• BMB Reports
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• v.44 no.5
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• pp.352-357
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• 2011

The effect of human MutY homolog (hMYH) on the activation of checkpoint proteins in response to hydroxyurea (HU) and ultraviolet (UV) treatment was investigated in hMYH-disrupted HEK293 cells. hMYH-disrupted cells decreased the phosphorylation of Chk1 upon HU or UV treatment and increased the phosphorylation of Cdk2 and the amount of Cdc25A, but not Cdc25C. In siMYH-transfected cells, the increased rate of phosphorylated Chk1 upon HU or UV treatment was lower than that in siGFP-transfected cells, meaning that hMYH was involved in the activation mechanism of Chk1 upon DNA damage. The phosphorylation of ataxia telangiectasia and Rad3-related protein (ATR) upon HU or UV treatment was decreased in hMYH-disrupted HEK293 and HaCaT cells. Co-immunoprecipitation experiments showed that hMYH was immunoprecipitated by anti-ATR. These results suggest that hMYH may interact with ATR and function as a mediator of Chk1 phosphorylation in response to DNA damage.

• Journal of Gastric Cancer
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• v.5 no.4 s.20
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• pp.260-265
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• 2005

Purpose: The protein kinase Chk1 is required for cell cycle arrest in response to DNA damage and is shown to play an important role in the G2/M checkpoint. The aim of this study was to investigate the relationship between microsatellite instability and frameshift mutation of the Chk1 gene in gastric cancers. Materials and Methods: The microsatellite instability was analyzed in 95 primary gastric carcinomas by using microdissection and 6 microsatellite markers. We also peformed single strand conformational polymorphism and sequencing to detect frameshift mutation of the Chk1 gene. Results: We found positive microsatellite instability in 19 (20%) of the 95 gastric cancers, 13 high- and 6 low-frequency microsatellite instability cases. The frameshift mutation of Chk1, which resulted in a truncated Chk1 protein, was detected in two high-frequency microsatellite instability cases. Conclusion: These data suggest that the microsatellite instability may contribute to the development of gastric carcinomas through inactivation of Chk1.

• Molecules and Cells
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• v.37 no.2
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• pp.126-132
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• 2014

As a tumor suppressor homologue during mitosis, Chk2 is involved in replication checkpoints, DNA repair, and cell cycle arrest, although its functions during mouse oocyte meiosis and early embryo development remain uncertain. We investigated the functions of Chk2 during mouse oocyte maturation and early embryo development. Chk2 exhibited a dynamic localization pattern; Chk2 expression was restricted to germinal vesicles at the germinal vesicle (GV) stage, was associated with centromeres at pro-metaphase I (Pro-MI), and localized to spindle poles at metaphase I (MI). Disrupting Chk2 activity resulted in cell cycle progression defects. First, inhibitor-treated oocytes were arrested at the GV stage and failed to undergo germinal vesicle breakdown (GVBD); this could be rescued after Chk2 inhibition release. Second, Chk2 inhibition after oocyte GVBD caused MI arrest. Third, the first cleavage of early embryo development was disrupted by Chk2 inhibition. Additionally, in inhibitor-treated oocytes, checkpoint protein Bub3 expression was consistently localized at centromeres at the MI stage, which indicated that the spindle assembly checkpoint (SAC) was activated. Moreover, disrupting Chk2 activity in oocytes caused severe chromosome misalignments and spindle disruption. In inhibitor-treated oocytes, centrosome protein ${\gamma}$-tubulin and Polo-like kinase 1 (Plk1) were dissociated from spindle poles. These results indicated that Chk2 regulated cell cycle progression and spindle assembly during mouse oocyte maturation and early embryo development.

• Journal of Life Science
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• v.23 no.6
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• pp.832-837
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• 2013

In this study, it was investigated the possible mechanisms by which glutamine deprivation exerts its anti-proliferative action in cultured human prostate carcinoma PC3 cells. Glutamine deprivation resulted in inhibition of growth and G2/M arrest of the cell cycle in a time-dependent manner without apoptosis induction, as determined by MTT assay, DAPI staining and flow cytometry analyses. The induction of G2/M arrest by glutamine deprivation was associated with the inhibition of expression of Cdc2, cyclin A and cyclin B1, and up-regulation of the expression of cyclin-dependent kinase (Cdk) inhibitor p21(WAF1/CIP1) in both transcriptional and translational levels. Moreover, glutamine deprivation increased the phosphorylation of checkpoint kinase (Chk)1 and Chk2; however, the levels of Cdc25C phosphorylation were decreased in response to glutamine deprivation in a time-dependent manner. Our data provide a first biochemical evidence that glutamine deprivation suppresses cell viability through G2/M phase arrest without induction of apoptosis in PC3 cells.

• Molecular & Cellular Toxicology
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• v.2 no.3
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• pp.159-165
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• 2006

To determine the anticancer effect of D-amygdalin (D-mandelinitrole-${\beta}$-D-gentiobioside) in human chronic myeloid leukemia cells K562, we profiled the gene expression between amygdalin treatment and control groups. Through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, the cytotoxicity of D-amygdalin was $57.79{\pm}1.83%$ at the concentration of 5 mg/mL for 24 h. We performed cDNA microarray analysis and compared the gene expression profiles between D-amygdalin (5 mg/mL, 24 h) treatment and control groups. Among the genes changed by D-amygdalin, we paid attention to cell cycle-related genes, and particularly cell cycle regulator genes; because arrest of cell cycle processing was ideal tactic in remedy for cancer. In our data, expressions of cyclin-dependent kinase inhibitor 1B (p27, Kip1) (CDKN1B), ataxia telangiectasia mutated (includes complementation groups A, C, and D) (ATM), cyclin-dependent kinase inhibitor 1C (p57, Kip2) (CDKN1C), and CHK1 checkpoint homolog (CHEK1, formally known as CHK1) were increased, while expressions of cyclin-dependent kinase 2 (CDK2), cell division cycle 25A (CDC25A), and cyclin E1 (CCNE1) were decreased. The pattern of these gene expressions were confirmed through RT-PCR. Our results showed that D-amygdalin might control cell cycle regulator genes and arrest S phase of cell cycle in K562 cells as the useful anticancer drug.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.287-294
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• 2016

The effect of kaempferol (3,5,7,4-tetrahydroxyflavone), a flavonoid compound that was identified in barnyard millet (Echinochloa crus-galli var. frumentacea) grains, on G₂-checkpoint and apoptotic pathways was investigated in human acute leukemia Jurkat T cell clones stably transfected with an empty vector (J/Neo) or a Bcl-xL expression vector (J/Bcl-xL). Exposure of J/Neo cells to kaempeferol caused cytotoxicity and activation of the ATM/ATR-Chk1/Chk2 pathway, activating the phosphorylation of p53 (Ser-15), inhibitory phosphorylation of Cdc25C (Ser-216), and inactivation of cyclin-dependent kinase 1 (Cdk1), with resultant G₂-arrest of the cell cycle. Under these conditions, apoptotic events, including upregulation of Bak and PUMA levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9, -8, and -3, anti-poly (ADP-ribose) polymerase (PARP) cleavage, and accumulation of apoptotic sub-G₁ cells, were induced without accompanying necrosis. However, these apoptotic events, except for upregulation of Bak and PUMA levels, were completely abrogated in J/Bcl-xL cells overexpressing Bcl-xL, suggesting that the G₂-arrest and the Bcl-xL-sensitive mitochondrial apoptotic events were induced, in parallel, as downstream events of the DNA-damage-mediated G₂-checkpoint activation. Together these results demonstrate that kaempferol-mediated antitumor activity toward Jurkat T cells was attributable to G₂-checkpoint activation, which caused not only G₂-arrest of the cell cycle but also activating phosphorylation of p53 (Ser-15) and subsequent induction of mitochondria-dependent apoptotic events, including Bak and PUMA upregulation, Bak activation, Δψm loss, and caspase cascade activation.

• Animal Bioscience
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• v.35 no.1
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• pp.126-137
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• 2022

Objective: Efficient gene editing technology is critical for successful knock-in in domestic animals. RAD51 recombinase (RAD51) gene plays an important role in strand invasion during homologous recombination (HR) in mammals, and is regulated by checkpoint kinase 1 (CHK1) and CHK2 genes, which are upstream elements of RAD51 recombinase (RAD51). In addition, mismatch repair (MMR) system is inextricably linked to HR-related pathways and regulates HR via heteroduplex rejection. Thus, the aim of this study was to investigate whether clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9)-mediated knock-in efficiency of human lactoferrin (hLF) knock-in vector in the bovine β-casein gene locus can be increased by suppressing DNA MMR-related genes (MSH2, MSH3, MSH6, MLH1, and PMS2) and overexpressing DNA double-strand break (DSB) repair-related genes (RAD51, CHK1, CHK2). Methods: Bovine mammary epithelial (MAC-T) cells were transfected with a knock-in vector, RAD51, CHK1, or CHK2 overexpression vector and CRISPR/sgRNA expression vector to target the bovine β-casein gene locus, followed by treatment of the cells with CdCl₂ for 24 hours. After 3 days of CdCl₂ treatment, the knock-in efficiency was confirmed by polymerase chain reaction (PCR). The mRNA expression levels of DNA MMR-related and DNA DSB repair-related genes were assessed by quantitative real-time PCR (RT-qPCR). Results: Treatment with CdCl₂ decreased the mRNA expression of RAD51 and MMRrelated genes but did not increase the knock-in efficiency in MAC-T cells. Also, the overexpression of DNA DSB repair-related genes in MAC-T cells did not significantly affect the mRNA expression of MMR-related genes and failed to increase the knock-in efficiency. Conclusion: Treatment with CdCl₂ inhibited the mRNA levels of RAD51 and DNA MMR-related genes in MAC-T cells. However, the function of MMR pathway in relation to HR may differ in various cell types or species.

• Journal of Life Science
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• v.29 no.6
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• pp.679-687
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• 2019

Litsea populifolia, a plant species of the Lauraceae family, is widely distributed in the tropical and subtropical areas of Asia. The phylogenetic relationships and botanical characteristics of L. populifolia have been reported; however, its anti-oxidative and anti-cancer activities remain unclear. In this study, we evaluated the anti-oxidative and anti-cancer effects of ethanol extracts of L. populifolia (EELP) together with the molecular mechanism of its anti-cancer activity in human lung adenocarcinoma A549 cells. EELP showed significant anti-oxidative effects with a 50% inhibitory concentration at $11.71{\mu}g/ml$, which was measured by the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay. EELP exhibited cytotoxic activity and induced cell cycle arrest at the G1 phase in A549 cells in a dose-dependent manner, whereas EELP did not have the cytotoxic effect on the normal human lung cell line IMR90. Treatment with EELP also resulted in a decreased expression of G1/S transition-related molecules-including cyclin-dependent kinase (CDK) 2, CDK6, cyclin D1, and cyclin E-both for the transcription and translation levels. EELP-induced G1 arrest was associated with the phosphorylation of checkpoint kinase 2 (CHK2), p53, cell division cycle 25 homolog A (CDC25A), and the reduction of CDC25A expression in A549 cells. Collectively, these results suggest that EELP may exert an anti-cancer effect by cell cycle arrest at the G1 phase through both p53-dependent and p53-independent (ATM/CHK2/CDC25A/CDK2) pathways in A549 cells.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.4
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• pp.1343-1348
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• 2015

Human UHRF1 (ubiquitin-like PHD and RING finger domain-containing 1) has been reported to be over-expressed in many cancers, but its role in ovarian cancer remains elusive. Here, we determined whether knockdown of UHRF1 by lentivirus-mediated shRNA could inhibit ovarian cancer cell growth. Lentivirus-mediated short hairpin RNAs (lv-shRNAs-UHRF1) were designed to trigger the gene silencing RNA interference (RNAi) pathway. The efficiency of lentivirus-mediated shRNA infection into HO-8910 and HO-8910 PM cells was determined using fluorescence microscopy to observe lentivirus-mediated GFP expression and was confirmed to be over 80 percent. UHRF1 expression in infected HO-8910 and HO-8910 PM was evaluated by real-time PCR and Western blot analysis. The Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability; flow cytometry and Hoechst 33342 assay was applied to measure cell cycle arrest and apoptosis. Cell invasion was assessed using transwell chambers. Our results demonstrated that the loss of UHRF1 promoted HO-8910 and HO-8910 PM cell apoptosis, while inhibiting cell proliferation. In addition, UHRF1 knockdown significantly inhibited the invasion of human ovarian cancer cells. In the present study, we also showed that depleting HO-8910 cells of UHRF1 caused activation of the DNA damage response pathway, with the cell cycle arrested in G2/M-phase. The DNA damage response in cells depleted of UHRF1 was illustrated by phosphorylation of CHK (checkpoint kinase) 2 on Thr68, phosphorylation of CDC25 (cell division control 25) on Ser 216 and phosphorylation of CDK1 (cyclin-dependent kinase 1) on Tyr 15.