γ Interferon treatment of cell lines. U937, a leukemia cell line, and Raji, a Burkitt’s lymphoma cell line, were treated with 1,000 U/mL of γ interferon or carrier control for 11 days. (A) U937 cell apoptosis. U937 cells were analyzed for apoptosis on days 3, 5, 7, 9, and 11. U937 cells underwent apoptosis when treated with γ interferon, with significant differences in cell death between treated cells and carrier control cells on each day (P < 10⁻⁴). The inset demonstrates U937 cells treated for 5 days with γ interferon or the carrier control; several of the γ interferon-treated cells are undergoing nuclear apoptotic changes (arrowheads point to apoptotic cells), whereas none of the carrier control-treated cells is. (B) Raji cell apoptosis. Raji cells were analyzed for apoptosis on days 3, 5, 7, 9, and 11. Raji cells did not undergo apoptosis when treated with γ interferon or the carrier control. Raji cells treated for 5 days with γ interferon show no cells undergoing apoptosis, and there are no apoptotic nuclei. (C) DAP kinase expression. Cells (10⁶) were lysed on days 1, 3, 5, 7, 9, and 11 of treatment with γ interferon or carrier control before Western analysis. U937 cells exposed to γ interferon upregulated the expression of DAP-Kinase from days 5 through 11. Carrier control cells expressed DAP-Kinase endogenously but did not upregulate the protein expression. Western analysis of U937 and Raji cells and RT-PCR analysis (far right) with GAPDH as controls demonstrate the lack of endogenous expression of DAP-Kinase in Raji at both the RNA and protein levels. DAP-Kinase RT-PCR amplifies a 343-bp product, whereas the GAPDH controls are 310 bp. + and − indicate the addition or the absence of reverse transcriptase (RT), respectively.