Limits...
HSV-tk/GCV gene therapy mediated by EBV-LMP1 for EBV-associated cancer.

Lifang Y, Min T, Midan A, Ya C - J. Exp. Clin. Cancer Res. (2008)

Bottom Line: To investigate the feasibility of gene therapy in treating Epstein-Barr virus (EBV)-associated cancer by employing the suicide gene, herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV), which uses the signaling pathway through the HIV-long terminal repeat (LTR) gene which is expressed from a nuclear factor-kappaB (NF-kappaB)-binding motif-containing promoter that is regulated by EBV-latent membrane protein 1 (LMP1) via NF-kappaB.The activation of TK was increased after transfection of the pVLTR-tk into the EBV-LMP1 positive cells.After GCV treatment, the clonogenicity and survival of the cells substantially declined, and a bystander effect was also observed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Research Center, Cancer Research Institute, XiangYa School of Medicine, Central South University, ChangSha, Hunan, 410078, PR China. anglifang99@hotmail.com

ABSTRACT

Background: To investigate the feasibility of gene therapy in treating Epstein-Barr virus (EBV)-associated cancer by employing the suicide gene, herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV), which uses the signaling pathway through the HIV-long terminal repeat (LTR) gene which is expressed from a nuclear factor-kappaB (NF-kappaB)-binding motif-containing promoter that is regulated by EBV-latent membrane protein 1 (LMP1) via NF-kappaB.

Methods: First, we constructed the plasmid pVLTR-tk, which was regulated by EBV-LMP1 via NF-kappaB, and then investigated the cytotoxic effect of the pVLTR-tk/GCV on cancer cells, using MTT assays, clonogenic assays, flow cytometry, and animal experiments.

Results: The activation of TK was increased after transfection of the pVLTR-tk into the EBV-LMP1 positive cells. After GCV treatment, the clonogenicity and survival of the cells substantially declined, and a bystander effect was also observed. The LMP1 positive cells exhibited remarkable apoptosis following pVLTR-tk/GCV treatment, and the pVLTR-tk/GCV restrained tumor growth in vivo for EBV-LMP1 positive cancers.

Conclusion: The pVLTR-tk/GCV suicide gene system may be used as a new gene targeting strategy for EBV-associated cancer.

Show MeSH

Related in: MedlinePlus

pVLTR-tk/GCV inhibit the cell proliferation in LMP1 positive cells. Cells were transfected with pVLTR-tk and treated with GCV. Cell proliferation was measured by MTT analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2562992&req=5

Figure 2: pVLTR-tk/GCV inhibit the cell proliferation in LMP1 positive cells. Cells were transfected with pVLTR-tk and treated with GCV. Cell proliferation was measured by MTT analysis.

Mentions: CNE1, CNE1-LMP1, HNE2-LMP1, B95-8, and Raji cells were transfected with the pVLTR-tk, and incubated with GCV at various concentrations. A decrease in cell viability was observed upon increasing GCV concentration, the highest toxicity being obtained at 500 μg/mL of GCV for all cell lines. These results suggested that for GCV concentrations higher than 500 μg/mL, the limiting factor affecting cytotoxicity is not related to the level of thymidine kinase expression. As illustrated in Figure 2, in CNE1-LMP1, HNE2-LMP1, B95-8, and Raji cells, the survival cell rates were about 80% with 50 μg/mL of GCV, about 40% with 100 μg/mL and 200 μg/mL of GCV, and about 20% with 300 μg/mL of GCV. When the effects at the same concentration of GCV are compared, these cells seem to be more sensitive to GCV than CNE1 cells. (The level of toxicity remained unchanged in CNE1 cells at 50 to 300 μg/mL of GCV.) These results indicated that transfection with the pVLTR-tk increases sensitivity to GCV in EBV-LMP1 positive cells.


HSV-tk/GCV gene therapy mediated by EBV-LMP1 for EBV-associated cancer.

Lifang Y, Min T, Midan A, Ya C - J. Exp. Clin. Cancer Res. (2008)

pVLTR-tk/GCV inhibit the cell proliferation in LMP1 positive cells. Cells were transfected with pVLTR-tk and treated with GCV. Cell proliferation was measured by MTT analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562992&req=5

Figure 2: pVLTR-tk/GCV inhibit the cell proliferation in LMP1 positive cells. Cells were transfected with pVLTR-tk and treated with GCV. Cell proliferation was measured by MTT analysis.
Mentions: CNE1, CNE1-LMP1, HNE2-LMP1, B95-8, and Raji cells were transfected with the pVLTR-tk, and incubated with GCV at various concentrations. A decrease in cell viability was observed upon increasing GCV concentration, the highest toxicity being obtained at 500 μg/mL of GCV for all cell lines. These results suggested that for GCV concentrations higher than 500 μg/mL, the limiting factor affecting cytotoxicity is not related to the level of thymidine kinase expression. As illustrated in Figure 2, in CNE1-LMP1, HNE2-LMP1, B95-8, and Raji cells, the survival cell rates were about 80% with 50 μg/mL of GCV, about 40% with 100 μg/mL and 200 μg/mL of GCV, and about 20% with 300 μg/mL of GCV. When the effects at the same concentration of GCV are compared, these cells seem to be more sensitive to GCV than CNE1 cells. (The level of toxicity remained unchanged in CNE1 cells at 50 to 300 μg/mL of GCV.) These results indicated that transfection with the pVLTR-tk increases sensitivity to GCV in EBV-LMP1 positive cells.

Bottom Line: To investigate the feasibility of gene therapy in treating Epstein-Barr virus (EBV)-associated cancer by employing the suicide gene, herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV), which uses the signaling pathway through the HIV-long terminal repeat (LTR) gene which is expressed from a nuclear factor-kappaB (NF-kappaB)-binding motif-containing promoter that is regulated by EBV-latent membrane protein 1 (LMP1) via NF-kappaB.The activation of TK was increased after transfection of the pVLTR-tk into the EBV-LMP1 positive cells.After GCV treatment, the clonogenicity and survival of the cells substantially declined, and a bystander effect was also observed.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Research Center, Cancer Research Institute, XiangYa School of Medicine, Central South University, ChangSha, Hunan, 410078, PR China. anglifang99@hotmail.com

ABSTRACT

Background: To investigate the feasibility of gene therapy in treating Epstein-Barr virus (EBV)-associated cancer by employing the suicide gene, herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV), which uses the signaling pathway through the HIV-long terminal repeat (LTR) gene which is expressed from a nuclear factor-kappaB (NF-kappaB)-binding motif-containing promoter that is regulated by EBV-latent membrane protein 1 (LMP1) via NF-kappaB.

Methods: First, we constructed the plasmid pVLTR-tk, which was regulated by EBV-LMP1 via NF-kappaB, and then investigated the cytotoxic effect of the pVLTR-tk/GCV on cancer cells, using MTT assays, clonogenic assays, flow cytometry, and animal experiments.

Results: The activation of TK was increased after transfection of the pVLTR-tk into the EBV-LMP1 positive cells. After GCV treatment, the clonogenicity and survival of the cells substantially declined, and a bystander effect was also observed. The LMP1 positive cells exhibited remarkable apoptosis following pVLTR-tk/GCV treatment, and the pVLTR-tk/GCV restrained tumor growth in vivo for EBV-LMP1 positive cancers.

Conclusion: The pVLTR-tk/GCV suicide gene system may be used as a new gene targeting strategy for EBV-associated cancer.

Show MeSH
Related in: MedlinePlus