Analysis of p53 function in primary MM tumor samples. (A) CD138 immunopurified MM cells were incubated overnight with either 10 μM nutlin-3a (A), 10 μM nutlin-3b (B), or DMSO (D). The status of p53, and of MDM2 and p21^WAF1, was assessed by Western analysis. The majority of tumor samples showed selective induction of p53 and of both downstream targets in response to nutlin-3a, confirming that these cells most likely contained wild-type p53. High constitutive and unmodified levels of p53 in patient samples no. 4 and 12, which represented cases of plasma cell leukemia (PCL) and pleural effusion (PE), respectively, indicate p53 mutations. (B) Nutlin-3a–induced apoptosis of primary MM cells. Cells from each of the tumor isolates represented in panel A were incubated with either 10 μM nutlin-3a (▪), 10 μM nutlin-3b (() or with DMSO (□), and cell death assessed through annexin-FITC/PI staining after 5 days. Height of the bars represents the percentage of live cells relative to the DMSO control. Nutlin-3a proved effective in all samples, except those shown to be defective in p53 signaling. An asterisk indicates that the apoptosis data shown derived from MM cells cocultured with BMSCs. (C) Dose-response curves for nutlin-3a–induced cell death, as assessed through annexin-FITC/PI staining after 5 days of exposure to the drug, in selected primary tumor samples. EC₅₀ values fell within the range identified for nutlin-3a–responsive MM cell lines (sample no. 5, 2.8 μM; sample no. 15, 4.4 μM).