Measurement of MAP kinase activation by flow cytometry using phospho-specific antibodies to MEK and ERK: Potential for pharmacodynamic monitoring of signal transduction inhibitors
Article first published online: 12 APR 2001
Copyright © 2001 Wiley-Liss, Inc.
Volume 46, Issue 2, pages 72–78, 15 April 2001
How to Cite
Chow, S., Patel, H. and Hedley, D. W. (2001), Measurement of MAP kinase activation by flow cytometry using phospho-specific antibodies to MEK and ERK: Potential for pharmacodynamic monitoring of signal transduction inhibitors. Cytometry, 46: 72–78. doi: 10.1002/cyto.1067
- Issue published online: 26 DEC 2002
- Article first published online: 12 APR 2001
- Manuscript Accepted: 1 FEB 2001
- Manuscript Received: 15 JAN 2001
- signal transduction;
- MAP kinase;
- phospho-specific antibody;
Cancer cells frequently show abnormal signaling via the mitogen activated protein kinase (MAP kinase) pathway due to increased activity of surface receptors for growth factors, or as a result of ras mutations. The development of potent anti-cancer agents that target this pathway prompts the need for analytical methods that allow pharmacodynamic monitoring of drug effects in patients during early phase clinical trial. We describe such a method, based on the activation of T-lymphocytes in undiluted peripheral blood using phorbol myristate acetate (PMA). Following rapid hypotonic lysis and formaldehyde fixation, activation of the MAP kinase pathway can then be demonstrated using phospho-specific antibodies that recognize the activated mediators MEK or ERK, followed by surface labeling with anti-CD3 to identify T-lymphocytes. This method was used to investigate the effects of a MEK inhibitor, U0126, and a new raf kinase inhibitor BAY 37-9751 in blood samples from normal donors. Dose-dependent inhibition of pERK activation was demonstrated for both agents. Furthermore, differential effects on pMEK activation allowed the molecular targets of the two inhibitors to be distinguished. In addition to monitoring drug effects in patients during treatment with inhibitors of the MAP kinase pathway, the general methodology described in this paper has the potential for wide application to the study of signal transduction at the single cell level using flow cytometry. Cytometry (Comm. Clin. Cytometry) 46:72–78, 2001. © 2001 Wiley-Liss, Inc.