ABSTRACT
The expression levels of programmed cell death 5 (PDCD5) are down-regulated in many malignancies. SG611-pdcd5, a recombinant conditionally replicative adenovirus carrying pdcd5 gene expression cassette, can evidently kill the leukemic cells and protect selectively the normal cells. The purpose of this study was to investigate the synergistic killing effect of SG611-pdcd5 and low-dose etoposide (VP-16) on K562 cells. K562 cells were treated with different concentrations of VP-16 or different multiplicities of infection (MOI) of SG611-pdcd5. After 48 hours of incubation the cell viability was determined by using MTT assay. The results showed that the cell viability of SG611-pdcd5 (MOI = 40) plus VP-16 (0.5 µg/ml) group significantly decreased as compared with single SG611-pdcd5 (MOI = 40) treatment group or single VP-16(0.5 µg/ml) treatment group (42.00 ± 5.75% vs 59.45 ± 4.12%; 42.00 ± 5.75% vs 82.91 ± 3.41%, respectively, both p < 0.05). The synergistic killing effect of SG611-pdcd5 plus VP-16 was higher than that of PDCD5 protein plus VP-16 or that of non-replicating adenovirus carrying pdcd5 (Ad-pdcd5) plus VP-16 (both p < 0.05). The cell viability of VP-16 (4.0 µg/ml) plus SG611-pdcd5 (MOI = 40) group, VP-16 (4.0 µg/ml) plus proPDCD5 (40 µg/ml) group and VP-16 (4.0 µg/ml) plus Ad-pdcd5 (MOI = 80) group was 37.09 ± 1.89%, 52.36 ± 1.64% and 73.64 ± 4.33%, respectively. It is concluded that SG611-pdcd5 can promote K562 cell death induced by low-dose VP-16. The combination of SG611-pdcd5 and VP-16 can enhance the killing effect on leukemic cells.
Subject(s)
Humans , Adenoviridae , Genetics , Antineoplastic Agents, Phytogenic , Pharmacology , Apoptosis , Apoptosis Regulatory Proteins , Genetics , Cell Survival , Etoposide , Pharmacology , Genetic Vectors , K562 Cells , Neoplasm Proteins , GeneticsABSTRACT
<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
The purpose of this study was to construct a recombinant conditionally replicating adenovirus (CRAd) expressing programmed cell death 5 (pdcd5). Pdcd5 gene was inserted in the E3 region of SG600-a CRAd in which the key genes for virus replication E1a and E1b were controlled under the human telomerase reverse transcriptase promoter (hTERTp) and the hypoxia response element (HRE) respectively, and with a deletion of 24 nucleotides within CR2 region of E1a. The insertion and orientation of all recombined plasmids were confirmed by restriction enzyme digestion and polymerase chain reaction (PCR). The infection efficiencies of a recombined virus carrying enhanced green fluorescent protein (EGFP) in leukemic cell lines were observed by using fluorescence microscope. The relative pdcd5 expression levels of K562 after being infected with SG611-pdcd5 were detected by real-time quantitative PCR. The results showed that the construction of SG611-pdcd5 was completed and confirmed. Pdcd5, hTERTp, HRE, skeleton and fiber11 of recombinant adenovirus SG611-pdcd5 were successfully amplified. The infection efficiencies of SG611-EGFP were all above 70% in both leukemic K562 and MEG-01 cell lines. SG611-pdcd5 expressed pdcd5 with high efficiency in leukemic cells as compared with Ad-pdcd5 or SG611 (p < 0.001). The expression level of pdcd5 increased gradually along with the increase of MOI. It is concluded that the triple-regulated adenovirus of SG611-pdcd5 containing the pro-apopro-tic gene pdcd5 has been successfully established with high pdcd5 expression level in leukemic cells, indicating that the recombinant adenovirus, SG611-pdcd5, promises further development of targeted tumor gene therapy.
Subject(s)
Adenoviridae , Genetics , Apoptosis Regulatory Proteins , Genetics , Genetic Therapy , Methods , Neoplasm Proteins , Genetics , Oncolytic Viruses , Genetics , Promoter Regions, Genetic , Telomerase , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate the factors influencing the success rate and stability of transient elastography(FibroScan)for assessment of liver fibrosis.</p><p><b>METHODS</b>Liver stiffness was assessed using transient elastography in totally 637 subjects including healthy subjects, asymptomatic hepatitis B virus (HBV) carriers, patients with chronic hepatitis B and patients with HBV-related cirrhosis. Of these subjects, 302 received 2 examinations and totalling 939 examinations were performed. In each case, one operator performed 2 consecutive series of 10 validated measurements, or 2 operators performed a series of 10 validated measurements. The factors including gender, age, body mass index (BMI) and the state of diseases were analyzed for their association with the success of the examination. Intraclass correlation coefficient (ICC) was used to evaluate the reproducibility of the operation.</p><p><b>RESULTS</b>Failure of the measurement occurred in 14 cases (2.2%), which was not associated with the age of the subjects and the state of diseases. The success rate of measurement decreased as the BMI increased (t=3.112, P=0.002), and was lower in female subjects (t=-2.193, P=0.029). The intra- and inter-operator stability of liver stiffness measurement was satisfactory, with ICC of 0.970 and 0.847, respectively. But for healthy subjects and asymptomatic HBV carriers, the stability was lower, with ICC of 0.736 and 0.639, respectively. Liver stiffness in patients with liver cirrhosis was positively correlated to complications and Child-Turcotte-Pugh (CTP) score.</p><p><b>CONCLUSION</b>Liver stiffness measurement has high stability with FibroScan, and high BMI could lower success rate of the measurement. Liver stiffness as measured by FibroScan allows prediction of the liver function and presence of complications in patients with liver cirrhosis.</p>