IFN-α induces apoptosis in U266 MM cells.

(A) At the indicated times following IFN-α treatment, cells were subjected to bivariate flow cytometry following Annexin V/PI staining. The data were obtained from the cell population from which debris were gated out against forward and side scatter. (B) Cells were treated with IFNs for 72 hours in the presence or absence of 75 μM z-VAD-fmk (added at the time of IFN addition, and every 24 hours thereafter), and apoptosis was determined at 72 hours by Hoechst 33258 staining and expressed as the percentage of the total cells. (C) Clonogenic survival was determined following treatment for 48 hours with IFN-α (100-2000 U/mL; left panel). A time course was determined for cells treated for 6, 24, or 48 hours (right panel) with IFN-α (o) or -β (Δ) (2000 U/mL) and then cultured as described in “Materials and methods.” Data shown represent the mean value of 2 duplicate experiments, normalized against untreated cells. (D) Plasma cells were isolated from bone marrow aspirates of consenting patients with MM as described in “Materials and methods,” treated with IFN-α, -β, -γ, and Apo2L before being examined by triple staining for CD38, CD45, and Annexin V, indicative of apoptosis. R3 and R4 represent CD38⁺/CD45^−/dim and CD45⁺ cells, respectively, with the gated regions being analyzed for Annexin V staining. (E) The response of patient plasma cells to INF-α and Apo2L was determined by Hoechst staining as above.