OBJECTIVETo explore the possible mechanism(s) of taurine-inhibiting the proliferation of hepatic stellate cells (HSC), this study investigated the effect of taurine on the HSC cell cycle and its regulatory protein expression.

METHODSCell proliferation was assessed by MTT assay. Cell cycle was analyzed by flow cytometry. Cell cycle regulatory protein Cyclin D1 and P21waf1 expression were determined by immunocytochemistry and image-analysis system, and real-time quantitative PCR.

RESULTSHSC proliferation was markedly inhibited when HSC were treated with taurine at concentrations of 5, 10, 20, 30, 40 and 50 mmol/L for 48 hours, and the inhibition rates were 6.7%, 14.4%, 23.3%, 32.2%, 36.7% and 45.6% respectively (P < 0.05-0.01). In the flow cytometry analysis, it was found that taurine could block HSC in the G0/G1 phase from entering the S phase, resulting in more cells in the G0/G1 phase and fewer in the S phase. The percentage of the cells in the G0/G1 phase and the S phase at the dosage of 40 mmol/L were 68.2%+/-1.4% and 26.2+/-1.3% respectively, which was significantly different in comparison to the controls (56.2%+/-1.7% and 38.5%+/-0.8% respectively, P < 0.01). HSC expressed cyclin D1 and P21waf1. Taurine inhibited cyclin D1 expression and induced P21waf1 expression. The cyclin D1 protein and mRNA in the HSC treated with 40 mmol/L taurine were significantly reduced compared with the controls [protein (optical density value): 0.13+/-0.02 versus 0.18+/-0.02, P < 0.01; mRNA: 5776.7+/-3345.0 versus 18,400.6+/-1374.8 copies/10(6) GAPDH, P < 0.01]; and the P21waf1 protein and mRNA were markedly increased compared with the controls [protein (optical density value): 0.19+/-0.02 versus 0.14+/-0.01, P < 0.01; mRNA: 44,866.7+/-3910.7 versus 16,933.3+/-960.9 copies/10(6) GAPDH, P less than 0.05].

CONCLUSIONSCyclin D1 and P21waf1 were cell cycle regulatory proteins in HSC, and taurine can inhibit the HSC cyclin D1 expression and stimulate P21waf1 expression, facilitate arresting cells in G0/G1 phase, and suppress cell proliferation.

Animals ; Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Line ; Cell Proliferation ; Cyclin D1 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Depression, Chemical ; Hepatocytes ; cytology ; Rats ; Taurine ; pharmacology

To examine the effects of ischemia and anoxia on cell activation and cell cycle of astrocytes in vitro, the cell cycles and the proliferation of astrocytes in different time points after ischemia and anoxia were studied by flow cytometry and BrdU labeling and the expression of GFAP and cyclin D1 was detected by the fluorescence immunochemistry. After ischemia and anoxia in vitro, the astrocytes in S phase were significantly increased as compared with those in the normal group and the proliferating ability of the astrocytes was highest 6 h after the treatment as revealed by BrdU pulse labeling, but the astrocytes in S phase and proliferating ability were decreased after 6 h. At the early stages of ischemia and anoxia, the positive staining intensity of GFAP was increased, peaked at 6th h, while 12 h after the ischemia and anoxia, the positive staining intensity of GFAP became weak, and the expression of cyclin D1 was gradually increased after the ischemic and anoxic damage. It is concluded that astrocytes are activated to proliferate and enter new cycle events by ischemia and anoxia, and cyclin D1 is implicated in the proliferation and repair of astrocytes. The cell cycle events are closely associated with the proliferation and activation of astrocytes.
Animals, Newborn ; Astrocytes/*cytology ; Cell Cycle ; Cell Hypoxia ; Cell Proliferation ; Cells, Cultured ; Cyclin D1/biosynthesis ; Cyclin D1/genetics ; Glial Fibrillary Acidic Protein/*biosynthesis ; Glial Fibrillary Acidic Protein/genetics ; Rats, Wistar

OBJECTIVETo study the role of cyclin D1 in malignant transformation of human embryonic lung diploid fibroblasts (HELF) induced by quartz.

METHODSpXJ41-cyclin D1 expressing sense and antisense cyclin D1 RNA were transinfected into malignant transformed HELF induced by quartz with DNA recombination and gene transduction. The expression of cyclin D1 was detected with hybridization in situ and immunohistochemistry methods to analyze changes in cell growth, double multiplication time, distribution of cell cycles, colony forming ability on soft agar, etc., before and after cyclin D1 transduction.

RESULTSDuring the process of malignant transformation of HELF induced by quartz, cyclin D1 gene was overexpressed. Antisense pXJ41-cyclin D1 RNA could suppress the growth and proliferation of malignant transformed cells induced by quartz. Growth speed of antisense pXJ41-cyclin D1 transinfected cells decreased by 58.69% on the 8th day in culture, as compared to malignant transformed cells induced by quartz, and its double multiplication time prolonged from 21.0 h to 31.4 h. Antisense cyclin D1 RNA led to cell cycle arrest, resulting in lengthened G1 phase (proportion of cells in phase G1 increased to 52.7% from 45.1% and that of cells in phase S decreased to 33.1% from 40.3%). Colony forming rate reduced significantly and size of colony became smaller.

CONCLUSIONSAbnormal expression of cyclin D1 in cells related to their malignant transformation induced by quartz. Highly expressed cyclin D1 could play an important role in maintaining the transformed phenotype of malignant cells.

Cell Transformation, Neoplastic ; Cells, Cultured ; Cyclin D1 ; biosynthesis ; genetics ; Embryo, Mammalian ; Fibroblasts ; cytology ; Humans ; Lung ; cytology ; Quartz ; toxicity

The effects of retinoic acid on the beta-catenin/TCF pathway in cultured porcine tracheobronchial epithelial cells (TBEC) were investigated. After TBEC were treated with retinoic acid at various concentrations, mRNA and protein changes of beta-catenin in cytoplasm, nucleus and whole cell of the TBEC were observed by immunocytochemical stain, RT-PCR and Western blotting. And the changes of the target gene cyclinD1 of beta-catenin/TCF pathway were also observed. It was found that there was no significant difference in beta-cat mRNA level after retinoic acid treatment. However, the expression of beta-catenin in the whole cell and cytoplasm was elevated with the increase of retinoic acid concentration (P<0. 01). The nuclear protein beta-catenin and target gene cyclinD1 of beta-catenin/TCF pathway was decreased (P<0.05). It was indicated that retinoic acid could increase beta-catenin level of the whole cell protein and decrease nuclear beta-catenin, downregulating beta-cat/TCF signaling activity and reducing target gene cyclinD1 protein level. As a result, retinoic acid can downregulate beta-catenin/TCF pathway in porcine tracheobronchial epithelial cell, suggesting that retinoic acid can inhibit the proliferation and accelerate differentiation of tracheobronchial epithelial cells.
Animals ; Bronchi ; cytology ; metabolism ; Cells, Cultured ; Cyclin D1 ; biosynthesis ; genetics ; Epithelial Cells ; cytology ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Signal Transduction ; Swine ; TCF Transcription Factors ; biosynthesis ; genetics ; Trachea ; cytology ; metabolism ; Tretinoin ; pharmacology ; beta Catenin ; biosynthesis ; genetics

OBJECTIVETo construct the eukaryotic expression vectors of human cyclin D1 gene and express them in poorly differentiated nasopharyngeal carcinoma cells (CNE-2Z cells).

METHODSThe full-length cyclin D1 was cloned from CNE-2Z cells by RT-PCR. The cDNA fragments were inserted into pIRES2-EGFP plasmids and pEGFP-C2 plasmids and confirmed by restriction enzyme digestion, PCR and sequencing. The recombinant vectors were transfected into CNE-2Z cells via Lipofectamine 2000, and the expression of cyclin D1 in the cells was examined by immunofluorescence and Western blotting.

RESULTSAgarose gel electrophoresis showed a 918 bp band of the RT-PCR products, which matched the expected size. Restriction enzyme digestion, PCR and sequencing demonstrated successful construction of the recombinant vectors. CNE-2Z cells transfected with the recombinant vectors expressed cyclin D1 protein or cyclin D1-GFP protein as were verified by immunofluorescence and Western blotting.

CONCLUSIONWe have cloned cyclin D1 gene and constructed its eukaryotic expression vectors that can be expressed in nasopharyngeal carcinoma cells, which may facilitate the study of the role of cyclin D1 in the development of nasopharyngeal carcinoma.

Cell Line, Tumor ; Cloning, Molecular ; Cyclin D1 ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

Carcinoma, Hepatocellular ; microbiology ; pathology ; Cell Line, Tumor ; Cyclin D1 ; biosynthesis ; genetics ; Helicobacter pylori ; physiology ; Humans ; Liver Neoplasms ; microbiology ; pathology ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo investigate the feasibility of detecting cyclin D1 mRNA in paraffin-embedded tissues by reverse transcriptase polymerase chain reaction (RT-PCR) and competitive RT-PCR and its diagnostic and differential diagnostic significance for mantle cell lymphoma (MCL).

METHODSParaffin-embedded samples of 36 cases of MCL, 71 cases of other small B-cell lymphomas and 20 cases of lymphoid reactive hyperplasia as control group were retrieved from archival materials. Cyclin D1 protein and its mRNA was detected by EnVision and RT-PCR and competitive RT-PCR in all samples. House-keeping gene PGK was choosen as internal control.

RESULTS(1) Cyclin D1 protein was expressed in 27 of the 38 MCL (71.1%). No cyclin D1 expression was found in the control group. (2) PGK was detected in 103 of the 116 cases (88.8%) and also detected in 34 of 36 MCL cases (94.7%). (3) cyclin D1 mRNA was detected in 34 nodal mantle cell lymphoma cases by RT-PCR in paraffin-embedded tissues. The positive rate of cyclin D1 mRNA was 94.4% in mantle cell lymphomas after exclusion of the 2 cases which were negative for both cyclin D1 mRNA and PGK. cyclin D1 mRNA was not detected in other nodal small B-cell lymphomas or lymphoid reactive hyperplasia, except 1 case of B-SLL. Sequencing analysis showed that sequences were identical to cyclin D1. (4) Cyclin D1 mRNA overexpression was detected in 27 cases of nodal mantle cell lymphoma by competitive RT-PCR in paraffin-embedded tissues. The positive rate of cyclin D1 mRNA overexpression was 75.0% in mantle cell lymphomas after exclusion of 2 cases which were negative for both cyclin D1 mRNA and PGK. cyclin D1 mRNA overexpression was not detected in other nodal small B-cell lymphomas or lymphoid reactive hyperplasia.

CONCLUSIONRT-PCR and competitive RT-PCR detection of cyclin D1 mRNA overexpression could be used for the diagnosis and differential diagnosis of mantle cell lymphoma in paraffin-embedded blocks.

Cyclin D1 ; biosynthesis ; genetics ; Diagnosis, Differential ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; metabolism ; pathology ; Lymphoma, Follicular ; genetics ; metabolism ; Lymphoma, Mantle-Cell ; genetics ; metabolism ; pathology ; Paraffin Embedding ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods

OBJECTIVETo compare the effects of TCM therapeutic principles of tonifying Shen (TS), benefiting Qi (BQ), invigorating Pi (IP) and activating blood circulation (ABC) herbs in regulating the gene expression in senescence related cell cycle.

METHODSDrug sera containing TCM herbs of the above-mentioned principles were used to treat the aged human diploid fibroblast cell line 2BS. The effect of TCM on the senescence related cell cycle and its related gene expression (P16INK4, Cyclin D1 and PCNA) were examined by means of cell proliferative doublings, flow cytometry, RT-PCR and Western blot analysis.

RESULTSTCM herbs of TS and BQ could improve the cell cycle, down-regulate the P16 and Cyclin D1 mRNA/protein expression, up-regulate PCNA mRNA/protein expression, while TCM herbs of IP and ABC showed insignificant effect on these indexes.

CONCLUSIONTCM herbs of TS and BQ have effect in improving cell cycle, it may be achieved through promoting the P16 pathway of gene expression.

Aging ; genetics ; Animals ; Cell Cycle ; drug effects ; Cells, Cultured ; Cellular Senescence ; genetics ; Cyclin D1 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; genetics ; Down-Regulation ; Drugs, Chinese Herbal ; pharmacology ; Fibroblasts ; cytology ; Gene Expression Regulation ; Lung ; cytology ; Male ; Medicine, Chinese Traditional ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the role of cyclin D1 and p27(kip1) in the occurrence and development of conventional renal cell carcinoma(CRCC).

METHODSRT-PCR and Western-blot were used to detect mRNA and protein contents of cyclin D1 and p27(kip1) in 25 CRCCs and 10 normal renal tissue distant to tumor. Immunohistochemistry was used to investigate the expression of cyclin D1 and p27(kip1) in pathological tissue sections of 76 CRCCs. The relationship between those index and clinicopathological parameters was analyzed statistically.

RESULTIn CRCC, the expression of cyclin D1 was higher than that of the control group. The higher cyclin D1 content was related to big tumor size (P<0.05); The expression of p27(kip1) was lower than that of the control group, and the lower p27(kip1) was related to higher nuclear grade and TNM stage (P<0.01). The 5-year cumulative survival rate of the p27(kip1) high expression group is longer than that of the low group (P<0.01).

CONCLUSIONThe excessive expression of cyclin D1 and lower expression of p27(kip1) play an important role in the carcinogenesis of CRCC. The lower expression of p27(kip1) may affect the progression of the tumors. The detection of p27(kip1) may be as a reference marker in the prognosis of CRCC.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Blotting, Western ; Carcinoma, Renal Cell ; genetics ; metabolism ; pathology ; Cyclin D1 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p27 ; biosynthesis ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Kidney Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

The effects of over-expression of testis-specific expressed gene 1 (TSEG-1) on the viability and apoptosis of cultured spermatogonial GC-1spg cells were investigated, and the immortal spermatogonial cell line GC-1spg (CRL-2053™) was obtained as the cell model in order to explore the function of TSEG-1. We transfected the eukaryotic vector of TSEG-1, named as pEGFP-TSEG-1 into cultured spermatogonial GC-1spg cells. Over-expression of TSEG-1 inhibited the proliferation of GC-1spg cells, and arrested cell cycle slightly at G0/G1 phase. Transfection of TSEG-1 attenuated the transcript levels of Ki-67, PCNA and cyclin D1. In addition, over-expression of TSEG-1 induced early and late apoptosis, and reduced the mitochondrial membrane potential of GC-1spg cells. Moreover, transfection of TSEG-1 significantly enhanced the ratio of Bax/Bcl-2 and transcript levels of caspase 9, and decreased the expression of Fas and caspase 8 in GC-1spg cells. These results indicated over-expression of TSEG-1 suppresses the proliferation and induces the apoptosis of GC-1spg cells, which establishes a basis for further study on the function of TSEG-1.
Animals ; Caspase 8 ; biosynthesis ; genetics ; Cell Line ; Cyclin D1 ; biosynthesis ; genetics ; G1 Phase ; physiology ; Histones ; genetics ; metabolism ; Ki-67 Antigen ; biosynthesis ; genetics ; Male ; Mice ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; Resting Phase, Cell Cycle ; physiology ; Spermatogonia ; cytology ; metabolism ; bcl-2-Associated X Protein ; biosynthesis ; genetics