1. MEL23 and MEL24 inhibit Mdm2 and p53 ubiquitination through the Mdm2-MdmX hetero-complex.
MEL23 and MEL24 inhibit Mdm2 and p53 ubiquitination through the Mdm2-MdmX hetero-complex. A, MEL23 inhibits Mdm2-MdmX E3 ligase activity in vitro. Bottom panel: Ubiquitination assays in the presence of increasing concentrations of MEL compounds were performed with 100 ng of full-length Flag-Mdm2 or Flag-Mdm2/HA-MdmX complexes, 32P-labeled ubiquitin, UbcH5C, and E1. Following 30 minutes' incubation at 37°C, reactions were terminated and resolved on a SDS-PAGE gel followed by autoradiography. Top panel, the gels were quantified by phosphorimaging using ImageQuant software. PC, positive control; NC, negative control with UbcH5C eliminated from the reaction. B, MdmX is needed for efficient MEL23 activity in cells. Top panel, RKO cells were transfected with siRNAs against MdmX or luciferase, as a control. Forty-eight hours after transfection, cells were treated with 10 μg/mL (28 μM) MEL23, 0.5 μg/mL (0.9 μM) doxorubicin, or DMSO for 4 hours before harvesting. Lysates were analyzed by Western blotting using anti-MdmX, anti-p53, and anti-eIF4E antibodies. Bottom left panel, fold increase in p53 levels of NT and MEL23 treated cells normalized to the NT control under two different siRNA co-treatment conditions. Bottom right panel, raw p53 levels in each condition. Protein levels were measured by densitometry using ImageJ and graphed using GraphPad Prism. Error bars represent the SEM of 3 samples. C, MEL23 does not inhibit Mdm2-p53 and Mdm2-MdmX complex formation. MCF7 cells were transfected with FLAG-Mdm2 (12 μg) and p53 (2.5 μg). After 24 hours, cells were treated with 10 μg/mL (28 μM) MEL23, 5.3 μg/mL (10 μM) Nutlin-3a, or DMSO for 6 hours. Cell lysates were incubated with anti-FLAG M2 antibody for 2 hours, followed by incubation with Protein-A/G sepharose beads for 1 hour at 4°C. Cells were lysed and analyzed by Western blotting using anti-Mdm2, anti-MdmX, and anti-p53 antibodies. NT, no-treatment control.
Ariel G. Herman et al. Cancer Discovery 2011;1:
©2011 by American Association for Cancer Research