Incorporation of?³²P into myelin basic protein (MBP) is assayed in the presence of a glutathione S-transferase fusion protein containing p44MAP kinase (GST-MAPK) and a glutathione S-transferase protein containing p45MEK (GST-MEK). The assay solution contained 20 mM HEPES, pH 7.4, 10 mM MgCl₂, 1 mM MnCl₂, 1 mM EGTA, 50 mM [gamma-³²P]ATP, 10 mg GST-MEK, 0.5 mg GST-MAPK and 40 mg MBP in a final volume of 100 mL. Reactions are stopped after 20 minutes by addition of trichloroacetic acid and filtered through a GF/C filter mat.?³²P retained on the filter mat is determined using a 1205 Betaplate. PD0325901 is assessed at various dose ranges in order to determine dose response curves.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

PTC cells (1×10⁴) are seeded in 24-well plates with 1 mL of medium for 4 days in a 37°C incubator. MEK inhibitor PD0325901 at varying concentrations is added to the cells in triplicate on day 0. MTT dissolved in 0.8% NaCl solution at 5 mg/mL is added to each well (0.2 mL) on day 2 to test GC₅₀ or every day for cell growth curves. The cells are incubated at 37°C for 3 hours with MTT. The liquid is then aspirated from the wells and discarded. Stained cells are dissolved in 0.5 mL of DMSO and their absorption at 570 nm is measured using a Synergy HT multidetection microplate reader. For GC₅₀, cell growth is calculated as 100×(T?T₀)/(C?T₀), where T is the optical density of the wells treated with inhibitors after a 48-hour period, T₀ is the optical density at time zero, and C is the control optical density with DMSO only.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice (10-14 per group) are anesthetized s.c. with a cocktail. K2 and TPC-1 cells stably infected with a retrovirus expressing luciferase (5×10⁵ cells in 5 μL RPMI1640 medium) are inoculated into the thyroid gland, and the mice are monitored weekly for tumor growth by Xenogen using Living Image 3.0 software. One week after inoculation, PD0325901 is dissolved in 80 mM citric buffer (pH 7) by sonication and given to mice daily by oral gavage (20-25 mg/kg) for 3 weeks (5 consecutive days/week). Mice are sacrificed only due to tumor burden or loss of 20% of body weight. Tumor sizes are measured with calipers and tumor volume (V) is calculated by the formula (V=length×width×depth). Control mice are given 80 mM citric buffer (pH 7) alone. All in vivo experiments are done at least twice.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Barrett SD, et al. The discovery of the benzhydroxamate MEK inhibitors CI-1040 and PD 0325901. Bioorg Med Chem Lett. 2008 Dec 15;18(24):6501-4.  [Content Brief]

  [2]. Judith S. Sebolt-Leopold, et al. The biological profile of PD 0325901: A second generation analog of CI-1040 with improved pharmaceutical potential

  [3]. Henderson YC, et al. MEK inhibitor PD0325901 significantly reduces the growth of papillary thyroid carcinoma cells in vitro and in vivo. Mol Cancer Ther. 2010 Jul;9(7):1968-76.  [Content Brief]