Antitumor effects of the fibroblasts transfected TNF-α gene and its mutants

Abstract

To compare the anti-tumor effects of transmembrane TNF-α (TM-TNF) and secreted TNF-α (S-TNF)in vivo, mouse fibroblasts NIH3T3 were transfected separately with three types of retrovirus containing wild type TNF-α (Wt-TNF), TM-TNF mutant (TM-TNFm), S-TNF mutant (S-TNFm). Southern blot, RT-PCR, FACS and bioassay were used to investigate TNF-α gene integration, expression and its biological activity. It was found that both fixed cells and supernatant of NIH3T3/Wt-TNF, the fixed cells of NIH3T3/TM-TNFm and the supernatant of NIH3T3/S-TNFm could express high level of TNF-α or its mutants and effectively kill H22in vitro. The trans-fected NIH3T3 were separately injected into the mice at the sites of H22 tumor cell inoculation according to a ratio of 5∶1 or 1∶1 (effector/target cells, E/T) after the third day of H22 challenge, respectively. At the E/T=5∶1, the NIH3T3/TM-TNFm induced the highest tumor regression, while NIH3T3/S-TNFm exerted the strongest tumor depressing effect at the E/T=1∶1in vivo. No obvious side effects were noted throughout the course of treatment. The results suggest that both TM-TNF and S-TNF could cause tumor regression. The anti-tumor effect of TM-TNF would be more powerful and safe than that of S-TNF at the proper E/T ratio.

Keywords

tumor gene therapy / transmembrane TNF-α / retroviral vector

References

[1] VilcekJ, LeeT H. Tumor necrosis factor: New insights into the molecular mechanism of its multiple actions. J Biol Chem, 1991, 2667313-7313

[2] LuettigB, DeckerT, Lohmann-MatthesM. Evidence for the existence of two forms of membrane tumor necrosis factor: an integral protein and a molecule attached to its receptor. J Immunol, 1989, 1434034-4034

[3] PerezC, AlbertIr, DeFayK, et al. . A nonsecretable cell surface mutant of tumor necrosis factor (TNF) kills by cell-to-cell contact. Cell, 1990, 63251-251

[4] FlickD A, GiffordG E. Comparison ofin vitro cell cytotoxic assays for tumor necrosis factor. J Immunol Methods, 1984, 68167-167