1.Vitamin C inhibits benzoapyrene-induced cell cycle changes partly via cyclin D1/E2F pathway in human embryo lung fibroblasts.
Ai GAO ; Bing-Ci LIU ; Xiang-Lin SHIT ; Chuan-Shu HUANG ; Xiao-Wei JIA ; Bao-Rong YOU ; Meng YE ; Fu-Hai SHEN ; Hong-Ju DU
Biomedical and Environmental Sciences 2006;19(3):239-244
OBJECTIVETo study the molecular mechanism of the inhibitory effects of vitamin C on benzo[a]pyrene (B[a]P)-induced changes of cell cycle in human embryo lung fibroblast (HELF) cells.
METHODSThe stable transfectants, HELF transfected with antisense cyclin D1 and antisense CDK4, were established. Cells were cultured and pretreated with vitamin C before stimulation with B[a]P for 24 h. The expression levels of cyclin D1, CDK4, E2F1, and E2F4 were determined by Western blot. Flow cytometric analysis was employed to detect the distributions of cell cycle.
RESULTSB[a]P significantly elevated the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. Vitamin C decreased the expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-stimulated HELF cells. Dose-dependent relationships were not found between the different concentrations of vitamin C (10, 100, 500, 1000, and 5000 micromol/L) and the expression levels of cyclin D1, E2F1, and E2F4 in HELF cells. The expression levels of cyclin D1, E2F1, and E2F4 in B[a]P-treated transfectants were lower than those in B[a]P-treated HELF cells. The expression levels of cyclin D1 and E2F4 treated with vitamin C and antisense cyclin D1 were decreased compared with those treated with antisense cyclin D1 alone. The effects of vitamin C combined with antisense CDK4 on the expression levels of cyclin D1 and E2F1/E2F4 were similar to those of antisense CDK4 alone. B[a]P progressed HELF cells from G1 to S phase. Both vitamin C and antisense cyclin D1 suppressed the changes of cell cycle progressed by B[a]P. However, antisense CDK4 did not attenuate the above changes. Vitamin C combined with antisense CDK4 markedly suppressed B[a]P-induced changes of cell cycle as compared with antisense CDK4. But the inhibitory effects of vitamin C combined with antisense cyclin D1 on B[a]P-induced changes of cell cycle were similar to those of vitamin C alone or antisense cyclin D1 alone.
CONCLUSIONSB[a]P progressed HELF cells from G1 to S phase via intracellular signaling pathway of cyclin D1/E2F. Vitamin C may modulate this signaling pathway to protect cells from injury caused by B[a]P.
Ascorbic Acid ; pharmacology ; Benzo(a)pyrene ; Blotting, Western ; methods ; Cell Cycle ; drug effects ; physiology ; Cells, Cultured ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Dose-Response Relationship, Drug ; E2F1 Transcription Factor ; metabolism ; Fibroblasts ; cytology ; drug effects ; metabolism ; G1 Phase ; drug effects ; physiology ; Humans ; Lung ; cytology ; embryology ; RNA, Antisense ; genetics ; S Phase ; drug effects ; physiology ; Transfection ; methods
2.P21(WAF1) inhibits the growth of leukemia cell line K562 and decreases its sensitivity to Vp16.
Hui YANG ; Shenwu WANG ; Huijun YIN
Chinese Journal of Hematology 2002;23(1):19-22
OBJECTIVETo explore the effect of p21(WAF1) on the proliferation and the sensitivity to Vp16 of leukemia cell line K562.
METHODSA p21(WAF1) retroviral expression vector pLXSN-p21(WAF1) was constructed by FuGENE 6, pLXSN-p21(WAF1) and pLXSN-neo, and transfected into p21(WAF1) defect leukemia cell line K562. After selected with G418, K562-p21(WAF1) cell clones that stably expressed p21(WAF1) were isolated. The ectopic expressions of p21(WAF1) mRNA and protein in K562-p21(WAF1) were identified by RT-PCR and Western b1ot. The cell growth rate was tested by trypan blue dye, the cell cycle by FCM and the sensitivity to Vpl6 by cell count and MTT assay.
RESULTSThe expression of p21(WAF1) protein and mRNA could be detected in K562-p21(WAF1) cells. A strong inhibition of cell proliferation was observed in K562-p21(WAF1) cell as compared with that of the control. The cell number in G(0)/G(1) phase was remarkably increased. The sensitivity to Vpl6 decreased, the IC (50) of K562-neo cells was (56.4 +/- 6.5) microgram/ml, and that of K562-p21(WAF1) cells was (131.0 +/- 8.7) microgram/ml (P < 0.01).
CONCLUSIONp21(WAF1) can inhibit the proliferation of leukemia cell and decrease its sensitivity to Vp16.
Antineoplastic Agents, Phytogenic ; pharmacology ; Blotting, Western ; Cell Division ; drug effects ; Cell Survival ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; genetics ; metabolism ; physiology ; Dose-Response Relationship, Drug ; Etoposide ; pharmacology ; G1 Phase ; drug effects ; Gene Expression ; Humans ; K562 Cells ; cytology ; drug effects ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Resting Phase, Cell Cycle ; drug effects ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors
3.Blockage of PI3K/PKB/P27kip1 signaling pathway can antagonize 17 beta-estradiol-induced Ishikawa proliferation and cell cycle progression.
Rui-xia GUO ; Li-hui WEI ; Yu-huan QIAO ; Jian-liu WANG ; Jian-min TANG
Chinese Medical Journal 2006;119(3):242-245
Apoptosis
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drug effects
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Cell Line, Tumor
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Cell Proliferation
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drug effects
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Chromones
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pharmacology
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Cyclin-Dependent Kinase Inhibitor p27
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antagonists & inhibitors
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physiology
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Endometrial Neoplasms
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drug therapy
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pathology
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Estradiol
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pharmacology
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Female
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G1 Phase
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drug effects
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Humans
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Intracellular Signaling Peptides and Proteins
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antagonists & inhibitors
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physiology
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Morpholines
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pharmacology
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Phosphatidylinositol 3-Kinases
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antagonists & inhibitors
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physiology
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Proto-Oncogene Proteins c-akt
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antagonists & inhibitors
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physiology
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Signal Transduction
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drug effects
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physiology
4.Telomerase activity,PI3K/AKT signaling pathway and cellular biological behavior in HeLa cell line.
Xiao-Xia YU ; Ying-Ai SHI ; Li-Hong ZHANG ; Yi-Shu WANG ; Shan WU
Chinese Journal of Pathology 2008;37(5):323-327
OBJECTIVETo investigate the telomerase activity and to document biological behaviors of HeLa cells upon treatment with specific PI3K/AKT signaling pathway inhibitor, LY294002.
METHODSCCK-8 assay was used to determine IC50 of LY294002. The expressions of total AKT and phosphorylation AKT (P-AKT) were determined using Western blot. Telomerase activity of cell was measured by TRAP-ELISA assay. Cell growth curve, flow cytometry technique and Hoechst33258 stain were used to evaluate the cell growth, cell cycle and apoptosis respectively. Cell migration was determined by cell wound healing assay.
RESULTSIC50 value of LY294002 of treated HeLa cells was 1.73 mg/L. Western blot showed that LY294002 enabled to decrease P-AKT activity in the presence of same total AKT protein. The cell telomerase activity was decreased to 36.72% in contrast to 98.61% of the control. LY294002 decreased the telomerase activity in HeLa cells, and the growth capacity of the cells was significantly suppressed. The number of cells at G0/G1 phases increased to 66.88% compared with that of the control cells (47.36%). The apoptosis rate also increased from 2.4% to 14.9%. The relative migration distance decreased to 24.6% compared with that of control (62.57%).
CONCLUSIONLY294002 inhibition of PI3K/AKT signaling pathway leads to alteration of telomerase activity along with changes of the biological behaviors of the HeLa cells suggesting that regulation of telomerase activity may be closely related to PI3K/AKT signaling pathway.
Apoptosis ; drug effects ; Blotting, Western ; Cell Line ; Cell Movement ; drug effects ; physiology ; Cell Proliferation ; drug effects ; Chromones ; pharmacology ; Enzyme Inhibitors ; pharmacology ; G1 Phase ; drug effects ; HeLa Cells ; Humans ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; Signal Transduction ; drug effects ; physiology ; Telomerase
5.Involvement of mitogen-activated protein kinases and p21Waf1 in hydroxyurea-induced G1 arrest and senescence of McA-RH7777 rat hepatoma cell line.
Seung Hee HONG ; Bum Sik HONG ; Dae Cheol KIM ; Mee Sook RHO ; Joo In PARK ; Soe Hee RHA ; Ho Sun JUN ; Jin Sook JEONG
Experimental & Molecular Medicine 2004;36(5):493-498
Hydroxyurea is commonly used to treat hematologic disorders and some type of solid tumors, but the mechanism for its therapeutic effect is not clearly known. In this study, we examined the effect of hydroxyurea on rat hepatoma McA-RH7777 cells, specifically, on the role of mitogen-activated protein (MAP) kinase signal transduction pathways and p21Waf1, p27Kip1 and p53. Rat hepatoma McA-RH7777 cells treated with hydroxyurea for 7 days, caused the inhibition of cell growth in a dose-dependent manner. But, this growth inhibition was not caused by necrosis or apoptosis but instead was associated with cell senescence-like change as evidenced by senescence associated-beta-galactosidase staining, and cells arrest at G1 phase of cell cycle. Phosphorylation of MAP kinases, such as ERK, JNK, and p38, was found to be decreased after treatment of cells with hydroxyurea. But, the expression of p21Waf1 was increased, while p27Kip1 and p53 were not detected in hydroxyurea treated rat hepatoma cells. Hydroxyurea treatment induced G1 arrest and a senescence-like changes in rat hepatoma McA-RH7777 cells may be the likely results of signal disruption of MAP kinases (ERK, JNK, and p38 MAP kinase) and p21Waf1 over-expression.
Animals
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Antineoplastic Agents/*pharmacology
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Cell Aging/drug effects
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Cell Cycle Proteins/analysis/metabolism/*physiology
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Cell Line, Tumor
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Cell Proliferation/drug effects
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G1 Phase/drug effects/physiology
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Hydroxyurea/*pharmacology
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Liver Neoplasms, Experimental/enzymology/*metabolism
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Mitogen-Activated Protein Kinases/analysis/*physiology
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Phosphorylation/drug effects
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Protein p53/analysis/metabolism
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Rats
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Research Support, Non-U.S. Gov't
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Tumor Suppressor Proteins/analysis/metabolism
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Up-Regulation
6.Dahuang zhechong pill containing serum inhibited platelet-derived growth factor-stimulated vascular smooth muscle cells proliferation by inducing G1 arrest partly via suppressing protein kinase C α-extracellular regulated kinase 1/2 signaling pathway.
Na LIU ; Jun-tian LIU ; Yuan-yuan JI ; Pei-pei LU
Chinese journal of integrative medicine 2012;18(5):371-377
OBJECTIVETo investigate effects of dahuang zhechong pill ( DHZCP) on the cell cycle and the related signal pathways in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF) with the method of serum pharmacology.
METHODSDNA synthesis in VSMCs was examined by detecting 5'-bromo-2'-deoxyuridine incorporation with the immunocytochemical method. The cycle of VSMCs was evaluated with flow cytometry. Expressions of cyclin D1, p27, protein kinase Cα (PKCα), and phosphorylated extracellular signal regulated kinase 1/2 (ERK1/2) were quantified by Western blot method.
RESULTSDHZCP containing serum significantly inhibited DNA synthesis of PDGF-stimulated VSMCs, arrested the cells in G G(1) phase, modulated the protein expressions of cyclin D D(1) and p27, and suppressed the activation of PKCα and ERK1/2.
CONCLUSIONDHZCP containing serum inhibits VSMCs proliferation via modulating the expressions of cell cycle proteins to arrest the cell in G G(1) phase, which is attributed to, at least in part, suppressing PKCα-ERK1/2 signaling in VSMCs.
Animals ; Aorta, Thoracic ; cytology ; Blood Proteins ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; DNA ; biosynthesis ; Drugs, Chinese Herbal ; pharmacology ; G1 Phase ; drug effects ; physiology ; MAP Kinase Signaling System ; physiology ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; enzymology ; Platelet-Derived Growth Factor ; pharmacology ; Protein Kinase C-alpha ; metabolism ; Rats ; Rats, Sprague-Dawley
7.Phosphatidylinositol 3-kinase inhibitor, LY294002, induced senescence-like changes in human diploid fibroblasts.
Shuping LI ; Zongyu ZHANG ; Tanjun TONG
Chinese Medical Journal 2003;116(6):901-905
OBJECTIVETo reveal the role of Phosphatidylinositol 3-kinases (PI3Ks) in regulating human diploid fibroblast (2BS cell) senescence as well as the possible mechanisms involved.
METHODSUsing a PI3Ks specific inhibitor, LY294002, cell cycle, apoptosis, proliferation, senescence association beta-galactosidase staining as well as senescence association CKIs, p16(INK4) and p21(Cip1) protein expressions were all measured in the low passages of 2BS cells.
RESULTSBoth 25 micro mol/L and 50 micro mol/L concentrations of LY294002 could cause a significant decrease in cells entering into S phase, and this cell cycle of G(1) phase arrest was dose-dependent. Meanwhile, LY294002 contributed to apoptosis, caused 2BS cell growth arrest, and activated senescence association beta-galactosidase (P < 0.05). In addition, LY294002 could induce time-course expressions of p16(INK4) and p21(Cip1) in 2BS cell lines.
CONCLUSIONSPI3Ks inhibitor LY294002 could induce senescence-like changes in 2BS cell lines. Two senescence associated CKIs, p16(INK4) and p21(Cip1), might be involved in this senescence phenotype proceeding in 2BS cell lines.
Cells, Cultured ; Cellular Senescence ; drug effects ; Chromones ; pharmacology ; Cyclin-Dependent Kinase Inhibitor p16 ; analysis ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; analysis ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Fibroblasts ; drug effects ; physiology ; G1 Phase ; drug effects ; Humans ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors
8.Changes in expression of cell cycle regulators after G1 progression upon repetitive thioacetamide treatment in rat liver.
Sook Hee HONG ; Gie Deug LEE ; Jun Young CHUNG ; Kyung Sook CHO ; Seok Hee PARK ; In Hoo KIM ; Jin Sook JEONG
Experimental & Molecular Medicine 2002;34(5):361-366
Repetitive low dose thioacetamide (TA) treatment of hepatocytes was found to induce cells in G2 arrest. In the present study, an attempt was made to investigate alterations in expression of cell cycle regulators after G1 progression in the same repetitive low dose TA treated hepatocytes system and to define the determinators involved in G2 arrest. TA was daily administered intraperitoneally, with a dose of 50 mg/kg for 7 days. Expression levels of cyclin E and CDK2 were similar, increased at day 1 and reached a peak at day 2. And they recycled from day 3 reaching a second peak at day 5. Expression level of cyclin A was similar to p27(Kip1) and p57(Kip2) but not to CDK2 and increased to a peak level at day 2. Expression levels of cyclin B1 and cdc2 were similar although the cyclin B1 level was generally low, decreased from day 1 to basal levels at day 3 and persisted at a low level till day 7. The expression level of cyclin G1 was similar to p53 that peaked at day 3 and again at day 6 elevated over basal level. BrdU-labeled hepatocytic nuclei increased from 12 h, reached a peak at day 2, then decreased, and were not detectable from day 6. The number of PCNA-labeled nuclei increased immediately, peaked at day 2, and maintained till day 7. These results suggest that G2 arrest induced by repeated TA treatment might be p53-dependent, via activation of cyclin G1, rather than inhibition of cyclin B1- cdc2 complex, and inhibitors holding S phase progression might be p27(Kip1) and p57(Kip2).
Animals
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Bromodeoxyuridine/metabolism
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CDC2 Protein Kinase/drug effects/metabolism
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*CDC2-CDC28 Kinases
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Cell Cycle/drug effects/*physiology
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Cell Cycle Proteins/drug effects/metabolism
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Cyclin-Dependent Kinases/antagonists & inhibitors/drug effects/metabolism
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Cyclins/drug effects/metabolism
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Dose-Response Relationship, Drug
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G1 Phase/drug effects/*physiology
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Liver/*drug effects/pathology
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Male
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Nuclear Proteins/drug effects/metabolism
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Proliferating Cell Nuclear Antigen/metabolism
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Protein p53/metabolism
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Protein-Serine-Threonine Kinases/antagonists & inhibitors/drug effects/metabolism
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Rats
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Rats, Sprague-Dawley
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Thioacetamide/administration & dosage/*pharmacology
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Tumor Suppressor Proteins/drug effects/metabolism
9.Sec13 induces genomic instability in U2OS cells.
Choong Ryoul SIHN ; Eun Jung SUH ; Kee Ho LEE ; Sang Hoon KIM
Experimental & Molecular Medicine 2005;37(3):255-260
Sec13p has been known as an endoplasmic reticulum-Golgi transport protein. Recently, it has also been shown to be required for the formation of septation in the fission yeast Schizosaccharomyces pombe. In the present study, we focused on the role of a human homolog of Saccharomyces cerevisiae SEC13, Sec13 protein during mitosis in U2OS cells. We found that the expression of Sec13 was constant throughout the cell cycle, and localized to the kinetochores at metaphase during mitosis. By using green fluorescent protein technology, we observed that Sec13 is required for evasion of mitotic arrest in response to spindle damage, leading to G1-like phase and apoptotic cell death. In addition, cells expressing exogenous Sec13 showed giant nuclei compared to endogenous ones in the absence of nocodazole. These results demonstrate that Sec13 is involved in the regulation of the metaphase/anaphase transition and may be functionally associated with mitotic machinery to maintain genomic stability during mitosis.
Anaphase
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Antineoplastic Agents/pharmacology
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Cell Line, Tumor/drug effects/metabolism/pathology
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*G1 Phase
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*Genomic Instability
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Green Fluorescent Proteins/metabolism
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Humans
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Kinetochores/metabolism
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Membrane Proteins/*genetics/metabolism
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Metaphase
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Mitosis/*physiology
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*Mitotic Spindle Apparatus
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Nocodazole/pharmacology
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Osteosarcoma/genetics/metabolism/pathology
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Research Support, Non-U.S. Gov't