ABSTRACT
<p><b>OBJECTIVE</b>To investigate the relationship between p16 expression and cell proliferation and prognosis in gastric cancer patients.</p><p><b>METHODS</b>Gastric cancer cell lines SGC-7901, MKN45, MKN28, human embryonic kidney cell line HEK293, human fibroblast cell line MRC-5, and surgical specimens of gastric carcinoma and adjacent normal gastric mucosa from 65 patients were included in this study. RT-PCR, MTT and FCM assays were used to detect p16 expression in gastric cancer cell lines and surgical specimens of gastric cancer. MTT assay was used to determine cancer cell viability and FCM to detect cell cycle. Kaplan-Meier survival curve and Log-Rank statistics were used to analyze the relationship between p16 expression and survival of petients with gastric cancer.</p><p><b>RESULTS</b>Gastric cancer cell lines were mostly negative for p16 expression, and p16 was re-expressed after the cells transfected with p16 gene by adenovirus AdCMV-p16. p16 re-expression resulted in the decrease of cancer cell viability and cancer cell cycle arrest with increased G(1) phase and decreased S phase. p16 expression in cancer specimens was 32.3% (21/65), significantly lower than the 81.5% (53/65) in normal mucosa (χ(2) = 32.124, P < 0.001). The disease-free survival was significantly shorter in p16-negative patients than that in p16-positive patients (P < 0.01), but not the overall survival (P > 0.05). p16 expression was significantly correlated with differentiation and lymph node metastasis, but not significantly correlated with sex, age, tumor size or invasion depth of the gastric cancer.</p><p><b>CONCLUSIONS</b>p16 gene is important for cancer cell proliferation. The inactivation gives cancer cells a high activity for proliferation and metastasis, and then influences the disease-free survival of gastric cancer patients.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Adenoviridae , Genetics , Cell Cycle Checkpoints , Cell Line, Tumor , Cell Proliferation , Cell Survival , Cyclin-Dependent Kinase Inhibitor p16 , Genetics , Metabolism , Disease-Free Survival , Gene Expression Regulation, Neoplastic , Genes, p16 , Lymphatic Metastasis , Neoplasm Invasiveness , RNA, Messenger , Metabolism , Recombinant Proteins , Genetics , Metabolism , Stomach Neoplasms , Metabolism , Pathology , Survival Rate , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To establish a human gallbladder carcinoma cell line derived from a metastatic gallbladder carcinoma and identify its biological characteristics.</p><p><b>METHODS</b>Tissue samples were separated from the surgical specimen obtained from a patient with metastatic carcinoma and single-cell suspension was prepared. Then the cells were cultured in DMEM medium supplemented with 15% fetal bovine serum. The morphology of tumor cells was observed under an electron microscope. The cell growth curve was plotted. The tumorigenicity of the cell line was studied by subcutaneous inoculation in SCID mice. The cells were infected by lentiviral vector carrying fluorescent report genes (lenti-GFP and lenti-Red2) separately for expressions of GFP and Red2, respectively.</p><p><b>RESULTS</b>A novel metastatic gallbladder carcinoma cell line was successfully established and named "EH-GB1". It could be passaged for over 20 generations with typical malignant epithelial morphology and a stable growth cycle of 24 h. Tumors were formed in all of the 10 SCID mice inoculated with EH-GB1 cells subcutaneously, and the tumor cells were tumor marker CA19-9-positive. Continuous expressions of fluorescent report genes were observed in EH-GB1 cells infected by lenti-GFP and lenti-Red2.</p><p><b>CONCLUSION</b>EH-GB1 cells might be the first stable cell line of human gallbladder carcinoma established from a metastatic focus of gallbladder carcinoma. This cell line with continuous expressions of GFP and Red2 might be a novel and perfect experimental model for clinical and basic research on gallbladder carcinoma.</p>
Subject(s)
Animals , Female , Humans , Mice , Middle Aged , Abdominal Neoplasms , Metabolism , Pathology , Abdominal Wall , Adenocarcinoma , Metabolism , Pathology , CA-19-9 Antigen , Metabolism , Cell Line, Tumor , Metabolism , Pathology , Gallbladder Neoplasms , Metabolism , Pathology , Genes, Reporter , Green Fluorescent Proteins , Metabolism , Mice, Nude , Mice, SCID , Neoplasm TransplantationABSTRACT
<p><b>OBJECTIVE</b>To construct an inducible eukaryotic vector carrying red fluorescent protein (DsRed) and evaluate the regulation of DsRed gene expression in vitro.</p><p><b>METHODS</b>The vector pRS17-RUDsRed containing DsRed gene, promoter and RU486-inducible system was constructed using molecular biological methods. To minimize potential interference, the two transcriptional elements were spaced with a 1.6 kb insulator. Fluorescence microscopy and flow cytometry were used to observe the activation of this regulatable vector after transfection in MFC cells.</p><p><b>RESULTS</b>The vector was identified by digestion with different restriction enzymes, sequencing and PCR. In the absence of RU486, the cells transfected with the vector exhibited very low DsRed protein expression, and the addition of RU486 induced efficient DsRed expression in the cells.</p><p><b>CONCLUSION</b>The RU486-inducible eukaryotic vector carrying DsRed protein allows effective regulation of the target gene expression in vitro, which provides a useful tool for gene regulation and gene therapy studies.</p>
Subject(s)
Humans , Gene Expression Regulation , Genetic Therapy , Methods , Genetic Vectors , Genetics , Luminescent Proteins , Genetics , Metabolism , Mifepristone , Pharmacology , Promoter Regions, Genetic , Genetics , Stomach Neoplasms , Genetics , Pathology , Tumor Cells, CulturedABSTRACT
<p><b>OBJECTIVE</b>To investigate the anti-tumor effect of a novel gene-viral therapeutic system CNHK300-murine endostatin (CNHK300-mE) on gastric cancer.</p><p><b>METHODS</b>SGC-7901 gastric cancer cells (5 x 10(7) cells/mouse) were injected s.c. into the right flank of Balb/c nude mice, grown to 4-5 mm to demonstrate tumor take, and 10(9) pfu/100 microl CNHK300-mE virus was injected into tumors. Tumor sizes were measured with calipers every other day. Serum samples were obtained by retro-orbital puncture and level of endostatin expression in serum was quantitated by ELISA. Fifteen days after treatment, all mice were sacrificed and tumors were excised for immunohistochemical staining of PCNA, hexon and vWF. Tumor cell apoptosis was detected by TUNEL method.</p><p><b>RESULTS</b>From the 7th day post-treatment, the bearing tumors of mice treated with CNHK300-mE were significantly smaller than those of control group treated with PBS. Seven days after treatment, expression of endostatin was (2115 +/- 770) ng/ml, significantly higher than that of control group. Immunohistochemical staining indicated that hexon was expressed in treated tumor cells, and PCNA LI (label index) [(55.0+/-1.4)% vs control (74.1 +/- 0.4)%, P<0.05], microvessel density (MVD) of CNHK300-mE treated tumors decreased significantly. Apoptosis obviously increased in tumor cells[(78.4 +/- 9.1)% vs control (15.2 +/- 0.5)%, P<0.01]. Apoptosis bodies and crystal grid were found in tumor cell nuclear by electron microscope.</p><p><b>CONCLUSIONS</b>Gene-viral therapeutic system CNHK300-murine endostatin can replicate in gastric cancer cells. The mouse endostatin gene cloned into CNHK300-mE expressed in high level. CNHK300-mE may induce tumor cells apoptosis, reduce the expression of PCNA and efficiently suppress gastric cancer growth through inhibiting tumor angiogenesis.</p>
Subject(s)
Animals , Female , Male , Mice , Adenoviridae , Genetics , Endostatins , Genetics , Genetic Therapy , Genetic Vectors , Mice, Inbred BALB C , Mice, Nude , Stomach Neoplasms , Therapeutics , Telomerase , Genetics , Transfection , Xenograft Model Antitumor AssaysABSTRACT
<p><b>OBJECTIVE</b>To develop a novel vector system, which combines the advantages of the gene therapy, antiangiogenic therapy and virus therapy, and to observe its effect on lung cancer.</p><p><b>METHODS</b>Human angiostatin gene hA(k1-5) was inserted into the genome of the replicative virus specific for the tumor cells by virus recombination technology. The expression of hA(k1-5), its effect on tumor growth in vitro and in vivo were studied.</p><p><b>RESULTS</b>A new kind of gene-viral vector system, designated as CNHK200-hA(k1-5), in which the E1b55 000 gene was deleted but the E1a gene of adenovirus preserved, was constructed. The novel vector system possessed the same property as the replicative virus ONYX-015, which replicates in p53- tumor cells but not in normal cells, thus specifically kills tumor cells. In vitro, CNHK200-hA and Ad-hA both could kill A549 tumor cells but the latter needed 100 times more MOI to achieve the same amplitude of cell killing. In vivo, the therapeutic effect of CNHK200-hA on human lung cancer A549 xenograft in nude mice was significantly better than that of Ad-hA and that of tumor-replicative virus ONYX-015.</p><p><b>CONCLUSION</b>CNHK200-hA(k1-5), a novel vector is constructed in which the angiostatin gene is inserted into the genome of the replicative adenovirus cytotoxic to p53-negative tumor cells. It has the advantages of specific tumor targeting, high level gene expression in tumor cells, and potent tumoricidal activity.</p>
Subject(s)
Animals , Female , Humans , Male , Mice , Adenoviridae , Genetics , Adenovirus E1A Proteins , Genetics , Angiostatins , Genetics , Physiology , Cell Line, Tumor , Cell Survival , Genetic Therapy , Genetic Vectors , Lung Neoplasms , Metabolism , Pathology , Therapeutics , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , TransfectionABSTRACT
Objective To construct an expression plasmid carrying the specific siRNA of survivin gene,and to evaluate its silencing effect on the expression of survivin gene and its inhibition effect on the growth of gastric cancer cells.Methods The specific siRNA of survivin gene was designed and synthesized,and an expression plasmid pAdGFP-siRNA was constructed.Gastric cancer cell line SGC-7901 was cuhured and transferred with pAdGFP-siRNA,then the silencing of survivin gene expression and the growth inhibition of cancer cell mediated by pAdGFP-siRNA were identified.Results The growth of gastric cancer cells was inhibited after transferring the pAdGFP-siRNA,with the inhibition rate of 68.2% compared to the control group.Immunohistochemistry showed that the specific siRNA markedly silenced the expression of survivin gene in cancer cells.Conclusions The overexpression of survivin gene in gastric cancer cells results in the high proliferation and the resistance to the chemo- and radio-therapy of the cancer cells.The specific siRNA can markedly silence the expression of survivin gene and inhibit the growth of cancer cells.