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

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

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 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 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 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

This paper focuses on the effect of the venom of the spider Macrothele raveni on the proliferation of human hepatocelluar carcinoma cell line HepG2 and the related molecular mechanism. XTT test showed that the proliferation of HepG2 cells in vitro was inhibited by the spider venom (P<0.05) in a concentration-dependent manner. By using flow cytometry, it was found that the spider venom caused selective G(2)/M cell cycle arrest in HepG2 cells. RT-PCR and Western blot indicated the expressions of p21 mRNA and protein in HepG2 cells were obviously up-regulated by the spider venom. The venom of the spider Macrothele raveni inhibited the proliferation of HepG2 cells. These results suggest that the possible mechanism of the spider venom is to activate the expressions of p21 gene and protein and to cause selective cell cycle arrest at G(2)/M phase, leading to HepG2 cell apoptosis.
Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Hep G2 Cells ; Humans ; RNA, Messenger ; genetics ; metabolism ; Spider Venoms ; pharmacology

BACKGROUNDMutation and expression change of p21WAF1/CIP1 may play a role in the growth of osteosarcoma. This study was to investigate the expression of the p21WAF1/CIP1 gene in human osteosarcoma, p21WAF1/CIP1 gene DNA sequence change and their relationships with the phenotype and clinical prognosis.

METHODSp21WAF1/CIP1 gene in 10 normal people and the tumours of 45 osteosarcoma patients were examined using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with silver staining. The PCR product with an abnormal strand was sequenced directly. The p21WAF1/CIP1 gene mRNA and P21 protein of 45 cases of osteosarcoma were investigated by using in situ hybridization and immunohistochemistry, respectively.

RESULTSThe occurrence of P21 protein in osteosarcoma was 17.78% (8/45), and p21WAF1/CIP1 mRNA expression in osteosarcoma was 42.22% (19/45). The p21WAF1/CIP1 gene DNA sequencing of amplified production showed that in p21WAF1/CIP1 gene exon 3 of 36 cases of human osteosarcoma, there were 17 cases (47.22%) with C-->T at position 609; 10 normal blood samples' DNA sequence analysis yielded 8 cases (80.00%) with C-->T at the same position.

CONCLUSIONSAlong with the increase of malignancy, the expression of p21WAF1/CIP1mRNA and P21 protein in osteosarcoma tends to decrease. It is uncommon for the p21WAF1/CIP1 gene mutation to occur in human osteosarcoma. As a result, the possible existence of tumour subtypes of p21WAF1/CIP1 gene mutation should be investigated. Our research leads to the location of p21WAF1/CIP1 gene polymorphism of Chinese osteosarcoma patients, which can provide a basis for further research.

Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; genetics ; Humans ; Osteosarcoma ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; RNA, Messenger ; analysis ; Sequence Analysis, DNA