Drug Metabolism and Disposition Fast Forward
First published on July 27, 2004; DOI: 10.1124/dmd.104.000737
0090-9556/04/3210-1178-1182$20.00
DMD 32:1178-1182, 2004
A CRUCIAL ROLE OF URIDINE/CYTIDINE KINASE 2 IN ANTITUMOR ACTIVITY OF 3'-ETHYNYL NUCLEOSIDES
Daigo Murata,
Yoshio Endo,
Tohru Obata,
Kazuki Sakamoto,
Yasuhiro Syouji,
Masakazu Kadohira,
Akira Matsuda, and
Takuma Sasaki
Department of Experimental Therapeutics, Cancer Research Institute, Kanazawa University, Kanazawa, Japan (D.M., Y.E., T.O., Y.S., M.K., T.S.); Tokushima Research Center, Taiho Pharmaceutical Co., Ltd., Tokushima, Japan (K.S.); and Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan (A.M.)
(Received June 1, 2004;
accepted July 21, 2004)
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Abstract
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The antitumor 3'-ethynyl nucleosides, 1-(3-C-ethynyl-ß-D-ribopentofuranosyl)cytosine (ECyd) and 1-(3-C-ethynyl-ß-D-ribopentofuranosyl)uridine (EUrd), are potent inhibitors of RNA polymerases and show excellent antitumor activity against various human solid tumors in xenograft models. ECyd is being investigated in phase I clinical trials as a novel anticancer drug possessing a unique antitumor action. ECyd and EUrd require the activity of uridine/cytidine kinase (UCK) to produce the corresponding active metabolite. The UCK family consists of two members, UCK1 and UCK2, and both UCKs are expressed in many tumor cells. It was unclear, however, whether UCK1 or UCK2 is responsible for the phosphorylation of the 3'-ethynyl nucleosides. We therefore established cell lines that are highly resistant to the 3'-ethynyl nucleosides from human fibrosarcoma HT-1080 and gastric carcinoma NUGC-3. All the resistant cell lines showed a high cross-resistance to ECyd and EUrd. As a result of cDNA sequence analysis, we found that UCK2 mRNA expressed in EUrd-resistant HT-1080 cells has a 98-base pair deletion of exon 5, whereas EUrd-resistant NUGC-3 cells were harboring the point mutation at nucleotide position 484 (C to T) within exon 4 of UCK2 mRNA. This mutation was confirmed by genome sequence analysis of the UCK2 gene. Moreover, the expression of UCK2 protein was decreased in these resistant cells. In contrast, no mutation in the mRNA or differences in protein expression levels of UCK1 were shown in the EUrd-resistant HT-1080 and NUGC-3 cells. These results suggest that UCK2 is responsible for the phosphorylation and activation of the antitumor 3'-ethynyl nucleosides.
The 3'-ethynyl nucleosides, 1-(3-C-ethynyl-ß-D-ribo-pentofuranosyl)cytosine (ECyd) and 1-(3-C-ethynyl-ß-D-ribo-pentofuranosyl)uracil (EUrd), are antitumor ribo-nucleoside analogs of cytidine and uridine, respectively. We previously reported that both ECyd and EUrd exhibit significant cytotoxicity and antitumor activity on various types of animal and human solid tumors in in vivo models (Hattori et al., 1996
; Tanaka et al., 1997; Takatori et al., 1999). ECyd, which is currently undergoing a phase I study, is a unique anticancer drug possessing a potent inhibitory effect on RNA polymerases. Both ECyd and EUrd incorporated into cells are rapidly phosphorylated to a monophosphate form by uridine/cytidine kinase (UCK; EC 2.7.1.48
[EC]
), and these monophosphates are subsequently phosphorylated to the corresponding di- (ethynyl cytidine diphosphate and ethynyl uridine diphosphate) and triphosphates (ethynyl cytidine triphosphate and ethynyl uridine triphosphate) (Shimamoto et al., 2002a). The triphosphates are active metabolites and inhibit RNA synthesis by blocking RNA polymerases I, II, and III (Tabata et al., 1997; Matsuda et al., 1999; Takatori et al., 1999). Intracellular accumulation of the triphosphates is therefore considered to be critical for the 3'-ethynyl nucleosides to exert a cytotoxic activity. We have reported that the sensitivity of tumor cells to EUrd is well correlated with the inhibition of cellular RNA synthesis by EUrd and that the inhibition of cellular RNA synthesis by EUrd is also associated with intracellular accumulation of ethynyl uridine triphosphate (Hattori et al., 1996).
We previously reported that EUrd-resistant human fibrosarcoma HT-1080 cells possessed diminished UCK activity. However, the mechanism of the enzymatic deficiency of UCK has not yet been clarified. We have also demonstrated that the decrease of deoxycytidine kinase activity leads tumor cells to acquire resistance to antitumor 2'-deoxycytidine analogs including 1-ß-D-arabinofuranosylcytosine and gemcitabine, and the enzymatic deficiency is frequently caused by mutation of the deoxycytidine kinase gene (Obata et al., 2001). Therefore, the EUrd-resistant HT-1080 may harbor a mutation in the UCK gene. More recently, the UCK family, which consists of at least two members, UCK1 and UCK2, has been found to be present in human cells (Van Rompay et al., 2001). The UCK1 gene encodes a 277-amino acid protein with a predicted molecular mass of 31 kDa. The UCK2 gene encodes a 261-amino acid protein (29 kDa). UCK1 and UCK2 share 68.8% identity at the deduced amino acid level. Although many tumors express both UCK1 and UCK2, their role in the phosphorylation of 3'-ethynyl nucleosides is not fully understood. To elucidate the resistance mechanisms of 3'-ethynyl nucleosides and the distinct roles of UCK1 and UCK2 in production of 3'-ethynyl nucleoside monophosphates, we investigated the expression of UCKs and underlying genetic mutations in resistant cells. We subsequently found that UCK2 mRNA expressed in the EUrd-resistant HT-1080 and human gastric cancer NUGC-3 cells harbored nonsense and missense mutations, respectively. In the present study, we demonstrate that UCK2 plays an exclusive role in the intracellular phosphorylation of 3'-ethynyl nucleosides and is a molecular target for acquired resistance to the nucleosides.
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Materials and Methods
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Chemicals. EUrd and ECyd were chemically synthesized as previously reported (Hattori et al., 1996). 1-ß-D-Arabinofuranosylcytosine was kindly provided by YAMASA Co. (Choshi, Japan).
Establishment of Resistant Tumor Cells. Human fibrosarcoma HT-1080 cells were obtained from the American Type Culture Collection (Manassas, VA), and human stomach carcinoma NUGC-3 cells were obtained from the Japanese Collection of Research Bioresources (National Institute of Health Sciences, Cell Bank, Tokyo, Japan). The cells were maintained as a monolayer culture in RPMI 1640 medium (Nissui Pharmaceutical, Osaka, Japan) containing 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA) and kanamycin (50 µg/ml). The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2.
EUrd-resistant and ECyd-resistant variants of HT-1080 (HT-1080/EUrd and HT-1080/ECyd) and NUGC-3 (NUGC-3/EUrd and NUGC-3/ECyd) were developed by continuous exposure of cells to EUrd or ECyd, starting with a concentration of 0.1 ng/ml (Tabata et al., 1997). When the cells grew logarithmically at the maximum growth rate, the concentration was increased incrementally. Finally, the cells could proliferate logarithmically in culture medium containing EUrd (10 µg/ml) and ECyd (10 µg/ml).
Cytotoxicity Assay. In vitro cytotoxicity was examined using a modified tetrazolium-based semiautomatic colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent (Sigma-Aldrich, St. Louis, MO) as reported previously (Carmichael et al., 1987; Tanaka et al., 1992). Briefly, 180-µl aliquots of an exponentially growing cell suspension (0.5-2 x 103 cells) were incubated in a 96-well microplate for 24 h; then, 20 µl of drugs were added at various concentrations. After exposure for 72 h, 25 µl of 2 mg/ml MTT was added to each well, and the cell cultures were incubated at 37°C for 4 h. The medium was carefully removed and 200 µl of dimethyl sulfoxide was added to each well to dissolve the formed formazan. After thorough mixing, the absorbance of each well was measured at 540 nm using a model NJ-2300 Immuno-Mini microplate reader (Nihon Intermed, Tokyo, Japan). Each experiment was performed using triplicate wells for each drug concentration and two or three independent experiments were carried out to confirm the reproducibility. The percentage of relative cell survival was calculated by applying the following formula: % of relative cell survival = T/C x 100, where C is the mean A540 of the control group and T is that of the treated group. The 50% inhibitory drug concentration (IC50 value) was measured graphically from the dose-response curve with at least three drug concentration points.
RT-PCR Analysis of UCK mRNA Expression. The RT-PCR analysis was conducted by a modification of the method of Conboy et al. (1988). Briefly, total RNA was extracted using ISOGEN (Nippon Gene, Tokyo, Japan) (Chomczynski and Sacchi, 1987). The prepared RNA was mixed with oligo(dT), incubated for 10 min at 68°C, and then quickly chilled in an ice bath for 5 min. The RNA samples were reverse-transcribed at 40°C for 90 min into the first-strand cDNA in RT solution [50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.5 mM each deoxynucleoside-5'-triphosphate, 225 µg/ml bovine serum albumin, 10 mM dithiothreitol, 4 units of RNasin (Promega, Madison, WI), and 50 units of Moloney murine leukemia virus reverse-transcriptase (Toyobo Co., Ltd., Osaka, Japan)] with a total volume of 20 µl. The cDNA samples were incubated at 95°C for 5 min to inactivate the reverse transcriptase and then chilled. The cDNA samples were amplified in PCR mixture [2 mM Tris-HCl (pH 8.0), 10 mM KCl, 2 mM MgCl2, 0.01 mM EDTA, 0.1 mM dithiothreitol, 0.05% Tween 20, 0.05% Nonidet P-40, 5% glycerol, 0.25 mM each deoxynucleoside-5'-triphosphate, 50 pmol of each sense and antisense primer, and 2.5 units of Ex Taq polymerase (Takara, Kyoto, Japan)] with a total volume of 50 µl. UCK1, UCK2, and the transferrin receptor gene were amplified for 1.5 min at 94°C, 2 min at 48°C, and 2 min at 72°C for three cycles, followed by 27 cycles of 40 s at 94°C, 1.5 min at 48°C, and 1.3 min at 72°C on a DNA thermal cycler (PerkinElmer Life and Analytical Sciences, Boston, MA). The PCR products were electrophoresed on a 1% agarose gel. Specific primers are presented in Table 1.
Cycle Sequencing. The PCR products of the UCK1 and UCK2 cDNA including a full-length coding region were purified using a QIAEX II Gel Extraction Kit (QIAGEN GmbH, Hilden, Germany). The purified PCR fragments were sequenced by a cycle-sequencing method using Gene Rapid (Amersham Biosciences AB, Uppsala, Sweden) and a Thermo Sequenase Cy5.5 Terminator Cycle Sequencing Kit (Amersham Biosciences AB).
Western Blot Analysis for UCK1 and UCK2 Proteins. Protein was extracted from HT-1080/EUrd and NUGC-3/EUrd cells with RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS]. After centrifugation for 10 min at 15,000 rpm at 4°C, the lysate supernatant was assayed for protein concentration using a BCA protein assay kit (Pierce, Rockford, IL). Samples were electrophoresed on a SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Bedford, MA) by electroblotting. Nonspecific binding was blocked by incubation with 5% nonfat dry milk in TBS-T [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20] overnight at 4°C. The membrane was washed with TBS-T and then incubated with anti-UCK1 or anti-UCK2 rabbit polyclonal antibody (a gift from Taiho Pharmaceutical Co., Ltd., Tokyo, Japan) (Shimamoto et al., 2002b) for 1 h at room temperature. After further washing with TBS-T and subsequent incubation with horseradish peroxidase-conjugated anti-rabbit IgG, specific complexes were detected by the ECL chemiluminescence technique (Amersham Biosciences AB) according to the manufacturer's instructions.
Creation of Recombinant UCK2 from NUGC-3/EUrd Cells. For recombinant protein production and purification, the mutant UCK2 (R162W) gene was amplified by PCR from NUGC-3/EUrd cells. The amplified PCR fragment was digested with both XhoI and NotI and the purified fragment was introduced into SalI/NotI-digested plasmid pPRO EX HTc. Finally, the mutant UCK2 gene of the resulting plasmid pPRO/UCK2 (R162W) was sequenced to confirm the entire nucleotide sequence. The expression plasmid pPRO/UCK2 (R162W) was transformed into the Escherichia coli BL21 and was induced by adding isopropyl ß-D-thiogalactoside. Recombinant UCK2 (R162W) was purified by a nickel-nitrilotriacetic acid column.
The Uridine Phosphorylation Catalytic Activity Assay. Uridine phosphorylation activity of wild-type and mutant recombinant UCK2 was measured using uridine as a substrate according to the method of Shimamoto et al. (2002b). The significance of differences between wild-type and mutant recombinant UCK2 was determined by the Student's t test. A P value lower than 0.05 was considered to be significant.
Transfection and Selection of the Expressing Cells. UCK2 cDNA was cloned into the pcDNA3.1/V5-His-TOPO vector to construct pcDNA3.1/UCK2. This plasmid was transfected into HT-1080 and HT-1080/EUrd cells using LipofectAMINE 2000 (Invitrogen). The transfectants named HT-1080/UCK2 and HT-1080/EUrd/UCK2 were isolated by growth in G418-containing medium, and G418-resistant clones were established. The pcDNA3.1 empty vector alone was also transfected into HT-1080 cells to generate control clones, designated as HT-1080/pcDNA3.1.
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Results
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Sensitivity to Antitumor Cytosine Nucleosides. Table 2 presents the IC50 values and relative resistance ratios to the IC50 value in the parental cells. The ECyd- and EUrd-resistant cell lines showed a high cross-resistance to both 3'-ethynyl nucleosides. The IC50 values of all resistant cells were over 50-fold higher than those of parental cells.
mRNA Expression of UCK1 and UCK2 in Resistant Cells. We examined the expression of UCK1 and UCK2 mRNA in HT-1080/EUrd and NUGC-3/EUrd cells by RT-PCR. HT-1080/EUrd cells produced an unexpected PCR product of UCK2 cDNA with a shorter size than expected (873 bp), but the intensity of the band was almost the same as that from the parental HT-1080. In NUGC-3/EUrd cells, the expected fragment was observed, but the expression level of UCK2 was significantly decreased compared with that of the parental NUGC-3 cells (Fig. 1). In contrast, the expression of UCK1 mRNA did not change in all the parental and resistant cells.
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FIG. 1. RT-PCR analysis of UCK mRNA expression. Total RNA from various cell lines resistant to antitumor 3'-ethynyl nucleosides was used to analyze UCK mRNA expression by RT-PCR. The amplified products were electrophoresed on a 1.0% agarose gel. The normal size (873 bp) and the aberrant form (775 bp) of UCK2, UCK1 (898 bp), and transferrin receptor (413 bp) are indicated at the right side. Lane 1, HT-1080; lane 2, HT-1080/EUrd; lane 3, NUGC-3; lane 4, NUGC-3/EUrd.
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Protein Expression of UCK1 and UCK2 in Resistant Cells. The expression of UCK1 and UCK2 proteins was examined by Western blot using specific polyclonal antibodies (Shimamoto et al., 2002b). The results showed that the expression level of UCK2 in HT-1080/EUrd and NUGC-3/EUrd was markedly decreased, but no change in UCK1 protein expression was shown between parental and resistant cells (Fig. 2).
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FIG. 2. Western blot analysis of UCK1 and UCK2 protein expression in EUrd-resistant cells. Western blot analysis was performed using anti-UCK1 and UCK2 polyclonal antibodies on cell lysates. Lane 1, HT-1080; lane 2, HT-1080/EUrd; lane 3, NUGC-3; lane 4, NUGC-3/EUrd.
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Mutations in EUrd-Resistant HT-1080 and NUGC-3 Cells. The RT-PCR products of UCK1 and UCK2 mRNA from HT-1080/EUrd and NUGC/EUrd cells were sequenced. UCK1 mRNA from both resistant cells had a normal sequence. In contrast, UCK2 mRNA from HT-1080/EUrd had a deletion at the nucleotide position 500-597 (98 bp) (Fig. 3). The deletion resulted in a frameshift mutation resulting in N-terminal miscoding and a truncated protein with 171 amino acids. Furthermore, UCK2 mRNA from NUGC-3/EUrd exhibited a one-base substitution (C to T) at nucleotide position 484 within the fourth exon (Fig. 4A). This mutation resulted in an amino acid change from Arg to Trp. This point mutation could be found in genomic DNA from EUrd-resistant NUGC-3 cells (Fig. 4B). NUGC-3/ECyd did not express UCK2 mRNA or protein but had no mutation in the coding region of UCK2 cDNA (data not shown).
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FIG. 3. Deletion mutation of UCK2 mRNA expressed in EUrd-resistant HT-1080 cells. In UCK2 mRNA from EUrd-resistant HT-1080 cells, the deletion was observed at the nucleoside position 500-597 (98 bp), resulting in a short form of UCK2.
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FIG. 4. A point mutation of the UCK2 gene in EUrd-resistant NUGC-3 cells. A, in UCK2 mRNA from resistant NUGC-3 cells, a one-base substitution (C to T) was observed at nucleotide position 484 within exon 4. B, the same point mutation was confirmed by genomic sequencing of the UCK2 gene in EUrd-resistant NUGC-3 cells. Top panel, 5'-sequence; bottom panel, 3'-sequence.
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Uridine Phosphorylation Activity of Recombinant UCK2 in NUGC-3/EUrd Cells. To investigate whether recombinant UCK2 expressed in NUGC-3/EUrd cells possesses uridine phosphorylation activity, we purified the recombinant mutant UCK2 (R162W) from E. coli. The results showed that UCK2 from NUGC-3/EUrd cells has no enzymatic activity (Fig. 5).
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FIG. 5. Catalytic activity of wild-type and mutant recombinant UCK2. Uridine phosphorylation activity of wild-type and mutant recombinant UCK2. Representative results are shown as mean ± S.D.
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Cytotoxicity of ECyd on HT-1080/EUrd cells. To examine whether sensitivity of HT-1080/EUrd cells to ECyd is restored by forced expression of normal UCK2, we transfected UCK2 cDNA into HT-1080/EUrd. After transfection and G418 selection, growing clones were examined by Western blotting to confirm UCK2 protein expression. HT-1080/EUrd cells transfected with UCK2 cDNA overexpressed V5-His-tagged UCK2 polypeptide (34 kDa). ECyd sensitivity of HT-1080/EUrd overexpressing normal UCK2 was restored to the level of that in parental cells (Fig. 6).
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FIG. 6. Restored sensitivity to ECyd in HT-1080/EUrd-UCK2 cells. The assay conditions are as described under Materials and Methods. The growth inhibition of HT-1080/EUrd-UCK2 cells (closed circle), HT-1080/EUrd-pcDNA3.1 cells (open circle), and HT-1080-pcDNA3.1 (open triangle) by ECyd is shown. Sensitivity was restored to levels present in the parental cells. The experiment was measured in triplicate and representative results are shown as mean ± S.D.
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Discussion
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The first phosphorylation of the 3'-ethynyl nucleosides, EUrd and ECyd, is catalyzed by UCK, and this step is thought to be rate-limiting. The level of cellular UCK activity therefore affects sensitivity to these 3'-ethynyl nucleosides. We have previously demonstrated that UCK activity was decreased concomitant with the progression of drug resistance to the 3'-ethynyl nucleosides (Tabata et al., 1997; Shimamoto et al., 2002a). However, it was not clear which isozyme of UCKs is responsible for the phosphorylation of 3'-ethynyl nucleosides. We have established human tumor cell lines highly resistant to EUrd and ECyd. These resistant cells showed a cross-resistance to both the 3'-ethynyl nucleosides. The results suggest that the resistant cell lines may harbor a mutation in UCK1 or UCK2. Eventually, we found a 98-bp deletion mutation in UCK2 mRNA from HT-1080/EUrd. Since the deletion site corresponded to exon 5 only, this mutation may result from a splicing disorder. However, we could not confirm a genomic DNA mutation upstream of exon 5 in the UCK2 gene by direct sequence analysis. We also analyzed the ±300-bp region of exons 4 and 6, but again, no mutation was shown. In the sequence analysis we performed, the primer pairs used targeted a short region between 5'- and 3'-flanking introns of each exon (exons 4, 5, or 6). Therefore, only the normal allele might be amplified if another allelic UCK2 gene has a gross deletion including exon 5 outside the annealing site of the primers used. Alternatively, the allelic UCK2 gene carrying a deletion mutation may be transcriptionally inactivated by genomic imprinting or by epigenetic regulation. In NUGC-3/EUrd cells, a point mutation at the nucleotide position 484 (C to T) was observed in UCK2 mRNA within exon 4. Genomic DNA from NUGC-3/EUrd showed the same point mutation within exon 4 of the UCK2 gene. In contrast, UCK1 mRNA expression in both NUGC-3/EUrd and HT-1080/EUrd cells was not changed from each parental cell, and no mutation was detected in mRNA from the resistant cells. The expression level of UCK2 protein in EUrd-resistant cells was markedly decreased as compared with that in parental cells, but no marked decrease of UCK1 protein was shown in EUrd-resistant cells. These mutant UCK2 proteins may have a short half-life due to their instability in cells. We examined the enzymatic activity of recombinant UCK2-R162W from NUGC-3/EUrd cells. The mutant UCK2 lacked Urd/Cyd kinase activity. We recently produced recombinant human UCK1 and UCK2 to investigate the kinetic parameters of UCK1 and UCK2 for the 3'-ethynyl nucleosides. Consequently, recombinant human UCK2 showed lower Km and higher Vmax values for the 3'-ethynyl nucleosides than did UCK1 (Km and Vmax for ECyd: UCK1, 889 µM and 0.215 nmol/mg/min; UCK2, 418 µM and 227 nmol/mg/min). The substrate efficiency (Vmax/Km) of UCK2 to ECyd was about 2000 times higher than that of UCK1 (data not shown). Thus, these 3'-ethynyl nucleosides might be preferentially phosphorylated by UCK2.
In this study, we demonstrate that UCK2 is an exclusive target for the acquisition of resistance to the antitumor 3'-ethynyl nucleosides, EUrd and ECyd. Although UCK2 is expressed in many types of tissues and tumor cells, the expression level of UCK2 appears to be very diverse in cells. Recently, the existence of single-nucleotide polymorphisms in the UCK2 gene was reported (Hasegawa et al., 2002). UCK2 may be a useful marker to predict chemosensitivity to antitumor uridine and cytidine analogs including EUrd and ECyd. In addition, UCK2 may be a novel molecular target for biochemical modulation to potentiate antitumor effects of such nucleoside analogs.
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Footnotes
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doi:10.1124/dmd.104.000737.
ABBREVIATIONS: ECyd, 1-(3-C-ethynyl-ß-D-ribo-pentofuranosyl)cytosine; EUrd, 1-(3-C-ethynyl-ß-D-ribo-pentofuranosyl)uridine; UCK, uridine/cytidine kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT-PCR, reverse transcriptase-polymerase chain reaction; TBS-T, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20; bp, base pair(s).
Address correspondence to: Dr. Takuma Sasaki, Department of Experimental Therapeutics, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa 920-0934, Japan. E-mail: takuma{at}kenroku.kanazawa-u.ac.jp
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Abstract
Materials and Methods
