Demonstration of synergistic apoptosis induction as measured by annexin V staining in cell lines and primary human myeloma cells that is associated with rapid caspase cleavage. (A) MM.1R cells were plated and concurrently treated with bortezomib (8 nm) and lonafarnib (5 μM). After 48 hours, cells were pooled and stained with annexin V. The percent annexin V-positive cells is represented as an index of apoptosis for either single-agent or combination therapy. (B) RPMI8226 cells were plated and concurrently treated with bortezomib (8 nM or 20 nM) and lonafarnib (5 μM). After 15 and 30 hours, Western blotting was performed for expression of caspase 8, caspase 9, caspase 3, and PARP. Antibodies were used to evaluate the amount of cleaved and uncleaved forms of these proteins. Lane 1, untreated control; lane 2, 8 nM bortezomib; lane 3, 20 nM bortezomib; lane 4, 5 μM lonafarnib; lane 5, 8 nM bortezomib + 5 μM lonafarnib; lane 6, 20 nm bortezomib + 5 μM lonafarnib. (C) Bone marrow samples collected from MM patients were treated with bortezomib and lonafarnib for 40 hours. Cells were pooled and stained with annexin V and PI as described in “Materials and methods.”