Hydrogen peroxide modulates MAPK traffic and cell cycle progression in tumoral cells.

GALLI S; PODEROSO C; JAITOVICH A; CARRERAS MC; PODEROSO JJ

Anahein, EEUU

Congreso; 96th Annual Meeting 2005. American Association for Cancer Research.; 2005

Mammalian cells express mitogen activated protein kinases (MAPKs) that integrate extracellular signals and transduction pathways regulating cell cycle progression. Mitochondria generate reactive oxygen species, implicated in physiological cell signaling. Hydrogen peroxide (H2O2) promotes tyrosine-phosphorylation of proteins, including ERK1/2, JNK1/2 and p38. Cell H2O2 steady-state concentration ([H2O2]ss) depends on the rates of mitochondrial production and degradation by cytosolic enzymes; we have previously shown that proliferating tissues (fetal liver and brain) and tumors exhibit diminished [H2O2]ss, while quiescent tissues (adult liver) display a hundred-fold higher [H2O2]ss. The aim of this work was to study H2O2 modulation of MAPK traffic in connection with cell cycle progression of tumoral cell line LP07. Subcellular distribution of kinases (nuclei, mitochondria and cytosol) was followed by western blot. Proliferation and apoptosis were evaluated by [H3]thymidine incorporation, and by flow cytometry either with Annexin V or propidium iodide staining. Mitochondria MAPK localization was assessed by confocal microscopy with Mitotracker Red and the appropriate antibodies, and by immune electron microscopy. In LP07 cells, 1 mM H2O2 promoted proliferation, and > 10 mM H2O2 cell cycle arrest or apoptosis, with differential set up of MAPKs pathways; at 1 mM H2O2, ERK1/2 activation and translocation to nucleus were persistent while 50 mM H2O2 only induced transient ERK1/2 activation, with parallel high and low cyclin D1 expression, respectively. Inhibition of MEK1/2 limited cyclin D1 expression at 1 mM H2O2, indicating that the proliferative signal is driven by ERK1/2. In contrast, 50 mM H2O2 induced JNK1/2 persistent activation and translocation to the nucleus, as well as a marked p38 translocation to the nucleus; cells became arrested with no signs of apoptosis. Both JNK and p38 inhibitors reverted cyclin D1 downregulation at 50 mM H2O2. Redox effects were associated to MAPK specific mitochondrial translocation and activation: at 50 mM H2O2 ERK1/2, MEK and p38, but not JNK1/2, where retained inside mitochondria. By co-immune precipitation we observed associations between ERK1/2 and MEK or p38, suggesting that complex formation in mitochondria may contribute to MAPK availability, so limiting ERK1/2 entrance to nuclei. The results suggest that H2O2 differentially activates MAPKs, and that this activation, and the traffic to mitochondria regulate cell cycle progression. Thus, persistent tumoral proliferation may depend on differential activation and traffic of MAPKs in connection with low [H2O2]ss in transformed cells.