CD40-ligation of CLL cells induces p53 and p53-regulated genes. CLL cells were cocultured with HeLa cells (-) or HeLa cells expressing CD154 (+) or cultured alone without treatment (-) or after treatment with 5 Gy (γ-radiation). After 24 hours the CLL cells were harvested and examined via flow cytometry or immunoblot analyses. For panels A and C, cell-surface expression of death receptors on CD19⁺ CLL cells was monitored by flow cytometry. Representative histograms of CLL samples that were stained with fluorochrome-conjugated mAbs specific for an irrelevant specificity (control; top row), CD95 (middle row), or DR5 (bottom row), as indicated on the left margin, are shown. The shaded histogram depicts the fluorescence of CLL cells cocultured with HeLa-CD154 (left column) or following γ-radiation (right column), as indicated at the top of each column. The open histograms depict the fluorescence of stained control-treated CLL cells following culture with (left column) or without (right column) HeLa cells. For panels B and D, cell lysates were prepared from CLL cells that had been cocultured with HeLa cells lacking CD154 (-) or HeLa-CD154 (+), or cultured alone after no treatment (-) or after 5 Gy ionizing radiation (+), as indicated at the top of each panel. Fifty μg of each lysate were resolved via PAGE for immunoblot analyses using antibodies specific for p53, Bid, p21, or β-actin, as indicated on the right-hand margin of each immunoblot. In panels A and B are the results from analyzing CLL cells with functional p53, as indicated at the top of the figure. In panels C and D are the results obtained using CLL cells that had a monoallelic, nonfunctional p53 gene, confirmed by DNA sequencing to harbor an inactivating mutation (eg, Met237Ile), as indicated at the top of the figure.