• Title/Summary/Keyword: reporter assay

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Virtual Screening and Testing of GSK-3 Inhibitors Using Human SH-SY5Y Cells Expressing Tau Folding Reporter and Mouse Hippocampal Primary Culture under Tau Cytotoxicity

  • Chih-Hsin Lin;Yu-Shao Hsieh;Ying-Chieh Sun;Wun-Han Huang;Shu-Ling Chen;Zheng-Kui Weng;Te-Hsien Lin;Yih-Ru Wu;Kuo-Hsuan Chang;Hei-Jen Huang;Guan-Chiun Lee;Hsiu Mei Hsieh-Li;Guey-Jen Lee-Chen
    • Biomolecules & Therapeutics
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    • v.31 no.1
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    • pp.127-138
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    • 2023
  • Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that implicates in multiple cellular processes and links with the neurodegenerative diseases including Alzheimer's disease (AD). In this study, structure-based virtual screening was performed to search database for compounds targeting GSK-3β from Enamine's screening collection. Of the top-ranked compounds, 7 primary hits underwent a luminescent kinase assay and a cell assay using human neuroblastoma SH-SY5Y cells expressing Tau repeat domain (TauRD) with pro-aggregant mutation ΔK280. In the kinase assay for these 7 compounds, residual GSK-3β activities ranged from 36.1% to 90.0% were detected at the IC50 of SB-216763. In the cell assay, only compounds VB-030 and VB-037 reduced Tau aggregation in SH-SY5Y cells expressing ΔK280 TauRD-DsRed folding reporter. In SH-SY5Y cells expressing ΔK280 TauRD, neither VB-030 nor VB-037 increased expression of GSK-3α Ser21 or GSK-3β Ser9. Among extracellular signal-regulated kinase (ERK), AKT serine/threonine kinase 1 (AKT), mitogen-activated protein kinase 14 (P38) and mitogenactivated protein kinase 8 (JNK) which modulate Tau phosphorylation, VB-037 attenuated active phosphorylation of P38 Thr180/ Tyr182, whereas VB-030 had no effect on the phosphorylation status of ERK, AKT, P38 or JNK. However, both VB-030 and VB-037 reduced endogenous Tau phosphorylation at Ser202, Thr231, Ser396 and Ser404 in neuronally differentiated SH-SY5Y expressing ΔK280 TauRD. In addition, VB-030 and VB-037 further improved neuronal survival and/or neurite length and branch in mouse hippocampal primary culture under Tau cytotoxicity. Overall, through inhibiting GSK-3β kinase activity and/or p-P38 (Thr180/Tyr182), both compounds may serve as promising candidates to reduce Tau aggregation/cytotoxicity for AD treatment.

Expression of TIMP1, TIMP2 Genes by Ionizing Radiation (이온화 방사선에 의한 TIMP1, TIMP2 유전자 발현 측정)

  • Park Kun-Koo;Jin Jung Sun;Park Ki Yong;Lee Yun Hee;Kim Sang Yoon;Noh Young Ju;Ahn Seung Do;Kim Jong Hoon;Choi Eun Kyung;Chang Hyesook
    • Radiation Oncology Journal
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    • v.19 no.2
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    • pp.171-180
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    • 2001
  • Purpose : Expression of TIMP, intrinsic inhibitor of MMP, is regulated by signal transduction in response to genotoxins and is likely to be an important step in metastasis, angiogenesis and wound healing after ionizing radiation. Therefore, we studied radiation mediated TIMP expression and its mechanism in head and neck cancer cell lines. Materials and Methods : Human head and neck cancer cell lines established at Asan Medical Center were used and radiosensitivity $(D_0)$, radiation cytotoxicity and metastatic potential were measured by clonogenic assay, n assay and invasion assay, respectively. The conditioned medium was prepared at 24 hours and 48 hours after 2 Gy and 10 Gy irradiation and expression of TIMP protein was measured by Elisa assay with specific antibodies against human TIMP. hTIMP1 promoter region was cloned and TIMP1 luciferase reporter vector was constructed. The reporter vector was transfected to AMC-HN-1 and -HN-9 cells with or without expression vector Ras, then the cells were exposed to radiation or PMA, PKC activator. EMSA was peformed with oligonucleotide (-59/-53 element and SP1) of TIMP1 promoter. Results : $D_0$ of HN-1, -2, -3, -5 and -9 cell lines were 1.55 Gy, 1.8 Gy, 1.5 Gt, 1.55 Gy and 2.45 Gy respectively. n assay confirmed cell viability, over $94\%$ at 24hrs, 48hrs after 2 Gy irradiation and over 73% after 10 Gy irradiation. Elisa assay confirmed that cells secreted TIMP1, 2 proteins continuously. After 2 Gy irradiation, TIMP2 secretion was decreased at 24hrs in HN-1 and HN-9 cell lines but after 10 Gy irradiation, it was increased in all cell lines. At 48hrs after irradiation, it was increased in HN-1 but decreased in HN-9 cells. But the change in TIMP secretion by RT was mild. The transcription of TIMP1 gene in HN-1 was induced by PMA but in HN-9 cell lines, it was suppressed. Wild type Ras induced the TIMP-1 transcription by 20 fold and 4 fold in HN-1 and HN-9 respectively. The binding activity to -59/-53, AP1 motif was increased by RT, but not to SP1 motif in both cell lines. Conclusions : We observed the difference of expression and activity of TIMPs between radiosensitive and radioresistant cell line and the different signal transduction pathway between in these cell lines may contribute the different radiosensitivity. Further research to investigate the radiation response and its signal pathway of TIMPs is needed.

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Transgenic Mutagenesis Assay to Elucidaate the Mechanism of Mutation at Gene Level (유전자수준에서 돌연변이 유발기전을 밝히는 Transgenic Mutagenesis Assay)

  • Ryu, Jae-Chun;Youn, Ji-Youn;Cho, Kyung-Hae;Chang, Il-Moo
    • Environmental Mutagens and Carcinogens
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    • v.18 no.1
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    • pp.15-21
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    • 1998
  • Transgenic animal and cell line models which are recently developed and used in toxicology fields combined with molecular biological technique, are powerful tools to study the mechanism of mutation in vivo and in vitro, respectively. Transgenic models, which have exogenous DNA incorporated into their genome, carry recoverable shuttle vector containing reporter genes to assess endogenous effects or alteration in specific genes related to disease processes. The lac I and lac Z gnee most widely used as a mutational target in transgenic systems. The assay is performed by treatment with putative mutagenic agents, isolation of genomic DNA from cells or tissues, exposure the isolated DNA to in vitro packaging extract, plating and sequencing. The results from these processes provide not only mutant frequency as quantitative evaluation but also mutational spectrum as qualitative evaluation of various agents. Therefore we introduce and review the principle, detailed procedure and application of transgenic mutagenesis assay system in toxicology fields especially in mutagenesis and carcinogenesis.

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Cell-Based Assay Design for High-Content Screening of Drug Candidates

  • Nierode, Gregory;Kwon, Paul S.;Dordick, Jonathan S.;Kwon, Seok-Joon
    • Journal of Microbiology and Biotechnology
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    • v.26 no.2
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    • pp.213-225
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    • 2016
  • To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as high-content screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

ESTABLISHMENT OF BIOASSAY TO DETECT ESTROGENIC FLAVONOIDS USING STABLE MCF-7-ERE CELL AND MCF-7 CELL PROLIFERASTION ASSAY

  • Joung, Ki-Eun;Kim, Yeo-Woon;Sheen, Yhun-Yhong
    • Proceedings of the Korean Society of Toxicology Conference
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    • 2001.10a
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    • pp.202-202
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    • 2001
  • Stable MCF-7-ERE cells, in which pERE-Luc reporter gene has been stably integrated into the genome of the MCF-7 cells, were used to detect the estrogenic activity of various dietary flavonoids.in either pure chemical or mixtures. Estradiol (E2) induced luciferase activity in dose dependent manner and this activity was inhibited by tamoxifen (Tam) concomitant treatment.(omitted)

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DNA Transfection in SK-N-BE(2)C Human Neuroblastoma Cells

  • Lee, Myung-Koo
    • Archives of Pharmacal Research
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    • v.16 no.2
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    • pp.155-157
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    • 1993
  • DNA transfection conditions were investigated by calcium phosphate-DNA co-precipitation in SK-N-BE(2)C human neuroblastoma cells. The DNA plasmid of TH2400CAT was used in which rat tyrosine hydroxylase gene was inserted into chloramphenicol acetyltransferase reporter gent. The transfection efficiency was 25-30% and the method was simple and reproducible. So, the method will be a good tool for transient transfection analysis.

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Estrogenic Activity, and Developmental Toxicity Studies of Pyrethroid Insecticides

  • Kim, Soon-Sun;Rhee, Gyu-Seek;Lee, Rhee-Da;Kwack, Seung-Jun;Lim, Kwon-Jo;Yhun, Hyo-Jung;Park, Kui-Lea
    • Proceedings of the Korea Society of Environmental Toocicology Conference
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    • 2003.10a
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    • pp.57-69
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    • 2003
  • It is well known that many pesticides possess hormonal activity, and affect the developments of wildlife and mammals including human. Currently, pyrethroid insecticides are in worldwide use to control in and outdoor pests, providing potential far environmental exposure. Hormonal activities of these pyrethroid insecticides, however, have been little studied, and the developmental effects of them were no reported. Therefore, we firstly examined the potential estrogenic activities of some pyrethroid insecticides (permethrin, cypermethrin, tetramethrin, deltamethrin, sumithrin, fenvalerate and bioallethrin) by immature rat uterotrophic assay, luciferase reporter gene assay and Calbindin-D$\sub$9k/ (CaBP-9k) gene expression assay. Uterine wet weights were increased by permethrin and the permethrin-induced weights were inhibited by ICI 182780 in the uterolrophic assay. On the other hand tetramethrin significantly reduced uterine and vaginal wet weights, and also inhibited the E2-induced weight increases at all doses tested. Cypermethrin and sumithrin had a tendency to increase uterine weights, although not statistically significant. Permethrin and cypermethrin dose-dependently increased the luciferase activity in reporter gene assay. Northern blot analysis showed that permethrin induced CaBP-9k mRNA expression whereas tetramethrin inhibted. Subsequent studies were conducted to investigate the possible developmental effects of four pyrethroid insecricides (permethrin, cypermethrin, sumithrin and teramethrin). Either diethlbestrol (DES) or 17${\beta}$ -estradiol (E2) was used as a reference control in this study. Pyrethroid insecticides were administered to Sprague Dawley rats via subcutaneous injection at 6 to 18 days of gestation or 1 to 5 days after birth. In utero treatment of permethrin (10mg/kg/day) in female rat resulted in significant increases in uterine and ovarian weights while significant decreases in serum E2 concentration, uterine and ovarian ER${\alpha}$ mRNA levels. Sumithrin and permethrin led to acceleration in vaginal opening of female rat, while delay in preputial separation of male after neonatal treatment. Anogenital distances of PND 18 were significantly reduced in sumthrin-treated, and permerhrin-treated male rats after neonatal treatment. All the pyrethroid insecticides tested caused significant increases in uterine weights on PND 18, while significant reductions in the first diestrus phase when neonataly treated. In addition, exposure to pyrethroids in neonatal period led to significant reduction in relative brain weight in female rat on PND 18, but its weight was recovered in diestrus phase. In summary, Our experimental data demonstrate the possibilities of developmental effects of pyrethroid insecticides via estrogenic or antiestrogenic activity.

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The Effect of Alpha Subunit of Go on Cell Growth

  • Won, Jung-Hee;Ghil, Sung-Ho
    • Biomedical Science Letters
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    • v.12 no.4
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    • pp.385-391
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    • 2006
  • Heterotrimeric GTP binding proteins (G proteins) mediate signals generated by neurotransmitters and hormones Among G proteins, Go is found in a large quantity in brain and growth cone membranes of neurons. In spite of its abundance in neurons, the role of Go is not fully understood. In our previous study, we identified promyelocytic leukemia zinc finger protein (PLZF) as an interacting partner of alpha subunit of Go ($Go{\alpha}$) and confirmed their interaction employing several biochemical assays. To date, it is reported that PLZF functioned as a cell growth suppressor and a transcription repressor. To determine effect of $Go{\alpha}$ and PLZF interaction on the cellular function of PLZF, we performed luciferase reporter gene assay and BrdU incorporation assay. Co-expression of $Go{\alpha}$ and PLZF synergistically increased the effect of PLZF alone. These results suggest that $Go{\alpha}$ may act as cellular activator of PLZF. This novel feature of Go may provide insights into understanding diverse role of Go-coupled receptor as well as its cellular actions.

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Thyroid Hormones Receptor/Reporter Gene Transcription Assay for Food Additives and Contaminants

  • Jeong Sang-Hee;Cho Joon-Hyoung
    • Toxicological Research
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    • v.21 no.4
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    • pp.333-338
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    • 2005
  • Many of thyroid hormones disrupting chemicals induce effects via interaction with thyroid hormone and retinoic acid receptors and responsive elements intrinsic in target cells. We studied thyroid hormones disrupting effects of food additives and contaminants including BHA, BHT, ethoxyquin, propionic acid, sorbic acid, benzoic acid, CPM, aflatoxin B1, cadmium chloride, genistein, TCDD, PCBs and TDBE in recombinant HeLa cells containing plasmid construct for thyroxin responsive elements. The limit of response of the recombinant cells to T3 and T4 was $1\times10^{-12}\;M$. BHA. genistein, cadmium and TBDE were interacted with thyroid receptors with dose-responsive pattern. In addition, BHA, BHT, ethoxyquin, propionic acid, benzoic acid, sorbic acid, and TBDE showed synergism while cadmium chloride antagonism for T3-induced activity. This study elucidates that recombinant HeLa cell is sensitive and high-throughput system for the detection of chemicals that induce thyroid hormonal disruption via thyroid hormone receptors and responsive elements. Also this study raised suspect of BHA. BHT, ethoxyquin, propionic acid, benzoic acid, sorbic acid, TBDE, genisteine and cadmium chloride as thyroid hormonal system disruptors.