lithium-chloride and Neuroblastoma

Neuroblastoma is the most common tumor amongst children amounting to nearly 15% of cancer deaths. This cancer is peculiar in its characteristics, exhibiting differentiation, maturation and metastatic transformation leading to poor prognosis and low survival rates among children. Chemotherapy, though toxic to normal cells, has shown to improve the survival of the patient with emphasis given more towards targeting angiogenesis. Recently, Tideglusib was designed as an 'Orphan Drug' to target the neurodegenerative Alzheimer's disease and gained significant momentum in its function during clinical trials. Duffy et al. recently reported a reduction in cell viability of human IMR32 neuroblastoma cells when treated with Tideglusib at varying concentrations. We investigated the effects of Tideglusib, at various concentrations, compared to Lithium chloride at various concentrations, on IMR32 cells. Lithium, a known GSK-3 inhibitor, was used as a standard to compare the efficiency of Tideglusib in a dose-dependent manner. Cell viability was assessed by MTT assay. The stages of apoptosis were evaluated by AO/EB staining and nuclear damage was determined by Hoechst 33258 staining. Reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were assessed by DCFDA dye and Rhodamine-123 dye, respectively. Tideglusib reported a significant dose-dependent increase in pro-apoptotic proteins (PARP, Caspase-9, Caspase-7, Caspase-3) and tumor-related genes (FasL, TNF-α, Cox-2, IL-8, Caspase-3). Anti-GSK3 β, pGSK3 β, Bcl-2, Akt-1, p-Akt1 protein levels were observed with cells exposed to Tideglusib and Lithium chloride. No significant dose-dependent changes were observed for the mRNA expression of collagenase MMP-2, the tumor suppressor p53, or the cell cycle protein p21. Our study also reports Tideglusib reducing colony formation and increasing the level of sub-G0/G1 population in IMR32 cells. Our investigations report the significance of Tideglusib as a promising apoptotic inducer in human neuroblastoma IMR32 cells. Our study also reports that LiCl reduced cell viability in IMR32 cells inducing apoptosis mediated by ROS generation.

Topics: Antineoplastic Agents; Apoptosis; Cell Culture Techniques; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; G1 Phase; Humans; Lithium Chloride; Membrane Potential, Mitochondrial; Neuroblastoma; Reactive Oxygen Species; Resting Phase, Cell Cycle; Thiadiazoles

Neuroblastoma is an embryonal tumor accounting for approximately 15% of childhood cancer deaths. There exists a clinical need to identify novel therapeutic targets, particularly for treatment-resistant forms of neuroblastoma. Therefore, we investigated the role of the neuronal master regulator GSK3 in controlling neuroblastoma cell fate. We identified novel GSK3-mediated regulation of MYC (c-MYC and MYCN) mRNA levels, which may have implications for numerous MYC-driven cancers. In addition, we showed that certain GSK3 inhibitors induced large-scale cell death in neuroblastoma cells, primarily through activating apoptosis. mRNA-seq of GSK3 inhibitor-treated cells was performed and subsequent pathway analysis revealed that multiple signaling pathways contributed to the loss of neuroblastoma cell viability. The contribution of two of the signaling pathways highlighted by the mRNA-seq analysis was functionally validated. Inhibition of the p53 tumor suppressor partly rescued the cell death phenotype, whereas activation of canonical Wnt signaling contributed to the loss of viability, in a p53-independent manner. Two GSK3 inhibitors (BIO-acetoxime and LiCl) and one small-molecule Wnt agonist (Wnt Agonist 1) demonstrated therapeutic potential for neuroblastoma treatment. These inhibitors reduced the viability of numerous neuroblastoma cell lines, even those derived from high-risk MYCN-amplified metastatic tumors, for which effective therapeutics are currently lacking. Furthermore, although LiCl was lethal to neuroblastoma cells, it did not reduce the viability of differentiated neurons. Taken together our data suggest that these small molecules may hold potential as effective therapeutic agents for the treatment of neuroblastoma and other MYC-driven cancers.

Topics: Blotting, Western; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Glycogen Synthase Kinase 3; Humans; Indoles; Lithium Chloride; Maleimides; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Oximes; Protein Kinase Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA Stability; RNA, Messenger; Time Factors; Tumor Suppressor Protein p53; Wnt Signaling Pathway

Amyloid precursor protein (APP) undergoes post-translational modification, including O- and N-glycosylation, ubiquitination, and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at threonine 668 (Thr-668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phospho-site-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, Thr-668 to alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the Thr-668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-β (GSK3β) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at Thr-668 in neurons. Our results show that the GSK3β kinase inhibitors LiCl, SB 216763, and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon, and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3β-dependent phosphorylation in SH-SY5Y cells.

Topics: Adjuvants, Immunologic; Aminophenols; Amyloid beta-Protein Precursor; Animals; Axons; Cell Line, Tumor; Copper; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippocampus; Humans; Indoles; Lithium Chloride; Maleimides; Mice; Mutation, Missense; Neuroblastoma; Phosphorylation; Protein Transport; Synapses

Neuroblastoma is a severe pediatric tumor characterized by poor prognosis. Identification of novel molecular targets and diversion of investigations on new drug trials is mandatory for cancer therapy. In this study, vinorelbine tartrate, lithium chloride, clomipramine, and medroxyprogesterone acetate are used for the possible new treatment modalities in neuroblastoma cells. Notch and c-kit are novel molecules in cancer research, and Notch pathway is one of the emerging molecules in the neuroblastoma pathogenesis. Cytotoxic effects of these drugs at different time points, with different doses were studied in the SH-SY5Y human neuroblastoma cell line. Analysis of Notch and c-kit signaling with immunohistochemistry were constituted in multicellular tumor spheroids, and morphologic investigation was performed for digital imaging of cancer stem cells (CSCs) with electron microscopy. Size kinetics of spheroids was also determined after drug treatment. Results showed that all drugs were cytotoxic for neuroblastoma cells. Yet, this cytotoxic action did not correlate with the inhibitory effects in cell signaling. Neuroblastoma spheroids showed increased immunoreactivity of Notch signaling and c-kit. Altered ultrastructural CSCs morphology was observed after clomipramine and medroxyprogesterone acetate treatment compared with other drugs. Lithium chloride showed cellular membrane destruction for both CSCs and the remaining population. In this study, independent effects of cytotoxicity in tumor cells with respect to CSCs were determined. Redundant cells, which are the bulk population in tumor a compound, destroyed with therapy, were neither a target for treatment nor a remarkable investigation of cancer.

Topics: Adjuvants, Immunologic; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Size; Cell Survival; Clomipramine; Humans; Lithium Chloride; Medroxyprogesterone Acetate; Microscopy, Electron, Transmission; Neuroblastoma; Proto-Oncogene Proteins c-kit; Receptor, Notch1; Signal Transduction; Spheroids, Cellular; Vinblastine; Vinorelbine

Topics: Cell Line, Tumor; Cyclic AMP; Cyclic Nucleotide Phosphodiesterases, Type 4; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Indoles; Lithium Chloride; Maleimides; Nerve Tissue Proteins; Neuroblastoma; Signal Transduction; Transfection

Glycogen synthase kinase 3β (GSK3β) activity is regulated by phosphorylation processes and regulates in turn through phosphorylation several proteins, including eukaryotic initiation factor 2B (eIF2B). Serine 9 phosphorylation of GSK3β (pGSK3βSer9), usually promoted by activation of the PI3K/Akt survival pathway, triggers GSK3β inhibition. By contrast, tyrosine 216 phosphorylation of GSK3β (pGSK3βTyr216) increases under apoptotic conditions, leading to GSK3β activation. Lithium chloride (LiCl) is usually described to increase pGSK3βSer9 through the PI3K/Akt pathway, resulting in GSK3β inhibition. The purpose of this study is to demonstrate that in some cases LiCl is also able to increase pGSK3βTyr216, resulting in GSK3β activation. For this, we used SH-SY5Y cells and primary neuronal cultures and investigated the effects of LiCl on the two phosphorylated forms of GSK3β under staurosporine (STS)-intoxicated conditions. The ratios between the phosphorylated and total forms of GSK3β and eIF2B were determined by Western blotting. Our results revealed that, besides its ability to increase pGSK3βSer9, LiCl is also able to increase pGSK3βTyr216 greatly in STS-intoxicated SH-SY5Y cells but not in STS-intoxicated primary neuronal cultures. This accumulation of both Ser9 and Tyr216 phosphorylation results in GSK3β activation in STS-intoxicated SH-SY5Y cells in spite of the presence of LiCl. These findings indicate that LiCl treatment is not necessarily correlated with GSK3β inhibition even though it generates Ser9 phosphorylation. Consequently, the ratio pGSK3βSer9/pGSK3βTyr216, which takes into account the balance between the two inactive (Ser9) and active (Tyr216) forms of GSK3β, could be more useful for predicting GSK3β inhibition.

Topics: Antimanic Agents; Cell Line, Tumor; Cells, Cultured; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lithium Chloride; Neuroblastoma; Neurons; Phosphorylation; Staurosporine

The aim of this work is to investigate whether clomipramine (CIM) and lithium chloride (LiCl) potentiate the cytotoxicity of vinorelbine (VNR) on SH-SY5Y human neuroblastoma cells in vitro and whether midkine (MK) can be a resistance factor for these treatments. Four groups of experiments were performed for 96 h using both monolayer and spheroid cultures of SH-SY5Y cells: (1) control group, (2) singly applied VNR, CIM, and LiCl, (3) VNR with CIM, and (4) VNR with LiCl. Their effects on monolayer and spheroid cultures were determined by evaluating cell proliferation, bromodeoxyuridine labeling index (BrdU-LI), apoptosis, cyclic adenosine monophosphate (cAMP) and midkine levels, colony-forming efficiency, spheroid size, and ultrastructure. In comparison with the control group, single and combination drug treatments significantly reduced the proliferation index (PI) for 96 h. The most potent reduction of PI was observed with VNR in combination with CIM and LiCl for all time intervals. VNR with CIM and LiCl seemed to be ineffective in reducing BrdU-LI of both monolayer cell and spheroid cultures, spheroid size, and cAMP level. VNR with LiCl increased apoptosis at 24 h, however VNR with CIM increased apoptosis at 96 h. VNR was the most potent drug in inhibiting colony-forming efficiency. The combination of VNR with CIM was the most potent in reducing midkine levels among all groups. Interestingly, the combination of VNR with LiCl led to both nuclear membrane breakdown and disappearance of the cellular membranes inside the spheroids. Both CIM and LiCl seemed to potentiate VNR-induced cytotoxicity, and MK was not a resistance factor for VNR, LiCl, and CIM.

Topics: Antidepressive Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Bromodeoxyuridine; Cell Line, Tumor; Cell Proliferation; Clomipramine; Cyclic AMP; Cytokines; Drug Synergism; Flow Cytometry; Humans; Lithium Chloride; Microscopy, Electron, Transmission; Midkine; Neoplastic Stem Cells; Neuroblastoma; Time Factors; Vinblastine; Vinorelbine

The mood stabilizing agents lithium chloride (LiCl) and sodium valproate (VPA) have recently gained interest as potential neuroprotective therapeutics. However, exploitation of these therapeutic applications is hindered by both a lack of molecular understanding of the mode of action, and a number of sub-optimal properties, including a relatively small therapeutic window and variable patient response. Human neuroblastoma cells (SH-SY5Y) were exposed to 1 mM lithium chloride or 1 mM sodium valproate for 6 h or 72 h, and transcriptomes measured by Affymetrix U133A/B microarray. Statistically significant gene expression changes were identified using SAM software, with selected changes confirmed at transcript (TaqMan) and protein (Western blotting) levels. Finally, anti-apoptotic action was measured by an in vitro fluorescent assay. Exposure of SH-SY5Y cells to therapeutically relevant concentrations of either lithium chloride or sodium valproate elicited 936 statistically significant changes in gene expression. Amongst these changes we observed a large (maximal 31.3-fold) increase in the expression of the homeodomain protein Six1, and have characterized the time- and dose-dependent up-regulation of this gene in response to both drugs. In addition, we demonstrate that, like LiCl or VPA treatment, Six1 over-expression protects SH-SY5Y cells from staurosporine-induced apoptosis via the blockade of caspsase-3 activation, whereas removal of Six1 protein via siRNA antagonises the ability of LiCl and VPA to protect SH-SY5Y cells from STS-induced apoptosis. These results provide a novel mechanistic rationale underlying the neuroprotective mechanism of LiCl and VPA, suggesting exciting possibilities for the development of novel therapeutic agents against neurodegenerative diseases such as Alzheimer's or Parkinsonism.

Topics: Antimanic Agents; Apoptosis; Cell Line, Tumor; Gene Expression Profiling; Gene Silencing; Homeodomain Proteins; Humans; Lithium Chloride; Neuroblastoma; Staurosporine; Up-Regulation; Valproic Acid

Ethanol is a potent teratogen for the developing central nervous system (CNS), and fetal alcohol syndrome (FAS) is the most common nonhereditary cause of mental retardation. Ethanol disrupts neuronal differentiation and maturation. It is important to identify agents that provide neuroprotection against ethanol neurotoxicity. Using an in vitro neuronal model, mouse Neuro2a (N2a) neuroblastoma cells, we demonstrated that ethanol inhibited neurite outgrowth and the expression of neurofilament (NF) proteins. Glycogen synthase kinase 3beta (GSK3beta), a multifunctional serine/threonine kinase negatively regulated neurite outgrowth of N2a cells; inhibiting GSK3beta activity by retinoic acid (RA) and lithium induced neurite outgrowth, while over-expression of a constitutively active S9A GSK3beta mutant prevented neurite outgrowth. Ethanol inhibited neurite outgrowth by activating GSK3beta through the dephosphorylation of GSK3beta at serine 9. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family rich in many edible berries and other pigmented fruits, enhanced neurite outgrowth by promoting p-GSK3beta(Ser9). More importantly, C3G reversed ethanol-mediated activation of GSK3beta and inhibition of neurite outgrowth as well as the expression of NF proteins. C3G also blocked ethanol-induced intracellular accumulation of reactive oxygen species (ROS). However, the antioxidant effect of C3G appeared minimally involved in its protection. Our study provides a potential avenue for preventing or ameliorating ethanol-induced damage to the developing CNS.

Topics: Adjuvants, Immunologic; Analysis of Variance; Animals; Anthocyanins; Antioxidants; Cell Lineage; Central Nervous System Depressants; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Ethanol; Gene Expression Regulation; Glucosides; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Lithium Chloride; Mice; Neurites; Neuroblastoma; Reactive Oxygen Species; RNA, Small Interfering; Transfection; Tretinoin

Lithium, a drug in the treatment of bipolar disorder, modulates many aspects of neuronal developmental processes such as neurogenesis, survival, and neuritogenesis. However, the underlying mechanism still remains to be understood. Here, we show that lithium upregulates the expression of sorting nexin 3 (SNX3), one of the Phox (PX) domain-containing proteins involved in endosomal sorting, and regulates neurite outgrowth in mouse N1E-115 neuroblastoma cells. The inhibition of SNX3 function by its knockdown decreases lithium-induced outgrowth of neurites. Transfection of the full-length SNX3 construct into cells facilitates the outgrowth. We also find that the C-terminus, as well as the PX domain, of SNX3 has a functional binding sequence with phosphatidylinositol monophosphates. Transfection of the C-terminal deletion mutant or only the C-terminus does not have an effect on the outgrowth. These results suggest that SNX3, a protein upregulated by lithium, is an as yet unknown regulator of neurite formation and that it contains another functional phosphatidylinositol phosphate-binding region at the C-terminus.

Topics: Amino Acid Sequence; Animals; Carrier Proteins; Gene Knockdown Techniques; Lithium Chloride; Mice; Molecular Sequence Data; Neurites; Neuroblastoma; Phosphatidylinositols; Protein Binding; Recombinant Proteins; RNA, Small Interfering; Sorting Nexins; Tumor Cells, Cultured; Up-Regulation; Vesicular Transport Proteins

Lithium is an effective mood stabilizer for bipolar disorder patients and its therapeutic effect may involve inhibition of inositol monophosphatase activity. In humans, the enzyme is encoded by two genes, IMPA1 and IMPA2. IMPA2 maps to 18p11.2, a genomic interval for which evidence of linkage to bipolar disorder has been supported by several reports. We performed a genetic association study in Japanese cohorts (496 patients with bipolar disorder and 543 control subjects). Interestingly, we observed association of IMPA2 promoter single nucleotide polymorphisms (SNPs) (-461C and -207T) with bipolar disorder, the identical SNPs reported previously in a different population. In vitro promoter assay and genetic haplotype analysis showed that the combination of (-461C)-(-207T)-(-185A) drove enhanced transcription and the haplotypes containing (-461C)-(-207T)-(-185A) contributed to risk for bipolar disorder. Expression study on post-mortem brains revealed increased transcription from the IMPA2 allele that harbored (-461C)-(-207T)-(-185A) in the frontal cortex of bipolar disorder patients. The examination of allele-specific expressions in post-mortem brains did not support genomic imprinting of IMPA2, which was suggested nearby genomic locus. Contrasting to a prior report, therapeutic concentrations of lithium could not suppress the transcription of IMPA2 mRNA, and the mood-stabilizing effect of lithium is, if IMPA2 was one of the targets of lithium, deemed to be generated via inhibition of enzymatic reaction rather than transcriptional suppression. In conclusion, the present study suggests that a promoter haplotype of IMPA2 possibly contributes to risk for bipolar disorder by elevating IMPA2 levels in the brain, albeit the genetic effect varies among populations.

Topics: Adult; Bipolar Disorder; Cell Line, Tumor; Cells, Cultured; Chromosomes, Human, Pair 18; Female; Gene Expression Regulation; Gene Frequency; Genetic Predisposition to Disease; Haplotypes; Humans; Lithium Chloride; Male; Middle Aged; Neuroblastoma; Phosphoric Monoester Hydrolases; Polymorphism, Single Nucleotide; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Risk; Transcription, Genetic; Transfection

Stress of the endoplasmic reticulum (ER), which is associated with many neurodegenerative conditions, can lead to the elimination of affected cells by apoptosis through only partially understood mechanisms. Thapsigargin, which causes ER stress by inhibiting the ER Ca(2+)-ATPase, was found to not only activate the apoptosis effector caspase-3 but also to cause a large and prolonged increase in the activity of glycogen synthase kinase-3beta (GSK3beta). Activation of GSK3beta was obligatory for thapsigargin-induced activation of caspase-3, because inhibition of GSK3beta by expression of dominant-negative GSK3beta or by the GSK3beta inhibitor lithium blocked caspase-3 activation. Thapsigargin treatment activated GSK3beta by inducing dephosphorylation of phospho-Ser-9 of GSK3beta, a phosphorylation that normally maintains GSK3beta inactivated. Caspase-3 activation induced by thapsigargin was blocked by increasing the phosphorylation of Ser-9-GSK3beta with insulin-like growth factor-1 or with the phosphatase inhibitors okadaic acid and calyculin A, but the calcineurin inhibitors FK506 and cyclosporin A were ineffective. Insulin-like growth factor-1, okadaic acid, calyculin A, and lithium also protected cells from two other inducers of ER stress, tunicamycin and brefeldin A. Thus, ER stress activates GSK3beta through dephosphorylation of phospho-Ser-9, a prerequisite for caspase-3 activation, and this process is amenable to pharmacological intervention.

Topics: Anti-Bacterial Agents; Apoptosis; Brefeldin A; Calcineurin; Calcineurin Inhibitors; Caspase 3; Caspases; Endoplasmic Reticulum; Enzyme Activation; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippocampus; Humans; Insulin-Like Growth Factor I; Lithium Chloride; Neuroblastoma; Neuroprotective Agents; Phosphorylation; Proteins; Signal Transduction; Thapsigargin; Tumor Cells, Cultured; Tunicamycin

Both lithium and valproate have been used in the treatment of manic-depressive illness with very limited understanding of their therapeutic mechanism of action. Recent literature suggests that blocking of potassium channels may be a common therapeutic mechanism of many antidepressant agents. To determine whether the commonly used antimanic agents could prevent potassium efflux-induced cell damage and apoptosis and the underlying mechanisms, we treated SH-SY5Y human neuroblastoma cells with the potassium ionophore, valinomycin (2-100 microM) and observed cell shrinkage, mitochondria damage, a significant increase in of lactate dehydrogenase (LDH) activity and caspase-3 protein expression. Cells treated with lithium (0.5-3 mM) or valproate (0.07-1.4 mM) alone produced no apoptotic morphological and biochemical changes while both mood stabilizers pretreatment reduced or prevented the apoptotic morphological changes. However, valinomycin-induced caspase-3 elevation was only prevented by lithium pretreatment while both lithium and valproate attenuated valinomycin-induced LDH release. Our results suggest that lithium and valproate share a common neuroprotective action against potassium efflux-induced cell apoptosis with different mechanisms.

Topics: Antimanic Agents; Apoptosis; Caspase 3; Caspases; Humans; Ionophores; L-Lactate Dehydrogenase; Lithium Chloride; Neuroblastoma; Tumor Cells, Cultured; Valinomycin; Valproic Acid

Mood stabilizing drugs decrease central nervous system cyclic AMP signaling. We report here that chronic, but not acute treatment with lithium chloride in human neuroblastoma SH-SY5Y cells, inhibits phosphorylation of cyclic AMP responsive element binding protein and cyclic AMP responsive element DNA binding induced by the adenylyl cyclase activator forskolin, but has no effect on constitutive expression of cyclic AMP responsive element binding protein. These results are consistent with an effect of lithium to blunt the cyclic AMP signal transduction pathway. Such an effect is not shared by the other commonly prescribed mood stabilizer, sodium valproate. Our results suggest that cyclic AMP responsive element binding protein regulated gene expression may be relevant to the long-term prophylactic effect of lithium. Furthermore, sodium valproate, which is also effective in bipolar disorder, would appear to act on other pathways to bring about its therapeutic effects.

Topics: Adenylyl Cyclases; Colforsin; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Humans; Kinetics; Lithium Chloride; Neuroblastoma; Phosphorylation; Signal Transduction; Tumor Cells, Cultured; Valproic Acid

Differential display PCR was used to identify genes regulated by mood stabilizer lithium in rat cerebral cortex. A differentially displayed lithium regulated gene fragment was isolated in rat cerebral cortex after chronic treatment with lithium (1.69 g/kg, p.o. ) for three weeks. A 1216-nucleotide cDNA for a novel lithium regulated gene (NLRG) was isolated from a rat brain cDNA library with RACE (rapid amplification of 5' cDNA end) PCR using a prime from the differentially displayed NLRG gene fragment. The deduced protein sequence was 321 amino acids long, and shows a significant homology with yeast nitrogen permease regulator 2 (NPR2). NLRG expression induced by lithium was confirmed by Northern and slot blot analysis in rat cerebral cortex and neuroblastomaxglioma NG108-15 cells, respectively. In situ hybridization revealed that chronic treatment with lithium increased NLRG gene expression in frontal cortex and hippocampus, but not in striatum, hypothalamus and thalamus regions of rat brain. These results suggest a novel target for lithium which may be relevant to its mechanism of action.

Topics: Amino Acid Sequence; Animals; Antimanic Agents; Base Sequence; Brain; Cerebral Cortex; DNA, Complementary; Gene Expression Regulation; Glioma; Hybrid Cells; In Situ Hybridization; Lithium Chloride; Male; Mice; Molecular Sequence Data; Nerve Tissue Proteins; Neuroblastoma; Polymerase Chain Reaction; Rats; Rats, Sprague-Dawley; Sequence Alignment; Sequence Homology, Amino Acid; Subtraction Technique; Trans-Activators; Tumor Cells, Cultured

The therapeutic efficacy of lithium in the treatment of mood disorders is delayed and only observed after chronic administration, a temporal profile that suggests alterations at the genomic level. Lithium has been demonstrated to modulate AP-1 DNA binding activity as well as the expression of genes regulated by AP-1, but the mechanisms underlying these effects have not been fully elucidated. In the present study, we found that the lithium-induced increases in AP-1 DNA binding activity were accompanied by increases in p-cJun and cJun levels in SH-SY5Y cells. Lithium also increased cJun-mediated reporter gene expression in a dose-dependent manner, with significant effects observed at therapeutically relevant concentrations. Lithium's effects on cJun-mediated reporter gene expression in SH-SY5Y cells were more pronounced in the absence of myo-inositol and were blocked by protein kinase C (PKC) inhibitors and by cotransfection with a PKCalpha dominant-negative mutant. Chronic in vivo lithium administration increased AP-1 DNA binding activity in frontal cortex and hippocampus and also increased the levels of the phosphorylated, active forms of c-Jun NH2-terminal kinases (JNKs) in both brain regions. These results demonstrate that lithium activates the JNK signaling pathway in rat brain during chronic in vivo administration and in human cells of neuronal origin in vitro; in view of the role of JNKs in regulating various aspects of neuronal function and their well-documented role in regulating gene expression, these effects may play a major role in lithium's long-term therapeutic effects.

Topics: Animals; Antimanic Agents; Cells, Cultured; DNA; Enzyme Activation; Enzyme Inhibitors; Frontal Lobe; Gene Expression Regulation; Genes, jun; Genes, Reporter; Hippocampus; Humans; Indoles; Inositol; Isoenzymes; JNK Mitogen-Activated Protein Kinases; Lithium Chloride; Male; Maleimides; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Neuroblastoma; Protein Kinase C; Protein Kinase C-alpha; Proto-Oncogene Proteins c-jun; Rats; Rats, Wistar; Transcription Factor AP-1; Tumor Cells, Cultured

Lithium and sodium valproate (VPA) are effective in the treatment of bipolar disorder (BD) and may function through the regulation of signal transduction pathways and transcription factors such as c-fos and c-Jun, which in turn results to changes in gene expression. The long-term efficacy of lithium and VPA in BD suggests that the regulation of gene expression may be an important target for these drugs. Preliminary evidence suggests that c-fos levels and AP-1 binding may be regulated by lithium and VPA, but the results are inconclusive. In the present study, we report differential effects of the two most commonly prescribed mood stabilizers used to treat BD on Fos/Jun protein levels and their AP-1 binding activity in human neuroblastoma SH-SY5Y cells. At therapeutically relevant concentrations, both drugs acutely (<24 h) induced c-Fos immunoreactivity and AP-1 binding. In contrast to lithium, chronic (1 week) treatment with VPA led to continued induction of c-Fos, in addition to induction of c-Jun immunoreactivity and a 33-35 kDa band previously identified as chronic FRA. AP-1 DNA binding activity was also increased after 1 week VPA treatment. These findings suggest that both these mood stabilizers may have an effect on neuronal gene expression of target genes containing the AP-1 consensus sequence in their promoter regions after acute treatment. The present results confirm and extend previous findings on the regulation of c-fos expression and AP-1 binding after administration of mood stabilizers, and further elucidate the mechanisms through which VPA increases AP-1 DNA binding.

Topics: Antimanic Agents; Base Sequence; Binding Sites; Consensus Sequence; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Lithium Chloride; Neuroblastoma; Nuclear Proteins; Oligodeoxyribonucleotides; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Time Factors; Transcription Factor AP-1; Tumor Cells, Cultured; Valproic Acid

Lithium, a monovalent cation, is the mainstay in the treatment of manic-depressive (MDI) illness, but despite extensive research, its mechanism of action remains to be elucidated. Since lithium requires chronic administration for therapeutic efficacy, and because its beneficial effects last well beyond its discontinuation, it has been postulated that lithium may exert major effects at the genomic level. In the present study we found that lithium, at therapeutically relevant concentrations, increases AP-1 DNA binding activity in human SH-SY5Y cells and rat C6 glioma cells. Additionally, in both SY5Y and C6 cells transiently transfected with a reporter gene vector driven by an SV40 promoter, lithium increased the activity of the reporter gene in a time- and concentration-dependent manner. Furthermore, mutations in the AP-1 sites of the reporter gene promoter significantly attenuated lithium's effects. These data indicate that lithium stimulates gene expression through the AP-1 transcription factor pathway, effects which may play a role in its long-term mood-stabilizing effects.

Topics: Animals; Base Sequence; Binding Sites; Chlorides; Consensus Sequence; Gene Expression Regulation, Neoplastic; Genes, Reporter; Glioma; Humans; Lithium Chloride; Luciferases; Molecular Sequence Data; Mutagenesis, Site-Directed; Neuroblastoma; Potassium Chloride; Promoter Regions, Genetic; Rats; Recombinant Fusion Proteins; Rubidium; Sequence Alignment; Sequence Homology, Nucleic Acid; Simian virus 40; Transcription Factor AP-1; Transfection; Tumor Cells, Cultured

In the present study transcriptional activities has been measured with different fragments of the 5'-flanking sequence of the human monoamine oxidase (MAO) genes linked to human growth hormone which was used as a reporter gene. SH-SY5Y neuroblastoma cells and 1242 MG glioma cells were compared under basal conditions as well as after treatments with different drugs. Under basal conditions, the relative reporter activities of the different promoter fragments were similar for both cell lines. No changes in promoter activities, were observed when cells were treated with L-deprenyl, lithium chloride or raclopride. In contrast, increases (2-3-fold) in both reporter gene expression and enzyme activity were observed after ethanol treatment of cells transfected with MAO-B fragments. Gel retardation analysis showed that ethanol caused changes in transcription factor binding to the MAO-B core promoter in both the SH-SY5Y and 1242 MG cell lines in a cell-type specific fashion.

Topics: Alcohol Drinking; Antimanic Agents; Antipsychotic Agents; Astrocytoma; Central Nervous System Depressants; Dose-Response Relationship, Drug; Electrophoresis; Ethanol; Humans; Lithium Chloride; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Neuroblastoma; Promoter Regions, Genetic; Psychotropic Drugs; Raclopride; Salicylamides; Selegiline; Transcription, Genetic; Tumor Cells, Cultured

In order to approach the molecular mechanism of Li+'s mood-stabilizing action, the effect of Li+ (LiCl) on inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] mass was investigated in human neuroblastoma SH-SY5Y cells, which express muscarinic M3 receptors, coupled to PtdIns hydrolysis. Stimulation of these cells, with the cholinergic agonist acetylcholine, resulted in a rapid and transient increase in Ins(1,4,5)P3 with a maximum at 10 s. This was followed by a rapid decline in Ins(1,4,5)P3 within 30 s to a plateau level above baseline, which gradually declined to reach a new steady state, which was significantly higher than resting Ins(1,4,5)P3 at 30 min. Li+ had no effect on Ins(1,4,5)P3 in resting cells, as well as on the acetylcholine-dependent peak of Ins(1,4,5)P3. However, Li+ caused a transient reduction (at 45 s), followed by a long lasting increase in the Ins(1,4,5)P3 (30 min), as compared with controls. The Li+ effects were dose-dependent and were observed at concentrations used in the treatment of bipolar disorders. Supplementation with inositol had no effect on the level of Ins(1,4,5)P3, at least over the time periods studied. Stimulation of muscarinic receptors with consequent activation of phospholipase C were necessary for the manifestation of Li+ effects in SH-SY5Y cells, Li+ did not interfere with degradation of Ins(1,4,5)P3 after receptor-blockade with atropine, suggesting that Li+ has no direct effect on the Ins(1,4,5)P3-metabolizing enzymes. A direct effect of Li+ on the phospholipase C also is unlikely. Blockade of Ca2+ entry into the cells by Ni2+, or incubation with EGTA, which reduces agonist-stimulated accumulation of Ins(1,4,5)P3, had no effect on the Li(+)-dependent increase in Ins(1,4,5)P3.

Topics: Acetylcholine; Atropine; Calcium; Egtazic Acid; Enzyme Activation; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Lithium Chloride; Neuroblastoma; Nickel; Receptors, Muscarinic; Second Messenger Systems; Tumor Cells, Cultured; Type C Phospholipases

We performed detailed chromatographic analyses on the molecular species of the major glycerophospholipids (GPLs) and free sn-1,2-diacylglycerols (DAGs) from SH-SY5Y human neuroblastoma cells following incubation with or without LiCl. For this comparison the inositol, choline, ethanolamine and serine GPLs were dephosphorylated with phospholipase C and the released sn-1,2-diacylglycerols along with the DAGs were subjected to high-temperature GLC on polar and non-polar capillary columns as their trimethylsilyl and tert.-butyl-dimethylsilyl ethers. A 30-min incubation with 10 mM LiCl increased the total amount of human neuroblastoma DAGs by 32-58% (P < 0.05) to 2.6 pmol/micrograms cell protein. This was accompanied by a limited qualitative shift in the molecular species pattern, the most obvious of which was the increase (13%) in the major saturated-polyunsaturated molecular species and the ca. 46% increase in the minor 18:1-18:1 species over control levels. The DAGs originated mainly from the inositol GPLs (IGPLs), as indicated by the high levels of the characteristic 18:0-20:4n6 (18:0-20:3n9) species in both IGPLs and DAGs, and to a lesser extent from the choline GPLs (CGPLs), as indicated by the high proportion in CGPLs of the oligoenoic species, which were largely absent from IGPLs. Alkenylacylglycerols were not detected in DAGs, although they made up some 60% of the total ethanolamine GPLs (EGPLs). No significant changes in the molecular species composition of the cellular GPLs, including IGPLs, were detected after exposure to LiCl.

Topics: Brain Neoplasms; Chromatography; Diglycerides; Glycerophosphates; Humans; Indicators and Reagents; Lithium Chloride; Neuroblastoma; Organosilicon Compounds; Trimethylsilyl Compounds; Tumor Cells, Cultured

Batrachotoxin (BTX), veratridine and monensin induced a time- and dose-dependent increase of [3H]-inositol monophosphate (3H-IP1) accumulation in the presence of lithium in prelabeled neurohybrid NCB-20 cells. A decrease of NaCl concentration to less than 30 mM markedly increased basal 3H-IP1 accumulation; however, the percentage of stimulation induced by these three agents remained unchanged even in the complete absence of sodium. The stimulation of phosphoinositide hydrolysis induced by these agents was detected in the absence of lithium but was largely prevented in the calcium-free medium. Tetradotoxin (TTX) blocked effects of BTX and veratridine (IC50 approximately 20nM), but not that stimulated by monensin. Thus, calcium-dependent activation of phospholipase C by these agents did not involve the entry of sodium or lithium. BTX and monensin also induced greater than additive effects on carbachol-induced 3H-IP1 accumulation. These effects were also TTX-sensitive and involved an increase in the Vmax and a decrease in the EC50 for carbachol. Veratridine provoked strikingly different effects on carbachol-dependent phosphoinositide turnover, depending on the passage number of the cells.

Topics: Batrachotoxins; Brain; Calcium; Carbachol; Chlorides; Hybrid Cells; Hydrolysis; Kinetics; Lithium; Lithium Chloride; Monensin; Neuroblastoma; Phosphatidylinositols; Sodium Channels; Sodium Chloride; Tetrodotoxin; Tumor Cells, Cultured; Type C Phospholipases; Veratridine; Veratrine

Because previous studies in whole-animal models have reported inconsistent results regarding the effect of chronic lithium on the regulation of the neuronal muscarinic acetylcholine receptor (mAChR) number, we examined the effect of chronic lithium on the regulation of mAChR in cell cultures of N1E-115, a mouse neuroblastoma clone. Li+ induced a concentration- and time-dependent increase in the mAChR number, with a 30% increase in specific [3H]quinuclidinyl benzilate binding in membrane homogenates induced by a 5-day incubation with 10 mM Li+. Agonist-induced down-regulation of the mAChR number was also inhibited by lithium: chronic treatment with 10 mM Li+ caused a 25-35% reduction in the magnitude of carbachol. In contrast, the decrease in the mAChR number induced by the synergistic action of A23187 (300 nM) and phorbol myristate acetate (300 nM) was unaffected by Li+. These results demonstrate that chronic treatment with Li+ increases the basal mAChR number and dampens the decrease in receptor number induced by a muscarinic agonist in neuroblastoma cells. The implications of these results in understanding the functional regulation of neuronal mAChR number are discussed.

Topics: Animals; Calcimycin; Carbachol; Cell Line; Cell Membrane; Chlorides; Kinetics; Lithium; Lithium Chloride; Mice; Neuroblastoma; Neurons; Receptors, Muscarinic; Tetradecanoylphorbol Acetate

The conversion of T4 to T3 in the brain and anterior pituitary gland contributes significantly to the T3 content of these tissues and appears to be an important modulator of thyroid hormone action. In the present study, the antimanic agent lithium was demonstrated in cultured neural and pituitary tissue to have a significant inhibitory effect on the activity of low Km (type II) iodothyronine 5'-deiodinase (I5'D), the enzyme mediating T3 formation. At medium lithium concentrations of 3.3-5 mM, 15'D activity was decreased 44 +/- 3% (P less than 0.001) in the NB41A3 mouse neuroblastoma cell line and 48 +/- 2% (P less than 0.001) in the GH3 rat pituitary tumor cell line. This inhibitory effect was only observed in intact cells. Significant inhibition of this enzymatic process was also noted in the anterior pituitary gland of thyroidectomized rats injected 3-24 h earlier with either 4 or 10 mmol/kg BW LiCl. This decrease in low Km I5'D activity was accompanied by significant decreases in the serum T3 concentration and the pituitary nuclear T3 content. Renal high Km (type I) I5'D activity was unaffected by lithium administration. These studies demonstrate that lithium, an agent of proven therapeutic benefit in patients with manic-depressive illness, can affect changes in T4 metabolism and cellular T3 content in neural and anterior pituitary tissue. Given the prominent mood changes that occur in patients with disordered thyroid function, this finding suggests that the therapeutic benefits of lithium in affective illness may be derived in part from alterations in thyroid hormone economy in the brain.

Topics: Animals; Cell Line; Cerebral Cortex; Chlorides; Iodide Peroxidase; Kidney; Kinetics; Lithium; Lithium Chloride; Male; Mice; Neuroblastoma; Pituitary Gland, Anterior; Pituitary Neoplasms; Rats; Rats, Inbred Strains; Thyroidectomy; Thyroxine; Triiodothyronine; Triiodothyronine, Reverse

Clonal neurohybridoma NCB-20 cells expressed muscarinic cholinergic receptors coupled to phospholipase C. Addition of carbachol in the presence of Li+ to cells prelabeled with 3H-inositol increased 3H-inositol-l-phosphate (3H-IP1) accumulation by more than 4-fold with an EC50 of about 50 microM. This carbachol-induced response was blocked by atropine and pirenzepine with a Ki of 0.5 and 25 nM, respectively. The EC50 of Li+ for the carbachol-induced phosphoinositide turnover was 17 +/- 1.2 mM compared with a value of 1.8 +/- 0.2 mM in brain slices, suggesting the presence of an unusual type of inositol-l-phosphatase in NCB-20 cells. Carbachol-induced IP1 accumulation in these cells was potently and noncompetitively inhibited by the biologically active phorbol esters, phorbol dibutyrate (PDB) and phorbol myristate diacetate (PMA), while the biologically inactive phorbol, 4 beta-phorbol, failed to affect this phosphoinositide breakdown. The basal IP1 accumulation was also significantly attenuated by PDB and PMA but not by 4 beta-phorbol.

Topics: Animals; Atropine; Benzodiazepinones; Brain; Carbachol; Cell Line; Chlorides; Cricetinae; Cricetulus; Embryo, Mammalian; Hybridomas; Inositol Phosphates; Lithium; Lithium Chloride; Mice; Neuroblastoma; Phorbol 12,13-Dibutyrate; Phorbol Esters; Pirenzepine; Sugar Phosphates; Tetradecanoylphorbol Acetate