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Objective To observe the effect of human telomerase reverse transcriptase-thymidine kinase/ganciclovir (phTERT-TK/GCV) system combined human telomerase reverse transcriptase-tumstatin (phTERT-tumstatin) system on apoptosis of human HepG2 and mRNA expression and protein content of AFP,RhoC related with cancer.Methods Fluorescence microscopy was used to observe expression of EGFP and MCHERRY in transfected HepG2 and L-02.Real-time PCR and Western blot were used to detect AFP and RhoC mRNA and protein content.Flow cytometry was used to detect the apoptosis of transfected HepG2.Results phTERT-tumstatin and phTERT-TK/GCV genes expressed in transfected HepG2.Real-time PCR showed that AFP and RhoC mRNA expression in different group were 0.76±0.09 and 0.80±0.04 (TK/GCV group),0.62±0.09 and 0.40±0.02 (TM group),0.49±0.07 and 0.54±0.03 (MK group).The differences were significant (P ＜ 0.01) except TK/GCV group compared with empty plasmid group.Western blot test results showed that protein content of AFP and RhoC were higher in TK/GCV group (0.97±0.02/1.17± 0.01),TM group (0.83±0.02/0.99±0.02),MK group (0.69±0.01/0.77±0.02) than in empty plasmid group (1.19±0.03/1.32±0.05) and non-transfected group (1.15±0.05/1.29±0.30) (P ＜ 0.01).Additionally,protein content of AFP and RhoC in MK group were significant difference with TK/GCV group and TM group (P ＜ 0.01).Flow cytometry showed that phTERT-tumstatin,phTERT-TK/GCV co-transfected HepG2 cells apoptosis rate was significantly higher than both individually transected group.Cells apoptosis rate of alone and co-transfected groups was significant difference compared with empty vector group and untransfected group.Conclusions The effect of phTERT-TK/GCV and phTERT-tumstatin on pro-apoptotic of HepG2 cells is significant.phTERT-TK/GCV combined with phTERT-tumstatin has strong therapeutic function.
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Liver cancer is one of the most common malignant tumors in China.The efficacy of traditional treatment for liver cancer is unsatisfactory,and the prognosis of the patients is poor.In recent 10 years,with the development of the molecular biological techniques,genetic therapy has become a new and promising approach for liver cancer.Of which,adenovirus mediated herpes simplex virus thymidine kinase (ADV-tk) for the treatment of liver cancer is widely applied.The enzyme secreted by ADV-tk transformed the prodrug gancyclovir (GCV) to the cytotoxic agents and thus to kill the liver cancer cells.The results of multiple animal and clinical experiments showed that ADV-tk/GCV is effective for the treatment of liver cancer.In this article,the recent progress of ADV-tk/GCV in the treatment of liver cancer was reviewed.
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Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elucidate the reason why the so-called "bystander effect" mediated by thymidine kinase (TK) suicide gene therapy on PCa cells is not of significance and to explore the role of GJIC in PCa carcinogenesis.mRNA and protein expression of Cx43 in a PCa cell line PC-3m was detected by reverse-transcription polymerase chain reaction (RT-PCR) and strapt-avidin-biotin-enzyme complex (SABC) immunohistochemical staining, and inherent GJIC of PC-3m cells was assayed by scrape-loading and dye transfer (SLDT) assay. The expression of Cx43 in human normal and malignant prostate tissues was determined by SABC immunohistochemistry as well. It was found that Cx43 mRNA and protein expression in PC-3m cells was slightly reduced as compared with positive controls and the location of Cx43 protein was aberrant in cytoplasm rather than on membrane. Assessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was abnormally located and markedly diminished as compared with normal prostatic epithelial ones, displaying a negative correlation to the pathological grade (χ2=4.025, P＜0.05). Additionally, capacity of inherent GJIC in PC-3m cells was disrupted, which was semi-quantified as (+) or (-). It was indicated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein participated in the mechanisms leading to deficient GJIC in PC-3m cells. Lack of efficient GJIC is a molecular event, which may contribute not only to limited extent of "bystander effect", but also to initiation and progression of prostatic neoplasm.
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The killing effects of herpes simplex virus thymidine kinase gene/ganciclovir (HSV-tk/GCV) approach by the addition of several commonly clinical chemotherapeutic agents on hormone refractory prostate cancer (HRPC) cells PC-3m were investigated. After transferring of the HSV-tk gene into PC-3m cells, mRNA and protein expression of HSV-tk was detected by reverse-transcript polymerase chain reaction (RT-PCR) and strept avidin-biotin complex (SABC) immunohistochemical method. The killing effect of GCV, cisplatin (CDDP), etoposide (VP-16), vincristine (VCR), methotrexate (MTX), 5-fluorouracil (5-Fu), and suramin on PC-3m cells was evaluated by morphological assessment analysis, trypan blue exclusion assay and MTT assay respectively. Additionally, the cooperative effect of HSV-tk/GCV system combined with the above agents on the target cancer cells was determined by MTT. Furthermore, apoptosis and necrosis induced by GCV plus 5-Fu or suramin was analyzed by flow cytometry (FCM). The results showed that that there was HSV-tk mRNA and protein expression in pDR2-tk plasmid transduced PC-3m cell. Combination of GCV with VP-16, VCR, 5-Fu or suramin led to an enhanced cellular killing effect, but with CDDP resulted in a reduced one and with MTX in an approximate one. FCM revealed that synergistic use of GCV and 5-fu or suramin resulted in a rather large proportion of apoptosis and necrosis with the apoptosis index being 36.38 % and 35.51%, and the proportion of necrosis being 33.05 % and 28.87 %, respectively. In conclusion, HSV-tk/CGV approach by addition of certain clinical available chemotherapeutic drugs brings on statistically significant enhanced cell killing over single-agent treatment.Our results highlight the potential for such new combination therapies for future treatments of HRPC.
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The potential therapeutic benefit of introducing IFN-gamma and GM-CSF genes in combination with the HSVtk suicide gene into subcutaneously implanted CT26 tumor cells was compared with that from each treatment alone. Cells, unmodified or retrovirally transduced with HSVtk or IFN-gamma/GM-CSF genes, were inoculated subcutaneously into syngeneic BALB/c mice in various combinations. HSVtk gene, with intraperitoneal ganciclovir treatment, reduced tumor volume by 81% at locally inoculated tumor sites (p<0.01) and by 25% at distantly inoculated tumor sites (p=0.052). IFN-gamma/GM-CSF genes showed a 56% tumor volume reduction at local tumor sites (p<0.01) and 15% volume reduction at remote tumor sites, although this was not statistically significant. The combination of HSVtk (with GCV) and IFN-gamma/GM-CSF genes showed an 81% volume reduction at local tumor sites (p<0.01) and a 43% volume reduction at remote tumor sites (p<0.01). Thus, the combination of HSVtk and IFN-gamma/GM-CSF gene therapy produced greater therapeutic efficacy than either treatment alone.
Subject(s)
Animals , Male , Mice , Cell Line , Cell Line, Tumor , Genetic Therapy/methods , Genes, Transgenic, Suicide , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , H-2 Antigens/metabolism , Interferon-gamma/biosynthesis , Mice, Inbred BALB C , Neoplasms, Experimental/therapy , Simplexvirus/enzymology , Thymidine Kinase/genetics , Transduction, GeneticABSTRACT
BACKGROUND: One of the important mechanisms responsible for a tumor escaping the immune response is an absence of the tumor associated antigen (TAA) on the cancer cell surface. To overcome this, combination gene therapy using a herpes simplex thymidine kinase (HSTK) gene, prototype of drug sensitizing gene, was conducted to enhance TAA release by cell destruction, as well as the cytokine genes for immune cell attraction. METHODS: We investigated whether or not transduction with the adenovirus-HSTK (Ad-HSTK) enhanced the sensitivity of Lewis lung carcinoma (LLC) to ganciclovir (GCV) and induced a bystander effect. A Tumor vaccine trial was performed using LLC with ad-HSTK ±ad-GM-CSF±ad-IL-2 to determine if they exhibit some antitumor effect on established lung cancer xenografts. RESULTS: LLC with ad-HSTK revealed a much higher sensitivity to ganciclovir (GCV). LLC transduced with ad-HSTK and/or ad-IL-2, ad-GM-CSF showed a lower in vivo tumorigenicity. In the treatment experiment, vaccination with LLC transduced with ad-HSTK, ad-IL-2, or ad-GM-CSF alone modestly suppressed the growth of an established tumor. Combined transduction with HSTK and GM-CSF induced stronger growth suppression of a established lung cancer, while HSTK and IL-2 combination transduction did not have any antitumor effect on individual transduction. Vaccination with LLC-HSTK-GM-CSF increased the infiltration of dendritic cells in the spleen. CONCLUSION: It was concluded that a tumor vaccine transduced with HSTK and GM-CSF induces strong antitumor immunity by activating the dendritic cells.
Subject(s)
Animals , Adenoviridae , Bystander Effect , Carcinoma, Lewis Lung , Cytokines , Dendritic Cells , Ganciclovir , Genetic Therapy , Granulocyte-Macrophage Colony-Stimulating Factor , Herpes Simplex , Heterografts , Interleukin-2 , Lung Neoplasms , Lung , Phosphotransferases , Simplexvirus , Spleen , Thymidine Kinase , United Nations , VaccinationABSTRACT
AIM:To investigate the in vitro killing effect of adenovirus-mediated herpes simplex virus thymidine kinase gene(HSV-TK) driven by hypoxic response element(HRE) on hepatoma cell line HepG2.METHODS:Recombinant adenoviral vector Ad-HRE-TK was constructed with HSV-TK under the control of HRE using AdEasy system.Then Ad-HRE-TK was transfected into hepatoma cell line HepG2 and the cells were cultured under normoxic or hypoxic conditions.After treated with GCV for 3 d,the sensitivity to GCV of HepG2 was measured by MTT method.RESULTS:Over 95% HepG2 cells infected with Ad-HRE-TK cultured under hypoxic condition were killed when the MOI was 100 and the concentration of GCV was 50 mg/L.On the contrary,no killing effect of GCV was observed in cells cultured under normoxic condition.CONCLUSION:HRE promotes the expression of HSV-TK specifically under hypoxic condition and induces the specific killing effect of GCV.
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0.05). The mean growth values in AdCMV-tk/GCV- and AdVEGF-tk/GCV-treated tumors were significantly lower than those in untreated tumors and AdVEGF-tk/saline-untreated tumors( P
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Transduction of retroviral-mediated herpes simplex virus thymidine kinase(HSVtk) gene into tumor cells and subsequent ganciclovir(GCV) treatment have been used as an experimental and clinical therapeutic strategy. Because non-transduced tumor cells can be killed by small proportion of transduced cells, known as bystander effect. Increasing bystander effect is useful strategy of suicidal gene therapy using HSVtk gene. To get a better bystander effect, we transduced T98G gliobastoma cells with HSVtk gene, single and double transduction with different marker genes respectively. Double HSVtk gene transduced cell lines showed significantly increased HSVtk activity(83%) by measuring the intracellular amount of phosphrylated 3H-GCV comparing to the single HSVtk gene transduced cell lines. In vitro bystander effect, examined by coculturing with HSVtk gene transduced cells and HSVtk- negative pa rental cells, was significantly increased on double HSVtk gene transduced cell lines. These results suggest that increasing herpes simplex virus thymidine kinase activity by double transfer of HSVtk gene into tumor cells can be a useful strategy for treating cancer with suicidal gene therapy.
Subject(s)
Bystander Effect , Cell Line , Ganciclovir , Genetic Therapy , Simplexvirus , Thymidine Kinase , ThymidineABSTRACT
OBJECTIVE: The bystander effect, the phenomenon that non-transduced tumor cells can be killed along with the transduced cells, enables suicidal gene therapy feasible in spite of low efficiency of gene transfer at present time. Increment of thymidine kinase activity in the cells through double copy insertion of HSVtk gene may lead to increased bystander effect in vitro and in vivo, therefore enhancing the therapeutic potential of suicidal gene therapy. To examnine this hypothesis, we did an experiment to improve bystander effect by using double transfer of HSVtk gene into 9L tuomr cells. METHODS: We transduced 9L glioblastoma cells which had one copy or two copies of HSVtk gene using retroviral vector. Two different retroviral vector plasmids containing HSVtk gene were made employing pBabePuro or LXSN plasmid whose selection markers were puromycin and G418, respectively(LtkSP and LtkSN). Recombinant retrovirus was produced from ecotrophic PA317 packaging cells. Infection of the 9L cells with LtkSP recombinant retrovirus and selection with puromycin was done in vitro to make 9L/LtkSP(9LtkS). These cells were infected again with LtkSN recombinant retrovirus and selected under G418 to establish cells containing two copies of HSVtk gene, 9L/LtkSP/ LtkSN(9LtkD). RESULTS: By measuring the intracellular amount of phosphorylated 3H-GCV, 9LtkD cell lines showed significantly increased HSVtk activity, being 70% higher than that of 9LtkS cell lines. The sensitivity to GCV was also markedly increased. In vitro bystander effect, examined by coculturing with HSVtk gene transduced cells and 9L cells, was significantly increased on 9LtkD cell lines. To evaluate the in vivo bystander effect, 9LtkD/9L(20%:80%) cells or 9LtkS/9L (20%:80%) cells were implanted into the brains of Fisher 344 inbred rats, followed by administration of ganciclovir. Rats implanted with 9LtkD/9L cells showed better tumor regression and longer survival than those of 9LtkS/9L after treatment with ganciclovir. CONCLUSION: These results suggest that increasing herpes simplex virus thymidine kinase activity by introducing double copy of HSVtk gene into tumor cells would improve in vitro and in vivo bystander effect, and lead to enhanced efficacy of suicidal gene therapy.