OBJECTIVETo study mechanisms of reduction of the malignant activities of human naso-pharyngeal carcinoma cell CNE-2L2 induced by ectopic expression of BCSC-1 gene.

METHODSDNA was stained with propidium iodide and assayed upon a flow cytometer. Chromosomes were stained with Hoechest 33258. Adhesion of CNE-2L2 cells was detected by cell aggregation test. Protein expression on CNE-2L2 cells was examined by Western blot.

RESULTSCell cycle analysis showed that the percentage of CNE-2L2 cells was 55.1%, 43.4%, and 39.4% in G0/G1 phase, 25.2%, 28.7%, and 30.9% in S phase, and 19.7%, 27.9%, and 29.7% in G2/M phase for the cell with ectopic expression of BCSC-1 gene, wild type cell (W cells), and the cell transduced with the mock (M cell). Many mitotic cells were found in W cells and M cells. In contrast, almost no mitotic cell was observed in the cells with ectopic expression of BCSC-1 gene. Ectopic BCSC-1 expression resulted in cell aggregation, enhanced expression of E-cadherin, cx-catenin, and p53.

CONCLUSIONSEctopic BCSC-1 expression causes enhancement of adhesion of CNE-2L2 cells associated with enhanced expression of E-cadherin and alpha-catenin, arrest of cell in G1 phase, which may be associated with enhanced expression of p53. These alteration may play a role in the reduction of malignant activities of the cells with ectopic expression of BCSC-1 gene.

Cell Adhesion ; Cell Cycle ; physiology ; Cell Line, Tumor ; Humans ; Nasopharyngeal Neoplasms ; Neoplasm Proteins ; biosynthesis ; genetics

The study was purposed to investigate the effect of small interference RNA (siRNA) targeting Polo-like kinase 1 (PLK1) gene on cell cycle progression, proliferation and drug resistance in K562/A02 cells. siRNA plasmid vector specifically targeting PLK1 gene with enhanced green fluorescence protein (EGFP) was transfected into K562/A02 cells. Expressions of PLK1 mRNA and protein were assayed by RT-PCR and Western-blot; cell proliferation was evaluated by direct cell counting after trypan blue staining. Cell cycle and intracellular adriamycin (ADM) accumulation was determined by flow cytometry; 50% inhibition concentration (IC50) of ADM on K562/A02 cells was determined by MTT method. The results showed that, as compared with control cells, siRNA plasmid reduced PLK1 mRNA expression by (34.7 +/- 2.1)% for 24 hours and by (56.6 +/- 1.5)% for 48 hours, PLK1 protein significantly decreased simultaneously by (49.9 +/- 3.2)% and by (62.1 +/- 1.7)%. After being transfected for 24 and 48 hours, the rate of survival cells decreased by 30% and 59% respectively. Forty-eight hours after transfection, the ratio of K562/A02 cells at G2/M increased by 2.77-fold, at the same time, intracellular ADM accumulation increased and the relative efficiency of K562/A02 cells to ADM was 73.8%. It is concluded that PLK1 gene silence can inhibit K562/A02 cell proliferation, induce cell cycle arrest at G2/M, and increase intracellular ADM accumulation, so that enhance cell sensitivity to ADM.
Cell Cycle ; Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Proliferation ; Daunorubicin ; pharmacokinetics ; Drug Resistance, Neoplasm ; genetics ; Gene Silencing ; Humans ; K562 Cells ; Protein-Serine-Threonine Kinases ; biosynthesis ; genetics ; Proto-Oncogene Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; pharmacology

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 construct the recombinant plasmid pcDNA3.0-RGC32 and evaluate the effect of the response gene to complement-32 (RGC32) on cell cytoskeleton in vitro.

METHODSThe full-length cDNA of RGC32 was obtained by RT-PCR and inserted into the eukaryotic expression vector pcDNA3.0 to generate the recombinant plasmid pcDNA3.0-RGC32. After transfection of the recombinant plasmid into SW480 cells, the expression of RGC32 in the cells was detected by Western blotting. The cytoskeleton of SW480 cells was visualized before and after the transfection, and the changes in the cell migration ability was assessed by wound-healing assay.

RESULTSThe recombinant plasmid pcDNA3.0-RGC32 was successfully constructed. The expression of RGC32 was significantly increased in SW480 cells after transfection with pcDNA3.0-RGC32. Before the transfection, the microfilaments of SW480 cells were few and short without obvious polarity, but after the transfection, the microfilaments were increased and elongated with also an obvious polarity, and the invasive structures of lamellae and lamellipodia occurred. The migration ability of the cells was enhanced after transfection with pcDNA3.0-RGC32.

CONCLUSIONOverexpression of RGC32 can cause the reorganization of cytoskeleton and promotes the cell migration, which can be an important mechanism of RGC32 in promoting cancer metastasis.

Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Line, Tumor ; Cell Movement ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Cytoskeleton ; chemistry ; metabolism ; Genetic Vectors ; Humans ; Muscle Proteins ; biosynthesis ; genetics ; Neoplasm Metastasis ; genetics ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

Ex vivo maintainance of human stem cells is crucial for many clinical applications. Current culture conditions provide some level support but cytokines induce most quiescent stem cells to proliferate and differentiate. Better control of primitive cells is needed to extend the time and range of manipulation of such cells. A recently identified plant lectin Flt3 receptor-interacting lectin (FRIL) present may a special ability to preserve primitive CB progenitors for extended periods in culture without exogenous cytokines. But the mechanisms of FRIL preserving quiescent primitive cells are still unknown. Recently a novel protein HTm4 and its alternatively spliced variant HTm4S, which serve as hematopoietic cell cycle regulators, have been identified. In this report we studied the effect of FRIL on the in vitro maintenance of quiescent human cord blood stem cells and the expression of the novel hematopoietic cell cycle regulator HTm4 and HTm4S in progenitor cells cultured in FRIL. We analyzed the proliferation and the HPP-CFC proportion of CD34(+) cells treated with FRIL. The human HTm4 and HTm4S mRNA expression was detected by semi-quantitative RT-PCR, and the cell cycle status of CB CD34(+) cells was analyzed by FACS. The results showed that incubation of CD34(+) cells in FRIL resulted in a low proliferation of progenitor cells and fewer cycling cells, but FRIL selectively maintained a higher number of primitive cells with proliferative potential in suspension culture. CB CD34(+) cells cultured in FRIL showed significant diversity in the expression of HTm4 and HTm4S during 0~14 d. On d 0, HTm4 was detected at high level, downregulated on d 1, but upregulated during d 3 to d 14, and reaching the highest level on d 7. But the expression levels of HTm4S changed little in the cells cultured in FRIL except the obviously increased expression on d 7. Exogenous expression showed that HTm4 was localized around the karyon while HTm4S scatted in the cytoplasm, respectively, which may be responsible for their difference in function. Thus, FRIL can preserve quiescent primitive CD34(+), and FRIL's ability to preserve quiescent primitive cells in a reversible manner may significantly expand the time and range of ex vivo manipulations of human stem cells for clinical applications. In other words, HTm4 and HTm4S may play a crucial role in the cell cycle modulation of CD34(+) progenitor cells maintained with FRIL in vitro.
Antigens, CD20 ; biosynthesis ; genetics ; Antigens, CD34 ; metabolism ; Cell Cycle ; Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Separation ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Mannose-Binding Lectins ; pharmacology ; Membrane Proteins ; biosynthesis ; genetics ; Plant Lectins ; pharmacology

Intimal hyperplasia is defined as the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) with deposition of extracellular matrix. However, the cell cycle regulatory mechanisms of injury-induced VSMC proliferation are largely unknown. To examine the expression kinetics of cell cycle regulatory factors which is known to be worked positively or negatively, we used rat balloon injury model. Marked induction of proliferating cell nuclear antigen (PCNA), G1/S cyclin-dependent kinase (cdk2), and its regulatory subunit (cyclin E) occurred between 1 and 3 days after balloon arterial injury, and this was sustained for up to 7 days and then declined. However, the induction of the negative regulators, p21 and p27, occurred between 3 and 5 days of injury, peaked after 7 and 14 days and was then sustained. VSMC proliferation after balloon catheter injury of the rat iliac artery is associated with coordinated expression of positive (cdk2, cyclin E and PCNA) and negative (p21, p27) regulators. Cell cycle regulators such as cdk2, cyclin E, p21, p27 may be suitable targets for the control of intimal hyperplasia.
Animals ; Arteries/*pathology ; Balloon Dilatation/*adverse effects ; Blotting, Western ; CDC2-CDC28 Kinases/biosynthesis ; Cell Cycle ; Cell Cycle Proteins/biosynthesis ; Cell Division ; Cyclin E/biosynthesis ; Cyclins/biosynthesis ; Endothelium, Vascular/pathology ; Extracellular Matrix/metabolism ; Hyperplasia/pathology ; Iliac Artery/pathology ; Immunohistochemistry ; Male ; Myocytes, Smooth Muscle/*cytology ; Proliferating Cell Nuclear Antigen/biosynthesis ; Rats ; Rats, Sprague-Dawley ; Support, Non-U.S. Gov't ; Time Factors ; Tumor Suppressor Proteins/biosynthesis

OBJECTIVETo study effects of alternative transcripts of ING1 transfection on human cancer cell lines.

METHODSp47/ING1A and p33/ING1B expression vehicles were constructed and introduced into a human breast cancer cell line MCF-7 and a human lung cancer cell line PAa, both expressing wild-type p53 protein. Growth characteristics of the transfectants and potentially related genes were analyzed.

RESULTSThe levels of p47/ING1A and p33/ING1B protein elevated respectively in tumor cells of MCF-7 and PAa after transfected with p47/ING1A and p33/ING1B, and the latter was much higher than that of the former. Ectopic overexpression of p33/ING1B effectively blocked tumor cell growth and arrested cells in the G(0) approximately G(1) phase of the cell cycle (P < 0.01), while p47/ING1A gave no effect on cell growth or cell cycle. Tumor cells overexpressing p33/ING1B contained more p21(WAF1) protein than that of the control cells, with undisturbed p53 protein level.

CONCLUSIONSExpression of two different transcripts of ING1 may have different effects on tumor cell growth. p33/ING1B may cooperate with p53 in stimulating expression of p21(WAF1) gene, thus to arrest cell cycle and to inhibit tumor cell growth. p33/ING1B may be considered to be a candidate as a partner of p53 in gene therapy.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Alternative Splicing ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Cycle ; Cell Cycle Proteins ; Cell Division ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; biosynthesis ; DNA-Binding Proteins ; Genes, Tumor Suppressor ; Humans ; Inhibitor of Growth Protein 1 ; Intracellular Signaling Peptides and Proteins ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Nuclear Proteins ; Protein Biosynthesis ; Proteins ; genetics ; Transfection ; Tumor Suppressor Protein p53 ; biosynthesis ; Tumor Suppressor Proteins

To study the mechanisms involved in the inhibition of chronic myeloid leukemic cells (K562) proliferation induced by arsenic trioxide (As2O3) and to explore the potential role of Survivin, an inhibitor of apoptosis protein, in the regulation of As2O3 induced cell apoptosis, K562 cells were cultured with As2O3 of different concentrations. Cells were collected for proliferation analysis by MTT assay. Cell cycle distribution and cell apoptosis were analyzed by flow cytometry. Expression of Survivin protein and mRNA were detected by flow cytometry and RT-PCR, respectively. Our results showed that As2O3(2-10 micromol/L) inhibited K562 cells growth effectively, but it did not induce cells apoptosis significantly. The percentage of K562 cells at G2/M phase increased in proportion to As2O3 concentrations, and the expression of Survivin mRNA and content of Survivin protein was up-regulated accordingly. It is concluded that As2O3 inhibited K562 cells growth by inducing cell cycle arrest mainly at G2/M phase. Over-expression of Survivin gene and protein might be one of the possible mechanisms contributing to K562 cells' resistance to As2O3-induced apoptosis.
Antigens, Neoplasm ; biosynthesis ; genetics ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Humans ; Inhibitor of Apoptosis Proteins ; K562 Cells ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; Oxides ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo study the expressions of p53 and Gadd45a proteins and their clinicopathological significance in human pancreatic cancer.

METHODSThe expression of p53 and Gadd45a proteins was detected with immunohistochemistry in a series of 59 pancreatic cancers. Their relationships with the clinicopathological parameters including gender, tumor site, TNM stage, histological differentiation, and the prognosis of pancreatic cancer patients were analyzed.

RESULTSThe positive expression rate of p53 protein was 67.8% (40/59) and that of Gadd45a protein was 42.4% (25/59). The positive expression rate of p53 protein is significantly higher in patients < 65 years than in patients > or = 65 years (chi squared = 4.711, P = 0.030). Gadd45a expression was not correlated to the age of the patients. No significant difference was found between the expression of p53 proteins and histological differentiation and TNM stage of the tumors. Gadd45a expression was correlated with histological differentiation of pancreatic cancer (chi squared = 10.052, P = 0.007), but not with TNM stage of the tumors. No significant differences in the prognosis were found between the groups with and without p53 expression (chi squared = 0.09, P = 0.764) and the groups with and without Gadd45a expression (chi squared = 0.14, P = 0.704).

CONCLUSIONSBoth p53 and Gadd45a are highly expressed in human pancreatic cancer and may be associated with biological features of pancreatic cancer. Their expression alone or co-expression may be not helpful to evaluate the prognosis of patients with pancreatic cancer.

Adult ; Aged ; Biomarkers, Tumor ; biosynthesis ; Carcinoma, Pancreatic Ductal ; metabolism ; pathology ; Cell Cycle Proteins ; biosynthesis ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Neoplasm Staging ; Nuclear Proteins ; biosynthesis ; Pancreatic Neoplasms ; metabolism ; pathology ; Tumor Suppressor Protein p53 ; biosynthesis

OBJECTIVETo explore the inhibitory effects of gene expression and functional activity of telomerase in leukemia cell lines by in vitro antisense hTERT treatment.

METHODSAn antisense hTERT eukaryotic expression vector was constructed by using gene recombination technique, targeting the 5' end mRNA sequence of the telomerase catalytic subunit. The vector expression in leukemia cell lines (HL60 and K562) was achieved by transfection using the SuperFect transfection reagent (Qiagen). After transfection, ectopic expression of the telomerase catalytic subunit was analyzed by quantitative fluorescence real-time RT-PCR, and cellular apoptosis and cell cycle parameters were evaluated by flow cytometry respectively.

RESULTSAn antisense pcDNA-hTERT eukaryotic expression vector was successfully constructed. Leukemia cell lines transfected with antisense hTERT constructed displayed a significant inhibition of gene expression of telomerase and its activity in vitro, as compared with the result of the control groups (without transfection and vector control).

CONCLUSIONIn-vitro antisense hTERT expression may down-regulate the gene expression and biological activity of telomerase in leukemia cells, suggesting a possibility of gene therapy against human malignancy through the telomerase-targeted molecular mechanism.

Apoptosis ; Cell Cycle ; DNA-Binding Proteins ; biosynthesis ; genetics ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; HL-60 Cells ; HeLa Cells ; Humans ; K562 Cells ; RNA, Antisense ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Telomerase ; biosynthesis ; genetics ; metabolism ; Transfection