In:
Molecular Cancer Therapeutics, American Association for Cancer Research (AACR), Vol. 10, No. 11_Supplement ( 2011-11-12), p. C120-C120
Abstract:
Background: To evaluate intracellular levels of phosphorylated proteins in many signaling pathways, Western blot, immunohistochemical and immufluorescence have been generally used until now. However, to establish pharmacokinetics-pharmacodynamics modeling of various drugs including antitumor agents, precise quantitative measurements of intracellular level of phosphorylated signaling molecules are critically needed. Methods and Materials: In this study, we investigated the ability of single cell phospho-specific flow cytometry to detect dynamic change of intracellular level of phosphorylated-ERK (pERK) as a pharmacodynamic marker of MEK/ERK inhibitor U0126 in human colon cancer cell lines. We also measured the intracellular concentration of U0126 in the cell lines using High-performance liquid chromatography. Results: The fluorescence intensity changes by pERK-specific single cell flow cytometry after U0126 treatment was corresponded to the pattern of results obtained by Western blot and immunofluorescence staining in dose- and time-dependent manner. In HCT116 and HT-29 human colon cancer cells, the basal fluorescence intensity of pERK was inhibited with a 0.2- to-0.8 fold decrease according to increasing concentration of U0126 (0.1 − 100 uM). We detected the rapid decrease of pERK activity within 15 min after U0126 treatment; this decrease was continued to 2 hours. And we observed the gradual recovery of pERK level closely basal status until 24 hours. The concentration of U0126 in the media was associated with decreased activity of pERK but the intracellular concentration of U0126 was not. Conclusion: Our data obtained by single cell phospho-specific flow cytometry are more sensitive, rapid, and quantitative in parallel compared with western blot. This accuracy allowed us to acquire precise inputs in order to establish pharmacokinetics-pharmacodynamics modeling of various anti-tumor agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C120.
Type of Medium:
Online Resource
ISSN:
1535-7163
,
1538-8514
DOI:
10.1158/1535-7163.TARG-11-C120
Language:
English
Publisher:
American Association for Cancer Research (AACR)
Publication Date:
2011
detail.hit.zdb_id:
2062135-8
SSG:
12
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