Cytotoxicity mediated by tumor necrosis factor in variant subclones of the ME-180 cervical carcinoma line: modulation by specific inhibitors of DNA topoisomerase II

The mechanism of tumor necrosis factor (TNF)-induced cytotoxicity has been investigated using two clonal variants of the ME-180 human cervical carcinoma cell line. The clonal lines were characterized with respect to their expression of TNF receptors, kinetics of cell death, and their ability to communicate intercellularly through gap junctions. The ME-180.4 and ME-180.8 clones were identified by their relative sensitivity to TNF induced lysis in a 24-h assay. The dose of TNF required to kill 50% of the target cells was 60 pM for the sensitive ME-180.4 and 2.5 nM for the ME-180.8. However, when assay times were extended, the dose response for both clones was the same, indicating that a difference in the kinetics of cell death and not absolute TNF sensitivity existed between the ME-180.4 and ME-180.8 clones. Both clones were gap junction deficient as judged by their inability to transfer Lucifer yellow or 6-carboxyfluorescein, a characteristic phenotype of cells sensitive to cytotoxicity by TNF. The level of surface receptor expressed on these clones was nearly identical with a Kd = 0.3 nM and 5,000 binding sites per cell. Measurement of the kinetics of cell death revealed that the time between the addition of TNF and the onset of observed cell death (induction phase) was much shorter for the ME-180.4 (32-55 h) than for the resistant ME-180.8 (55-80 h). Mitomycin C, a DNA alkylating agent, significantly reduced the length of the induction phase for both clones, although the kinetic difference between the clones remained unchanged. Two epipodophyllotoxins, VP-16 and VM-26, which specifically inhibit the rejoining activity of DNA topoisomerase II, showed a 10-100-fold synergistic effect when combined with TNF as shown by isobologram analysis. VM-26 when added to the resistant ME-180.8 clones decreased the length of induction phase and abolished the kinetic difference observed with the ME-180.4 clone. These results indicate that the variance in the TNF response of these two clones was closely associated with DNA topoisomerase II, and suggest that this enzyme may play an important role in TNF mediated cytotoxicity.