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: jaegal@ncc.re.kr *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 [...]... 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Targeted deletion of both thymidine phosphorylase and uridine phosphorylase and consequent disorders in mice Mol Cell Biol 22, 5212–5221 21 Kim S & Shim J (2008) A forward genetic approach for analyzing the mechanism of resistance to the anti-cancer drug, 5-fluorouracil, using Caenorhabditis elegans Mol Cells 25, 119–123 22 Kim S, Park DH & Shim J (2008) Thymidylate synthase and dihydropyrimidine dehydrogenase... 5-(phenylselenenyl)acyclouridine, an inhibitor of uridine phosphorylase: relevance to chemotherapy Cancer Chemother Pharmacol 45, 351–361 15 Al Safarjalani ON, Rais R, Shi J, Schinazi RF, Naguib FN & el Kouni MH (2006) Modulation of 5-fluorouracil host-toxicity and chemotherapeutic efficacy against human colon tumors by 5-(phenylthio)acyclouridine, a uridine phosphorylase inhibitor Cancer Chemother Pharmacol 58,... 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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. Functional analysis of pyrimidine biosynthesis enzymes using the anticancer drug 5-fluorouracil in Caenorhabditis elegans Seongseop Kim 1, *, Dae-Hun Park 2, *,. 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