Atypical Apoptotic Cell Death Induced in L929 Targets by Exposure to Tumor Necrosis Factor

The mechanism by which tumor necrosis factor (TNF) induces cytotoxicity of murine fibroblasts was investigated. Electrophoresis of DNA extracted from TNF-treated L929 targets showed fragmentation of DNA into a ladder-like pattern, typical of cells dying by apoptosis. Morphologic analysis also indicated apoptotic cell death, demonstrating clumping and crescentic condensation of chromatin. In contrast, chromatin condensation and ladder-like DNA fragmentation were not detected in L929 targets dying by necrosis from exposure to heat, repeated cycles of freeze-thaw, and sodium azide. Chromatin condensation was an early event, detected as early as 6 h of incubation. However, DNA fragmentation (assayed by double-stranded fragmentation assay and gel electrophoresis), as well as the apoptotic changes detected by Hoechst fluorescence, both occurred later and did not precede TNF cytotoxicity (membrane permeabilization detected by trypan blue or propidium iodide staining). This atypical pattern of apoptosis was a characteristic of L929 target cells rather than a generalized cytotoxic response to TNF because TNF-treated squamous cancer cells showed typical features of apoptosis (DNA fragmentation before cytotoxicity) and etoposide-treated L929 cells demonstrated the same atypical kinetics as TNF-treated cells. Zinc significantly inhibited TNF cytotoxicity as well as DNA fragmentation of L929. However, because DNA fragmentation occurred belatedly in TNF-treated targets, lagging behind cytotoxicity, the protection by zinc against TNF appears mediated by events that occur before the ultimate endonuclease-induced cleavage of DNA into small fragments.