Multiple agents rescue PC12 cells from serum-free cell death by translation- and transcription-independent mechanisms

Abstract

Past studies revealed that NGF and fibroblast growth factor (FGF) prevent the death of PC 12 pheochromocytoma cells that otherwise occurs in serum-free medium. Additional agents were tested here for their abilities to promote long-term survival of naive and NGF-pretreated (primed) PC 12 cells in serum-free conditions. Forskolin and permeant cAMP analogs effectively prevented serum-free cell death, as did micromolar levels of insulin and 10–100-nM levels of insulin-like growth factors I and II. In contrast to NGF and FGF, none of these agents caused neuronal differentiation of naive cells or neurite regeneration by primed cells. Each of the agents also prevented rapid cell death in a balanced salt solution, thus apparently ruling out a mechanism dependent on regulation of nutrient uptake. Epidermal growth factor and elevated K+ appeared to slow the rate of cell death, but did not promote long-term survival; phorbol ester, dexamethasone, or vanadate did not prevent cell death. Each of the survival-promoting agents was effective even when macromolecular synthesis was blocked. Because the synthesis inhibitors themselves did not significantly prevent cell death, such findings indicate that survival was promoted by mechanisms that do not require synthesis of RNA or protein. In addition, various lines of experimental evidence (using the kinase inhibitor K-252a or PC 12 cell variants deficient either in protein kinase A activity or in responsiveness to NGF) further suggested that the effective agents maintain survival by independent initial pathways. Regulation of protein kinase activity appears to be a common feature of each pathway and may therefore play a key convergent role in mediating prevention of cell death.

The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic

“…Thus one possible explanation for the differences between the cell types could therefore be due to differential expression of high and low affinity Trk\NGF receptors with different signalling properties to downstream signal transduction components. This possibility has been suggested by others for the differential capabilities of the low and high affinity NGF receptors to protect cells from apoptosis [37,38]. A further possible explanation for Isolated hepatocytes were infected with an SV40 promoter driven ∆Raf-1 : ER plasmid conjugated via poly-L-lysine to a replication defective adenovirus (250 m.o.i.)…”

Nedd2 Is Required for Apoptosis after Trophic Factor Withdrawal, But Not Superoxide Dismutase (SOD1) Downregulation, in Sympathetic Neurons and PC12 Cells

“…NGF-primed (neuronally differentiated) PC12 cells were grown for at least 7 d in RPMI 1640 medium plus 1% horse serum and NGF (100 ng/ml). For cell survival assays involving trophic factor deprivation, cells (either naive or NGF-pretreated) were washed extensively in serum-free RPMI 1640 medium and replated on fresh collagen-coated 24-well dishes, as previously described (Rukenstein et al, 1991), in RPMI 1640 medium lacking serum or NGF. For SOD1 downregulation survival assays, cells were replated in complete medium with V-ASOD1 (vector-linked antisense oligonucleotide to SOD1, 50 nM), as previously described (Troy et al, 1996a).…”

“…Various concentrations of V-ANedd (vector-linked antisense oligonucleotide to Nedd2) were included in the medium as indicated. Numbers of viable cells per culture were determined by quantifying intact nuclei, as previously described (Rukenstein et al, 1991). Counts were performed in triplicate and reported as mean Ϯ SEM.…”

“…In the rat pheochromocytoma PC12 line, a commonly used model for neuronal differentiation and cell death, apoptosis may be triggered by either trophic factor/nerve growth factor (NGF) withdrawal (Greene, 1978;Batistatou and Greene, 1991;Rukenstein et al, 1991;Mesner et al, 1992;Pittman et al, 1993;Lindenboim et al, 1995) or oxidative stress induced by downregulation of Cu 2ϩ /Zn 2ϩ superoxide dismutase (SOD1) (Troy and Shelanski, 1994;Troy et al, 1996a-c). The initiating mechanisms of death seem to be distinct in each instance.…”

“…The initiating mechanisms of death seem to be distinct in each instance. Apoptosis triggered by NGF deprivation is blocked by cAMP analogs (Rydel and Greene, 1988;Rukenstein et al, 1991) and high concentrations of N-acetylcysteine (Ferrari et al, 1995), whereas these agents do not inhibit death induced by downregulation of SOD1 (Troy et al, 1996a,c). In contrast, the latter is blocked by vitamin E (Troy and Shelanski, 1994) and inhibitors of nitric oxide (NO) synthase (Troy et al, 1996a), which have no effect on apoptosis evoked by NGF withdrawal (Ferrari et al, 1995;Farinelli et al, 1996).…”