Combined antitumor effects of an adenoviral cytosine deaminase/thymidine kinase fusion gene in rat C6 glioma

JinWoo Chang, Heuiran Lee, Eunhee Kim, Yong Lee, Sang Sup Chung, Joo Hang Kim

Research output: Contribution to journalArticle

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Abstract

OBJECTIVE: In this study, we investigated the feasibility of a double-suicide gene/prodrug therapy, involving direct introduction of the herpes simplex virus Type I thymidine kinase (TK) gene and the Escherichia coli cytosine deaminase (CD) gene, via a recombinant adenoviral vector, and ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) treatment, in a rat C6 glioma model. METHODS: Efficient gene transfer and transduction of C6 glioma cells via a recombinant adenovirus were evaluated by infecting cells with adenovirus bearing the β-galactosidase gene and then staining cells with X-5-bromo-4-chloro-3-indolyl-13-D-galactoside. CD/TK expression in cells infected with adenovirus bearing the CD/TK gene (ad-CD/TK) was examined by immunoblotting analysis. For in vitro cytotoxicity experiments, the cells were infected with ad-CD/TK or ad-ΔE1 (as a control). After the addition of a variety of concentrations of GCV and 5-FC, either separately or in combination, cell viability was determined by staining the cells with crystal violet solution 6 days after infection. For in vivo antitumor experiments, 1 × 105 cells were stereotactically injected into the right caudate-putamen of female Wistar rat brains. At 3 days after implantation, 1 × 108 plaque-forming units of ad-CD/TK or ad-δE1 (as a control) were stereotactically injected into the tumors and GCV (25 mg/kg) and 5-FC (250 mg/kg), alone or in combination, were intraperitoneally administered. Animals were then killed, and tumor volumes were measured by determining the tumor area in every fifth section, using a light microscope. RESULTS: C6 glioma cells were efficiently transduced with recombinant adenoviral vector at multiplicities of infection of 200 or more. In vitro cytotoxicity of GCV and/or 5-FC, either alone or in combination, was exclusively observed in the cells transduced with ad-CD/TK. Obvious cytotoxicity (>50% inhibition) was observed in the presence of 5-FC at concentrations greater than 30 μg/ml or GCV at concentrations greater than 0.3/μg/ml at a multiplicity of infection of 100. Additionally, cytotoxicity in the presence of both GCV and 5-FC was greater than that after single-prodrug treatments, indicating additive effects of the prodrug treatments. In in vivo experiments, the tumor volumes of the rats treated with GCV or 5-FC alone after ad-CD/TK injection (59.1 ± 4.6 and 57.4 ± 7.1 mm3, respectively) were significantly smaller than that of the control rats (157 ± 8.9 mm3, P < 0.05). Furthermore, the tumor volume of the rats treated with GCV and 5-FC in combination was 14.7 ± 1.8 mm3. CONCLUSION: These results demonstrated the efficient transduction of C6 glioma cells with a recombinant adenovirus and the additive effects of CD/TK fusion gene/GCV/5-FC treatment, compared with single-gene therapy with the TK or CD gene. Therefore, our data suggest that the direct administration of a double-suicide gene/prodrug therapy has great potential in the treatment of brain tumors.

Original languageEnglish
Pages (from-to)931-939
Number of pages9
JournalNeurosurgery
Volume47
Issue number4
DOIs
Publication statusPublished - 2000 Jan 1

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Cytosine Deaminase
Flucytosine
Thymidine Kinase
Gene Fusion
Ganciclovir
Glioma
Adenoviridae
Genes
Prodrugs
Tumor Burden
Genetic Therapy
Suicide
Infection
Galactosidases
Staining and Labeling
Gentian Violet
Therapeutics
Galactosides
Putamen
Feasibility Studies

All Science Journal Classification (ASJC) codes

  • Surgery
  • Clinical Neurology

Cite this

Chang, JinWoo ; Lee, Heuiran ; Kim, Eunhee ; Lee, Yong ; Chung, Sang Sup ; Kim, Joo Hang. / Combined antitumor effects of an adenoviral cytosine deaminase/thymidine kinase fusion gene in rat C6 glioma. In: Neurosurgery. 2000 ; Vol. 47, No. 4. pp. 931-939.
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abstract = "OBJECTIVE: In this study, we investigated the feasibility of a double-suicide gene/prodrug therapy, involving direct introduction of the herpes simplex virus Type I thymidine kinase (TK) gene and the Escherichia coli cytosine deaminase (CD) gene, via a recombinant adenoviral vector, and ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) treatment, in a rat C6 glioma model. METHODS: Efficient gene transfer and transduction of C6 glioma cells via a recombinant adenovirus were evaluated by infecting cells with adenovirus bearing the β-galactosidase gene and then staining cells with X-5-bromo-4-chloro-3-indolyl-13-D-galactoside. CD/TK expression in cells infected with adenovirus bearing the CD/TK gene (ad-CD/TK) was examined by immunoblotting analysis. For in vitro cytotoxicity experiments, the cells were infected with ad-CD/TK or ad-ΔE1 (as a control). After the addition of a variety of concentrations of GCV and 5-FC, either separately or in combination, cell viability was determined by staining the cells with crystal violet solution 6 days after infection. For in vivo antitumor experiments, 1 × 105 cells were stereotactically injected into the right caudate-putamen of female Wistar rat brains. At 3 days after implantation, 1 × 108 plaque-forming units of ad-CD/TK or ad-δE1 (as a control) were stereotactically injected into the tumors and GCV (25 mg/kg) and 5-FC (250 mg/kg), alone or in combination, were intraperitoneally administered. Animals were then killed, and tumor volumes were measured by determining the tumor area in every fifth section, using a light microscope. RESULTS: C6 glioma cells were efficiently transduced with recombinant adenoviral vector at multiplicities of infection of 200 or more. In vitro cytotoxicity of GCV and/or 5-FC, either alone or in combination, was exclusively observed in the cells transduced with ad-CD/TK. Obvious cytotoxicity (>50{\%} inhibition) was observed in the presence of 5-FC at concentrations greater than 30 μg/ml or GCV at concentrations greater than 0.3/μg/ml at a multiplicity of infection of 100. Additionally, cytotoxicity in the presence of both GCV and 5-FC was greater than that after single-prodrug treatments, indicating additive effects of the prodrug treatments. In in vivo experiments, the tumor volumes of the rats treated with GCV or 5-FC alone after ad-CD/TK injection (59.1 ± 4.6 and 57.4 ± 7.1 mm3, respectively) were significantly smaller than that of the control rats (157 ± 8.9 mm3, P < 0.05). Furthermore, the tumor volume of the rats treated with GCV and 5-FC in combination was 14.7 ± 1.8 mm3. CONCLUSION: These results demonstrated the efficient transduction of C6 glioma cells with a recombinant adenovirus and the additive effects of CD/TK fusion gene/GCV/5-FC treatment, compared with single-gene therapy with the TK or CD gene. Therefore, our data suggest that the direct administration of a double-suicide gene/prodrug therapy has great potential in the treatment of brain tumors.",
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Combined antitumor effects of an adenoviral cytosine deaminase/thymidine kinase fusion gene in rat C6 glioma. / Chang, JinWoo; Lee, Heuiran; Kim, Eunhee; Lee, Yong; Chung, Sang Sup; Kim, Joo Hang.

In: Neurosurgery, Vol. 47, No. 4, 01.01.2000, p. 931-939.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Combined antitumor effects of an adenoviral cytosine deaminase/thymidine kinase fusion gene in rat C6 glioma

AU - Chang, JinWoo

AU - Lee, Heuiran

AU - Kim, Eunhee

AU - Lee, Yong

AU - Chung, Sang Sup

AU - Kim, Joo Hang

PY - 2000/1/1

Y1 - 2000/1/1

N2 - OBJECTIVE: In this study, we investigated the feasibility of a double-suicide gene/prodrug therapy, involving direct introduction of the herpes simplex virus Type I thymidine kinase (TK) gene and the Escherichia coli cytosine deaminase (CD) gene, via a recombinant adenoviral vector, and ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) treatment, in a rat C6 glioma model. METHODS: Efficient gene transfer and transduction of C6 glioma cells via a recombinant adenovirus were evaluated by infecting cells with adenovirus bearing the β-galactosidase gene and then staining cells with X-5-bromo-4-chloro-3-indolyl-13-D-galactoside. CD/TK expression in cells infected with adenovirus bearing the CD/TK gene (ad-CD/TK) was examined by immunoblotting analysis. For in vitro cytotoxicity experiments, the cells were infected with ad-CD/TK or ad-ΔE1 (as a control). After the addition of a variety of concentrations of GCV and 5-FC, either separately or in combination, cell viability was determined by staining the cells with crystal violet solution 6 days after infection. For in vivo antitumor experiments, 1 × 105 cells were stereotactically injected into the right caudate-putamen of female Wistar rat brains. At 3 days after implantation, 1 × 108 plaque-forming units of ad-CD/TK or ad-δE1 (as a control) were stereotactically injected into the tumors and GCV (25 mg/kg) and 5-FC (250 mg/kg), alone or in combination, were intraperitoneally administered. Animals were then killed, and tumor volumes were measured by determining the tumor area in every fifth section, using a light microscope. RESULTS: C6 glioma cells were efficiently transduced with recombinant adenoviral vector at multiplicities of infection of 200 or more. In vitro cytotoxicity of GCV and/or 5-FC, either alone or in combination, was exclusively observed in the cells transduced with ad-CD/TK. Obvious cytotoxicity (>50% inhibition) was observed in the presence of 5-FC at concentrations greater than 30 μg/ml or GCV at concentrations greater than 0.3/μg/ml at a multiplicity of infection of 100. Additionally, cytotoxicity in the presence of both GCV and 5-FC was greater than that after single-prodrug treatments, indicating additive effects of the prodrug treatments. In in vivo experiments, the tumor volumes of the rats treated with GCV or 5-FC alone after ad-CD/TK injection (59.1 ± 4.6 and 57.4 ± 7.1 mm3, respectively) were significantly smaller than that of the control rats (157 ± 8.9 mm3, P < 0.05). Furthermore, the tumor volume of the rats treated with GCV and 5-FC in combination was 14.7 ± 1.8 mm3. CONCLUSION: These results demonstrated the efficient transduction of C6 glioma cells with a recombinant adenovirus and the additive effects of CD/TK fusion gene/GCV/5-FC treatment, compared with single-gene therapy with the TK or CD gene. Therefore, our data suggest that the direct administration of a double-suicide gene/prodrug therapy has great potential in the treatment of brain tumors.

AB - OBJECTIVE: In this study, we investigated the feasibility of a double-suicide gene/prodrug therapy, involving direct introduction of the herpes simplex virus Type I thymidine kinase (TK) gene and the Escherichia coli cytosine deaminase (CD) gene, via a recombinant adenoviral vector, and ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) treatment, in a rat C6 glioma model. METHODS: Efficient gene transfer and transduction of C6 glioma cells via a recombinant adenovirus were evaluated by infecting cells with adenovirus bearing the β-galactosidase gene and then staining cells with X-5-bromo-4-chloro-3-indolyl-13-D-galactoside. CD/TK expression in cells infected with adenovirus bearing the CD/TK gene (ad-CD/TK) was examined by immunoblotting analysis. For in vitro cytotoxicity experiments, the cells were infected with ad-CD/TK or ad-ΔE1 (as a control). After the addition of a variety of concentrations of GCV and 5-FC, either separately or in combination, cell viability was determined by staining the cells with crystal violet solution 6 days after infection. For in vivo antitumor experiments, 1 × 105 cells were stereotactically injected into the right caudate-putamen of female Wistar rat brains. At 3 days after implantation, 1 × 108 plaque-forming units of ad-CD/TK or ad-δE1 (as a control) were stereotactically injected into the tumors and GCV (25 mg/kg) and 5-FC (250 mg/kg), alone or in combination, were intraperitoneally administered. Animals were then killed, and tumor volumes were measured by determining the tumor area in every fifth section, using a light microscope. RESULTS: C6 glioma cells were efficiently transduced with recombinant adenoviral vector at multiplicities of infection of 200 or more. In vitro cytotoxicity of GCV and/or 5-FC, either alone or in combination, was exclusively observed in the cells transduced with ad-CD/TK. Obvious cytotoxicity (>50% inhibition) was observed in the presence of 5-FC at concentrations greater than 30 μg/ml or GCV at concentrations greater than 0.3/μg/ml at a multiplicity of infection of 100. Additionally, cytotoxicity in the presence of both GCV and 5-FC was greater than that after single-prodrug treatments, indicating additive effects of the prodrug treatments. In in vivo experiments, the tumor volumes of the rats treated with GCV or 5-FC alone after ad-CD/TK injection (59.1 ± 4.6 and 57.4 ± 7.1 mm3, respectively) were significantly smaller than that of the control rats (157 ± 8.9 mm3, P < 0.05). Furthermore, the tumor volume of the rats treated with GCV and 5-FC in combination was 14.7 ± 1.8 mm3. CONCLUSION: These results demonstrated the efficient transduction of C6 glioma cells with a recombinant adenovirus and the additive effects of CD/TK fusion gene/GCV/5-FC treatment, compared with single-gene therapy with the TK or CD gene. Therefore, our data suggest that the direct administration of a double-suicide gene/prodrug therapy has great potential in the treatment of brain tumors.

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