Alternation of retinoic acid induced neural differentiation of P19 embryonal carcinoma cells by reduction of reactive oxygen species intracellular production.

OBJECTIVES

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.