• Environmental Mutagens and Carcinogens
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• v.19 no.1
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• pp.7-13
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• 1999

The mouse lymphoma thymidine kinase (tk+/-) gene assay (MOLY) using L5178Y tk+/- mouse lymphoma cell line is one of the mammalian forward mutation assays. It is well known that MOLY has many advantages and more sensitive than the other mammalian forward mutation assays such as x-linked hyposanthine phosphoribosyltransferase (hprt) gene assay. The target gene of MOLY is a heterozygous tk+/- gene located in 11 chromosome of L5178Y tk+/- cell, so it is able to detect the wide range of genetic changes like point mutation, deletion, rearrangement, and mitotic recombination within tk gene or deletion of entire chromosome 11. MOLY has relatively short expression time (2-3 days) compared to 1 week of hprt gene assay. MOLY can also induce relatively high mutant frequency so a large number of events can be recorded. The bimodal distribution of colony size which may indicate gene mutation and chromosome breakage potential of chemicals according to mutation scale such as large normal-growing mutants and small slow-growing mutants can be observed in this assay. The statistical analysis of data can be performed using the MUTANT program developed by York Electronic Research in association with Hazelton as recommended by the UKEMS (United Kingdom Environmental Mutagen Society) guidelines. This report reviewed MOLY using the microtiter cloning technique (microwell assay).

• BMB Reports
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• v.28 no.1
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• pp.12-16
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• 1995

The branched-chain ${\alpha}$-keto acid dehydrogenase (BCKAD) complex is a rate limiting enzyme which catalyzes the oxidative decarboxylation of branched-chain ${\alpha}$-keto acids. Numerous studies have suggested that BCKAD is subject to covalent modification in vitro via phosphorylation and dephosphorylation, which are catalyzed by a specific kinase and phosphatase, respectively. The biggest difficulty in the assay of BCKAD activity is to arrest the interconversion between the active and inactive forms. BCKAD activity was determined from fresh rat heart and liver tissues using homogenizing and assay buffers containing inhibitors of phosphatase and kinase. The results suggest that a radiochemical assay using ${\alpha}$-keto[1-$^{14}C$]-isovalerate as a substrate for the enzyme can be applied as a reliable method to determine in vitro enzyme activity with arrested interconversion between the active and inactive forms of the BCKAD complex.

• Journal of Ginseng Research
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• v.40 no.4
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• pp.400-408
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• 2016

Background: Ginsenoside Rh2 (GRh2) is the main bioactive component in American ginseng, a commonly used herb, and its antitumor activity had been studied in previous studies. PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK), a serine/threonine protein kinase, is highly expressed in HCT116 colorectal cancer cells. Methods: We examined the effect of GRh2 on HCT116 cells ex vivo. Next, we performed in vitro binding assay and in vitro kinase assay to search for the target of GRh2. Furthermore, we elucidated the underlying molecular mechanisms for the antitumor effect of GRh2 ex vivo and in vivo. Results: The results of our in vitro studies indicated that GRh2 can directly bind with PBK/TOPK and GRh2 also can directly inhibit PBK/TOPK activity. Ex vivo studies showed that GRh2 significantly induced cell death in HCT116 colorectal cancer cells. Further mechanistic study demonstrated that these compounds inhibited the phosphorylation levels of the extracellular regulated protein kinases 1/2 (ERK1/2) and (H3) in HCT116 colorectal cancer cells. In vivo studies showed GRh2 inhibited the growth of xenograft tumors of HCT116 cells and inhibited the phosphorylation levels of the extracellular regulated protein kinases 1/2 and histone H3. Conclusion: The results indicate that GRh2 exerts promising antitumor effect that is specific to human HCT116 colorectal cancer cells through inhibiting the activity of PBK/TOPK.

• YAKHAK HOEJI
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• v.37 no.6
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• pp.598-604
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• 1993

The calyx extract of Campsis grandiflora displayed inhibitory activity against protein kinase C from the bovine brain. Separation guided by protein kinase C enzyme assay and bleb forming assay led to isolation of a potent protein kinase C inhibitor that was identified as a known phenylpropanoid glycoside, verbascoside. It suppressed completely bleb-formation of K562 cell surface induced by phorbol 12,13-dibutylate at the concentration of 60 $\mu\textrm{g}$/ml and IC$_{50}$ of the protein kinase C occured at 20 $\mu{M}$. This compound was tested for cytotoxic activity against ten human tumor cell lines in vitro. it exhibited moderate cytotoxic activity against skin tumor cell line M14 (IC$_{50}$ 2.2 $\mu\textrm{g}$/ml) and very weak cytotoxicity against other cell lines (IC$_{50}$>10 $\mu\textrm{g}$/ml)

• Bulletin of the Korean Chemical Society
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• v.34 no.9
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• pp.2829-2832
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• 2013

• Journal of Microbiology and Biotechnology
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• v.5 no.2
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• pp.105-109
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• 1995

Anticancer activity of a fraction of the ethanol extract from the root of Korean angelica (Angelica gigas Nakai) was recognized in human cancer cell lines HeLa $S_3$, K-562, and Hep $G_2$. The extract blocked the phorbol ester-inducing megakaryocytic differentiation of K-562 cells, which indicated the modification of protein kinase C (PKC) activity. In vitro assay showed the activation of PKC by the extract. An effective fraction of the Angelica gigas extract, of which $R_f$ value was 0.64 in a thin layer chromatography, was a different component from those of European angelicas. The $ED_50$ value of the fraction was 8, 9, and $16\;\mu\textrm{m}/ml$ against HeLa $S_3\;Hep\;G_2$, and K-562 cells, respectively, while the fraction showed higher $ED_50$ values against normal cell lines.

• YAKHAK HOEJI
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• v.50 no.1
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• pp.52-57
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• 2006

The activation of cyclin dependent kinase 4 (CDK4) is found in more than half of all human cancers. Therefore CDK4 is an attractive target for the development of a novel anticancer agent. For mass screening of CDK4 inhibitor, we set up in vitro kinase assay for CDK4 activity using a cyclin D1-CDK4 fusion protein, which is constitutively active and exhibits enhanced stability. From the screening of representative compound library of Korea Chemical Bank, we found that 7-chloro-4-nitro-benzo[1,2,5]oxadiazole 1-oxide (FBP-1248) selectively inhibited CDK4 activity in vitro by ATP competitive manner. This compound prevented the phosphorylation of retinoblatsoma tumor suppressor protein, Rb, and inhibited cell growth through cell cycle arrest. In summary, we developed an efficient assay system for CDK4 activity in vitro and identified the CDK4 inhibitory compound, FBP-1248.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.23 no.3
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• pp.11-32
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• 2010

Objective : The aim of this study is to determine the effects of Dendrobii herba extract and Punica granatum extract on skin disease and skin beauty. Methods : To investigate in vitro anti-oxidant activity assay, ethanol extracts of medicinal plants tested by DPPH radical, xanthine oxidase activity. In the next experiment, to investigate anti-inflammatory activity assay, examined by relations in NO synthesis, IL-$1{\beta}$, IL-6, TNF-${\alpha}$, NF-${\kappa}B$, COX-2, MAP kinase. To study Skin wrinkle formation effect, we were examined by tyrosinase activities, melanin synthesis in MNT-1 cell. Results : 1. In an anti-oxidant test, Dendrobii and Punica granatum extract showed high radical scavenging activity. 2. In an anti-inflammatory test, Dendrobii herba and Punica granatum extract weakly inhibited the lipopolysaccharide(LPS)-induced nitric oxide(NO) release from RAW 246.7 macrophage cells. Dendrobii herba and Punica granatum extract also inhibited LPS-induced IL-$1{\beta}$ and COX-2 expressions. The inhibitory effect of Dendrobii herba and Punica granatum extract on macrophage activation were via the inhibition of NF-${\kappa}B$, evidenced by transient transfection assay. however, Dendrobii herba and Punica granatum extract did not have any effects about activation of Jun-N-terminal kinase(JNK) and inhibition of p38 MAP kinase in RAW 264.7 cells. 3. In the skin wrinkle formation assay, Dendrobii herba and Punica granatum extract weakly inhibited collagenase and elastase, however it was not statistically significant. 4. In the skin whitening assay, Dendrobii herba and Punica granatum extract weakly inhibited tyrosinase activity, however, it was not statistically significant. They did not have any effect on melanin synthesis, indicating that they could not be applicable for skin whitening. Conclusion : Dendrobii herba extract and Punica granatum extract may play a significant role in skin disease and skin beauty.

• Molecular & Cellular Toxicology
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• v.2 no.4
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• pp.244-250
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• 2006

The detection of many synthetic chemicals used in industry that may pose a genetic hazard in our environment is of great concern at present. Since these substances are not limited to the original products, and enter the environment, they have become widespread environmental pollutants, thus leading to a variety of chemicals that possibly threaten the public health. In this respect, to regulate and to evaluate the chemical hazard will be important to environment and human health. The genotoxicity of 3 synthetic chemicals was evaluated in L5178Y $tk^{+/-}$ mouse lymphoma cells in vitro. 9H-carbazole (CAS No. 86-74-8) did not induce significant mutation frequencies both in the presence and absence of metabolic activation system. 1, 3-Dichloro-2-propanol (CAS No. 96-23-1) revealed a significant increase of mutation frequencies in the range of $625-373\;{\mu}g/mL$ in the absence of metabolic activation system and $157-79\;{\mu}g/mL$ in the presence of metabolic activation system. And also, fenpropathrin (CAS No. 64257-84-7) appeared the positive results only in the absence of metabolic activation system. Through the results of MLA tk assay with 3 synthetic chemicals in L5178Y cells in vitro, we may provide the important clues on the genotoxic potentials of these 3 chemicals.

• Tuberculosis and Respiratory Diseases
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• v.51 no.5
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• pp.437-447
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• 2001

Background: TNF-alpha is related to the generation of lung fibrosis in patients with UIP. The precise mechanism leading to lung fibrosis by TNF-alpha is unknown. However, the activation of a transcription factor like AP-1(down stream of c-jun N-terminal kinase, JNK) by TNF-alpha may be related to the induction of fibrogenic cytokines like PDGF or IGF-I. Furthermore, JNK was reported to be activated in the radiation-induced lung fibrosis model. This study examined JNK activity in patients with UIP. Methods : The expression of phosphorous JNK(p-JNK), macrophage/monocyte specific markers, CD68, and cytokeratin was evaluated by immunohistochemical(IHC) staining of lung tissues from patients with UIP and lung cancer. An in vitro kinase assay was performed with alveolar macrophages obtained by a bronchol-avleolar lavage from patients with UIP and healthy persons as the control. Results : The IHC stain showed that p-JNK is expressed in the almost all of the alveolar macrophages and smooth muscle cells in patients with UIP. In case of the normal areas of the lung from patients with lung cancer, the alveolar macrophages showed little p-JNK expression. Interestingly, increased JNK activity was not found in the in vitro kinase assay of the alveolar macrophages obtained from both patients with UIP and healthy persons as the control. Furthermore, 10 ng/mL of TNF-alpha failed to increase the JNK activity of the alveolar macrophages in both patients with UIP and healthy people. Conclusion : The JNK was activated constitutionally in patients with UIP. However, the role of JNK in the pathogenesis of lung fibrosis needs to be clarified.