Good progress has been made in the researches on correlating genes of breast cancer in recent years. Quite a few kinds of genes such as susceptibility gene, oncogene and tumor suppressor genes have been found with implications for diagnosis, therapy and prognosis. Abnormality of breast cancer susceptibility gene (BRCA) is of great significance, especially in the development of breast cancer.
BRCA1 Protein ; genetics ; BRCA2 Protein ; genetics ; Breast Neoplasms ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; genetics ; Female ; Humans ; Mutation ; Neoplasm Proteins ; genetics ; Proto-Oncogene Proteins ; genetics ; Tumor Suppressor Protein p53 ; genetics

OBJECTIVETo investigate the biological function of RIG-G gene by establishing a cell line stably expressing RIG-G protein.

METHODSEctopic RIG-G gene was transfected into U937 cells by using Tet-off expression system. Changes before and after RIG-G expression were detected for cell growth, cell morphology, cell surface antigen and cell cycle regulating proteins.

RESULTSRIG-G protein arrested the cells at G0/G1 phase and inhibited cell growth by increasing the cell cycle inhibitors P21 and P27. As compared to that in control group, the proportion of cells at G0/G1 phase in RIG-G-expressing cell group increased from (43.9 +/- 5.6)% to (63.9 +/- 2.3)% (P < 0.01). The rate of growth inhibition was (68.7 +/- 0.2)%. In addition, an increase in CD11C expression [(61.3 +/- 1.1)% vs. (18.0 +/- 0.4)% (P < 0.01)] and in cells with morphologic features of partial differentiation (smaller cell size, reduced nucleus-cytoplasm ratio, notched nucleus and coarse chromatin) was also observed in RIG-G-expressing cells.

CONCLUSIONSRIG-G has potential abilities to inhibit cell proliferation and promote cell differentiation.

Cell Cycle ; genetics ; Cell Differentiation ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; physiology ; Plasmids ; genetics ; Transfection ; U937 Cells

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

OBJECTIVETissue kallikrein cleaves kininogen substrate to produce vasoactive kinin peptides that have been implicated in the proliferation of vascular smooth muscle cells. We investigated the effects of adenovirus-mediated human tissue kallikrein (Ad-hKLK1) gene delivery on the proliferation of vascular smooth muscle cells of SHR (VSMCs(SHR)) induced by platelet derived growth factor-BB (PDGF-BB).

METHODSPrimary VSMCs(SHR) were isolated and cultured from thoracic aorta of male SHR. The VSMCs(SHR) proliferation induced by PDGF-BB was accessed by cell counting and methyl thiazolyl tetrazolium (MTT). Western blot was used to determine the protein expression of hKLK1, the cycle-independent kinase inhibitors p27(Kip1) and p21(Cip1). The mRNA expressions of bradykinin B1 receptor and B2 receptor were detected by RT-PCR in VSMCs(SHR).

RESULTSProliferation of VSMCs(SHR) induced by PDGF-BB was significantly inhibited post transfection of Ad-hKLK1 (20-100 MOI) in a MOI-dependent manner. The peak inhibition titer of Ad-hKLK1 was 100 MOI with peak inhibition rate of 39.3% (cell counting, n = 3, P < 0.01), 30.2% (MTT, n = 3, P < 0.01) and 36.4% (peak stunning rate of cell-cycle in phase G(0)/G(1)). The inhibitory effects of proliferation and cell-cycle caused by hKLK1 gene delivery could be abolished by Hoe140, a bradykinin B2 receptor antagonist. The protein expression of p27(Kip1) and p21(Cip1) increased significantly after the hKLK1 gene delivery, whereas Hoe140 nearly completely blocked these effects (n = 3, P < 0.001, respectively). PDGF-BB also significantly upregulated the mRNA expression of B2 receptor but not B1 receptor in VSMCs(SHR).

CONCLUSIONThe hKLK1 gene delivery could inhibit PDGF-BB induced proliferation in VSMCs(SHR) through Bradykinin B2 receptor and up-regulate expression of p27(Kip1) and p2l(Cip1).

Animals ; Cell Division ; drug effects ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Humans ; Kallikreins ; genetics ; pharmacology ; Male ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Rats ; Rats, Inbred SHR ; Recombination, Genetic

OBJECTIVETo observe the effects of N-acetylcysteine (NAC) on aging in neonatal SD rat cardiomyocytes and explore related mechanisms.

METHODSCultured cardiomyocytes were randomized assigned to 6 groups: 1-day, 5-day, 10-day, 1-day + NAC (1 mmol/L), 5-day + NAC (1 mmol/L) and 10-day + NAC (1 mmol/L). Flow cytometry was used to examine cell cycle. Real-time quantitative PCR and Western blot were used to determine mRNA and protein expression of p16INK4a, p21WAF1 and Rb gene. beta-galactosidase staining kit was used to investigate beta-galactosidase activity.

RESULTSNumbers of cardiomyocytes resided in G(0)/G(1) phase were significantly higher in the group of 5-day + NAC and 10-day + NAC compared with 5-day, 10-day, respectively (P < 0.05). The mRNA and protein expression of p16INK4a and p21WAF1 were also significantly higher in the group of 5-day + NAC and 10-day + NAC compared with 5-day, 10-day, respectively (P < 0.05 or P < 0.01). The mRNA and protein expression of Rb was significantly lower in the group of 5-day + NAC and 10-day + NAC compared with 5-day, 10-day, respectively (P < 0.01). beta-galactosidase activity was not affected by NAC in the 1-day + NAC group but was significantly higher in 5-day + NAC and 10-day + NAC groups compared with the 5-day, 10-day groups (all P < 0.05).

CONCLUSIONNAC could promote aging through upregulating the expression of p16INK4a and p21WAF1 and inhibiting Rb phosphorylation in neonatal SD rat cardiomyocytes.

Acetylcysteine ; pharmacology ; Animals ; Cell Cycle ; Cells, Cultured ; Cellular Senescence ; drug effects ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Myocytes, Cardiac ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

Carcinoma, Hepatocellular ; pathology ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Humans ; Liver Neoplasms ; pathology ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; Transfection ; Tumor Cells, Cultured ; Xeroderma Pigmentosum ; genetics

OBJECTIVETo study the p53/p21 fusion gene as a potential fusion gene for the gene therapy of human oral squamous cell carcinoma.

METHODSp21 cDNA was obtained from normal human embryonic lung cells by RT-PCR, fusing with p53 gene. The recombinant plasmid pcDNA-p53/p21 was constructed by inserting the p53/p21 fusion gene into eukaryotic expression vector pcDNA3.1 and subsequently transfected into human oral squamous cell carcinoma cell line (Tca8113) with lipofectamine. RT-PCR and Western blot were used to demonstrate the expression of p53/p21 fusion gene. Using clonal formation experiment and (3)H-TdR incorporation assay were used to evaluate the clonal formation and proliferation ability of Tca8113 cells.

RESULTSIt was observed that p53/p21 fusion gene could inhibit clonal formation and proliferation of human oral carcinoma. RT-PCR and Western blot demonstrated that it was the expression of exogenous p53/p21 fusion gene that led to the above results.

CONCLUSIONSTransfection of p53/p21 fusion gene to Tca8113 cells could inhibit the tumor cell proliferation and clone formation in vitro, and make itself a potential fusion gene for the gene therapy of human oral squamous cell carcinoma.

Carcinoma, Squamous Cell ; therapy ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Gene Fusion ; genetics ; Genes, p53 ; genetics ; Genetic Therapy ; Humans ; Mouth Neoplasms ; therapy

OBJECTIVETo study the effects of arsenic trioxide (As(2)O(3)) on cell cycle and expression of cyclin dependent kinase inhibitors (CDKIs) in multiple myeloma (MM) cells, and explore its pharmacological mechanism.

METHODSThe DNA content of MM cells line HS-Sultan was analyzed by flow cytometry after exposure to As(2)O(3), the effects on expression of CDKI P15, P16 AND P21 were studied by reverse transcriptase PCR.

RESULTSDNA flow cytometric analysis showed that As(2)O(3) induced most of HS-Sultan cells, arrest at G(0)/G(1) phase and a small fraction at G(2)/M phase and apoptosis occurred mainly in S phase. There was no expression of P15 and P16 mRNA in untreated HS-Sultan cells and 1.0 micromol/L As(2)O(3) could make them expressed after exposed 24 or 48 hours respectively. Expression of P12 mRNA was obviously elevated by As(2)O(3) comparing with that of control.

CONCLUSIONOne of the pharmacological mechanisms of As(2)O(3) is to activate the expression of CDKI P15, P16 and P21, and consequently affect cell proliferation cycle.

Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Cell Cycle ; drug effects ; physiology ; Cyclin-Dependent Kinase Inhibitor p15 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Humans ; Multiple Myeloma ; drug therapy ; metabolism ; pathology ; Oxides ; pharmacology ; RNA, Messenger ; genetics ; Tumor Cells, Cultured

OBJECTIVETo study the regulatory effect of TGF-beta1 on growth of rhabdomyosarcoma RD cell line.

METHODSAfter various durations of TGF-beta1 treatment, the viability of RD cell line was examined by growth rate measurement, MTT assay and (3)H-thymidine incorporation. The cell cycle was analyzed by flow cytometry. Immunofluorescent staining was used to localize p15, p21 and p27 in RD cell line under laser scanning confocal microscope. The protein and mRNA of p15, p21 and p27 in RD cell line were detected by western blot and reverse transcriptase-polymerase chain reaction respectively.

RESULTSThe viability of RD cell line treated with TGF-beta1 was obviously decreased. RD cell line was arrested in G(1) phase by TGF-beta1. There was increased expression of p21 and p27 in RD cell line with TGF-beta1 treatment at protein and mRNA levels. The expression of p21 in RD cell line was seen in both nucleus and cytoplasm after 24 hours of TGF-beta1 treatment. The expression of p15 showed no obvious changes upon TGF-beta1 treatment.

CONCLUSIONSTGF-beta1 inhibits growth of RD cell line and induces G(1)-arrest. It up-regulates protein and mRNA of p21 and p27 and shows no obvious influence on p15 expression. The growth arrest of RD cell line may result from the up-regulation of p21 and p27 by TGF-beta1.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p15 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p27 ; biosynthesis ; genetics ; G1 Phase ; drug effects ; Humans ; RNA, Messenger ; biosynthesis ; genetics ; Rhabdomyosarcoma ; pathology ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo clarify the role of RhoC in the growth of hepatocellular carcinoma cells and its molecular mechanism, so as to explore the molecular target of tumor cell growth.

METHODSsiRNA-RhoC plasmid was constructed and RhoC gene silencing the cell-line of hepatocellular carcinoma was setup. Cell growth was assessed by MTT assay. AgNORs staining was applied to determine cell proliferation. Plate cell clone test was conducted to examine the capacity of cell clone formation. FACS was adopted to measure the course of cell cycle and semi-quantitative RT-PCR was used to determine the expression of cell cycle proteins. In order to further determine the effect of RhoC expression on cell growth, a RhoC over-expression human hepatocellular cell line was setup by PcDNA3-RhoC plasmid transfection.

RESULTSThe inhibition rate of RhoC was 82.3%. From the fourth day of cell culture, the growth of cells in RNAi group was significantly slower than that in parental Bel7402 and negative control groups (0.41 ± 0.10 vs. 0.73 ± 0.11 and 0.71 ± 0.07 respectively, P < 0.05). AgNORs staining showed that average cell stained particles in RNAi group was significantly lower than that in parental Bel7402 and negative control(1.23 ± 0.35 vs. 3.47 ± 0.93 and 3.17 ± 0.78, P < 0.01). Plate clone formation test showed that clone formation efficiency in the RNAi group was notably lower than that in the control group [(20.33 ± 5.42)% vs. (70.58 ± 10.10)% and (69.83 ± 14.77)%, respectively, P < 0.01]. Cell cycle analysis by FACS showed that G(0)/G(1) cell percentage in the RNAi group was significantly higher than that in the control group [(73.14 ± 5.93)% vs. (57.05 ± 5.97)% and (52.99 ± 4.80)%, P < 0.05]. Compared with Bel7402 and negative control groups, the expression of following growth associated genes was significantly decreased: cyclin D1(0.45 ± 0.21 vs. 1.25 ± 0.24 and 1.12 ± 0.15, respectively, P < 0.05)and CDK4 (0.55 ± 0.08 vs. 1.18 ± 0.32 and 1.10 ± 0.29, respectively, P < 0.05); the following genes were notably increased: p16(1.07 ± 0.23 vs. 0.36 ± 0.12 and 0.35 ± 0.13, respectively, P < 0.01)and p21(0.42 ± 0.12 vs. 0.17 ± 0.06 and 0.19 ± 0.08, respectively, P < 0.05). RhoC was highly expressed in PcDNA3-RhoC transfected hepatocellular cell line. From the third day on of the cell culture, cell growth in PcDNA3-RhoC group was remarkably higher than that in the HL7702 and PcDNA3 groups (0.83 ± 0.10 vs. 0.54 ± 0.11 and 0.58 ± 0.55, respectively, P < 0.05).

CONCLUSIONSRhoC is the key molecule in promoting hepatocellular cell growth, and is a promising target for tumor cell growth controlling.

Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Humans ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Plasmids ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection ; rho GTP-Binding Proteins ; genetics ; metabolism ; rhoC GTP-Binding Protein