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Possible Requirement of Internalization in the Mechanism of in Vitro Cytotoxicity in Tumor Necrosis Serum¹

Wellcome Research Laboratories, Department of Molecular Biology, Research Triangle Park, North Carolina 27709

The mechanism of in vitro cytotoxicity by tumor necrosis serum (TNS) and a purified fraction was examined using sensitive L-M cells. Cell death was assessed by uptake of the dye trypan blue and/or by release of radiolabeled chromium. Cell killing was time and dose dependent. Cell survival was assessed by counting the number of survivors via their ability to internalize the dye neutral red and/or by adherent cell protein. The cytotoxin was not cytostatic. Survival was found to be inversely proportional to cell death. The change in survival was used to estimate the number of cells killed. The number of cells killed was logarithmically related to the amount of toxin. The ability to kill a fixed number of cells was inversely related to the number of cells in the assay well. It was estimated that, at ideal cell seed numbers, 1 µg of TNS protein per 250 µl killed about 50,000 cells in a 20-hr period. Sensitivity was equated with the amount of TNS required to kill 35,000 cells in 20 hr. Inhibitors of RNA and protein synthesis and also elevated temperatures enhanced sensitivity. The combined treatment of 1 µM actinomycin D and 40° enhanced sensitivity by 15-fold. Resistant normal and tumorigenic cell lines (including human) were rendered sensitive by concomitantly treating them with TNS and cycloheximide or actinomycin D. Cytoskeletal-disrupting agents (colchicine, Colcemid, and cytochalasin B), inhibitors of lysosome activity (chloroquine, methylamine, and leupeptin), and 32° all depressed sensitivity. Sensitivity was nearly equivalent in calcium-free medium. Results substituting partially purified cytotoxin were similar. The results suggest that a toxic factor(s) may need to be internalized and that lysosomal activity may be necessary for cell killing. Comparisons with lymphotoxin are discussed.

¹ Presented in part at the annual meetings of the Federation of the American Societies of Experimental Biology, 1980 (16).

² To whom requests for reprints should be addressed.

Received 12/10/80. Accepted 8/21/81.