p53 and caspases drive CD200 expression during DC apoptosis. (A) The first and second putative p53REs (p53RE no. 1 and p53RE no. 2) or randomly chosen sequence (control) from the first intron of the human CD200 gene were cloned into a luciferase expression vector containing a minimal promoter element. NCI-H1299 cells were cotransfected with either of these constructs with or without a p53 expression vector (pFC-p53). Results shown are fold increases in luciferase activity over no pFC-p53 cotransfection for each vector. ^*P < .005 compared with control by Mann-Whitney U test. Results are representative of 5 replicate experiments. (B) CD11c⁺ DCs were isolated from wild-type (WT) and p53^-/-C57BL/6 mice and cultured in complete DMEM for 48 hours to induce apoptosis. Cells were stained with CaspACE and anti-CD200 mAb in the presence of 7-AAD at various time points. Shaded and unshaded histograms represent staining with anti-CD200 and isotype control mAbs, respectively. Cells shown are gated on early apoptotic cells (CaspACE⁺/7AAD^-). Results are representative of 2 replicate experiments. (C) Quantitative real-time RT-PCR was performed on cultured CD11c⁺ cells derived from either WT or p53^-/- mice. Total RNA was normalized to 18S rRNA at each time point. Results are representative of 2 experimental replicates. (D) CD11c⁺ DCs were cultured in medium for 48 hours in the presence or absence of 100 μM Z-VAD-FMK and assayed for CD200 expression. ^*P < .05 as compared with Z-VAD-FMK treatment. Results are combined data from 3 replicate experiments. (E) DCs from WT and p53^-/- mice were isolated as described earlier, cultured in medium with or without 100 μM Z-VAD-FMK, and examined for CD200 expression by flow cytometry. Error bars indicate ±1 SD (A, C-D).