We have tested the ability of various compounds to raise intracellular cyclic AMP (cAMP) levels and, either alone or in combination with retinoic acid (RA), to promote differentiation of two “RA-resistant” sublines of LA-N-5 human neuroblastoma cells, designated LA-N-5HP and LA-N-5R9. Direct activation of adenylate cyclase by forskolin and cholera toxin increased intracellular cAMP levels over 10-fold in both cell lines after 1 h of treatment, after which the levels slowly declined for the next 16 to 24 h. After 5 days of continuous treatment, cAMP levels still remained 2- to 7-fold elevated above controls and were accompanied by a decrease in cell proliferation and an increase in neurite outgrowth. All these effects were exaggerated when the agents were combined with phosphodiesterase enzyme inhibitors. Increasing cAMP levels (up to 24-fold) with N6,O2′-dibutyryl cyclic AMP (dbcAMP) or 8-bromo-cAMP also resulted in decreased proliferation and an increase in morphological differentiation. Isoproterenol and epinephrine did not alter cAMP levels and had no discernible biological effects. Of the agents that raised cAMP levels, only dbcAMP caused an increase in acetylcholinesterase activity. This effect was duplicated with sodium butyrate and prostaglandin E1 in the absence of an increase in cAMP. RA promoted differentiation but also had little effect on cAMP levels. Combination treatment of cells with RA plus agents that raised cAMP levels resulted in greater degrees of differentiation than seen with single agent treatments. We conclude that: (a) the cAMP synthetic and degradative pathways are functional in LA-N-5HP and LA-N-5R9 cells; (b) elevation of cAMP is sufficient for inhibiting proliferation and promoting neurite outgrowth from these cells, but is not a necessary condition for inducing differentiation; and (c) elevation of intracellular cAMP potentiates the differentiation-inducing activity of RA.
This work was supported by Grants CA43503, CA30515, and CA39515 awarded by the NIH, Department of Health and Human Services, and by the Goldwyn Foundation, Los Angeles, CA.