Effect of 8-bromo-cyclic AMP on neuron specific enolase, heat shock protein, nitric oxide, nitric oxide synthase and nitric oxide synthase mRNA in human retinoblastoma HXO-Rb44 cells and cell differentiation

Objective To study the effect of 8-bromo-cyclic AMP (8-Br-cAMP) on nitric oxide synthase (NOS) mRNA, NOS and nitric oxide (NO) product, heat shock protein (hsp)70 and neuron specific enolase (NSE) in human retinoblastoma HXO-Rb44 cells and the effect related to cell differentiation. Methods Cultured human retinoblastoma HXO-Rb44 cells were divided into two aliquots. One was cultured with 2×10(-5)mol/L of 8-Br-cAMP for 24 hours as the experiment group; the other was treated with no 8-Br-cAMP as the control group. The cell suspensions in concentration of 1×10(7)/ml in both groups were dropped onto the nitrocellulose membrane (NCM). The NOS mRNA was detected with the biotin-labeled NOS cDNA probe by RNA dot blot. The NOS activity was detected by protein dot blot. The immunoreactivity (IR) of hsp70 and NSE was detected by protein dot blot. The NO was detected by nitrate reductase method. NCM specimens were analyzed by a TLC scanner for detection of the dot blot signal intensity. Results The signals of NOS mRNA, NOS activity, hsp70-IR, NSE-IR, and NO content in the experiment group were higher than those in the control group (P<0.05-0.01).Conclusions 8-Br-cAMP could increase NO product and the expression of NOS mRNA, NOS , NSE and hsp70. The results indicate that 8-Br-cAMP could facilitate synthesis of NO in the neuroblastoma HXO-Rb44 cells which could have tendency toward neuron development, suggesting that the increased hsp70, NO and NOS may involve cell differentiation of the retinoblastoma HXO-Rb44.