Abstract
AbstractIt has been shown that the cytokine immunotherapy, which can enhance immune response to fight tumor, is a powerful tool for cancer therapy. Interleukin 3 (IL-3)-transduced into tumor, for example, can suppress tumor growth and generate the specific immunity against parental tumor. Although animal experiments and clinic trials show promise results, high concentration of IL3 circulating in body causes side effects. The most significant appearance resulted from high concentration of IL-3 in the blood is splenomegaly. The aim of this study is to examine whether the interrupting of the pathway that leads to IL-3 secretion could reduce the side effect during IL3 immunotherapy. Using gene recombination techniques, we construct pIRESpuro-IL3, pIRESpuro-IL3(64), pIRESpuro-IL3(66), pIRESpuro-IL3(168) and pIRESpuro-IL3(169) plasmids, which have different regions of signal sequence truncated. These plasmids were then transfected into NFsa and Fsa cells. Following puromycin selection, the mRNA expression of each construct was detected by RT-PCR. ELISA assay demonstrates that the deletion of signal sequence (clones from the transfection of pIRESpuro-IL3(64), pIRESpuro-IL3(66), pIRESpuro-IL3(168) and pIRESpuro-IL3(169) plasmids) prevents the secretion of IL-3 into medium. Immunocytochemistry further confirms that non-secreted form of IL-3 was indeed produced in cytosol region. To investigate whether the transfection of various forms of IL-3 constructs can induce the functional alteration, we performed TNF-alpha responsive assay and intrinsic radiosensitivity assay. The TNF-alpha responsive data show that the cells transfected with secretory form of IL-3 become more sensitive to TNF-alpha killing than those transfected with non-secreted form of IL-3. Furthermore, cells transfected with non-secreted form, like parental tumor, can proliferate under low concentration of TNF-alpha. The result from intrinsic radiosensitivity assay shows that the transfection of non-secreted form of IL-3 do not alter radiosensitivity. In animal experiments, mice bearing tumors transfected with non-secreted form of IL-3 do not develop splenomegaly. This suggests that the side effects reported from previous IL-3 gene immunotherapy can be reduced by this approach. In conclusion, this study suggests that it is possible to establish the model of non-secreted form of IL-3 by changing IL-3 signal sequence although its biological function needs further investigation. The potential of non-secreted form of IL-3 model in cancer therapy is in combining with radiotherapy. The aim of this approach does not only intend to enhance tumor curing rate, but it can also reduce the side effect.