Apoptosis induced by 5-fluorocytosine in human pancreatic cancer cells genetically modified to express cytosine deaminase

AIM:To elucidate the pattern of 5-fluorocytosine(5-FC) induced apoptosis and its role in gene therapy for human pancreatic cancer. METHODS: The human pancreatic cancer SW1990 cells(CEA-producing) were infected with recombinant adenoviruses(Adex1CEA-prCD or Adex1CEA-prZ).Cytosine deaminase(CD) expression was examind by western blot. Apoptosis induced by 5-FC in human pancreatic cancer SW1990 cells genetically modified to express cytosine deaminase was investigated by applying electron microscopy, DNA electrophoresis and flow cytometry analysis techniques. RESULTS: The SW1990 cells infected with Adex1CEA-prCD were treated with 5-FC at 100 μmol·L^-1 for 48 h, cell apoptosis occurred. Typical apoptosis morphological feature appeared and DNA ladder could be demonstrated on DNA electrophoresis. Apoptosis peak was also showed by flow cytometry. Apoptotic cells accounted for 34.6% of the cell population. Cells in G₁, S and G₂/M phase of cell cycle were 64%, 11% and 7%, respectively. CONCLUSION: The apoptosis induced by 5-FC may be a primary mechanism in CD gene therapy for pancreatic cancer.     

Treatment of hepatocellular carcinoma in vitro and in vivo with yCD/TK double suicide gene driven by AFP promoter

AIM: To study the effect and mechanism of yeast cytosine deaminase/ thymidine kinase (yCD/TK) double suicide gene driven by alpha fetoprotein (AFP) promoter on hepatocellular carcinoma (HCC) in vitro and in vivo. METHODS: The expression plasmid with yCD/TK double suicide gene, which was driven by AFP promoter, was constructed. HepG2 (AFP positive) and SMMC7721 (AFP negative) human HCC cell lines were both transfected with the above-mentioned expression plasmid through cationic liposome. The cells were treated with 5-fluorocytosine (5-FC) and/or ganciclovir (GCV) at different concentrations. The cell proliferation and cell cycle phase were evaluated by MTT test and flow cytometry respectively. The effect of double suicide gene on HCC xenografts in nude mice was observed through measuring the tumor size and the number of apoptosis cells. RESULTS: The double suicide gene was expressed selectively on HepG2 cells, rather than on SMMC7721 cells. The 5-FC and/or GCV inhibited effectively the proliferation of HepG2 cells in a dose-dependent manner, but had no influence on SMMC7721 cells. The inhibitory effect on HepG2 cells among different treatments was GCV+5-FC>5-FC>GCV. In vivo, the treatments inhibited markedly the growth of HepG2 cell xenografts in nude mice, transfected with yCD/TK gene. More apoptotic cells were found in HepG2 xenografts after the treatment. However, the growth of SMMC7721 cell xenografts could not be inhibited by this double suicide gene therapy, and few apoptotic cells were found. CONCLUSION: yCD/TK double suicide gene driven by AFP promoter has a significant efficacy in treatment of AFP positive HCC. Cell apoptosis may be an important mechanism of yCD/TK double suicide gene-inhibiting the growth of HCC.     

Antitumor effects of HSV-tk and cytosine deaminase double suicide gene mediated by adenovirus on cholangiocarcinoma cells in vitro

AIM: To study the antitumor effects of HSV-tk and cytosine deaminase double suicide gene system on cholangiocarcinoma cells in vitro. METHODS: Recombinant adenovirus vector carring HSV-tk and cytosine deaminase double suicide gene was constructed and was transfected into QBC939 huamn cholangiocarcinoma cells. The antitumor effects were observed in vitro in comparison with a single suicide gene system. RESULTS: By transfection, HSV-tk and (or) cytosine deaminase double suicide gene gained high expression in tumor cells. Compared with single suicide gene system, this double gene system presented higher sensitivity, stronger antitumor effect and bystander effect. CONCLUSION: HSV-tk and cytosine deaminase double suicide gene system has a powerful antitumor effect on cholangiocarcinoma cells.     

CD147 monoclonal antibody-mediated nanoparticles for gene therapy to target lung cancer cells

AIM: In this study, CD147 antibody was used to carry out targeted modification of nanoparticles for protein kinase Cε (PKCε)-siRNA gene therapy to target lung cancer cells. The inhibitory effects of the nanoparticles on the proliferation and invasion of the lung cancer cells were observed. METHODS: The magnetic nanoparticles targeting CD147 protein were assembled as gene vector. The expression of CD147 in the lung cancer cells was observed under laser scanning confocal microscope. The cells were divided into CP group, CN group and LP group as the experimental groups. Targeted nanoparticles were used as CA group. Non-transfected cells were used as control group. The cell transfection was carried out with 250 ng plasmids/well in 6-well plate. The effect of nanocontrast agent on the cell endocytosis was observed under laser scanning confocal microscope. The mRNA expression of PKCε was detected by RT-qPCR. The protein expression of Ki67, MMP3, PKCε, Wnt1 and GAPDH was determined by Western blot. The cell proliferation ability was detected with colony formation assay. The cell invasion ability was detected by Transwell method. RESULTS: The expression of CD147 protein in the human lung cancer A549 cells was confirmed by immunofluorescence staining. The endocytosis of siRNA into the A549 cells in CP group was observed with the highest efficiency as compared with CN group and LP group. The relative mRNA expression of PKCε in the A549 cells of CP group, CN group, LP group and CA group were (9.76±0.18)%, (98.51±0.32)%, (99.17±0.16)% and (99.68±0.11)%, respectively. The difference between CP group and control group was statistically significant (P<0.05). No significant difference among CN group, LP group and control group was observed. The protein expression of PKCε, Ki-67, MMP3 and Wnt1 in CP group was significantly reduced, and the protein expression levels among CN group, LP group and control group had no significant difference. The colony number in CP group was significantly smaller than that in control group (P<0.05). The effective colony numbers in CN group, LP group and CA group had no significant difference as compared with control group. The number of the invading cells in CP group was significantly less than that in control group (P<0.05). The numbers of the invading cells in CN group, LP group and CA group had no significant difference as compared with control group. CONCLUSION: Nanogene vector targeting CD147 can carry PKCε-siRNA to conduct gene therapy efficiently on the lung cancer cells to achieve effective inhibitory effects on the proliferation and invasion of the lung cancer cells.     

Construction and in vitro analysis of recombinant adenovirus vector carrying red fluorescent protein reporter gene

AIM: To provide important tools for gene therapy and gene vaccine research by constructing an adenovirus vector containing red fluorescent protein ( RFP ) reporter gene with the approach of in vitro recombinant ligation. METHODS: The RFP gene fragment of pTurboRFP-N was digested and ligated into pShuttle transfer vector to construct recombinant vector pShuttle-TurboRFP-N. I- Ceu I/PI- Sce I were used to double digest recombinant vector pShuttle-TurboRFP-N and backbone of vector pH5'040.pkGFP-II. The target fragment was collected and ligated, and recombinant adenovirus vector AdH5'.040.CMV.RFP-N was obtained. After linearization, the vector was transfected into AD293 cells by liposome for virus packaging. The efficiency of virus packaging and RFP expression level in AD293 cells were examined using fluorescent microscope. In addition, the biological activity and titer of the virus were tested. Human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were infected with recombinant adenovirus vector AdH5'.040.CMV.RFP-N and control adenovirus vector AdH5.CMV.EGFP respectively. The infection efficiencies of the 2 vectors to different cell lines were compared by evaluating the expression levels of RFP and enhanced green fluorescent protein (EGFP). RESULTS: The recombinant adenovirus vector AdH5'.040.CMV.RFP-N was correctly constructed and confirmed by enzyme digestion. The virus was packaged by the vector in AD293 cells and had the ability to infect the target cells. The target gene in eukaryotic cells was also expressed. The number of recombinant adenoviruses and the titer of the virus after amplification and purification were 3.6×10¹⁵ vp/L and 1×10¹³ pfu/L,respectively. The infection efficiencies of recombinant adenovirus vector Ad5'.040.CMV.RFP-N to human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were higher than those in control adenovirus vector AdH5.CMV.EGFP (P<0.05). CONCLUSION: We have constructed recombinant virus vector carrying RFP reporter gene and provide an important tool for gene therapy and gene vaccine research. The reporter gene can be highly expressed in AD293 cells and has high infection efficiency to cancer cells. RFP is a good substitution and supplement to green fluorescent protein.     

Effect of B7-H4 gene transfection on growth of ovarian cancer cells

AIM: To observe the effect of B7-H4 gene transfection on human ovarian cancer cell growth and its tumor formation. METHODS: Human ovarian cancer cell line SKOV3 was transfected with PEGFP-N1/B7-H4 and PEGFP-N1 by LipofectamineTM2000 method. The expression of B7-H4 gene was detected by RT-PCR. The high expression cell strain was selected. The growth curve was drawn by MTT methods. The tumor size was observed after SKOV3/B7-H4 cells, SKOV3/neo cells and SKOV3 cells were injected subcutaneously into SCID mouse. RESULTS: The expressions of B7-H4 mRNA and fusion protein in SKOV3/B7-H4 cells were positive. Compared with the other two groups, transfection significantly promoted cell proliferation in vitro. In addition, facilitated tumor formation and enhanced tumor growth were observed in SKOV3/B7-H4 group. No difference in tumor growth between SKOV3 group and SKOV3/N1 group was observed. CONCLUSION: B7-H4 may be a candidate gene for gene therapy of ovarian cancer.     

Therapeutic effect of MCP-1 mediated macrophages on ovarian epithelial carcinoma

AIM: To investigate the therapeutic effect of MCP-1 mediated macrophages on ovarian epithelial carcinoma and its mechanisms.METHODS: Retorviral expression vectors pLXSN/MCP-1 was transfected into the packaging cell line PA317 by lipofectin-mediated gene transfer system. The virus particles containing MCP-1 gene were collected to infect NuTu-19. RT-PCR and Boyden Chamber were used to confirm the expression of MCP-1. Rat Fischer344 spleen macrophages were isolated. MTT method was applied to investigate the tumoricidal effect of macrophages. The survival time of the intraperitoneal disseminating ovarian cancer animal model was observed, and flow cytometry method was applied to analyze the expression of CD25 or CD44v6, and then the anti-tumor mechanisms of gene modified tumor cell lines were discussed. RESULTS: Stable MCP-1 expression in the cell line NuTu-19/MCP-1 possessed the chemotatic activity. The maximum killing ratio of macrophages on NuTu-19/MCP-1 cells was 28%. In the animal models immunized by MCP-1 expressing cells, prolonged survival time was showed which had statistical significance compared with that in the control group (P<0.05). The expression rate of CD25 (25.82%) in the NuTu-19/MCP-1 cells was higher than that in NuTu-19/neo cells (8.73%). The expression of CD44v6 in NuTu-19/MCP-1 cells was significantly lower than that in control NuTu-19/neo cells. CONCLUSION: MCP-1 mediates macrophages and suppresses the growth of NuTu-19. MCP-1 gene modified tumor cells can induce anti-tumor immunity. This strategy would be used as a promising approach for the treatment of ovarian cancer.     

Breast cancer stem cells cultured without insulin and effect of estrogen induction

AIM:To determine whether suspension culture medium without insulin can be used to feed breast cancer tumorsphere, or not. METHODS:MCF7 cells were used to build tumorsphere. The morphological changes, CD44+ CD24- expression, aldehyde dehydrogenase 1 (ALDH1) expression and multiple division ability were measured to identify the breast cancer stem cells and to detect the function of 17β-estradiol (E2β) in tumorsphere of MCF7 cells. RESULTS:The tumorshere, each containing 30 to 60 cells, was obtained by the method of insulin-removal suspension culture. These cells were cytokeratin 18 and CD10 proteins positive, and the number of CD44+ CD24- cells and ALDH1 protein expression were significantly higher than the adherent cultured cells (P<0.05). Using 10-10 mol/L E2β to treat the tumorshere for 7 d, the tumor cell number and volume were significantly increased. Using 10-10 mol/L E2β to treat the tumorshere for 24 h, the CD44+ CD24-cells and ALDH1 protein expression were significantly higher than those in non-treatment group (P<0.05). CONCLUSION: Suspension culture medium without insulin can be used to feed breast cancer tumorsphere. These tumorsphere could be used as a model to determine the function of E2β in breast cancer stem cell research.     

The inhibitory effects of endostatin gene transfer on the growth of breast cancer cells in vivo and in vitro

AIM: To investigate the therapeutic action of secreted endostatin (ES) on breast cancer cells. METHODS: Retroviral-mediated endostatin gene was transferred to breast cancer cell line MDA-MB-231. The ES biological properties and function were evaluated by polymerase chain reaction (PCR), MTT and a murine xenograft model. RESULTS: After retroviral transduction, endostatin genetically modified breast tumor cells were confirmed by PCR, and the integration and durative expression of endostatin gene was successfully committed. Compared with controls, endostatin secreted by genetically modified cells markedly inhibited endothelial cell proliferation (P<0.05) while the influences on the growth of MDA-MB-231 cell line in vitro were not found (P>0.05). The results of the transplanted subcutaneous tumor model in nude mice suggested that the subcutaneous growth of MDA-MB-231 was significantly inhibited by the expression of endostatin gene (P<0.05). In experimental groups, the tumor microvascular density (MVD) and VEGF expression were decreased. CONCLUSION: Retroviral- mediated overexpression of endostatin inhibits the proliferation of vascular endothelial cells and angiogenesis that associated with tumor growth in vivo via the paracrine pathway, which has a potential effect in the angiostatic gene therapy for breast cancer.     

Effects of different chemotherapeutic agents on reversing the acquired resistance to TRAIL gene in DLD1 colon cancer cells

AIM:To evaluate effects of different chemotherapeutic agents on reversing the acquired resistance to TRAIL gene and clarify the involved mechanisms in DLD1-TRAIL/R colon cancer cells. METHODS:Human colon cancer cell line DLD1-TRAIL/R cells that were resistant to TRAIL-expressing adenovector (Ad/gTRAIL) were treated with Ad/gTRAIL combined with different chemotherapeutic agents. Then, the cell viability was measured by MTT method, and apoptotic signaling conditions, including activation of caspase-3 and caspase-8, expression of Bax and Bcl-XL, were measured by Western blotting analysis. RESULTS:In vitro data showed that several chemotherapeutic agents, including 5-fluorouracil (5-FU) and mitomycin c (MMC), overcome the acquired resistance to TRAIL gene in DLD1-TRAIL/R colon cancer cells. The combination of Ad/gTRAIL and 5-FU effectively suppressed tumor growth in vivo in subcutaneous tumors established from DLD1-TRAIL/R cells. Further data showed that treatment with the combination of Ad/gTRAIL and 5-FU or MMC led to enhance the activation of caspase-3. Moreover, MMC but not 5-FU induced overexpression of Bax gene that was sufficient to overcome the resistance to TRAIL gene in DLD1-TRAIL/R cells. CONCLUSION:Chemotherapeutic agents, such as 5-FU and MMC, overcome the acquired resistance to TRAIL gene in DLD1-TRAIL/R cells. The candidate mechanisms for MMC but not 5-FU to overcome this resistance might involve the induction of over-expressed Bax protein in DLD1-TRAIL/R cells.     

Effects of RNA interference of FABP5 on subcutaneous tumor of human hepatocellular carcinoma cell line HepG 2 transplanted in nude mice

AIM: To investigate the effect of recombinant lentiviral vector for RNA interference (RNAi) on the expression of fatty acid-binding protein 5 (FABP5) gene in hepatocellular carcinoma HepG2 cells and tumor formation in nude mice.METHODS: RNAi lentiviral vector was used in the experiment. Human hepatocellular carcinoma HepG2 cells were divided into 3 groups:the HepG2 cells in experimental group were transfected with the recombinant lentivirirus vector LV-shRNA-FABP5, the cells in negative control group were transfected with a control lentiviral vector LV-shRNA-NC, and the cells in normal control group were without any treatment. The nude mice were randomly divided into 3 groups. The growth of the transplanted tumor cells in the nude mice was observed. The tumor growth curve, volume and weight were determined 4 weeks after the cell inoculation. The expression of FABP5 was detected by real-time PCR, Western blot and immunohistochemical staining.RESULTS: Transfection of the lentiviral vector FABP5-shRNA obviously reduced FABP5 expression in the HepG2 cells. Tumor formation was all positive in the 3 groups of the nude mice inoculated with the tumor cells. Compared with normal control group and negative control group, the tumor growth slowed significantly in experimental group with smaller volume and weight. FABP5 expression in the transplanted tumor tissues was significantly down-regulated at mRNA and protein levels in experimental group as compared with normal control group and negative control group.CONCLUSION: RNAi-induced down-regulation of FABP5 effectively inhibits the growth of transplanted hepatocellular carcinoma, suggesting that FABP5 gene may be an effective target for gene therapy in treating liver cancer.     

Influences of wild-type p53 gene overexpression on the differentiation,apoptosis and expression of scavenger receptor CD36 in U937 cells*

AIM: To study the effect of wild-type p53 gene on the differentiation, apoptosis and expression of scavenger receptor CD36 in U937 cells. METHODS： Recombinant adenovirus vector with wild-type p53 gene was constructed and used to transfect U937 cells. With the expression of wild-type p53 gene following adenoviral infection, transfected U937 cells were largely promoted to differentiate into macrophages. RESUITS： Trypanblue-staining test demonstrated that the percentage of positive cells increased from (14.2±5.5)% to (64.6±9.2)% and nitroblue tetrazolium (NBT) reduction test reached similar results (6.3±1.8)% vs (49.7±12.6)%. Furthermore, CD36 mRNA was up-regulated as confirmed by RT-PCR. The increased expression level of CD 36 was also detected by flow cytometry analysis. CONCLUSION： These results suggest that wild-type p53 gene can affect U937 cells differentiation and apoptosis, up-regulate expression of scavenger receptor CD36. It may have a potential significance on atherogenesis.     

Anti-tumor efficacy and immune mechanism of gene vaccine expressing PSMA

AIM: To observe both the anti-tumor efficacy induced by gene vaccine expressing PSMA in mice and the feather of the specific immune responses, which may offer some theoretical knowledge and experimental foundation of the gene vaccine therapy for the prostate cancer.METHODS: pcDNA3.0-PSMA plasmids were injected into the hind-leg quadriceps muscle of the BALB/c mice. Anti-PSMA antibody was detected in sera of the animals. The proliferation and cytotoxicity of the spleen cells were observed. The immunized mice were inoculated subcutaneously in the right flank with sp2/0-PSMA cells. The anti-tumor efficacy of the gene vaccine was determined through the ratio of the tumor formation, tumor volume, tumor weight and the survival rate of immunized mice.RESULTS: High levels of anti-PSMA antibody were induced in the immunized mice. The splenocytes from vaccination group were stimulated to produce strong proliferative responses and significant cytotoxic T-cells (CTL) activity. Moreover, during a period of time, the antibody concentration, proliferation index and CTL activity appeared to go up as the time went by and the numbers of immunization increased. After DNA vaccine immunization, tumor occurrence decreased, the tumor-free time was prolonged and the growth velocity of tumor was markedly reduced (P<0.05).CONCLUSION: The humoral and cellular immunity is induced by PSMA gene vaccine. The anti-tumor efficacy in the immunized mice is produced strikingly.     

Microarray analysis of tumor metastasis-related gene expression in PSMA-silencing prostate cancer cells

AIM: To investigate the functions of prostate-specific membrane antigen (PSMA) in prostate cancer metastasis. METHODS: Specific siRNA to knock down PSMA expression was designed and transfected into LNCaP cells. The tumor metastasis gene chip was also used to analyze the differential expression of 84 genes related to cancer metastasis. RESULTS: Specific siRNA was successfully designed and constructed and the gene expression of PSMA in LNCaP cells was knocked down. The RNAi efficiency was more than 75% at mRNA level and more than 68% at protein level. The results of the tumor metastasis gene chip indicated that 10 genes were up-regulated (such as CDH6 and CXCL12) and 4 genes were down-regulated (such as CCL7 and MDM2) in the LNCaP cells treated with PSMA siRNA. CONCLUSION: The PSMA is involved in the regulatory pathways in prostate cancer metastasis.     

Effect of transfection of tumor necrosis factor α gene and its mutants on tumorigenicity of H22 tumor cells in vivo

AIM: To compare the tumorigenecity of H22 cells transfected with TNF-α gene and its mutants(secreted TNF-α mutant, S-TNF^m, transmembrane TNF-α mutant, TM-TNF^m and wild type of TNF-α, Wt-TNF) in vivo. METHODS: Three kinds of mouse liver cancer cell line H22 expressing TNF-α and its two mutants were mixed with untransfected H22 at different effector/target ratio separately. The growth of tumor was examined after injection of 2.5×10⁵(100 μL) mixed H22 tumor cells into mice. The lymphocyte infiltration in the site of tumor and the expression of Fas on tumor cells were detected by immunohistochemistry. RESULTS: The tumorigenecity of H22 cells transfected with TNF-α gene and its mutants was significantly weakened( P <0.01). Besides the cytotoxicity of the both forms of TNF, TM-TNF was found to induce the expression of death receptor Fas by tumor cells and S-TNF was shown to promote frank lymphocyte infiltration in the site of tumor. Furthermore, a transient decrease in body weight was found in mice inoculated with H22/S-TNF^m. CONCLUSION: The tumorigenecity of tumor cells was reduced by transfection with TM-TNF or S-TNF gene. It results from the cytotoxicity of TNF and their activation of tumorcidal mechanisms in vivo. TM-TNF may induce tumor cell apoptosis via the Fas pathway while S-TNF may exert its antitumor effect by recruiting and activating lymphocytes in the site of tumor.     

Effects of inhibition of Cripto gene siRNA on vascular endothelial growth factor of colon cancer cell line LS-174T

AIM:To study the effects of Cripto gene on vascular endothelial growth factor (VEGF) of colon carcinoma cells.METHODS:Cripto siRNA was designed and constructed. Colon cancer LS-174T cells were divided into 4 groups: control group and different dose (3.125, 6.25 and 12.5 nmol/L) of siRNA groups. After transfected for 24, 48 and 72 h, colon cancer cells were harvested to carry on the next tests. Expression of Cripto mRNA was determined with real-time PCR, and immunofluorescence isothiocyanate (FITC) labeling assay and Northern blotting were performed to examine the expression of protein and mRNA of VEGF, respectively. The cells in control group and cells transfected with 12.5 nmol/L siRNA were inoculated into nude mice respectively. 30 days after inoculated, the mice of two groups were executed, and immunohistochemical (ICH) assay was used to evaluate the VEGF protein of mice tumor. RESULTS:siRNA down-regulated the Cripto mRNA in a dose and time dependent manner. Protein and mRNA of VEGF in transfected cells reduced in a dose and time dependent manner. Compared to control, the expression of VEGF protein from ICH assay was lowered significantly (P<0.05).CONCLUSION:Cripto gene might contribute to the regulation of angiogenesis of colon carcinoma. The down-regulation of Cripto gene by siRNA can suppress angiogenesis of human colon cancer.     

Effect of CD97 gene silencing by small interfering RNA on migration and invasion of gastric carcinoma cell lines

AIM: To investigate the effect of CD97 gene silencing by small interfering RNA(siRNA) on migration and invasion of gastric carcinoma cell lines. METHODS: Gastric carcinoma cell lines AGS and MGC803 were used in the study. Four pairs of siRNA were designed according to the sequence of CD97 gene and synthesized chemically. The siRNAs were transfected into the gastric carcinoma cell lines. Forty-eight hours after transfection, the total RNA was extracted and the mRNA expression of CD97 was detected by real-time RT-PCR so as to screen the most effective siRNA. The protein level of CD97 was also measured by fluorescence-activated cell sorting (FACS) 72 h after Transfection. The abilities of migration and invasion were evaluated by Transwell test. The viability of the cells was measured by MTT method. RESULTS: Real-time RT-PCR and FACS revealed that CD97-siRNA notably down-regulated CD97 expression at both mRNA and protein levels. The mRNA level decreased by (89.34±9.95)% and (95.42±1.93)% in AGS and MGC803 cells,respectively. The protein levels of CD97^EGF and CD97^stalk in AGS cells decreased by (19.29±3.45)% and (30.11±5.93)%,respectively. The protein levels of CD97^EGF and CD97^stalk in MGC803 cells decreased by (26.25±5.73)% and (16.22±3.23)%,respectively. No change of the cell viability after siRNA transfection was observed. The cell number of migration and invasion in AGS cells was decreased by (67.63±12.03)% and (68.02±15.63)%,respectively. The cell number of migration and invasion in MGC803 cells was decreased by (14.92±2.03)% and (22.09±5.43)%,respectively. CONCLUSION: The siRNA effectively inhibits CD97 expression and restrains the migration and invasion capacities of gastric carcinoma cell lines, suggesting that CD97 plays an important role in the metastasis of gastric cancer.     

Transfection efficiency and cytotoxicity in human T lymphocytes infected with Ad5 adenoviral vector

AIM:To determine the transfection efficiencies and to evaluate the cytotoxicity of infection with Ad5 adenoviral vector in human T lymphocytes. METHODS:The T-lymphoma Jurkat cell line, normal CD3+ T cells and pre-stimulated CD3+ T cells were transfected with Ad5 adenovirus at multiplicities of infection (MOI) ranging from 20 to 400. GFP expression was analyzed by flow cytometry 48 h after transfection. The cells were harvested 24 h, 48 h and 72 h after transfection. The cell cycle was analyzed using propidium iodide staining and the apoptosis of T lymphocytes was detected by annexin V/7-AAD staining. Trypan blue exclusion assay was used to determine the survival cell numbers after 48 h or 72 h of transfection. RESULTS:The transfection of primary human T cells by the Ad5 virus was less efficient than that of a T-lymphoma cell line. Similar transfection efficiency was observed in both CD4+ T lymphocytes and CD8+ T lymphocytes. The activation of T lymphocytes resulted in a decrease in Ad5 transfection efficiency in CD8+ T cells. Following transfection (24 h, 48 h and 72 h), the percentages of G 0/G 1-phase cells, S-phase cells, G 2/M-phase cells and apoptotic cells at all MOI did not change significantly. Therefore, transfection by the Ad5 adenoviral vector did not influence the cell cycle and survival cell numbers. CONCLUSION:The Ad5 adenoviral vector, which shows little cytotoxicity to T lymphocytes, may be a valuable tool for T cell receptor gene therapy.