Abstract A quantitative in vitro assay is described for the cytotoxic activity in tumor necrosis serum. The assay, which is based on the ability of survivor cells to incorporate the dye neutral red, measures the volume test substance killing 50,000 mouse L-M cells with an accuracy of ±6%. It detects cytotoxic/cytostatic effects at dilutions of tumor necrosis serum as great as 1 part in 64,000. The activity appears to segregate upon purification into multiple size forms that share similar physicochemical properties. Cytotoxic activity is not inhibited by the protease inhibitors diisopropylfluorophosphate, phenylmethylsul-fonylfluoride, and bovine pancreatic trypsin inhibitor; however, 20% normal serum in the assay medium inhibits cytotoxicity 66%. The activity is inactivated by treatment with serine proteases, sodium periodate, and dithiothreitol. The activity is markedly pH labile, stable at 56°C, and inactivated at 70°C. Multiple forms fractionate on gel filtration in a m.w. range of 225,000 to 100,000. These forms migrate electrophoretically in the α-globulin region. Another form fractionates with an apparent size of 50,000 and migrates electrophoretically anodal to albumin. Different size fractions are partially purified such that the cytotoxic activity per volume exceeds the parent serum. Two fractions (m.w. 225,000 and 50,000) fail to induce in vivo tumor necrosis of an intradermal tumor implant. A fraction with an intermediate size (m.w. 160,000) induces in vivo tumor necrosis. We suggest the cytotoxic and the necrotizing activities in tumor necrosis serum may be distinct.
Kull et al. (Wed,) studied this question.