Báo cáo khoa học: Functional analysis of pyrimidine biosynthesis enzymes using the anticancer drug 5-fluorouracil in Caenorhabditis elegans docx

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

5-fluorouracil; C. elegans; UMPK; UMPS;
uridine phosphorylase
Correspondence
J. Shim, Cancer Experimental Resources
Branch, National Cancer Center, 809 Madu
1-dong, Goyang-si, Gyeonggi-do, 411-769,
Korea
Fax: +82 31 920 2002
Tel: +82 31 920 2262
E-mail:
*These authors contributed equally to this
work
(Received 19 May 2009, revised 22 June
2009, accepted 24 June 2009)
doi:10.1111/j.1742-4658.2009.07168.x
Pyrimidine biosynthesis enzymes function in many cellular processes and
are closely associated with pyrimidine antagonists used in cancer chemo-
therapy. These enzymes are well characterized from bacteria to mammals,
but not in a simple metazoan. To study the pyrimidine biosynthesis path-
way in Caenorhabditis elegans, we screened for mutants exhibiting resis-
tance to the anticancer drug 5-fluorouracil (5-FU). In several strains,
mutations were identified in ZK783.2, the worm homolog of human uridine
phosphorylase (UP). UP is a member of the pyrimidine biosynthesis family
of enzymes and is a key regulator of uridine homeostasis. C. elegans UP
homologous protein (UPP-1) exhibited both uridine and thymidine phos-
phorylase activity in vitro. Knockdown of other pyrimidine biosynthesis
enzyme homologs, such as uridine monophosphate kinase and uridine
monophosphate synthetase, also resulted in 5-FU resistance. Uridine
monophosphate kinase and uridine monophosphate synthetase proteins are
redundant, and show different, tissue-specific expression patterns in C. ele-

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].

Uridine is a pyrimidine nucleoside that is essential
for the synthesis of RNA and biomembranes and is
involved in the regulation and function of the cardio-
circulatory, reproductive, nervous and respiratory sys-
tems [7]. Furthermore, it modulates the cytotoxic
effects of fluoropyrimidines in both normal and neo-
plastic tissues [8]. The concentration of uridine in
plasma and tissues is tightly regulated by cellular
transport mechanisms and by UP activity [7]. UP cata-
lyzes the reversible phosphorolysis of uridine, yielding
uracil and Rib1P, and is an important enzyme in the
pyrimidine salvage pathway. Human UP and TP each
exhibit both uridine and thymidine phosphorylase
activities. Pyrimidine phosphorylases differ in activity
and substrate specificity, and play different roles in flu-
oropyrimidine sensitivity [9]. UP is the major phos-
phorylase that regulates uridine homeostasis, but TP
also acts on uridine as a substrate to a certain extent.
At least two UPs and one TP are present in humans.
The first human UP was cloned in 1995 [10], and this
was followed by the cloning and expression analysis of
UPP-2 [11]. UP expression is controlled transcription-
ally by oncogenes, tumor suppressor genes, and cyto-
kines [12]. UP activity is typically upregulated in
various tumor tissues, conferring a therapeutic advan-
tage for 5-FU in cancer patients [13]. Beyond tran-
scriptional regulation, UP activity is modulated by
specific inhibitors, such as 5-phenylthioacyclouridine
[14,15]. During oncogenesis, ectopic expression of UP
is reported to support anchorage-independent cell

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

5-FU. Uncovering the mechanism of 5-FU resistance
and characterization of pyrimidine biosynthesis
enzymes in C. elegans will help to further our under-
standing of pyrimidine biosynthesis enzymes by fill-
ing in a missing link between bacteria and higher
organisms.
Results
UPP-1 mediates 5-FU functions in C. elegans
We performed a genetic screen for 5-FU-resistant
mutant C. elegans strains by scoring for larval growth
in the presence of 5-FU. One of these mutants (jg1)
was mapped to identify the mutated gene by using
single nucleotide polymorphisms (SNPs). The jg1
mutation mapped near SNP uCE3-1087, which is in
the )0.07 region of chromosome III (Fig. 1G) and
could be rescued with a single cosmid, ZK783. It
could also be rescued by expression of a single ORF
(ZK783.2) encoding UPP-1. Transgenic worms
expressing these rescue constructs exhibited 5-FU
sensitivity (Fig. 1H).
Although the upp-1 mutant grew slowly on the 5-FU
plate, it advanced to late larval and adult stages
(Fig. 1C), unlike wild-type worms, which arrested at
L1 or L2 (Fig. 1B,E). Larvae were evaluated 60 h after
egg transfer from normal plates to 5-FU plates, during
which time wild-type worms grown on control plates
exhibit the vulval invagination typical of L4 larva
(Fig. 1A,D). Because early embryogenesis is more
Characterization of worm UPP-1, UMPK, and UMPS S. Kim et al.
4716 FEBS Journal 276 (2009) 4715–4726 ª 2009 The Authors Journal compilation ª 2009 FEBS

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).

Fig. 1. The upp-1 mutant is highly resistant to 5-FU. The upp-1 (jg1) mutant grows well on the 5-FU plate as compared with wild-type
C. elegans (A–C). Although the growth of upp-1 animals on the 5-FU plate is slower than that of wild-type animals on normal plates, this
mutant survives up to stage L4 and adulthood (D–F). The arrowhead (A) and asterisk (D) indicate vulval invagination, which is a character-
istic of the L4 stage. The arrow indicates turning of the gonad, which occurs at the early L4 larval stage (F). Wild-type worms growing
on the 5-FU plates are arrested at the L2 stage (B, E). Growth tests were done on plates containing 800 n
M 5-FU (A–F). (A–C) Dissect-
ing microscope images. (D–F) Nomarski images from a high-resolution differential interference contrast microscope. (A, D) Wild-type
worms on control plates. (B, E) Wild-type worms on 5-FU plates. (C, F) upp-1 (jg1) mutant worms on 5-FU plates. All pictures were
taken 60 h after egg transfer. Scale bars: 100 lm (A–C) and 10 lm (D–F). (G) The SNP mapping method using the Hawaiian strain
CB4856 was used for upp-1 (jg1) mutant cloning. The upp-1 mutation mapped near the SNP uCE3-1087 in the )0.07 region of LG III.
(H) Fifteen cosmids in that region were obtained from the Sanger Center, and the phenotype of the mutant was rescued with a single
ZK783 cosmid. The upp-1 mutant was also rescued with a genomic PCR product including a single ORF (ZK783.2). The upp-1 mutant
worm carrying only the control pRF4 (rol-6gf) plasmid [upp-1; Ex (pRF4)] was the same as the nontransgenic upp-1 mutant. Transgenic
worms carrying the ZK783 cosmid [upp-1; Ex (ZK783; pRF4)] or ZK783.2 PCR product [upp-1; Ex (ZK783.2 ; pRF4)] were sensitive to
5-FU. The upp-1 mutant worm expressing human UPP-1 under the control of the worm upp-1 promoter [upp-1; Ex (hUPP1; pRF4)] also
exhibited 5-FU sensitivity. Coinjection of the marker pRF4 was used to identify transgenic worms. Error bars represent standard error of
the mean (SEM). *P < 0.001 as compared with control Ex (pRF4) worms on the 5-FU plate, determined by unpaired Student’s t-test.
**P > 0.1.
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 4717
mutants (jg1, jg2, jg3, jg7, jg8, and jg11) revealed mis-
sense mutations in all but jg3, which had a nonsense
mutation at glutamine 203 (Fig. 2A).
As upp-1 mutants exhibited strong 5-FU resistance,
and as there is only one UPP gene in the C. elegans
genome, we hypothesized that the expression of UPP-1
was ubiquitous. Transgenic worms expressing UPP-
1::green fluorescent protein (GFP) showed bright GFP
signal in the hypodermis, pharynx, and spermatheca
(Fig. 2C). UPP-1::GFP was also expressed in the

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).

The pyrimidine biosynthesis pathway is well
conserved between C. elegans and humans
5-FU is converted to FdUMP by several sequential
steps of the pyrimidine salvage pathway, which
involves several metabolic enzymes. UP mediates the
first step of 5-FU conversion. We searched for homo-
logs of the human pyrimidine biosynthesis enzymes,
including uridine kinase, UMPK, and UMPS, in
C. elegans to test their roles in 5-FU function and
were able to identify homologs for most of them in
C. elegans.
To explore the functional relationships of these
pyrimidine biosynthesis enzymes using 5-FU, we used
Fig. 3. C. elegans UPP-1 exhibits TP and UP activities. (A) UPP-1
converted both Rib1P and dRib1P to uridine and thymidine in the
presence of uracil and thymine, respectively. The enzymatic activi-
ties of three mutant UPP-1 proteins were very low as compared
with that of wild-type UPP-1 in vitro. The TP activity of UPP-1 is
two times higher than the UP activity. No differences in enzymatic
activity were observed between the three UPP-1 mutant proteins.
Both *P and **P (as compared with wild-type UPP-1 activity) are
< 0.001. (B) The ratios of L4 and adult worms compared with the
total for the three upp-1 mutants on 5-FU plates are shown. No dif-
ferences in growth were observed among three the upp-1 alleles.
*P-values (as compared with wild-type worms on 5-FU plates) cal-
culated by unpaired Student’s t-test were < 0.001. (C) The upp-1
(jg1) mutant also grew well on 5dFUR plates. 5dFUR, a precursor
of 5-FU, is converted by UP and TP activities. Growth test results
are reported as percentages of L4 and adult animals out of total
progeny (y-axis). *P-values (as compared with wild-type worms on

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

F40F8.1 share 82% identity (Fig. S4), but knockdown
of C29F7.3 results in 5-FU resistance, whereas knock-
down of F40F8.1 does not (Fig. 4). In order to evalu-
ate how these proteins function differently in the 5-FU
pathway, we studied their expression patterns using
GFP reporter fusion constructs (Fig. 5A).
C29F7.3::GFP is expressed in the hypodermis, intes-
tine, and pharynx, whereas F40F8.1::GFP signal is
observed mostly in neurons and the pharynx. These
distinct expression patterns may account for the differ-
ences between these two proteins in the 5-FU treat-
ment and RNAi experiments. The activities of these
enzymes in the hypodermis and intestine may be
important for mediating 5-FU effects.
As C29F7.3 and F40F8.1 share considerable
sequence identity, we wished to rule out the possibility
of RNAi cross-effects. C29F7.3 RNAi was very effec-
tive, with most GFP disappearing in the hypodermis
and intestine, but neuronal expression of
C29F7.3::GFP appeared (data not shown). In general,
C. elegans neurons are resistant to RNAi [23]. It was
difficult to dissect the effects of F40F8.1 RNAi,
because F40F8.1::GFP was expressed strongly in neu-
rons and the pharynx, but weakly in the intestine.
F40F8.1 RNAi resulted in a slightly decreased GFP
signal. We made a transgenic worm expressing
F40F8.1::GFP under the control of the C29F7.3 pro-
moter to further evaluate the F40F8.1 RNAi efficiency
and specificity. Ectopically expressed F40F8.1::GFP
was diminished by F40F8.1 RNAi, but not by

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

and C29F7.3 by RNAi resulted in 5-FU resistance. Error bars
represent SEM, and *P-values (as compared with wild-type worms
on 5-FU plates, calculated by unpaired Student’s t-test) were
< 0.001.
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 4719
decarboxylase (OMPDC). T07C4.1 and R12E2.11 both
have an OPRT domain, but their functional roles are
unclear. Bacteria and fungi have separate genes for
OPRT and OMPDC, but animals and plants have a
single UMPS protein comprising both OPRT and OM-
PDC [24]. Interestingly, worms have both UMPS
(OPRT plus OMPDC) and OPRT forms (Fig. 6A).
Sequence alignments indicate that T07C4.1 protein is
very similar to human UMPS, and R12E2.11 is very
similar to the OPRT domain of human UMPS and
T07C4.1 (Fig. S5). The T07C4.1::GFP fusion construct
is expressed in neurons and intestinal cells, but
R12E2.11::GFP is expressed in the body wall muscle,
spermatheca, intestine, and vulval muscle (Fig. 6B).
Finally, we verified the OPRT and OMPDC activi-
ties of T07C4.1 and R12E2.11 proteins in vitro. Both
proteins can synthesize UMP from uracil and phos-
phoribosyl pyrophosphate, but only T07C4.1 exhibited
OMPDC activity (Fig. 6C). The single UMPS of
higher organisms is more efficient than the separate
OPRT and OMPDC system for UMP synthesis [24].
T07C4.1 shows stronger OPRT activity than
R12E2.11, and mixing the two proteins has an additive
effect on UMP synthesis. Interestingly, R12E2.11 itself

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-

pressing DPD and TS, however, showed only small
increases in survival ratios on 5-FU plates [22]. In
addition, 5-FU-induced cell death is dependent on p53
[27], but the C. elegans cep-1 ⁄ p53 mutant exhibited
only minimal improvement in germ cell death as com-
pared with the upp-1 mutant [21]. Thus, UPP-1 is a
key player mediating 5-FU functions in C. elegans.
TP and UP participate in both uridine and thymi-
dine synthesis, and humans possess at least two
different UPs and one TP. This complex redundancy
makes the relationship between UP⁄ TP and 5-FU sen-
sitivity in humans difficult to decipher. In contrast,
C. elegans has only one UP, which functions as both
UP and TP (Fig. 3A). Human UPP1 can rescue the
C. elegans upp-1 mutant phenotype (Fig. 1H), suggest-
ing that UP function is evolutionarily conserved and
mediates 5-FU function in vivo in humans. Addition-
ally, the upp-1 mutant showed similar resistance to
5dFUR and 5-FU (Fig. 3C). These results also indicate
that the single upp-1 gene in C. elegans plays a key role
in the pyrimidine salvage pathway.
Fig. 6. Characteristics of UMPS and OPRT
homologs in C. elegans. (A) Domains of
UMP synthesis enzymes. Bacteria and fungi
have separate OPRT and OMPDC proteins,
but higher animals have a single protein
with both OPRT and OMPDC functions.
C. elegans has a long UMPS homolog and a
short OPRT homolog. (B) The expression
patterns of T07C4.1::GFP and

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

UMP (Fig. 5), but their knockdown produces different
5-FU responses (Fig. 4). This difference is probably
due to the distinct expression patterns of these genes in
the intestine and hypodermis, which appears to be
important for 5-FU function.
Another gene mediating 5-FU function is that encod-
ing T07C4.1, which contains OPRT and OMPDC
domains. As the OPRT activity of T07C4.1 is impor-
tant for mediating 5-FU function and the salvage path-
way of pyrimidine biosynthesis, the differences in 5-FU
metabolism between T07C4.1 and another OPRT pro-
tein, R12E2.11, is puzzling. Both proteins have OPRT
enzymatic activity (Fig. 6C), but knockdown of only
the gene encoding T07C4.1 resulted in 5-FU resistance.
The expression patterns of these proteins differ, but not
enough to explain the RNAi results. It has been
reported that UMPS, which is a single protein with
both OPRT and OMPDC domains, is more stable and
has higher activity than separate OPRT and OMPDC
proteins [28,29]. R12E2.11 exhibited lower activity than
T07C4.1 in vitro, and it is possible that this difference is
amplified in vivo . The higher OPRT activity and strong
intestinal expression of T07C4.1 may account for the
difference. In addition, knockdown of R12E2.11 pro-
moted T07C4.1::GFP expression (data not shown),
indicating a complex relationship between these two
proteins in vivo.
As UP ⁄ uridine kinase and OPRT both synthesize
UMP from uracil in the pyrimidine salvage pathway,
the strong 5-FU resistance resulting from single gene

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.

Although C. elegans has been used as a model sys-
tem in pharmacogenetics and chemical genetics, it has
only recently begun to be used to study anticancer
Fig. 7. Comparison of the human and C. elegans 5-FU conversion
and pyrimidine biosynthesis pathways. Two pathways allow the
conversion of 5-FU to FdUMP in humans. The C. elegans genome
has homologs for the enzymes in these pathways. Humans show
redundancy of pyrimidine synthesis enzymes, including two UPs
and one TP, but C. elegans has only one uridine and thymidine
phosphorylase, UPP-1. The C. elegans uridine kinase has not been
identified yet, but C29F7.3 and F40F8.1 proteins were identified by
their UMPK activities. Two OPRT homologs, T07C4.1 and
R12E2.11, mediate conversion of uracil to UMP in C. elegans.
Characterization of worm UPP-1, UMPK, and UMPS S. Kim et al.
4722 FEBS Journal 276 (2009) 4715–4726 ª 2009 The Authors Journal compilation ª 2009 FEBS
drugs, such as farnesyl transferase inhibitors [30].
Here, we evaluate how the C. elegans upp-1 mutant
interacts with the anticancer drug 5-FU. As C. elegans
is a simple metazoan, interpreting the relationships
between anticancer drugs and gene function may be
less complex than in higher organisms. At the same
time, the example of a single, dual-function protein in
humans that takes on the roles that two separate
enzymes play in worms underscores the challenges and
discoveries that await us in C. elegans. Our findings
support a close relationship between pyrimidine
salvage enzymes in 5-FU function and resistance in
both C. elegans and humans.
Experimental procedures
C. elegans strains and culture

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

calculated.
Sequence alignment
Amino acid sequences of human and C. elegans proteins
were aligned using macvector (MacVector Inc., Cary, NC,
USA). The GenBank accession number of human UPP1 is
AAH07348, and that of UMPS is CAG33068. The amino
acid sequences of UPP-1 (ZK783.2), C29F7.3, F40F8.1,
T07C4.1 and R12E2.11 are from wormbase (http://
www.wormbase.org).
Plasmid construction and protein purification
Rescue experiments were performed using PCR-amplified
upp-1 genomic DNA and the ZK783 cosmid obtained from
the Sanger Institute (Cambridge, UK). The following primers
were used to amplify upp-1 genomic DNA: 5¢-AGC ATC
TGC AGC AAC CAC C-3¢ and 5¢-TGG ATC CGA TCC
CGG TCT GCT TGC G-3¢. To construct the upp-1::gfp
fusion construct, the GFP expression vector pPD95.77
(obtained from A. Fire, Stanford University, CA, USA) was
used. The following primers were used to amplify upp-1 geno-
mic DNA (4793 bp): 5¢-TTT CTG CAG GAG AGT TGT
ACC TAA AGG CGC G-3¢ and 5¢-TTT GGT ACC ATC
CCG GTC TGC TTG CGA A TG-3¢. Amplified PCR frag-
ments were digested with PstIandKpnI, and u sed as insert DNA.
To generate the human UPP1 rescue construct, the
C. elegans upp-1 promoter region was fused with the human
UPP1 cDNA in pPD95.77. To amplify the C. elegans upp-1
promoter region (3105 bp), the following primers were
used: 5¢-TTC TGC AGG TGA TGC CTT TGA GCA CT
T AGC-3¢ and 5¢-TTT TCT AGA CTT GAT GGA TCT
GAA AAA ATT CC-3¢. Amplified PCR fragments were

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-3¢ (XbaI) for R12E2.11::GFP.
To construct the plasmid expressing the maltose-binding
protein (MBP)::UPP-1 fusion protein, full-length upp-1
cDNA (888 bp) was cloned from first-strand worm cDNA
by PCR and inserted in-frame, downstream of the MBP
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 4723
sequence in the E. coli expression vector pMAL-c2X (New
England Biolabs, Ipswich, MA, USA). PCR was performed
using the following primers: 5¢-T
GG ATC CAT GAA
CGGACT TGT CAA GAA CGG-3¢ and 5¢-TTT
AAG
CTT TTA GAT CCC GGT CTG CTT GC-3¢. The ampli-
fied PCR fragments were digested with BamHI and
HindIII, and were ligated into pMAL-c2X. The same vector
and methods were used to make MBP::C29F7.3,
MBP::F40F8.1, MBP::B0001.4, MBP::T07C4.1 and
MBP::R12E2.11 constructs. The primers and restriction
enzyme sites used were as follows: 5¢-TT
A GAT CTA TGT
ACA ACG TCG TCT TTG TTC-3¢ (BglII) and 5¢-AA
G
GTA CCC TAT TTT TGG TTG GCA GCC G-3¢ (KpnI)
for C29F7.3, 5¢-AA
G GAT CCA TGC ACA ACG TGG
TTT TTG TTC-3¢ (BamHI) and 5¢-AA
G GTA CCT TAT
TTT TTG TTG GCA GCG TC-3¢ (KpnI) for F40F8.1,
5¢-AA

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

upp-1::gfp fusion construct was injected (75 lgÆmL
)1
) into
adult N2 animals along with the pRF4 plasmid
(75 lgÆmL
)1
). The C29F7.3::GFP, F40F8.1::GFP,
B0001.4::GFP, T07C4.1::GFP and R12E2.11::GFP plas-
mids were injected using the same method and at the same
DNA concentration.
RNAi by bacterial feeding was performed as previously
described [33]. Briefly, synchronized L4 larvae were
transferred onto plates containing 1 mm isopropyl thio-b-
d-galactoside and the HT115-RNAi bacterial clone. The
next day, adult worms were transferred to new RNAi
plates. Embryos from RNAi plates were transferred to
both a control plate without 5-FU and an experimental
plate containing 5-FU (800 nm). After 60 or 72 h,
L4 ⁄ adult animals and total worms were counted, and the
ratio of L4 and adult animals to total worms was
calculated. The empty vector L4440 was used as a
control.
In vitro enzymatic assays
To induce the MBP–UPP1 fusion protein, 0.3 lm IPTG
was added to the culture. Induced fusion proteins were
purified using amylose resin (New England Biolabs),
according to the manufacturer’s protocol, and the UPP-1
proteins were cleaved and eluted by factor Xa digestion
(New England Biolabs). Modified methods described by
Kouni et al. [34] were used, and the activity assay mixture

(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

out on a Waters 2695 Separation Module. The separation
system consisted of a Prevail C-18 column (250 · 4.6 mm,
5 lm particle size) (Alltech Associates, Inc., Deerfield, IL,
USA) and a mobile phase developed with buffer A (10 mm
KH
2
PO
4
, and 8 mm tetrabutyl ammonium hydrogen sulfate
as the ion-pairing reagent, pH 7.0) and buffer B (100 mm
KH
2
PO
4
,10mm tetrabutyl ammonium hydrogen sulfate,
30% MeOH, pH 5.3). The gradient was formed as follows:
6 min with 100% buffer A; 1 min with 75% buffer A; 7 min
with 58% buffer A; 2 min with 45% buffer A; 16 min with
20% buffer A; and 10 min with 100% buffer B. The flow
rate was 1.0 mLÆmin
)1
, and absorbance was monitored at
260 nm with a 2996 Photodiode Array Detector (Waters
Corporation, Milford, MA, USA).
Microscopy and photography
Images of worms were captured using an AxioCam HRc
digital camera attached to a Zeiss Axio Imager M1 micro-
scope (Zeiss Corporation, Jena, Germany). axiovision
Release 4.6 software (Zeiss) was used for image acquisition
and processing.

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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Supporting information
The following supplementary material is available:
Fig. S1. Hatching tests of wild-type and upp-1(jg1)

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.
This supplementary material can be found in the
online article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
by the authors. Such materials are peer-reviewed and
may be re-organized for online delivery, but are not
copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
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