| The TNF Receptor Expressions in Cancer Cells Transfected with TNF-alpha cDNA Using Retroviral Vector. |
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Hyuk Pyo Lee, Chul Gyu Yoo, Young Whan Kim, Young Soo Shim, Sung Koo Han |
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Department of Internal Medicine, College of Medicine and Lung Institute, Medical Research Center, Seoul National University, Seoul, Korea. |
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| Abstract |
BACKGROUND
Tumor necrosis factor(TNF) has been considered as an important candidate for cancer gene therapy based on its potent anti-tumor activity. However, since the efficiency of current techniques of gene transfer is not satisfactory, the majority of current protocols is aiming the in vitro gene transfer to cancer cells and re-introducing genetically modified cancer cells to host. In the previous study, it was shown that TNF-sensitive cancer cells transfected with TNF-alpha cDNA would become highly resistant to TNF, and the probability was shown that the acquired resistance to TNF might be associated with synthesis of some protective protein. Understanding the mechanisms of TNF-resistance in TNF-alpha cDNA transfected cancer cells would be an important step for improving the efficacy of cancer gene therapy as well as for better understandings of tumor biology. This study was designed to evaluate whether the levels of TNF receptor mRNA expression and soluble TNF receptor release from cancer cells are changed after TNF-alpha cDNA transfection. METHODS: We transfected TNF-alpha c-DNA to WEHI164(murine fibrosarcoma cell line), NCI-H2058(human mesothelioma cell line), A549(human non-small cell lung cancer cell line), ME180(human cervix cancer cell line) cells using retroviral veetor(pLT12SN(TNF)) and confirm the expression of TNF with PCR, ELISA, MTT assay. Then we determined the TNF resistance of TNF-alpha cDNA transfected cells(WEHI164-TNF, NCI-H2058-TNF, A549-TNF, ME180-TNF) and evaluated the TNF receptor mRNA expression with Northern blot analysis and soluble TNF receptor release with ELISA. RESULTS: The TNF receptor mRNA expressions of parental cells and genetically modified cells were not significanfly different The soluble TNF receptor levels of media from genetically modified cells were lower than those from parental cells. CONCLUISON: The acquired resistance to TNF after TNF-alpha cDNA transfection may not be associated with the change in the TNF receptor and the soluble TNF receptor expression. |
| Key Words:
Tumor necrosis factor(TNF), Gene transfection, Resistance, Mechanism, Protective protein, TNF receptor, Soluble TNF receptor |
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