). were taken at 24 and 48 h after PHA stimulation, including negative To characterize dUTPase expression in human tissues, we devel- controls. Untreated PBLs express the DUT-M isoform at both 24- and oped an immunohistochemical assay using a dUTPase-specific MAb, 48-h time points, whereas the DUT-N isoform is undetectable in these DUT415. To account for the expression of both isoforms during quiescent cells. However, after PHA addition, DUT-N undergoes a immunohistochemistry analysis, it is essential to demonstrate that the dramatic induction as PBLs are stimulated to proliferate. Analysis of antibody used in these experiments is able to recognize both protein DNA replication by [3H] thymidine incorporation indicates that PBLs species. To demonstrate cross-reactivity, dUTPase protein was first immunoprecipitated from total HeLa cell extract with DUT415 MAb and fractionated by 15% SDS-PAGE. The protein isoforms were then visualized by immunoblot analysis using dUTPase-specific polyclonal antisera. We used the polyclonal antibody in this experiment because DUT415 is not useful for immunoblot analysis. The Western blot shown in Fig. 2B demonstrates that DUT415 is able to immunopre- cipitate both nuclear and mitochondrial isoforms of dUTPase. The processed DUT-M protein (Mr 23,000) is readily resolved from the

DUT-N protein, which has an apparent molecular weight of Mr 22,000. These data demonstrate the cross-reactivity of DUT415, which is likely attributable to the large region of homology common to these isoproteins. Expression of dUTPase Isoforms in PBLs. Prior to this study, our knowledge of the expression of dUTPase isoforms in human cells relied primarily on cell culture models. Using normal human fibroblasts (34Lu cells), our laboratory has investigated dUTPase isoform expression as a function of cellular proliferation status. Data from these experiments demonstrate that DUT-N is regulated in a growth- dependent manner, with expression correlating with the onset of DNA replication at both the protein and mRNA levels. In contrast, DUT-M is expressed in a constitutive fashion, mimicking the pattern of mtDNA replication that occurs independently of nuclear DNA replication (32, 33). To establish the distribution of dUTPase protein expression in human tissues, we have developed methodologies to Fig. 3. Analysis of dUTPase expression in resting and activated PBLs. A, to determine detect dUTPase by immunocyto- and immunohistochemistry using the pattern of dUTPase isoform expression in quiescent and replicating cells, a time- DUT415. Initially, PBLs were examined as a primary cell culture course experiment of PBL activation was performed. Samples were fractionated by 15% SDS-PAGE, and dUTPase protein was detected by Western blot analysis using dUTPase- model. To provide a basis for the interpretation of isoform expression specific polyclonal antibodies. PBLs were treated with and without PHA (as indicated), in human tissues, immunoblot analysis and immunohistochemistry and time points were taken at 24 and 48 h after treatment. B, immunocytochemistry was performed using DUT415 on PBLs isolated from whole blood. This analysis was per- were used in tandem to investigate the differential expression of formed on quiescent cells and cells stimulated with PHA for 48 h. dUTPase protein dUTPase isoforms after mitogenic stimulation of quiescent PBLs. expression is indicated by the presence of red chromogen. 3496

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2000 American Association for Cancer Research. dUTPase EXPRESSION IN NORMAL AND NEOPLASTIC TISSUES begin DNA synthesis ϳ24 h after stimulation, suggesting that DUT-N tubules of the kidney and thyroid follicular cells. The cytoplasmic is up-regulated just prior to the onset of DNA replication in these cells expression of dUTPase does not correlate with proliferation status and (data not shown). These data are in agreement with the 34Lu fibro- is found in both replicating and nonreplicating cell types. This obser- blast model, where DUT-M is constitutively expressed and DUT-N vation is in agreement with dUTPase expression observed in 34Lu expression is growth dependent (32). cells and PBLs in which expression of the DUT-M isoform occurs To determine whether we could detect dUTPase directly within independent of proliferation status. Together these data suggest that these cells, the DUT415 antibody was used in immunocytochemistry the observed cytoplasmic staining in normal tissues represents the experiments on either quiescent or stimulated PBLs. Fig. 3B illustrates mitochondrial isoform of dUTPase. dUTPase expression in PBLs that were either left untreated or mito- Fig. 4E illustrates dUTPase expression in normal colonic mucosa. genically stimulated with PHA for 48 h. The quiescent PBLs exhibit Immunohistochemical staining of colon tumors will be described in no detectable staining using this methodology, suggesting that the the next section; therefore, representative staining of DUT415 in level of DUT-M protein in these cells is below the sensitivity of this normal colon is included here as a reference. Nuclear staining is assay. In contrast, the PHA-stimulated cells demonstrate strong nu- observed in the replicating cells of the bottom half of the crypts of clear expression of dUTPase, as indicated by the presence of red Lieberku¨hn. There is also significant dUTPase staining in the cyto- chromogen. Preincubating the DUT415 antibody with a 5-fold excess plasm of these cells. Staining of colonic mucosa with Mito-M anti- of recombinant antigen abolishes staining in the PHA treated cells serum demonstrates that these cells are rich in mitochondria, suggest- (data not shown). These observations are in agreement with the ing that the cytoplasmic staining of dUTPase in these cells represents immunoblot analysis, where DUT-N is dramatically up-regulated in DUT-M (data not shown). stimulated cells. These data also demonstrate the ability of the To demonstrate the specificity of the DUT415 antibody in the DUT415 antibody to detect dUTPase protein in normal human cells immunohistochemistry assay, we performed antigen competition anal- by immunocytochemical techniques. ysis on a serial section of normal colon tissue. DUT415 was prein- Immunohistochemical Localization of dUTPase in Normal Hu- cubated with 5-fold molar excess of purified recombinant dUTPase man Tissues. To determine the expression of dUTPase in normal protein, and immunohistochemistry was performed as described. Pre- tissues, we developed methodology to detect dUTPase in formalin- incubation with antigen completely abrogates staining with DUT415 fixed and paraffin-embedded human tissues by immunohistochemis- in both the nucleus and cytoplasm as illustrated in Fig. 4F. Compe- try. In addition to dUTPase, the proliferation marker Ki67 (MIB-1 tition experiments were also performed with several other tissues clone) and the mitochondrial marker [113-1 clone (Mito-M)] were including, tonsil, kidney, breast cancer, colon cancer, and melanoma. used to stain serial sections of normal tissue. These markers were used Identical to normal colon tissue, staining with DUT415 was effec- as controls in these experiments to identify proliferating cells (Ki67) tively competed with recombinant antigen (data not shown). and cells that possess a rich population of mitochondria (Mito-M; The overall analysis of dUTPase expression in both cell culture and Refs. 34 and 35). To establish the pattern of dUTPase expression normal human tissues suggests that the pattern of intracellular distri- throughout the human body, we surveyed a panel of different normal bution of dUTPase protein reflects the physiological status of the cell. human tissues with the DUT415 antibody. Results from these exper- Nuclear staining (DUT-N) is observed in proliferating cells, corre- iments reveal both nuclear and cytoplasmic staining by DUT415 (Fig. sponding to an up-regulation of S phase-dependent gene products. In 4). An ABC-peroxidase technique was used in these experiments, and contrast, cytoplasmic staining (DUT-M) appears to reflect rich mito- protein expression is indicated by the presence of brown chromogen. chondrial content within the cell that is indicative of a high metabolic Nuclear dUTPase staining parallels Ki67 in highly proliferative cell status. types in all cases throughout the human body. Examples include Immunohistochemical Detection of dUTPase in Neoplastic Tis- proliferative cells of the immune system and replicating cells of sues. In an effort to establish the distribution and intracellular local- epithelia. Fig. 4A illustrates nuclear staining by DUT415 in palantine ization of dUTPase expression in human cancers, we performed tonsil. The proliferating B cells of the germinal center and stratum immunohistochemistry on colon and gastric tumor samples. This basale layer of the associated stratified squamous epithelium stain in analysis was performed on formalin-fixed and paraffin-embedded the nucleus with the DUT415 antibody. The proliferation marker Ki67 tumor tissue using the DUT415 antibody. Results from these exper- demonstrates a similar pattern of expression, as shown in Fig. 4B. The iments reveal that dUTPase expression varies dramatically in different nuclear staining of dUTPase in proliferating cells of normal human tumor specimens, not only in the quantity of expression but also in the tissues is consistent with the studies of DUT-N expression in both intracellular localization. Our data show varied phenotypes of expres- 34Lu cells and PBLs, where DUT-N protein is expressed in response sion ranging from exclusive cytoplasmic expression to exclusive to proliferation status. In addition, DUT415 staining was consistent nuclear expression. In addition, there is evidence suggesting that within and among individual tissue samples. For example, reproduc- dUTPase is aberrantly expressed in certain tumors. Photomicrographs ible staining patterns were observed in 10 different tonsil specimens, of dUTPase and Ki67 staining in two colon tumors are presented to and repeated staining of the same tissues yielded similar results. illustrate examples of the observed expression patterns. Cytoplasmic expression of dUTPase was observed throughout the In Fig. 5A, dUTPase expression is highly expressed in both the human body in metabolically active tissues such as the kidney, heart, cytoplasm and nucleus within the tumor cells, while the surrounding thyroid, and adrenal glands. An example of cytoplasmic dUTPase stromal cells exhibit very little dUTPase expression. A serial section expression is shown in Fig. 4C. In this image, the DUT415 MAb was of the same tumor, stained with the proliferation marker Ki67, dem- used to stain the nonproliferative cells of the myocardium. Although onstrates a characteristic nuclear staining pattern in the tumor cells the nuclei are readily visible and lack detectable nuclear dUTPase indicating a high proliferative status within this cancer (Fig. 5B). staining, these cells stain strongly in the cytoplasm with DUT415. In contrast, a different pattern of dUTPase expression is exhibited Staining of a serial section of myocardium with the Mito-M poly- in Fig. 5C. This photomicrograph depicts the margin of the tumor clonal antibody reveals a similar staining pattern as DUT415 (Fig. where both neoplastic tissue (left) and normal colonic mucosa (right) 4D). In all cases tested, cytoplasmic expression of dUTPase is corre- can be observed. In this case, dUTPase expression within the tumor lated with a high mitochondrial content, as determined by Mito-M cells is predominantly cytoplasmic (Fig. 5C). The colonic mucosa on immunoreactivity. Specific examples include the proximal convoluted the right side of this image demonstrates characteristic nuclear 3497

Fig. 4. Immunohistochemical localization of dUTPase in normal human tissues. A, routinely processed human tonsil stained with DUT415. All immunohistochemical staining was detected using DAB as a chromogen. B, routinely processed human tonsil stained with the proliferation marker, Ki67 (MIB-1). C, routinely processed human myocardium stained with DUT415. D, routinely processed human myocardium stained with the mitochondrial marker (Mito-M), clone 113-1. E, routinely processed human colon stained with DUT415. F, this is a serial section of the tissue observed in E. Preincubation of DUT415 with 5-fold molar excess of purified recombinant dUTPase protein abolishes staining, thereby demonstrating the specificity of this antibody.

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Fig. 5. Immunohistochemical localization of dUTPase in neoplastic human tissues. A, routinely processed human colon cancer (case 1) stained with DUT415. B, routinely processed human colon cancer (case 1) stained with Ki67. C, routinely processed human colon cancer (case 2) stained with DUT415. D, routinely processed human colon cancer (case 2) stained with Ki67. dUTPase staining in the replicating cells in the bottom half of the negative for dUTPase staining (data not shown). Additionally, the crypts of Lieberku¨hn, providing an internal positive control for nu- phenotype of expression appears to be stable within a given tumor clear dUTPase staining (see Fig. 4E for comparison). Interestingly, a specimen. A tumor sample exhibiting exclusive cytoplasmic expres- serial section of the same tissue stained for Ki67 demonstrates strong sion within one field of view retains this phenotype throughout the expression in the nucleus of both the tumor cells and the replicating specimen and tends not to display heterogeneous phenotypes. It is cells of the crypts (Fig. 5D). These data demonstrate that nuclear currently unknown whether metastatic lesions exhibit different pat- dUTPase staining and Ki67 do not always correlate within tumor terns of expression relative to the primary tumor. The expression tissues, suggesting that dUTPase isoforms may be aberrantly ex- patterns of dUTPase within colon and gastric cancers provide distinct pressed or incorrectly localized in certain tumors. and recognizable phenotypes that together or independently may be of To further illustrate the variable intracellular distribution of significant prognostic value. dUTPase in tumors, photomicrographs are presented in Fig. 6 of colon dUTPase Expression in Patients with Metastatic Colon Cancer. cancers stained with the DUT415 MAb. This panel of images illus- To explore the possible role of dUTPase expression as a prognostic trates exclusive nuclear expression (Fig. 6, A and B), exclusive cyto- marker, we conducted a retrospective study of 20 patients with met- plasmic expression (Fig. 6, C and D), and a combination of nuclear astatic colorectal cancer who were treated with protracted infusion of and cytoplasmic expression (Fig. 6, E and F). It is significant to note 5-FU/LV. Patient samples were scored according to the percent cells that the different patterns of dUTPase expression illustrated are com- staining and the pattern of dUTPase staining within the tumor cells monly observed in colon and gastric cancers. For example, a study of (i.e., nuclear or cytoplasmic). Tumor samples were scored as positive dUTPase expression in 41 gastric tumors revealed that 27% had for nuclear dUTPase expression if Ͼ10% of cells exhibited nuclear nuclear and cytoplasmic staining, 12% had exclusive nuclear expres- staining. The cutoff level of 10% was chosen arbitrarily based on sion, 39% had exclusive cytoplasmic expression, and 22% were cutoff levels of other markers such as p53 and p21. This threshold was 3499

Fig. 6. Immunohistochemical localization of dUTPase in human colon cancers. Colon cancer tumor specimens were routinely processed and stained with DUT415. A and B illustrate exclusive nuclear dUTPase expression. C and D illustrate exclusive cytoplasmic expression. E and F illustrate a combination of nuclear and cytoplasmic expression.

chosen prior to the statistical evaluation of the data and appeared to be titative reverse transcription-PCR and used as a comparative marker high enough to rule out any background staining. Independent, larger in these samples according to the methods detailed by Lenz et al. (Ref. patient studies will be required to identify the clinical significance of 22and references therein). different levels of positive staining, as well as to determine the Of the 20 samples tested, we found that 8 patients’ tumors con- optimal cut point for predicting response. Cytoplasmic staining was tained positive nuclear dUTPase staining (Ͼ10% positive nuclear scored on a percentage basis; however, these results were not included expression). There was a variable amount of cytoplasmic staining because there was no apparent association between cytoplasmic stain- (ranging from Ͼ75% to Ͻ5% tumor cells demonstrating cytoplasmic ing and response to chemotherapy, time to progression, or survival in staining). None of these patients responded (0%) to 5-FU-based this limited study. TS gene expression was also determined by quan- chemotherapy. Only 1 patient had stable disease, and 7 patients 3500

Table 2 Intracellular localization of dUTPase expression in advanced colon cancers: association with response, median survival, time to progression, and TS expression No. of Response to Median survival Median time to progression Median TS expressionc dUTPase IHCa patients chemotherapy (95% CI)b (95% CI)b (95% CI)b Ϫ Nuclear ϩ 8 Responders 0 6.9 mo 2.7 mo 5.4 ϫ 10 3 Stable disease 1 (5.1–11.4) (2.5–3.5) (3.8–8.9) Progression 7 Ϫ Nuclear Ϫ 12 Responders 6 8.5 mo 6.3 mo 2.2 ϫ 10 3 Stable disease 4 (7.4–17.9) (5.1–11.2) (1.5–4.2) Progression 2 Pd (Nucϩ vs. NucϪ) 0.005 0.09 0.017 0.06 a IHC, immunohistochemistry. b 95% CI, confidence interval. c TS expression is presented as a ratio of TS:␤-actin. d Two-sided P are based on Fisher’s exact test (response), the log-rank test (survival and progression), and the Wilcoxon rank sum test (TS). Nucϩ, nuclear ϩ; NucϪ, nuclear Ϫ. demonstrated progressive disease. From these 8 nuclear-positive of dUTPase, the chief regulator of dUTP pools, may be a critical patients, the overall median TS gene expression was 5.35 ϫ 10Ϫ3 factor in determining the efficacy of a broad range of chemothera- (expressed as a ratio of TS:␤-actin). The median survival in this peutic agents that target de novo thymidylate biosynthesis. The model group was 6.9 months, and time to tumor progression was 2.7 of uracil-DNA-mediated cytotoxicity predicts that overexpression of months (Table 2). dUTPase induces resistance to TS-directed chemotherapeutics by We found that 12 patients’ tumors were negative for nuclear limiting drug-induced dUTP pool accumulation, thereby preventing dUTPase staining. Again, there was a variable amount of cytoplasmic uracil misincorporation into DNA. In contrast, low dUTPase expres- dUTPase expression associated with these tumors. Of these 12 nucle- sion would promote dUTP accumulation, thereby inducing greater ar-negative tumors, 6 responded to chemotherapy (at least 50% re- sensitivity to these agents. To test this hypothesis and establish the duction of tumor size), 4 had stable disease, and 2 showed tumor value of dUTPase expression as a prognostic marker, we have devel- progression. From these 12 tumors, the median TS expression was oped methodology to measure dUTPase expression by immunohisto- Ϫ3 2.20 ϫ 10 . The median survival in this group was 8.5 months, and chemistry using the MAb DUT415. This investigation is the first to time to tumor progression was 6.3 months (Table 2). characterize dUTPase isoform expression in normal and neoplastic Comparative analysis of these data indicates that nuclear dUTPase tissues and to correlate nuclear dUTPase expression with response and expression may be a statistically significant predictor of response to survival in colorectal cancer. ϭ 5-FU-based chemotherapy (P 0.005), time to progression The data presented in this study establish the expression patterns of ϭ ϭ (P 0.017), and overall survival (P 0.09) in metastatic colon dUTPase isoforms in normal tissues. Immunohistochemical analysis cancer. High nuclear dUTPase expression is associated with poor of dUTPase throughout the human body demonstrates that nuclear response, shorter time to progression, and poorer survival. Con- staining occurs in replicating cell types. Examples include the stratum versely, low nuclear dUTPase staining is associated with response to basale of epithelial tissue, cells at the base of the crypts of Lieberku¨hn chemotherapy, longer time to progression, and greater overall sur- in the mucosa of the gastrointestinal tract, and proliferating lympho- vival. cytes. This observation is confirmed by identical staining with the proliferation marker Ki67. These data are consistent with the expres- DISCUSSION sion of DUT-N in cell culture models (e.g., PBLs and 34Lu human lung fibroblasts) where DUT-N protein and message are regulated in Inhibitors of thymidylate metabolism (i.e., the fluoropyrimidines a growth-dependent manner (32). Taken together, these data strongly and anitfolates) represent an important class of antineoplastic agents used for the treatment of head and neck, breast, and gastrointestinal suggest that nuclear staining by DUT415 in normal human tissues is cancers (1, 2). Clinical response to 5-FU-based regimens is typically indicative of DUT-N expression. between 20 and 30%, and drug resistance, either intrinsic or acquired, dUTPase staining is also observed in the cytoplasm of normal is a common phenomenon. Because response is difficult to predict tissues that have a high mitochondrial content. Examples include using anatomical staging alone, considerable effort has been directed myocardial tissue and the cells of the proximal convoluted tubules in toward understanding mechanisms of drug action and identifying the kidney. Similar staining with the polyclonal antibody Mito-M that markers that are better able to predict for response to therapy. Recent immunoreacts with mitochondria supports this observation. The cy- advances in this field have focused on determining intracellular levels toplasmic expression of dUTPase is independent of cellular prolifer- of relevant enzymes, thereby generating a predictive molecular profile ation status and is found in both replicating and nonreplicating cell for each tumor specimen. Important markers of survival and response types. For example, the replicating cells at the base of the crypts of to 5-FU-based chemotherapy include not only the target enzyme TS Lieberku¨hn in the colon demonstrate cytoplasmic dUTPase staining as but also enzymes involved in drug bioactivation and deactivation (i.e., well as the nonreplicating cells of the myocardium (Fig. 4). These data TP and dihydropyrimidine dehydrogenase) and critical protein deter- are in agreement with the constitutive expression of DUT-M observed minants of DNA damage response (p53 and p21; Refs. 22 and 36–39). in both quiescent and replicating PBLs and 34Lu cells. We have also Despite these advances, there has been no clinical evaluation of shown that DUT-M mRNA levels remain constant in both quiescent enzymes involved in uracil-DNA-mediated cytotoxicity as prognostic and replicating cells (32). Taken together, these data suggest that the markers of fluoropyrimidine-based therapy. cytoplasmic staining detected in normal human tissues is indicative of Investigation of the underlying molecular mechanisms of cell kill- DUT-M expression. Thus, DUT415 may be a useful marker in normal ing induced by inhibitors of thymidylate metabolism suggest that tissues to simultaneously identify replicating cell populations by nu- aberrant uracil-DNA metabolism plays a significant role in initiating clear staining and mitochondrial content through cytoplasmic staining. DNA damage and cell death (as reviewed by Aherne and Brown 4). Immunohistochemical staining of dUTPase in colon cancers dem- A growing body of evidence suggests that variation in the expression onstrates that dUTPase expression varies dramatically in different 3501

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2000 American Association for Cancer Research. dUTPase EXPRESSION IN NORMAL AND NEOPLASTIC TISSUES tumor samples, both in magnitude and intracellular localization. Pat- cases, mtDNA degradation and not nuclear DNA degradation may be terns of dUTPase expression range from exclusive nuclear or exclu- critical for response to chemotherapy. sive cytoplasmic staining to tandem expression of both. A recent Within this study, nuclear dUTPase expression was the important report by Fleischmann et al. (40) confirms these data, where these variable in predicting response and survival, suggesting that drug- authors observed variable levels of dUTPase expression in colorectal induced nuclear DNA damage may be the ultimate mediator of tumors. Thus, dUTPase expression among patient tumor samples is cytotoxicity. Although this limited study failed to correlate cytoplas- highly variable, exhibiting distinct and recognizable phenotypes of mic dUTPase expression with clinical response or survival, the strik- staining that may be of significant prognostic value. ing variability of dUTPase expression in the cytoplasm of tumor cells To examine the association between dUTPase expression and re- derived from different patients suggest that overexpression of DUT-M sponse to 5-FU-based chemotherapy and overall survival, we initiated may play a significant role in mediating protection of mtDNA from a retrospective study including tumor specimens from 20 patients who uracil-DNA-mediated degradation. The role of mtDNA degradation in had received protracted infusion of 5-FU and LV for treatment of overall fluoropyrimidine cytotoxicity and DUT-M-induced resistance metastatic colon cancer. In these colon cancer cases, nuclear dUTPase awaits further investigation. expression was associated with resistance to 5-FU-based chemother- The results of this study demonstrate that DUT415 is capable of apy (P ϭ 0.005), shorter time to progression (P ϭ 0.017), and a detecting human dUTPase isoforms in formalin-fixed, paraffin- shorter median survival (P ϭ 0.09). In contrast, tumors lacking embedded tissues by immunohistochemistry. In normal tissues, nu- nuclear dUTPase expression were more responsive to chemotherapy clear expression of dUTPase is observed in replicating cell types, and had a longer time to progression and longer overall survival whereas cytoplasmic expression is observed in mitochondria-rich period (see Table 2). Significantly, no patients with nuclear positive tissues. In addition, we have measured dUTPase expression in colon dUTPase expression responded to 5-FU-based chemotherapy, and all cancer tumor specimens and correlated these data with clinical out- responders demonstrated a nuclear negative phenotype. These data come and response to chemotherapy. Although additional studies will suggest that elevated expression of dUTPase in the nucleus of tumor be needed to further establish the clinical utility of this marker, these cells may protect cells from the cytotoxic effect of uracil misincor- data strongly implicate dUTPase as a significant predictor of survival poration induced by inhibition of thymidylate metabolism. Variable and response to 5-FU-based chemotherapy in metastatic colon cancer. cytoplasmic expression of dUTPase was also observed in these tumor The ability to measure dUTPase expression in patient tumor specimens by immunohistochemistry will aid in determining the role of specimens; however, no association between cytoplasmic staining and dUTPase in drug resistance and may lead to improved therapeutic response or survival was evident in this limited study. strategies for patients treated with inhibitors of thymidylate The expression of nuclear dUTPase was also associated with TS metabolism. gene expression within these tumors (P ϭ 0.06). This observation may be expected because TS gene expression has already been shown to be a predictive marker of response to 5-FU-based chemotherapy in REFERENCES gastrointestinal cancers (19, 21, 28). Larger studies will be required to 1. Moertel, C. G. Chemotherapy for colorectal cancer. N. Engl. J. 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Robert D. Ladner, Frank J. Lynch, Susan Groshen, et al.

Cancer Res 2000;60:3493-3503.

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