Real Time RT-PCR Shows Correlation between Retinoid-Induced Apoptosis and NGF-R mRNA Levels

Abstract

Neurotrophins and retinoic acid have a critical role in the differentiation and the survival of neurons. All-trans-, 9-cis-retinoic acid (10−6 M) or NGF (50–100 ng/ml) induced morphologic differentiation and inhibited cell growth in SH-SY5Y neuroblastoma cells after 7 days of culture. Continuous treatment of undifferentiated cells with all-trans- or 9-cis-retinoic (10−6 M) did not induce apoptosis, whereas NGF-differentiated cells showed dramatic apoptosis after 2 to 4 days of retinoic acid treatment as evidenced by TUNEL reaction and flow cytometry analysis following propidium iodide staining. Addition of Ro41-5253 blocked all-trans-retinoic-induced apoptosis, suggesting that the apoptotic signaling pathway was mediated by RARs. The effects of all-trans- or 9-cis-retinoic acid on the expression of NGF receptors was evaluated using real-time fluorescence reverse transcription-PCR. A slight transient increase in the expression of p75NGFR mRNA was observed by 2 to 4 h after retinoid treatment of undifferentiated cells, whereas a larger increase in the expression of both TrkA and p75NGFR mRNA up to threefold the basal level, was observed by 2 to 6 h after retinoid treatment of NGF-differentiated cells. Our results suggest that NGF-differentiated cells may be more susceptible to retinoid-induced apoptosis that undifferentiated cells.