Cell cycle-dependent tumor necrosis factor apoptosis.
To determine if tumor necrosis factor (TNF)-mediated apoptosis affects cells at defined stages of the cell cycle, WEHI-164/2F (WEHI) cells were synchronized at G0-G1 after 3-day cultures in medium containing RPMI 1640 and 0.5% FCS (RPMI-0.5% FCS). The arrested WEHI cells (60-75% in G0-G1) showed increased sensitivity to TNF killing, measured as 48-h 3-(5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assays, and 15-h apoptosis by propidium iodide staining and flow cytometry analysis. The TNF killing kinetics of G0-G1-arrested cells was similar to controls, and TNF did not accelerate or retard cell cycle progression of the arrested cells after feeding with fresh RPMI-0.5% FCS. However, TNF inhibited WEHI DNA synthesis as early as 1 h after treatment, and inhibition was proportionate to sensitivity to TNF-induced apoptosis. WEHI cells treated with TNF showed a higher percentage of cells in S phase with concomitant decrease in G0-G1 and G2-M. When cultured for 3-18 h in fresh RPMI-0.5% FCS to allow progression of the G0-G1-arrested cells toward the G1-S boundary, WEHI cells became more sensitive to TNF killing, especially at the 3-9 h time points. Moreover, TNF did not degrade [125I]5-iodo-2'-deoxyuridine-labeled WEHI DNA if the labeled cells were precultured for 9 h in fresh RPMI-0.5% FCS to allow them to pass S phase before the addition of TNF. These results show that TNF-induced apoptosis of WEHI cells is connected to cell cycle events; WEHI targets receive the TNF cytotoxic signal mainly at the G1-S boundary and begin to die by apoptosis as they exit from S phase.
Shih
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The Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
Stutman
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D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle; D004249:DNA Damage; D004261:DNA Replication / Q000187:drug effects; D016193:G1 Phase; D006801:Humans; D016192:Resting Phase, Cell Cycle; D016196:S Phase; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D014409:Tumor Necrosis Factor-alpha