Normal Neuronal Cells Research Articles

Increased Dosage of Dyrk1A Alters Alternative Splicing Factor (ASF)-regulated Alternative Splicing of Tau in Down Syndrome

Two groups of tau, 3R- and 4R-tau, are generated by alternative splicing of tau exon 10. Normal adult human brain expresses equal levels of them. Disruption of the physiological balance is a common feature of several tauopathies. Very early in their life, individuals with Down syndrome (DS) develop Alzheimer-type tau pathology, the molecular basis for which is not fully understood. Here, we demonstrate that Dyrk1A, a kinase encoded by a gene in the DS critical region, phosphorylates alternative splicing factor (ASF) at Ser-227, Ser-234, and Ser-238, driving it into nuclear speckles and preventing it from facilitating tau exon 10 inclusion. The increased dosage of Dyrk1A in DS brain due to trisomy of chromosome 21 correlates to an increase in 3R-tau level, which on abnormal hyperphosphorylation and aggregation of tau results in neurofibrillary degeneration. Imbalance of 3R- and 4R-tau in DS brain by Dyrk1A-induced dysregulation of alternative splicing factor-mediated alternative splicing of tau exon 10 represents a novel mechanism of neurofibrillary degeneration and may help explain early onset tauopathy in individuals with DS.

Clinical and Cellular Manifestations of OSTM1-Related Infantile Osteopetrosis

Infantile ARO is a genetic disorder characterized by osteoclast dysfunction that leads to osteopetrosis. We describe a novel mutation affecting the OSTM1 locus responsible for ARO. In addition to common clinical features of osteopetrosis, the patient developed a unique neuronal pathology that provided evidence for an essential role of OSTM1 in normal neuronal cell development. Infantile autosomal recessive osteopetrosis (ARO) is a genetic disorder characterized by osteoclast dysfunction that leads to osteopetrosis. We describe a novel mutation affecting the OSTM1 locus responsible for ARO. In addition to common clinical features of osteopetrosis, the patient developed a unique neuronal pathology that provided evidence for an essential role of OSTM1 in normal neuronal cell development. We report a new case of ARO caused by an homozygous mutation in OSTM1. In addition to osteopetrosis and bone marrow failure, this patient also had neurological impairment not related to bone entrapment. Retinal dystrophy with absent evoked visual potentials and sensorineural deafness were documented, as well as cerebral atrophy and bilateral atrial subependymal heterotopias. The patient developed generalized seizures and had a profound developmental delay. Nerve biopsy failed to show inclusion material suggestive of neuroaxonal dystrophy. Bone marrow transplantation was declined considering the severe neurological compromise. The patient died at 1 yr of age. Osteoclasts derived from peripheral blood were mature and multinucleated. Expression analysis showed that the amount of OSTM1 cDNA transcript was significantly lowered but not absent. These results support the role of OSTM1 in osteoclast function and activation. However, they also suggest that OSTM1 has a primary role in neural development not related to lysosomal dysfunction.

Functional Domains of the BACE1and BACE2 Promoters and Mechanisms of Transcriptional Suppression of the BACE2 Promoter in Normal Neuronal Cells

The beta-amyloid (Abeta) protein present in the neuritic plaques of Alzheimer's disease is cleaved from Abeta precursor protein (APP) by beta- and gamma-secretases. Following identification of beta-APP cleaving enzyme (BACE1) as the beta-secretase, a homologous beta-secretase 2 (BACE2) was described. Our goal is to characterize the regulatory region of the BACE genes. We compare functional domains within the BACE1 and BACE2 regulatory regions. Both BACE genes lack canonical TATAand CAAT boxes, but they contain distinguishing transcription start sites and transcription factor-binding sites. The BACE1 sequence contains more repetitive elements than does BACE2 (no elements). Regulatory domains do not overlap strongly between the two promoter regions. The BACE1upstream sequence contains both negative and positive domains, separated from the transcription seat by a long neutral domain. The corresponding BACE2sequence consists of a weakly positive domain directly upstream of a strongly positive domain, near a functionally active domain. DNA-protein interaction was corroborated by functional data. In primary rat cortical cultures, BACE1-driven reporter protein's expression was twice that of BACE2- driven reporter. The BACE2 gene promoter relatively reduced function in neuronal cells compared with BACE1. The BACE1 gene might operate through a single transcriptional control site. BACE2 operates through dual transcriptional control sites. Two (or more) regulatory pathways might control transcription in BACE2. Thus, BACE2 is partially suppressed in normal neuronal cells and likely to be a highly regulated gene expressed in a particularly tissue-specific fashion.

Wild-type PINK1 Prevents Basal and Induced Neuronal Apoptosis, a Protective Effect Abrogated by Parkinson Disease-related Mutations

Mutations in the PTEN-induced kinase 1 (PINK1) gene have recently been implicated in autosomal recessive early onset Parkinson Disease (1, 2). To investigate the role of PINK1 in neurodegeneration, we designed human and murine neuronal cell lines expressing either wild-type PINK1 or PINK1 bearing a mutation associated with Parkinson Disease. We show that under basal and staurosporine-induced conditions, the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive cells was lower in wild-type PINK1 expressing SH-SY5Y cells than in mock-transfected cells. This phenotype was due to a PINK1-mediated reduction in cytochrome c release from mitochondria, which prevents subsequent caspase-3 activation. We show that overexpression of wild-type PINK1 strongly reduced both basal and staurosporine-induced caspase 3 activity. Overexpression of wild-type PINK1 also reduced the levels of cleaved caspase-9, caspase-3, caspase-7, and activated poly(ADP-ribose) polymerase under both basal and staurosporine-induced conditions. In contrast, Parkinson disease-related mutations and a kinase-inactive mutation in PINK1 abrogated the protective effect of PINK1. Together, these results suggest that PINK1 reduces the basal neuronal pro-apoptotic activity and protects neurons from staurosporine-induced apoptosis. Loss of this protective function may therefore underlie the degeneration of nigral dopaminergic neurons in patients with PINK1 mutations.

Biological effects of TrkA and TrkB receptor signaling in neuroblastoma

The Trk family consists of three receptor tyrosine kinases, each of which can be activated by one or more of four neurotrophins—NGF, BDNF, NT3 and NT4. Neurotrophins mediate their multiple effects through a number of distinct intracellular signaling cascades regulating such diverse biological responses as cell survival, proliferation and differentiation in normal and neoplastic neuronal cells. Expression of Trk receptors also plays an important role in the biology and clinical behavior of neuroblastomas. High expression of TrkA is present in neuroblastomas with favorable biological features and highly correlated with patient survival, whereas TrkB is mainly expressed on unfavorable, aggressive neuroblastomas. This short review discusses recent data on the biological roles of TrkA and TrkB signaling in neuroblastoma.

Ryanodine receptor-mediated interference of neuronal cell differentiation by presenilin 2 mutation

Neuronal cell differentiation alterations induced by mutant presenilin 2 (PS2) were investigated in transgenic mice expressing wild-type or mutant-type PS2. Progressive increases in differentiation and marker protein expression were found in neuronal cells expressing wild-type PS2, whereas these processes were much perturbed in mutant-type PS2 with elevated ryanodine-receptor (RyR) expression and intracellular calcium levels. Moreover, dantrolene, a blocker of RyR reduced the PS2 mutation-induced interference of cell differentiation and calcium release, but caffeine, an activator of RyR, exacerbated PS2 mutation-induced interference with cell differentiation. Our results indicate that mutant PS2 inhibits normal neuronal cell differentiation and that RyR-mediated calcium overrelease may be a significant factor.

In vitro uptake of a new cholesteryl carborane ester compound by human glioma cell lines

The cellular uptake and retention of a new cholesteryl carborane ester compound, cholesteryl 1,12‐dicarba‐closo‐dodecaboranel‐carboxylate (BCH), by two human glioma cell lines, glioblastoma multiforme SF‐763 and SF‐767, was evaluated. BCH, which is an extremely hydrophobic compound, was formulated into liposomes and incubated with two human glioma tumor cell lines and one human normal neuron cell line. The amount of BCH uptake by the cells was measured by high performance liquid chromatography. The effects of BCH concentration in the culture medium and the incubation time on the cellular uptake of BCH were studied. In addition, BCH uptake by tumor cells was examined in the presence and absence of lipoprotein in the culture medium. It was found that the amount of BCH taken by the glioma cell lines was much more (up to 14 times) than that by the normal neuron cell line. The cellular uptake of BCH was related to the amount of BCH in the medium as well as the incubation time. The cellular uptake of BCH by SF‐763 and SF‐767 cells after 16h of incubation was 283.3 ± 38.9 and 264.0 ± 36.5 μg boron/g cells, respectively. The majority of BCH taken up in tumor cells was retained after the subsequent incubation. In the presence of lipoprotein, the cellular uptake of BCH by SF‐767 tumor cells was about four times as much as that in the absence of lipoprotein. In conclusion, the cellular uptake of BCH by glioma cells was about 14 times higher than by normal neuron cells. The uptake in glioma cells was up to 10 times higher than that required for successful cancer treatment and BCH was well retained in the tumor cells. Lipoprotein seemed to have an important role in the BCH uptake by glioma cells. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:13–19, 2004

Neuroprotection by selective nitric oxide synthase inhibition at 24 hours after perinatal hypoxia-ischemia.

Perinatal hypoxia-ischemia is a major cause of neonatal morbidity and mortality. Until now no established neuroprotective intervention after perinatal hypoxia-ischemia has been available. The delay in cell death after perinatal hypoxia-ischemia creates possibilities for therapeutic intervention after the initial insult. Excessive nitric oxide and reactive oxygen species generated on hypoxia-ischemia and reperfusion play a key role in the neurotoxic cascade. The present study examines the neuroprotective properties of neuronal and inducible but not endothelial nitric oxide synthase inhibition by 2-iminobiotin in a piglet model of perinatal hypoxia-ischemia. Twenty-three newborn piglets were subjected to 60 minutes of hypoxia-ischemia, followed by 24 hours of reperfusion and reoxygenation. Five additional piglets served as sham-operated controls. On reperfusion, piglets were randomly treated with either vehicle (n=12) or 2-iminobiotin (n=11). At 24 hours after hypoxia-ischemia, the cerebral energy state, presence of vasogenic edema, amount of apparently normal neuronal cells, caspase-3 activity, amount of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL)-positive cells, and degree of tyrosine nitration were assessed. A 90% improvement in cerebral energy state, 90% reduction in vasogenic edema, and 60% to 80% reduction in apoptosis-related neuronal cell death were demonstrated in 2-iminobiotin-treated piglets at 24 hours after hypoxia- ischemia. A significant reduction in tyrosine nitration in the cerebral cortex was observed in 2-iminobiotin-treated piglets, indicating decreased formation of reactive nitrogen species. Simultaneous and selective inhibition of neuronal and inducible nitric oxide synthase by 2-iminobiotin is a promising strategy for neuroprotection after perinatal hypoxia-ischemia.

The integrin alpha(v)beta8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-beta1.

Întegrins, matrix metalloproteases (MMPs), and the cytokine TGF-β have each been implicated in homeostatic cell behaviors such as cell growth and matrix remodeling. TGF-β exists mainly in a latent state, and a major point of homeostatic control is the activation of TGF-β. Because the latent domain of TGF-β1 possesses an integrin binding motif (RGD), integrins have the potential to sequester latent TGF-β (SLC) to the cell surface where TGF-β activation could be locally controlled. Here, we show that SLC binds to αvβ8, an integrin expressed by normal epithelial and neuronal cells in vivo. This binding results in the membrane type 1 (MT1)-MMP–dependent release of active TGF-β, which leads to autocrine and paracrine effects on cell growth and matrix production. These data elucidate a novel mechanism of cellular homeostasis achieved through the coordination of the activities of members of three major gene families involved in cell–matrix interactions.

Gene expression profiling of exposure to TZT-1027, a novel microtubule-interfering agent, in non-small cell lung cancer PC-14 cells and astrocytes.

Clinical use of TZT-1027, a microtubule-interfering agent that inhibits the polymerization of tubulin, is expected because of its potent effects on solid tumors. TZT-1027 is thought to act directly on cellular microtubules, and arrest cell mitosis, however, the molecular mechanisms of the microtubule damage by TZT-1027 have not been fully identified. To investigate the possible novel mechanisms of action of TZT-1027, we used the cDNA macroarray technique to examine its effect on the expression of hundreds of tightly transcriptionally controlled genes. We used two cell lines, one was human non-small cell lung carcinoma PC-14 cells as a model for cancer cells, and the other was human astrocytes as a model for normal neuronal cells, because the dose-limiting-factor of microtubule-interfering agents is mainly peripheral neurotoxicity. mRNAs prepared from the PC-14 and astrocyte cell lines treated with TZT-1027 were compared with 588 genes spotted onto the filter, and which gene groups TZT-1027 modulated between the two cell lines was investigated. TZT-1027 exposure modulated expression of a variety of genes including the genes encoding cell-cycle and growth regulators, receptors, angiogenesis and invasion regulators, rho family small GTPases and their regulators and growth factors and cytokines. However, the way of gene regulation by TZT-1027 exposure was different between PC-14 cells and astrocytes. Genes up-regulated in both PC-14 cells and astrocytes were those for RAR-epsilon, TNFR 1 and 2 and so on. Specifically altered genes in PC-14 cells, such as the genes coding for cytokeratin 8, XPG, fau and the genes regulated only in PC-14 cells may be involved in the antitumor activity of TZT-1027. On the other hand, growth factor receptor precursors was upregulated specifically in astrocytes by TZT-1027 and this gene regulation only in astrocytes may be candidates related with neurotoxicity.

Effect of 0.2 T static magnetic field on human neurons: remodeling and inhibition of signal transduction without genome instability

We describe the effect of the static magnetic field generated by a 0.2 T magnetic resonance tomograph on a normal human neuronal cell culture (FNC-B4). After 15 min exposure cells showed dramatic changes of morphology: they formed vortexes of cells and exposed branched neurites featuring synaptic buttons. At the same time, thymidine incorporation and inositol lipid signaling were significantly reduced. Control (sham exposed) or non-neuronal cells (mouse leukemia, and human breast carcinoma cells) did not show any alteration following exposure. Endothelin-1 release from FNC-B4 cells was also dramatically reduced after 5 min exposure. However, PCR analysis of 12 DNA microsatellites selected as gauges of genome instability, did not reveal any alteration following exposure, thus ruling out a direct effect of the magnetic field on DNA stability. These data can be interpreted as a specific effect of the static magnetic field on human neuronal cells and are consistent with the induction of remodeling and differentiation; they demonstrate that fields below 0.5 T have significant biological effects on human neurons.

The effect of rapid preconditioning on the microglial, astrocytic and neuronal consequences of global cerebral ischemia.

Previous studies indicated preconditioning of the brain with sublethal ischemic insults separated by many hours, protected tissues from a subsequent lethal insult. We recently reported neuroprotection by a rapid preconditioning paradigm where a sublethal ischemic insult preceded test ischemia by only 30 min. We hypothesize that neuroprotection caused by the rapid ischemic preconditioning (IPC) will result in lowered microglial, reactive astrocytes and increased normal neuronal cell counts. Wistar rats underwent normothermic (36.5-37 degrees C) global cerebral ischemia, produced by bilateral carotid artery ligation after lowering mean systemic blood pressure. The preconditioning ischemic insult lasted 2 min and was associated with a sufficient amount of time to provoke anoxic depolarization. After a 30-min reperfusion period, 10-min test ischemia was produced, and histopathology was assessed 3 and 7 days later. Normal neuronal cell counts for control rats at 3 days survival were significantly lower (by 58%) than in IPC animals. Although there was a trend toward protection in IPC rats at 7 days, the difference in normal neuronal cell count between the IPC and control groups was not significant. IPC rats at 3 days but not 7 days of survival showed a significantly lower microglial cell count (by 56%) than control rats. These results showed that the protection induced through IPC at 3 days of survival produced lower numbers of microglia, while maintaining normal neuronal cells. No significant differences between control and IPC groups were found in astrocytic cell count at any time of reperfusion in any region of the hippocampus studied. The beneficial effects of IPC may, therefore, involve anti-inflammatory processes that target microglial activation after cerebral ischemia.

Inhibition of p75 Tumor Necrosis Factor Receptor by Antisense Oligonucleotides Increases Hypoxic Injury and β-Amyloid Toxicity in Human Neuronal Cell Line

Recent evidence indicates that tumor necrosis factor-alpha (TNF-alpha) is up-regulated following brain injury and in neurodegenerative disorders such as stroke, multiple sclerosis, Parkinson's disease, and Alzheimer's disease. TNF-alpha elicits its biological effects through two distinct TNF receptor (TNFR) subtypes: p55 TNFR (TNFR1) and p75 TNFR (TNFR2). Studies have demonstrated that the p55 TNFR contributes to cell death, whereas the role of the p75 TNFR in neuronal viability is unclear. To better understand the role of p75 TNFR, we treated human neuronal SH-SY5Y cells with phosphorothioate-modified antisense oligonucleotides (ASO) for p75 TNFR and established that ASO inhibited p75 TNFR expression. Treatment of SH-SY5Y cells with ASO alone did not affect cell viability, whereas treatment with both ASO and human TNF-alpha significantly increased cell death relative to treatment with TNF-alpha alone. Moreover, addition of ASO significantly increased the level of cell injury observed following hypoxic conditions or exposure of beta-amyloid peptide. These results indicate that inhibition of p75 TNFR using ASO increases the vulnerability of neurotypic cells to insults and suggest that the p75 TNFR may not be required for normal neuronal cell viability but rather plays a protective role following injury.

Experimental brain glioma: growth arrest and destruction by a blood-group-related tetrasaccharide.

A synthetic tetrasaccharide (TS4), structurally related to blood groups, inhibited the proliferation of the C6 glioma cells in culture and the growth of tumors formed after intracerebral transplantation of C6 cells. TS4-treated tumors were substantially smaller than controls, as expected from TS4 cytostatic action on C6 glioma cells in culture. However, in vivo treatment also caused extensive tumor destruction. This effect appeared to be caused by indirectly, either by activation of natural killer cells, cytotoxic lymphocytes, or by inhibition of tumor vascularization. Enhanced antigenicity of TS4-treated glioma may be related to the increased expression of connexin 43 observed in glioma cell cultures treated with the oligosaccharide. Because concentrations of up to 20 mg/ml of TS4 were not toxic for normal neuronal or glial cells, specific oligosaccharides such as TS4 offer the possibility of selective tumor treatment.

Structural changes of anterior horn neurons and their synaptic input caudal to a low thoracic spinal cord hemisection in the adult rat: a light and electron microscopic study.

Structural changes in lumbosacral ventral horn neurons and their synaptic input were studied at 3, 10, 21, 42, and 90 days following low thoracic cord hemisection in adult rats by light microscopic examination of synaptophysin immunoreactivity (SYN-IR) and by electron microscopy. There was an ipsilateral transient decrease in SYN-IR at the somal and proximal dendritic surfaces of anterior horn neurons which extended caudally from the site of injury over a postoperative (p.o.) period of 42 days. Concomitantly, at 21 days p.o., perineuronal SYN-IR started to recover in upper lumbar segments. By 90 days p.o., a normal staining pattern of SYN was noted in upper and mid lumbar segments, but the perineuronal SYN-IR was still slightly below normal levels in low lumbar and sacral segments. Electron microscopy revealed ultrastructural changes coincident with the alterations in SYN-IR. At 3 days p.o., phagocytosis of degenerating axon terminals by activated microglial cells was observed at the somal and proximal dendritic surfaces of ventral horn neurons. These changes were most prominent up to two segments caudal to the lesion. At 10 days p.o., advanced stages of bouton phagocytosis were still detectable in all lumbosacral motor nuclei. Additionally, abnormal axon terminals, with a few dispersed synaptic vesicles and accumulations of large mitochondria, appeared at the scalloped somal surfaces of anterior horn neurons. At 21 days p.o., several large lumbosacral motoneurons had developed chromatolysis-like ultrastructural alterations and motoneuronal cell bodies had become partially covered by astrocytic lamellae. At 42 days p.o., there was a transient appearance of polyribosomes in some M-type boutons. In addition, at 42 and 90 days p.o., a few degenerating motoneurons were detected in all lumbosacral segments, but most displayed normal neuronal cell bodies contacted by numerous intact synapses as well as by astrocytic processes. In contrast to these striking alterations of synaptic input at somal and proximal dendritic surfaces of motoneurons, relatively few degenerating boutons were detected in the neuropil of motor nuclei at all the p.o. times studied. We suggest that the preferential disturbance of the predominantly inhibitory axosomatic synapses on ventral horn neurons may be involved in the mechanisms which influence the well-established increase in motoneuronal excitability after spinal cord injury.

Maturation of cerebellar granule cells is delayed in cultures derived from ethanol-treated chick embryos: Survival and proliferation studies

Previous studies from this laboratory have shown that ethanol administration to chick embryos during embryonic days 1–3, a critical period of neuroembryogenesis, differentially affects primordial CNS structures. In this study, chick embryos were treated in ovo with ethanol (10 mg/50 μl/day) at E1 to E3. At 14 days of embryonic age cerebellar (E14CE) granule cell cultures were prepared from both control and ethanol-treated embryos. Growth patterns were evaluated morphologically and the neuronal nature of these cultures was evaluated immunocytochemically. E14CE granule cell cultures exhibited neurofilament immunoreactivity demonstrating the neuronal-nature of these cultures. In addition E14CE granule cultures contained numerous glutamateric neurons as assessed by positive glutamate immunoreactivity and also some GABAergic neurons as assessed by positive GABA immunoreactivity. Cultures derived from both control and ethanol-treated embryos were labeled with 3H-thymidine and assessed for effects on survival and proliferation in culture. Cultures derived from ethanol-treated embryos showed a higher rate of proliferation and survival during the first 3 days in culture as compared to those derived from controls. However, after 3 days in culture, survival was lower in the cultures from ethanol-treated embryos as compared to those derived from controls. We interpret these findings to mean that (a) ethanol arrested cerebellar granule cell development at an immature state; (b) immature neurons have a higher survival capacity than differentiated neurons; and (c) ethanol accelerates normal neuronal cell death as previously reported.

The granin-(chromogranin/secretogranin) family

The chromogranins/secretogranins, referred to in abbreviated form as granins, are a family of acidic secretory proteins that are found in the secretory granules of a wide variety of endocrine cells and neurons, being stored together with many different peptide hormones and neuropeptides. The recent elucidation of their primary structure has provided insights into possible functions of these proteins. Moreover, the granins have been successfully used as markers for normal and neoplastic endocrine and neuronal cells, as well as model proteins to understand the sorting mechanism involved in the formation of secretory granules.

Introduction of macromolecules into primary cultured neuronal cells by fusion with erythrocyte ghosts

The macromolecules were injected into primary cultured neuronal cells by fusion with human erythrocyte ghosts containing macromolecules. The injection efficiency was about 30% and 80% of the cells extended neurites like normal neuronal cells. In order to succeedd in this method, phytohaemagglutinin E and polyethylene glycol 1000 were essential, and monolayer culture cells were used.

Normal neuronal cell bodies of the nucleus tractus mesencephalici nervi trigemini react with antibodies against phosphorylated epitopes on neurofilaments

Reactivity to antibodies directed against phosphorylated neurofilament epitopes is normally present in axons. Phosphorylated neurofilament immunoreactivity is not generally found in neuronal perikarya, except in abnormal states such as Alzheimer's disease. We found that cytoplasm of neurons of the nucleus tractus mesencephalici nervi trigemini in normal mice and rats reacts with monoclonal antibodies against phosphorylated epitopes on neurofilaments. This suggests either that phosphorylated epitopes on neurofilaments are localized in the perikarya of some normal neurons or that the antibodies that were used (SMI 31 and SMI 34) recognize more than phosphorylated epitopes.

L-Ascorbic acid-induced DNA strand breaks and cross links in human neuroblastoma cells

The effect of high concentrations of l-ascorbic acid on the in vivo and in vitro growth of human neuroblastoma has been investigated. Directly implemented into cell culture it decreased the DNA, RNA and protein synthesis, and mitosis of neuroblastoma cells, without affecting normal neuronal cells. In vivo treatment of young nude mice bearing human neuroblastoma with 500 mg/kg l-ascorbic acid for the first 10 days markedly inhibited the growth of tumor mass. As determined by alkaline elution, both DNA strand breaks and DNA cross links were observed in tumor cells treated with 1 × 10 −4 M l-ascorbic acid for 2 h. DNA-DNA and DNA-protein cross links in cells treated with l-ascorbic acid were revealed by the proteinase potassium assay. The results indicated that l-ascorbic acid can be a very effective and selective agent against human neuroblastoma.