-
[
Mol Cell Biol,
2005]
Male germ cell-associated kinase (MAK) and intestinal cell kinase (ICK) are nuclear Cdc2-related kinases with nearly identical N-terminal catalytic domains and more divergent C-terminal noncatalytic domains. The catalytic domain is also related to mitogen-activated protein kinases (MAPKs) and contains a corresponding TDY motif. Nuclear localization of ICK requires subdomain XI and interactions of the conserved Arg-272, but not kinase activity or, surprisingly, any of the noncatalytic domain. Further, nuclear localization of ICK is required for its activation. ICK is activated by dual phosphorylation of the TDY motif. Phosphorylation of Tyr-159 in the TDY motif requires ICK autokinase activity but confers only basal kinase activity. Full activation requires additional phosphorylation of Thr-157 in the TDY motif. Coexpression of ICK with constitutively active MEK1 or MEK5 fails to increase ICK phosphorylation or activity, suggesting that MEKs are not involved. ICK and MAK are related to Ime2p in budding yeast, and cyclin-dependent protein kinase-activating kinase Cak1p has been placed genetically upstream of Ime2p. Recombinant Cak1p phosphorylates Thr-157 in the TDY motif of recombinant ICK and activates its activity in vitro. Coexpression of ICK with wild-type CAK1 but not kinase-inactive CAK1 in cells also increases ICK phosphorylation and activity. Our studies establish ICK as the prototype for a new group of MAPK-like kinases requiring dual phosphorylation at TDY motifs.
-
[
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.
-
[
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.
-
[
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.
-
[
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.
-
[
Cancer Discov,
2017]
Bacteria alter the response to the chemotherapeutics 5-FU, FUDR, capecitabine, and CPT in C. elegans.
-
[
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.
-
[
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.
-
Vargas, ML, Andersen, EF, Strome, S, Ketel, CS, Suh, J, Simon, JA
[
Mol Cell Biol,
2005]
The ESC-E(Z) complex of Drosophila melanogaster Polycomb group (PcG) repressors is a histone H3 methyltransferase (HMTase). This complex silences fly Hox genes, and related HMTases control germ line development in worms, flowering in plants, and X inactivation in mammals. The fly complex contains a catalytic SET domain subunit, E(Z), plus three noncatalytic subunits, SU(Z)12, ESC, and NURF-55. The four-subunit complex is > 1,000-fold more active than E(Z) alone. Here we show that ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. We also show that loss of ESC disrupts global methylation of histone H3-lysine 27 in fly embryos. Subunit mutations identify domains required for catalytic activity and/or binding to specific partners. We describe missense mutations in surface loops of ESC, in the CXC domain of E(Z), and in the conserved VEFS domain of SU(Z)12, which each disrupt HMTase activity but preserve complex assembly. Thus, the E(Z) SET domain requires multiple partner inputs to produce active HMTase. We also find that a recombinant worm complex containing the E(Z) homolog, MES-2, has robust HMTase activity, which depends upon both MES-6, an ESC homolog, and MES-3, a pioneer protein. Thus, although the fly and mammalian PcG complexes absolutely require SU(Z)12, the worm complex generates HMTase activity from a distinct partner set.
-
[
EMBO J,
2021]
Germ granules are biomolecular condensates that form in germ cells of all/most animals, where they regulate mRNA expression to promote germ cell function and totipotency. In the adult Caenorhabditis elegans germ cell, these granules are composed of at least four distinct sub-compartments, one of which is the Z granule. To better understand the role of the Z granule in germ cell biology, we conducted a genetic screen for genes specifically required for Z granule assembly or morphology. Here, we show that
zsp-1, which encodes a low-complexity/polyampholyte-domain protein, is required for Z granule homeostasis. ZSP-1 localizes to the outer surface of Z granules. In the absence of ZSP-1, Z granules swell to an abnormal size, fail to segregate with germline blastomeres during development, and lose their liquid-like character. Finally, ZSP-1 promotes piRNA- and siRNA-directed gene regulation and germline immortality. Our data suggest that Z granules coordinate small RNA-based gene regulation to promote germ cell function and that ZSP-1 helps/is need to maintain Z granule morphology and liquidity.