Human Astrocytoma Cell Line 1321N1 Research Articles

Modulation of Cytokine-Induced Astrocytic Endothelin-1 Production as a Possible New Approach to the Treatment of Multiple Sclerosis.

Background: In the human central nervous system (CN), resting astrocytes do not visually show endothelin-1 (ET-1)-like immunoreactivity. In patients with multiple sclerosis (MS), an inflammatory disorder of the CNS, high levels of ET-1 are found in reactive astrocytes in demyelinated plaques. ET-1 may contribute to the pathology of MS by interrupting the blood-brain-barrier, enhancing inflammatory responses, excitotoxicity and reducing cerebral blood flow. Methods: We used the human astrocytoma cell line 1321N1 to investigate the role of inflammatory cytokines involved in MS lesions (IL-1β, TNF-α, IFN-γ, LPS, IL-10, TGF-β) on astrocytic ET-1 upregulation. Prucalopride, rolipram, fenofibrate, fluoxetine, simvastatin, daglutril, and resveratrol were investigated as potential candidate drugs to suppress cytokine-induced astrocytic ET-1 production. Effects on ET-1 production were measured using both ELISA and RT-qPCR. Results and Conclusions: ET-1 secretion by astrocytoma cells was only stimulated by the pro-inflammatory cytokines IL-1β and TNF-α. Fluoxetine, simvastatin, and resveratrol significantly inhibited this IL-1β- and TNF-α-induced ET-1 production. Simvastatin and resveratrol significantly reduced ET-1 mRNA levels, indicating an effect at the level of transcription. Fluoxetine significantly reduced endothelin converting enzyme-1 mRNA levels, suggesting and effect at the level of protein-processing. The required concentrations of simvastatin (>0.1 µM) and resveratrol (>10 µM) cannot be achieved in humans using pharmacologically accepted doses. Fluoxetine exerted a significant inhibitory effect on ET-1 secretion at a concentration of 5 µM, which is pharmacologically achievable in human brain, but the effect was modest (<50% suppression) and probably not sufficient to obtain a clinically relevant ET-1 effect. Our in vitro model can be a useful screening tool in the development of new drugs to suppress astrocytic ET-1 production. The effect of simvastatin was for the most part mediated via the mevalonate pathway, suggesting that this might be an interesting target for further drug development.

Histamine elicits glutamate release from cultured astrocytes

Astrocytes play key roles in regulating brain homeostasis and neuronal activity. This is, in part, accomplished by the ability of neurotransmitters in the synaptic cleft to bind astrocyte membrane receptors, activating signalling cascades that regulate concentration of intracellular Ca2+ ([Ca2+]i) and gliotransmitter release, including ATP and glutamate. Gliotransmitters contribute to dendrite formation and synaptic plasticity, and in some cases, exacerbate neurodegeneration. The neurotransmitter histamine participates in several physiological processes, such as the sleep-wake cycle and learning and memory. Previous studies have demonstrated the expression of histamine receptors on astrocytes, but until now, only a few studies have examined the effects of histamine on astrocyte intracellular signalling and gliotransmitter release. Here, we used the human astrocytoma cell line 1321N1 to study the role of histamine in astrocyte intracellular signalling and gliotransmitter release. We found that histamine activated astrocyte signalling through histamine H1 and H2 receptors, leading to distinct cellular responses. Activation of histamine H1 receptors caused concentration-dependent release of [Ca2+]i from internal stores and concentration-dependent increase in glutamate release. Histamine H2 receptor activation increased cyclic adenosine monophosphate (cAMP) levels and phosphorylation of transcription factor cAMP response-element binding protein. Taken together, these data emphasize a role for histamine in neuron-glia communication.

Comparison and optimization of transient transfection methods at human astrocytoma cell line 1321N1

Gene delivery to eukaryotic cells is the technique to study the regulation of gene expression. Human astrocytoma cell line 1321N1 could be useful to study G-protein-coupled receptors (GPCRs). Different transient transfection methods, namely calcium phosphate, Lipofectamine, FuGENE, Arrest-In, and microporation (Microporator), were investigated. Results were analyzed by fluorescence-activated cell sorting and fluorescence microscope using green fluorescent protein (GFP) as a reporter gene. To verify the transfection efficiency of these techniques, the expression of human GPR17 gene ( hgpr17) was analyzed by transcription quantitative polymerase chain reaction. Microporation resulted in the best method to promote enriched hgpr17 delivery into the human astrocytoma cell line.

Secreted PLA2 induces proliferation in astrocytoma through the EGF receptor: another inflammation-cancer link

We have investigated mechanisms that contribute to reinforce the relationship between inflammation and cancer. Secreted phospholipase A(2) group IIA (sPLA(2)-IIA) is a molecule relevant in inflammatory events and has been proposed as a marker for some of these. Previously, we reported the mitogenic properties of this sPLA(2) in the human astrocytoma cell line 1321N1. Here, we go deeper into the mechanisms that link this inflammatory protein with proliferation in one of the most aggressive types of tumors. We found that phosphorylation of the extracellular regulated kinase (ERK) was preceded by the activation of the small GTPase Ras, and both failed to be activated by inhibiting protein kinase C (PKC). Fractionation and immunofluorescence studies revealed translocation of PKC alpha, delta, and epsilon to the membrane fraction upon stimulation with sPLA(2)-IIA. Immunoprecipitation analysis showed that sPLA(2)-IIA induces phosphorylation of the epidermal growth factor receptor (EGFR) through a PKC-dependent pathway. We found that phosphorylation of this receptor contributed to Ras and ERK activation and that inhibition of ERK, PKC, and EGFR blocked the mitogenic response induced by sPLA(2)-IIA. This study showed that sPLA(2)-IIA is able to bring into play EGFR to trigger its signaling and that PKC leads the distribution of resources. Interestingly, we found that this is not a cell-specific response, because sPLA(2)-IIA was also able to transactivate EGFR in MCF7 human breast cancer cells. Therefore, this mechanism could contribute to worsen the prognosis of a tumor in an inflammatory microenvironment. We also present more links of the tumor chain possibly susceptible to targeting.

Differential sensitivity and responsiveness of three human cell lines HepG2, 1321N1 and HEK 293 to cadmium

Cadmium (Cd) is a toxic heavy metal with no uniform mechanism of toxicity. In this the present study, the toxic effect of 5, 10 and 50 microM of Cd chloride was compared in three human cell lines; a human hepatoma cell line HepG2, a human astrocytoma cell line 1321N1, and a human embryonic kidney cell HEK 293. The results indicate a decrease in the viability of all three cell lines following exposure to Cd with HepG2 cells (IC50=13.96 microM) showing the most sensitivity when measured using the MTT assay. There was significant increase in lactate dehydrogenase leakage, DNA damage, malondialdeyde and antioxidant enzymes activities in all three cell lines especially at 50 microM Cd. Significant decreases in ATP production were also observed at all Cd concentrations in HepG2 and HEK 293 cell lines. ROS levels significantly increase and GSH/GSSG ratio significantly decrease in all the three cell lines after Cd exposure, but these effects were attenuated by the presence of N-acetylcysteine (NAC). The present study therefore shows that ROS production and glutathione (GSH) depletion may play a role in Cd-induced toxicity in all the three cell lines. The endogenous levels of protective enzymes as well as their responsiveness are likely to determine a cell's susceptibility to Cd.

Expression of corticosteroid-binding globulin in human astrocytoma cell line.

Glial tumor cells are known to be sensitive to glucocorticoids (GC) in vivo and in vitro. Here we studied the expression of corticosteroid-binding globulin (CBG) in the low-grade malignant human astrocytoma cell line 1321N1. CBG was observed in cytoplasm of most of these cells with immunocytochemistry. RT-PCR revealed the presence of the respective mRNA. Only scattered cells contained nuclear immunoreactivity for glucocorticoid receptor as visualized by double immunostaining. Immunoreactive CBG could be recovered from the supernatant of cultures that had been exposed to 10(-5) M cortisol. Our observations indicate the endogenous expression of CBG in 1321N1 cells which may occur independently from classical glucocorticoid receptor pathways. Cortisol seems to facilitate liberation of CBG in a paracrine manner, perhaps through membrane action of the steroid. Effects of adrenal steroids on proliferation and apoptosis of certain glial tumors may in part depend on these mechanisms.

Effect of 4-trifluoromethyl derivatives of salicylate on nuclear factor kappaB-dependent transcription in human astrocytoma cells.

1. The effect of two derivatives of salicylate, 2-hydroxy-4-trifluoromethylbenzoic acid (HTB) and 2-acetoxy-4-trifluoromethylbenzoic acid (triflusal), on the expression of several proteins displaying pro-inflammatory activities the regulation of which is associated to the transcription factor NF-kappaB, was assayed in the human astrocytoma cell line 1321N1. 2. Tumour necrosis factor-alpha (TNF-alpha) activated NF-kappaB as judged from both the appearance of kappaB-binding activity in the nuclear extracts, the degradation of IkappaB proteins in the cell lysates, and the activation of IkappaB kinases using an immunocomplex kinase assay with glutathione S-transferase (GST)-IkappaB proteins as substrates. 3. HTB up to 3 mM did not inhibit the nuclear translocation of NK-kappaB/Rel proteins as judged from electrophoretic mobility-shift assays; however, HTB inhibited the degradation of IkappaBbeta without significantly affecting the degradation of both IkappaBalpha and IkappaBepsilon. 4. In keeping with their inhibitory effect on IkappaBbeta degradation in the cell lysates, both HTB and triflusal inhibited the phosphorylation of GST-IkappaBbeta elicited by TNF-alpha, without affecting the phosphorylation of GST-IkappaBalpha. 5. The effect of both HTB and triflusal on kappaB-dependent trans-activation was studied by assaying the expression of both cyclo-oxygenase-2 (COX-2) and vascular cell adhesion molecule-1 (VCAM-1). HTB and triflusal inhibited in a dose-dependent manner the expression of COX-2 and VCAM-1 mRNA and the induction of COX-2 protein at therapeutically relevant concentrations. 6. These findings show the complexity of the biochemical mechanisms underlying the activation of NF-kappaB in the different cell types and extend the anti-inflammatory effects of HTB and triflusal to neural cells.

Lysophosphatidic acid inhibits Ca2+ signaling in response to epidermal growth factor receptor stimulation in human astrocytoma cells by a mechanism involving phospholipase C(gamma) and a G(alphai) protein.

The effect of the lysophospholipid mediators lysophosphatidic acid (LPA) and sphingosine 1-phosphate and the polypeptide growth factor epidermal growth factor (EGF) on the human astrocytoma cell line 1321N1 was assessed. These agonists produced a rapid and transient increase of the intracellular Ca(2+) concentration. When LPA was perfused before addition of EGF, the EGF-dependent Ca(2+) transient was abrogated, whereas this was not observed when EGF preceded LPA addition. This inhibitory effect was not found for other EGF-mediated responses, e.g., activation of the mitogen-activated protein kinase cascade and cell proliferation, thus pointing to the existence of cross-talk between LPA and EGF for only a branch of EGF-induced responses. As 1321N1 cells expressed mRNA encoding the LPA receptors endothelial differentiation gene (Edg)-2, Edg-4, and Edg-7 and as sphingosine 1-phosphate did not interfere with LPA signaling, Edg-2, Edg-4, and/or Edg-7 could be considered as the LPA receptors mediating the aforementioned cross-talk. Attempts to address the biochemical mechanism involved in the cross-talk between the receptors were conducted by the immunoprecipitation approach using antibodies reacting with the EGF receptor (EGFR), phosphotyrosine, phospholipase Cgamma (PLCgamma)-1, and G(alphai) protein. LPA was found to induce coupling of PLCgamma-1 to the EGFR by a mechanism involving a G(alphai) protein, in the absence of tyrosine phosphorylation of both PLCgamma and the EGFR. These data show a cross-talk between LPA and EGF limited to a branch of EGFR-mediated signaling, which may be explained by a LPA-induced, G(alphai)-protein-mediated translocation of PLCgamma-1 to EGFR in the absence of detectable tyrosine phosphorylation of both proteins.

Secretory Phospholipase A2 Induces Phospholipase Cγ-1 Activation and Ca2+ Mobilization in the Human Astrocytoma Cell Line 1321N1 by a Mechanism Independent of Its Catalytic Activity

The effect of secretory phospholipase A2 (sPLA2) on intracellular Ca2+ signaling in human astrocytoma cells was studied. sPLA2 increased cytosolic [Ca2+] ([Ca2+]c) in both Ca2+-containing and Ca2+-free medium, thus suggesting Ca2+ release from intracellular stores. The activation by sPLA2 of arachidonate release via cytosolic PLA2 (cPLA2) was also independent of extracellular Ca2+. As sPLA2 requires Ca2+ for activity, these results indicate that both Ca2+ mobilization and cPLA2 activation induced by sPLA2 are unrelated to phospholipase activity but dependent on signaling mechanisms. The sPLA2-induced [Ca2+]c peak was sensitive to Bordetella pertussis toxin and inhibited by caffeine, suggesting its mediation by inositol 1,4,5-trisphosphate (IP3). sPLA2 induced tyrosine phosphorylation and membrane targeting of phospholipase Cγ-1 (PLCγ-1). Moreover, the Ca2+ peak was sensitive to protein tyrosine kinase inhibitors. sPLA2 activates two signaling pathways: one leading to the activation of the MAP kinase/cPLA2 cascade and another leading to PLCγ activation and Ca2+ release.

Secretory Phospholipase A2 Activates the Cascade of Mitogen-activated Protein Kinases and Cytosolic Phospholipase A2 in the Human Astrocytoma Cell Line 1321N1

The biological effects of type IIA 14-kDa phospholipase A2 (sPLA2) on 1321N1 astrocytoma cells were studied. sPLA2 induced a release of [3H]arachidonic acid ([3H]AA) similar to that elicited by lysophosphatidic acid (LPA), a messenger acting via a G-protein-coupled receptor and a product of sPLA2 on lipid microvesicles. In contrast, no release of [1-14C]oleate could be detected in cells labeled with this fatty acid. As these findings pointed to a selective mechanism of [3H]AA release, it was hypothesized that sPLA2 could act by a signaling mechanism involving the activation of cytosolic PLA2 (cPLA2), i.e. the type of PLA2 involved in the release of [3H]AA elicited by agonists. In keeping with this view, stimulation of 1321N1 cells with sPLA2 elicited the decrease in electrophoretic mobility that is characteristic of the phosphorylation of cPLA2, as well as activation of p42 mitogen-activated protein (MAP) kinase, c-Jun kinase, and p38 MAP kinase. Incubation with sPLA2 of quiescent 1321N1 cells elicited a mitogenic response as judged from an increased incorporation of [3H]thymidine. Attempts to correlate the effect of extracellular PLA2 with the generation of LPA were negative. Incubation with pertussis toxin prior to the addition of either sPLA2 or LPA only showed abrogation of the response to LPA, thus suggesting the involvement of pertussis-sensitive Gi-proteins in the case of LPA. Treatments with inhibitors of the catalytic effect of sPLA2 such as p-bromophenacyl bromide and dithiothreitol did not prevent the effect on cPLA2 activation. In contrast, preincubation of 1321N1 cells with the antagonist of the sPLA2 receptor p-aminophenyl-alpha-D-mannopyranoside-bovine serum albumin, blocked cPLA2 activation with a EC50 similar to that described for the inhibition of binding of sPLA2 to its receptor. Moreover, treatment of 1321N1 cells with the MAP kinase kinase inhibitor PD-98059 inhibited the activation of both cPLA2 and p42 MAP kinase produced by sPLA2. In summary, these data indicate the existence in astrocytoma cells of a signaling pathway triggered by engagement of a sPLA2-binding structure, that produces the release of [3H]AA by activating the MAP kinase cascade and cPLA2, and leads to a mitogenic response after longer periods of incubation.

Thrombin produces phosphorylation of cytosolic phospholipase A2 by a mitogen-activated protein kinase kinase-independent mechanism in the human astrocytoma cell line 1321N1.

The release of [3H]arachidonic acid was studied in the 1321N1 astrocytoma cell line upon stimulation with thrombin. The effect of thrombin was antagonized by hirudin only when both compounds were added simultaneously, which suggests activation of thrombin receptor. Evidence that the cytosolic phospholipase A2 (cPLA2) takes part in thrombin-induced arachidonate release was provided by the finding that thrombin induced retardation of the mobility of cPLA2 in SDS/polyacrylamide gels, which is a feature of the activation of cPLA2 by mitogen-activated protein (MAP) kinases. Thrombin induced activation of two members of the MAP kinase family whose consensus primary sequence appears in cPLA2, namely p42-MAP kinase and c-Jun kinase. However, the activation of c-Jun kinase preceded the phosphorylation of cPLA2 more clearly than the activation of p42-MAK kinase did. Both cPLA2 and c-Jun kinase activation were not affected by PD-98059, a specific inhibitor of MAP kinase kinases, which indeed completely blocked p42-MAP kinase shift. Heat shock, a well-known activator of c-Jun kinase, also phosphorylated cPLA2 but not p42-MAP kinase. These data indicate the existence in astrocytoma cells of a signalling pathway triggered by thrombin receptor stimulation that activates a kinase cascade acting on the Pro-Leu-Ser-Pro consensus primary sequence, activates cPLA2, and associates the release of arachidonate with nuclear signalling pathways.

Cytosolic phospholipase A2 is coupled to muscarinic receptors in the human astrocytoma cell line 1321N1: characterization of the transducing mechanism

The cholinergic agonist carbachol induced the release of arachidonic acid in the 1321N1 astrocytoma cell line, and this was blocked by atropine, suggesting the involvement of muscarinic receptors. To assess the mechanisms of signalling involved in the response to carbachol, a set of compounds characterized by eliciting responses through different mechanisms was tested. A combination of 4beta-phorbol 12beta-myristate 13alpha-acetate and thapsigargin, an inhibitor of endomembrane Ca2+-ATPase that induces a prolonged elevation of cytosolic Ca2+ concentration, induced an optimal response, suggesting at first glance that both protein kinase C (PKC) and Ca2+ mobilization were involved in the response. This was consistent with the observation that carbachol elicited Ca2+ mobilization and PKC-dependent phosphorylation of cytosolic phospholipase A2 (cPLA2; phosphatide sn-2-acylhydrolase, EC 3.1.1.4) as measured by a decrease in electrophoretic mobility. Nevertheless, the release of arachidonate induced by carbachol was unaltered in media containing decreased concentrations of Ca2+ or in the presence of neomycin, a potent inhibitor of phospholipase C which blocks phosphoinositide turnover and Ca2+ mobilization. Guanosine 5'-[gamma-thio]triphosphate added to the cell-free homogenate induced both [3H]arachidonate release and cPLA2 translocation to the cell membrane fraction in the absence of Ca2+, thus suggesting the existence of an alternative mechanism of cPLA2 translocation dependent on G-proteins and independent of Ca2+ mobilization. From the combination of experiments utilizing biochemical and immunological tools the involvement of cPLA2 was ascertained. In summary, these data indicate the existence in the astrocytoma cell line 1321N1 of a pathway involving the cPLA2 which couples the release of arachidonate to the occupancy of receptors for a neurotransmitter, requires PKC activity and G-proteins and might operate in the absence of Ca2+ mobilization.