OBJECTIVETo observe the effect of recombinant adenovirus-mediated wild-type p53 gene on the number and proteins of centrosome in K562 cells. To explore the possibility of application of wild-type p53 gene therapy in the treatment of chronic myeloid leukemia.

METHODSThe recombinant adenoviruses carrying wild-type p53 gene (Ad5 wtp53), mutant p53 gene (Ad5 mtp53) or the green fluorescent protein (GFP) gene was repeatedly amplified and co-infected into K562 cells with cation polybrene. The optimal infection titer and infection time of the recombinant adenoviruses were determined by MTT assay, p53 mRNA and protein expression were determined by RT-PCR and Western blot respectively. The centrosomal structural protein gamma-tubulin and the spindle protein alpha-tubulin were marked simultaneously by indirect immunofluorescence staining, and the expression of the centrosomal gamma-tubulin protein, the mitosis and the number of centrosome were observed under the laser confocal microscopy.

RESULTSInfection efficiency with recombinant adenoviruses was facilitated by polybrene in K562 cells, and 4 microg/ml polybrene was chosen. The optimal adenovirus infection titer was 20,000 MOI and the optimal infection time was 72 hours. p53 mRNA and P53 protein can be expressed in K562 cells by Ad5wtp53 and Ad5mtp53. Both the expression of the centrosomal gamma-tubulin protein and the number of centrosomes were decreased after Ad5wtp53 infection.

CONCLUSIONThere is sustained expression of P53 protein in K562 cells after its infection by Ad5wtp53. Wild-type P53 protein can lead to the down-regulation of the number of centrosomes and the expression of centrosomal gamma-tubulin protein in K562 cells.

Adenoviridae ; genetics ; Centrosome ; metabolism ; Genes, p53 ; genetics ; Genetic Vectors ; Humans ; K562 Cells ; Transfection ; Tubulin ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo elucidate the possible role of p53-p21(waf1) pathway for centrosome amplification in oral squamous cell carcinoma (OSCC).

METHODSFormalin-fixed, paraffin-embedded tissues of 8 cases of normal oral epithelium tissues and 27 cases of OSCC tissues were examined for the expression of p21(waf1) and mutated p53 proteins by flow cytometry and immunohistochemistry, and centrosome status was investigated by indirect immunofluorescence double staining with antibodies to centrosome protein gamma-tubulin and cytokeratin. The correlation between p21(waf1), p53 and centrosome amplification in OSCC was statistically analyzed by SPSS 12.0.

RESULTSAll normal oral epithelium tissues showed normal centrosomes (1-2 centrosomes per cell)in epithelium cells, while 21 out of 27 cases (78%) of OSCC showed the evidence of centrosome amplification characterized by supernumerary centrosomes ( >2 centrosomes per cell) in a fraction of tumor cells. The quantity of p21(waf1) protein was lower in OSCC with centrosome amplification [(0.878 +/- 0.081)] than that in OSCC without centrosome amplification [(0.952 +/- 0.018), t = 3.838, P < 0.01], and negative correlations were found between the quantity of p21(waf1) protein and the degree of centrosome amplification (r = -0.472, P < 0.05), as well as the positive staining of p53 (r = -0.491, P < 0.01).

CONCLUSIONSp53-p21(waf1) pathway might involve in centrosome duplication cycle in OSCC. Down-regulated p21(waf1) protein, via p53 transactivation-dependent mechanism, was likely a contributing factor towards centrosome amplification in OSCC.

Carcinoma, Squamous Cell ; metabolism ; pathology ; Centrosome ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Humans ; Mouth Neoplasms ; metabolism ; pathology ; Tumor Suppressor Protein p53 ; genetics ; metabolism

OBJECTIVETo analyze the centrosome abnormalities in the malignant transformation of human bronchial epithelial cells (BEAS-2B) induced by coal tar pitch smoke extracts and to investigate the role and action mechanism of centrosome in the lung cancer induced by coal tar pitch.

METHODSMedium-temperature coal tar pitch smoke extracts were used to treat immortalized human bronchial epithelial cells (BEAS-2B) and establish a malignant transformation model. The treated BEAS-2B cells were used as exposure group, and solvent control group and normal control group were also set for passage culture. The changes of centrosome in BEAS-2B cells seeded on coverslips were evaluated by indirect immunofluorescence assay. The mRNA expression of p53, p21, and cyclin E in BEAS-2B cells was measured by real-time quantitative RT-PCR, and their protein levels in BEAS-2B cells seeded on coverslips were measured by semiquantitative immunohistochemical analysis.

RESULTSThe overall rate of centrosome abnormalities in BEAS-2B cells at passage 20 was 6.56±1.01% in the exposure group, significantly higher than those in the normal control group (3.40±0.86%) and solvent control group (3.14±0.59%) (P < 0.05). In addition, the exposure group had a significantly higher overall rate of centrosome abnormalities in BEAS-2B cells at passage 30 compared with the normal control group and solvent control group (22.39±9.5% vs 4.34±1.04%, P < 0.05; 22.39±9.5% vs 4.33±1.20%, P < 0.05). Compared with the normal control group and solvent control group, the exposure group had significantly decreased mRNA and protein expression of p53 and significantly increased mRNA and protein expression of cyclin E in BEAS-2B cells at passages 20 and 30 (P < 0.05).

CONCLUSIONCentrosome abnormalities occur before the malignant transformation in BEAS-2B cells treated with coal tar pitch smoke extracts, and they may be mediated by the p53/p21/cyclin E signaling pathway.

Cell Line ; Cell Transformation, Neoplastic ; metabolism ; pathology ; Centrosome ; metabolism ; pathology ; Coal Tar ; Cyclin E ; metabolism ; Epithelial Cells ; cytology ; metabolism ; Humans ; Signal Transduction ; Smoke ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo construct a mammalian expression plasmid of the BC022687 gene and investigate the expression and localization of the fusion protein in Chinese hamster ovary (CHO) cells.

METHODSThe BC022687 coding sequence was amplified by polymerase chain reaction (PCR) and subcloned into the pEGFP-C1 vector carrying the gene of green fluorescence protein (GFP). After the target region was sequenced, the recombinant plasmid was transfected into CHO cells, and its expression in the CHO cells was determined by Western blot. The localization of GFP-tagged BC022687 in the CHO cells was observed by laser scanning confocal microscopy.

RESULTSBC022687 was successfully cloned into the mammalian expression vector pEGFP-C1, with the restriction fragment length of 950 bp. The expression of the fusion protein was confirmed, with the relative molecular weight of 64 000. The GFP-tagged BC022687 protein was mainly localized in the cytoplasm, and also presented in the centrioles in the transfected CHO cells.

CONCLUSIONThe successful construction of the plasmid expressing BC022687 in CHO cells has laid a foundation for further studies on the role of this protein in ciliogenesis.

Animals ; CHO Cells ; Centrosome ; metabolism ; Cilia ; metabolism ; Cricetinae ; Cricetulus ; DNA, Complementary ; Genetic Vectors ; Male ; Mice ; Plasmids ; Recombinant Fusion Proteins ; genetics ; Transfection

OBJECTIVETo study the correlation between cyclin E protein overexpression and centrosome amplification in oral squamous cell carcinoma (OSCC).

METHODSFormalin-fixed, paraffin-embedded tissues from 12 normal oral epithelium cases and 46 cases of OSCC were studied. Their centrosome status was analyzed by indirect immunofluorescence double staining with antibodies to centrosome protein gamma-tubulin and cytokeratin. The expression of cyclin E protein was studied by immunohistochemical methods. The correlation between cyclin E protein expression and centrosome amplification in OSCC was statistically analyzed by SPSS 12.0.

RESULTSThirty-seven of the 46 OSCC cases (80.4%) studied showed evidence of centrosome amplification, as signified by enlargement and/or increase in number of centrosomes, while normal oral epithelium possessed centromeres of normal size and number. Positive staining for cyclin E protein was observed in 30 of the 46 OSCC cases (65.2%), while all the normal oral epithelium cases were cyclin E protein-negative. The percentage of centrosome amplification in OSCC with positive cyclin E protein staining (90.0%, 27/30) was higher than that in OSCC with negative cyclin E protein staining (62.5%, 10/16) (chi(2) = 5.014, P < 0.05). Centrosome amplification showed positive correlation with cyclin E protein overexpression (r = 0.330, P < 0.05).

CONCLUSIONUp-regulation of cyclin E protein may represent one of the possible mechanisms for centrosome amplification in OSCC.

Carcinoma, Squamous Cell ; metabolism ; pathology ; surgery ; Centrosome ; pathology ; ultrastructure ; Cyclin E ; metabolism ; Epithelium ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Microscopy, Confocal ; Mouth Mucosa ; metabolism ; pathology ; Mouth Neoplasms ; metabolism ; pathology ; surgery ; Up-Regulation

OBJECTIVETo study interaction between a novel centrosomal protein TACP1 and mitotic kinase Nek2A.

METHODSNek2A305-446 protein was expressed and purified in E.coli and TACP1 protein was expressed in transfected 293T cells. Pull-down assay was used to examine the interaction between Nek2A305-446 and TACP1. TACP1 and Nek2A complex was tested by co-immunoprecipitation assay with polyclonal anti-TACP1 antibody. The localization of those two proteins in Hela cells was verified by immunofluorescence.

RESULTSTACP1 was pulled down by Nek2A305-446 protein but not by GST control. Nek2A was co-precipitated with TACP1 protein by polyclonal anti-TACP1 antibody but not by pre-immunization serum. The Immunofluorescence test showed that these two proteins formed a complex at centrosome during mitosis.

CONCLUSIONCentrosomal protein TACP1 is a novel interacting protein with Nek2A, both of which are localized in centrosome during mitosis.

Cell Line ; Centrosome ; metabolism ; Escherichia coli ; genetics ; Fluorescent Antibody Technique ; HeLa Cells ; Humans ; Immunoprecipitation ; Mitosis ; NIMA-Related Kinases ; Protein Binding ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Telomere-Binding Proteins ; genetics ; metabolism ; Transfection

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.
Amino Acid Motifs/physiology ; Amino Acid Sequence ; Antigens, Neoplasm/chemistry/*metabolism ; Cells, Cultured ; Centrosome/*metabolism ; Fluorescent Antibody Technique ; Humans ; Leucine Zippers/*physiology ; Molecular Sequence Data ; Mutation ; Nuclear Proteins/chemistry/*metabolism ; Protein Conformation ; Protein Structure, Tertiary ; Sequence Analysis, Protein

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.
Cell Line ; Cell Nucleus/*drug effects/*metabolism ; Centrosome/*drug effects/*metabolism ; Gene Amplification ; Humans ; Hydrogen Peroxide/*pharmacology ; MAP Kinase Signaling System ; Mitotic Spindle Apparatus/drug effects ; Phenotype ; Reactive Oxygen Species/metabolism ; Research Support, Non-U.S. Gov't

Antineoplastic Agents ; therapeutic use ; BRCA2 Protein ; metabolism ; Breast Neoplasms ; drug therapy ; metabolism ; pathology ; Cell Cycle Proteins ; metabolism ; physiology ; Centrosome ; metabolism ; Drug Screening Assays, Antitumor ; Female ; Humans ; Microtubule-Associated Proteins ; metabolism ; Neoplasm Invasiveness ; Nuclear Proteins ; metabolism ; Phosphorylation ; Prognosis ; Protein-Serine-Threonine Kinases ; metabolism ; physiology ; Proto-Oncogene Proteins ; metabolism ; physiology ; RNA, Small Interfering ; pharmacology ; Tumor Suppressor Protein p53 ; metabolism