Kim S et al. (2008) Mol Cells "Thymidylate synthase and dihydropyrimidine dehydrogenase levels are associated with response to 5-fluorouracil in Caenorhabditis elegans."

Kim S, Park DH, Shim J

[Mol Cells, 2008]

5-Fluorouracil (5-FU), a pyrimidine antagonist, has a long history in cancer treatment. The targeted pyrimidine biosynthesis pathway includes dihydropyrimidine dehydrogenase (DPD), which converts 5-FU to an inactive metabolite, and thymidylate synthase (TS), which is a major target of 5-FU. Using Caenorhabditis elegans as a model system to study the functional and resistance mechanisms of anti-cancer drugs, we examined these two genes in order to determine the extent of molecular conservation between C. elegans and humans. Overexpression of the worm DPD and TS homologs (DPYD-1 and Y110A7A.4, respectively) suppressed germ cell death following 5-FU exposure. In addition, DPYD-1 depletion by RNAi resulted in 5-FU sensitivity, while treatment with Y110A7A.4 RNAi and 5-FU resulted in similar patterns of embryonic death. Thus, the pathway of 5-FU function appears to be highly conserved between C. elegans and humans at the molecular level.

Jaegal Shim et al. (2007) International Worm Meeting "Genetic study of uridine phosphorylase in the function and toxicity of the anti-cancer drug, 5-Fluorouracil using C. elegans."

Seongseop Kim, Jaegal Shim

[International Worm Meeting, 2007]

5-Fluorouracil (5-FU) is a widely used anti-cancer drug for treatment of stomach, pancreatic, breast, colorectal, head and neck cancers. 5-FU has been used more than 40 years but it is still a mainstay drug of colorectal cancer. The anti-cancer drugs of pyrimidine antagonists including 5-FU are inserted into DNA and RNA or inhibit DNA synthesis. Therefore, DNA damage is occurred, and cancer cells become arrested or enter the apoptotic pathway. One of the obstacles of chemotherapy is the anti-cancer drug resistance. To study the resistant mechanism of cancer cells, we used C. elegans as a model system. We treated 5-FU to C. elegans and observed germ-line cell death and larval growth inhibition. Then we searched and studied C. elegans homologues of two enzymes in the 5-FU metabolic pathway. They are dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS). DPD converts 5-FU to inactive molecule, and TS is a major target of 5-FU. Overexpression of DPD and TS gives C. elegans resistance against 5-FU. From this result, we did genetic screen to find out genes related to the 5-FU resistant mechanism. We selected 15 mutants from 72,000 F1 screening. Six mutants are revealed to have mutations in ZK783.2 ORF, and ZK783.2 is uridine phosphorylase homologue. Uridine phosphorylase is one of enzymes in the pyrimidine salvage pathway. Uridine phosphorylase is expressed in most cells from early embryo to late adult. We also tested homologous genes of this pathway whether they are related to 5-FU resistance or not. Using RNAi, we know that some genes are related to 5-FU resistance but others are not. Now we are doing in vitro and in vivo enzyme activity of wild and mutant type uridine phosphorylases, and rescue experiment with human uridine phosphorylase. In addition, we are also doing mapping and cloning of another 5-FU resistant mutant.

Seongseop Kim et al. (2006) East Asia C. elegans Meeting "Genetic study for the anti-cancer drug, 5-Fluorouracil resistant mechanism using C. elegans"

Jaegal Shim, Seongseop Kim

[East Asia C. elegans Meeting, 2006]

5-Fluorouracil (5-FU), which is pyrimidine antagonist, is a widely used anti-cancer drug for treatment of stomach, pancreatic, breast, colorectal, head and neck cancers. 5-FU has been used more than 40 years but it is still a mainstay drug of colorectal cancer. The anti-cancer drugs of pyrimidine antagonists are inserted into DNA and RNA or inhibit DNA synthesis pathway. Therefore, DNA damage is occurred and cancer cells become arrested or enter the apoptotic pathway. 5-FU is basically a pro-drug, and it is converted to an active drug by the several metabolic enzymes. Fluorodeoxyuridylate (5-FdUMP), which is the main metabolite of 5-FU, inhibits DNA synthesis through the binding with thymidylate synthase and induces apoptosis of cell. Another metabolite of 5-FU, 5-fluorouridine monophosphate (5-FUMP) is inserted into RNA and inhibits processing and function of RNA. One of the most critical points of chemotherapy is resistance of cancer cells against anti-cancer drugs. To study the resistant mechanism of cancer cells, we used C. elegans as a model system. We treated 5-FU to C. elegans and observed germ-line cell death and larval growth inhibition. Next, we studied C. elegans homologous genes of two main enzymes in the 5-FU metabolic pathway. They are dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS). DPD converts 5-FU to inactive molecule, and TS is a major target of 5-FU. Both two genes seem to be related 5-FU resistance, although there are several controversial reports. DPD and TS of C. elegans are identical over 60% to their human homologues. Overexpression of them inhibited germ cell death caused by 5-FU treatment. From these results, we concluded that C. elegans and human have similar conserved pathways of 5-FU metabolism. From these results, we started mutant screen to elucidate the 5-FU resistant mechanism. We selected 9 mutants from 72,000 F<SUB>1</SUB> screening, and they belonged to 2 complementation groups. Now, we are trying to clone these two mutants. We expect that one mutant should be cloned in a month, because we are doing rescue experiment with cosmids.

Kim S et al. (2008) Mol Cells "A forward genetic approach for analyzing the mechanism of resistance to the anti-cancer drug, 5-fluorouracil, using Caenorhabditis elegans."

Shim J, Kim S

[Mol Cells, 2008]

Pyrimidine antagonists including 5-Fluorouracil (5-FU) have been used in chemotherapy for cancer patients for over 40 years. 5-FU, especially, is a mainstay treatment for colorectal cancer. It is a pro-drug that is converted to the active drug via the nucleic acid biosynthetic pathway. The metabolites of 5-FU inhibit normal RNA and DNA function, and induce apoptosis of cancer cells. One of the major obstacles to successful chemotherapy is the resistance of cancer cells to anti-cancer drugs. Therefore, it is important to elucidate resistance mechanisms to improve the efficacy of chemotherapy. We have used C. elegans as a model system to investigate the mechanism of resistance to 5-FU, which induces germ cell death and inhibits larval development in C. elegans. We screened 5-FU resistant mutants no longer arrested as larvae by 5-FU. We obtained 18 mutants out of 72,000 F1 individuals screened, and mapped them into three complementation groups. We propose that C. elegans could be a useful model system for studying mechanisms of resistance to anti-cancer drugs.

(2017) Cancer Discov "Bacterial Metabolism Alters the Efficacy of Chemotherapeutics."

[Cancer Discov, 2017]

Bacteria alter the response to the chemotherapeutics 5-FU, FUDR, capecitabine, and CPT in C. elegans.

Hwang IT et al. (2007) Biochem Biophys Res Commun "Drug resistance to 5-FU linked to reactive oxygen species modulator 1."

Kim HJ, Hwang IT, Kim BS, Kim JJ, Yoo YD, Kim JS, Chung YM, Chung JS

[Biochem Biophys Res Commun, 2007]

While acute oxidative stress triggers cell apoptosis or necrosis, persistent oxidative stress induces genomic instability and has been implicated in tumor progression and drug resistance. In a previous report, we demonstrated that reactive oxygen species modulator 1 (Romo1) expression was up-regulated in most cancer cell lines and suggested that increased Romo1 expression might confer chronic oxidative stress to tumor cells. In this study, we show that enforced Romo1 expression induces reactive oxygen species (ROS) production in the mitochondria leading to massive cell death. However, tumor cells that adapt to oxidative stress by increasing manganese superoxide dismutase (MnSOD), Prx I, and Bcl-2 showed drug resistance to 5-FU. To elucidate the relationship between 5-FU-induced ROS production and Romo1 expression, Romo1 siRNA was used to inhibit 5-FU-triggered Romo1 induction. Romo1 siRNA treatment efficiently blocked 5-FU-induced ROS generation, demonstrating that 5-FU treatment stimulated ROS production through Romo1 induction. Based on these results we suggest that cellular adaptive response to Romo1-induced ROS is another mechanism of drug resistance to 5-FU and Romo1 expression may provide a new clinical implication in drug resistance of cancer chemotherapy.

Kumar S et al. (2010) Reprod Toxicol "Anticancer drug 5-fluorouracil induces reproductive and developmental defects in Caenorhabditis elegans."

Michaux G, Morel F, Kumar S, Aninat C

[Reprod Toxicol, 2010]

In order to examine the chronic effects of anticancer drug 5-fluorouracil (5-FU) on reproduction and development, we exploited Caenorhabditis elegans as a model system. We demonstrate that 5-FU induces cell-cycle arrest and apoptosis of germline cells and reduces by approximately 30-40% the number of mitotic nuclei per gonad arm when compared to untreated worms. This drug also affects vulva development, some animals being vulvaless, as well as dysfunction of vulval and egg laying muscles leading to an 8-10 days delay in reproductive time. Interestingly, 5-FU represses levels of mRNA encoding LIN-29, a transcription factor that affects vulva development and egg laying system. Finally, we demonstrate that RNAi-dependent repression of ung-1 gene, which encodes a uracil-DNA glycosylase, partially abolishes 5-FU effects on embryo hatching. Thus, we proposed that C. elegans could be a useful model system for studying the mechanisms by which 5-FU might affect either embryo, adult or organ development.

Maria Martinez-Iniesta et al. (2008) European Worm Meeting "Identification of response and resistance genes to 5-fluroracil (5-FU) in Caenorhabditis elegans."

Maria Martinez-Iniesta, Xavi Sole, Nuria Baixeras, Alberto Villanueva, Laura Padulles, Ramon Salazar, Victor Moreno, J Ramon Germa, Gabriel Capella

[European Worm Meeting, 2008]

Introduction: Colorectal cancer (CRC) is one of the leading causes of. cancer-related mortality in the Western world. In Spain is the second type. of cancer in incidence and mortality in both sexes and 50% of total. diagnostic cases death. Despite the improvement of diagnostic and treatment. only a few decreases have been observed. About 60% of all patients. diagnosed with CRC will present localized disease, being the surgery the. mainstay therapy. The rest of patients (Duke''s C and D) are diagnosed with. distal metastasis. 5-FU and oxaliplatin based chemotherapy (QT) are the. base of the standard treatment of colorectal cancer patients in both. adjuvant and metastasic setting. Initially, more patients did not respond. to QT treatments and others relapse after a short period of response. In. spite of the clinical relevance of 5-FU, little is known about the. mechanisms of resistance to this drug commonly observed in colorectal. cancer patients. Aim: To identify the genes involved in the mechanism of. resistance to 5-FU in the pluricellular model organisms Caenorhabditis. elegans (C.elegans), and posterior validation of these genes as a markers. of response/sensitivity to 5-FU chemotherapy treatments in tumor series.. Methods and Results: A 5-FU plate toxicity assay has been established. We. are characterizing the ceTS-1 gene, C.elegans homolog of human thymidylate. synthase gene, and temporal and cellular expression patterns during. development are studying in GFP transgenic worms. Different alternative. splicing isoforms are generating in CeTS-1 with unknown biological. implications that we are evaluating. Different phenotypes have been. associated with the worm 5-FU treatment. N2 wild-type worms and cib-1(e300). mutant, harboring a point mutation in Thymidilate Synthase gene (Schnabel. R), were synchronized, and both strains of worms were growth at different. drug concentrations and at different times. Profiles of 5-FU response are. being determined in C.elegans Affymetrix microarrays. To identify response. genes (we expected around 100-150 genes) we are performing a feeding double-. stranded RNA-mediated interference (dsRNAi) screening for response genes.. Conclusions: Identification in C. elegans of genes involved in the. resistance/sensitivity to 5-FU drug and later validation of their. predictive value of response in colorectal cancer series may permit to. dispose of the some genetic markers that finally can improve the CRC. patient treatment.. avillanueva@iconcologia.net

Koneru, Sneha Latha et al. (2017) International Worm Meeting "Revisiting the role of eff-1 in epidermal seam cell patterning."

Koneru, Sneha Latha, Barkoulas, Michalis, Quah, Fu Xiang, Ghose, Ritobrata, Gritti, Nicola, van Zon, Jeroen Sebastiaan

[International Worm Meeting, 2017]

Developmental patterning in Caenorhabditis elegans proceeds in a stereotyped manner and is considered highly invariant. We use the terminal number of seam cells as an experimental model to understand mechanisms of developmental robustness to stochastic noise. To this end, we have conducted mutagenesis screens to identify mutants showing increased variance in seam cell number without necessarily displaying changes in the mean. One of the mutations we have recovered represents a null mutation in the fusogen gene eff-1, which has been previously implicated in most cell fusion events in C. elegans including the fusion of the anterior cell daughter of the asymmetric seam cell divisions with the surrounding hyp7 syncytium. To understand the developmental basis of cell number variability in eff-1 mutants, we performed long-term time-lapse imaging of post-embryonic seam cell divisions. We found that despite the fusion defects in eff-1 animals, seam cell division and differentiation patterns were mostly unaffected. This is largely because anterior cell daughters, despite their inability to fuse, differentiate normally by adopting a hypodermal fate, as evidence by the acquisition of hypodermal (dpy-7, elt-3) rather than seam cell fate marker expression (egl-18, nhr-73). We show that acquisition of hypodermal fate in anterior daughters correlated with breaks in adherens junctions, allowing their cytoplasmic continuity with the hyp7 syncytium. Surprisingly, we found that the rare decrease in seam cell number was explained by a loss of seam cell identity in early larval stages due to inappropriate differentiation of both daughter cells to hypodermis. However, these errors were mostly compensated at later developmental stages, due to loss of anterior daughter cell differentiation to hypodermis, which contributes towards an increase in seam cell number. Taken together, these results refine the role of eff-1 in epidermal seam cell patterning.

Kim S et al. (2009) FEBS J "Functional analysis of pyrimidine biosynthesis enzymes using the anticancer drug 5-fluorouracil in Caenorhabditis elegans."

Hwang M, Park DH, Kim S, Kim TH, Shim J

[FEBS J, 2009]

Pyrimidine biosynthesis enzymes function in many cellular processes and are closely associated with pyrimidine antagonists used in cancer chemotherapy. These enzymes are well characterized from bacteria to mammals, but not in a simple metazoan. To study the pyrimidine biosynthesis pathway in Caenorhabditis elegans, we screened for mutants exhibiting resistance 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 phosphorylase 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. elegans. 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.