1. MEL23 and MEL24 inhibit Mdm2 and p53 ubiquitination in cells.
MEL23 and MEL24 inhibit Mdm2 and p53 ubiquitination in cells. A, MEL compounds inhibit Mdm2 ubiquitination in cells. Mdm2(wt)-luciferase cells were transfected with HA-ubiquitin (5 μg) and treated 8 hours later with 10 μg/mL (28 μM) MEL23, 10 μg/mL (31 μM) MEL24, or a DMSO control. After 16 hours, cells were additionally treated with 10 μM MG132 or a DMSO control. Three hours later cells were lysed and immunoprecipitated for 3 hours with an anti-HA affinity matrix. Total lysates and immunoprecipitated samples were analyzed by Western blotting using an anti-Mdm2 antibody. B, MEL23 increases the half-life of p53 and Mdm2 proteins. RKO cells were treated with 5 μg/mL (14 μM) MEL23, 10 μM MG132, or NT control for 4 hours, followed by treatment with 5 μg/mL (18 μM) cycloheximide for 30 minutes or 1, 2, 4, or 6 hours. Cell lysates were analyzed by Western blotting with anti-Mdm2, anti-p53 and anti-eIF4E antibodies. Protein levels were measured by densitometry using ImageJ and normalized to eIF4E. Half-life was calculated by plotting the protein levels in the treated cells to the protein level in the cells not treated with cycloheximide. C, MEL compounds inhibit p53 ubiquitination in cells. H1299 cells were transfected with Flag-Mdm2 (5 μg), p53 (0.5 μg), and HA-ubiquitin (2 μg). After 8 hours, cells were treated with 10 μg/mL (28 μM) MEL23, 10 μg/mL (31 μM) MEL24, or a DMSO control. After 16 hours cells were additionally treated with 10 μM MG132 or a DMSO control. Three hours later cells were lysed and analyzed by Western blotting using an anti-p53 antibody. Brackets indicate the slower migrating species of the protein that are the ubiquitin-conjugated forms. NT, no-treatment control.
Ariel G. Herman et al. Cancer Discovery 2011;1:
©2011 by American Association for Cancer Research