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KMID : 0391320000100010037
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Korean Journal of Biological Response Modifiers
2000 Volume.10 No. 1 p.37 ~ p.48
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Effects of Transforming Growth Factor-β (TGF-β) Type Ⅱ Receptor Introduced into JC Mammary Tumor Cells by Retroviral Vector on their Responsiveness to TGF-β
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Park Byung-Kiu
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Abstract
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Background: Transforming growth factor-β (TGF-β) type Ⅱ receptor (RⅡ) has been considered as a tumor suppressor in a variety of human cancers. The aim of this study was to assess the efficiency of retroviral mediated RⅡ gene transfer and investigate whether the responsiveness of JC, a murine mammary tumor cell line, to TGF-β-induced growth inhibitory effects could be restored following retroviral transfection of wild type (WT) RⅡ.
Methods: Recombinant retroviral vectors were constructed by cloning of either WT R Ⅱ or dominant negative (DN) RⅡ cDNA lacking intracellular domain along with internal ribosome entry site (IRES) and neomycin resistance gene into MFG vector. Transfected JC cells were evaluated for their expression of introduced RⅡ by Northern blot assay, and their responsiveness to exogenous as well as endogemous TGF-β1 by [3H]thymidine uptake assay.
Results: Parental JC cells exhibited much less RⅡ expression compared to CCL-64, normal lung epithelial cells. Transfected JC cells, selected in G418 containing media, expressed huge WT or DN RⅡ, indicating efficient transfection. The growths of parental and DN RⅡ trassfected JC cells (DN-RⅡ-JC) were stimulated in the presence
of exogenous TGF-β1 with their [3H]thymidine uptakes at 10 ng/ml of TGF-β1 being 120% and 123% of non-TGF-β1 treated control values, respectively. However, [3H]thymidine uptake of WT RⅡ tranfected cells (WT-RⅡ-JC) at 10 ng/ml of TGF-β1 was equal to control value. When neutraling antibodies against TGF-β1 were applied to the media to block endogenously produced TGF-β1, [3H]thymidine uptakes of parental and DN-RⅡ-JC cells were less than IgG1-treated control values. In contrast, anti-TGF-β1 antibodies raised [3H]thymidine uptake of WT-RⅡ-JC cells by 1.2-fold relative to control.
Conclusion: RⅡ and neomycin-resistance genes were simultaneously and efficiently transfected and expressed by retroviral vector containing IRES. The responsiveness of WT RⅡ transfected JC cells to TGF-β was restored in some degree, but not considerably, suggesting that other abnormalities in the TGF-β system might coexist in
JC cells.
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KEYWORD
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TGF-β, TGF-β type Ⅱ receptor, JC cells, Responsiveness, Retroviral transfection,
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