Nerve growth factor regulates neuroectodermal tumor cell responses to mitogenic growth factors

Previous studies showed that nerve growth factor (NGF) decreases the proliferation of neuroectodermal tumor (NET) cells (C-1300 and Neuro2A murine neuroblastoma, PC12 rat pheochromocytoma) within 5-7 days in a dose-dependent manner. This effect is regulated by the concentration of serum in the culture medium. Therefore, we hypothesized that NGF exerts its antimitogenic activities by interfering with the proliferative action of other growth factors. We studied the effects of short-term vs. long-term as well as endogenous vs. exogenous NGF on NET cell proliferation in response to various mitogenic growth factors. Retrovirus-mediated transfer of the beta-NGF gene into NET cells activated TrkA and consistently decreased their proliferative responses to insulin-like growth factor (IGF)-I, IGF-II, fibroblast growth factor-2, and epidermal growth factor (EGF), down-regulating EGF and IGF-I binding sites. It also decreased tyrosine phosphorylation of ERK-1, STAT3, and EGF or IGF receptors after treatment with IGF-I or EGF. Long-term incubation of NET cells with NGF mimicked the responses induced by beta-NGF gene transfer, albeit in a reversible manner. Short-term NGF treatment augmented the proliferative responses to IGF-I or EGF by enhancing cell survival. It also increased tyrosine phosphorylation of signal transducing proteins after exposure to IGF or EGF, an effect opposite to that of long-term NGF treatments. Hence, long-term NGF exposure in vitro might better reproduce the effects of NGF in vivo than short-term treatments. Only long-term exposure to NGF decreased the responses of NET cells to mitogenic growth factors by down-regulating their receptors and attenuating signal transduction events required for cell proliferation. These results suggest that NGF could exert similar actions on cellular responses to growth factors in vivo.     

Effect of nerve growth factor on C-1300 murine neuroblastoma tumor growth and catecholamine content in neonatally sympathectomized mice

The in situ C-1300 murine neuroblastoma (MNB) tumor model was used to investigate the influence of exogenously administered nerve growth factor (NGF) on tumor growth and tissue catecholamine concentration in mice sympathectomized with 6-hydroxy-dopamine (6-OHDA) on postnatal days 4-10. Mice were implanted with 1 x 10(6) disaggregated MNB cells 3 days after termination of 6-OHDA administration. NGF (12-15 micrograms/mouse/day) treatment was initiated at the time of MNB cell implantation and continued until sacrifice of the animal. The time interval between tumor cell implantation and detection of palpable tumor (tumor onset time), transverse tumor diameter, tumor weight, tumor weight to body weight ratio, and tumor catecholamine concentration were determined. Neonatal sympathectomy caused a decrease in myocardial norepinephrine concentration of 88% compared with vehicle-treated animals as well as a significant reduction in total body and organ weight. Average body, brain, heart, and spleen weights were decreased 31%, 16%, 25%, and 42%, respectively, below control values. The daily injection of NGF, from the time of MNB tumor implantation to sacrifice, did not prevent these effects of chemical sympathectomy from being expressed. Tumor onset time following implantation of MNB cells was significantly increased in neonatally sympathectomized mice and was not altered by treatment with NGF. In contrast, the decrease in MNB tumor growth rate observed in sympathectomized mice was reversed by administration of NGF. Mean tumor weight and mean tumor to body weight ratio were 89% and 115% of comparable control values, respectively, in sympathectomized mice receiving exogenous NGF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bioassay detection of mouse nerve growth factor (mNGF) in the brain of adult mice

Two pools of seven brains each from adult Swiss-Webster mice were homogenized, and supernatants were collected for bioassay. PC-12 cells were placed in a bioassay plate at time zero, at a concentration of 10⁴ cells per well, and primed for 48 hours in a medium containing 50 ng/ml of mNGF. The PC-12 cell bioassay for neurite outgrowth was conducted after primed cells were exposed to an NGF-free medium for 24 hours. Suitable controls for serum toxicity and cell viability were established. The sensitivity of the bioassay approximates 100 pg NGF/ml. The results showed 80–100% neurite outgrowth in wells exposed to brain pool supernatant (BPS) alone, and control level outgrowth (3–8%) in wells containing BPS and specific anti-β-NGF antibody. Therefore, the brains of Swiss-Webster adult mice contain an NGF-like substance which promotes neurite outgrowth in PC-12 cells. The substance probably is NGF itself, since the effect is blocked by specific NGF antiserum.     

Axotomized frog sciatic nerve releases diffusible neurite-promoting factors.

Using the bullfrog (Rana catesbeiana) dorsal root ganglia (DRG) and its sciatic nerve (ScN) as a model system, we have previously described neuronal and non-neuronal molecular changes associated with the early regenerative response of DRG neurons to axotomy. Since diffusible molecular factors, released by axotomized ScN, might function to stimulate axon regrowth, we have assayed the ability of ScN-conditioned bath to promote in vitro neurite outgrowth from PC-12 cells. Diffusible ScN proteins were collected by incubating segments of normal or axotomized ScN in a small volume of RPMI media for 4 h (nerve bath). The nerve baths, supplemented with serum, were then added to PC-12 cell cultures to assay for the presence of neurite growth factors released by ScN. Results showed that nerve baths, collected from sham-operated or axotomized ScN, could not induce the differentiation of PC-12 into neurite-bearing cells. Therefore, in all subsequent neurite growth assay experiments, an exogenous source of nerve growth factor (NGF) (50 ng/ml) was added to the nerve baths or unconditioned media to generate and maintain PC-12 neuritic structure. We found that nerve baths, collected from previously axotomized (at least 3 days post-injury) nerve, contained diffusible factors which enhanced PC-12 neurite growth, relative to unconditioned media. No neurite growth factors were observed to be released by sham-operated ScN or 1-day post-axotomized ScN. Further experiments were conducted to identify the diffusible neurite growth factors released from axotomized ScN. We showed that the release (if any) of endogenous diffusible NGF or laminin from axotomized nerve could not have accounted for the facilitation of neurite growth. Analysis of radiolabelled ScN proteins by two-dimensional polyacrylamide gel could not have accounted for the facilitation of neurite growth. Analysis of radiolabelled ScN proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed that the relative abundance of two diffusible proteins (M(r) approximately 35 and 70 kDa) in the nerve bath was directly correlated with the ability of the nerve bath to facilitate PC-12 neurite growth.     

Neuritogenic and metabolic effects of individual gangliosides and their interaction with nerve growth factor in cultures of neuroblastoma and pheochromocytoma

The 4 major ganglioside species, GM1, GD1a, GD1b and GT1b (200 micrograms/ml), were tested individually for the ability to stimulate neuronal trophic responses. The growth parameters measured were: morphologic changes, quantitated by computer-assisted morphometry of neurite length and number per soma, and metabolic changes, indicated by alterations in ornithine decarboxylase activity (ODC). In addition, the interaction of each ganglioside with nerve growth factor (NGF) was investigated with an NGF-responsive pheochromocytoma PC12 cell line and NGF-insensitive neuroblastoma Neuro-2a cultures. PC12 cells responded to gangliosides only in the presence of NGF (20 micrograms/ml): GM1 produced the greatest morphologic response, but did not alter metabolic levels; GT1b increased both parameters. The presence (5 micrograms/ml) or absence of NGF did not have an effect on the ganglioside-mediated morphologic responses of Neuro-2a cells to each species: GD1b elicited the greatest increase in neurite length, while GD1a and GT1b stimulated both length and number. In contrast, while GT1b alone was able to elevate ODC activity independently of NGF, the simultaneous exposure of Neuro-2a cultures to NGF and GM1 or GD1a resulted in a stimulation of cellular metabolism. These results indicate that each ganglioside species has a specific target action in the stimulation of different trophic responses and that performance in one category is not a predictor of the result in another. In addition, it is possible to confer a sensitivity to NGF by simultaneous treatment with specific gangliosides. This indicates that membrane gangliosides may modulate the actions of neurotrophic factors.     

Herpes simplex virus replication in pheochromocytoma cell line that responds to nerve growth factor

Summary Cultured cells of neural origin (PC-12, pheochromocytoma cell line) respond to nerve growth factor (NGF) by extending neurites. These cells whether treated with NGF or not can be infected with herpes simplex virus and produce progeny virus. Viral antigens are detected on the cell surface and fusion of cells to form polykaryocytes takes place. Nucleocapsids are found within the cell nucleus and enveloped virus in present both in the cytoplasm and extracellular space. Virus was not observed within the neurites but alterations in the neurite microtubular structure occurred after infection.     

p21(ras) stimulates pathways in addition to ERK, p38, and Akt to induce elongation of neurites in PC12 cells

Initiation and elongation of neurites in PC12 cells has been shown to be stimulated by nerve growth factor (NGF). Initiation of NGF-stimulated neurites in a PC12 subclone (PC12-N09) is rapid, giving rise to short neurites that do not elongate after 1 day. To determine whether increasing activation of p21(ras) could restore neurite elongation in these cells and whether it would affect the phosphorylation of signaling proteins, the subclone PC12-N09 was transfected with constitutively active p21(ras61L) (PC12-N09ras61L) and neurite outgrowth with or without NGF was determined. Overexpression of wild-type p21(ras) (PC12-N09rasWT) did not lead to spontaneous neurite initiation but restored the ability of NGF to stimulate continuous neurite elongation. However, NGF-stimulated phosphorylation of ERK, p38, and Akt in PC12-N09rasWT cells is similar in duration to that in PC12-N09 cells, indicating that the p21(ras) signaling through ERK, p38, and Akt was not involved in the restoration of normal neurite elongation in PC12-N09 cells. These results show that p21(ras)-activated pathways other than ERK, p38, and Akt are necessary for appropriate NGF-stimulated neurite elongation in PC12 cells.     

Autocrine differentiation of PC12 cells mediated by retroviral vectors

Rat pheochromocytoma PC12 cells have been modified genetically by the use of replication-defective retroviral vectors containing either the bacterial gene for beta-galactosidase (lac Z) or cDNAs for mouse beta-nerve growth factor (NGF) and the bacterial gene for neomycin resistance. Using the lac Z vector, clonal lines of PC12 cells were obtained in which almost 100% of cells stably expressed this histochemical marker. Infection of PC12 cells or the derived subclone PC12-BAG, which expresses beta-galactosidase, with the NGF vectors resulted in autocrine differentiation as assessed by extensive neurite formation, which occurred within hours after infection and was maintained for weeks in culture. Neurite formation could be partially blocked by antibodies to NGF. The percentage of cells expressing neurite outgrowth was greater than that of PC12 cells treated with exogenous NGF. PC12 cells infected with the NGF vectors were shown to release this trophic factor into the medium using a two-site enzyme immunoassay and a bioassay on 'naive' PC12 cells. PC12 cells genetically modified using these vectors provide a means to: follow the fate of the cells after transplantation into animals; test for delivery in vivo of NGF and catecholamines by grafted, autocrine-differentiated PC12 cells; and study the long-term actions of NGF on responsive cells without adding exogenous NGF.     

On the role of the low-affinity neurotrophin receptor p75LNTR in nerve growth factor induction of differentiation and AP 1 binding activity in PC12 cells

Three clones of PC12 cells that differ with respect to their nerve growth factor (NGF) receptors were examined: wild-type PC12 cells that have both trkA and p75^LNTR receptors; the MR-1 clone that possesses a normal trkA receptor and a truncated form of p75^LNTR without the extracellular NGF-binding part; and a new PC12 variant, called v-clone, that is partly characterized here. The v-clone had no demonstrable binding to trkA, but displayed binding to p75^LNTR as assessed by chemical crosslinking. NGF did not induce any change in the tyrosine phosphorylation of phosphatidy-3′-kinase in the v-clone. NGF induced neurite extension in wild-type cells, induced it more rapidly in mR-1, but not at all in v-clone cells. The v-clone lacked the b-form of protein kinase C, but transfection with this enzyme did not restore responsiveness to NGF. Neurite extension in response to staurosporine and basic fibroblast growth factor was equal in wild-type and v-clone cells. All three clones responded to forskolin, with the mR-1 clone the most responsive. NGF stimulated AP 1 binding activity in all clones. The response was transient in the MR-1 clone but prolonged in the wild-type and v-clone cells. In the wild-type and MR-1 clone cells, AP 1 binding activity was reduced by a tyrphostin analog, whereas in the v-clone cells it was inhibited by staurosporine. NGF increased inositol (1,4,5)-trisphosphate (InsP₃) formation in all clones. In the wild-type and v-clone cells the InsP₃ responses were followed by [Ca²⁺]_i increases. It is concluded that although trkA is required for differentiation in response to NGF in PC12 cells, the concomitant stimulation, by NGF, of p75^LNTR may affect phospholipase C and AP 1. This may be important for the reported ability of p75^LNTR to modify the phenotypic changes induced in PC12 cells by NGF.     

PC12 neurite regeneration and long-term maintenance in the absence of exogenous nerve growth factor in response to contact with Schwann cells

We have investigated mouse and rat ganglionic Schwann cells as possible sources of neurite outgrowth-promoting factors by co-culturing Schwann cells with nerve growth factor (NGF)-responsive PC12 pheochromocytoma cells primed by pretreatment with NGF. NGF-primed PC12 cells are capable of neurite regeneration when provided with an appropriate neurite promoting factor such as NGF. When primed PC12 cells were co-cultured with Schwann cells in the absence of exogenous NGF, PC12 cells that directly contacted Schwann cells became enlarged and flattened, attaining a neuron-like morphology within one day. When contact with Schwann cells was established, PC12 cells regenerated neurites by the first day of co-culture and these were maintained throughout the experiments (7 weeks). Most PC12 cells cultured in the same collagen-coated dishes with Schwann cells, but not directly in contact with them, failed to regenerate neurites. Instead, they began to proliferate, forming cell clusters. Neurite regeneration by PC12 cells in contact with Schwann cells was not blocked by antibody to NGF. These results demonstrate the presence of a neurite-promoting activity localized to the vicinity of the Schwann cell surface which is capable of eliciting regeneration and long-term maintenance of PC12 neurites in the absence of exogenous NGF. This activity does not appear to be due to NGF.     

Requirement for enzymatically active lipoprotein lipase in neuronal differentiation: a site-directed mutagenesis study

Lipoprotein lipase (LPL) is well known for its role in the catabolism of plasma triglyceride (Tg)-rich lipoproteins, such as very low density lipoproteins (VLDL) and chylomicrons. The action of LPL on Tg-rich lipoproteins provides free fatty acids to skeletal muscle and adipose tissues, the main sites of LPL synthesis. Several studies have demonstrated that LPL is widely expressed in the parenchyma of brain tissues. We have recently shown that LPL expression is essential for promoting VLDL-stimulated differentiation of Neuro-2A cells. In the present study, we have generated stably transfected Neuro-2A cell lines expressing either wild-type LPL or various LPL mutants, including three enzymatically inactive variants (Asp156Asn, Gly188Glu and Pro207Leu), an enzymatically defective variant (Asn291Ser) and a variant known to express increased LPL activity (Ser447Ter). In Neuro-2A cells expressing enzymatically inactive LPL variants, VLDL-stimulated differentiation and neurite extension were not observed. However, in Neuro-2A cells expressing partially active or overactive LPL variants, VLDL added to the cultured medium was able to induce the phenotypic differentiation similar to that observed in Neuro-2A cells expressing wild-type LPL. In summary, these data show that the availability of fatty acids, resulting from the catabolism of VLDL by LPL, is required to promote the phenotypical differentiation of neuroblastoma cells. These findings may have significant relevance to lipoprotein metabolism in the brain as well as to the maturation and regeneration of nervous tissues in carriers of mutant LPL.     

Fast and slow nerve growth factor binding sites in human neuroblastoma and rat pheochromocytoma cell lines: relationship of sites to each other and to neurite formation

We studied (a) the distribution and properties of fast and slow 125I-nerve growth factor (125I-NGF) binding sites in cultured human neuroblastoma (NB) cell lines that were categorized as responsive (N+) or unresponsive (N-) to NGF by neurite outgrowth, (b) whether fast or slow sites mediate actions of NGF, and (c) whether NGF-mediated conversion of fast to slow sites occurs in human NB and pheochromocytoma PC 12 cells. In human NB SH-SY5Y cells, the slow sites were trypsin resistant and binding was of high affinity. Loss of binding to the slow sites had a half-time of 25 to 30 min at 37 degrees C and was very slow at 4 degrees C. In contrast, the fast sites were trypsin sensitive and binding was of lower affinity; its dissociation half-time was less than 1 min at 4 degrees C and 37 degrees C. The association rate constants of both sites were about 0.8 to 1.2 X 10(7) M-1 sec-1. Some human NB cells had both fast and slow sites. The N+ human NB lines SH-SY5Y and LA-N-5 had only slow sites. Despite the virtual elimination of fast sites by trypsin in NB MC-IXC cells, remaining slow sites could still efficiently bind 125I-NGF. These observations showed that fast sites are not required for slow site binding, neurite outgrowth, or other demonstrated actions of NGF in some NB cells. In PC 12 cells, 125I-NGF initially bound to fast sites was not directly transferred to slow sites as required for NGF-mediated conversion. The association rate constants of fast and slow sites in PC12 cells were both about 2 X 10(7) M-1 sec-1. The association kinetics were consistent with simple bimolecular reactions in both NB and PC12 cells. The combined evidence in NB and PC12 cells did not support the hypothesis of NGF-mediated conversion of fast to slow sites.     

A quantitative bioassay for nerve growth factor,using PC12 clones expressing different levels of trkA receptors

Nerve growth factor (NGF) is a neurotrophin required for differentiation, development, and survival of the sympathetic nervous system, with many of its biological effects being mediated via trkA receptors. There is a need for a standard quantitative bioassay for NGF, to be used in basic research and in pharmaceutical studies. The objective of the present research was to develop a selective, quantitative, and reliable bioassay for NGF, using a morphological criterion: neurite cell outgrowth. In addition, we aimed to apply the aforementioned bioassay to measure NGF administered to mice. Pheochromocytoma PC12 cell variants including wild-type cultures, and a trkA-overexpressing stable transfectant PC12-6.24-I, PC12nnr5, and PC12EN lacking trkA receptors, were used. Dose-response curves were generated with NGF β-subunit (2.5S) purified from mouse submaxillary glands. Our results demonstrated that the bioassay was sensitive to 0.3–20 ng/mL, and selective, as neurite outgrowth was not seen by any other growth factor other than NGF. In addition, variant clones PC12nnr5 and PC12EN, lacking trkA receptors, did not respond to NGF. The bioassay detected NGF in serum of mice injected with NGF. This novel developed bioassay can serve as a model system for various neuroscience purposes.     

Second-generation antipsychotic drugs, olanzapine, quetiapine, and clozapine enhance neurite outgrowth in PC12 cells via P13K/AKT, ERK, and pertussis toxin-sensitive pathways

Second-generation antipsychotic drugs, olanzapine, quetiapine, and clozapine, were found to enhance neurite outgrowth induced by nerve growth factor (NGF) in PC12 cells. These drugs increased the number of cells bearing neurites, the length of primary neurites, and the size of the cell body of NGF-differentiated PC12 cells. In addition, the drugs induced sprouting of neurite-like processes in PC12 cells in the absence of NGF. Olanzapine, quetiapine, and clozapine enhanced the phosphorylation of Akt and ERK in combination with NGF, and specific inhibitors of these pathways attenuated these effects. Pretreatment of cells overnight with pertussis toxin had no effect on NGF-induced differentiation but significantly decreased the effects of the antipsychotic drugs on neurite outgrowth, suggesting that G_i/G_o-coupled receptors are involved in the response to drug. A better understanding of the mechanisms underlying the effects of the second-generation drugs might suggest new therapeutic targets for enhancement of neurite outgrowth.     

Nerve growth factor and a fibroblast growth factor-like neurotrophic activity in cerebrospinal fluid of brain injured human patients

We report here the presence of nerve growth factor (NGF) in the cerebrospinal fluid (CSF) of some brain-injured human patients soon after injury. The NGF was quantified against a recombinant human NGF standard in a two-site enzyme-linked immunoabsorbant assay using antibodies against murine B NGF. None of the samples collected more than 2 days after injury contained detectable levels of NGF. When the CSF was assayed for the ability to promote neurite outgrowth from PC12 cells, neurite outgrowth was reduced, but not completely blocked, by antibodies to B NGF, suggesting that there were other biologically active factors present. Fibroblast growth factor (FGF) also promotes neurite outgrowth in PC12 cells. In an initial screening for the presence of FGF, we employed PC12 cells and NR119 cells, PC12 variants in which recombinant human B NGF, but not recombinant human basic FGF, promotes neurite outgrowth. CSF from brain injury patients promoted greater neurite outgrowth from PC12 cells than from NR119 cells, suggesting that some of the biological activity associated with the injury CSF may be due a FGF. This possibility is further supported by the observation that the biological activity of the injury CSF significantly reduced by batch absorption with heparin Sepharose, suggesting the presence of a heparin binding neurotrophic factor. Neurotrophic factors appear in CSF as a consequence of diverse types of brain injury, including head trauma, intracerebral hemorrhage and subarachnoid hemorrhage. The appearance of these factors may reflect important common elements in the complex series of cellular changes occuring in response to acute brain injury.     

Characterization of a Distinctive Motif of the Low Molecular Weight Neurotrophin Receptor that Modulates NGF-mediated Neurite Growth

The cytoplasmic region of the common neurotrophin receptor (p75^NGFR) (rat, human, chick) contains a putative membrane-associating domain implicated in intracellular signalling. A peptide (R3) identical to this domain (p75^NGFR 367–379) and various analogues of this peptide displayed circular dichroism spectra in aqueous and non-polar environments identical to the amphiphilic tetradecapeptide mastoparan (MP) and were internalized by PC12 rat pheochromocytoma cells. The R3 peptide enhanced neurite growth in PC12 cells, embryo chick primary sensory neurons and fetal rat primary sensory neurons in vitro in the presence of sub-saturating concentrations of NGF. Peptide analogues of R3 not faithful to the distance and angular relationships of ionic groups and the putative amphiphilic structure of p75^NGFR 367–379 displayed reduced potency to enhance NGF-mediated neurite growth. Exposure of NGF and the R3 peptide to a cell line displaying predominantly p75^NGFR (PC12^nnr5), had no influence on neurite growth. The R3 peptide had no effects on cell survival, cell binding or uptake of [¹²⁵I]NGF, affinity cross-linking of [¹²⁵I]NGF to p75^NGFR or trkA monomers and homodimers, or NGF-mediated trkA monomer tyrosine phosphorylation. The studies implicate a role for a highly conserved motif of p75^NGFR in the downstream modulation of NGF-mediated neurite growth.