Neural Differentiation of Human Neuroblastoma GOTO Cells via a Rho-Rho Kinase-Phosphorylation Signal Transduction and Continuous Observation of a Single GOTO Cell during Differentiation

Abstract

Nerve growth factor, retinoic acid, dibutyryl cAMP, ganglioside G(Q1b), and botulinum C3 exoenzyme were evaluated for their neural differentiating potential on human neuroblastoma GOTO cells. C3 exoenzyme is an ADP-ribosyltransferase inactivating Rho protein (a small GTP-binding protein). C3 exoenzyme caused the fastest differentiation of GOTO cells into neural cells and induced the strongest network of the cells. Fasudil, an inhibitor of Rho-kinase, induced outgrowth of the neurites in the GOTO cells. Calyculin A, an inhibitor of phosphatases including myosin phosphatase, counteracted C3 exoenzyme-induced neurite outgrowth of the cells. These findings suggest that differentiation of GOTO cells triggered by C3 exoenzyme is attained via inactivation of Rho protein, inhibition of Rho-kinase, and activation of myosin phosphatase. Because of the strong differentiating potential of C3 exoenzyme, the transduction pathway consisting of Rho protein, Rho-kinase, and myosin phosphatase seems to be main stream in the neural differentiation of GOTO cells. A single GOTO cell was observed continuously after treatment with C3 exoenzyme. The cell started to change shape from its original morphology only 15 min after treatment with C3 exoenzyme, and it was completely spherical within 60 min. Neurites appeared on the membrane of the cell 2 hr after the treatment and then gradual outgrowth began. These observations are fundamental information in elucidating the mechanism of neural differentiation, especially at an early stage.