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Cell Cycle-dependent Tumor Necrosis Factor Apoptosis¹

The Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

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.

¹ This work was supported by Grants R01 CA15988 and CA55068 from the NIH (to O. S.) and Cancer Center Support Grant Ca08748.

² To whom requests for reprints should be addressed, at Cellular Immunology Program, Memorial Sloan-Kettering Cancer Center, Box 51, 1275 York Avenue, New York, NY 10021.

Received 9/11/95. Accepted 1/26/96.

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