In vitro effects of ABT-888 and bortezomib in MM cells. (A) Cells were cultured in control medium or in the presence of ABT-888 (2.5μM) and/or bortezomib (1.25nM) for 24-48 hours. Viability was determined by annexin V/PI staining. Results shown are means ± SD of 3 independent sets of experiments. *P < .05. (B) Effects of ABT-888 and bortezomib on CD138⁺ primary myeloma cells. BM mononuclear cells isolated by Ficoll gradient from the BM aspirate of MM patients and then cultured in 20% medium with or without ABT-888 (5μM) and/or bortezomib (2.5nM) for 24 hours. MM cells were identified by CD138 staining and flow cytometric analysis. Viability of CD138⁺ cells was determined by annexin V/PI staining. A representative experiment is shown here. (C) Effects of the combination of ABT-888 and bortezomib in 8 different MM patients. Viability was determined by annexin V/PI staining. (D) Effects of ABT-888 and bortezomib on CD34⁺ peripheral blood stem cells. Cells were cultured in 20% medium with or without ABT-888 (5-10μM) and/or bortezomib (2.5nM) for 24 hours. Viability was determined by annexin V/PI staining. Two representative samples are shown here. (E) Effects of the combination of ABT-888 and bortezomib on CD34⁺ peripheral blood stem cell function using a GM-CFU assay (CytoSelect; Cell Biolabs). At 10 days, colonies were quantified using a fluorescence plate reader (485/520 nm filter set) according to the assay protocol. (F) Cleavage of PARP, caspase 3, and caspase 8 was examined after exposure of MM cells to bortezomib (2.5nM) with or without ABT-888 (5μM) for 16 hours. (G-H) Effects of ABT-888 and bortezomib on γ-H2AX foci formation in MM cells treated with ABT-888 (5μM) and bortezomib (2.5nM) for 24 hours. Treatment with radiation (4 Gy for 30 minutes) was used as positive control for γ-H2AX foci formation in response to DNA damage.