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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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| Biochemical kinase assays |
c-Met catalytic activity is quantitated using a continuous-coupled spectrophotometric assay in which the time-dependent production of ADP by c-Met is determined by analysis of the rate of consumption of NADH. NADH consumption is measured by a decrease in absorbance at 340 nm by spectrophotometry at designated time points. To determine Ki values, PF-2341066 is introduced into test wells at various concentrations in the presence of assay reagents and incubated for 10 minutes at 37 °C. The assay is initiated by the addition of the c-Met enzyme. |
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Cell Assay
[1]
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| Cell Lines |
GTL-16 gastric carcinoma cells and T47D breast carcinoma cells |
| Concentrations |
0-256 nM |
| Incubation Time |
1 hour |
| Methods |
Cells including GTL-16 gastric carcinoma cells and T47D breast carcinoma cells are seeded in 96-well plates in media supplemented with 10% fetal bovine serum (FBS) and transferred to serum-free media [with 0.04% bovine serum albumin (BSA)] after 24 hours. In experiments investigating ligand-dependent RTK phosphorylation, corresponding growth factors are added for up to 20 minutes. After incubation of cells with PF-2341066 for 1 hour and/or appropriate ligands for the designated times, cells are washed once with HBSS supplemented with 1 mM Na3VO4, and protein lysates are generated from cells. Subsequently, phosphorylation of selected protein kinases is assessed by a sandwich ELISA method using specific capture antibodies used to coat 96-well plates and a detection antibody specific for phosphorylated tyrosine residues. Antibody-coated plates are (a) incubated in the presence of protein lysates at 4 °C overnight; (b) washed seven times in 1% Tween 20 in PBS; (c) incubated in a horseradish peroxidase–conjugated anti–total-phosphotyrosine (PY-20) antibody (1:500) for 30 min; (d) washed seven times again; (e) incubated in 3,3′,5,5′-tetramethyl benzidine peroxidase substrate to initiate a colorimetric reaction that is stopped by adding 0.09 N H2SO4; and (f) measured for absorbance in 450 nm using a spectrophotometer. |
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Animal Study
[1]
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| Animal Models |
Female or male nu/nu mice bearing NCI-H441,or DLD-1, or MDA-MB-231 |
| Formulation |
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| Doses |
12.5 mg/kg/day, 25 mg/kg/day, and 50 mg/kg/day |
| Administration |
Administered via p.o. |
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References |
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[1] Zou HY, et al. Cancer Res. 2007, 67(9), 4408-4417.
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[2] Christensen JG, et al. Mol Cancer Ther. 2007, 6(12 Pt 1), 3314-3322.
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[3] Sampson ER, et al. J Bone Miner Res. 2011, 26(6), 1283-1294.
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[4] Cullinane C, et al. J Nucl Med. 2011, 52(8), 1261-1267.
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[5] Gong HC, et al. Int J Proteomics. 2011, 2011, 215496.
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