Figure 1
Figure 1. The MEK1/2 inhibitor AZD6244 blocks ERK1/2 activation induced by the novel Chk1 inhibitor AZD7762 in human MM cells, leading to synergistic induction of apoptosis and diminished clonogenicity. (A) H929 cells were pretreated (24 hours) with the indicated concentrations of the MEK1/2 inhibitor AZD6244, followed by 200nM or 300nM AZD7762 for another 48 hours, after which time cell survival was monitored with the MTT assay. **P < .01 and ***P < .002 versus values for cells treated with AZD7762 alone. (B-C) H929 cells were sequentially treated as described in panel A with AZD6244 in either the presence or absence of the indicated concentrations of AZD7762 (B), or with the indicated concentrations of AZD7762 in either the presence or absence of 2.5μM AZD6244 (C). After drug treatment, the percentage of apoptotic (annexin V+) cells were determined by flow cytometry. *P < .05, **P < .01, and ***P < .001 versus values for cells treated with AZD6244 or AZD7762 alone. (D) H929 cells were incubated for the indicated intervals with 300nM AZD7762 after preadministration (24 hours) of 2.5μM AZD6244. Cell death was then monitored by 7-AAD staining and flow cytometry. *P < .01 and **P < .001 versus values for cells treated with AZD7762 alone. (E) H929 cells were treated with a range of AZD6244 concentrations for 24 hours and a range of AZD7762 concentrations for another 48 hours, alone or in combination at a fixed ratio (50:1). At the end of this period, 7-AAD+ cells were determined by flow cytometry. Median-dose effect analysis was used to characterize the nature of the interaction. Two additional studies yielded equivalent results. (F) H929 cells were treated with 2.5μM AZD6244 for 24 hours ± 300nM AZD7762 for another 48 hours, and then stained on Cytospin slides by TUNEL. (G) Alternatively, after being treated with the indicated concentrations of AZD6244 for 24 hours and AZD7762 for another 48 hours, cells were washed free of drug and plated in soft agar. After incubation for 14 days, colonies, consisting of groups of > 50 cells, were scored, and colony formation for each condition was expressed relative to untreated controls. Results represent the means ± SD for 3 separate experiments performed in triplicate. **P < .005 versus single-drug treatment. (H) In parallel, H929 cells were pretreated for 24 hours with the indicated concentrations of AZD6244 and with 300nM AZD7762 for another 48 hours, after which time Western blot analysis was performed to evaluate ERK1/2 phosphorylation and caspase-3 cleavage. Each lane was loaded with 20 μg of protein; blots were stripped and reprobed with anti-tubulin Ab to ensure equal loading and transfer. Results are representative of 3 separate experiments.

The MEK1/2 inhibitor AZD6244 blocks ERK1/2 activation induced by the novel Chk1 inhibitor AZD7762 in human MM cells, leading to synergistic induction of apoptosis and diminished clonogenicity. (A) H929 cells were pretreated (24 hours) with the indicated concentrations of the MEK1/2 inhibitor AZD6244, followed by 200nM or 300nM AZD7762 for another 48 hours, after which time cell survival was monitored with the MTT assay. **P < .01 and ***P < .002 versus values for cells treated with AZD7762 alone. (B-C) H929 cells were sequentially treated as described in panel A with AZD6244 in either the presence or absence of the indicated concentrations of AZD7762 (B), or with the indicated concentrations of AZD7762 in either the presence or absence of 2.5μM AZD6244 (C). After drug treatment, the percentage of apoptotic (annexin V+) cells were determined by flow cytometry. *P < .05, **P < .01, and ***P < .001 versus values for cells treated with AZD6244 or AZD7762 alone. (D) H929 cells were incubated for the indicated intervals with 300nM AZD7762 after preadministration (24 hours) of 2.5μM AZD6244. Cell death was then monitored by 7-AAD staining and flow cytometry. *P < .01 and **P < .001 versus values for cells treated with AZD7762 alone. (E) H929 cells were treated with a range of AZD6244 concentrations for 24 hours and a range of AZD7762 concentrations for another 48 hours, alone or in combination at a fixed ratio (50:1). At the end of this period, 7-AAD+ cells were determined by flow cytometry. Median-dose effect analysis was used to characterize the nature of the interaction. Two additional studies yielded equivalent results. (F) H929 cells were treated with 2.5μM AZD6244 for 24 hours ± 300nM AZD7762 for another 48 hours, and then stained on Cytospin slides by TUNEL. (G) Alternatively, after being treated with the indicated concentrations of AZD6244 for 24 hours and AZD7762 for another 48 hours, cells were washed free of drug and plated in soft agar. After incubation for 14 days, colonies, consisting of groups of > 50 cells, were scored, and colony formation for each condition was expressed relative to untreated controls. Results represent the means ± SD for 3 separate experiments performed in triplicate. **P < .005 versus single-drug treatment. (H) In parallel, H929 cells were pretreated for 24 hours with the indicated concentrations of AZD6244 and with 300nM AZD7762 for another 48 hours, after which time Western blot analysis was performed to evaluate ERK1/2 phosphorylation and caspase-3 cleavage. Each lane was loaded with 20 μg of protein; blots were stripped and reprobed with anti-tubulin Ab to ensure equal loading and transfer. Results are representative of 3 separate experiments.

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