Measurement of MAP kinase activation by flow cytometry using phospho-specific antibodies to MEK and ERK: Potential for pharmacodynamic monitoring of signal transduction inhibitors

Cancer cells frequently show abnormal signaling via the mitogen activated p rotein kinase (MAP kinase) pathway due to increased activity of surface rec eptors 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 i n patients during early phase clinical trial. We describe such a method, ba sed on the activation of T-lymphocytes in undiluted peripheral blood using phorbol myristate acetate (PMA), Following rapid hypotonic lysis and formal dehyde fixation, activation of the MAP kinase pathway can then be demonstra ted using phospho-specific antibodies that recognize the activated mediator s MEK or ERK, followed by surface labeling with anti-CD3 to identify T-lymp hocytes, This method was used to investigate the effects of a MEK inhibitor , UO126, and a new raf kinase inhibitor BAY 37-9751 in blood samples from n ormal donors. Dose-dependent inhibition of pERK activation was demonstrated for both agents. Furthermore, differential effects on pMEK activation allo wed the molecular targets of the two inhibitors to be distinguished. In add ition to monitoring drug effects in patients during treatment with inhibito rs of the MAP kinase pathway, the general methodology described in this pap er has the potential for wide application to the study of signal transducti on at the single cell level using flow cytometry, (C) 2001 Wiley-Liss. Inc.