WEHI-164 clone 2F: in vitro antitumor effects of tumor necrosis factor and gamma-interferon

Based on their enhanced susceptibility to tumor necrosis factor (TNF), sublines of the BALB/c fibrosarcoma WEHI-164 are more and more frequently being used to measure levels of TNF in cell-free supernatants. In studies aimed at characterizing the cytostatic and cytolytic effects of TNF and gamma-interferon (IFN-gamma) on a highly TNF-sensitive subline of WEHI-164, we show that: (1) the MTT assay which measures viable cell numbers (i.e. both cytostasis and cytolysis) at appropriate target cell numbers is sensitive to less than 0.1 pg/ml of recombinant murine TNF (rmTNF) without the need to add protein synthesis inhibitors such as actinomycin D; (2) using irradiated cells, the MTT assay is significantly more sensitive and measures cell lysis but not stasis; (3) clone 2F is sensitive to the cytostatic, but not cytolytic effects of recombinant murine IFN-gamma; (4) although lysis of 2F by rmTNF requires more than 5 h, a short exposure (30-60 min) to rmTNF is sufficient to trigger the lytic process, and (5) lysis of 2F is not cell-cycle-dependent. The use of highly sensitive indicator cell lines such as 2F allow for the measurement of low levels of TNF present in culture supernatants and for studies into the mechanism of action of TNF. However, sensitivity to other agents such as IFN supports the need to confirm that the antitumor effects seen are due to TNF via neutralizing antibody.