Inhibition of Hsp90 induces degradation of ZAP-70 in CLL cells but not in T cells. (A) ZAP-70⁺ CLL cells were treated in vitro with increasing concentrations of 17-AAG (▴), 17-DMAG (×) and the analog EC116 (▪) for 48 hours. Cells incubated in media with DMSO 1% final concentration were used as a control (⋄). ZAP-70 expression was evaluated by intracellular staining. (B) ZAP-70 expression in CLL cells after treatment with 17-AAG (100 nM) was evaluated by immunoblot at different time points. In addition, immunoblots were probed with anti-Hsp70 and anti-poly ADP-ribose polymerase (PARP-1) monoclonal antibodies.β-actin was used as a protein loading control. Apoptosis after treatment with 17-AAG was 30% and 50% at 24 and 72 hours, respectively. (C) Peripheral blood mononuclear cells from ZAP-70⁺ patients were treated for 48 hours with 17-AAG (300 nM). The cells were stained with specific antibodies and analyzed by flow cytometry. The panel shows a density plot of cells labeled with anti-CD3 and anti-ZAP-70 antibodies. In each quadrant the percentage of cells is shown. The ZAP-70 mean florescence intensity (MFI) prior to treatment with 17-AAG in CLL cells was 110 and in T cells was 210. The posttreatment MFI value for ZAP-70 was 25 for CLL cells and 190 for T cells.