The Role of MnSOD in the Mechanisms of Acquired Resistance to TNF

Lee, Hyuk Pyo; Yoo, Chul Gyu; Kim, Young Whan; Han, Sung Koo; Shim, Young Soo

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 eDNA 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 the role of MnSOD, an antioxidant enzyme, in the acquired resistance to TNF of TNF-alpha cDNA transfected cancer cells.
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 vector(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 the changes of MnSOD mRNA expressions with Northern blot analysis.
RESULTS
The MnSOD mRNA expressions of parental cells and genetically modified cells of WEHI164 and ME180 cells(both are naturally TNF sensitive) were not significantly different The MnSOD mRNA expressions of genetically modified cells of NCI-H2058 and A549(both are naturally TNF resistant) were higher than those of the parental cells, while those of parental cells with exogenous TNF were also elevated. CONCLUISON: The acquired resistance to TNF after TNF-alpha cDNA transfection may not be associated with the change in the MnSOD expression, but the difference in natural TNF sensitivity of each cell may be associated with the level of the MnSOD expression.