PRIMA-1Met induced cell death in primary cells, irrespective of TP53 status, and synergized with BSO. (A) PRIMA-1Met induced cell death in primary cells. Purified myeloma cells (sample 10; Table 1) were treated overnight with PRIMA-1Met (10 μM) or nutlin3a (10 μM) and stained with CD138-PE (cytogram), control-PE, or anti-DR5-PE (histogram) mAbs to assess cell death (loss of CD138 staining) or DR5 expression level. The thin line represents control staining, and the thick line is DR5 staining. (B) PRIMA-1Met induced primary myeloma cell death and synergized with BSO but was antagonized by NAC or GSH-MEE. Primary myeloma cells were isolated from the bone marrow or peripheral blood of patients with MM or plasma cell leukemia. Cells were incubated overnight in the presence or absence of PRIMA-1Met (10 μM), BSO (0.5 mM), NAC (5 mM), GSH-MEE (5 mM), or nutlin3a (10 μM). BSO, NAC, and GSH-MEE did not induce any myeloma cell death (data not shown). Cell death was assessed by the loss of CD138 staining, as described previously. (C) PRIMA-1Met-induced cell death was independent of del17p status. The TP53 status of primary cells was assessed, using fluorescence in situ hybridization, by defining the percentage of myeloma cells lacking the short arm of chromosome 17 (del17p), as previously reported.24 In this cohort, deletion was considered negative when a minority of cells harbored a 17p deletion (range, 0%-39%), and positive when more than 50% of cells harbored the deletion (range, 50%-97%; Table 1). Samples 6′ and 22′ were excluded from this analysis to analyze independent samples. (D-F) PRIMA-1Met, in contrast to nutlin3a, did not increase DR5 expression in primary cells. Cells were treated overnight with PRIMA-1Met (10 μM) or nutlin3a (10 μM) and were stained with control-PE or anti-DR5-PE mAbs. DR5 expression was calculated by dividing the specific mean fluorescence (DR5) of untreated and treated cells by the control staining of untreated and treated cells, respectively. The variation of DR5 expression was analyzed in the whole samples (D) and in samples without (E) or with (F) del17p. (G) Modulation in DR5 expression induced by PRIMA-1Met was not correlated with del17. The fold-increase in DR5 expression was plotted against the percentage of cells with del17p within the samples. (H) Modulation in DR5 expression induced by nutlin3a was inversely correlated to del17p. The fold-increase in DR5 expression was plotted against the percentage of cells with del17p within the samples. (I) Modulation in DR5 expression induced by nutlin3a, but not by PRIMA-1Met, was significantly higher in samples without del17p than in samples with del17p. *P < .05; ***P < .001.