Patupilone induces G2M arrest of MM cells, followed by apoptosis. (A) MM.1S, RPMI 8226, and U266 cells were incubated in culture medium containing 10 nM patupilone for 0, 8, and 24 hours; cell cycle profile was obtained as described in “Materials and methods.” By 48 hours, remaining viable cells were in G2M phase. Gray histograms indicate G1/S phase; black histograms, G2M phase; and white outline, all phases. (B) Cell lines were exposed to 0, 1, or 10 nM patupilone. DNA laddering, indicative of apoptosis, was observed after 24-hour exposure of MM.1S, RPMI 8226, and U266. (C) Western blot analysis of protein lysates obtained from MM.1S cells treated for 0, 2, 4, 8, 24, 48, and 72 hours with 10 nM patupilone were probed with antibodies to bcl-2, bax, caspase 3, and PARP. Treatment for 8 and 24 hours led to emergence of an extra bcl-2 band (*), consistent with phosphorylated bcl-2. Cleaved forms of caspase 3 and PARP, indicative of caspase activation and apoptosis, respectively, appeared by 24 hours. Dexamethasone-treated cells served as a control for apoptosis, and actin served as a protein loading control.
