Functional analysis of pyrimidine biosynthesis enzymes
using the anticancer drug 5-fluorouracil in
Caenorhabditis elegans
Seongseop Kim
1,
*, Dae-Hun Park
2,
*, Tai Hoon Kim
1
, Moogak Hwang
1
and Jaegal Shim
1
1 Cancer Experimental Resources Branch, National Cancer Center, Gyeonggi-do, Korea
2 College of Pharmacy, Kangwon National University, Gangwon-do, Korea
Introduction
Enzymes responsible for pyrimidine biosynthesis play
critical roles in cellular metabolism, because they pro-
vide the pyrimidine nucleosides that are key compo-
nents of many biomolecules, such as RNA and DNA.
Pyrimidine metabolism disorders can cause diseases
such as orotic aciduria, which results from uridine
monophosphate synthetase (UMPS) deficiency [1].
There are two routes for synthesizing pyrimidines:
de novo and salvage pathways. Many genes encoding
pyrimidine salvage pathway enzymes are genetic fac-
tors influencing pyrimidine antagonist-based cancer
chemotherapy [2].
5-Fluorouracil (5-FU) is a major pyrimidine anta-
gonist that has been used for more than 40 years in
Keywords

gans. Whereas pyrimidine biosynthesis pathways are highly conserved
between worms and humans, no human thymidine phosphorylase homolog
has been identified in C. elegans. UPP-1 functions as a key regulator of the
pyrimidine salvage pathway in C. elegans, as mutation of upp-1 results in
strong 5-FU resistance.
Abbreviations
5dFUR, 5¢-deoxy-5-fluorouridine; 5-FU, 5-fluorouracil; DPD, dihydropyrimidine dehydrogenase; dRib1P, 2-deoxy-a-
D-ribose 1-phosphate; GFP,
green fluorescent protein; MBP, maltose-binding protein; OMPDC, orotate monophosphate decarboxylase; OPRT, orotate phosphoribosyl
transferase; PRPP, phosphoribosyl pyrophosphate; RNAi, RNA interference; SEM, standard error of the mean; SNP, single-nucleotide
polymorphism; TK, thymidine kinase; TP, thymidine phosphorylase; TS, thymidylate synthase; UMPK, uridine monophosphate kinase; UMPS,
uridine monophosphate synthetase; UP, uridine phosphorylase.
FEBS Journal 276 (2009) 4715–4726 ª 2009 The Authors Journal compilation ª 2009 FEBS 4715
cancer chemotherapies. 5-FU and other pyrimidine
antagonists, such as capecitabine and tegafur, have
been used to treat various cancers, including colo-
rectal, stomach, ovarian, head and neck cancers. In
particular, 5-FU is a primary therapy for colorectal
cancer [2]. Like other pyrimidine antagonists, 5-FU is
a prodrug that is converted to the active form via
the pyrimidine biosynthesis pathway [3]. Therefore,
the function of this drug is closely associated with
the activity of pyrimidine synthesis enzymes, includ-
ing dihydropyrimidine dehydrogenase (DPD), thymi-
dylate synthase (TS), uridine phosphorylase (UP),
thymidine phosphorylase (TP), uridine monophosphate
kinase (UMPK), and orotate phosphoribosyl trans-
ferase (OPRT). Expression levels of these enzymes
in cancer cells are linked to 5-FU sensitivity and
resistance [2–6].

growth [16]. Thus, UP is a possible prognostic factor
for several cancers, including breast cancer and oral
squamous cell carcinoma [13,17].
As a core enzyme of the pyrimidine salvage path-
way, UP is conserved across kingdoms, and many
studies on UP have been carried out in Escherichia
coli. However, given that UP function is important for
both normal physiology and cancer therapy, animal
models are increasingly being used to study this
enzyme. Disruption of UP activity in mouse embryonic
stem cells leads to increased 5-FU concentrations in
plasma and reduced incorporation of 5-FU into
nucleic acids [18]. Moreover, UP
) ⁄ )
mice exhibit
increased uridine concentrations in the plasma, lung,
gut, liver and kidney as compared with wild-type mice
[19]. The inhibition of TP activity also results in ele-
vated pyrimidine levels in plasma and axonal swelling
in the brains of mice [20].
Previously, we demonstrated that 5-FU induces
germ cell death and inhibits development in Caenor-
habditis elegans [21]. We also observed that C. elegans
DPD and TS expression levels are associated with
5-FU function [22]. Here, we describe the results
obtained from a 5-FU-resistant mutant screen in
C. elegans, from which we identified upp-1 mutations
from several 5-FU-resistant mutants. In addition, we
characterized C. elegans UMPK and UMPS ⁄ OPRT
homologs using RNA interference (RNAi) and

sensitive to 5-FU than late larval development, we
decreased the 5-FU concentration (5 nm) to compare
the hatching ratios of wild-type and upp-1 mutant
worms. Wild-type worms on the 5-FU plate exhibited
a low hatching ratio (10% of total eggs), whereas the
upp-1 mutant exhibited a high hatching ratio (over
90%) (Fig. S1). Observation of upp-1 mutants under a
dissection microscope and a high-resolution differential
interference contrast microscope revealed that, with
the exception of 5-FU resistance, they did not differ
from wild-type worms in either morphology or behav-
ior. However, the lifespan of upp-1 mutant worms was
reduced by about 30% as compared with wild-type
worms (Fig. S2).
ZK783.2 encodes a protein with amino acid homol-
ogy to human UP (UPP-1 and UPP-2) (46% identical;
Fig. S3). In order to verify the functional conservation
between human and worm UPP-1, we expressed
human UPP-1 under the control of the worm upp-1
promoter. Human UPP-1 was able to rescue the upp-
1(jg1) mutant (Fig. 1H). Sequencing of six upp-1
Fig. 2. UPP-1 is highly conserved from C. elegans to humans. (A)
The ZK783.2 ORF encodes a homolog of human UP. Six upp-1
mutants were sequenced, and their mutations are indicated by an
asterisk on the ZK783.2 genomic diagram. Asterisks indicate the
location of each mutation, and Q203* indicates that the gluta-
mine 203 was changed to a stop codon. (B, C) The UPP-1::GFP
fusion proteins were expressed in several tissues, including the
hypodermis, pharynx, spermatheca, and gonad. Scale bars: 100 lm
(B) and 10 lm (C).

gonad (Fig. 2D), consistent with our observation that
germ cell death normally induced by 5-FU is sup-
pressed in upp-1 mutants [21].
UPP-1 has both UP and TP activities
As the upp-1 mutant is resistant to 5-FU, and no TP
homolog has been identified in C. elegans, we hypoth-
esized that C. elegans UPP-1 functions as both a UP
and a TP. Thus, a single mutation in upp-1 may con-
fer strong 5-FU resistance. Indeed, C. elegans UPP-1
exhibited both UP and TP activity in vitro, with TP
activity being greater than UP activity (Fig. 3A). We
also tested the activities of several mutant UPP-1
proteins (T128I, Y209F, and Q203*), and compared
larval growth of these mutant strains on 5-FU plates.
All three UPP-1 mutant proteins exhibited very low
levels of enzyme activity ( 20% of that of wild-type
UPP-1) in vitro (Fig. 3A). The growth rates of these
upp-1 mutants were also similar to each other
(Fig. 3B).
Next, we treated wild-type and upp-1 mutant worms
with 5¢-deoxy-5-fluorouridine (5dFUR) to further
examine the function of UPP-1 in the pyrimidine bio-
synthesis pathway. 5dFUR is converted to 5-FU by
UP and TP [9]. The effects of 5dFUR in the upp-1
mutant are questionable, as no TP homolog has been
discovered in the C. elegans genome. Both 5dFUR and
5-FU, however, exhibited similar effects on wild-type
worms, and growth of the upp-1 mutant on the
5dFUR plate resembled that on the 5-FU plate
(Fig. 3C).

5-FU or 5dFUR plates) calculated by unpaired Student’s t-test were
< 0.001. Error bars represent SEM.
Characterization of worm UPP-1, UMPK, and UMPS S. Kim et al.
4718 FEBS Journal 276 (2009) 4715–4726 ª 2009 The Authors Journal compilation ª 2009 FEBS
RNAi to knock down these genes by the bacterial
feeding method. Some genes, such as T23G5.1 (rnr-1)
and C03C10.3 (rnr-2), which are homologs of the
genes encoding ribonucleotide reductase a and b
subunits, respectively, exhibited a lethal phenotype, so
we could not test the growth of these worms on 5-FU
plates. Knockdown of T07C4.1 (UMPS) or C29F7.3
(UMPK) resulted in 5-FU resistance, whereas RNAi
for other genes had no effect on 5-FU sensitivity
(Fig. 4). Our results indicate that the pyrimidine
biosynthesis and 5-FU functional pathways are well
conserved between humans and C. elegans.
Two UMPK homologs were expressed in
different tissues
Using homology searches and RNAi, we determined
that C29F7.3 and T07C4.1 were associated with 5-FU
function (Fig. 4). Interestingly, C29F7.3 shows amino
acid similarity to human uridine kinase, UMPK, and
UDPK. Uridine kinases are downstream of UP in the
pyrimidine salvage pathway. Homology searches
revealed that several uridine kinases exist in the C. ele-
gans genome. Three of these were selected on the basis
of length and sequence homology, and their expression
patterns and enzymatic activities were characterized.
Deletion (tm2740) or knockdown of B0001.4 did not
result in altered 5-FU sensitivity. C29F7.3 and

C29F7.3 RNAi (data not shown), indicating that
knockdown of these two genes is very specific.
To more precisely understand the functions of these
uridine kinase homologs, we analyzed their enzyme
activity in vitro. The proteins purified from the bacte-
rial induction system and several substrates were incu-
bated together, and products were detected by HPLC.
Both C29F7.3 and F40F8.1 exhibited only UMPK
activity, but B0001.4 showed no uridine kinase activity
(Fig. 5B). C29F7.3 and F40F8.1 exhibited similar
UDP peaks when UMP was added as a substrate.
Both C29F7.3 and F40F8.1 may function downstream
of UPP-1, but show different responses in mediating
5-FU function, probably because of their different
expression patterns.
T07C4.1 and R12E2.11 proteins have OPRT
function
Both de novo synthesis and salvage pathways are used
to synthesize UMP. The salvage pathway includes
UP ⁄ uridine kinase and OPRT, and the de novo path-
way includes enzymes such as orotate monophosphate
Fig. 4. The pyrimidine biosynthesis pathway is conserved from
humans to C. elegans. Some enzymes of the pyrimidine biosynthe-
sis pathway are also involved in 5-FU resistance. Growth tests
were performed on 5-FU plates following RNAi for worm homologs
of various human genes. F25H2.5 is a putative homolog of uridine
diphosphate kinase, and Y43C5A.5 is TK. R12E2.11 and T07C4.1
are OPRT domain proteins. F19B6.1, B0001.4, C29F7.3 and
F40F8.1 are putative uridine kinase or UMPK homologs. ZK783.2
(upp-1) RNAi was used as a positive control. Depletion of T07C4.1

has no OMPDC activity, but mixing R12E2.11 and
T07C4.1 results in a higher UMP peak than observed
with T07C4.1 protein alone. This suggests that
R12E2.11 and T07C4.1 may cooperate to synthesize
UMP in C. elegans intestinal cells.
Discussion
The upp-1 mutant is highly resistant to 5-FU, even
when compared with other 5-FU resistant mutant and
Fig. 5. Characterization of UMPKs in C. ele-
gans. (A) The expression patterns of
C29F7.3::GFP and F40F8.1::GFP are shown.
C29F7.3::GFP expression is robust in the
pharynx, hypodermal cells, and intestine,
whereas F40F8.1::GFP is expressed
strongly in neurons and the pharynx, but
weakly in the intestine. Scale bars: 100 lm.
(B) In vitro enzymatic assays of three uridine
kinase homologs using analytical HPLC. No
proteins exhibited uridine kinase activity
when uridine was used as a substrate, but
both C29F7.3 and F40F8.1 produced UDP
when UMP was used as a substrate.
Arrows indicate UDP peaks. Detection
times are shown on the x-axes, and UV
absorbance at 260 nm on the y-axes.
Characterization of worm UPP-1, UMPK, and UMPS S. Kim et al.
4720 FEBS Journal 276 (2009) 4715–4726 ª 2009 The Authors Journal compilation ª 2009 FEBS
transgenic worms. Expression levels of DPD and TS
are closely related to the 5-FU response and sensitivity
in human cancers [25,26]. Transgenic worms overex-

R12E2.11::GFP are shown. T07C4.1::GFP
expression is robust in neurons and the
intestine, whereas R12E2.11::GFP is
expressed strongly in the body wall muscle,
spermatheca, and vulval muscle. Scale bars:
100 lm. (C) Enzymatic activities of T07C4.1
and R12E2.11 proteins in vitro. OPRT (left)
and OMPDC (right) activities were mea-
sured by adding phosphoribosyl pyropho-
sphate (PRPP) with uracil (Ura) and orotate
(Oro), respectively, as a substrate. Both
T07C4.1 and R12E2.11 have OPRT activity,
but only T07C4.1 has OMPDC activity, as
expected from the protein domain struc-
tures. R12E2.11 itself has no OMPDC activ-
ity, but it promotes the OMPDC activity of
T07C4.1. Detection times are shown on the
x-axes, and UV absorbance at 260 nm on
the y-axes.
S. Kim et al. Characterization of worm UPP-1, UMPK, and UMPS
FEBS Journal 276 (2009) 4715–4726 ª 2009 The Authors Journal compilation ª 2009 FEBS 4721
RNAi for other pyrimidine biosynthesis pathway
enzymes revealed that the depletion of only three genes
resulted in 5-FU resistance. One explanation for the
observed results is that knockdown of a single gene is
not sufficient to abolish pyrimidine biosynthesis, owing
to the existence of redundant genes or pathways. Inter-
estingly, two UMPK genes, C29F7.3 and F40F8.1, are
very similar in amino acid sequence, and their protein
products show similar abilities to synthesize UDP from

knockdown for UPP-1 or UMPS was unexpected.
However, UPP-1 is strongly expressed in the hypo-
dermis, and T07C4.1 is mainly expressed in the intes-
tine. Knockdown of C29F7.3, on which both UP ⁄
uridine kinase and OPRT converge, resulted in high
5-FU resistance. As C29F7.3 is expressed in the hypo-
dermis and intestine, both UP ⁄ UMPK in the hypo-
dermis and OPRT ⁄ UMPK in the intestine are essential
for mediating 5-FU function in C. elegans. It is also
possible that UPP-1 and OPRT cooperate to mediate
5-FU function and UMP synthesis, because knock-
down of T07C4.1 in upp-1 mutants resulted in similar
5-FU resistance as knockdown of either T07C4.1 or
UPP-1 (data not shown).
On the basis of these results, we propose a model of
pyrimidine biosynthesis and 5-FU conversion in
humans and in C. elegans (Fig. 7). In a human cancer
model, the conversion of 5-FU to FdUMP is mediated
by three independent pathways involving UPP, OPRT,
and TPP. In contrast, the C. elegans genome does not
include a TP homolog, and downstream signaling via
tyrosine kinase (TK) does not appear to be associated
with 5-FU function, given the lack of 5-FU resistance
following knockdown of the worm TK candidate gene,
Y43C5A.5 (Fig. 4). Our data do not explain all of the
similarities and differences in the pyrimidine salvage
pathway and 5-FU function between humans and
C. elegans, but it is clear that UP and OPRT activity
mediated by UMPK is a major 5-FU conversion path-
way in C. elegans.

The Bristol strain N2 was used as a wild-type strain. The
Hawaiian strain CB4856 was used as a reference strain for
mapping mutant genes by SNPs [31]. The B0001.4 deletion
mutant (tm2740) was a gift from S. Mitami (Tokyo
Women’s Medical University, Japan). Animals were cul-
tured as described by Brenner [32].
Chemicals
Rib1P, 2-deoxy-a-d-ribose 1-phosphate (dRib1P), uridine,
UMP, UDP, UTP, ATP, 2-deoxyuridine, 5-FU, 5dFUR,
orotidine 5¢-phosphate and PPRP were purchased from
Sigma-Aldrich Chemicals (St Louis, MO, USA). [6-
14
C]5-FU
(specific activity 52 mCiÆmmol
)1
) was purchased from
Moravek Biochemicals, Inc. (Brea, CA, USA).
5-FU sensitivity and mutant phenotype analysis
Analysis of 5-FU sensitivity was performed on plates con-
taining 5-FU (800 nm). Synchronized embryos were trans-
ferred to 5-FU plates. After 60 or 72 h, the numbers of L4
larvae ⁄ adult worms and total worms were counted, and the
ratios of L4 larvae ⁄ adult worms to total worms were calcu-
lated. 5-FU plates were kept in the dark during experiments
to avoid fluorine degradation by light. 5dFUR sensitivity
was tested using the same method. For upp-1 (jg1) mutant
rescue experiments, total L4 and adult animals from trans-
genic worms carrying additional genes, such as the ZK732
cosmid, were counted, and the ratio of roller worms con-
taining coinjected pRF4 plasmid to nonroller worms was

digested with PstI and XbaI, and ligated into pPD95.77.
Human UPP1 cDNA (995 bp) was then amplified, using a
human cDNA library (Clontech Laboratories, Inc., Moun-
tain View, CA, USA) as a template and the following prim-
ers: 5¢-TTT CCC GGG CAC TGC AGA CGT CTG TCC
G-3¢ and 5¢-TTT GGT ACC CAG GCC TTG CTC AGT
TTC TTC-3¢. PCR products were digested with SmaI and
KpnI, and ligated to the amplified C. elegans upp-1 pro-
moter. The same vector and methods were used to make
C29F7.3::GFP, F40F8.1::GFP, T07C4.1::GFP, and
R12E2.11::GFP. The primers and restriction enzyme sites
used were as follows: 5¢-TTT
AAG CTT CTT TAT CAG
TAG TTT TGA GGC CG-3¢ (HindIII) and 5¢-AAT
CTG
CAG TTT TTG GTT GGC AGC CGC GAA TAC-3¢
(PstI) for C29F7.3::GFP, 5¢-TT
G TCG ACC AGT CTT
CAA AAT AGC GCA GG-3¢ (SalI) and 5¢-TTT
TCT
AGA TTT TTT GTT GGC AGC GTC G-3¢ (XbaI) for
F40F8.1::GFP, 5¢-AAT GGG
CTG CAG AAG AAA
AGG GTG GC-3¢ (PstI) and 5¢-T
GG ATC CAA TGC
TAT CGT CGC TTC TCG-3¢ (BamHI) for T07C4.1::GFP,
5¢-TTT
CTG CAG TTG TCC TTG ATA TCT C-3¢ (PstI)
and 5¢-AA
T CTA GAA GCA GAT GAG CAA TAA TCT

G GAT CCA TGA AAA ACA CTC TGA AAT
TGC-3¢ (BamHI) and 5¢-AA
G GTA CCT TAA TGT GGA
CGG GAG AAT GG-3¢ (KpnI) for B0001.4, 5¢-AA
G GAT
CCA TGC ACA ACG TGG TTT TTG TTC-3¢ (BamHI)
and 5¢-TTT
TCT AGA TCA AAT GCT ATC GTC GCT
TCT CG-3¢ (XbaI) for T07C4.1, and 5¢-TTT
GAA TTC
ATG ACC GCC GCC ACC G-3¢ (EcoRI) and 5¢-AA
G
GTA CCT TAA TGT GGA CGG GAG AAT GG-3¢
(KpnI) for R12E2.11.
Microinjection and RNAi
All transgenic strains were generated by microinjection to
achieve germline transformation. For rescue experiments,
the ZK783 cosmid carrying the upp-1 (ZK783.2) PCR prod-
uct and the construct containing the C. elegans upp-1 pro-
moter fused with human UPP1 cDNA were injected
(75 lgÆmL
)1
) along with the marker pRF4 (75 lgÆmL
)1
)
into upp-1 (jg1) mutants. Control transgenic worms were
injected with pRF4 plasmid DNA (100 lgÆmL
)1
) only. To
generate transgenic worms that express UPP-1::GFP, the

(35 lL) consisted of 1 lg of purified UPP-1 fusion protein,
10 mm Tris ⁄ HCl buffer (pH 7.4), 0.8 mm EDTA, 2.5 mm
Rib1P or dRib1P,5mm MgCl
2
, and 192 lm [6-
14
C]5-FU.
The reaction was incubated at 37 °C for 1 h. After incuba-
tion, samples were boiled for 3 min to stop the enzymatic
reaction, and then chilled on ice. Compounds were sepa-
rated by TLC. All assay mixtures were spotted onto PEI
cellulose sheets with 4 lL of nonradioactive tracer (100 lg
of 5-FU and 100 lg of uridine for the UP assay mixture;
100 lg of 5-FU and 100 lg of deoxyuridine for the TP
assay mixture). After development with distilled water,
spots were excised using 254 nm UV light. The activity was
counted after addition of 4 mL of scintillation fluid.
Evaluation of the enzymatic activity of C29F7.3, F40F8.1,
B0001.4, T07C4.1 and R12E2.11 was performed as described
by Li et al. [35] and Krungkrai et al. [36], with a few modifi-
cations. All reaction mixtures contained 10 lg of recombi-
nant proteins in a total reaction volume of 100 lL. The
reaction mixture was incubated for 12 h at room tempera-
ture, and then boiled at 100 °C for 3 min to stop the reaction.
The 10· reaction buffer mixture contained 500 mm Tris ⁄ HCl
buffer (pH 7.4), 100 mm MgCl
2
, 2.5 nm dithiothreitol, and
10 mm EDTA. Analytical HPLC using the method described
by Di Pierro et al. [37], with some modifications, was carried

Characterization of worm UPP-1, UMPK, and UMPS S. Kim et al.
4724 FEBS Journal 276 (2009) 4715–4726 ª 2009 The Authors Journal compilation ª 2009 FEBS
Acknowledgements
This work was supported by research grants from the
National Cancer Center (NCC-0510583 and NCC-
0810070) of South Korea. We thank the Sanger Center
for providing cosmids, and S. Mitami for several
mutants, including the B0001.4 deletion mutant
(tm2740). We also thank the Caenorhabditis elegans
Genetics Center (CGC) for providing reference mutant
worms, such as lon-1.
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worms.
Fig. S2. Lifespan tests of wild-type worms and three
upp-1 mutants.
Fig. S3. Sequence alignments of C. elegans UPP-1 and
human UPP-1.
Fig. S4. Sequence alignments of C29F7.3 and F40F8.1.
Fig. S5. Sequence alignments of human UMPS and
worm OPRT homologs.
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Characterization of worm UPP-1, UMPK, and UMPS S. Kim et al.
4726 FEBS Journal 276 (2009) 4715–4726 ª 2009 The Authors Journal compilation ª 2009 FEBS