Human Neuroblastoma Lines Research Articles

Chiral Skeletons of Mesoporous Silica Nanospheres to Mitigate Alzheimer's β-Amyloid Aggregation.

Chiral mesoporous silica (mSiO2) nanomaterials have gained significant attention during the past two decades. Most of them show a topologically characteristic helix; however, little attention has been paid to the molecular-scale chirality of mSiO2 frameworks. Herein, we report a chiral amide-gel-directed synthesis strategy for the fabrication of chiral mSiO2 nanospheres with molecular-scale-like chirality in the silicate skeletons. The functionalization of micelles with the chiral amide gels via electrostatic interactions realizes the growth of molecular configuration chiral silica sols. Subsequent modular self-assembly results in the formation of dendritic large mesoporous silica nanospheres with molecular chirality of the silica frameworks. As a result, the resultant chiral mSiO2 nanospheres show abundant large mesopores (∼10.1 nm), high pore volumes (∼1.8 cm3·g-1), high surface areas (∼525 m2·g-1), and evident CD activity. The successful transfer of the chirality from the chiral amide gels to composited micelles and further to asymmetric silica polymeric frameworks based on modular self-assembly leads to the presence of molecular chirality in the final products. The chiral mSiO2 frameworks display a good chiral stability after a high-temperature calcination (even up to 1000 °C). The chiral mSiO2 can impart a notable decline in β-amyloid protein (Aβ42) aggregation formation up to 79%, leading to significant mitigation of Aβ42-induced cytotoxicity on the human neuroblastoma line SH-ST5Y cells in vitro. This finding opens a new avenue to construct the molecular chirality configuration in nanomaterials for optical and biomedical applications.

Proximal Protein Interaction Landscape of RIT2 Neuronal GTPase

RIT2 is a small Ras-like GTPase expressed exclusively in neurons. RIT2 expression is first detected late in embryonic development (~e16), increasing rapidly after birth and plateauing in the adult mouse brain, suggesting a more important role for RIT2 in adult neurological function than in developmental processes. Ras-related proteins serve as GDP/GTP-regulated binary switches which function to relay extracellular signals to signaling pathways that regulate diverse cellular processes, including essential roles in neuronal regulation. Genome-wide association studies have linked RIT2 to Parkinson's disease, autism spectrum disorder, and schizophrenia; but its physiological function(s) remain obscure and known interaction partners are extremely limited. To begin to address these deficiencies, we employed proximity-dependent biotin identification technology (BioID) to define the proximal protein-protein interaction landscape of RIT2. This system allows weak or transient interactions in living cells to be captured. RIT2 was fused to a re-engineered version of the bacterial BirA biotin ligase, TurboID, which conjugates biotin to proteins within the immediate vicinity (~10nm) of its active site. Stable SK-N-DZ cells (a human neuroblastoma line known to express RIT2) expressing either active Turbo-RIT2 or unconjugated Turbo (control) were treated in triplicate with biotin. Biotinylated proteins were captured with streptavidin-resin and subjected to quantitative bioanalysis by LC-mass spectrometry. RIT2 interactors include Brn3a (POU4F1), a previously described RIT2 effector, as well as other transcription factors and chromatin modification factors. This study provides new information about the potential function(s) of the poorly characterized RIT2. These newly discovered protein interaction networks open many paths to greater understanding of RIT2 and cell signaling.

Modulation of Nerve Cell Differentiation: Role of Polyphenols and of Contactin Family Components.

In this study the mechanisms are explored, which modulate expression and function of cell surface adhesive glycoproteins of the Immunoglobulin Supergene Family (IgSF), and in particular of its Contactin subset, during neuronal precursor developmental events. In this context, a specific topic concerns the significance of the expression profile of such molecules and their ability to modulate signaling pathways activated through nutraceuticals, in particular polyphenols, administration. Both in vitro and in vivo approaches are chosen. As for the former, by using as a model the human SH-SY5Y neuroblastoma line, the effects of grape seed polyphenols are evaluated on proliferation and commitment/differentiation events along the neuronal lineage. In SH-SY5Y cell cultures, polyphenols were found to counteract precursor proliferation while promoting their differentiation, as deduced by studying their developmental parameters through the expression of cell cycle and neuronal commitment/differentiation markers as well as by measuring neurite growth. In such cultures, Cyclin E expression and BrdU incorporation were downregulated, indicating reduced precursor proliferation while increased neuronal differentiation was inferred from upregulation of cell cycle exit (p27–Kip) and neuronal commitment (NeuN) markers as well as by measuring neurite length through morphometric analysis. The polyphenol effects on developmental parameters were also explored in vivo, in cerebellar cortex, by using as a model the TAG/F3 transgenic line, which undergoes delayed neural development as a consequence of Contactin1 adhesive glycoprotein upregulation and premature expression under control of the Contactin2 gene (Cntn-2) promoter. In this transgenic line, a Notch pathway activation is known to occur and polyphenol treatment was found to counteract such an effect, demonstrated through downregulation of the Hes-1 transcription factor. Polyphenols also downregulated the expression of adhesive glycoproteins of the Contactin family themselves, demonstrated for both Contactin1 and Contactin2, indicating the involvement of changes in the expression of the underlying genes in the observed phenotype. These data support the hypothesis that the complex control exerted by polyphenols on neural development involves modulation of expression and function of the genes encoding cell adhesion molecules of the Contactin family and of the associated signaling pathways, indicating potential mechanisms whereby such compounds may control neurogenesis.

Abstract 5485: The combined effects of polyamine and GSH inhibition on the viability of human cancer cell lines

Abstract Polyamines play essential roles in DNA stability and cell proliferation, making polyamine metabolism a potential target for cancer therapeutics. The spermine analog N1,N11- Diethylnorspermine (DenSpm) reduces levels of ODC, the enzyme responsible for polyamine biosynthesis, and also induces the enzyme SSAT, responsible for the catabolism of polyamines. Polyamines have been shown to inactivate reactive oxygen species (ROS), including peroxides, protecting mammalian cells from oxidative damage to nucleic acids. The catabolism of polyamines by SSAT, in contrast, leads to the production of ROS, which results in cellular death and limits the progression of cancer. Buthionine sulfoximine (BSO) selectively depletes stores of intracellular glutathione GSH, an antioxidant that neutralizes ROS. We hypothesized that treating cancer cell lines with both DenSpm and BSO will have a synergistic cytotoxic effect on the cells, due to the depletion of ROS-scavenging polyamines, the generation of ROS caused by DenSpm and the absence of GSH. Treatment of the four cell lines, MCF-7, MDA-MD-231, Caco-2 and PANC-1, with DenSpm resulted in moderate reductions in cell viability as measured using the Cell Titer Blue assay (Promega, Inc.). As has been widely reported in other mammalian systems, DenSpm (10 µM) reduced intracellular polyamine concentrations (HPLC of benzoylated polyamine derivatives) and induced SSAT (Western Blots). Treatment of cells with BSO (500 µM) caused a significant decrease in intracellular GSH levels. Similar findings have been reported in previous studies with human neuroblastoma lines (Anderson, et al. 1999). The combination of BSO and DenSpm had effects markedly greater than either compound alone. Pretreatment of cells with BSO (500 µM) for 24 hours with subsequent removal of BSO before DenSpm (10, 100, 1000 µM) addition resulted in significantly reduced viability in all cell lines. DenSpm was found to be most effective in the continued presence of BSO as compared to a 24-hour BSO pretreatment. The compounds used in combination also enhanced ROS production, further demonstrating their synergistic effects. Citation Format: John Grady, Davey Holzer, Kaci Keleher, Emma Meetz, Russ Feirer. The combined effects of polyamine and GSH inhibition on the viability of human cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5485. doi:10.1158/1538-7445.AM2017-5485

Selective depletion of tumour suppressors Deleted in Colorectal Cancer (DCC) and neogenin by environmental and endogenous serine proteases: linking diet and cancer.

BackgroundThe related tumour suppressor proteins Deleted in Colorectal Cancer (DCC) and neogenin are absent or weakly expressed in many cancers, whereas their insertion into cells suppresses oncogenic behaviour. Serine proteases influence the initiation and progression of cancers although the mechanisms are unknown.MethodsThe effects of environmental (bacterial subtilisin) and endogenous mammalian (chymotrypsin) serine proteases were examined on protein expression in fresh, normal tissue and human neuroblastoma and mammary adenocarcinoma lines. Cell proliferation and migration assays (chemoattraction and wound closure) were used to examine cell function. Cells lacking DCC were transfected with an ectopic dcc plasmid.ResultsSubtilisin and chymotrypsin selectively depleted DCC and neogenin from cells at nanomolar concentrations without affecting related proteins. Cells showed reduced adherence and increased migration, but after washing they re-attached within 24 h, with recovery of protein expression. These effects are induced by chymotryptic activity as they are prevented by chymostatin and the soybean Bowman-Birk inhibitor typical of many plant protease inhibitors.Conclusions Bacillus subtilis, which secretes subtilisin is widely present in soil, the environment and the intestinal contents, while subtilisin itself is used in meat processing, animal feed probiotics and many household cleaning agents. With chymotrypsin present in chyme, blood and tissues, these proteases may contribute to cancer development by depleting DCC and neogenin. Blocking their activity by Bowman-Birk inhibitors may explain the protective effects of a plant diet. Our findings identify a potential non-genetic contribution to cancer cell behaviour which may explain both the association of processed meats and other factors with cancer incidence and the protection afforded by plant-rich diets, with significant implications for cancer prevention.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2795-y) contains supplementary material, which is available to authorized users.

Abstract 2648: Chimeric antigen receptor T-cell therapy against anaplastic lymphoma kinase (ALK) is limited by target antigen density and CAR surface expression

Abstract ALK is overexpressed on the surface of neuroblastoma (NB) and is associated with high risk disease. We developed a second generation CAR based on a monoclonal antibody against ALK. ALK CAR T-cells significantly delay the growth of human NB cell lines in murine xenograft models, but animals eventually succumb to tumors. In order to understand how target antigen density limits the effectiveness of this CAR, we created a library of NALM-6 B-cell leukemias with variable amounts of ALK expressed on the surface of each clone. In vitro, ALK CAR T-cells lyse high ALK expressing leukemias but have reduced activity against leukemias with low expression of ALK. In co-culture assays, we found that there is a minimum target antigen density required for CAR T-cells to produce appreciable amounts of Th1 cytokines. This threshold of ALK expression is above the expression on human NB lines. There is also a threshold target antigen density at which maximum cytokine production occurs. Above this density, no additional cytokines are produced. In xenograft models, ALK CAR T-Cells are significantly more effective against high ALK expressing leukemia than against low ALK expressing leukemia. To our knowledge, this is the first report of greater in vivo efficacy of CAR T-cells against tumors with higher antigen expression. The level of surface expression of the CAR on T-cells also alters the function of CAR T-cells. We have identified a phenomenon in which both ALK and CD19 CAR T-cells lose surface expression of CAR over time in culture. Additionally, both the ALK CAR and the CD19 CAR downregulate quickly after T-cells are exposed to antigen. Using a fluorescently tagged CD19 CAR, we demonstrate that CARs are rapidly internalized upon antigen encounter. We created an assay to understand the interplay of target density and CAR surface expression. We transduced T-cells with different amounts of supernatant to achieve different ALK CAR surface densities and then exposed these T-cells to varying amounts of immobilized protein-L in order to crosslink the CAR. T-cell activation (measured by an NFAT reporter) is greatest when both CAR surface expression and target antigen density are highest. As both variables are decreased, there is a significant loss of T-cell activity. Thus, diminished CAR surface density due to antigen-dependent and independent CAR downmodulation may limit CAR T-cell efficacy, especially in the context of low tumor antigen expression. In conclusion, we have created a novel CAR targeting ALK that demonstrates in vivo efficacy against NB. Efficacy is limited by low target antigen density on NB and low CAR surface expression. We have identified phenomena in which CAR T-cells lose surface expression of CAR over time and also quickly internalize their receptors in response to antigen. These factors contribute to the efficacy of other CAR T-cells and this data provides important insights into future CAR target selection. Citation Format: Robbie G. Majzner, Alec J. Walker, Meera Murgai, Ling Zhang, Adrienne H. Long, Kelsey M. Wanhainen, Rimas J. Orentas, Crystal L. Mackall. Chimeric antigen receptor T-cell therapy against anaplastic lymphoma kinase (ALK) is limited by target antigen density and CAR surface expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2648.

The Proliferative Response of NB69 Human Neuroblastoma Cells to a 50 Hz Magnetic Field is mediated by ERK1/2 Signaling

A number of studies have reported that extremely low frequency magnetic fields (ELF-MF) can modulate proliferative processes in vitro; however, the transduction mechanisms implicated in such phenomena remain to be identified. The present study was aimed to determine whether a 50 Hz, 100 µT MF can induce cell proliferation in the human neuroblastoma line NB69, and whether the signaling pathway MAPK-ERK1/2 (Mitogen-Activated Protein Kinase - Extracellular-Signal-Regulated Kinase 1 and 2) is involved in that proliferative response. The cultures were exposed intermittently or continuously to the MF for a 63-hour duration. The continuous treatment did not induce significant changes in cell proliferation. In contrast, intermittent exposure caused statistically significant increase in the percent of cells in phase S of the cell cycle, followed by a significant increase in cell number. The intermittent treatment also induced an early, transient and repetitive activation of ERK1/2 that could be involved in the proliferative effects. In fact, both the proliferative response and the repeated activation of ERK1/2 were blocked by PD98059, the specific inhibitor of MEK (ERK kinases 1 and 2). Taken together, the described results indicate that a 50 Hz, 100 µT MF can stimulate proliferation in NB69 cells by triggering MAPK-ERK1/ 2 signaling at each of the “On” periods of an intermittent exposure.

Synergistic Activity of Fenretinide and the Bcl-2 Family Protein Inhibitor ABT-737 against Human Neuroblastoma

Fenretinide (4-HPR) is a cytotoxic retinoid with minimal systemic toxicity that has shown clinical activity against recurrent high-risk neuroblastoma. To identify possible synergistic drug combinations for future clinical trials, we determined whether ABT-737, a small-molecule BH3-mimetic that inhibits most proteins of the antiapoptotic Bcl-2 family, could enhance 4-HPR activity in neuroblastoma. Eleven neuroblastoma cell lines were tested for the cytotoxic activity of 4-HPR and ABT-737 as single agents and in combination using the DIMSCAN fluorescence digital imaging cytotoxicity assay. The effect of these agents alone and in combination on mitochondrial membrane depolarization and apoptosis (by flow cytometry), cytochrome c release, caspases, Bax-α, t-Bid, and Bak activation, and subcutaneous xenografts in nu/nu mice was also determined. Multilog synergistic cytotoxicity was observed for the drug combination in all of the 11 neuroblastoma cell lines tested, including MDR lines and those insensitive to either drug as single agents. 4-HPR + ABT-737 induced greater mitochondrial membrane depolarization and mitochondrial cytochrome c release, greater activation of caspases, Bax-α, t-Bid, and Bak, and a higher level of apoptosis than either drug alone. In vivo, 4-HPR + ABT-737 increased the event-free survival of the MDR human neuroblastoma line CHLA-119 implanted subcutaneously in nu/nu mice (194.5 days for the combination vs. 68 days for ABT-737 and 99 days for 4-HPR). Thus, the combination of 4-HPR with a BH3-mimetic drug warrants clinical trials in recurrent neuroblastoma.

Effects of paraoxon on neuronal and lymphocytic cholinergic systems

The cholinergic system in lymphocytes is hypothesized to be a key target for neurotoxic organophosphates (OPs). The present study determined the comparative effects of paraoxon, the active metabolite of OP-parathion, which is detected in the human neuroblastoma line, SH-SY5Y, and leukemic T-lymphocytes, MOLT-3, in vitro. Paraoxon induced cytotoxic effects in a dose- and time-dependent manner in both cells. Further, the paraoxon-induced modulatory effects were comparable despite different cell types, including over-expression of N-terminus acetylcholinesterase (N-AChE) protein, a marker of apoptosis, down-regulations of mRNA encoding M1, M2, and M3 muscarinic acetylcholine receptors (mAChRs), and induction in expression of c-Fos gene, an indication of certain mAChR subtype(s) activation. Furthermore, the non-selective cholinergic antagonist atropine partially attenuated the paraoxon-induced N-AChE and c-Fos activations in both types of cells. These results provide initial and additional information that OPs may similarly induce neuro- and immuno-toxic effects through mAChRs activation, and they underline the potential of using lymphocytes for assessing OPs-induced neurotoxicity.

Tacrine–Melatonin Hybrids as Multifunctional Agents for Alzheimer's Disease, with Cholinergic, Antioxidant, and Neuroprotective Properties

Tacrine-melatonin hybrids were designed and synthesized as new multifunctional drug candidates for Alzheimer's disease. These compounds may simultaneously palliate intellectual deficits and protect the brain against both beta-amyloid (A beta) peptide and oxidative stress. They show improved cholinergic and antioxidant properties, and are more potent and selective inhibitors of human acetylcholinesterase (hAChE) than tacrine. They also capture free radicals better than melatonin. Molecular modeling studies show that these hybrids target both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. At sub-micromolar concentrations they efficiently displace the binding of propidium iodide from the PAS and could thus inhibit A beta peptide aggregation promoted by AChE. Moreover, they also inhibit A beta self-aggregation and display neuroprotective properties in a human neuroblastoma line against cell death induced by various toxic insults, such as A beta(25-35), H(2)O(2), and rotenone. Finally, they exhibit low toxicity and may be able to penetrate the central nervous system according to an in vitro parallel artificial membrane permeability assay for the blood-brain barrier (PAMPA-BBB).

Hypoxanthine-guanine phosphoribosyl transferase regulates early developmental programming of dopamine neurons: implications for Lesch-Nyhan disease pathogenesis

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency results in Lesch-Nyhan disease (LND), where affected individuals exhibit a characteristic neurobehavioral disorder that has been linked with dysfunction of dopaminergic pathways of the basal ganglia. Since the functions of HPRT, a housekeeping enzyme responsible for recycling purines, have no direct relationships with the dopaminergic pathways, the mechanisms whereby HPRT deficiency affect them remain unknown. The current studies demonstrate that HPRT deficiency influences early developmental processes controlling the dopaminergic phenotype, using several different cell models for HPRT deficiency. Microarray methods and quantitative PCR were applied to 10 different HPRT-deficient (HPRT(-)) sublines derived from the MN9D cell line. Despite the variation inherent in such mutant sublines, several consistent abnormalities were evident. Most notable were increases in the mRNAs for engrailed 1 and 2, transcription factors known to play a key role in the specification and survival of dopamine neurons. The increases in mRNAs were accompanied by increases in engrailed proteins, and restoration of HPRT reverted engrailed expression towards normal levels, demonstrating a functional relationship between HPRT and engrailed. The functional relevance of the abnormal developmental molecular signature of the HPRT(-) MN9D cells was evident in impoverished neurite outgrowth when the cells were forced to differentiate chemically. To verify that these abnormalities were not idiosyncratic to the MN9D line, HPRT(-) sublines from the SK-N-BE(2) M17 human neuroblastoma line were evaluated and an increased expression of engrailed mRNAs was also seen. Over-expression of engrailed occurred even in primary fibroblasts from patients with LND in a manner that suggested a correlation with disease severity. These results provide novel evidence that HPRT deficiency may affect dopaminergic neurons by influencing early developmental mechanisms.

Apoptotic and necrotic effects of hexanedione derivatives on the human neuroblastoma line SK-N-SH

The potential cytotoxicity of two hexanedione food additives (2,3 and 3,4 isomers) was evaluated in comparison with the neurotoxic hexane metabolite 2,5-hexanedione in the human SK-N-SH neuroblastoma line using the MTT assay to indicate mitochondrial dehydrogenase activity and flow cytometry to monitor the cell cycle over 48 h. The IC 50s of the 2,3-hexanedione (3.3 ± 0.1 mM) and 3,4-hexanedione (3.5 ± 0.1 mM), indicated that the sensitivity of the cells was approximately seven-fold greater to these toxins compared with the 2,5 derivative (IC 50 of 22.4 ± 0.2 mM). Comparison between the respective IC 50s of the 2,3-hexanedione and 3,4-hexanedione revealed no difference between the two isomers in terms of their effects on MTT turnover. With flow cytometry analysis, all three hexanediones showed increases in apoptosis within their respective concentration ranges of toxicity shown previously by MTT. In the presence of 2,5-hexanedione, between 8.5 and 17 mM concentrations, there was a significant increase in apoptotic nucleoids which was accompanied by a significant fall in the percentage of nucleoids in the G0/G1 phase (72.4 ± 0.3–45.3 ± 0.6%,), and a rise in the numbers of cells in the G2/M phase. This is likely to indicate growth arrest at cell cycle G2/M checkpoint in response to toxin damage. G2/M accumulation was also shown with 3,4 and 2,3 HD, which was maximal at much lower concentrations (approximately 4 and 3 mM, respectively). Arrest at G1 and G2/M phase is indicative of inhibition of the cell cycle at the stages of DNA replication and chromosome segregation, respectively. It was also apparent that flow cytometry, rather than the MTT assay, did distinguish between the effects of the α-diketones 2,3-hexanedione and 3,4-hexanedione on the cell cycle. At a concentration of 5.8 mM 3,4-hexanedione, the percentage of apoptotic nucleoids was 10.9 ± 0.8% whilst apoptosis induced by 3,4-hexanedione had already reached a maximal level of 60.4 ± 0.5%. In summary, flow cytometry indicated that the 3,4-hexanedione derivative was more toxic than its 2,3 isomer and that both food additives caused interruption in the neuroblastoma cell cycle and further investigation may be required to assess if these α-diketones present in diets pose any possible risks to human health.

Herpes Simplex Virus Type 1 Enters Human Epidermal Keratinocytes, but Not Neurons, via a pH-Dependent Endocytic Pathway

Herpes simplex virus (HSV) enters some laboratory cell lines via a pH-dependent, endocytic mechanism. We investigated whether this entry pathway is used in human cell types relevant to pathogenesis. Three different classes of lysosomotropic agents, which raise endosomal pH, blocked HSV entry into primary and transformed human keratinocytes, but not into human neurons or neuroblastoma lines. In keratinocytes, incoming HSV particles colocalized with markers of endocytic uptake. Treatment with the isoflavone genistein, an inhibitor of protein tyrosine kinases, reduced the delivery of incoming viral particles to the nuclear periphery and virus-induced gene expression in keratinocytes but not neurons. Moreover, in keratinocyte monolayer islets, HSV infected both the inner and outer cells in a genistein-sensitive manner, suggesting viral endocytosis from both basolateral and apical plasma membrane surfaces. Together, the results indicate that HSV enters human epidermal keratinocytes, but not neurons, by a low-pH, endocytic pathway that is dependent on host tyrosine phosphorylation. Thus, HSV utilizes fundamentally different cellular entry pathways to infect important target cell populations.

The neurogene BTG2TIS21/PC3 is transactivated by ΔNp73α via p53 specifically in neuroblastoma cells

The p53 gene and its homologue p73 are rarely mutated in neuroblastoma. In recent studies, we showed that overexpression of DeltaNp73alpha, an isoform lacking the N-terminal transactivation (TA) domain, surprisingly induces p53 protein accumulation in the wild-type (wt) p53 human neuroblastoma line SH-SY5Y. As can be expected owing to its dominant-negative effect, DeltaNp73alpha inhibits Waf1/p21 gene expression, but equally importantly, it upregulates BTG2TIS21/PC3, another p53 target gene. This effect is not observed in neuroblastoma cells that express a mutated p53. To better understand the DeltaNp73-mediated transactivation of the BTG2TIS21/PC3 gene we performed luciferase assays with two reporter plasmids harboring long and short BTG2 promoter sequences in three human neuroblastoma cell lines and one breast cancer cell line. Our results demonstrate that BTG2TIS21/PC3 transactivation by DeltaNp73alpha depends on both p53 status (as it is not observed in a p53-/- neuroblastoma cell line) and cellular context (as it occurs in a p53+/+ neuroblastoma cell line but not in a p53+/+ breast tumor cell line). The fact that DeltaNp73alpha may either inhibit or stimulate wt-p53 transcriptional activity, depending on both the p53 target gene and the cellular context, was confirmed by real-time quantitative PCR. Moreover, transactivation of the BTG2TIS21/PC3 promoter requires a complete DeltaNp73alpha C-terminus sequence as it is not observed with DeltaNp73beta, which lacks most of the C-terminal domain. We have previously shown that DeltaNp73alpha is the only p73 isoform expressed in undifferentiated neuroblastoma tumors. In light of all these findings, we propose that DeltaNp73alpha not only acts as an inhibitor of p53/TAp73 functions in neuroblastoma tumors, but also cooperates with wt-p53 in playing a physiological role through the activation of BTG2TIS21/PC3 gene expression.

Guanine nucleotide depletion induces differentiation and aberrant neurite outgrowth in human dopaminergic neuroblastoma lines: a model for basal ganglia dysfunction in Lesch–Nyhan disease

Lesch–Nyhan disease (LND), caused by complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT), is characterized by a neurological deficit, the etiology of which is unknown. Evidence has accumulated indicating that it might be related to dysfunction of the basal ganglia with a prominent loss of striatal dopamine fibers. Guanine nucleotide depletion has been shown to occur in cells from Lesch–Nyhan patients. In this study we demonstrate that chronic guanine nucleotide depletion induced by inhibition of inosine monophosphate dehydrogenase with low levels (50 nM) of mycophenolic acid (MPA) lead human neuroblastoma cell lines to differentiate toward the neuronal phenotype. The MPA-induced morphological changes were more evident in the dopaminergic line LAN5, than in the cholinergic line IMR32. MPA-induced differentiation, unlike that induced by retinoic acid, caused a less extensive neurite outgrowth and branching (similar to that observed in cultured HPRT-deficient dopaminergic neurons) and involved up-regulation of p53, p21 and bax, and bcl-2 down-regulation without p27 protein accumulation. These results suggest that guanine nucleotide depletion following HPRT deficiency, might lead to earlier and abnormal brain development mainly affecting the basal ganglia, displaying the highest HPRT activity, and could be responsible for the specific neurobehavioral features of LND.

Aβ production as consequence of cellular death of a human neuroblastoma overexpressing APP

In human brain the Aβ peptide is produced mainly by neurons and the overexpression of amyloid precursor protein (APP) that involves an increase in Aβ secretion, has been observed in some areas of the Alzheimer's disease patients brain. We have generated two stably transfected human neuroblastoma lines which overexpress APP; both of them secreted Aβ and showed morphological changes and cell death with apoptotic program characteristics. Interestingly, coculture experiments with the untransfected human neuroblastoma cell line showed that the Aβ peptide was not responsible for the death in those cell lines; additionally, we indicate that upon cell death, Aβ peptide is secreted into cell medium.

Homozygous deletion and expression of PTEN and DMBT1 in human primary neuroblastoma and cell lines.

Neuroblastoma is the most common pediatric solid tumor. Although many allelic imbalances have been described, a bona fide tumor suppressor gene for this disease has not been found yet. In our study, we analyzed 2 genes, PTEN and DMBT1, mapping 10q23.31 and 10q25.3-26.1, respectively, which have been found frequently altered in other kinds of neoplasms. We screened both genes for homozygous deletions in 45 primary neuroblastic tumors and 12 neuroblastoma cell lines. Expression of these genes in cell lines was assessed by RT-PCR analysis. We could detect 2 of 41 (5%) primary tumors harboring PTEN homozygous deletions. Three of 41 (7%) primary tumors and 2 of 12 cell lines presented homozygous losses at the g14 STS on the DMBT1 locus. All cell lines analyzed expressed PTEN, but lack of DMBT1 mRNA expression was detected in 2 of them. We tried to see whether epigenetic mechanisms, such as aberrant promoter hypermethylation, had any role in DMBT1 silencing. The 2 cell lines lacking DMBT1 expression were treated with 5-aza-2'-deoxycytidine; DMBT1 expression was restored in only one of them (MC-IXC). From our work, we can conclude that PTEN and DMBT1 seem to contribute to the development of a small fraction of neuroblastomas, and that promoter hypermethylation might have a role in DMBT1 gene silencing.

Functional coupling between heterologously expressed dopamine D(2) receptors and KCNQ channels.

Activation of KCNQ potassium channels by stimulation of co-expressed dopamine D(2) receptors was studied electrophysiologically in Xenopus laevis oocytes and in mammalian cells. To address the specificity of the interaction between D(2)-like receptors and KCNQ channels, combinations of KCNQ1-5 channels and D(2)-like receptors (D(2L), D(3), and D(4)) were investigated in Xenopus oocytes. Activation of either receptor with the selective D(2)-like receptor agonist quinpirole (100 nM) stimulated all the KCNQ currents, independently of the subunit combination, indicating a common pathway of receptor-channel interaction. The KCNQ4 current was investigated in further detail and was increased by 19.9+/-1.6% ( n=20) by D(2L) receptor stimulation. The effect could be mimicked by injection of GTPgammaS and prevented by injection of Bordetella pertussis toxin, indicating that channel stimulation was mediated via a G protein of the G(alphai/o) subtype. Cells of the human neuroblastoma line SH-SY5Y were co-transfected transiently with KCNQ4 and D(2L) receptors. Stimulation of D(2L) receptors increased the KCNQ4 current ( n=6) as determined in whole-cell patch-clamp recordings. The specificity of the dopaminergic activation of the KCNQ channels was confirmed by co-expression of other neuronal K(+) channels (BK, K(V)1.1, and K(V)4.3) with the D(2L) receptor in Xenopus oocytes. None of these K(+) channels responded to stimulation of the D(2L) receptor. In the mammalian brain, dopamine D(2) receptors and KCNQ channels co-localise postsynaptically in several brain regions, so modulation of neuronal excitability by dopamine release could in part be mediated via an effect on KCNQ channels.

BDNF dependence in neuroblastoma

Neuroblastomas are heterogeneous tumors arising from sympathetic precursors in the neural crest. Growth factor stimulation of neuroblastomas promote diverse biological responses (mitogenesis, differentiation, cell death) depending on the particular tumor studied. Here we show that brief treatment with retinoic acid (RA) rendered the human neuroblastoma lines SY5Y, NGP, SMS-KCNR, and SK-N-SH dependent on brain-derived neurotrophic factor (BDNF) for survival. The BDNF- and trkB-expressing line SMS-KCN was dependent on an autocrine BDNF/trkB survival without exposure to RA. We conclude that the BDNF/trkB pathway plays an important role in neuroblastoma survival and speculate on a possible role in tumor pathogenesis.

Induction of major histocompatibility complex class I molecules on human neuroblastoma line cells by a flavoid antioxidant

Human major histocompatibility complex (MHC) class I expression is usually suppressed in neuronal cells and neuroblastoma cells. In the present study, we analyzed the effect of a flavonoid antioxidant, silymarin, on the induction of MHC class I molecules in human neuroblastoma line cells. Treatment of neuroblastoma cells with silymarin resulted in the expression of MHC class I molecules. Silymarin treatment enhanced the transcriptional activity of the reporter construct containing MHC class I promoter truncated within −428 bp of transcription initiation, but not the construct containing the promoter truncated within −284 bp. Because an E-box element is located between −428 and −285 bp of the transcription initiation, results suggest that silymarin acts on the enhancer activity of the E-box in the MHC class I promoter. Our findings indicate that silymarin induces the transcriptional factors to enhance the MHC class I promoter through the class I E-box element.