Quantitative analysis of ERK signaling inhibition in colon cancer cell lines using phospho-specific flow cytometry

To evaluate the activity of U0126, a MEK1/2 inhibitor, in downregulating the phosphorylation of ERK in colon cancer cell lines and to explore the correlation of phospho-flow cytometry with standardized methods to validate its use in clinical settings. Phospho-specific flow cytometry provides an optimal platform for the analysis of signaling abnormalities in cancer. In this study, we used phospho-specific flow cytometry to monitor intracellular signaling in cells stimulated with phorbol 12-myristate 13-acetate (PMA).

Multiparametric flow cytometry was performed on two colon cancer cell lines, HCT116 and HT29. PMA-stimulated cells were treated with U0126, and phospho-specific antibodies were used to monitor ERK signaling. The resulting data were compared to western blotting and immunofluorescence staining.

HCT116 and HT29 cells were treated with increasing amounts of U0126 after PMA stimulation. The western blot analysis revealed that increasing the amount of U0126 resulted in inhibition of phospho-ERK (p-ERK). Fluorescence-activated cell sorting plots of phosphorylation of ERK demonstrated that the levels of p-ERK decreased with increasing concentrations of U0126. Results of immunofluorescence staining indicated that the staining density of the immunofluorescent dye decreased as the concentration of U0126 increased from 0.1 microM to 100 microM.

Quantitative and correlated expression profiles for ERK signaling suggest that phospho-specific flow cytometry will provide new insights into mechanisms underlying defective signaling in cancer and enable us to predict drug responses in cancer cell lines.