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Research Area
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Description
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Cancer |
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Protocol
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Kinase Assay
[1]
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| In vitro cascade assay |
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. |
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Cell Assay
[3]
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| Cell Lines |
PTC cells |
| Concentrations |
0.1 nM- 1 μM |
| Incubation Time |
48 hours |
| Methods |
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 GI50 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 GI50, 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. |
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Animal Study
[3]
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| Animal Models |
Ncr-nu/nu mice bearing PTC cells |
| Formulation |
80 mM citric buffer (pH 7) |
| Doses |
20-25 mg/kg |
| Administration |
Oral gavage |
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References |
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[1] Barrett SD, et al. Bioorg Med Chem Lett, 2008, 18(24), 6501-6504.
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[2] Judith SS, et al. Proc Amer Assoc Cancer Res.2004,45, Abstract #4003
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[3] Henderson YC, et al. Mol Cancer Ther, 2010, 9(7), 1968-1976.
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