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Atypical apoptotic cell death induced in L929 targets by exposure to tumor necrosis factor
1Department of Medicine, Veterans Administration Wadsworth-University of California, Los Angeles Medical Center 90073, USA.
Abstract:
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.
Insights
Tumor necrosis factor (TNF) induces a unique form of apoptosis in L929 cells, where cell death occurs before DNA fragmentation. Zinc protects against TNF cytotoxicity by acting before DNA fragmentation.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Tumor necrosis factor (TNF) is a key cytokine involved in inflammation and immunity.
- Understanding the precise mechanisms of TNF-induced cell death is crucial for therapeutic development.
Purpose of the Study:
- To investigate the mechanism of TNF-induced cytotoxicity in murine fibroblasts (L929 cells).
- To characterize the temporal relationship between TNF-induced apoptosis and DNA fragmentation.
Main Methods:
- DNA electrophoresis and morphologic analysis to detect apoptosis.
- Assessing cytotoxicity via membrane permeabilization (trypan blue, propidium iodide staining).
- Investigating the effect of zinc on TNF-induced cell death and DNA fragmentation.
Main Results:
- TNF induced apoptosis in L929 cells, characterized by chromatin condensation but delayed DNA fragmentation.
- Cytotoxicity (membrane permeabilization) preceded DNA fragmentation in TNF-treated L929 cells.
- Zinc inhibited both TNF cytotoxicity and DNA fragmentation, suggesting a protective mechanism upstream of DNA cleavage.
Conclusions:
- L929 cells exhibit an atypical apoptotic response to TNF, with cytotoxicity preceding DNA fragmentation.
- This atypical apoptosis is specific to L929 cells under TNF treatment, not a general response.
- Zinc's protective effect against TNF involves mechanisms acting before the final DNA fragmentation stage.