Synergistic Differentiation by Chronic Exposure to Cyclic AMP and Nerve Growth Factor Renders Rat Phaeochromocytoma PC12 Cells Totally Dependent upon Trophic Support for Survival

Abstract

Chronic dibutyryl cAMP (dbcAMP) treatment was observed not only to potentiate the differentiating actions of nerve growth factor (NGF) in PC12 cells, but to render them completely dependent on trophic support for survival even in the presence of serum proteins. When both NGF and dbcAMP were withdrawn from doubly differentiated PC12 cultures, degenerative events occurred after a lag period of 12-18 h, and by 48 h < 5-10% of the cells remained viable. Reduction in [3H]dopamine uptake, an index of cell function and neurite integrity, paralleled cell demise. At the cellular level, approximately 20-30% of the nuclei exhibited clear signs of chromatin fragmentation, as characterized by propidium iodide staining, suggesting that degeneration occurred by apoptosis. The cells could be rescued completely from degeneration by dbcAMP or by other 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 stopped by restoration of initial culture conditions. Degenerative changes produced by deprivation and recovery processes were not inhibited by macromolecular synthesis inhibitors, e.g. cycloheximide and actinomycin-D. However, chronic addition of cycloheximide prior to deprivation greatly 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 PC12 cells.

PDE4 inhibitors activate a mitochondrial apoptotic pathway in chronic lymphocytic leukemia cells that is regulated by protein phosphatase 2A

“…Elevation of cAMP inhibits apoptosis in neural (rat pheochromocytoma PC12 and primary rat cerebellar granule neurons), hepatic (primary rat hepatocytes), and myeloid (MC/9 and primary neutrophils) cells. [22][23][24][25][26] Comparable treatment of thymocytes, resting human B cells, CLL cells, or acute lymphoblastic leukemia (ALL) cells induces apoptosis, while peripheral T cells are generally resistant to cAMP-mediated apoptosis. 1,[27][28][29][30] The mechanisms underlying these striking differences remain unclear.…”

Pressure related apoptosis in neuronal cell lines

“…These cells were differentiated in vitro to produce neurons that had exited the cell cycle (Michel et al, 1995). Undifferentiated PC12 cultures were maintained in DMEM with 5% fetal calf serum and 10% heat inactivated horse serum (Gibco).…”

Mitochondrial Free Radical Signal in Ceramide‐Dependent Apoptosis: A Putative Mechanism for Neuronal Death in Parkinson's Disease

“…Pheochromocytoma-derived PC12 cells (Greene and Tischler, 1976), which acquire a neuronal phenotype in the presence of nerve growth factor (NGF) and both synthesize and take up dopamine, were plated at a density of 2,000-3,000/cm 2 in 24-well culture dishes coated with polyethylenimine (1 mg/mi) in Leibovitz modified L15 medium supplemented with 2% horse serum and 150 ng/ml NGF (grade II; Alomone Labs), as previously described (Michel et al, 1995). Apoptosis was induced, after 8 days in the presence of NGF, with the cell-permeant C 2 analogue of ceramide, N-acetylsphingosine (Biomol Research Laboratory), which mimics endogenous ceramide in the sphingomyelin-dependent signaling pathway, in nonneuronai cells (Obeid et al, 1993) and in primary cultures of mesencephalon (Brugg et al, 1996).…”