Alternation of retinoic acid induced neural differentiation of P19 embryonal carcinoma cells by reduction of reactive oxygen species intracellular production.
OBJECTIVE
Intracellularly generated reactive oxygen species (ROS) are thought to modulate redox sensitive signaling pathways and thus regulate cell physiology including proliferation and differentiation. However, the role of ROS in neuronal differentiation of embryonic pluripotent cells is unknown. For this reason, the modification of retinoic acid (RA) induced neuronal differentiation of mouse embryonal carcinoma cells P19 by selected ROS scavengers and flavoprotein inhibitor was evaluated.


METHODS
Intracellular ROS was evaluated by flowcytometry. Cellular redox status was evaluated based on total levels of reduced thiol groups in cells. The activity of the RA responsive element (RARE) was evaluated by luciferase reporter assay. The RA-induced neuronal differentiation was determined based on changes in the expression of protein markers characteristic for undifferentiated (Oct-4) and neuron-like cell differentiated cells (N-cadherin and III-beta tubulin).


RESULTS
RA increased the intracellular ROS production that was accompanied by a decrease in thiol groups in cells. The ROS scavengers and flavoprotein inhibitor reduced RA-induced ROS production, RA-induced activity of RARE, and it decreased the RA-induced expression of N-cadherin and III-beta tubulin.


CONCLUSIONS
Our data outline a role of ROS as important molecules in the transduction of an intracellular signal during the neuronal differentiation of ES cells.
Konopka
Roman
R
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
Kubala
Lukas
L
Lojek
Antonin
A
Pacherník
Jirí
J
D000098:Acetophenones / Q000494:pharmacology; D000111:Acetylcysteine / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects*; D054278:Embryonal Carcinoma Stem Cells / Q000187:drug effects*; D005434:Flow Cytometry; D016166:Free Radical Scavengers / Q000494:pharmacology; D017930:Genes, Reporter / Q000187:drug effects; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D009474:Neurons / Q000187:drug effects*; D009861:Onium Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014212:Tretinoin / Q000494:pharmacology*; D014815:Vitamins / Q000494:pharmacology*
D000098:Acetophenones; D016166:Free Radical Scavengers; D009861:Onium Compounds; D017382:Reactive Oxygen Species; D013438:Sulfhydryl Compounds; D014815:Vitamins; D014212:Tretinoin; C007517:diphenyleneiodonium; C056165:acetovanillone; D005978:Glutathione; D001205:Ascorbic Acid; D000111:Acetylcysteine