• BMB Reports
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• v.46 no.3
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• pp.163-168
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• 2013

The AML1 gene is an essential transcription factor regulating the differentiation of hematopoietic stem cells into mature blood cells. Though at least 12 different alternatively spliced AML1 mRNAs are generated, three splice variants (AML1a, AML1b and AML1c) have been characterized. Here, using the reverse transcription-polymerase chain reaction with outward-facing primers, we identified a novel non-polyadenylated transcript from the AML1 gene, with exons 5 and 6 scrambled. The novel transcript resisted RNase R digestion, indicating it is a circular RNA structure that may originate from products of mRNA alternative splicing. The expression of the novel transcript in different cells or cell lines of human and a number of other species matched those of the canonical transcripts. The discovery provides additional evidence that circular RNA could stably exist in vivo in human, and may also help to understand the mechanism of the regulation of the AML1 gene transcription.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.427-434
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• 2011

Mitochondria diseases have been reported to involve structural and functional defects of complex I-V. Especially, many of these diseases are known to be related to dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I). The dysfunction of mitochondria complex I is associated with neurodegenerative disorders, such as Parkinson's disease, Huntington's disease, and Leber's hereditary optic neuropathy (LHON). Mammalian mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) is largest and consists of at least 46 different subunits. In contrast, the NDI1 gene of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. The Saccharomyces cerevisiae NDI1 gene using a recombinant adeno-associated virus vector (rAAV-NDI1) was successfully expressed in AML12 mouse liver hepatocytes and the NDI1-transduced cells were able to grow in media containing rotenone. In contrast, control cells that did not receive the NDI1 gene failed to survive. The expressed Ndi1 enzyme was recognized to be localized in mitochondria by confocal immunofluorescence microscopic analyses and immunoblotting. Using digitonin-permeabilized cells, it was shown that the NADH oxidase activity of the NDI1-transduced cells was not affected by rotenone which is inhibitor of complex I, but was inhibited by antimycin A. Furthermore, these results indicate that Ndi1 can be functionally expressed in the AML12 mouse liver hepatocytes. It is conceivable that the NDI1 gene is powerful tool for gene therapy of mitochondrial diseases caused by complex I deficiency. In the future, we will attempt to functionally express the NDI1 gene in mouse embryonic stem (mES) cell.

• Molecules and Cells
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• v.41 no.5
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• pp.444-453
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• 2018

Aberrations in histone modifications are being studied in mixed-lineage leukemia (MLL)-AF9-driven acute myeloid leukemia (AML). In this study, we focused on the regulation of the differentiation of the MLL-AF9 type AML cell line THP-1. We observed that, upon phorbol 12-myristate 13-acetate (PMA) treatment, THP-1 cells differentiated into monocytes by down-regulating Aurora kinase A (AURKA), resulting in a reduction in H3S10 phosphorylation. We revealed that the AURKA inhibitor alisertib accelerates the expression of the H3K27 demethylase KDM6B, thereby dissociating AURKA and YY1 from the KDM6B promoter region. Using Flow cytometry, we found that alisertib induces THP-1 differentiation into monocytes. Furthermore, we found that treatment with the KDM6B inhibitor GSK-J4 perturbed the PMA-mediated differentiation of THP-1 cells. Thus, we discovered the mechanism of AURKA-KDM6B signaling that controls the differentiation of THP-1 cells, which has implications for biotherapy for leukemia.

• Journal of Life Science
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• v.22 no.11
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• pp.1500-1506
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• 2012

Transforming growth factor (TGF)-${\beta}$-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues, especially in liver, in vivo. To investigate which gene expressions are critical for TGF-${\beta}$-induced apoptosis in hepatocytes, gene expression profiling experiments were performed with TGF-${\beta}$-treated and non-treated mouse hepatocytes AML12 cells. Findings showed that serum and glucocorticoid-inducible protein kinase1 (SGK1) expression is markedly downregulated during TGF-${\beta}$-induced apoptosis. Findings confirmed that expression of SGK1 protein, as well as mRNA, is also markedly decreased with TGF-${\beta}$ treatment. Infection of adenoviral vector encoding constitutively active SGK1 (CA-SGK1), but not kinase dead SGK1 (KD-SGK1), attenuated TGF-${\beta}$-induced apoptosis. All of these results suggest that downregulation of SGK1 expression is critical for TGF-${\beta}$-induced apoptosis in AML12 cells.

• BMB Reports
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• v.28 no.4
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• pp.281-289
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• 1995

Homogeneous human deoxycytidine kinase was purified in one step from a variety of spontaneous human leukemic cells (T-ALL, B-ALL, B-CLL, AML, CML), and from cultured T-lymphoblast cells (MOLT-4) using the newly developed affinity medium, $dCp_4$-Sepharose. Starting with an ammonium sulfate fraction, purification was achieved in one step with the kinase being eluted from a column by the end product inhibitor, dCTP. The purified deoxycytidine kinase from T-ALL cells phosphorylated deoxyadenosine and deoxyguanosine, as well as deoxycytidine. The enzyme purified from T-ALL and B-CLL cells yielded one major band with a molecular weight of 52 kDa determined by SDS-polyacrylamide gel electrophoresis. AML and CML cells yielded one 52 kDa band and an extra band of 30 kDa molecular weight. On the other hand, B-ALL and MOLT-4 cells showed a low molecular weight band of 30 kDa only. However, the electrophoretic mobilities of enzymatic activity in 12% non-denaturing gels were identical for the dCyd kinase from all different kinds of leukemic cell lines, except that the B-ALL, B-CLL, and MOLT-4 cell preparations had an extra minor peak, all at the same position. dAdo and dCyd phosphorylating activities comigrated indicating that these activities are all associated with the same protein. Two new methods, a disk implantation method and a nitrocellulose powder method were used with a small amount of enzyme protein to raise polyclonal antibodies against dCyd kinase purified from T-ALL cells.

• Toxicological Research
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• v.33 no.3
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• pp.219-224
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• 2017

Lipin1 was identified as a phosphatidate phosphatase enzyme, and it plays a key role in lipid metabolism. Since free radicals contribute to metabolic diseases in the liver, this study investigated the effects of free radicals on the regulation of Lipin1 expression in Huh7 and AML12 cells. Hydrogen peroxide induced mRNA and protein expression of Lipin1 in Huh7 cells, which was assayed by quantitative RT-PCR and immunoblotting, respectively. Induction of Lipin1 by hydrogen peroxide was confirmed in AML12 cells. Hydrogen peroxide treatment significantly increased expression of sterol regulatory element-binding protein (SREBP)-2, but not SREBP-1. Moreover, nuclear translocation of SREBP-2 was detected after hydrogen peroxide treatment. Hydrogen peroxide-induced Lipin1 or SREBP-2 expression was significantly reduced by N-acetyl-$\small{L}$-cysteine treatment, indicating that reactive oxygen species (ROS) were implicated in Lipin1 expression. Next, we investigated whether the hypoxic environments that cause endogenous ROS production in mitochondria in metabolic diseases affect the expression of Lipin1. Exposure to hypoxia also increased Lipin1 expression. In contrast, pretreatment with antioxidants attenuated hypoxia-induced Lipin1 expression. Collectively, our results show that ROS activate SREBP-2, which induces Lipin1 expression.

• The Journal of Korean Medicine Ophthalmology and Otolaryngology and Dermatology
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• v.20 no.1 s.32
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• pp.80-98
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• 2007

Objectives : This study was carried out to evaluate anti-oxydative, anti-tumor effect for clinical application of Whakijogyungtang (WJT) Results : 1. DPPH radical scavenging activities of WJT water extracts(Exts) were in proportion as concentration of WJT.(3 ${\mu}g/ml:12.6{\pm}2.3$ %) 2. ABTS+ scavenging activities of WJT water Exts were more effective in high density.(3 ${\mu}g/ml:4.3{\pm}1.6$ %, 10 ${\mu}g/ml$: $11.8{\pm}2.5$ %, 30 ${\mu}g/ml:45.3{\pm}3.2%$ 100 ${\mu}g/ml$: $62.7{\pm}4.8%$) 3. Hydrogen peroxide$(H_2O_2)$ scavenging activities of WJT water Exts were effective.(3 ${\mu}g/ml:4.7{\pm}0.8$ %, 10 ${\mu}g/ml: $8.2{\pm}1.6$ %, 30 ${\mu}g/ml:19.5{\pm}3.2$ % 100 ${\mu}g/ml$: $24.6{\pm}3.8$ %) 4. Anti oxidative effects against linoleic acid were not effective. 5. The generation of $O_2\;^-$ in S-180 cells were according to the concentration of WJT water Exts, specially effective over 100 ${\mu}g/ml$ concentration. 6. The SOD activities in S-180 cells were in proportion as cytotoxicity against S-180 cells of WJT water Exts. 7. The GPx activities in S-180 cells were in proportion as cytotoxicity against S-180 cells of WJT water Exts(more effective on 300 ${\mu}g/ml$ and 1000 ${\mu}g/ml$ concentration), but the catalase activities in S-180 cells were not effective. 8. The results of activites against multi-drug-resistance(MDR) of WJT were as follows. 1) In water Exts from WJT, cytotoxicity against AML-2/D100 with vincristine($IC_{50:}39.78$ ${\mu}g/ml$) was more effective than without vincristine($IC_{50:}$ 183.58 ${\mu}g/ml$), Cross resistance(CR:3.85) was not effective, and anti-MDR activites(RF) was effective.(RF:3.85) 2) In hexane fraction, cytotoxicity against AML-2/D100 with vincristine ($IC_{50:}130.88$ ${\mu}g/ml$) was more effective than without vincristine ($IC_{50:}293.10$ ${\mu}g/ml$) and anti-MDR activites(RF) was effective.(RF:4.61) 3) In chloroform fraction, the cytotoxicity against AML-2/WT and AML-2/D100 was not effective. 4) In ethyl acetate fraction, cytotoxicity against AML-2/D100 with vincristine($IC_{50:}36.43$ ${\mu}g/ml$) was more effective than without vincristine ($IC_{50:}73.07$ ${\mu}g/ml$), Cross resistance(CR:0.53) was not effective, and anti-MDR activites(RF) was effective.(RF:2) 5) In butanol fraction, the cytotoxicity against AML-2/WT and AML-2/D100 was not effective. 6) In $H_2O$ fraction, the cytotoxicity against AML-2/WT and AML-2/D100 was not effective. Conclusion : These result suggest that WJT has antioxidative effects, anti-tumor effects by apoptosis of free radical$(O_2\;^-)$ activity, and anti-MDR activites(especially hexane and ethyl acetate fraction).

• Journal of Life Science
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• v.25 no.3
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• pp.317-322
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• 2015

Alcohol-induced fatty liver (steatosis) results from excessive generation of reducing equivalents by ethanol metabolism. Generally, chronic ethanol treatment causes hepatosteatosis by regulating sterol regulatory element-binding protein 1c (SREBP-1c), which increases the synthesis of hepatic lipids. The effect of ethanol on SREBP-1c is mediated through mammalian sirtuin-1 (SIRT-1), a NAD⁺-dependent protein deacetylase that regulates hepatic lipid metabolism. Ginseng is a widely used herbal medicine that is used in Asia for its anti-diabetes and anti-obesity effects. The pharmacological and therapeutic effects of ginseng are primarily produced by bioactive constituents known as ginsenosides. Here, we examined the regulatory effects of Korean red ginseng (KRG) extracts on SREBP-1c and SIRT-1 on lipid homeostasis in AML-12 mouse hepatocytes. AML-12 cells were treated with ethanol and/or KRG extracts (0 - 1,000 μg/ml). Lipid droplets were assayed using Oil red O staining, and western blotting was used to measure SIRT-1 and SREBP-1 expression. Treatment with KRG extracts restored SIRT-1 expression and reduced SREBP-1c expression in ethanol-treated cells. We also showed that KRG extract and ginsenosides Rb₂ and Rd significantly decreased SREBP-1 acetylation in ethanol-treated cells. These results show that treatment with KRG extract and its active ginsenoside constituents Rb₂ and Rd protected against alcohol-related hepatosteatosis via regulation of SIRT-1 and downstream acetylation of SREBP-1c, which altered hepatic lipid metabolism.

• Biomolecules & Therapeutics
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• v.32 no.2
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• pp.214-223
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• 2024

Metabolic abnormalities in the liver are closely associated with diverse metabolic diseases such as non-alcoholic fatty liver disease, type 2 diabetes, and obesity. The aim of this study was to evaluate the ameliorating effect of robinetin (RBN) on the significant pathogenic features of metabolic failure in the liver and to identify the underlying molecular mechanism. RBN significantly decreased triglyceride (TG) accumulation by downregulating lipogenesis-related transcription factors in AML-12 murine hepatocyte cell line. In addition, mice fed with Western diet (WD) containing 0.025% or 0.05% RBN showed reduced liver mass and lipid droplet size, as well as improved plasma insulin levels and homeostatic model assessment of insulin resistance (HOMA-IR) values. CD38 was identified as a target of RBN using the BioAssay database, and its expression was increased in OPA-treated AML-12 cells and liver tissues of WD-fed mice. Furthermore, RBN elicited these effects through its anti-histone acetyltransferase (HAT) activity. Computational simulation revealed that RBN can dock into the HAT domain pocket of p300, a histone acetyltransferase, which leads to the abrogation of its catalytic activity. Additionally, knock-down of p300 using siRNA reduced CD38 expression. The chromatin immunoprecipitation (ChIP) assay showed that p300 occupancy on the promoter region of CD38 was significantly decreased, and H3K9 acetylation levels were diminished in lipid-accumulated AML-12 cells treated with RBN. RBN improves the pathogenic features of metabolic failure by suppressing the p300-CD38 axis through its anti-HAT activity, which suggests that RBN can be used as a new phytoceutical candidate for preventing or improving this condition.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.1
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• pp.357-360
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• 2016

Background: Acute myeloid leukemia (AML) is an acquired clonal frequent malignant disorder of myeloid progenitor cells. Our aim was to study demographical and clinicopathological features of adult Pakistani AML patients at presentation. Materials and Methods: In this single centre study extending from January 2010 to December 2014, data were retrieved from the patient records with a predetermined performa and analyzed with SPSS version 22. Results: Overall 125 patients were diagnosed at our institution with de novo AML during the study period. There were 76 males and 49 females (ratio 1.5:1), with an age range between 15 and 85 years and a mean age of $38.8{\pm}20.1years$. The major complaints were fever (72.8%), generalized weakness (60%), bleeding (37.6%) and dyspnea (12%). Physical examination revealed pallor in 56.8%, splenomegaly and hepatomegaly in 16% and 12.8%, respectively, and lymphodenopathy in 10.4%. The mean hemoglobin was $8.19{\pm}2.12g/dl$ with a mean MCV of $86.0{\pm}9.83fl$, a mean total leukocyte count of $43.1{\pm}68.5{\times}10^9/l$, an ANC of $3.09{\pm}6.66{\times}10^9/l$ and a mean platelet count of $62.3{\pm}78.6{\times}10^9/l$. Conclusions: AML in Pakistani patients is seen in a relatively very young population with male preponderance, compared with the west. However, clinico-pathological features appear comparable to published data.