![Figure 1. Src inhibitors block ERK1/2 activation in human MM cells exposed to UCN-01, in association with synergistic induction of apoptosis. (A-B) Human MM cells were exposed (24 hours) to UCN-01 (U266, 150nM; RPMI 8226, MM.1S, and MM.1R, 100nM) ± BMS354825 (0.5-2.5μM [A]) or SKI606 (U266 and RPMI 8226, 2μM; MM.1S and MM.1R, 1μM [B]), after which Western blot analysis was performed to detect expression of total and/or phosphorylated c-Src (Y418) and ERK1/2. (C) RPMI 8226 and U266 cells were treated as described in panel A for 24 hours and 48 hours, respectively. (D) MM.1S and MM.1R cells were exposed to 100nM UCN-01 ± 1μM BMS354825 for 24 hours. (E) MM cell lines were treated as described in (B) for 24 hours (RPMI 8226, MM.1S, and MM.1R) or 48 hours (U266). For C-E, after drug treatment, the percentage of apoptotic (annexin V+) cells was determined by flow cytometry (means ± SD, *P < .01 and **P < .001 vs UCN-01 alone). (C) Inset: RPMI 8226 cells were exposed to a range of BMS354825 (0.5-1.25μM) and UCN-01 (50-125nM) concentrations alone and in combination at fixed ratio (10:1) for 24 hours. At the end of this period, the percentage of annexin V+ cells was determined for each condition. Fractional effect values were obtained by comparing results with those of untreated controls, and median dose-effect analysis was used to characterize the nature of the interaction between these agents. Combination index values less than 1.0 denote a synergistic interaction. The results of a representative experiment are shown; 2 additional studies yielded equivalent results. (F) CD138+ cells isolated from the bone marrow of a patient (patient #2) with MM were exposed (24 hours) to 100nM UCN-01 ± 0.5 or 1.0μM BMS354825, after which the extent of apoptosis was determined by annexin V/propidium iodide (PI) staining and flow cytometry. Values shown represent the percentage of annexin V+ cells including early (annexin V+/PI−, bottom right quadrants) and late apoptosis (annexin V+/PI+, top right quadrants). Two additional experiments yielded equivalent results.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/117/6/10.1182_blood-2010-06-291146/4/zh89991066390001.jpeg?Expires=1735843042&Signature=41rceR6hQUeTORBZgDWBe51oSl2iZAs11k4PyZS4P8sp4b6vsYEsZRgm9wot-pjEmLkWd8o9qSrD~U4siyWEPA8tHWFNFNamwxB3GcbvLq3L5ZczMuZD-gcgKLSqk3hkOx28IvNgmi-QyNTCqVW87kbEPAtxJQEUwSeUo3qkym1uGSnsfuWehh2UheffMlroFPSDiHya7e~aYfjVUuzB4XrZpH9YKCC3JkP~uJhOUXe2NvTfjGfGoqXkPfc2xyI6ZhgEcHd1gqpwkEdtMmcZem5o80KmstFPhDEScrobkf~LNH2LO32iGQUu3wntdv8-ht76aDbn1qGF6cjZEVDt9w__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Src inhibitors block ERK1/2 activation in human MM cells exposed to UCN-01, in association with synergistic induction of apoptosis. (A-B) Human MM cells were exposed (24 hours) to UCN-01 (U266, 150nM; RPMI 8226, MM.1S, and MM.1R, 100nM) ± BMS354825 (0.5-2.5μM [A]) or SKI606 (U266 and RPMI 8226, 2μM; MM.1S and MM.1R, 1μM [B]), after which Western blot analysis was performed to detect expression of total and/or phosphorylated c-Src (Y418) and ERK1/2. (C) RPMI 8226 and U266 cells were treated as described in panel A for 24 hours and 48 hours, respectively. (D) MM.1S and MM.1R cells were exposed to 100nM UCN-01 ± 1μM BMS354825 for 24 hours. (E) MM cell lines were treated as described in (B) for 24 hours (RPMI 8226, MM.1S, and MM.1R) or 48 hours (U266). For C-E, after drug treatment, the percentage of apoptotic (annexin V+) cells was determined by flow cytometry (means ± SD, *P < .01 and **P < .001 vs UCN-01 alone). (C) Inset: RPMI 8226 cells were exposed to a range of BMS354825 (0.5-1.25μM) and UCN-01 (50-125nM) concentrations alone and in combination at fixed ratio (10:1) for 24 hours. At the end of this period, the percentage of annexin V+ cells was determined for each condition. Fractional effect values were obtained by comparing results with those of untreated controls, and median dose-effect analysis was used to characterize the nature of the interaction between these agents. Combination index values less than 1.0 denote a synergistic interaction. The results of a representative experiment are shown; 2 additional studies yielded equivalent results. (F) CD138+ cells isolated from the bone marrow of a patient (patient #2) with MM were exposed (24 hours) to 100nM UCN-01 ± 0.5 or 1.0μM BMS354825, after which the extent of apoptosis was determined by annexin V/propidium iodide (PI) staining and flow cytometry. Values shown represent the percentage of annexin V+ cells including early (annexin V+/PI−, bottom right quadrants) and late apoptosis (annexin V+/PI+, top right quadrants). Two additional experiments yielded equivalent results.
