Cloning of Thymidine Kinase Gene of Duck Plague Virus Using Degenerate PCR

The DNA of duck plague virus （DPV） thymidine kinase （TK） gene was cloned and sequenced from a vaccine virus in the study. Degenerate oligonucleotide primers for the consensus site of herpesvirus UL24, TK, and glycoprotein H（gH） gene were used in the polymerase chain reaction （PCR） to amplify DNA product with 3 741-base-pairs （bp） in size. DNA sequence analysis revealed a 1 077-base-pairs （bp） open reading frame （ORF） encoding a 358 amino acid polypeptide homologous to herpesvirus TK proteins. The predicted TK protein shared 31.2, 41.3, 35.7, 37.4, and 28.4% identity with herpes simplex virus typel, equine herpesvirus type 4, Marek＇s disease virus 2, herpesvirus turkey, and infectious laryngotracheitis virus, respectively. Comparison of the amino acid sequences of other herpesvirus TK proteins showed that these proteins were not conserved on the whole, otherwise the portion of the TK proteins corresponding to the nucleotide binding domain and the nucleoside binding site were highly conserved among herpesvirus. Comparison with the amino acid sequences of the conserved nucleotide and nucleoside binding domains of other eleven herpesvirus TK proteins to the predicted DPV peptide confirmed its identity as the DPV TK protein.

Retrovirus-mediated herpes simplex virus thymidine kinase gene therapy approach for hepatocellular carcinoma

The therapeutic effect of herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV) system on hepatocellular carcinoma was studied in this experiment. The tk-containing retroviral recombinants were used to infect hepatoma cells (BEL-7402) and the cells were treated with ganciclovir (0-1000 microg/ml). The results showed that HSV-tk gene could be efficiently transferred in vitro into hepatoma cells and stably expressed. The growth potential of the tk-containing cells was significantly inhibited by GCV (P

Adenovirus-Mediated Herpes Simplex Virus Thymidine Kinase Gene Transfer Driver by KDR Promoter in Treatment of Experimental Human HepatocelLular Carcinoma in Nude Mice

Objective： To investigate the therapeutic efficacy of adenovirus-mediated herpes simplex virus thymidine kinase （HSV-tk） gene transfer under the driving of KDR promoter （AdKDR-tk） in combination of ganciclovir （GCV） against human hepatocellular carcinoma in nude mice. Methods： HepG2 cell line was implanted subcutaneously into 32 nude mice, which were subsequently divided into 4 groups （n=8 each group）： Ganciclovir group （Ⅰ）, Ad group （Ⅱ）, AdCMV-tk/GCV group （under the driving of CMV promoter） （Ⅲ） and AdKDR-tk/GCV group （Ⅳ）. Then intratumoral injection of recombinant adenovirus or Ad was performed in all nude mice, and repeated 24 h later. For the following 10 d GCV was given at a dose of 100 mg/（kg· d）, ip. All the treated animals were killed to evaluate the tumor weight and the histopathological changes and the microvessel density of tumors after the treatment was determined. Results Compared with group Ⅰ, the tumor inhibitory rate was 12.3% in group Ⅲ and 24.5% in group Ⅳ; the inhibition rates were significantly different between group Ⅲand Ⅳ （P〈0.05）. The mean MVDs in group Ⅰ, Ⅱ, Ⅲ and Ⅳ were 37.4±8.6, 30.6±7.8, 27.6±7.1, and 10.7±4.1 （microvessels/mm^2）, respectively. Significant differences were found between group Ⅲ and Ⅱ （P〈0.05）, Ⅳ and Ⅱ （P〈0.01）, and Ⅳ and Ⅲ （P〈0.01）. Conclusion： Intratumoral injection of AdKDR-tk results in marked inhibition of HCC growth through inhibition angiogenesis in nude mice. It may be a new treatment approach for human HCC,

Vascular damage and anti-angiogenic effects of tumor vessel-targeted adenovirus-mediated herpes simplex virus thymidine kinase gene

AIM： To explore the therapeutic efficacy and mechanism of herpes simplex virus-thymidine kinase （HSV-tk） targeting angiogenesis against hepatocellular carcinoma in vivio and in vitro. METHODS： Recombinant adenovirus containing kinase domain insert with receptor （KDR） or cytomegalovirus （CMV） promoter-controlled HSV-tk gene （AdKDR-tk and AdCMV-tk） was constructed using pAdeasy system. The expression of KDR antigen in human umbilical venous endothelial cells （HUVEC） and HepG2 was detected with histological analysis of cells. The virus was used to infect HUVEC and HepG2. Following administration of ganciclovir （GCV）, the survival rate of gene-transfected HUVEC and HepG2 was evaluated by MTT method. To develop hepatocarcinomas in 32 Balb/C mice with HepG2 cells, the mice were divided into four groups： ganciclovir group （Ⅰ）, Ad group （Ⅱ）, AdCMV-tk group （Ⅲ） and AdKDR-tk group （Ⅳ）. Then selective administration of recombinant adenovirus or Ad via the intratumorial was given to all rats. Ganciclovir （GCV） was given at a dose of 100 mg·kg^-1·d^-1 （ip） started on the following day and lasted 10 d. Microvessel density （MVD） of tumor in all the treated animals were examined by the immunohistochemical methods and tumor burden was evaluated 10 d before and alter the last GCV dose.RESULTS： Immunocytochemical staining indicated the expression of KDR antigen in HUVEC. Under adenovirus infection index of 100, with increasing GCV concentration from 0 up to 50 mg/L, the survival rate of AdKDR-tk- transfected HUVEC and HepG2 decreased from 100% to （28.94 ± 5.67）% and （75.45 ± 2.91）% at proper order, respectively （P 〈 0.01）, while the survival rate of AdCMV- tk-transfected HUVEC and HepG2 declined from 100% to （17.56 ± 2.48）% and （23.15± 5.72）%, respectively （P 〉 0.05）. Compared with group I, there was a decrease of tumor weight by 14.7% in group Ⅲ and by 23.6% in group Ⅳ. And there was a distinct difference between group M and Ⅳ （P 〈 0.05）. The median MVD for all groups was 37.4 ± 8.6, 30.6 ± 7.8, 27.6 ± 7.1, and 10.7 ± 4.1 （microvessels/mm^2） in group Ⅰ, Ⅱ, M and IV, respectively. And there was a marked difference between group M and Ⅱ （P 〈 0.05）, Ⅳ and Ⅱ （P 〈 0.01）, and Ⅳ and M （P 〈 0.01）. CONCLUSION： KDR promoter-HSV-tk gene may effectually restrain the growth of tumor via targeting angiogenesis for hepatocellular carcinoma with treatment of GCV.

HE TREATMENT OF HEPATIC CARCINOMA WITH HERPES SIMPLEX THYMIDINE KINASE GENE/ACYCLOVIR SYSTEM

A retroviral vector(LNHcTL)containing the herpes simplex virus type 1 thymldine kinase(HSVI-tk)gene was constructed and used for transduction of the gene into human hepatocellular carcinoma cells(SMMC-7721).Xenografted tumor on nude mice was produced with the injection of the transduced cells(SMMC- 7721/LN HcTL) inoculated subcutaneously and showed regression when treated with Acyclovir.The mean weight of the residual tumors was six times less than that of the controls'tumors. Patients with liver carcinoma were given an intratumoral injection of ampbotropic packing cells(PA317/LNHcTL)producing HSV1-tk recombinant retroviral particles,and then treated with Acyclovir intravenously, which showed a marked regression of the tumor.Our preliminary data suggest that HSV1-tk gene/Acyclovir system might be a useful therapeutic approach for the treatment of hepatic carcinoma in humans.

Cooperative Therapeutic Effects of Herpes Simplex Virus Thymidine Kinase Gene/Ganciclovir System and Chemotherapeutic Agents on Prostate Cancer in vitro

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） im- munohistochemical 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.

Construction of the recombinant vector carrying herpes simplex virus thymidine kinase and cytokine genes expressed in cell line Tca8113

Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell line Tca8113. Methods: IL-2 cDNA was obtained by reverse transcription. Hsv-tk, IL-2 and IRES genes were amplified by PCR. The purified amplification products were inserted into pGEM-T-Easy, and transformed into E.coli JM109. The purified recombinant plasmids were identified by restriction endonucleases. The recombinant plasmids were digested and pEGFP-N 3 were linearized, DNA fragments of Hsv-tk, IRES and IL-2 were ligated into linearized pEGFP-N 3, and then transferred into E.coli JM109. The recombinant tk-IL-2 genes were cloned separately and introduced into the expression vector pEGFP-N 3 containing GFP. The recombinant vectors were identified by their restriction sites through PCR. The plasmids pEGFP-TI was also transfected into Tca8113 cells by calcium phosphate method for the expression of fusion proteins. Fusion genes expressing vector PL(TI)SN was generated by the fusion of HSV-tk, IRES and IL-2 with the use of DNA recombination technology. The recombinant retroviruses were transferred into Tca8113 cells by lipofectamine. The positive clones were obtained after G418 selection and named Tca/TI respectively. Results: The pEGFP-TI pasmid was identified respectively by restriction endonucleases, and their fragment sizes were 1 120 bp and 450 bp. The pEGFP-TI pasmid as templates were amplified respectively by PCR, and their PCR products were 1 120 bp and 450 bp. The pEGFP-TI vectors were used to transfect Tca8113 cell, and the cells with fluorescence accounted for 60% of the total amount. Conclusion: pFGFP-tk-IRES-IL-2 expressing vector is easy to assess the expression of tk-IRES-IL-2-GFP fusion protein localization in transfected cells. The successful construction of expressing vector containing fusion genes of Hsv-tk, IRES and IL-2 may be beneficial for gene therapy in cell line Tca8113.

High efficient generation of replication-defective adenoviruses containing thymidine kinase by homogeneous recombination in bacteria

Background Suicide gene therapy is a widely used molecular treatment for head and neck cancer. In this study, we try to use the method of homogenous recombination in bacteria to clone thymidine kinase gene （tk）-a kind of suicide gene to adenovirus backbone vectors for the construction of replication-defective adenoviruses. Methods pAdTrack-CMV/tk was constructed through subclone of a restriction endonuclease fragment including thymidine kinase gene from plasmid pCMV-tk to another plasmid pAdTrack-CMV, and then co-transfected with supercoiled pAdEasy-1, which was an adenoviral backbone vector except for deletions of E1 and E3, to competent E. coli BJ5183 for homogenous recombination using electroporation procedure. With the same method, pAdTrack-CMV was also co-transformed with pAdEasy-1 for homogenous recombination in BJ5183. Identified with restriction endonuclease Pad and polymerase chain reaction （PCR）, plasmids pAd-GFP/tk and pAd-GFP were successfully constructed. Each of them was digested with Pacl and sequently transfected into human embryo kidney 293 cells （HEK293） using Lipofectamine 2000. Results Comet-like adenovirus-producing foci of Ad-GFP/tk and Ad-GFP were observed after 5 to 7 days of cell culture After twelve days of packaging, the replication-defective adenoviruses were collected. Identified with PCR, thymidine kinase gene was successfully constructed into Ad-GFP/tk. Conclusion The replication-defective adenoviruses containing thymidine kinase can be constructed more easily by homogenous recombination in bacteria than conventional techniques.

Evaluation of miR-122-regulated suicide gene therapy for hepatocellular carcinoma in an orthotopic mouse model

Objective： Intratumoral administration of adenoviral vector encoding herpes simplex virus （HSV） thymidine kinase （TK） gene （Ad-TK） followed by systemic ganciclovir （GCV） is an effective approach in treating experimental hepatocellular carcinoma （HCC）. However, hepatotoxicity due to unwanted vector spread and suicide gene expression limited the application of this therapy, miR-122 is an abundant, liver-specific microRNA whose expression is decreased in human primary HCC and HCC-derived cell lines. These different expression profiles provide an opportunity to induce tumor-specific gene expression by miR-122 regulation. Methods： By inserting miR-122 target sequences （miR-122T） in the 3＇ untranslated region （UTR） ofTK gene, we constructed adenovirus （Ad） vectors expressing miR-122-regulated TK （Ad-TK-122T） and report genes. After intratumoral administration of Ad vectors into an orthotopic miR-122-deficient HCC mouse model, we observed the miR-122-regulated transgene expression and assessed the antitumor activity and safety of Ad-TK-122T. Results： Insertion of miR-122T specifically down-regulated transgene expression in vitro and selectively protected the miR-122-positive cells from killing by TK/GCV treatment. Insertion of miR-122T led to significant reduction of tansgene expression in the liver without inhibition of its expression in tumors in vivo, resulting in an 11-fold improvement of tumor-specific transgene expression. Intratumoral injection of Ad vectors mediated TK/GCV system led to a vector dosage-dependent regression of tumor. The insertion of miR-122T does not influence the antitumor effects of suicide gene therapy. Whereas mice administrated with Ad-TK showed severe lethal hepatotoxicity at the effective therapeutic dose, no liver damage was found in Ad-TK-122T group. Conclusions： miR-122-regulated TK expression achieved effective anti-tumor effects and increased the safety of intratumoral delivery of adenovirus-mediated TK/GCV gene therapy for miR-122-deficient HCC.

Altered Expression of Connexin-43 and Impaired Capacity of Gap Junctional Intercellular Communication in Prostate Cancer Cells

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 elu- cidate 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 re- verse-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 malig- nant 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. As- sessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was ab- normally located and markedly diminished as compared with normal prostatic epithelial ones, dis- playing 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 indi- cated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein par- ticipated 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.

HSVTK Gene Therapy for CarcinoembryonicAntigen-Producing Human Lung Cancer Cells

The long-term success of gene therapy for cancer relies heavily on the development of effective targeting systems. We investigate the possibility of targeted gene therapy using promoter of carcinoembryonic antigen (CEA) gene. By using luciferase reporter gene, we found that CEA promoter exhibit 16 times high activity in CEA-producing lung cancer cells, A549 than in nonproducing cells, Hela. We also constructed a recombinant expression plasmid pCEATK, in which CEA promoter drives the effector gene, thymidine kinase gene of Herpes Simplex Virus (HSVTK). A549 cells transfected with pCEATK became 865 times more sensitive to ganciclovir (GCV) than the control cells. However, Hela cells transfected with this plasmid remained resistant to GCV. These data indicate the potential for targeted gene therapy using the CEA promoter against CEA-producing tumor cells, such as lung cancer cells.

Construction of plasmid vector pAFP-HSVtk-IRES2-EGFP and its effect on the cytotoxicity of ganciclovir to hepatocellular carcinoma

Background Herpes simplex virus thymidine kinase phosphorylates ganciclovir to ganciclovir monophosphate,which is then converted to ganciclovir triphosphate by endogenous cellular nucleoside kinases.The ganciclovir triphosphate acts as a DNA chain terminator due to the lack of a functional 3＇-OH group and terminates the process of DNA replication,hence leading to cell apoptosis.At present,HSVtk gene usually acts as suicide gene to kill tumor cells.The aim of this study was to investigate the selective cytotoxicity of the herpes simplex virus thymidine kinase/ganciclovir （HSVtK/GCV） suicide gene system controlled by the α-fetoprotein （AFP） promoter on hepatocellular carcinoma （HCC） cells in vitro.Methods pAFP-HSVtk-IRES2-EGFP recombinant plasmid vectors driven by the AFP promoter were constructed.HL-7702 liver cells,HUH-7 HCC,and HepG2 HCC were transfected with the recombinant plasmids.HSVtK gene expression was detected using Western blotting analysis.HepG2 cells line stably expressing HSVtk gene was selected by G418 reagent.The cytotoxicity of HSVtK/GCV suicide gene system on hepatoma cells was measured by CCK-8 reagents when different doses of ganciclovir were added.Results Plasmid pAFP-TK-IRES2-EGFP-expressed HSVtk gene was constructed successfully.HSVtk gene expression level was significantly higher in AFP-positive hepatoma cells than in AFP-negative liver cells.After G418 selection,a HepG2 cells line stably expressing HSVtk gene was acquired.With the increase of the dose of ganciclovir the optical density at 450 nm of HepG2 cells stably expressing HSVtk gene gradually decreased （P ＜0.05）.Conclusion The HSVtK gene-specific expression in hepatoma cells as well as the cytotoxicity of the suicide gene system in HepG2 cells provided the basis for the targeted gene therapy of HCC.

Characteristics of ovarian cancer cells transduced by the bicistronic retroviral vector containing GM-CSF and HSV-TK genes

Objectives To explore whether HSV-TK (herpes simplex virus thymidine kinase) and GM-CSF (granulocyte-macrophage colony-stimulating factor) genes could be linked by internal ribosome entry site (IRES) in one retroviral vector and expressed by ovarian cancer cells following transfection, and to observe the characteristics of the transduced cells.Methods Retroviral vector pLGM-I-TK was constructed by linking the HSV-TK gene and GM-CSF gene with the IRES sequence. By using the “ping-pong' technique, pLGM-I-TK was transfected into the packaging cell line, PA317, to produce a PA317/TK-GM cell line. Using the resulting viral supernatant to infect the ovarian cancer cell line SKOV3, PCR and RT-PCR were used to explore the integration and transcription of HSV-TK and GM-CSF genes. The cytotoxicity of GCV (gancyclovir) on SKOV3/TK-GM was determined by MTT assay and the bystander effect of the HSV-TK/GCV system was also assessed. ELISA was used to measure the expression of GM-CSF by the transgene cells.Results The bicistronic retroviral vector constructed could be successfully transduced into PA317 and the titer of the retroviral vector was about 8.6×105?cfu/ml. PCR and RT-PCR demonstrated the successful integration and transcription of HSV-TK and GM-CSF genes transduced into the SKOV3 cell. SKOV3/TK-GM cells could be killed by GCV, and the IC50 was 0.7?μg/ml. The bystander effect was demonstrated. The expression level of GM-CSF in SKOV3/TK-GM was 60.4?ng*ml-1*106 cells-1*2 days-1.Conclusion The IRES sequence can be used to construct retroviral vectors to facilitate co-transfection of two genes. SKOV3/TK-GM cells can simultaneously express the HSV-TK and GM-CSF genes with biological activities which could be useful for enhancing the function of immune cells on the basis of suicide gene therapy.