Synergism between MDM2 antagonists and chemotherapeutic drugs. (A) Effect on the viability of B cells and T cells in B-CLL. Cells were untreated (C) or treated with 0.2 μM doxorubicin (D), 2.5 μM chlorambucil (CH), or 0.4 μM fludarabine (F) without (□) or with (▪)1 μM nutlin-3a for 48 hours. Viability was measured as nonapoptotic CD3^–/CD19⁺ B cells or CD3⁺/CD19^– T cells as described in “Patients, materials, and methods” and expressed as the percentage of the viability of untreated cells. Data are shown as the mean value ± SD of 5 patients (patients 3, 6, 7, 12, and 13). (B) Effect on the stability and accumulation of p53 targets. B-CLL cells were untreated (C) or treated with 0.2 μM doxorubicin (D), or 2.5 μM chlorambucil (CH), 0.4 μM fludarabine (F), or 0.5 mM acadesine without or with 1 μM nutlin-3a for 48 hours. Cells were lysed and analyzed by Western blot as described in “Patients, materials, and methods.” Total levels of p53, MDM2, p21, PUMA, and Bcl-2 were determined. (C) Effect of nutlin-3a on chemotherapeutic drugs dose responses. B-CLL cells (patient 2) were cultured with increasing doses of chemotherapeutic drugs without (○, doxorubicin; □, chlorambucil; ▵, fludarabine; ⋄, acadesine) or with (•, doxorubicin; ▪, chlorambucil; ▴, fludarabine; ♦, acadesine) 1 μM nutlin-3a for 48 hours. (D) Combination index (CI) determined in relation to the fraction of cells affected using median dose effect analysis to characterize interactions of nutlin-3a with the chemotherapeutic drugs (•, doxorubicin; ▪, chlorambucil; ▴, fludarabine; ♦, acadesine). CI values < 1.0 correspond to synergistic interactions. Viability was measured by analysis of phosphatidylserine exposure and PI uptake as described in “Patients, materials, and methods,” and it is expressed as the percentage of the viability of untreated cells.