title: Quantitative analysis of ERK signaling inhibition in colon cancer cell lines using phospho-specific flow cytometry.
abstract: OBJECTIVE
  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).
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Internal Medicine, Cancer Metastasis Research Center, Yonsei Cancer Center, Yonsei University College of Medicine, 250 Seongsan-no, Seodaemun-gu, Seoul 120-752, Korea.
authors.forename: Sun Min
authors.initials: SM
authors.lastname: Lim
authors.forename: Jee Won
authors.initials: JW
authors.lastname: Hwang
authors.forename: Soo Kyung
authors.initials: SK
authors.lastname: Bae
authors.forename: Soo Hyeon
authors.initials: SH
authors.lastname: Bae
authors.forename: Joong Bae
authors.initials: JB
authors.lastname: Ahn
authors.forename: Sun Young
authors.initials: SY
authors.lastname: Rha
authors.forename: Jae Kyung
authors.initials: JK
authors.lastname: Roh
authors.forename: Hyun Cheol
authors.initials: HC
authors.lastname: Chung
authors.forename: Sang Joon
authors.initials: SJ
authors.lastname: Shin
mesh_terms: D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology*; D002273:Carcinogens / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards*; D005455:Fluorescent Antibody Technique; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000502:physiology; D009570:Nitriles / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D002070:Butadienes; D002273:Carcinogens; D004791:Enzyme Inhibitors; D009570:Nitriles; C113580:U 0126; D013755:Tetradecanoylphorbol Acetate