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

Adult ; Aged ; Biomarkers, Tumor ; biosynthesis ; genetics ; Carcinoma, Hepatocellular ; metabolism ; Cell Cycle Proteins ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p27 ; Female ; Humans ; Liver Cirrhosis ; metabolism ; Liver Neoplasms ; metabolism ; Male ; Middle Aged ; Retinoblastoma Protein ; biosynthesis ; genetics ; Tumor Suppressor Proteins ; biosynthesis ; genetics

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

This study was purposed to investigate the significance of mitosis checkpoint gene chfr expression in acute leukemia (AL). 2 ml of bone marrow were extracted from each of 46 AL patients and 10 normal donors as control and their mononuclear cells were isolated. Then, their chfr expression was detected by using RT-PCR and immunohistochemistry. Normal control blood samples were also analyzed. The results showed that in 15 out of 28 cases of acute non-lymphocytic leukemia and 13 out of 18 cases of acute lymphocytic leukemia expression of chfr gene mRNA and protein significantly decreased as compared with control. The cytogenetic analysis of patients with a decreased Chfr expression revealed abnormal chromosome. In conclusion, Chfr gene is a leukemia-related gene and may play an important role in leukemia pathogenesis.
Bone Marrow Cells ; metabolism ; Cell Cycle Proteins ; biosynthesis ; genetics ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Mitosis ; Neoplasm Proteins ; biosynthesis ; genetics ; Poly-ADP-Ribose Binding Proteins ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; metabolism ; Ubiquitin-Protein Ligases

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

To study the chemosensitivity and the mechanisms of recombinant adenovirus vector expressing ING4 and IL-24 (Ad-ING4-IL-24) on lung adenocarcinoma in vitro and in vivo, the expression of ING4 and IL-24 in A549 cells was detected by RT-PCR and Western blotting. The growth inhibition, apoptosis rate and apoptosis body were measured by MTT, flow cytometry and Hoechst staining respectively. For in vivo study, we first established the A549 tumor model by grafting A549 cells in athymic nude mice; and then injected Ad-ING4-IL-24 into the tumors. Two weeks after injection, we killed the mice, removed the tumors, weighted and calculated the ratios of tumor-suppression. We also detected the expressions of ING4, IL-24, bax, bcl-2, VEGF with immunohistochemistry. The results indicated that ING4 and IL-24 were proved successfully transcription and expression in A549 cells. More interestingly, the joint group inhibited the growth of A549 cells and induced apoptosis. The in vivo data showed that the joint group suppressed the tumor growth conspicuously through up-regulating the expression of bax, and down-regulating the expression of bcl-2, VEGF. The study proved that Ad-ING4-IL-24 significantly enhanced the chemosensitivity to anticancer drug DDP in lung adenocarcinoma, which may related with cell apoptosis and antiangiogenesis.
Adenocarcinoma ; drug therapy ; metabolism ; Adenoviridae ; genetics ; metabolism ; Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; Homeodomain Proteins ; biosynthesis ; genetics ; Humans ; Interleukins ; biosynthesis ; genetics ; Lung Neoplasms ; drug therapy ; metabolism ; Mice ; Mice, Nude ; Neoplasms, Experimental ; drug therapy ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Transfection ; Tumor Suppressor Proteins ; biosynthesis ; genetics

OBJECTIVEE2F-1 and Rb are involved in cell cycle regulation. This study was to illustrate the mechanism of transformation from benign papillomatosis to ductal carcinoma in situ (DCIS) of the breast in relation to E2F-1 and Rb expression.

METHODSIn situ hybridization (ISH) was used to determinate the expression of E2F-1 and Rb mRNA of mild papillomatosis (MP, n = 40), severe papillomatosis (SP, n = 40) and DCIS (n = 40). Immunohistochemistry (IHC) was used to examine the expression of E2F-1 and Rb protein.

RESULTSThe positive rate of E2F-1 mRNA expression in MP, SP and DCIS was 17.5%, 45.0% and 80.0%, and that of E2F-1 protein expression was 20.0%, 47.5% and 77.5%, respectively. There were significant differences among the three groups (P < 0.01), and between any two groups (P < 0.01). The positive rate of Rb mRNA expression in MP, SP and DCIS was 90.0%, 50.5% and 20.0%, and that of Rb protein expression was 85.0%, 52.5% and 22.5%, respectively, with statistically significant difference similar with that of E2F-1. With the progression of papillomatosis to DCIS, the expression of E2F-1 mRNA and protein increased, while that of Rb decreased. The protein expression by IHC was positively correlated with the mRNA expression by ISH. However, that of E2F-1 was negatively correlated with Rb.

CONCLUSIONE2F-1 and Rb might provide a valuable basis for screening high risk papillomatosis and new target of gene therapy for pre-cancerous lesions of the breast.

Breast Neoplasms ; genetics ; metabolism ; Carcinoma, Intraductal, Noninfiltrating ; genetics ; metabolism ; Cell Cycle Proteins ; biosynthesis ; genetics ; DNA-Binding Proteins ; biosynthesis ; genetics ; E2F Transcription Factors ; E2F1 Transcription Factor ; Female ; Gene Expression Regulation, Neoplastic ; Genes, Retinoblastoma ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Papilloma ; genetics ; metabolism ; Precancerous Conditions ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Retinoblastoma Protein ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics

OBJECTIVETo study the expression of targeting protein for Xklp2 (TPX2) and its significance in squamous cell carcinoma (SCC) of the lung.

METHODTwo SCC cell lines and 4 immortalized bronchial epithelial cell lines (as a precancerous model) were examined by Western blot for TPX2 expression. Reverse transcription-polymerase chain reaction analysis for TPX2 was also performed using tumor tissues from 21 patients with SCC of the lung. The expression of TPX2 was studied by immunohistochemistry (using tissue microarray) on paraffin-embedded sections of pulmonary SCC and corresponding precancerous lesions from a group of 319 patients.

RESULTSTPX2 was variably expressed in all the cell lines studied. Compared with matched controls using normal lung tissue, high level of TPX2 mRNA was detected in 16 of the 21 SCC tumor tissue samples analyzed. Immunohistochemical study showed that TPX2 was mainly present in tumor tissues but not in normal controls. The expression of TPX2 correlated with tumor grade, stage and nodal status. As for precancerous lesions, the level of TPX2 was also increased, in accordance with the degree of dysplasia.

CONCLUSIONSExpression of TPX2 may play a role in carcinogenesis of bronchial epithelium and tumor progression of pulmonary SCC. It may also represent a potential biomarker for surveillance of SCC of lung.

Blotting, Western ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; Lung ; metabolism ; pathology ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Nuclear Proteins ; biosynthesis ; genetics ; Precancerous Conditions ; genetics ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Array Analysis

OBJECTIVETo study the effects of antisense Bmi-1 (B cell-specific moloney murine leukemia virus insertion site 1) RNA on the growth, cell cycle and apoptosis of lung cancer cell line A549.

METHODSRecombinant plasmids carrying antisense Bmi-1 RNA were transfected into A549 cells, which expressed a high level of endogenous Bmi-1. The mRNA level of A549 cell was analyzed by real time quantitative RT-PCR and the protein level was determined using Western blot. MTT growth curve and plate colony forming assay were used to measure the effect of antisense Bmi-1 RNA expression on the growth of A549. Flow cytometry was used to analyze cell cycle and apoptosis.

RESULTSAntisense Bmi-1 RNA reduced the Bmi-1 expression at the protein level, but did not alter the mRNA level in A549 cells. Compared with the control cells, A549 cells transfected with antisense Bmi-1 RNA showed a strong inhibition of the cell growth. The number of plate colony formation of the antisense Bmi-1 transfected cells (0.67 +/- 0.50) was less than those of the control (73.0 +/- 4.1) and cells transfected with empty vector (67.0 +/- 4.0, P < 0.01). Transfection of antisense Bmi-1 RNA arrested the A549 cells at G₀/G₁ phase of the cell cycle and did not increase the apoptosis.

CONCLUSIONAntisense Bmi-1 RNA expression inhibits A549 cells proliferation, likely through the interference of Bmi-1 leading to an arrest of the proliferating cells at the G₀/G₁ phase.

Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Nuclear Proteins ; biosynthesis ; genetics ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; biosynthesis ; genetics ; RNA, Antisense ; pharmacology ; RNA, Messenger ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Repressor Proteins ; biosynthesis ; genetics ; Transfection