Lung Cancer (2005) 49, 345—351

The role of thymidylate synthase anddihydropyrimidine dehydrogenase in resistanceto 5-fluorouracil in human lung cancer cells

Tetsuya Oguri ∗, Hiroyuki Achiwa, Yuji Bessho, Hideki Muramatsu,Hiroyoshi Maeda, Takashi Niimi, Shigeki Sato, Ryuzo Ueda

Department of Internal Medicine and Molecular Science, Nagoya City University Graduate School ofMedical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan

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eceived 8 March 2005; received in revised form 29 April 2005; accepted 2 May 2005

KEYWORDSNon-small-cell lungcancer;5-Fluorouracil;Thymidylate synthase;Dihydropyrimidinedehydrogenase;Thymidinephosphorylases;Orotate phosphoribosyl-transferase

Summary The expressions of thymidylate synthase (TS) and intracellularmetabolic enzymes have been reported to be associated with the sensitivity and/orresistance to 5-fluorouracil (5-FU). However, since the role of these enzymes in themechanism of resistance to 5-FU has not been fully examined in lung cancer, in thepresent study we measured the expression levels of TS, dihydropyrimidine dehydro-genase (DPD), thymidine phosphorylase (TP), and orotate phosphoribosyltransferase(OPRT) genes in lung cancer cell lines by real-time PCR, and the sensitivity to 5-FUusing the MTS assay. The expression of DPD was significantly correlated with theconcentration of 5-FU for 50% cell survival in 15 non-small-cell lung cancer (NSCLC)cell lines (p < 0.05), but the expressions of TS, TP, and OPRT were not. Treatmentwith 5-chloro-2,4-dihydroxypyridine, an inhibitor of DPD, altered the sensitivity to5-FU in DPD-expressing RERF-LC-MT cells, indicating that modulation of DPD activ-ity could increase the 5-FU sensitivity in lung cancer. In contrast, TS expressionwas dramatically higher in a 5-FU-resistant small-cell lung cancer cell line than inthe parent cell line, whereas the expressions of DPD, TP, and OPRT genes were notmarkedly different. In order to examine the effect of other cytotoxic agents on TSand DPD expression, we compared the expressions of both genes between cisplatin-,paclitaxel-, gemcitabine-, or 7-ethyl-10-hydroxycamptothecin-resistant lung cancercells and their respective parent cells, but found no differences between any pairof resistant subline and the corresponding parent cell line. Our results indicate thatdegradation of 5-FU due to DPD is an important determinant in 5-FU sensitivity,while induction of TS contributes to acquired resistance against 5-FU in lung cancer.Therefore, the expression levels of TS and DPD genes may be useful indicators of5-FU activity in lung cancer.© 2005 Elsevier Ireland Ltd. All rights reserved.

∗ Corresponding author. Tel.: +81 52 853 8216; fax: +81 52 852 0849.E-mail address: t-oguri@med.nagoya-cu.ac.jp (T. Oguri).

169-5002/$ — see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.lungcan.2005.05.003

346 T. Oguri et al.

1. Introduction

5-Fluorouracil (5-FU) and its derivatives areantimetabolite drugs that are widely used incancer chemotherapy. The effects of 5-FU havebeen attributed to inhibition of thymidylate syn-thase (TS) and incorporation of its metabolitesinto RNA and DNA. 5-FU enters the cells rapidlyand is converted intracellularly by metabolicenzymes. Dihydropyrimidine dehydrogenase (DPD)is the first and rate-limiting enzyme for convert-ing 5-FU into an inactive metabolite, dihydrofluo-rouracil. In contrast, other first metabolic enzymes,namely orotate phosphoribosyltransferase (OPRT)and thymidine phosphorylase (TP), catalyze synthe-sis of active metabolites, resulting in disruption ofthe action of TS and DNA or RNA synthesis [1].

Lung cancer is one of the most common malig-nancies worldwide, and several randomized clinicaltrials and meta-analyses have demonstrated thatsurvival in patients with advanced non-small-celllung cancer (NSCLC) can be slightly but significantlyprolonged with chemotherapy [2,3]. Although 5-FU derivatives are not usually administered as afirst-line chemotherapy, a recent study showed that

QG56), and 2 large-cell carcinomas (NCI-H460and SK-LC-6). Cells from a human small-cell lungcancer (SCLC) cell line, PC-6, and those fromtheir 5-FU-resistant subline PC-6/FU23-26, 7-ethyl-10-hydroxycamptothecin (SN-38)-resistant sublinePC-6/SN2-5, and paclitaxel (TAX)-resistant sublinePC-6/TAX1-1 were kindly provided by Dr. AkikoTohgo (Daiichi Pharmaceutical Co., Tokyo, Japan[12]). The human lung adenocarcinoma cell linesPC-9 and PC-14, and cells from the cisplatin (CDDP)-resistant human lung adenocarcinoma cell linePC-9/CDDP were kindly provided by Dr. KazutoNishio (National Cancer Institution, Tokyo, Japan[13]). Cells from the gemcitabine (GEM)-resistanthuman lung adenocarcinoma cell line H23/GEM-R were established as described previously [14].Cells were cultured in RPMI 1640 (or, for A549, Dul-becco’s modified Eagle’s medium) supplementedwith 10% heat-inactivated FBS and 1% (v/w) peni-cillin/streptomycin. 5-FU was provided from KyowaHakko Kogyo (Tokyo, Japan), and DPD inhibitor 5-chloro-2,4-dihydroxypyridine (CDHP) was providedfrom Taiho Pharmaceutical (Tokyo, Japan).

2.2. Total RNA extraction and RT-PCR

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postoperative oral administration of uracil-tegafurimproves survival among patients with completelyresected stage I lung adenocarcinomas [4]. Further,a novel oral fluorouracil S-1 was shown to exertpromising effects against advanced NSCLC [5,6].These results indicate the effectiveness of 5-FUderivatives in NSCLC treatment.

Biomarkers for the prediction of the sensitiv-ity and/or resistance to 5-FU have been identifiedpreviously, including TS and intracellular metabolicenzymes [1,7,8]. It was demonstrated that TS andDPD gene expression and/or activity are associatedwith the efficacy of 5-FU in NSCLC [9—11]. However,the role of TS and intracellular metabolic enzymesin the mechanism of resistance to 5-FU has not beenfully examined in lung cancer. Therefore, we exam-ined the expression levels of TS, DPD, TP, and OPRTgenes to clarify the mechanism of sensitivity andresistance to 5-FU in lung cancer.

2. Materials and methods

2.1. Cell lines and chemicals

The following human NSCLC cell lines were usedin this study: 11 adenocarcinomas (A549, NCI-H23, PC-9, PC-14, VMRC-LCD, VMRC-LCF, RERF-LC-MT, RERF-LC-OK, RERF-LC-MS, ACC-LC-176, andSK-LC-10), 2 squamous-cell carcinomas (PC10 and

otal RNA was extracted with TRIZOL reagentInvitrogen, USA) according to the manufacturer’snstructions. cDNA was synthesized using a ran-om hexamer (Amersham, UK) with SuperscriptNase H-reverse transcriptase (Gibco-BRL, USA).he reverse-transcribed cDNA from each sampleas subjected to PCR amplification using Taq poly-erase (Promega, USA) and primers. The sequencesf the TS, DPD, TP, OPRT, and GAPDH primers, andhe PCR conditions were as described previously7,8,15,16]. Amplified products were separated by% agarose gel electrophoresis, and bands wereisualized by staining with ethidium bromide. Welso performed quantitative RT-PCR with the Light-ycler FastStart DNA SYBR Green kit (Roche Appliedcience, USA). We conducted a melting-curve anal-sis to control for specificity of the amplificationroducts. The number of transcripts was calcu-ated from a standard curve obtained by plottinghe known input of six different concentrations tohe PCR cycle number at which the detected fluo-escence intensity reached a fixed value. For eachample, results were normalized by the housekeep-ng gene GAPDH.

.3. Chemosensitivity assay

ells were cultured at 5000 cells/well in 96-wellissue culture plates. To assess cell viability, step-ise 10-fold dilutions of the anticancer drug were

The role of thymidylate synthase and dihydropyrimidine dehydrogenase in resistance 347

added 2 h after plating, and the cultures were incu-bated at 37 ◦C for 96 h. At the end of the cultureperiod, 20�l of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] solution (CellTiter 96®

AQueous One Solution Cell Proliferation Assay,Promega) was added, the cells were incubatedfor a further 4 h and the absorbance was mea-sured at 490 nm using an ELISA plate reader.Mean values were calculated from three inde-pendent experiments performed in quadruplicate.Chemosensitivity is expressed here as the drug con-centration for 50% cell survival (IC50), determinedfrom the concentration—effect relationship usingGraphPad Prism version 4 (GraphPad Software,USA).

2.4. Statistical analysis

Spearman’s test was used for correlation analysisbetween the expressions of TS, DPD, TP, and OPRTgenes and the IC50 values for 5-FU. The differencesin the gene expression levels between samples wereevaluated with Student’s paired t-test. The level ofsignificance was set at 5%, using two-sided analysis.

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Fig. 1 Relationship between 5-FU sensitivity and thebasal expression levels of TS, DPD, TP, and OPRT genesin NSCLC lines. The DPD expression and 5-FU sensitiv-ity were clearly correlated in the 15 NSCLC cell lines(r = 0.571, p = 0.026), but no significant correlation wasobserved between the expression of the other genesinvestigated and 5-FU sensitivity. Each IC50 value is themean of three independent sensitivity tests performedin quadruplicate. Expression levels are relative to expres-sion by GAPDH. Statistical significance of the correlationswas determined by Spearman’s correlation test.

cell lines: PC-14 cells (IC50 of 5-FU 1�M), whichexpress a very low level of DPD, and RERF-LC-MTcells (IC50 of 5-FU 58.54�M), which express a highlevel of DPD (Fig. 2A). As shown in Fig. 2B, treat-ment with CDHP at 1 or 10�M produced no signifi-cant change in the IC50 values of PC-14 cells to 5-FU.In contrast, the IC50 of 5-FU in RERF-LC-MT cells was58% (CDHP 1�M) and 65% (CDHP 10�M) lower thanthat in cells not treated with CDHP, and CDHP treat-ment at both concentrations significantly decreasedthe IC50 value to 5-FU (CDHP 1�M, p = 0.011; CDHP10�M, p = 0.002).

3.3. Expressions of TS, DPD, TP, and OPRTgenes in a 5-FU-resistant lung cancer cellline

We compared the expression levels of TS, DPD, TP,and OPRT genes in cells of the 5-FU-resistant SCLCcell line PC-6/FU23-26 with those in parent PC-6cells. The IC50 values of PC-6 and PC-6/FU23-26were 0.78 and 19.65�M, respectively (Fig. 3A). TheTS gene expression was dramatically higher in PC-6/FU23-26 cells than in PC-6 cells, whereas the

. Results

.1. Relationship between cytotoxicity of-FU and expressions of TS, DPD, TP, andPRT genes

sing quantitative real-time RT-PCR, the expres-ion levels of TS, DPD, TP and OPRT genes wereetermined in 15 NSCLC cell lines. The standardurves of TS, DPD, TP, OPRT, and GAPDH werebtained (data not shown), and real-time PCR effi-acies were calculated from the slopes. The corre-ponding real-time PCR efficacy of one cycle in thexponential phase was calculated using the equa-ion E = 10|−1/slope|. IC50 values of all cell lines to-FU were compared with their relative expressionsf TS, DPD, TP, and OPRT. We found a significantorrelation between the DPD gene expression and-FU sensitivity in 15 NSCLC cell lines (r = 0.579,= 0.026; Fig. 1), but no significant correlation wasbserved between TS, TP, or OPRT gene expressionnd 5-FU sensitivity (Fig. 1).

.2. Inhibition of DPD activity by CDHP

o elucidate whether DPD actually affects 5-FU sen-itivity, we added the DPD inhibitor CDHP at a non-ytotoxic concentration with 5-FU to two NSCLC

348 T. Oguri et al.

Fig. 2 Modification of 5-FU sensitivity by CDHP treatment in lung cancer cell lines. (A) Expression of DPD genes inPC-14 and RERF-LC-MT cells. (B) CHDP concentration-dependent 5-FU cytotoxicity in PC-14 and RERF-LC-MT cells. Eachcell line was incubated in different concentrations of CDHP and a gradient of 5-FU for 96 h. Each 5-FU IC50 value isnormalized to that for vehicle-treatment cells. The IC50 of 5-FU in RERF-LC-MT cells treated with CDHP was significantlylower than that in cells not treated with CDHP (CDHP 1�M, p = 0.011; CDHP 10�M, p = 0.002; Student’s paired t-test).

Fig. 3 IC50 value and gene expression levels in 5-FU-resistant SCLC cells and their parent cells. (A) Comparisonof IC50 values between cells of the 5-FU-resistant SCLCcell line PC-6/FU23-26 and parent PC-6 cells. (B) Expres-sions of TS, DPD, TP, and OPRT genes in PC-6/FU23-26and PC-6 cells. Lung adenocarcinoma cell line NCI-H23was used as a positive control.

expressions of DPD and OPRT were not higher inPC-6/FU23-26 cells than in PC-6 cells (Fig. 3B). TPgene expression was not detected by our PCR condi-tions in either PC-6 or PC-6/FU23-26 cells (Fig. 3B).We also confirmed the significant difference in TSgene expression by real-time PCR, and its absencein DPD, OPRT, and TP (data not shown).

3.4. Expressions of TS and DPD genes inCDDP-, TAX-, GEM-, and SN-38-resistant lungcancer cell lines

We compared the expression levels of TS and DPDgenes in CDDP-, TAX-, GEM-, and SN-38-resistantcell lines with those in the respective parent celllines. The IC50 of 5-FU did not differ markedlybetween TAX-, GEM-, and SN-38-resistant cells andtheir respective parent cells, whereas the IC50 of5-FU in CDDP-resistant cells was decreased com-pared with that in the parent cells (Fig. 4A). Incontrast, the expression levels of TS and DPD genesdid not differ between each pair of resistant cellsand parent cells (Fig. 4B). We confirmed the expres-sion levels of TS and DPD genes by real-time RT-PCR(data not shown).

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. Discussion

n this study we analyzed the relationship betweenhe expressions of TS, DPD, TP, and OPRT genes and

The role of thymidylate synthase and dihydropyrimidine dehydrogenase in resistance 349

Fig. 4 IC50 value and expression levels of TS and DPD genes in CDDP-, TAX-, GEM-, and SN-38-resistant cells, and inthe respective parent cells. (A) Comparison of IC50 values between CDDP-, TAX-, GEM-, and SN-38-resistant cells andthe respective parent cells. (B) Expression levels of TS and DPD genes in CDDP-, TAX-, GEM-, and SN-38-resistant cells,and those in the respective parent cells.

the sensitivity to 5-FU in NSCLC cell lines. We foundthat the basal level of DPD expression was signif-icantly correlated with 5-FU sensitivity in NSCLCcells. In addition, treatment with CDHP, which isan inhibitor of DPD, improves the sensitivity to 5-FU in NSCLC cells. These data suggest that cellularDPD expression is an important determinant of 5-FUsensitivity in NSCLC cells.

TS and DPD were previously reported to besensitivity-limiting factors of 5-FU in gastrointesti-nal cancers [8,15,16]. DPD was also associated with5-FU sensitivity in NSCLC, while the results for TShave been controversial [9—11]. Previous reportshave shown that there was a significant correla-tion between the gene expression levels and theenzymatic activities in both TS and DPD [17,18];therefore, we examined gene expression levels in

this study. We found that the basal level of DPDexpression was significantly correlated with 5-FUsensitivity in NSCLC, whereas that of TS was not.Since TS and DPD were considered to be indepen-dent sensitivity-limiting factors of 5-FU, this dif-ference may reflect differences in the populationsof TS and DPD among various cancers. NSCLC wascharacterized by high DPD and low TS activity [19],indicating that DPD levels may dominantly affectthe sensitivity to 5-FU in NSCLC cells.

A previous report has shown that inhibition ofDPD by CDHP enhances the sensitivity to 5-FU incancer cells [20]. In the present study we haveconfirmed in lung cancer cells that inhibiting DPDactivity modifies 5-FU sensitivity in highly DPD-expressing RERF-LC-MT cells, but not in low-DPD-expressing PC-14 cells. Recent clinical trials have

350 T. Oguri et al.

shown that a novel oral fluorouracil S-1 containinga prodrug of 5-FU and DPD inhibitor CDHP exhibitedpromising results against advanced NSCLC [5,6].Further, DPD activity levels have been shown to besignificantly higher in NSCLC tissues than in normallung tissues [9,11]. Taken together, these resultsindicate that DPD is a useful indicator in 5-FU activ-ity in NSCLC, and that inhibition of DPD activitypotentially increases the antitumor effect of 5-FUon NSCLC.

We next compared the expressions of TS, DPD,TP, and OPRT genes in 5-FU-resistant SCLC cellswith those in their parent cells in order to eluci-date factors for the acquired resistance to 5-FU.In contrast with the relationship between basalexpression and 5-FU sensitivity, TS expression wasdramatically higher in 5-FU-resistant cells than inthe parent cells. Previous reports have indicatedthat TS expression is easily induced by exposureto TS-targeting drugs (including 5-FU), concomi-tant with the development of resistance to them[21,22]. Our data are consistent with these previousresults. While several possible mechanisms for theupregulation of TS have been suggested, includingtranscription, translation, and stability of TS mRNA

in salvage synthesis of thymidylate, although TSactivity was not different between them. Platinum-containing chemotherapy is now standard first-linechemotherapy in patients with advanced NSCLC,and docetaxel is currently approved as a second-line chemotherapy agent [24]. Although 5-FUderivatives have not been shown to be effectiveas a second-line chemotherapy in NSCLC, the mul-titargeted antifolate pemetrexed whose mode ofaction is primarily the inhibition of TS showed clin-ical efficacy equivalent to that of docetaxel as asecond-line chemotherapy in patients with NSCLC[25]. Taken together with our results, TS-targetingdrugs may be potentially useful as not only first-linebut also second-line chemotherapies in advancedNSCLC. Clarifying the molecular determinants ofresistance to 5-FU derivatives may result in a newcombined chemotherapy in NSCLC.

We demonstrated that increased DPD expres-sion is a determinant of 5-FU sensitivity, and thatincreased TS expression is associated with acquiredresistance to 5-FU in NSCLC cells. The metabolismof 5-FU is very complicated, and recent studieshave identified a newly cloned drug-efflux pumpas a potential cyclic nucleotide-efflux pump and aritmatpc

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and/or protein, the induction of TS expression inresponse to 5-FU may be attributable to maintainedthymidylate and DNA biosynthesis in the face of acytotoxic stress against 5-FU treatment [20].

5-FU is converted to active forms after glucosyla-tion, in which OPRT and TP catalyze different path-ways [1]. Several studies have shown an associationbetween 5-FU sensitivity and TP or OPRT expres-sion in cancer tissues [7,8,15,16]. We believe thatthe present study is the first to examine the rela-tionship between 5-FU sensitivity and TP or OPRTexpression in lung cancer, and to find no correlationbetween them. Because TP is also an angiogeneticendothelial-cell growth factor [1], it is possiblethat TP acts not just as a 5-FU-converting enzyme.Nonetheless, our results indicate that the expres-sions of TP and OPRT genes are not useful markersof 5-FU activity in lung cancer.

5-FU derivatives are clinically used as a com-bined chemotherapy in cancer treatment, andhence it is important to identify the potential druginteractions in the action of 5-FU. We found thatCDDP-resistant cells are more sensitive than theparent cells to 5-FU, whereas we did not find differ-ences in TS or DPD expression between CDDP-, TAX-,GEM-, and SN-38-resistant cells and the respectiveparent cells. The present results suggest that noneof these agents affect the action of 5-FU. Previ-ously, Ohe et al. [23] showed that CDDP-resistanthuman lung cancer cells were more sensitive to 5-FU than their parental cells due to the decrease

esistance factor for 5-FU [26]. Usually a decreasen the intracellular concentration of drugs dueo drug-efflux pumps is considered as a deter-inant for drug resistance. While further studiesre required to clarify the mechanism for resis-ance to 5-FU, we consider that TS and DPD areotentially useful indicators of 5-FU activity in lungancer.

cknowledgments

e thank Mrs. Yukiko Nagao for their technicalssistance and Dr. Noriko Hattori for helpful discus-ions.

eferences

[1] Longley DB, Harkin DP, Johnston PG. 5-Fluorouracil: mech-anisms of action and clinical strategies. Nat Rev Cancer2003;3:330—8.

[2] Non-Small Cell Lung Cancer Collaborative Group.Chemotherapy in non-small cell lung cancer: a meta-analysis using updated data on individual patients from 52randomized clinical trials. BMJ 1995;311:899—909.

[3] Schiller JH, Harrington D, Belani CP, Langer C, SandlerA, Krook J, et al. Comparison of four chemotherapy regi-mens for advanced non-small-cell lung cancer. N Engl J Med2002;346:92—8.

[4] Kato H, Ichinose Y, Ohta M, Hata E, Tsubota N, Tada H, et al.,Japan Lung Cancer Research Group on Postsurgical AdjuvantChemotherapy. A randomized trial of adjuvant chemother-

The role of thymidylate synthase and dihydropyrimidine dehydrogenase in resistance 351

apy with uracil-tegafur for adenocarcinoma of the lung. NEngl J Med 2004;350:1713—21.

[5] Kawahara M, Furuse K, Segawa Y, Yoshimori K, Matsui K,Kudoh S, et al., S-1 Cooperative Study Group (Lung Can-cer Working Group). Phase II study of S-1, a novel oralfluorouracil, in advanced non-small-cell lung cancer. Br JCancer 2001;85:939—43.

[6] Ichinose Y, Yoshimori K, Sakai H, Nakai Y, Sugiura T, Kawa-hara M, et al. S-1 plus cisplatin combination chemother-apy in patients with advanced non-small cell lung can-cer: a multi-institutional phase II trial. Clin Cancer Res2004;10:7860—4.

[7] Yoshinare K, Kubota T, Watanabe M, Wada N, Nishibori H,Hasegawa H, et al. Gene expression in colorectal cancerand in vitro chemosensitivity to 5-fluorouracil: a study of88 surgical specimens. Cancer Sci 2003;94:633—8.

[8] Salonga D, Danenberg KD, Johnson M, Metzger R, GroshenS, Tsao-Wei DD, et al. Colorectal tumors responding to 5-fluorouracil have low gene expression levels of dihydropy-rimidine dehydrogenase, thymidylate synthase, and thymi-dine phosphorylase. Clin Cancer Res 2000;6:1322—7.

[9] Huang CL, Yokomise H, Kobayashi S, Fukushima M, HitomiS, Wada H. Intratumoral expression of thymidylate synthaseand dihydropyrimidine dehydrogenase in non-small cell lungcancer patients treated with 5-FU-based chemotherapy. IntJ Oncol 2000;17:47—54.

[10] Higashiyama M, Kodama K, Yokouchi H, Takami K,Fukushima M, Minamigawa K, et al. Thymidylate synthaseand dihydropyrimidine dehydrogenase activities in non-small cell lung cancer tissues: relationship with in vitro

[

[

[

[

[

gastrointestinal cancer cells to 5-fluorouracil and 5-fluoro-2′-deoxyuridine. World J Gastroenterol 2004;10:172—6.

[16] Ichikawa W, Uetake H, Shirota Y, Yamada H, Takahashi T,Nihei Z, et al. Both gene expression for orotate phosphori-bosyltransferase and its ratio to dihydropyrimidine dehy-drogenase influence outcome following fluoropyrimidine-based chemotherapy for metastatic colorectal cancer. BrJ Cancer 2003;89:1486—92.

[17] Ishikawa Y, Kubota T, Otani Y, Watanabe M, Teramoto T,Kumai K, et al. Dihydropyrimidine dehydrogenase activityand messenger RNA level may be related to the antitumoreffect of 5-fluorouracil on human tumor xenografts in nudemice. Clin Cancer Res 1999;5:883—9.

[18] Fujiwara H, Terashima M, Irinoda T, Takagane A, Abe K,Kashiwaba M, et al. Quantitative measurement of thymidy-late synthase and dihydropyrimidine dehydrogenase mRNAlevel in gastric cancer by real-time RT-PCR. Jpn J CancerRes 2002;93:1342—50.

[19] Fukushima M, Morita M, Ikeda K, Nagayama S. Populationstudy of expression of thymidylate synthase and dihydropy-rimidine dehydrogenase in patients with solid tumors. Int JMol Med 2003;12:839—44.

[20] Takechi T, Fujioka A, Matsushima E, Fukushima M. Enhance-ment of the antitumour activity of 5-fluorouracil (5-FU) by inhibiting dihydropyrimidine dehydrogenase activ-ity (DPD) using 5-chloro-2,4-dihydroxypyridine (CDHP) inhuman tumour cells. Eur J Cancer 2002;38:1271—7.

[21] Chu E, Callender MA, Farrell MP, Schmitz JC. Thymidy-late synthase inhibitors as anticancer agents: from benchto bedside. Cancer Chemother Pharmacol 2003;52(Suppl.

[

[

[

[

[

sensitivity to 5-fluorouracil. Lung Cancer 2001;34:407—16.

11] Shintani Y, Ohta M, Hirabayashi H, Tanaka H, Iuchi K, Naka-gawa K, et al. Thymidylate synthase and dihydropyrimidinedehydrogenase mRNA levels in tumor tissues and the effi-cacy of 5-fluorouracil in patients with non-small-cell lungcancer. Lung Cancer 2004;45:189—96.

12] Ishii M, Iwahana M, Mitsui I, Minami M, Imagawa S, Tohgo A,et al. Growth inhibitory effect of a new camptothecin ana-log, DX-8951f, on various drug-resistant sublines includingBCRP-mediated camptothecin derivative-resistant variantsderived from the human lung cancer cell line PC-6. Anti-cancer Drugs 2000;11:353—62.

13] Morikage T, Ohmori T, Nishio K, Fujiwara Y, Takeda Y, SaijoN. Modulation of cisplatin sensitivity and accumulation byamphotericin B in cisplatin-resistant human lung cancer celllines. Cancer Res 1993;53:3302—7.

14] Achiwa H, Oguri T, Sato S, Maeda H, Niimi T, Ueda R. Deter-minants of sensitivity and resistance to gemcitabine: theroles of human equilibrative nucleoside transporter 1 anddeoxycytidine kinase in non-small cell lung cancer. CancerSci 2004;95:753—7.

15] Ma T, Zhu ZG, Ji YB, Zhang Y, Yu YY, Liu BY, et al. Cor-relation of thymidylate synthase, thymidine phosphorylaseand dihydropyrimidine dehydrogenase with sensitivity of

1):S80—9.22] Longley DB, Ferguson PR, Boyer J, Latif T, Lynch M, Maxwell

P, et al. Characterization of a thymidylate synthase (TS)-inducible cell line: a model system for studying sensitivityto TS- and non-TS-targeted chemotherapies. Clin CancerRes 2001;7:3533—9.

23] Ohe Y, Sugimoto Y, Saijo N. Collateral sensitivity ofcisplatin-resistant human lung cancer cell lines to thymidy-late synthase inhibitors. Cancer J 1990;3:332—6.

24] Pfister DG, Johnson DH, Azzoli CG, Sause W, Smith TJ, BakerJr S, et al. American Society of Clinical Oncology. AmericanSociety of Clinical Oncology treatment of unresectable non-small-cell lung cancer guideline: update 2003. J Clin Oncol2004;22:330—53.

25] Hanna N, Shepherd FA, Fossella FV, Pereira JR, De MarinisF, von Pawel J, et al. Randomized phase III trial of peme-trexed versus docetaxel in patients with non-small-cell lungcancer previously treated with chemotherapy. J Clin Oncol2004;22:1589—97.

26] Guo Y, Kotova E, Chen ZS, Lee K, Hopper-Borge E,Belinsky MG, et al. MRP8, ATP-binding cassette C11(ABCC11), is a cyclic nucleotide efflux pump and a resis-tance factor for fluoropyrimidines 2′,3′-dideoxycytidineand 9′-(2′-phosphonylmethoxyethyl) adenine. J Biol Chem2003;278:29509—14.