• BMB Reports
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• 제51권6호
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• pp.261-262
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• 2018

Aurora B is an important kinase involved in dynamic cellular events in mitosis. Aurora B activity is controlled by several post-translational modifications (PTMs). Among them, E3 ubiquitin ligase-mediated ubiquitination plays crucial roles in controlling the relocation and degradation of Aurora B. Aurora B, ubiquitinated by different E3 ligases, moves to the exact site for its mitotic function during metaphase-anaphase transition and is then degraded for cell cycle progression at the end of mitosis. However, how the stability of Aurora B is maintained until its degradation has been poorly understood. Recently, we have found that USP35 acts as a deubiquitinating enzyme (DUB) for Aurora B and affects its stability during cell division, thus being involved in the regulation of mitosis. In this review, we discuss the USP35-mediated deubiquitination of Aurora B and the regulation of mitotic progression by USP35.

• Journal of Ginseng Research
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• 제46권3호
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• pp.481-488
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• 2022

Background: Although the tumor-suppressive effects of ginsenosides in cell cycle have been well established, their pharmacological properties in mitosis have not been clarified yet. The chromosomal instability resulting from dysregulated mitotic processes is usually increased in cancer. In this study, we aimed to investigate the anticancer effects of ginsenoside Rg1 on mitotic progression in cancer. Materials and methods: Cancer cells were treated with ginsenoside Rg1 and their morphology and intensity of different protein were analyzed using immunofluorescence microscopy. The level of proteins in chromosomes was compared through chromosomal fractionation and Western blot analyses. The location and intensity of proteins in the chromosome were confirmed through immunostaining of mitotic chromosome after spreading. The colony formation assays were conducted using various cancer cell lines. Results: Ginsenoside Rg1 reduced cancer cell proliferation in some cancers through inducing mitotic arrest. Mechanistically, it inhibits the phosphorylation of histone H3 Thr3 (H3T3ph) mediated by Haspin kinase and concomitant recruitment of chromosomal passenger complex (CPC) to the centromere. Depletion of Aurora B at the centromere led to abnormal centromere integrity and spindle dynamics, thereby causing mitotic defects, such as increase in the width of the metaphase plate and spindle instability, resulting in delayed mitotic progression and cancer cell proliferation. Conclusion: Ginsenoside Rg1 reduces the level of Aurora B at the centromere via perturbing Haspin kinase activity and concurrent H3T3ph. Therefore, ginsenoside Rg1 suppresses cancer cell proliferation through impeding mitotic processes, such as chromosome alignment and spindle dynamics, upon depletion of Aurora B from the centromere.

• BMB Reports
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• 제48권7호
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• pp.413-418
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• 2015

DEPDC1 is a recently identified novel tumor-related gene that is upregulated in several types of cancer and contributes to tumorigenesis. In this study, we have investigated the expression pattern and functional implications of DEPDC1 during cell cycle progression. Expression studies using synchronized cells demonstrated that DEPDC1 is highly expressed in the mitotic phase of the cell cycle. Immunofluorescence assays showed that DEPDC1 is predominantly localized in the nucleus during interphase and is redistributed into the whole cell upon nuclear membrane breakdown in metaphase. Subsequently, siRNA-mediated knockdown of DEPDC1 caused a significant mitotic arrest. Moreover, knockdown of DEPDC1 resulted in remarkable mitotic defects such as abnormal multiple nuclei and multipolar spindle structures accompanied by the upregulation of the A20 gene as well as several cell cycle-related genes such as CCNB1 and CCNB2. Taken together, our current observations strongly suggest that this novel cancerous gene, DEPDC1, plays a pivotal role in the regulation of proper mitotic progression. [BMB Reports 2015; 48(7): 413-418]

• Journal of Microbiology
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• 제39권4호
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• pp.286-292
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• 2001

During mitosis. the proper segregation of duplicated chromosomes is corrdinated by a spindle check-point. The bifurcated spindle checkpoint blocks cell cycle progression at metaphase by monitoring unattached kinetochores and inhibits mitotic exit in response to the misorientation of the mitotic spin- dle Ibd1p/Bfa1p is a spindle checkpoint regulator of budding yeast in the Bub2p checkpoint pathway for mitotic exit and its disruption abolishes mitotic arrest when proper organization of the mitotic spin-dls inhibited. Ibd1p/Bfa1p localizes to the spindle pole body, a microtublue-organizing center in yeast, and its overexpression arrests the cell cycle in 80% of cells with an enlarged budy at mitosis and in 20 % of cells with multiple buds. In this study, we found that the C-terminus of Ibd1p/Bfa1p phys-ically interacts with Skp1p, a key component of SCF (Skp1/cullin/F-box) complex for ubiquition-medi-ated proteolysis of cel cycle regulatores as well as an evolutionally conserved kinetochore protein for cell cycle progression. A putative F-box motif was found in the C-terminus of Ibd1p/Bfa1p and its function was investigated by making mutants of conserved residues in the motif. These Ibd1p/Bfa1p mutants of a putative F-box interacted with SKp1p in vitro by two-hybrid assays as wild type Ibd1p/Bfa1p. Also these Ibd1p/Bfa1p utants displayed the overexpression phenotypes of wild type Ibd1p, when over-expressed under inducible promoters . These results suggest that a putative F-box motif of Ibd1p/Bfa1p is not essential for the interaction with SKp1p and its function in mitotic exit and cytokinesis.

• Journal of Microbiology and Biotechnology
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• 제25권5호
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• pp.598-605
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• 2015

The cohesin complex holds sister chromatids together and prevents premature chromosome segregation until the onset of anaphase. Mcd1 (also known as Scc1), the α-kleisin subunit of cohesin, is a key regulatory subunit of the mitotic cohesin complex and is required for maintaining sister chromatid cohesion, chromosome organization, and DNA repair. We investigated the function of Mcd1 in meiosis by ectopically expressing Mcd1 during early meiotic prophase I in Saccharomyces cerevisiae. Mcd1 partially regulated the progression of meiotic recombination, sister chromatid separation, and nuclear division. DNA physical analysis during meiotic recombination showed that Mcd1 induced double-strand breaks (DSBs) but negatively regulated homologous recombination during DSB repair; Mcd1 expression delayed post-DSB stages, leading to inefficiencies in the DSB-to-joint molecule (JM) transition and subsequent crossover formation. These findings indicate that meiotic cells undergo Mcd1-mediated DSB formation during prophase I, and that residual Mcd1 could regulate the progression of JM formation during meiotic recombination.

• Molecules and Cells
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• 제41권8호
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• pp.733-741
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• 2018

Mutations in spliceosome components have been implicated in carcinogenesis of various types of cancer. One of the most frequently found is U2AF1 S34F missense mutation. Functional analyses of this mutation have been largely limited to hematological malignancies although the mutation is also frequently seen in other cancer types including lung adenocarcinoma (LUAD). We examined the impact of knockdown (KD) of wild type (wt) U2AF1 and ectopic expression of two splice variant S34F mutant proteins in terms of alternative splicing (AS) pattern and cell cycle progression in A549 lung cancer cells. We demonstrate that induction of distinct AS events and disruption of mitosis at distinct sub-stages result from KD and ectopic expression of the mutant proteins. Importantly, when compared with the splicing pattern seen in LUAD patients with U2AF1 S34F mutation, ectopic expression of S34F mutants but not KD was shown to result in common AS events in several genes involved in cell cycle progression. Our study thus points to an active role of U2AF1 S34F mutant protein in inducing cell cycle dysregulation and mitotic stress. In addition, alternatively spliced genes which we describe here may represent novel potential markers of lung cancer development.

• 생명과학회지
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• 제23권5호
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• pp.622-628
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• 2013

본 연구에서는 마늘에서 유래된 생리활성 물질인 diallyl trisulfide (DATS) 처리에 따른 U937 인체혈구암세포의 증식억제가 apoptosis 및 cell cycle arrest 유발과 관련이 있는지 조사하였다. U937 세포증식은 DATS에 의해 농도 및 시간 의존적으로 감소함을 확인 하였고, 이는 apoptosis에 의한 직접적인 세포죽음과 CDK1 및 cyclin B1의 발현 증가 및 histone H3의 인산화와 연관된 mitotic arrest와 관련이 있음을 알 수 있었다. 또한 DATS 처리 초기에 reactive oxygen species (ROS)의 생성이 매우 증가되었으나, ROS scavenger (N-acetyl-l-cysteine)에 의한 인위적 ROS 생성의 억제는 DATS에 의한 apoptosis 및 mitotic arrest를 완벽하게 차단시켰다. 이는 U937 세포에서 DATS에 의해 유도된 apoptosis 및 mitotic arrest가 ROS에 의해 매개된다는 것을 의미하며, 본 연구의 결과는 DATS가 인체혈구암세포에서 세포증식억제와 관련된 항암기전을 이해할 수 있는 기초자료로서 매우 유용하게 사용될 것이라 생각된다.

• BMB Reports
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• 제46권6호
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• pp.289-294
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• 2013

To maintain cellular homeostasis against the demands of the extracellular environment, a precise regulation of kinases and phosphatases is essential. In cell cycle regulation mechanisms, activation of the cyclin-dependent kinase (CDK1) and cyclin B complex (CDK1:cyclin B) causes a remarkable change in protein phosphorylation. Activation of CDK1:cyclin B is regulated by two auto-amplification loops-CDK1:cyclin B activates Cdc25, its own activating phosphatase, and inhibits Wee1, its own inhibiting kinase. Recent biological evidence has revealed that the inhibition of its counteracting phosphatase activity also occurs, and it is parallel to CDK1:cyclin B activation during mitosis. Phosphatase regulation of mitotic kinases and their substrates is essential to ensure that the progression of the cell cycle is ordered. Outlining how the mutual control of kinases and phosphatases governs the localization and timing of cell division will give us a new understanding about cell cycle regulation.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.912-918
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• 2003

Mitotic centromere-associated kinesin (MCAK), which is a novel kinesin with a central motor domain, is believed to playa role in mitotic segregation of chromosome during the M phase of the cell cycle. In the present study, it is shown that a rabbit polyclonal antibody has been produced using the N-terminal region (187 aa) of human MCAK expressed in E. coli as the antigen. To express the N-terminal region in E. coli, the MCAK cDNA fragment encoding N-terminal 187 aa was obtained by PCR and was then inserted into the pET 3d expression vector. Molecular mass of the N-terminal region overexpressed in the presence of IPTG was 23.2 kDa on SDS-PAGE, and the protein was insoluble and mainly localized in the inclusion body that could be easily purified from the other cellular proteins. The N-terminal region was purified by electro-elution from the gel after the inclusion body was resolved on the SDS-PAGE. The antiserum obtained after tertiary immunization with the purified protein specifically recognized HsMCAK when subjected to Western blot analysis, and showed a fluctuation of the protein level during the cell cycle of human Jurkat T cells. Synchronization of the cell-cycle progression required for recovery of cells at a specific stage of the cell cycle was performed by either hydroxyurea or nocadazole, and subsequent release from each blocking at 2, 4, and 7 h. Northern and Western analyses revealed that both mRNA and protein of HsMCAK reached a maximum level in the S phase and declined to a basal level in the G1 phase. These results indicate that a polyclonal antibody raised against the N-terminal region (187 aa) of HsMCAK, overexpressed in E. coli, specifically detects HsMCAK (81 kDa), and it can analyze the differential expression of HsMCAK protein during the cell cycle.

• Journal of Microbiology and Biotechnology
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• 제30권2호
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• pp.279-286
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• 2020

A novel compound named 'kanakugiol' was recently isolated from Lindera erythrocarpa and showed free radical-scavenging and antifungal activities. However, the details of the anti-cancer effect of kanakugiol on breast cancer cells remain unclear. We investigated the effect of kanakugiol on the growth of MCF-7 human breast cancer cells. Kanakugiol affected cell cycle progression, and decreased cell viability in MCF-7 cells in a dose-dependent manner. It also enhanced PARP cleavage (50 kDa), whereas DNA laddering was not induced. FACS analysis with annexin V-FITC/PI staining showed necrosis induction in kanakugiol-treated cells. Caspase-9 cleavage was also induced. Expression of death receptors was not altered. However, Bcl-2 expression was suppressed, and mitochondrial membrane potential collapsed, indicating limited apoptosis induction by kanakugiol. Immunofluorescence analysis using α-tubulin staining revealed mitotic exit without cytokinesis (4N cells with two nuclei) due to kanakugiol treatment, suggesting that mitotic catastrophe may have been induced via microtubule destabilization. Furthermore, cell cycle analysis results also indicated mitotic catastrophe after cell cycle arrest in MCF-7 cells due to kanakugiol treatment. These findings suggest that kanakugiol inhibits cell proliferation and promotes cell death by inducing mitotic catastrophe after cell cycle arrest. Thus, kanakugiol shows potential for use as a drug in the treatment of human breast cancer.