MEK inhibitor U0126 interferes with immunofluorescence analysis of apoptotic cell death.
BACKGROUND
Binding of extracellular growth factors to cell surface receptors often results in activation of the mitogen-activated protein kinase (MAPK). MAPK is regulated by MAPK kinase, also called MEK. Deprivation of growth factors during cell culture or intracellular MEK inhibition leads to inhibition of proliferation and apoptotic cell death. Besides other techniques, apoptotic cells can be identified by phosphatidylserine (PS) exposure and exclusion of membrane-impermeant propidium iodide (PI). We investigated the limitations of detection of apoptotic cell death and cytofluorometry in cells cultured in the presence of the MEK inhibitor U0126.


METHODS
Apoptotic cell death was induced in the plasmacytoma cell line INA-6, in peripheral blood mononuclear cells (PBMC), and in cultured T lymphoblasts by deprivation of interleukin-6 (IL-6) or by incubation with the MEK inhibitor U0126. Apoptotic cell death was quantified by flow cytometry using annexin V/propidium iodide (AxV/PI) double staining.


RESULTS
U0126-treated cells dramatically changed their fluorescence pattern during cell culture. If AxV/PI staining is employed to detect apoptotic cell death, the background fluorescence mimicks PS exposure on viable cells. The compound itself has no intrinsic fluorescence in vitro but develops an intensive fluorescence during cell culture which can be observed in all fluorescence channels with a predominance in the FL1 channel (525 nm). We further demonstrate that at least some of the U0126-induced background fluorescence is dependent on cellular uptake and intracellular modifications or cellular responses.


CONCLUSIONS
These results demonstrate that appropriate controls for every single time point are necessary if fluorescence analyses are performed in the presence of chemical enzyme inhibitors. In the case of MEK inhibitors, either the use of PD098059 or PD184352 as an alternative for U0126 or nonfluorometric methods for detection of apoptosis should be considered.
Blank
Norbert
N
Institute for Clinical Immunology, Department of Medicine III, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany.
Burger
Renate
R
Duerr
Birgit
B
Bakker
Frank
F
Wohlfarth
Anika
A
Dumitriu
Ingrid
I
Kalden
Joachim R
JR
Herrmann
Martin
M
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002070:Butadienes / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005455:Fluorescent Antibody Technique / Q000592:standards*; D006801:Humans; D015850:Interleukin-6 / Q000032:analysis / Q000494:pharmacology; D048728:MAP Kinase Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009336:Necrosis; D009570:Nitriles / Q000494:pharmacology*; D010954:Plasmacytoma; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015203:Reproducibility of Results; D012542:Scattering, Radiation; D013194:Staining and Labeling; D013601:T-Lymphocytes / Q000737:chemistry / Q000166:cytology; D014407:Tumor Cells, Cultured
D017304:Annexin A5; D002070:Butadienes; D004791:Enzyme Inhibitors; D015850:Interleukin-6; D009570:Nitriles; C113580:U 0126; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human