SYNERGISTIC DIFFERENTIATION BY CHRONIC EXPOSURE TO CYCLIC-AMP AND NERVE GROWTH-FACTOR RENDERS RAT PHEOCHROMOCYTOMA PC12 CELLS TOTALLY DEPENDENT UPON TROPHIC SUPPORT FOR SURVIVAL

Chronic dibutyryl cAMP (dbcAMP) treatment was observed not only to pot entiate the differentiating actions of nerve growth factor (NGF) in PC 12 cells, but to render them completely dependent on trophic support f or survival even in the presence of serum proteins. When both NGF and dbcAMP were withdrawn from doubly differentiated PC12 cultures, degene rative events occurred after a lag period of 12-18 h, and by 48 h <5-1 0% of the cells remained viable. Reduction in [H-3]dopamine uptake, an index of cell function and neurite integrity, paralleled cell demise, At the cellular level, similar to 20-30% of the nuclei exhibited clea r signs of chromatin fragmentation, as characterized by propidium iodi de staining, suggesting that degeneration occurred by apoptosis, The c ells could be rescued completely from degeneration by dbcAMP or by oth er cAMP analogues, whereas NGF and depolarization were also effective, but only partially. Phorbol 12-myristate-13-acetate failed to afford protection. If deprivation was interrupted, cell demise could be stopp ed by restoration of initial culture conditions, Degenerative changes produced by deprivation and recovery processes were not inhibited by m acromolecular synthesis inhibitors, e.g, cycloheximide and actinomycin -D. However, chronic addition of cycloheximide prior to deprivation gr eatly impaired the differentiation of NGF/dbcAMP cells, allowing these cells to withstand trophic support withdrawal, Altogether our results indicate that the cAMP transduction pathway plays a crucial role not only in the differentiation but also in the survival of NGF/dbcAMP PC1 2 cells.