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.
The luminous intensity of dark variant (S1) separated from photobacterium phosphoreum (A2) was 1/10 000 less than that of wild-type. Ethidium bromide (EB) (0.6 mg/L), Mytomycin C (MC, 0.05 mg/L), 2-amino fluorene (2-AF, 1.0 mg/L) all could strongly induce reversion mutation for S1 within 24 h and increase reversion ratio significantly. The results of experiments indicated that these revertants had stable genetic characteristic and the mutation may take place at gene levels. The mutagenesis to S1 caused by EB, MC and 2-AF was detected and it may be used as a new rapid, simple and sensitive method for gene toxicant monitoring.