Response of freshly isolated CD138⁺ cells to culture in actinomycin D or flavopiridol.

CD138⁺ cells were isolated from the BM of patients with relapsed MM. (A) Cells were cultured for the indicated times in the presence or absence of IL-6 (1 ng/mL) or actinomycin D (1 μg/mL), and protein extracts were subjected to Western blot analysis by means of antibodies with the specificities shown along the left margin. In the lower part of panel A, aliquots of CD138⁺ cells were cultured for 24 hours in medium containing IL-6 with the addition of actinomycin D (1 μg/mL) or dexamethasone (1 μM) for the final 6 hours. Cytospin preparations were analyzed by TUNEL with Hoechst 33342 counterstain. (B) CD138⁺ MM cells were cultured for 20 hours in the presence or absence of IL-6 (1 ng/mL) as indicated. Actinomycin D (1 μg/mL) or flavopiridol (200 nM) was added to the indicated cultures for the entire 20-hour incubation period, after which some cells were harvested for Western blot analysis while others were used in TUNEL assays (lower portion of the panel). (C) CD138⁺ BM cells from a third MM patient were treated as described in panel B, except that the cells were cultured for a 6-hour period.