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

Sun Min Lim, Jee Won Hwang, Soo Kyung Bae, Soo Hyeon Bae, Joong Bae Ahn, Sun Young Rha, Jae Kyung Roh, Hyun Cheol Chung, Sang Joon Shin

Research output: Contribution to journalArticle

3 Citations (Scopus)

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. Phosphospecific 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). Study Design: 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 μM to 100 μM. Conclusion: 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.

Original languageEnglish
Pages (from-to)309-316
Number of pages8
JournalAnalytical and Quantitative Cytology and Histology
Volume34
Issue number6
Publication statusPublished - 2012 Dec 1

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Colonic Neoplasms
Flow Cytometry
Cell Line
Acetates
Staining and Labeling
Fluorescent Antibody Technique
Western Blotting
Phosphorylation
Phospho-Specific Antibodies
HCT116 Cells
Neoplasms
HT29 Cells
U 0126
Coloring Agents
Down-Regulation
Pharmaceutical Preparations
phorbol-12-myristate

All Science Journal Classification (ASJC) codes

  • Anatomy
  • Histology

Cite this

Lim, Sun Min ; Hwang, Jee Won ; Bae, Soo Kyung ; Bae, Soo Hyeon ; Ahn, Joong Bae ; Rha, Sun Young ; Roh, Jae Kyung ; Chung, Hyun Cheol ; Shin, Sang Joon. / Quantitative analysis of ERK signaling inhibition in colon cancer cell lines using phospho-specific flow cytometry. In: Analytical and Quantitative Cytology and Histology. 2012 ; Vol. 34, No. 6. pp. 309-316.
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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. Phosphospecific 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). Study Design: 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 μM to 100 μM. Conclusion: 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.",
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Quantitative analysis of ERK signaling inhibition in colon cancer cell lines using phospho-specific flow cytometry. / Lim, Sun Min; Hwang, Jee Won; Bae, Soo Kyung; Bae, Soo Hyeon; Ahn, Joong Bae; Rha, Sun Young; Roh, Jae Kyung; Chung, Hyun Cheol; Shin, Sang Joon.

In: Analytical and Quantitative Cytology and Histology, Vol. 34, No. 6, 01.12.2012, p. 309-316.

Research output: Contribution to journalArticle

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AU - Hwang, Jee Won

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