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[
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.
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[
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.
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[
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.
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[
Proc Natl Acad Sci U S A,
2006]
DJ-1/PARK7, a cancer- and Parkinson''s disease (PD)-associated protein, protects cells from toxic stresses. However, the functional basis of this protection has remained elusive. We found that loss of DJ-1 leads to deficits in NQO1 [NAD(P)H quinone oxidoreductase 1], a detoxification enzyme. This deficit is attributed to a loss of Nrf2 (nuclear factor erythroid 2-related factor), a master regulator of antioxidant transcriptional responses. DJ-1 stabilizes Nrf2 by preventing association with its inhibitor protein, Keap1, and Nrf2''s subsequent ubiquitination. Without intact DJ-1, Nrf2 protein is unstable, and transcriptional responses are thereby decreased both basally and after induction. This effect of DJ-1 on Nrf2 is present in both transformed lines and primary cells across human and mouse species. DJ-1''s effect on Nrf2 and subsequent effects on antioxidant responses may explain how DJ-1 affects the etiology of both cancer and PD, which are seemingly disparate disorders. Furthermore, this DJ-1/Nrf2 functional axis presents a therapeutic target in cancer treatment and justifies DJ-1 as a tumor biomarker.
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[
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.
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[
Cancer Discov,
2017]
Bacteria alter the response to the chemotherapeutics 5-FU, FUDR, capecitabine, and CPT in C. elegans.
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[
J Neural Transm,
2010]
DJ-1 is a neuroprotective gene mutated in recessive Parkinson's disease (PD). In addition to direct protective functions in neurons, DJ-1 regulates neuroinflammatory signaling in primary mouse brain astrocytes. To assess the influence of DJ-1 on innate immunity signaling in vivo, we have generated
djr-1 knockout Caenorhabditis elegans. When grown on pathogenic gram-negative bacteria,
djr-1 (-/-) worms showed stronger phosphorylation of
p38 mitogen-activated protein kinase (PMK-1) and hyper-induction of PMK-1 target genes. Thus, PD-associated DJ-1 contributes to regulation of innate immunity.
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[
Hum Mol Genet,
2012]
Human DJ-1 is a genetic cause of early-onset Parkinson's disease (PD), although its biochemical function is unknown. We report here that human DJ-1 and its homologs of the mouse and Caenorhabditis elegans are novel types of glyoxalase, converting glyoxal or methylglyoxal to glycolic or lactic acid, respectively, in the absence of glutathione. Purified DJ-1 proteins exhibit typical Michaelis-Menten kinetics, which were abolished completely in the mutants of essential catalytic residues, consisting of cysteine and glutamic acid. The presence of DJ-1 protected mouse embryonic fibroblast and dopaminergically derived SH-SY5Y cells from treatments of glyoxals. Likewise, C. elegans lacking cDJR-1.1, a DJ-1 homolog expressed primarily in the intestine, protected worms from glyoxal-induced death. Sub-lethal doses of glyoxals caused significant degeneration of the dopaminergic neurons in C. elegans lacking cDJR-1.2, another DJ-1 homolog expressed primarily in the head region, including neurons. Our findings that DJ-1 serves as scavengers for reactive carbonyl species may provide a new insight into the causation of PD.
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[
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.
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Mieyal JJ, Lin J, Currran PL, Wilson MA, Johnson WM, Ray A, Zhu X, Gorelenkova Miller O, Choe K, Yao C, Ravindranath V, Chen SG, Milkovic NM, Wang W, Golczak M, Wilson-Delfosse AL
[
Biochemistry,
2016]
Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide, caused by the degeneration of the dopaminergic neurons in the substantia nigra. Mutations in PARK7 (DJ-1) result in early onset autosomal recessive PD, and oxidative modification of DJ-1 has been reported to regulate the protective activity of DJ-1 in vitro. Glutathionylation is a prevalent redox modification of proteins resulting from the disulfide adduction of the glutathione moiety to a reactive cysteine-SH; and glutathionylation of specific proteins has been implicated in regulation of cell viability. Glutaredoxin 1 (Grx1) is the principal deglutathionylating enzyme within cells, and it has been reported to mediate protection of dopaminergic neurons in C. elegans, however many of the functional downstream targets of Grx1 in vivo remain unknown. Previously, DJ-1 protein content was shown to decrease concomitantly with diminution of Grx1 protein content in cell culture of model neurons (SH-SY5Y and Neuro-2A lines). In the current study we aimed to investigate the regulation of DJ-1 by Grx1 in vivo and characterize its glutathionylation in vitro. Here, with Grx-/- mice we provide evidence that Grx1 regulates protein levels of DJ-1 in vivo. Furthermore, with model neuronal cells (SH-SY5Y) we observed decreased DJ-1 protein content in response to treatment with known glutathionylating agents; and with isolated DJ-1 we identified two distinct sites of glutathionylation. Finally, we found that overexpression of DJ-1 in the dopaminergic neurons partly compensates for the loss of the Grx1 homolog in a C. elegans in vivo model of PD. Therefore; our results reveal a novel redox modification of DJ-1 and suggest a novel regulatory mechanism for DJ-1 content in vivo.