Figure 3
Figure 3. Romidepsin-resistant cells exhibit exquisite sensitivity to MEK inhibitors and reduction of Bim plays a role in resistance. (A) HuT78 parental, DpVp50, and DpP75 cells were incubated with the noted concentrations of the MEK inhibitors AS703026, PD0325901, and AZD6244 for 48 hours. Cells were then harvested and incubated with anti-annexin V antibody and propidium iodide as outlined in “Materials and methods.” Results are from at least 3 independent experiments. C, control. (B) DpVp50 or (C) DpP75 cells were treated with increasing concentrations of PD0325901 or AZD6244, respectively, for 48 hours. Cells were then harvested and whole-cell lysates were subjected to PAGE, transferred to nitrocellulose membranes, and probed with antibodies against PARP, cleaved PARP (c-PARP), phosphorylated ERK (p-ERK), total ERK, and GAPDH. The experiment was repeated three times. (D) HuT78 cells were treated with 25 ng/mL romidepsin (Dp) for 48 hours, and percent annexin-positive cells were determined as outlined in (A). DpVp50 cells were treated for 48 hours with 25 ng/mL romidepsin alone (Dp) or with 1, 3, or 5 nM PD0325901 (PD), 20 µM OSI-906 (OSI), or 10 µM LY294002 (LY) alone or in combination with 25 ng/mL romidepsin (Dp). Percent annexin-positive cells were determined as in (A). Bars denote the average percentage of cells that stained positively for annexin. Whole-cell lysates were prepared from HuT78 and the romidepsin-resistant lines, subjected to PAGE, and transferred to a nitrocellulose membrane that was probed for the apoptosis-associated proteins (E) Bim as well as (F) Bax, Bid, Bak, Bcl-xL, and Mcl-1. GAPDH served as a loading control. The romidepsin-resistant sublines (G) DpVp50 and (H) DpP75 were treated with the MEK inhibitors AZD6244 and PD0329501, respectively, for 48 hours after which whole-cell lysates were subjected to PAGE and transferred to nitrocellulose. HuT78 parental cells were included as a positive control for Bim in (G). Blots were subsequently probed for PARP, cleaved PARP (c-PARP), Bim, and GAPDH. C, control.

Romidepsin-resistant cells exhibit exquisite sensitivity to MEK inhibitors and reduction of Bim plays a role in resistance. (A) HuT78 parental, DpVp50, and DpP75 cells were incubated with the noted concentrations of the MEK inhibitors AS703026, PD0325901, and AZD6244 for 48 hours. Cells were then harvested and incubated with anti-annexin V antibody and propidium iodide as outlined in “Materials and methods.” Results are from at least 3 independent experiments. C, control. (B) DpVp50 or (C) DpP75 cells were treated with increasing concentrations of PD0325901 or AZD6244, respectively, for 48 hours. Cells were then harvested and whole-cell lysates were subjected to PAGE, transferred to nitrocellulose membranes, and probed with antibodies against PARP, cleaved PARP (c-PARP), phosphorylated ERK (p-ERK), total ERK, and GAPDH. The experiment was repeated three times. (D) HuT78 cells were treated with 25 ng/mL romidepsin (Dp) for 48 hours, and percent annexin-positive cells were determined as outlined in (A). DpVp50 cells were treated for 48 hours with 25 ng/mL romidepsin alone (Dp) or with 1, 3, or 5 nM PD0325901 (PD), 20 µM OSI-906 (OSI), or 10 µM LY294002 (LY) alone or in combination with 25 ng/mL romidepsin (Dp). Percent annexin-positive cells were determined as in (A). Bars denote the average percentage of cells that stained positively for annexin. Whole-cell lysates were prepared from HuT78 and the romidepsin-resistant lines, subjected to PAGE, and transferred to a nitrocellulose membrane that was probed for the apoptosis-associated proteins (E) Bim as well as (F) Bax, Bid, Bak, Bcl-xL, and Mcl-1. GAPDH served as a loading control. The romidepsin-resistant sublines (G) DpVp50 and (H) DpP75 were treated with the MEK inhibitors AZD6244 and PD0329501, respectively, for 48 hours after which whole-cell lysates were subjected to PAGE and transferred to nitrocellulose. HuT78 parental cells were included as a positive control for Bim in (G). Blots were subsequently probed for PARP, cleaved PARP (c-PARP), Bim, and GAPDH. C, control.

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