A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy.
Protein kinases play important roles in many disease processes and are primary targets for drug development. Because cellular phosphorylation cascades are complex multidirectional pathways, the behavior of a drug in a biochemical enzyme assay may not accurately reflect its performance in the context of a whole cell. We have developed a near-infrared cytoblot assay that can be used to investigate both kinase signaling and effects of kinase inhibitors. Adherent cells were grown in either 96- or 384-well plates. Following stimulation, protein phosphorylation was detected immunohistochemically by simultaneous staining with two primary antibodies: a phospho-specific primary and normalization antibody that recognized either the target protein regardless of phosphorylation status (pan protein) or a housekeeping protein. Secondary antibodies labeled with two spectrally distinct near-infrared dyes were used for visualization. Nuclear staining with TO-PRO-3 was also used in place of the normalization antibody. Normalization for well-to-well variability was accomplished by ratiometric analysis of the two wavelengths. The near-infrared cytoblot was used to analyze phosphorylation of EGFR, Akt, Stat3, MEK 1, and ERK1/2. This assay format was also able to simultaneously assess the phosphorylation of multiple signaling proteins in response to known kinase inhibitors. We observed that the IC50 for the EGFR inhibitor PD168393 was similar for EGFR and Stat3 but was significantly higher for ERK1/2, a downstream modulator of EGFR function. The observation that the receptor and its effectors show different IC50 values for the same inhibitory drug could be important for target selection in drug development.
Chen
Huaxian
H
LI-COR Biosciences, Lincoln, NE 68504, USA.
Kovar
Joy
J
Sissons
Sean
S
Cox
Karen
K
Matter
William
W
Chadwell
Fred
F
Luan
Peng
P
Vlahos
Chris J
CJ
Schutz-Geschwender
Amy
A
Olive
D Michael
DM
D000818:Animals; D002070:Butadienes / Q000494:pharmacology; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000032:analysis; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D019085:Fluorescent Antibody Technique, Direct / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D041681:NIH 3T3 Cells; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000032:analysis*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D050796:STAT3 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013055:Spectrophotometry, Infrared; D015534:Trans-Activators / Q000378:metabolism
D002070:Butadienes; D002867:Chromones; D004268:DNA-Binding Proteins; D009025:Morpholines; D009570:Nitriles; D010982:Platelet-Derived Growth Factor; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse; D015534:Trans-Activators; C113580:U 0126; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004815:Epidermal Growth Factor; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases