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At least 127 records · Page 7Linked to original sources
PubMed2006

Oxidative stress reveals heterogeneity of FMRP granules in PC12 cell neurites.

PC12 cells are a well-known model of parasympathetic neurons. They have also been used to study the dynamics of heterologously expressed fragile X mental retardation (FMRP) granule trafficking down neurites. Here, we demonstrate that undifferentiated and differentiated PC12 cells harbor endogenous FMRP-containing granules. These granules are not stress granules because they do not associate with an authentic stress granule marker protein T-cell internal antigen 1 (TIA-1). Treatment with sodium arsenite induces stress granule formation in undifferentiated and differentiated PC12 cells. In NGF-treated cells, FMRP-containing stress granules are observed in the soma, neurites and growth cones by co-immunostaining with anti-TIA-1 antibody. These data demonstrate that all three microdomains respond similarly to oxidative stress. Nevertheless, we find significantly less co-localization of FMRP and TIA-1 and FMRP and its homologs in the neurites of differentiated PC12 cells treated with sodium arsenite than in the soma or growth cones. The heterogeneity of these granules suggests that FMRP has multiple roles in neurites.

Animals↗
PubMed2001

Peroxide mediates ethanol-induced cytotoxicity in PC12 cells.

Pheochromocytoma (PC12) cell cultures exhibited a loss of cells and increase in intracellular oxidative stress when exposed to ethanol (EtOH) for 24 h. Catalase, an enzyme that hydrolyzes hydrogen peroxide (H(2)O(2)) to O(2) and H(2)O can attenuate EtOH-induced cell loss and oxidative stress in PC12 cells. This study provides the first clear evidence that oxidative stress in the form of elevated intracellular H(2)O(2) is a primary mechanism of EtOH neurotoxicity in PC12 cells.

Animals↗
PubMed1997

Different mechanisms for inhibition of cell proliferation via cell cycle proteins in PC12 cells by nerve growth factor and staurosporine.

PC12 cells have previously been shown to cease cell division during nerve growth-factor (NGF)-induced differentiation by affecting specific cell cycle proteins. Staurosporine, a protein kinase inhibitor, also causes PC12 cell differentiation, independently of neurotrophins or plasma membrane receptors. We have investigated the relationship of the tumor suppressor protein, p53, and other cell cycle proteins to the antiproliferative effects of NGF and staurosporine in PC12 cells. NGF treatment of PC12 cells stimulated an increase of p53 protein in the nucleus and, more slowly, an increase in total cellular p53 protein. Levels of the cyclin-kinase inhibitor p21/WAF1, cyclin D1, and cyclin G, all downstream transcriptional targets of p53, increased after short times of NGF treatment. Cessation of replication and differentiation occurred more rapidly in defined medium (2 days) than in serum medium (6 days), in correspondence with the more rapid changes in both p53 and p21/WAF1 levels in defined medium (1 hour) than in serum (1 day). Levels of p34cdc2 and p33cdk2 kinase dropped after 6 to 10 days treatment with NGF in serum, close to the time of terminal differentiation. Staurosporine, on the other hand, inhibited DNA replication of PC12 cells in a time- and dose-dependent fashion by affecting cyclin-dependent kinases. Staurosporine had no effect on the protein levels of p53, p21/WAF1, or cyclin G. The kinase activity of both p34cdc2 and p33cdk2 were inhibited in vitro with IC50 values of 20 nM and 75 nM, respectively. In vivo p34cdc2 kinase activity was inhibited within 1 day, before the decrease in the levels of p34cdc2 protein at days 2 to 3. In contrast, in vivo p33cdk2 kinase activity only decreased in concert with protein levels. Although both NGF and staurosporine inhibit DNA replication concomitant with induction of differentiation by affecting the activity of p34cdc2 and p33cdk2, the mechanism of the two agents is quite different. NGF achieves inhibition of activity of these cyclin-dependent kinases by signalling through the TrkA receptor to the tumor suppressor protein p53 and then to p21/WAF1. In contrast, staurosporine directly inhibits the activity of p34cdc2 and p33cdk2 by binding to them and also indirectly by alteration of their phosphorylation through other regulatory kinases.

Animals↗
PubMed1987

Further characterization of dopamine release by permeabilized PC12 cells.

Rat pheochromocytoma cells (PC12) permeabilized with staphylococcal alpha-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC12 cells. Permeabilization with alpha-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC12 cells.

Adenosine Triphosphate↗
PubMed2004

RNA interference-mediated silencing of synaptotagmin IX, but not synaptotagmin I, inhibits dense-core vesicle exocytosis in PC12 cells.

Although PC12 cells express three synaptotagmin isoforms (Syts I, IV and IX), all of which have been proposed to regulate dense-core vesicle exocytosis, it remains unknown which of the Sytisoforms acts as the major Ca2+ sensor for dense-core vesicle exocytosis. In the present study, it has been shown by immunoaffinity purification and immunocytochemistry that Syts I and IX, but not Syt IV, are present on the same secretory vesicles in PC12 cells. Silencing of Syt IX with specific small interfering RNA significantly reduced high KCl-dependent neuropeptide Y secretion from PC12 cells, whereas silencing of Syt I with specific small interfering RNA had no significant effect. The results indicate that Syts I and IX are not functionally equivalent and that Syt IX, and not Syt I, is indispensable for the regulation of Ca2+-dependent dense-core vesicle exocytosis in PC12 cells.

Animals↗
PubMed1998

Overexpression of cation-dependent mannose 6-phosphate receptor prevents cell death induced by serum deprivation in PC12 cells.

PC12 cells express well cation-independent mannose 6-phosphate receptors (CI-MPR), but not cation-dependent (CD)-MPR as much. To examine CD-MPR dependency of transport of cathepsins B and D to lysosomes in PC12 cells, we prepared the cells overexpressing CD-MPR. Immunoreactivity for cathepsin B became more distinct and larger in size in the transfected cells than in wild-type cells. No difference in the distribution of cathepsin D was seen between these two cells. The viability of the cells following serum deprivation was significantly higher in the transfected cells than in wild-type cells. This increased viability of the transfected cells was blocked by CA074, a specific inhibitor of cathepsin B, while pepstatin A suppressed the action of CA074. The results suggest that CD-MPR preferentially transport cathepsin B in PC12 cells, and cathepsins B and D participate in the regulation of PC12 cell apoptosis.

Amino Acid Sequence↗
PubMedDate not supplied

Inhibition of growth factor-induced differentiation of PC12 cells by microinjection of antibody to ras p21.

The protein products (p21) of the ras cellular proto-oncogenes are thought to transduce membrane signals necessary for the induction of cell division. However, there is uncertainty as to the precise role of ras p21 in mediating ligand-membrane receptor signals leading to cell differentiation. Treatment of rat phaeochromocytoma cells (PC12) with nerve growth factor (NGF) results in the induction of a number of phenotypic characteristics of sympathetic neurones, including cessation of cell division and outgrowth of neuronal processes (neurites). Here we report that microinjection of antibody to ras p21 into PC12 cells inhibited neurite formation and resulted in temporary regression of partially extended neurites, an effect which was observed up to 36 h after initiation of NGF treatment. Neurite formation induced by cyclic AMP was unaffected by injection of anti-p21 antibody. These results indicate that p21 is involved in the initiation phase of NGF-induced neurite formation in PC12 cells and has a role in hormone-mediated cellular responses distinct from cell proliferation.

Animals↗
PubMed2000

Mediation of nerve growth factor-driven cell cycle arrest in PC12 cells by p53. Simultaneous differentiation and proliferation subsequent to p53 functional inactivation.

Upon stimulation with nerve growth factor (NGF), PC12 cells extend neurites and cease to proliferate by influencing cell cycle proteins. Previous studies have shown that neuritogenesis and a block at the G(1)/S checkpoint correlate with the nuclear translocation of and an increase in the p53 tumor suppressor protein. This study was designed to determine if p53 plays a direct role in mediating NGF-driven G(1) arrest. A retroviral vector that overexpresses a temperature-sensitive p53 mutant protein (p53ts) was used to extinguish the function of endogenous p53 in PC12 cells in a dominant-negative manner at the nonpermissive temperature. NGF treatment led to transactivation of a p53 response element in a luciferase reporter construct in PC12 cells, whereas this response to NGF was absent in PC12(p53ts) cells at the nonpermissive temperature. With p53 functionally inactivated, NGF failed to activate growth arrest, as measured by bromodeoxyuridine incorporation, and also failed to induce p21/WAF1 expression, as measured by Western blotting. Since neurite outgrowth proceeded unharmed, 50% of the cells simultaneously demonstrated neurite morphology and were in S phase. Both PC12 cells expressing SV40 T antigen and PC12 cells treated with p53 antisense oligonucleotides continued through the cell cycle, confirming the dependence of the NGF growth arrest signal on a p53 pathway. Activation of Ras in a dexamethasone-inducible PC12 cell line (GSRas1) also caused p53 nuclear translocation and growth arrest. Therefore, wild-type p53 is indispensable in mediating the NGF antiproliferative signal through the Ras/MAPK pathway that regulates the cell cycle of PC12 cells.

Animals↗
PubMed1995

Interleukin 6 mediated differentiation and rescue of cell redox in PC12 cells exposed to ionizing radiation.

The differentiation of PC12 cells to a neuron-like morphology was induced by ionizing radiation in the presence of serum. This effect was detectable at 5 grays (Gy) and reached a maximum at 10-20 Gy. Increases in the DNA binding activity of nuclear factor kappa B (NF-kappa B) and increased Interleukin 6 (IL-6) mRNA levels were observed at a dose of 15 Gy. Neutralization of supernatant IL-6 by the addition of anti-IL-6 antibody inhibited the neuronal differentiation and decreased cellular redox. Ionizing radiation and serum may act synergistically as neurotropic factors.

Animals↗
PubMed2004

Protective effect of 1-methylated beta-carbolines against 3-morpholinosydnonimine-induced mitochondrial damage and cell viability loss in PC12 cells.

The present study investigated the effect of 1-methylated beta-carbolines (harmaline, harmalol and harmine) on change in the mitochondrial membrane permeability and cell death due to reactive nitrogen species in differentiated PC12 cells. beta-Carbolines, caspase inhibitors (z-LEHD.fmk and z-DQMD.fmk) and antioxidants (N-acetylcysteine, dithiothreitol, melatonin, carboxy-PTIO and uric acid) depressed cell viability loss due to 3-morpholinosydnonimine (SIN-1) in PC12 cells. beta-Carbolines inhibited the nuclear damage, the decrease in mitochondrial transmembrane potential, the cytochrome c release, the formation of reactive oxygen species and the depletion of GSH caused by SIN-1 in PC12 cells. beta-Carbolines decreased the SIN-1-induced formations of 3-nitrotyrosine, malondialdehyde and carbonyls in PC12 cells. The results show that 1-methylated beta-carbolines attenuate SIN-1-induced mitochondrial damage. This results in the inhibition of caspase-9 and -3 and apoptotic cell death in PC12 cells by suppressing the toxic actions of reactive oxygen and nitrogen species, including the GSH depletion.

Animals↗
PubMed2005

Synergistic effects of hydrogen peroxide and ethanol on cell viability loss in PC12 cells by increase in mitochondrial permeability transition.

The promoting effect of ethanol against the cytotoxicity of hydrogen peroxide (H2O2) in differentiated PC12 cells was assessed by measuring the effect on the mitochondrial membrane permeability. Treatment of PC12 cells with H2O2 resulted in the nuclear damage, decrease in the mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c, activation of caspase-3, increase in the formation of reactive oxygen species (ROS) and depletion of GSH. In PC12 cells and dopaminergic neuroblastoma SH-SY5Y cells, the promoting effect of ethanol on the H2O2-induced cell death was increased with exposure time. Ethanol promoted the nuclear damage, change in the mitochondrial membrane permeability, ROS formation and decrease in GSH contents due to H2O2 in PC12 cells. Catalase, carboxy-PTIO, Mn-TBAP, N-acetylcysteine, cyclosporin A and trifluoperazine inhibited the H2O2 and ethanol-induced mitochondrial dysfunction and cell injury. The results show that the ethanol treatment promotes the cytotoxicity of H2O2 against PC12 cells. Ethanol may enhance the H2O2-induced viability loss in PC12 cells by promoting the mitochondrial membrane permeability change, release of cytochrome c and subsequent activation of caspase-3, which is associated with the increased formation of ROS and depletion of GSH. The findings suggest that ethanol as a promoting agent for the formation of mitochondrial permeability transition may enhance the neuronal cell injury caused by oxidants.

Animals↗
PubMed2005

Modulation of delayed rectifier potassium channels by alpha1-adrenergic activation via protein kinase C zeta and p62 in PC12 cells.

When PC12 cells are exposed to nerve growth factor (NGF), they extend neurites and express autonomic ganglion cell properties. We have previously shown that NGF is capable of inducing p62 expression, enabling the formation of the protein kinase C zeta (PKCzeta)-p62-Kvbeta (beta-subunit of delayed rectifier K+ channel) complex, a Kv channel-modulating complex. The formation of this complex results in the shifting of the Kv channel activation curve to the left via PKCzeta activity. During the experiments, we noted that PC12 cells in a high-density culture exhibited a Kv channel activation curve shift similar to that observed in the NGF-treated cells. Therefore, we hypothesized that catecholamines released from PC12 cells may induce p62 expression. In order to test this idea, cells in a low-density culture were treated for 24h with norepinephrine (NE). In these cells, we noted a leftward shift of the activation curve. The presence of the alpha1-adrenergic antagonist specifically prevented the effects of NE. Pre-treatment of the low-density cells with alpha1-agonists induced changes similar to those associated with NE, confirming that NE modulates Kv channels via the alpha1-adrenergic receptor. NE's effects were blocked by treatment with PKCzeta specific inhibitors. Using Western blotting, we observed increased levels of p62 expression in both the high-density cells and the NE-treated low-density cells. These results suggest that locally secreted NE induces an increase in p62 expression, and also exerts a modulatory effect on Kv channels via the PKCzeta-p62-Kvbeta channel modulating complex.

Adrenergic alpha-Agonists↗
PubMed1995

Inhibition of drug-induced apoptosis by survival factors in PC12 cells.

Pheochromocytoma (PC12) cells have been shown to undergo apoptosis (programmed cell death) when deprived of serum and to be rescued by nerve growth factor, fibroblast growth factor, dibutyryl cyclic AMP, aurintricarboxylic acid, or exogenous expression of bcl-2. We show here that the cytotoxic drugs cycloheximide, actinomycin D, colchicine, and EGTA also induce apoptosis in PC12 cells. These findings prompted us to investigate whether apoptosis induced by these drugs involves similar pathways in each case, and whether the factors preventing the apoptotic death of serum-deprived PC12 cells can also protect the cells from apoptosis induced by the cytotoxic drugs. Nerve growth factor, dibutyryl cyclic AMP, and expression of bcl-2 inhibited apoptosis induced by all four cytotoxic drugs. Fibroblast growth factor inhibited apoptosis induced by EGTA or colchicine. Aurintricarboxylic acid inhibited apoptosis induced by EGTA. These results suggest that apoptosis induced by treatments with the various drugs is mediated by different initiating pathways, all of which converge into a final, common pathway. Nerve growth factor, dibutyryl cyclic AMP, and bcl-2 appear to affect the final common pathway, whereas fibroblast growth factor and aurincarboxylic acid appear to be more specific and affect only some of the pathways.

Animals↗
PubMed1993

Protein kinase C isozymes that mediate enhancement of neurite outgrowth by ethanol and phorbol esters in PC12 cells.

Using PC12 cells to study ethanol's effects on growth of neural processes, we found that ethanol enhances NGF- and basic FGF-induced neurite outgrowth. Chronic ethanol exposure selectively up-regulates delta and epsilon protein kinase C (PKC) and increases PKC-mediated phosphorylation in PC12 cells. Since PKC regulates differentiation, we investigated the role of PKC in enhancement of neurite outgrowth by ethanol. Like ethanol, 0.3-10 nM phorbol 12-myristate, 13-acetate (PMA) increased NGF-induced neurite outgrowth. However, higher concentrations did not, and immunoblot analysis demonstrated that 100 nM PMA markedly depleted cells of beta, delta and epsilon PKC. PMA (100 nM) also down-regulated beta, delta and epsilon PKC in ethanol-treated cells and completely prevented enhancement of neurite outgrowth by ethanol. In contrast, the cAMP analogue 8-bromoadenosine cAMP did not completely mimic the effects of ethanol on neurite outgrowth, and ethanol was able to enhance neurite formation in mutant PC12 cells deficient in protein kinase A (PKA). These findings implicate beta, delta or epsilon PKC, but not PKA, in the neurite-promoting effects of ethanol and PMA. Since chronic ethanol exposure up-regulates delta and epsilon, but not beta PKC, these findings suggest that delta or epsilon PKC regulate neurite outgrowth.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Dexamethasone-induced effects on B-50/GAP-43 expression and neurite outgrowth in PC12 cells.

Undifferentiated PC12 cells contain detectable levels of the nervous-specific protein B-50/GAP-43. Upon treatment with NGF or change of culture medium, B-50/GAP-43 levels remained unchanged during the first 12 hours while neuritogenesis starts. Both, B-50/GAP-43 levels and neurite outgrowth peak at 24 hours. These results suggest that in PC12 cells the amount of B-50 already present is sufficient to support the start of NGF-induced neuritogenesis, presumably by translocation from cytosolic compartments to the membrane. Addition of DEX reversed the rise in B-50/GAP-43 levels induced by either the change of medium or by NGF. In contrast, neurite outgrowth was inhibited to a lesser extent, although after 36 hours of pretreatment with DEX neurite length was lower than control. NGF was capable of enhancing B-50/GAP-43 levels both in the presence and absence of DEX. This corroborates data from others, who concluded that DEX and NGF exert their effects through different mechanisms, e.g., transcription versus mRNA stabilization, respectively. The inhibitory effect of DEX under various conditions on both B-50 expression and neurite outgrowth in the normal PC12 cell line demonstrates the tight coupling of these parameters that might be indicative of a threshold effect of B-50 levels on neurite outgrowth.

Animals↗
PubMed2000

Activation of signal transducers and activators of transcription by alpha(1A)-adrenergic receptor stimulation in PC12 cells.

In PC12 cells stably expressing alpha(1A)-adrenergic receptors (ARs), norepinephrine (NE) activates several mitogen-activated protein kinase pathways and causes differentiation (). Using retroviral luciferase reporters, we found that NE also activated both signal transducers and activators of transcription (Stat) and gamma-interferon-activated sequence-mediated transcriptional responses, with maximal effects similar to those caused by interleukin-6 (IL-6). UTP and epidermal growth factor had no effect, whereas nerve growth factor caused a small Stat activation. Responses to NE were blocked by prazosin and depended on receptor density. Responses to NE were not blocked by inhibitors of mitogen-activated protein kinase kinase (PD98059), protein kinase C (GFX203290), Src (PP2), Jak2 (AG490), or the calcium chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. The p38 mitogen-activated protein kinase inhibitors SB202190 and SB203580 blocked Stat activation by NE, the epidermal growth factor receptor inhibitor AG1478 caused a small inhibition, but the phosphoinositide 3 kinase inhibitor LY294002 potentiated both responses. Gel shifts confirmed formation of nuclear factors binding to both Stat and gamma-interferon-activated sequence consensus sequences in response to NE and IL-6. Immunoprecipitation experiments showed that IL-6 increased tyrosine phosphorylation of Stat1 and Stat3 in PC12 cells, whereas NE caused a sustained increase in tyrosine phosphorylation of Stat1. These results suggest that alpha(1A)-AR stimulation causes Stat-mediated transcriptional responses in PC12 cells that are not downstream of known second messenger or tyrosine kinase pathways.

Animals↗
PubMed1993

Regulation of muscarinic stimulation of norepinephrine release and PI hydrolysis during cell growth in PC12 cells.

We have studied the relationships of cell growth to muscarinic stimulation of norepinephrine release and phosphoinositide hydrolysis in the rat pheochromocytoma PC12 cells. The ability of these cells to release norepinephrine in response to muscarinic agonists was maximal during the early phase of exponential growth, and then rapidly decreased to undetectable levels as the cells approached stationary phase. In contrast, muscarinic stimulation of phosphoinositide hydrolysis was low in the early exponential phase of growth, increased to a maximum during late exponential growth and then dramatically dropped in the stationary phase. The number of muscarinic receptors, as measured by antagonist-binding studies, also varied during cell growth with maximal levels at days 2 and 8, corresponding to the maxima in muscarinic-stimulated norepinephrine release and phosphoinositide hydrolysis, respectively.

Animals↗
PubMed2002

Reduced pteridine derivatives induce apoptosis in PC12 cells.

In cerebrospinal fluid of patients with cerebral infections, elevated concentrations of the pteridine compounds neopterin and 7,8-dihydroneopterin were detected. Here, the potential of pteridines to induce apoptosis of the rat pheochromocytoma cells (PC12) was investigated. In contrast to aromatic pteridines like neopterin, the reduced forms 7,8-dihydroneopterin, 5,6,7,8-tetrahydrobiopterin and 7,8-dihydrobiopterin led to a significant increase of apoptotic cells. After terminal differentiation, cells were less sensitive to incubation with pteridines. A noticeable augmentation of apoptosis was observed upon incubation with 7,8-dihydroneopterin and 7,8-dihydrofolic acid. Antioxidants partly protected PC12 cells from pteridine-induced apoptosis, suggesting the involvement of reactive oxygen intermediates. Exposure of cells to 7,8-dihydroneopterin led to activation of the mitogen-activated protein (MAP) kinase and to a lesser degree also of JUN/SAP kinase. Results implicate that high concentrations of reduced pteridines, might contribute to the pathogenesis involved in neurodegeneration.

Animals↗