• 한국환경성돌연변이발암원학회지
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• 제21권2호
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• pp.82-88
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• 2001

Recently, there has been significant progress in understanding the control process of the cell division cycle. To investigate the influence of toxic substances on the cell cycle, the effect of benzo(a)pyrene (BAP) and mitomycine C (MMC) on synchronized HeLa cells was analyzed during the cell cycle. To synchronize the HeLa cells, 10$^{6}$ cells were grown for 1 day and then treated with 1 mM hydroxyurea for 14 h. The arrested cells were then allowed to proceed through their cell cycle by removing the hydroxyurea and resupplying a fresh medium. The arrested cells in the G1/S transition then proceeded to the S phase after 4 h, the G2/M phase after 8h, and the G1 phase after 12 h, subsequent to the resupply of a fresh medium. In the untreated HeLa cells, the p34$^{cdc2}$ kinase activity, measured using a p34$^{cdc2}$ specific peptide, peaked after 8h (G2/M) and then declined after 12 h (G1). However, treatment with 30 $\mu$M BAP delayed the peak of the p34$^{cdc2}$ kinase activity. The amount of p34$^{cdc2}$ remained unchanged in the untreated, BAP-, and MMC-treated cells throughout the cell cycle. The cyclin B level peaked after 8 h in the untreated cells, yet peaked after 10-12 h in the BAP-treated cells. There was no significant change in the cyclin B level in the MMC-treated cells.

• Asian Pacific Journal of Cancer Prevention
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• 제17권3호
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• pp.1299-1308
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• 2016

Our previous studies clearly demonstrated that a combination of WP 631 and Epo B has higher activity against ovarian cancer cells than either of these compounds used separately. In order to fully understand the exact mechanism of action in combination, we assessed effects on the cell cycle of SKOV-3 cells. We evaluated three control points essential for WP 631 and Epo B action to determine which cell cycle-regulating proteins (CDK1/cyclin B complex, EpCAM or HMGB1) mediate activity. The effects of the drug on the cell cycle were measured based on the nuclear DNA content using flow cytometry. Expression of cell cycle-regulating genes was analyzed using real-time PCR. It was discovered that WP 631, at the tested concentration, did not affect the SKOV-3 cell cycle. Epo B caused significant G2/M arrest, whereas the drug combination induced stronger apoptosis and lower mitotic arrest than Epo B alone. This is very important information from the point of view of the fight against cancer, as, while mitotic arrest in Epo B-treated cells could be overcame after DNA damage repair, apoptosis which occurs after mitotic slippage in combination-treated cells is irreversible. It clearly explains the higher activity of the drug combination in comparison to Epo B alone. Epo B acts via the CDK1/cyclin B complex and has the ability to inhibit CDK1, which may be a promising strategy for ovarian cancer treatment in the future. The drug combination diminishes EpCAM and HMGB1 expression to a greater degree than either WP 631 and Epo B alone. Owing to the fact that the high expression of these two proteins is a poor prognostic factor for ovarian cancer, a decrease in their expression, observed in our studies, may result in improved efficacy of cancer therapy. The presented findings show that the combination of WP 631 and Epo B is a better therapeutic option than either of these drugs alone.

• Tuberculosis and Respiratory Diseases
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• 제44권4호
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• pp.796-805
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• 1997

연구배경 : 세포주기의 활성화, 그 중에서도 특히 $G_1$/S 이행에 관여하는 세포주기관련 단백질들은 암발생에 있어서 매우 중요한 역할을 하는 것으로 알려져 있다. $G_1$ 세포주기 관련 단백질 중의 하나인 cdk4 (cyclin dependent kinase 4)의 억제제로 알려져 있는 p16 유전자는 최근에 밝혀진 종양억제유전자중의 하나로서 MTS1 (multiple tumor suppressor 1)이라고도 불린다. p16 유전자는 지금까지 알려진 어느 종양관련 유전자보다도 유전자변이의 빈도가 높은 암억제유전자인데, 특히 비소세포폐암인 경우는 70% 이상의 세포주에서 p16 단백질의 발현이 없는 것으로 밝혀져 있어 p16 유전자는 비소세포폐암 발생에 매우 중요한 역할을 할 것이라고 알려져 있다. 본 연구에서는 비소세포폐암에서 p16을 이용한 유전자치료의 타당성을 입증하기 위하여 다음과 같은 연구를 시행하였다. 방 법 : p16이 결여된 비소세포폐암 세포주 (NCI-H441)에, 정상섬유아세포에서 총 RNA를 추출하여 역전사효소 및 DNA 중합효소반응으로 증폭된 p16 cDNA를 유핵세포 발현 vector인 pRC-CMV plasmid에 subcloning하여 구축된 pRC-CMV-p16 plasmid vector를 lipofectin을 이용하여 유전자 이입한 후, 단백질을 추출하여 Western blot 분석과 면역침전법으로 $G_1$ 세포주기관련 단백질의 변동을 관찰하고, colony 형성능을 비교함으로써 암억제효과를 확인하였다. 결 과 : p16이 유전자주입된 NCI-H441 세포주에서 p16과 cdk4가 복합체를 형성하고 있고 인산화 Rb가 대조 세포주에 비해 감소되어 있음을 확인할 수 있어, p16이 cdk4와 결합함으로써 cdk4에 의한 Rb의 인산화를 방해하고 이에 따른 $G_1$ 세포주기 정체에 의해 종양억제효과가 나타난다는 설명을 뒷받침할 수 있었다. Clonogenic assay 결과는 p16 유전자주입된 NCI-H441 세포주의 colony 형성능이 대조 세포주에 비하여 현격히 감소함을 관찰하였다. 결 론 : 이상의 결과로 p16(MTS1) 유전자를 p16 단백질을 발현하지 못하는 비소세포폐암 세포주에 주입할 경우, 주입한 유전자에서 생성되는 p16 단백질이 cdk와 결합하여 Rb 단백질의 인산화를 저하시켜 궁극적으로 암억제 효과를 일으킬 수 있음이 확인되었고, 이는 향후 비소세포폐암의 유전자치료에 있어서 p16 유전자의 이용 가능성을 확인한 기초자료가 된다고 생각된다.

• 한국동물학회:학술대회논문집
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• 한국동물학회 1999년도 한국생물과학협회 학술발표대회
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• pp.182.1-182
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• 1999

No Abstract, See Full Text

• Asian-Australasian Journal of Animal Sciences
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• 제16권1호
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• pp.15-22
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• 2003

Experiments were conducted to evaluate the effect of blastomere diameters and cell cycle stages on the subsequent development of nuclear transplant rabbit embryos (NT-embryos) using nuclei derived from the 16- or 32-cell stage embryos. All blastomeres and NT-embryos were cultured individually in modified Ham's F-10 medium supplemented with 10% rabbit serum (RS) at $38^{\circ}C$ and 5% $CO_2$ in air. The diameter of blastomeres from 16-cell stage embryos was found twice of those from 32-cell stage (51 vs 27 ${\mu}m$). Significant differences were observed in cleavage rates ($\geq$3 divisions) in the isolated single blastomeres (54 vs 48 for 16-cell; 28 vs 14 for 32-cell, p<0.05), but the fusion rates of oocytes with transferred nuclei were similar between small and large single blastomeres derived from either 16-cell or 32-cell stage embryos. When 16-cell stage blastomeres were used as nuclear donors, cleavage rates ($\geq$3 divisions) of the NT-embryos were greater in the small nuclear donors than in the large donors (73 vs 55%, p<0.05). On the contrary, significantly higher cleavage (43 vs 6%, p<0.05) and developmental rates (14 vs 0%, p<0.05) were observed in the large blastomere nuclear donor group of the 32-cell stage embryos. When the cell cycle stages were controlled by a microtubule polymerization inhibitor (Demicolcine, DEM) or the combined treatment of DEM and Aphidicolin (APH), a DNA polymerase inhibitor, fusion rates were 88-96% for the 16-cell donor group (without DEM treatment), which were greater than the 32-cell donor group (54-58%). Cleavage rates were also greater in the transplants derived from G1 nuclear donor group (93-95%) than those from the DEM and APH combined treatment (73%) for the 16-cell donor group (p<0.05). No significant difference was detected in the morula/blastocyst rates in either donor cell stage (p>0.05). In conclusion, it appeared that no difference in the developmental competence between large and small isolated blastomeres was observed. When smaller 16-cell stage blastomeres were used as nuclear donor, the cleavage rate or development of NT-embryos was improved and was compromised when 32-cell stage blastomeres were used. Therefore, control nuclear stage of the donor cell at $G_1$ phase in preactivated nuclear recipients seemed to be beneficial for the cleavage rate of the reconstructed embryo in the 16-cell transplant, but not for subsequent morula or blastocyst development.

• Asian Pacific Journal of Cancer Prevention
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• 제16권4호
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• pp.1361-1365
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• 2015

Background: We aimed to discover potential gene biomarkers for gastric cancer (GC) diagnosis. Materials and Methods: Genechips of 10 GC tissues and 10 gastric mucosa (GM, para-carcinoma tissue, normal control) tissues were generated using an exon array of Affymetrix containing 30,000 genes. The differentially expressed genes (DEGs) between GC tissues and normal control were identified by the Limma package and analyzed by hierarchical clustering analysis. Gene ontology (GO) and pathway enrichment analyses were performed for investigating the functions of DEGs. Receiver operating characteristics (ROC) analysis was performed to measure the effects of biomarker candidates for diagnosis of GC. Results: Totals of 896 up-regulated and 60 down-regulated DEGs were identified to be differentially expressed between GC samples and normal control. Hierarchical clustering analysis showed that DEGs were highly differentially expressed and most DEGs were up-regulated. The most significantly enriched GO-BP term was revealed to be mitotic cell cycle and the most significantly enriched pathway was cell cycle. The intersection analysis showed that most significant DEGs were cyclin B1 (CCNB1) and cyclin B2 (CCNB2). The sensitivities and specificities of CCNB1 and CCNB2 were both high (p<0.0001). Areas under the ROC curve for CCNB1 and CCNB2 were both greater than 0.9 (p<0.0001). Conclusions: CCNB1 and CCNB2, which were involved in cell cycle, played significant roles in the progression and development of GC and these genes may be potential biomarkers for diagnosis and prognosis of GC.

• Journal of Microbiology and Biotechnology
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• 제12권3호
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• pp.470-475
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• 2002

In the course of screening for a novel cell cycle inhibitor, a potent Cdk 1 inhibitor, HY558, was found from the culture broth of Penicillium minioluteum F558 isolated from a soil sample. The molecular ion of HY558 was identified at m/z 329 (MH+) with a molecular formula of $C_20H_44ON_2$. HY558 exhibited selective antiproliferative effects on various human cancer cell lines. Its $IC_50$ values were estimated to be 0.29 mM on HepG2, 0.30 mM on HeLa, 0.30 mM on HL6O, 0.33 mM on HT-29, and 0.25 mM on AGS cells. Interestingly, Hy558 demonstrated no antiproliferative effect with normal lymphocytes used as the control, and a low level of inhibition on the proliferation of A549 cancer cells. A flow cytometric analysis of HepG2 cells revealed an appreciable arrest of cells at the G1 and G2/M phases of the cell cycle following treatment with Hy558. furthermore, DNA fragmentation due to apoptosis was observed in HeLa cells treated with 0.46 mM of HY558.

• Asian Pacific Journal of Cancer Prevention
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• 제14권6호
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• pp.3669-3676
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• 2013

Faithful transmission of genetic information depends on accurate chromosome segregation as cells exit from mitosis, and errors in chromosomal segregation are catastrophic and may lead to aneuploidy which is the hallmark of cancer. In eukaryotes, an elaborate molecular control system ensures proper orchestration of events at mitotic exit. Phosphorylation of specific tyrosyl residues is a major control mechanism for cellular proliferation and the activities of protein tyrosine kinases and phosphatases must be integrated. Although mitotic kinases are well characterized, phosphatases involved in mitosis remain largely elusive. Here we identify a novel variant of mouse protein tyrosine phosphatase containing domain 1 (Ptpcd1), that we named Ptpcd2. Ptpcd1 is a Cdc14 related centrosomal phosphatase. Our newly identified Ptpcd2 shared a significant homology to yeast Cdc14p (34.1%) and other Cdc14 family of phosphatases. By subcellular fractionation Ptpcd2 was found to be enriched in the cytoplasm and nuclear pellets with catalytic phosphatase activity. By means of immunofluorescence, Ptpcd2 was spatiotemporally regulated in a cell cycle dependent manner with cytoplasmic abundance during mitosis, followed by nuclear localization during interphase. Overexpression of Ptpcd2 induced mitotic exit with decreased levels of some mitotic markers. Moreover, Ptpcd2 failed to colocalize with the centrosomal marker ${\gamma}$-tubulin, suggesting it as a non-centrosomal protein. Taken together, Ptpcd2 phosphatase appears a non-centrosomal variant of Ptpcd1 with probable mitotic functions. The identification of this new phosphatase suggests the existence of an interacting phosphatase network that controls mammalian mitosis and provides new drug targets for anticancer modalities.

• Imaging Science in Dentistry
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• 제30권4호
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• pp.275-279
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• 2000

Purpose: Radiation damage is produced and viable cell number is reduced. We need to know the type of cell death by the ionizing radiation and the amount and duration of cell cycle arrest. In this study, we want to identified the main cause of the cellular damage in the oral cancer cells and normal keratinocytes with clinically useful radiation dosage. Materials and Methods: Human gingival tissue specimens obtained from healthy volunteers were used for primary culture of the normal human oral keratinocytes (NHOK). Primary NHOK were prepared from separated epithelial tissue and maintained in keratinocyte growth medium containing 0.15 mM calcium and a supplementary growth factor bullet kit. Fadu and Hep-2 cell lines were obtained from KCLB. Cells were irradiated in a /sup 137/Cs γ-irradiator at the dose of 10 Gy. The dose rate was 5.38 Gy/min. The necrotic cell death was examined with Lactate Dehydrogenase (LDH) activity in the culture medium. Every 4 day after irradiation, LDH activities were read and compared control group. Cell cycle phase distribution and preG1-incidence after radiation were analyzed by flow cytometry using Propidium Iodine staining. Cell cycle analysis were carried out with a FAC Star plus flowcytometry (FACS, Becton Dickinson, USA) and DNA histograms were processed with CELLFIT software (Becton Dickinson, USA). Results: LDH activity increased in all of the experimental cells by the times. This pattern could be seen in the non-irradiated cells, and there was no difference between the non-irradiated cells and irradiated cells. We detected an induction of apoptosis after irradiation with a single dose of 10 Gy. The maximal rate of apoptosis ranged from 4.0% to 8.0% 4 days after irradiation. In all experimental cells, we detected G2/M arrest after irradiation with a single dose of 10 Gy. Yet there were differences in the number of G2/M arrested cells. The maximal rate of the G2/M ranges from 60.0% to 80.0% 24h after irradiation. There is no significant changes on the rate of the G0/G1 phase. Conclusion: Radiation sensitivity was not related with necrosis but cell cycle arrest and apoptosis. These data suggested that more arrested cell is correlated with more apoptosis.

• Asian Pacific Journal of Cancer Prevention
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• 제15권13호
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• pp.5437-5442
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• 2014

Esophageal squamous cell carcinoma (ESCC) is one of the most malignancies with a poor prognosis. The phospholipase $C{\varepsilon}$ gene (PLCE1) encodes a novel ras-related protein effector mediating the effects of R-Ras on the actin cytoskeleton and membrane protrusion. However, molecular mechanisms pertinent to ESCC are unclear. We therefore designed PLCE1-special small interfering RNA and transfected to esophageal squamous cell (EC) 9706 cells to investigat the effects of PLCE1 gene silencing on the cell cycle and apoptosis of ESCC and indicate its important role in the development of ESCC. Esophageal cancer tissue specimens and normal esophageal mucosa were obtained and assayed by immunohistochemical staining to confirm overexpression of PLCE1 in neoplasias. Fluorescence microscopy was used to examine transfection efficiency, while the result of PLCE1 silencing was examined by reverse transcription (RT-PCR). Flow cytometry and annexin V apoptosis assays were used to assess the cell cycle and apoptosis, respectively. Expression of cyclin D1 and caspase-3 was detected by Western-blotting. The level of PLCE1 protein in esophageal cancer tissue was significantly higher than that in normal tissue. After transfection, the expression of PLCE1 mRNA in EC 9706 was significantly reduced, compared with the control group. Furthermore, flow cytometry results suggested that the PLCE1 gene silencing arrested the cell cycle in the G0/G1 phase; apoptosis was significantly higher than in the negative control group and mock group. PLCE1 gene silencing by RNAi resulted in decreased expression of cyclin D1 and increased expression of caspase-3. Our study suggests that PLCE1 may be an oncogene and play an important role in esophageal carcinogenesis through regulating proteins which control cell cycling and apoptosis.