Overexpression of the orotate phosphoribosyl-transferase gene enhances the effect of 5-Fluorouracil in head and neck squamous cell carcinoma in vitro.
ABSTRACT 5-Fluorouracil (5-FU) is a widely used drug in head and neck squamous cell carcinoma (HNSCC). In the anabolic pathway of 5-FU, the first step in activation of the drug is phosphorylation of 5-FU by orotate phosphoribosyltransferase (OPRT), which directly metabolizes 5-FU to 5-fluorouridine monophosphate (FUMP) in the presence of 5-phosphoribosyl-1-pyrophosphate. To date, OPRT expression in the tumors has been related to the clinical response or survival of cancer patients receiving 5-FU-based chemotherapy. In this study, we examined whether OPRT expression correlates with the chemosensitivity to 5-FU and cell proliferation in HNSCC. We constitutively expressed an OPRT cDNA in an HNSCC cell line. The effects of OPRT expression on in vitro cell growth and 5-FU cytotoxicity were examined. OPRT transfection increases the cytotoxicity of 5-FU without affecting cell proliferation of HNSCC cells in vitro. These results indicate that OPRT expression plays an important role in the sensitivity of HNSCC to 5-FU chemotherapy.
Article: Chemotherapy for colorectal cancer.New England Journal of Medicine 05/1994; 330(16):1136-42. · 51.66 Impact Factor
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ABSTRACT: Activation of 5-fluorouracil into its nucleotides requires phosphorylation by three pathways involving orotate phosphoribosyl-transferase (OPRT), uridine phosphorylase (UP), or thymidine phosphorylase (TP). In this study, we investigated the association between gene expressions of these three enzymes and antitumour effect. Gene expressions in primary colorectal tumours were analysed by a real-time reverse transcriptional-polymerase chain reaction method in 37 patients receiving oral treatment of tegafur-uracil and leucovorin for metastatic diseases. The median values of OPRT mRNA expressions were 1.39 and 0.85 for responding tumours and nonresponding tumours, respectively, showing a statistically significant difference (P=0.0008). Responding tumours had statistically lower expressions of TP mRNA than nonresponding tumours (P=0.006). However, there was no difference in UP mRNA expression between responding and nonresponding tumours. Patients with high OPRT (>/=1.0) gene expression survived longer than those with low OPRT (<1.0) expression. Dihydropyrimidine dehydrogenase (DPD) gene expressions were measured. Responding tumours had a statistically higher OPRT/DPD ratio than the nonresponding ones (P=0.003). When the median value of the OPRT/DPD ratio was selected as the cutoff value, patients with a high OPRT/DPD ratio survived statistically longer than those with a low ratio (P=0.0014). In conclusion, both the expression of OPRT gene and the OPRT/DPD ratio might be useful as predictive parameters for the efficacy of fluoropyrimidine-based chemotherapy for metastatic colorectal cancer.British Journal of Cancer 10/2003; 89(8):1486-92. · 5.08 Impact Factor
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ABSTRACT: Six cell lines differing in histological origin were studied regarding the growth inhibitory effect of fluoropyrimidines in relation to their metabolism. The human colon carcinoma cell line WiDr was most sensitive to 5-fluorouracil (FUra) (50% growth inhibitory concentration, 0.7 microM) and to its analogue 5'deoxy-5-fluorouridine (5'dFUR) (50% growth inhibitory concentration, 18 microM). The murine B16 melanoma cell line was moderately sensitive to FUra but least sensitive to 5'dFUR. The 50% growth inhibitory concentration values in the human melanoma cell lines IGR3 and M5, the transformed human intestine cell line intestine 407 and the rat hepatoma cell line H35 varied for FUra between 1.7 and 5.0 microM, and for 5'dFUR between 54 and 160 microM. Several enzymes from pyrimidine metabolism responsible for FUra metabolism were measured with FUra as a substrate. The activity of uridine phosphorylase, which catalyzes the conversion of 5'dFUR to FUra, was lowest in B16 cells correlating with the low sensitivity to 5'dFUR. When adenosine 5'-triphosphate was included in the reaction mixture for uridine phosphorylase, FUra was rapidly channeled into FUra nucleotides via its nucleoside. The rate of channeling appeared to correlate with the nucleoside phosphorylase activity in the various cell lines. In several cell lines activities of nucleotide-degrading enzymes were rather high and interfered with the measurement of orotate phosphoribosyl transferase (OPRT) with FUra as substrate. Addition of the phosphatase inhibitor glycerol-2-phosphate partly prevented breakdown of the newly formed 5-fluorouridine 5'-monophosphate and enabled measurement of OPRT. The WiDr cell line had a relatively high OPRT activity which could explain its sensitivity to FUra. The activity of thymidylate synthase was measured at a suboptimal concentration of 1 microM and at the optimal concentration of 10 microM deoxyuridine 5'-phosphate. With all cell lines the ratio between the activities at 10 and 1 microM was between 2.3 and 3.6. The activity of thymidylate synthase was lowest in WiDr and IGR3 cells and 3-4 times higher in M5 and Intestine 407 cells. The inhibition of 0.01 microM 5-fluorodeoxyuridine 5'-monophosphate was 80-90% at 1 microM deoxyuridine 5'-phosphate and 50-70% at 10 microM deoxyuridine 5'-phosphate with all cell lines. At 0.1 microM 5-fluorodeoxyuridine 5'-monophosphate enzyme activity was inhibited by 95-100%. The incorporation of FUra into RNA was relatively low in IGR3 cells and 3-5 times higher in all other cell lines.(ABSTRACT TRUNCATED AT 400 WORDS)Cancer Research 02/1986; 46(1):20-8. · 8.65 Impact Factor
Hindawi Publishing Corporation
Journal of Oncology
Volume 2012, Article ID 649605, 4 pages
Overexpressionof theOrotate Phosphoribosyl-Transferase
GeneEnhancesthe Effectof 5-FluorouracilinHead andNeck
RyujiYasumatsu,Torahiko Nakashima,and Shizuo Komune
Department of Otorhinolaryngology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan
Correspondence should be addressed to Ryuji Yasumatsu, firstname.lastname@example.org
Received 26 October 2011; Accepted 29 January 2012
Academic Editor: Dirk Rades
Copyright © 2012 Ryuji Yasumatsu et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
5-Fluorouracil (5-FU) is a widely used drug in head and neck squamous cell carcinoma (HNSCC). In the anabolic pathway of
5-FU, the first step in activation of the drug is phosphorylation of 5-FU by orotate phosphoribosyltransferase (OPRT), which
directly metabolizes 5-FU to 5-fluorouridine monophosphate (FUMP) in the presence of 5-phosphoribosyl-1-pyrophosphate.
To date, OPRT expression in the tumors has been related to the clinical response or survival of cancer patients receiving 5-FU-
based chemotherapy. In this study, we examined whether OPRT expression correlates with the chemosensitivity to 5-FU and cell
proliferation in HNSCC. We constitutively expressed an OPRT cDNA in an HNSCC cell line. The effects of OPRT expression on
in vitro cell growth and 5-FU cytotoxicity were examined. OPRT transfection increases the cytotoxicity of 5-FU without affecting
cell proliferation of HNSCC cells in vitro. These results indicate that OPRT expression plays an important role in the sensitivity of
HNSCC to 5-FU chemotherapy.
5-Fluorouracil (5-FU) has been used most frequently for
treating head and neck squamous cell carcinoma (HNSCC)
in a form of single agent or in combination with cisplatin
 and the drug of choice for systemic therapy in colorectal
cancer . However, nowadays 5-FU resistance during the
course of treatment has become common, which is an
important cause of failure for cancer therapies .
It has been reported that response rate of 5-FU and
its derivatives are due to interindividual difference in the
enzyme activities for anabolism and catabolism. In the
anabolic pathway of 5-FU, the first step in activation of the
drug is phosphorylation of 5-FU by orotate phosphoribo-
syltransferase (OPRT), which directly metabolizes 5-FU to
5-fluorouridine monophosphate (FUMP) in the presence
of 5-phosphoribosyl-1-pyrophosphate . This step is the
most important mechanism of 5-FU activation. To date,
OPRT expression in the tumors has been related to the
clinical response or survival of cancer patients receiving
5-FU-based chemotherapy [5, 6]. However, no study has
confirmed directly whether the regulation of intratumoral
OPRT expression level affects the efficacy of 5-FU and the
cell activity in HNSCC. We therefore investigated whether
overexpression of the OPRT enhances sensitivity to 5-FU.
In this study, to assess the role of OPRT in the biological
regulation of HNSCC, we constitutively expressed the OPRT
of OPRT on in vitro cell growth and 5-FU cytotoxicity was
2.1. Cell Line. The human head and neck squamous cell
carcinoma cell line, YCU-H, which was generously provided
by Dr. M. Tsukuda, was cultured in RPMI 1640 medium
and supplemented with 10% fetal bovine serum (FBS) and
penicillin/streptomycin 1000IU/mL (Invitrogen, Carlsbad,
CA, USA). Cells were maintained in a humidified incubator
at 37◦C under 5% CO2.
2Journal of Oncology
OPRT cDNA (kindly provided by Taiho Pharmaceutical
Co. Ltd., Tokyo, Japan) that contained the entire coding
sequence was subcloned in its sense orientation into Eco
RI-Kpn I sites of the expression vector pTARGET. Human
YCU-H cells were stably transfected with the pTARGET-
OPRT plasmid and the pTARGET vector control plasmid
via liposome-mediated transfection using Lipofectamine
2000 (Invitrogen), according to the conditions described
by the supplier. Forty eight hours after transfection, trans-
duced cells were selected in complete medium containing
500μg/mL Geneticin (Invitrogen) for 2 to 3 weeks. After
selection, single independent clones were randomly isolated
using cloning rings, and each clone was plated separately.
2.3. Western Blot Analysis of Cultured Cells. Western blot
analyses were performed as reported previously [7, 8] to
detect the OPRT expression in the YCU-H cell line. Total
cellular protein was extracted and quantified using the M-
Per Mammalian Protein Extracted Reagent and “Coomassie”
Protein Assay Reagent Kit (Pierce, Rockford, IL). Equal
amounts (10μg) of cell lysates were subjected to SDS-
polyacrylamide gel electrophoresis and transferred to a
nitrocellulose membrane. The membrane was blocked for
1h with 5% nonfat dry milk in PBS and then incubated
with the purified polyclonal antibody against OPRT (kindly
provided by Taiho Pharmaceutical Co. Ltd., Tokyo, Japan)
or an anti-β-actin antibody (Sigma Chemical Company, St.
Louis, MO, USA) for loading control for 1h at 37◦C. The
membranes were then incubated with the HRP-conjugated
goat anti-rabbit Ig G secondary antibody for 1h at room
temperature, followed by the detection with the enhanced
chemiluminescence (ECL) system (Amersham International,
2.4. In Vitro Proliferation Assays. Transfected clone and
vector control clone cells (1 × 104cells/dish) were seeded
onto 35 mm dishes in RPMI 1640 medium plus 10% FBS.
The number of the cells was counted every 48 hours for 8
subsequent days, in triplicate assays, using a Coulter Counter
(Beckman Coulter, Fullerton, CA, USA). The mean values
were used to generate growth curves.
were plated in 96-well plates at a density of 104cells/well and
further incubated for 24h. The medium was then removed
and replaced with fresh medium containing 5-FU (kindly
provided by Kyowa Hakko Co. Ltd.) for another 48h. Then,
10μL sterile MTT dye (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide, 5mg/mL; Sigma) was added
to the culture medium to a final concentration of 0.5mg/mL
and incubated at 37◦C for 2h. After that, the formazan
crystals were solubilized with 100μL of dimethylsulfoxide
(DMSO) for 10min. Spectrometric absorbance at 550nm
was measured with microplate reader. The IC50 value was
Figure 1: Western blot analysis indicating OPRT expression levels
in human head and neck squamous cell carcinoma YCU-H cells
transfected with OPRT cDNA. The OPRT protein level was
increased 35 times in the OPRT transfected clones H-OPRT in
comparison with the parental cell line, YCU-H cells.
2.6. Statistical Analysis. Statistical analyses were performed
using the Mann-Whitney U test. The Kaplan-Meier method
was used for analysis of survival data. The significance of
differences of survival plots was analyzed by the log-rank
test. Differences with a P value <0.05 were considered to be
3.1. Transfection of OPRT cDNA in YCU-H Human Head and
Neck SCC Cell Line. Four independent clones were selected
after 2 to 3 weeks of growth in medium supplemented with
Geneticin (500μg/mL). Representative clone, H-OPRT, was
selected for use in subsequent experiments. The levels of
Figure 1. The level of OPRT protein was increased 35 times
in the H-OPRT cells compared to control cells, respectively.
3.2. In Vitro Growth of the OPRT Overexpressing Cell Line.
When grown in 10% fetal bovine serum medium, the vector
control cell lines and OPRT transfected cell line showed
similar doubling times. There were no significant differences
in in vitro growth between OPRT overexpressing clone and
control clones (Figure 2).
3.3. Correlation between the Level of OPRT Expression in
HNSCC Cells and Their Sensitivity to 5-FU. Cell cytotoxic
assays were performed using MTT assay to examine whether
the transfected OPRT cDNA increased 5-FU sensitivity in
the OPRT overexpressing cells. The increased sensitivity of
the OPRT transfected cells to 5-FU was observed in H-OPRT
cells. The 50% growth inhibitory (IC50) value to 5-FU in H-
cells (IC50: 150μM) (Figure 3).
For decades, 5-FU and its derivatives such as 5?-DFUR and
tegafur have been used to treat cancer patients, and the
effectiveness of 5-FU is well proven in HNSCC. However,
the presence of drug-resistant tumor cells, which occurs with
other chemotherapeutic agents as well, causes poor response
to 5-FU-based chemotherapy .
Journal of Oncology3
(Cell number ×104)
Figure 2: In vitro growth curve of the OPRT transfectants. There
were no significant differences in in vitro growth between OPRT
clone and control clones. ?: YCU-H, ?: pTARGET, ?: H-OPRT.
5-FU concentration (μM)
Cell viability (%)
Figure 3: The cytotoxic effect of 5-FU was measured by MTT assay.
of concentration versus growth inhibition rate. Twenty-four hours
after plating, the cells were exposed to 5-FU for 2 days. Each value
represents the mean of triplicate plates; bars, SD. ?: pTARGET, ?:
To phosphorylate 5-FU into its nucleotides, the fol-
lowing 3 metabolic pathways have been reported: path-
way 1: phosphorylation to 5-fluorouridine monophosphate
(FUMP) by OPRT; pathway 2: phosphorylation to 5-fluo-
rodeoxyuridine (FdUR) by TP and a sequent conversion
to 5-fluorodeoxyuridine-monophosphate (FdUMP) by thy-
midine kinase (TK); pathway 3: phosphorylation to 5-fluor-
ouridine (FUR) by UP and a sequent conversion to FUMP
by uridine kinase (UK) [9, 10]. Thus, OPRT is one of the
main enzymes responsible for the phosphorylation of 5-FU
in human cancer cells. In addition, several clinical reports
have demonstrated a relationship between OPRT activity in
the tumors and clinical response of cancer patients receiving
5-FU-based chemotherapy [5, 6]. However, the role of OPRT
expression in HNSCC has not been established and, to the
best of our knowledge, there are no studies investigating the
relationship between the in vitro OPRT expression and 5-FU
cytotoxicity in HNSCC.
To directly confirm whether the overexpression of OPRT
affects the sensitivity to 5-FU and to review the role of
OPRT in HNSCC cells, we transfected OPRT cDNA in a
human HNSCC cell line, YCU-H. As a result, we found a
35-fold higher expression of the endogeneous OPRT protein
in OPRT transfected YCU-H cells. The present study also
demonstrated that the increased sensitivity to 5-FU was
observed in OPRT overexpressing cells. Inaba et al. reported
a significantly low activity of OPRT in the 5-FU-resistant cell
line by measuring the enzyme activities in the human cancer
cell lines, . Taomoto et al. also indicated that OPRT
overexpression plays an important role in the increased
sensitivity of gastric carcinomas to 5-FU chemotherapy .
The present result is compatible with those of the earlier
studies. These results, combined with other reports, strongly
suggest that overexpression of the OPRT in cancer cells
enhances the sensitivity to 5-FU treatment.
There were no significant differences in the in vitro
growth between OPRT overexpressing cells and control cells
protein is not linked to rapid tumor cell proliferation of
HNSCC cells. On the other hand, in bladder carcinoma, it
was reported that OPRT activity was upregulated compared
with the activity in normal bladder and that OPRT may
be of prognostic value . Miyake et al. also reported
that OPRT may play a potential role in regulating the
malignant potential of pancreatic cancer . Because the
regulation of OPRT expression and its involvement with
tumor proliferation remains unclear, we could not reach any
conclusion about the relationship between this enzyme and
In conclusion, our data suggest that OPRT affects the
present results strongly indicate that OPRT overexpression
plays an important role in the sensitivity of HNSCC to 5-FU
chemotherapy. Because the level of OPRT expression could
be used as a predictive indicator for 5-FU efficacy against
select patients for more intensive treatment including CDDP
Conflict of Interests
The authors declare no conflict of interests.
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