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14-3-3e and 14-3-3c localize to the centrosome and regulate centrosome duplication, by inhibiting cdc25C function. As14-3-3c and 14-3-3e form a complex with centrosomal proteins, we asked if this ability was required to regulate centrosomeduplication. The results in this report demonstrate that 14-3-3e and 14-3-3c form a complex with Centrin2 and that thebinding site is located in the N-terminal EF hand in Centrin2, EF1. A Centrin2 mutant that does not form a complex with14-3-3 proteins displays a punctate cytoplasmic localization and does not localize to the centrosome. These results suggestthat in addition to negatively regulating centrosome duplication as previously reported, 14-3-3 proteins might also berequired for centriole biogenesis by regulating the localization of Centrin2 at the centrosome.
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Objective To explore the influence of miR-200 c on the biological behavior of laryngeal carcinoma Hep-2 cells and determine whether miR-200 c exerts its biological function through peptidyl-prolyl cis/trans isomerase (PIN1) in laryngeal carcinoma. Methods A qRT-PCR assay for the expression of miR-200 c was performed in laryngeal carcinoma tissues. Hep-2 cells were transfected with miR-200 c related small RNAs. Transwell assay detected the migration ability of the cells. Immunofluorescence assay was used to detect the abnormal amplification of the centrosome. A dual luciferase reporter gene system was used to detect the binding ability between miR-200 c and PIN1. Western blotting detected the protein expression level of PIN1. Results The expression of miR-200 c in laryngeal carcinoma was significantly increased. miR-200 c inhibited the migration of Hep-2 cells and could weaken the abnormal amplification of centrosome.PIN1 was confirmed as one of the target genes of miR-200 c. miR-200 c inhibited the expression of PIN1 at the translation level and could inhibit Hep-2 cell migration and abnormal centrosome amplification by regulating PIN1. Conclusion miR-200 c can inhibit the migration ability of laryngeal carcinoma cells and abnormal centrosome amplification by regulating PIN1.
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Oligoasthenozoospermia, teratozoospermia or low sperm motility is the main cause of male infertility. Low sperm motility can be induced by abnormalities of the sperm tail structure and sperm function. The outer dense fiber protein 2 (ODF2) is a protein fiber maintaining cytoskeleton, as a major component of the mammalian sperm tail and centrosome, and its abnormality is closely related to asthenospermia. Recent studies indicate that ODF2 includes many proteins of the same name and homologous splices located in the sperm centrosomes and spindles of cleaved-embryos, necessary for animal ciliogenesis and associated with sperm capacitation. The features of ODF2 indicate that it is not a single-structural protein. This paper reviews the known functions of ODF2, paving a ground for further studies of the relationship between the ODF2 protein and fertilization.
Subject(s)
Animals , Humans , Male , Asthenozoospermia , Azoospermia , Centrosome , Chemistry , Cytoskeleton , Chemistry , Heat-Shock Proteins , Physiology , Infertility, Male , Sperm Motility , Physiology , Sperm Tail , Spermatozoa , PhysiologyABSTRACT
Objective To explore the Cep70 by adjusting the stability of acetylated alpha tubulin,participate in breast cancer drug resistance mechanisms.Methods (1) In order to induce taxol drug resistance cell line Michigan cancer foundation-7 (MCF-7)/pac,high-dose shock treatments taxol MCF-7 was used for 6 months,until the cells can grow in 3.5 μmol/L of paclitaxel.(2) The 3-(4,5-dimenthylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method was used to detect inhibition rate by taxol to MCF-7 and MCF-7/pac cell.(3) Immunofluorescence and Western blot were used to test acetylated alpha-tubulin and Cep70 expression levels in MCF-7 and MCF-7/pac cells.(4) Chemical intervention was used to acetylate apha-tubulin expression,Western blot and polymerase chain reaction (PCR) were used to detect the change of acetylated alpha-tubulin and Cep70 in MCF-7 and MCF-7/pac groups.Flow cytometry and Western blot were used to detect the change of cell cycle.Results (1) IC50 of MCF-7 and MCF-7/pac was 22.47 μ mol/L and 31.38 μmol/L,respectively.(2) Immunofluorescence and Western blot results showed that the expression of acetylation of alpha-tubulin in resistant MCF-7 cell/pac was obviously decreased.(3) Real time polymerase chain reaction (RT-PCR) and Western blot showed Cep70 expression was consistent of acetylation of alpha-tubulin.(4) After incubation with paclitaxel for 24 hours,the expressions of acetylation of alpha-tubulin and Cep70 in MCF-7 and MCF-7/pac were increased,but the extent of MCF-7 cell was much higher.Instead,incubation with nocodazole after 24 hours,the acetylation of alpha-tubulin and Cep70 in MCF-7 and MCF-7/pac cells were obviously lowered.(5) After paclitaxel intervention,compared to the same group MCF-7 cells,the G2 phase ratio in MCF-7/pac cells was lower.In addition,given nocodazole after the intervention,compared to the same group MCF-7 cells,the ratio of G2 phase in MCF-7 cell/pac was significantly decreased.Conclusions Cep70 decreased the expression of the acetylated alpha-tubulin,reduced the stability of microtubules,which could be an important mechanism of taxol drug resistance.
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Aurora A plays a key role in cellular mitosis. It is located in the centrosome and spindle, and is mainly involved in the processes of centrosome maturation and separation, bipolar spindle assembly, and the regulation of mitotic progression. Recent studies have suggested that Aurora A is involved in tumorigenesis and tumor development through multiple mechanisms. Overexpression of Aurora A could cause abnormal centrosome amplification, aneuploidy formation, and G2/M checkpoint defects, which result in chromosome instability and imbalance between cell division and apoptosis, and eventually leads to abnormal cell proliferation. Aurora A also participates in the regulation of the p53 and BRCA1 pathways, leading to suppressor gene dysfunction and changes in cell viability, and it induces telomerase activity by upregulating c-Myc, resulting in tumorigenesis. In addition, Aurora A also induces drug resistance in liver cancer cells. Thus, Aurora A has gradually become a new target for cancer therapy in recent years. This paper has summarized the recent studies on Aurora A, and reviewed its biological functions in cell mitosis and roles in liver tumorigenesis.
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Objective The objectives of this study were to investigate the expression of Aurora-A pro-tein in cervical cancer and precancerous lesions,and its relationship with human papilloma virus( HPV) infection, and to analyze the role of Aurora-A in the pathogenesis of cervical cancer. Methods One hundred cases of cer-vical biopsy or surgical resection specimens were collected from high-risk HPV( HR-HPV) test. There were 20 cases of normal cervical tissues,20 cases of CIN grade 1 ( CIN1 ) ,20 cases of CIN grade 2 ( CIN2 ) ,20 cases of CIN grade 3(CIN3),and 20 cases of cervical squamous cell carcinoma. The expression of Aurora-A protein was detected by immunohistochemistry and the correlation between Aurora-A expression and HR -HPV infection was analyzed. Results Aurora-A was highly expressed in cervical intraepithelial neoplasia and cervical cancer (P<0. 05),and its positive expression rate increased with the degree of cervical lesions. There was a positive correlation between Aurora-A expression and cervical cancer(r=0. 475,P<0. 001). There was a positive cor-relation between Aurora-A expression and HR-HPV infection in CIN2 and CIN3(V=0. 591,P<0. 05). Con-clusion Aurora-A may be associated with the development of cervical cancer. Aurora-A can be used as an important biomarker for the early diagnosis of cervical intraepithelial neoplasia or cervical cancer. It is also a po-tential therapeutic target for cervical cancer.
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Cell polarity is a common feature of many different types of cells,and it is essential to the normal differentiation and function of cells. Partitioning defective 6( PAR6)gene encodes PAR6 protein, which is crucial to asymmetric cell division and polarized growth. PAR6 protein as a member of the PAR6 polarity complex,affects the synthetic of centrosome and protein recruitment to the centrosome. The abnormal number of centrosomal and the loss of cell polarity may eventually lead to the occurrence of tumor.
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The hyperamplification in centrosomes give rise to a series of biochemical events including formation of multipolar spindle, abnormal segregation of chromosome and aneuploid, promoting the performance of chromosome instability, which is the main pathogenesis of multiple human malignancies.STIL participates in the formation of centrioles, activates CDK1/CyclinB1 complex and promotes mitotic entry.Moreover, it regulated expression of related downstream genes through Sonic Hedgehog signal pathway.Thus, STIL correlates with gastric and pancreatic carcinogenesis and progression.We aim to review the structure and function of STIL, association with malignancies and its potential mechanism.
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BACKGROUND: Mutations in centrosomal protein genes have been identified in a number of genetic diseases in brain development, including microcephaly. Centrosomal P4.1-associated protein (CPAP) is one of the causal genes implicated in primary microcephaly. We previously proposed that CPAP is essential for mother centriole maturation during mitosis. METHODS: We immunostained CPAP-depleted cells using the ninein antibody, which selectively detects subdistal appendages in mature mother centrioles. RESULTS: Ninein signals were significantly impaired in CPAP-depleted cells. CONCLUSION: The results suggest that CPAP is required for mother centriole maturation in mammalian cells. The selective absence of centriolar appendages in young mother centrioles may be responsible for asymmetric spindle pole formation in CPAP-depleted cells.
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Humans , Brain , Cell Cycle , Centrioles , Centrosome , Microcephaly , Mitosis , Mothers , Spindle PolesABSTRACT
BACKGROUND: Hyperdiploid pre‑B‑cell acute lymhoblastic leukemia (pre‑B‑ALL) is a common form of childhood leukemia with very good prognosis with present day chemotherapy. However, the chromosomal composition of the hyperdiploidy has not been extensively studied and possible mechanism for this pathology remains so far conjectural. OBJECTIVE: To analyze the pattern of chromosome involvement in a cohort of childhood hyperdiploid pre‑B‑ALL from India and from this pattern to develop an understanding on the causation of such pathology. Whether such patients also carry translocations and FLT3 mutations in addition to their hyperdiploid karyotype. MATERIALS AND METHODS: One hundred and twenty‑six childhood pre‑B‑ALL patients were studied. Bone marrow aspirate of these patients were evacuated for morphology, FAB classification, immunophenotyping and both conventional and molecular cytogenetics. RESULTS: Of 126 patients with pre‑B‑ALL (age 2-15 years), 90 patients with abnormal karyotype showed 50 with hyperdiploid karyotype (50/90 i.e. 55.5%). Chromosomes 9, 10, 14, 17, 18, 20 and 21 were more often involved in hyperdiploidy. Chromosome 21 duplication was present in 92% of the cases. Chromosomes 5, 15, 16, 17 and Y were less often involved (18-20%) in hyperdiploidy. About 44% of patients with hyperdiploidy had additional karyotypic abnormality of which TEL‑AML1 was predominant (24%). Chromosome loss was rare and accounted for 20% of the cases only. We did not find any FLT3 mutation in our patients. CONCLUSION: In this study, the pattern of chromosome involvement in hyperdiploid karyotype of ALL patients is same as other studies except some chromosomes like 1, 6, 11, 12, 19 and 22 have some more frequent involvement than other studies. This study also showed the occurrence of TEL/AML1 fusion is more (19.8%) than other reports from India.
Subject(s)
Centrosome/pathology , Child , Chromosomes/genetics , Cytogenetics/methods , Female , Humans , India/epidemiology , Male , Mitosis/abnormalities , Mitosis/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Uniparental Disomy/geneticsABSTRACT
Objective: This study proposed a possible molecular mechanism underlying centrosome amplification in oral squa-mous cell carcinoma (OSCC) by analyzing the relationship of centrosome amplification with the expression of STK15 and p53 in OS-CC tissues. Methods: The expression of STK15 and p53 in formalin-fixed, paraffin-embedded tissues from 8 patients with normal oral epithelium and 43 with OSCC were quantitatively analyzed by immunohistochemistry. Centrosome status was investigated by an indi-rect-immunofluorescence double-staining method to determine the message of centrosome amplification in OSCC. The correlation of the expression of the two proteins with centrosome amplification in OSCC was statistically analyzed with SPSS13.0. Results: Normal oral epithelium showed normal centrosomes in epithelium cells, whereas 33 of the 43 OSCC cases (76.74%) showed evidence of centro-some amplification. STK15 was undetectable in normal oral epithelium. The percentage of STK15 overexpression was 67.44% in OS-CC (P=0.028). The percentage of STK15 overexpression was significantly higher in OSCC with positive p53 staining than in OSCC with negative p53 staining (P=0.01). Spearman correlation analysis indicated a correlation between STK15/p53 positive co-expression and centrosome amplification of OSCC (P=0.019). Conclusion: Centrosome amplification is a common abnormal phenomenon in OS-CC. The p53/STK15 trans-activation-independent pathway plays a role in the systemic molecular regulation of centrosome in OSCC, which leads to the occurrence of OSCC.
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Objective To explore the molecular mechanism of BRAFV600E inducing chromosome instability in Sbcl2 and SK-MEL31 melanoma cells.Methods The endogenous Mps1 in stable Sbcl2-and SK-MEL31-B-RafV600E expression cells were depleted by siRNA approach.To test the effect of B-RafV600E on the centrosome amplification and the formation of multipolar spindles,cells at S-phase with HU-treatment were arrested and then the centrosomes and mitotic spindles structure were detected through immunofluoresence.Results The percentage of B-RafV600E expressing Sbcl2 and SK-MEL31 cells (Sbcl2-B-RafV600E and SKMEL31-B-RafV600E) with centrosome amplification and multipolar spindle was reduced from 36 ％ to 6 ％ when Mps1 was absent.Conclusion B-RafV600E leads to centrosome amplification and multipolar spindle through Mps1,thus results in chromosome instability in Sbcl2 and SK-MEL31 melanoma cells.
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The centrosome plays a crucial role in the maintaining of cell conformation,mitosis and chromosome seg-regation.It responds to the DNA damage and keeps the genome stability via cross-talking with the intranuclear DNA damage repair system.The apoptosis of tumor cells can be induced through inhibition of the duplication of centro-some.So the inhibitors of centrosomal proteins will be a potential anti-tumor therapy.
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Objective To study the effect of wild type p53 gene on centrosome hyperamplification in bladder cancer cells.Methods A wild type p53 gene recombinant adenovirus vector AdCMVp53 was constructed,and then trangfected into the human bladder cancer cell line T24.The cells were stained with the monoclonal antibody against pericentrin by indirect immunofluorescence method.The change of centrosome hyperamplification was observed under the fluorescence microspcope.Results Introduction of wild type p53 could suppress the centrosome amplification of T24 cell line.Conclusion p53 might play an important role in the regulation of centrosome hyperamplification.The loss of p53 might be one of the mechanisms involved in chromosome instability and contribute to the genesis and development of the bladder carcinoma.
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This study was conducted to evaluate the microtubule distribution following control of nuclear remodeling by treatment of bovine somatic cell nuclear transfer (SCNT) embryos with caffeine or roscovitine. Bovine somatic cells were fused to enucleated oocytes treated with either 5 mM caffeine or 150 micrometer roscovitine to control the type of nuclear remodeling. The proportion of embryos that underwent premature chromosome condensation (PCC) was increased by caffeine treatment but was reduced by roscovitine treatment (p < 0.05). The microtubule organization was examined by immunostaining beta- and gamma-tubulins at 15 min, 3 h, and 20 h of fusion using laser scanning confocal microscopy. The gamma-tubulin foci inherited from the donor centrosome were observed in most of the SCNT embryos at 15 min of fusion (91.3%) and most of them did not disappear until 3 h after fusion, regardless of treatment (82.9-87.2%). A significantly high proportion of embryos showing an abnormal chromosome or microtubule distribution was observed in the roscovitine-treated group (40.0%, p < 0.05) compared to the caffeine-treated group (22.1%). In conclusion, PCC is a favorable condition for the normal organization of microtubules, and inhibition of PCC can cause abnormal mitotic division of bovine SCNT embryos by causing microtubule dysfunction.
Subject(s)
Animals , Female , Male , Pregnancy , Caffeine/pharmacology , Cattle/embryology , Cell Nucleus/drug effects , Fertilization in Vitro/veterinary , Microscopy, Confocal/veterinary , Microtubules/drug effects , Nuclear Transfer Techniques/veterinary , Oocytes/physiology , Purines/pharmacologyABSTRACT
Objective:To explore the relation of the abnormal condition of centrosome and p53 genetic mutation of bladder cancer cells,its effect on the onset and progress of the transitional cell carcinoma of bladder because of p53 genetic mutation. Methods: 76 transitional cell carcinoma of bladder were examined.The centrosome was stained by indirect immunofluorescence methods,and the primary antibody was monoclonal antibody against pericentrin.The p53 was stained by SP immunohistochemical methods and the primary antibody was monoclonal antibody against p53.Results: Through statistical analysis,detection of CH or p53 expression was useful in providing prognostic information in bladder cancer. There existed a positive correlated(r=0.707,P
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Objective:To investigate the expressions of ?-tubulin,?-tubulin and ?-tubulin of centrosome in immortalized hu-man squamous epithelial cel(lH8)and cervical cancer cel(lCaski)and the role of them in occurrence and development of cer-vical cancer.Methods:Immunocytochemistry was used to analyze the protein expressions of ?-tubulin and ?-tubulin in im-mortalized human cervical cells and carcinoma cells.The skeleton protein was extracted by ProteoExtract Subcellular Proteome Extraction Kit,and then Western blotting was used to determine the protein expressions of ?-tubulin,?-tubulin and ?-tubulin semi-quantitatively.Results:The protein expressions of ?–tubulin,?-tubulin and ?-tubulin in Caski were higher than those in H8(P
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KM-HN-1 is a C-terminal coiled-coil domain containing protein previously referred to as image clone MGC33607. This protein has been previously identified as a cancer/testis antigen and reported as nuclear and chromatin localizing protein. We raised polyclonal antisera with the GST fusion protein and identified them as a 105 kDa protein. Motif analysis showed that this protein harbors the leucine zipper motif in internal 1/3 region and the coiled-coil domain in the C-terminal region. Using the full length and various deletion mutants, we determined the motif that governs the subcellular localization of KM-HN-1. Immunofluorescence staining of the endogenous KM-HN-1 and various kinds of GFP-tagged KM-HN-1 revealed that KM-HN-1 localizes to the centrosomes as well as nucleus. The centrosomal localization-determining region of this protein is C-terminal coiled-coil domain in which the leucine zipper motif and the nuclear export signal (NES) harbor.
Subject(s)
Humans , Amino Acid Motifs/physiology , Amino Acid Sequence , Antigens, Neoplasm/chemistry , Cells, Cultured , Centrosome/metabolism , Fluorescent Antibody Technique , Leucine Zippers/physiology , Molecular Sequence Data , Mutation , Nuclear Proteins/chemistry , Protein Conformation , Protein Structure, Tertiary , Sequence Analysis, ProteinABSTRACT
Multinucleated cells resulted from mitosis defect have been noted in pathophysiological states of the cells such as inflammation, senescence and cancer. Since oxidative stress has been known to correlate with these pathophysiological conditions, we tested the effect of H2O2 on the cell cycle progression and formation of multinucleated cells. H2O2 induced a significant delay in cell cycle progression in Chang liver cells. Interestingly, H2O2 actively induced hyperamplification of centrosomes (> or =3) and multipolar spindle formation during mitosis and subsequently increased the generation of multinucleated cells. A significant increase of the phospho-ERK level was observed upon H2O2 treatment but PD98059, an MEK1/2 inhibitor, didn't reduce the frequency of cells with hyperamplified centrosomes. On the other hand, treatment of either H2O2 or adriamycin increased intracellular ROS levels and multinucleated cells, which were significantly suppressed by antioxidants, N-acetylcysteine and PDTC. Thus, our results suggest that oxidative stress can trigger centrosome hyperamplification and multinucleated cell formation, which may promote pathophysiological progression.
Subject(s)
Humans , Cell Line , Cell Nucleus/drug effects , Centrosome/drug effects , Gene Amplification , Hydrogen Peroxide/pharmacology , MAP Kinase Signaling System , Spindle Apparatus/drug effects , Phenotype , Reactive Oxygen Species/metabolismABSTRACT
Objective To compare and analyze the difference of ?-tubulin and aurora-A expression in human cervical cancer cells (CasKi ) and immortalized human cervical squamous H8 cells with positive HPV16 E6E7.Methods Difference of ?-tubulin and aurora-A expression in CasKi and H8 cells was analyzed by showing the fluorescence intensity of ?-tubulin with indirect immunofluorescence.Expression level of aurora-A mRNA was detected by RT-PCR.Expression level of ?-tubulin and aurora-A in CasKi and H8 cells was semi-quantitatively analyzed by Western blot.Results The immunofluorescence signal of ?-tubulin was stronger in Caski cells than in H8 cells (57.78?3.13 vs 37.37?2.37,P