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.

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This work was supported by Grants R01 CA15988 and CA55068 from the NIH (to O. S.) and Cancer Center Support Grant Ca08748.

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