Neural Cell Adhesion Molecule mRNA Research Articles

Hyperfunction of the stress response system and novelty-induced hyperactivity correlate with enhanced cocaine-induced conditioned place preference in NCAM-deficient mice.

Several studies in humans and rodents suggest an association between impulsivity and activity of the stress response on the one hand and addiction vulnerability on the other. The neural cell adhesion molecule (NCAM) has been related to several neuropsychiatric disorders in humans. Constitutively NCAM-deficient (-/-) mice display enhanced novelty-induced behavior and hyperfunction of the hypothalamic-pituitary-adrenal axis. Here we hypothesize that NCAM deficiency causes an altered response to cocaine. Cocaine-induced behaviors of NCAM-/- mice and wild-type (+/+) littermates were analyzed in the conditioned place preference (CPP) test. c-fos mRNA levels were investigated by quantitative polymerase chain reaction (qPCR) to measure neural activation after exposure to the cocaine-associated context. NCAM-/- mice showed an elevated cocaine-induced sensitization, enhanced CPP, impaired extinction, and potentiated cocaine-induced hyperlocomotion and CPP after extinction. NCAM-/- showed no potentiated CPP as compared with NCAM+/+ littermates when a natural rewarding stimulus (ie, an unfamiliar female) was used, suggesting that the behavioral alterations of NCAM-/- mice observed in the CPP test are specific to the effects of cocaine. Activation of the prefrontal cortex and nucleus accumbens induced by the cocaine-associated context was enhanced in NCAM-/- compared with NCAM+/+ mice. Finally, cocaine-induced behavior correlated positively with novelty-induced behavior and plasma corticosterone levels in NCAM-/- mice and negatively with NCAM mRNA levels in the hippocampus and nucleus accumbens in wild-type mice. Our findings indicate that NCAM deficiency affects cocaine-induced CPP in mice and support the view that hyperfunction of the stress response system and reactivity to novelty predict the behavioral responses to cocaine.

MiR-30a-5p is induced by Wnt/β-catenin pathway and promotes glioma cell invasion by repressing NCAM

Wnt/β-catenin signaling pathway is frequently dysregulated in human tumors and plays a critical role in tumorigenesis; however, the roles of microRNAs in mediating Wnt/β-catenin pathway are not well understood. Herein, we show that miR-30a-5p is activated by Wnt/β-catenin pathway through direct binding of β-catenin/TCF4 to two sites in the promoter region of miR-30a-5p. We also found that miR-30a-5p represses neural cell adhesion molecule (NCAM) expression by directly targeting two sites in the 3′ untranslated region (3′-UTR) of NCAM mRNA. Moreover, Wnt/β-catenin pathway represses NCAM expression in glioma cells, which depends on miR-30a-5p. Finally, we found that miR-30a-5p promotes glioma cell growth invasion by repressing NCAM. Our findings demonstrate a novel Wnt/β-catenin–miR-30a-5p–NCAM regulatory axis which plays important roles in controlling glioma cell invasion and tumorigenesis.

Neural cell adhesion molecule (cluster of differentiation 56) in health and disease

Cluster of differentiation (CD) 56, a member of the immunoglobulin superfamily, and an isoform of neural cell adhesion molecule (NCAM), was the first cell adhesion molecule to be identified. NCAM (CD56) plays an important role both in human health and in disease. Human NCAM gene is located on chromosome 11q23. CD56 antigen is a 175–185-kD cell surface glycoprotein expressed on all subsets of human natural killer (NK) cells except a small minority of CD56− NK-cell, on subsets of CD4+/CD8+ T-cells, interleukin-2-activated thymocytes, bone marrow macrophages, osteoclasts, and on adrenal gland and neural tissues. NCAM is important in calcium independent cell-cell interactions that mediate homotypic and heterotypic cell-cell and cell-matrix adhesions. At least 27 alternatively spliced NCAM mRNAs are produced giving a wide diversity to NCAM isoforms sharing a similar structural organization. NCAM in the cerebellum and cerebral cortex mediates homophilic adhesion of neural cells, and plays an important role in brain development, emotions, and memory functions. While CD56+ NK-cells play an important role in defense against infections, tumor remission, normal pregnancy and graft rejection. Malignancies expressing CD56 are usually aggressive, with more potential for metastasis and extramedullary/central nervous system involvement, and may respond to new CD56-linked targeted therapies.

Effects of Chronic Social Isolation on Wistar Rat Behavior and Brain Plasticity Markers

Chronic stress is a contributing risk factor in the development of psychiatric illnesses, including depressive disorders. The mechanisms of their psychopathology are multifaceted and include, besides others, alterations in the brain plasticity. Previously, we investigated the effects of chronic social stress in the limbic brain structures of Wistar rats (hippocampus, HIPPO, and prefrontal cortex, PFC) and found multiple characteristics that resembled alterations described in some clinical studies of depression. We extended our investigations and followed the behavior of stressed animals by the open field test (OFT) and forced swimming test (FST), and the expression and polysialylation of synaptic plasticity markers, neural cell adhesion molecule (NCAM) and L1, in the HIPPO and PFC. We also determined the adrenal gland mass and plasma corticosterone (CORT) as a terminal part of the hypothalamic-pituitary-adrenal axis activity. Our data indicated that stressed animals avoided the central zone in the OFT and displayed decreased swimming, but prolonged immobility in the FST. The animals exhibited marked hypertrophy of the adrenal gland cortex, in spite of decreased serum CORT. Simultaneously, the stressed animals exhibited an increase in NCAM mRNA expression in the HIPPO, but not in the PFC. The synaptosomal NCAM of the HIPPO was markedly polysialylated, while cortical PSA-NCAM was significantly decreased. The results showed that chronic social isolation of Wistar rats causes both anxiety-like and depression-like behavior. These alterations are parallel with molecular changes in the limbic brain, including diminished NCAM sialylation in the PFC. Together with our previous results, the current observations suggest that a chronic social isolation model may potentially be used to study molecular mechanisms that underlie depressive symptomatology.

Effect of maternal subclinical hypothyroidism during pregnancy on brain development in rat offspring

Objective To explore whether maternal subclinical hypothyroidism gives rise to poor performance in the offspring and whether this is associated with the expression of several genes that are under the control of thyroid hormones. Methods Sixty female rats were divided into three groups （ each group n = 20）： （ 1 ）maternal subclinical hypothyroidism （ total thyroidectomy with T4 infusion）, （2） maternal hypothyroidism （ total thyroidectomy without T4 infusion）, and （3） control （sham operated）. All rats were mated 10 days after the start of infusion. The infusion continued until 10 days postpartum. Pups were sacrificed at postnatal day 3, 7, and 21. The hippocampus was collected and tested for brain-derived neurotrophic factor （BDNF） and Rap1 protein expression by Western blotting and for BDNF and neural cell adhesion molecule （ NCAM ） mRNA expression by real-time polymerase chain reaction. On day 41-49, rat pups explored the Morris water maze. Time spent in the quadrant previously containing the plat form was recorded. Results The present study found decreases in BDNF mRNA （on day 3 ） and protein levels （on day 3 and 7 ） in hippocampi of pups from subclinical hypothyroidism dams （P〈0.05）. No change was observed in the levels of NCAM mRNA, whereas at day 21, expression of Rap1 protein was higher than that of control offspring. In addition, pups of subclinical hypothyroidism dams showed a trend toward depression in short-term memory （P〉0.05）, and long-term memory testing revealed a trend toward subclinical hypothyroidism group pups being less able to remember a fixed platform position than controls, spending less time in the proper quadrant （ P〈0. 05 ）. Conclusion The long-term memory deficits of pups born to maternal subclinical hypothyroidism dams are likely related with decrease in BDNF expression as well as increase in Rap1 expression in hippocampi. Key words: Subclinical hypothyroidism; Pregnancy; Brain development

GDNF stimulates the proliferation of cultured mouse immature Sertoli cells via its receptor subunit NCAM and ERK1/2 signaling pathway

BackgroundThe proliferation and final density of Sertoli cells in the testis are regulated by hormones and local factors. Glial cell line-derived neurotrophic factor (GDNF), a distantly related member of the transforming growth factor-β superfamily, and its receptor subunits GDNF family receptor alpha 1 (GFRα1), RET tyrosine kinase, and neural cell adhesion molecule (NCAM) have been reported to be expressed in the testis and involved in the regulation of proliferation of immature Sertoli cells (ISCs). However, the expression patterns of these receptor subunits and the downstream signaling pathways have not been addressed in ISCs.ResultsIn the present study, we have reported that the proliferation of cultured ISCs was significantly enhanced by GDNF. The receptor subunits GFRα1 and NCAM but not RET were expressed in ISCs, and the stimulatory effect of GDNF on the proliferation of ISCs was significantly reduced by anti-NCAM antibody blocking or siRNA that specifically targets NCAM mRNA. Additionally, the ERK1/2 inhibitor, PD98059, completely abolished the mitogenic effect of GDNF on ISCs.ConclusionsGDNF stimulates the proliferation of ISCs via its receptor subunit NCAM and the consequent activation of the ERK1/2 signaling pathway.

The Effect of Maternal Subclinical Hypothyroidism During Pregnancy on Brain Development in Rat Offspring

Maternal thyroid hormones (THs), especially thyroxine (T(4)), are crucial to early brain development in the mammalian embryo. Epidemiological studies and case reports have shown that maternal subclinical hypothyroidism may result in significant negative effects on pregnancy and neurodevelopment of the fetus. To understand the mechanism responsible for these neurological alterations, we induced maternal subclinical hypothyroidism in pregnant rats. Behavior and several genes that are under the control of THs were evaluated in the offspring of TH-deficient rats. A total of 60 female rats were divided into three groups: (i) maternal subclinical hypothyroidism (total thyroidectomy with T(4) infusion), (ii) maternal hypothyroidism (total thyroidectomy without T(4) infusion), and (iii) control (sham operated). All rats were mated 10 days after the start of infusion. The infusion continued until 10 days postpartum. Pups were sacrificed at postnatal day 3 (PND 3), PND 7, and PND 21. The hippocampus was collected and tested for brain-derived neurotrophic factor (BDNF) and Rap1 protein expression by Western blotting and for BDNF and neural cell adhesion molecule mRNA expression by real-time polymerase chain reaction. On PND 41-PND 49, rat pups explored the Morris water maze. Time spent in the quadrant previously containing the platform was recorded. This study found decreases in BDNF mRNA expression (on PND 3) and protein level (on PND 3 and PND 7) in hippocampi of pups from subclinical hypothyroidism dams (p < 0.05). Rap1 protein expression was higher in maternal subclinical hypothyroidism offspring than in control offspring at PND 7 and PND 21. No change was observed in neural cell adhesion molecule mRNA expression in the maternal subclinical hypothyroidism offspring. In addition, results from the Morris water maze revealed that pups from the subclinical hypothyroidism dams showed deficits in long-term memory, spending less time in the platform quadrant (p < 0.05) during testing. There was a trend toward a deficit in short-term memory (p > 0.05) in this group as well. The long-term memory deficits of pups born to maternal subclinical hypothyroidism dams likely related with decreasing in BDNF mRNA expression and protein level as well as increasing in Rap1 protein expression in hippocampi.

Role of the neural cell adhesion molecule (NCAM) in amygdalo-hippocampal interactions and salience determination of contextual fear memory

Evidence suggests that the neural cell adhesion molecule (NCAM) is an important molecular constituent of adaptive and maladaptive circuit (re-)organization in the central nervous system. Here, we further investigate its putative involvement in amygdala and hippocampus functions during context fear memory formation. Using laser capture microdissection and quantitative RT-PCR, we show high NCAM mRNA expression levels in the lateral and basolateral subnuclei of the amygdala, as well as their training intensity- and context-dependent regulation during fear memory consolidation. Moreover, we demonstrate that deficits of NCAM-/- mice in context fear memory can be overcome through contextual pre-exposure, i.e. by reducing the modulatory influence of the amygdala on this hippocampus-dependent memory. On the contrary, NCAM-/- mice failed to increase contextual fear memory after salient overtraining, although they adequately increased their response to auditory-cued fear stimuli. Finally, we demonstrate a reduction of amygdalo-hippocampal theta synchronization in NCAM-/- mice during fear memory retrieval. Together, these results suggest an involvement of NCAM-mediated cell recognition processes in information processing of the amygdalo-hippocampal system and in the amygdala-mediated modulation of context fear memory according to stimulus salience.

Developmental regulation of neural cell adhesion molecule in human prefrontal cortex

Neural cell adhesion molecule (NCAM) is a membrane-bound cell recognition molecule that exerts important functions in normal neurodevelopment including cell migration, neurite outgrowth, axon fasciculation, and synaptic plasticity. Alternative splicing of NCAM mRNA generates three main protein isoforms: NCAM-180, -140, and -120. Ectodomain shedding of NCAM isoforms can produce an extracellular 105–115 kilodalton soluble neural cell adhesion molecule fragment (NCAM-EC) and a smaller intracellular cytoplasmic fragment (NCAM-IC). NCAM also undergoes a unique post-translational modification in brain by the addition of polysialic acid (PSA)-NCAM. Interestingly, both PSA-NCAM and NCAM-EC have been implicated in the pathophysiology of schizophrenia. The developmental expression patterns of the main NCAM isoforms and PSA-NCAM have been described in rodent brain, but no studies have examined NCAM expression across human cortical development. Western blotting was used to quantify NCAM in human postmortem prefrontal cortex in 42 individuals ranging in age from mid-gestation to early adulthood. Each NCAM isoform (NCAM-180, -140, and -120), post-translational modification (PSA-NCAM) and cleavage fragment (NCAM-EC and NCAM-IC) demonstrated developmental regulation in frontal cortex. NCAM-180, -140, and -120, as well as PSA-NCAM, and NCAM-IC all showed strong developmental regulation during fetal and early postnatal ages, consistent with their identified roles in axon growth and plasticity. NCAM-EC demonstrated a more gradual increase from the early postnatal period to reach a plateau by early adolescence, potentially implicating involvement in later developmental processes. In summary, this study implicates the major NCAM isoforms, PSA-NCAM and proteolytically cleaved NCAM in pre- and postnatal development of the human prefrontal cortex. These data provide new insights on human cortical development and also provide a basis for how altered NCAM signaling during specific developmental intervals could affect synaptic connectivity and circuit formation, and thereby contribute to neurodevelopmental disorders.

IgIII (270–280)-fragment-like H 2N-DDSDEEN-COOH peptide modulates N-CAM expression via Ca 2+-dependent ERK signaling during “in vitro neurogenesis”

The two major isoforms (180 kDa and 140 kDa) of the neural cell adhesion molecule (N-CAM) are crucially involved in neurogenesis and brain repair via activation of the mitogen-activated protein kinase (MAPK) cascade. Modification by glycosylation, and homophilic and heterophilic interactions regulate the function of N-CAM, but little is known about the interplay of these processes. In the neuron-like PC12 cell line, extracellular small acidic peptides have been shown to modulate the expression of N-CAM mRNA and protein and regulate its translocation to the plasma membrane. Among these peptides, a synthetic Ig-III-like short sequence (H 2N-DDSDEEN-COOH), designated sSP, was particularly potent. In this study, we analyzed the cross-talk between nerve growth factor (NGF) and extracellular sSP in native and N-CAM-transfected PC12 cells to determine if these systems interact to modulate transduction pathways and regulate early steps of neurogenesis in vitro. Our results indicate that sSP accelerated the phosphorylation of extracellular regulated kinase-1 (ERK1) and -2 (ERK2) and promoted plasma membrane translocation of 180 kDa N-CAM. By stabilizing cell–cell contacts and promoting cell cluster formation, these events, which were mediated via a significant increase in intracellular Ca 2+, regulated some of the early stages of the NGF-induced differentiation process.

Neural cell adhesion molecule (NCAM) isoform expression is associated with neuroblastoma differentiation status

NCAM is a member of the immunoglobulin superfamily of cell adhesion molecules. While highly expressed on neuroblastoma cells, the relative contribution of the three major NCAM isoforms (120, 140, and 180 kDa) to neuroblastoma biology has not been investigated. NCAM protein expression was measured in a neuroblastic tumor tissue microarray (N = 185) by immunohistochemistry. Relative expression of NCAM mRNA isoforms was measured in a panel of 24 human neuroblastomas and compared to fetal and adult human brain using real-time quantitative PCR and Western blot analysis. Associations with clinical and tumor biological co-variates were performed. NCAM protein was detected on all neuroblastic tumors and was highly expressed in all but 7/167 cases. The mRNA species predicted to encode the 120 kDa protein species was the most abundant isoform in adult brain, ganglioneuromas and ganglioneuroblastomas (P = 0.0007), but the mRNA predicted to encode the 180 kDa species was predominant in neuroblastomas (P = 0.043). Microdissected ganglion and neuroblast cells from human primary tumors confirmed these findings. Ganglioneuromas and ganglioneuroblastomas express the adhesive 120 kDa NCAM isoform, while neuroblastomas preferentially express the 180 kDa isoform classically involved in cell motility. These data suggest a mechanism for the enhanced metastatic potential of undifferentiated neuroblastomas.

Effects of fluoride on neural cell adhesion molecules mRNA and protein expression levels in primary rat hippocampal neurons

To investigate the effects of fluoride on the growth and viability, and mRNA and protein expression levels of neural cell adhesion molecules (NCAM) in primary rat hippocampal neurons. The growth and development, the rate of cell survivor, and the mRNA and protein expression levels of NCAM were measured by MTT, RT-PCR, and Western blot respectively after the hippocampal neurons were incubated with 20, 40, and 80 microg/ml sodium fluoride for 24 hours in vitro. As compared with the control group, the number of cells, the length and number of neuritis, and rate of cell survivor were significantly decreased in 80 microg/ml fluoride-treated group (P < 0.05). The mRNA expression levels of NCAM in 40 and 80 microg/ml fluoride-treated groups were significantly lower than that in the control group and decreased with the increasing fluoride concentration. Compared with the control group, the mRNA expression level of NCAM in 20 microg/ml fluoride-treated group was decreased, but the difference was not statistically significant (P > 0.05). The NCAM-180 protein expression levels in 40 and 80 microg/ml fluoride-treated groups, the NCAM-140 protein expression levels in all fluoride-treated groups, and NCAM-120 protein expression level in 80 microg/ml fluoride-treated group were significantly lower than those in the control group (P < 0.05, P < 0.05, P < 0.05, respectively). Fluoride might restrain the growth and survival of rat hippocampal neurons, and decrease mRNA and protein expression levels of NCAM. The impairment of developmental hippocampus might be one of the neurotoxicant target sites for fluoride toxicity.

Effects of fluoride on the expression of NCAM, oxidative stress, and apoptosis in primary cultured hippocampal neurons

The mechanisms underlying the neurotoxicity of endemic fluorosis still remain unknown. To investigate the expression level of neural cell adhesion molecules (NCAM), oxidative stress, and apoptosis induced by fluoride, the primary rat hippocampal neurons were incubated with 20, 40, and 80 mg/l sodium fluoride for 24 h in vitro. The results showed that the cell survival rate in the 80 mg/l fluoride-treated group was significantly lower than that of the control group. Forty and 80 mg/l of fluoride induced significantly increased lactate dehydrogenase release, intracellular reactive oxygen species, and the percentage of apoptosis. Compared with control group, the malondialdehyde levels were significantly elevated while glutathione levels and glutathione peroxidase activities were decreased in all fluoride-treated groups, accompanied by the markedly reduced superoxide dismutase activity in 80 mg/l fluoride-treated group. With respect to NCAM mRNA expression levels, a significant dose-dependent decrease was observed in 40 and 80 mg/l fluoride-treated groups against the control group. In addition, as compared to the control group, the protein expression levels of NCAM-180 in 40 and 80 mg/l fluoride-treated groups, NCAM-140 in all fluoride-treated groups, and NCAM-120 in the 80 mg/l fluoride-treated group were significantly decreased. Our study herein suggested that fluoride could cause oxidative stress, apoptosis, and decreased mRNA and protein expression levels of NCAM in rat hippocampal neurons, contributing to the neurotoxicity induced by fluoride.

Cimetidine induces apoptosis of human salivary gland tumor cells

It has been reported that cimetidine, a histamine type-2 receptor (H2R) antagonist, inhibits the growth of glandular tumors such as colorectal cancer. However, its effects against salivary gland tumors are still unknown. We demonstrated previously that human salivary gland tumor (HSG) cells spontaneously express the neural cell adhesion molecule (NCAM) and also that HSG cell proliferation could be controlled via a homophilic (NCAM-NCAM) binding mechanism and that NCAM may be associated with perineural invasion by malignant salivary gland tumors. In the present study, we investigated the effects of cimetidine via the expression of NCAM on tumor growth and perineural/neural invasion in salivary gland tumor cells. Expression of both NCAM mRNA and protein was found to decrease in a dose-dependent manner upon treatment with cimetidine for 24 h. The MTT assay and confocal laser microscopy clearly showed that HSG cells underwent apoptosis after treatment with cimetidine. Activation of caspases 3, 7, 8 and 9 was observed in HSG cells after cimetidine treatment, thus confirming that the apoptosis was induced by the activated caspases. Apaf-1 activity was also detected in HSG cells in a dose-dependent manner after treatment with cimetidine. We also found that the cimetidine-mediated down-regulation of NCAM expression in HSG cells did not occur via blocking of the histamine receptor, even though H2R expression was observed on HSG cells, as two other H2R antagonists, famotidine and ranitidine, did not show similar effects. We demonstrated for the first time that cimetidine can induce significant apoptosis of salivary gland tumor cells, which express NCAM, at least in part by down-regulation of NCAM expression on the cells. These findings suggest that the growth, development and perineural/neural invasion of salivary gland tumor cells can be blocked by cimetidine administration through down-regulation of NCAM expression, as well as induction of apoptosis.

Expression of nectin-1, nectin-3, N-cadherin, and NCAM in spinal motoneurons after sciatic nerve transection

We here study the expression patterns of the cell adhesion molecules nectin-1, nectin-3, N-cadherin, and neural cell adhesion molecule (NCAM) in motoneurons after sciatic nerve transection (SNT). Nectins are a newly discovered family of adhesion molecules that colocalize with N-cadherin in synapses and are expressed in axons during development. By in situ hybridization (ISH), we found nectin-3, N-cadherin, and NCAM mRNA in uninjured motoneurons. In uninjured animals, nectin-3 mRNA was present in a few vesicular acetylcholine transporter (VAChT)-positive cells of small motoneuron size in lamina IX of the spinal cord. SNT induced a significant increase of nectin-1, nectin-3, and NCAM mRNA, but the signal for N-cadherin mRNA was not affected. After SNT, signal for nectin-3 mRNA appeared over most motoneurons. We next investigated the presence of N-cadherin and nectin protein in synapses on spinal motoneurons by immunohistochemistry. Only N-cadherin immunoreactivity was seen in close relation to synaptophysin staining, while nectin-1 and nectin-3 immunoreactivity did not display such proximity. SNT resulted in decreased immunoreactivity for N-cadherin around the motoneuron soma, while nectin-1 and nectin-3 immunoreactivity remained unchanged. In the peripheral sciatic nerve, nectin-3 immunoreactivity was observed both in controls and following injury and nectin-3 colocalized with both neurofilament and the Schwann cell marker S100. In addition, an increased ISH signal for nectin-3 mRNA could be seen over the proximal stump of the sciatic nerve after SNT. We conclude that motoneuron injury induces complex changes in the spatiotemporal expression pattern of the investigated cell adhesion molecules.

Hypoxic Preconditioning Improves Spatial Cognitive Ability in Mice

Although it has been reported in a lot of studies that hypoxic preconditiong could protect the brain from hypoxic/ischemic injury, it is not clear whether hypoxic preconditioning could affect brain functions such as cognitive ability. This work aims at investigating the effect of hypoxic preconditioning on spatial cognitive ability in mice after acute and repeated hypoxic exposures. The mice were randomly divided into 3 groups: a control group in which mice were not exposed to hypoxia (H0) and experimental groups in which mice encountered hypoxia either once (H1) or 4 times (H4). Neural cell adhesion molecule (NCAM) expression, long-term potentiation (LTP) recording and Morris water maze test were used to measure the animals’ cognitive ability. The tolerance time was progressively prolonged as exposure went on. The expression of both NCAM mRNA and NCAM protein as well as the LTP induction rate decreased in group H1, but recovered to control level in group H4. The performance of mice in the maze test was improved in H4 in comparison with that in both H1 and H0. These findings may indicate that spatial cognitive ability is improved in adult mice by their hypoxic preconditioning.

Induction of apoptosis in human salivary gland tumor cells by anti-NCAM antibody

Neural cell adhesion molecule (NCAM) is a type of cell surface glycoprotein and a member of the immunoglobulin superfamily. It has been reported that NCAM may be associated with perineural invasion by malignant salivary gland tumors such as adenoid cystic carcinoma. We have previously demonstrated that NCAM is constitutively expressed in the human salivary gland tumor cell line HSG, in vitro. In the present study, we have aimed to clarify the hypothesis that NCAM-mediated inhibition of salivary gland tumor proliferation is caused by homophilic binding and involves the prevention of signal transduction for perineural invasion using HSG cells. NCAM mRNA and protein expression was found to decrease in a dose-dependent manner upon treatment with the anti-NCAM antibody (MAb NCAM) for 24 h. The MTT assay showed a significant reduction in the number of viable HSG cells. Confocal laser microscopy showed that HSG cells underwent apoptosis after treatment with MAb NCAM. The activation of caspases 3, 7 and 9 was observed in HSG cells after treatment with MAb NCAM, thus confirming that apoptosis was induced by the activated caspases. Apaf-1 activity was also detected in HSG cells in a dose-dependent manner after treatment with MAb NCAM. The up-regulation of TGF-beta1-mediated NCAM expression appeared to lead to the activation of homophilic NCAM binding, further accelerating HSG cell proliferation. In addition, the localization of NCAM in adenoid cystic carcinomas (ACCs) was examined using an immunohistochemical method. NCAM was slightly to moderately positive in 9 of 13 cases (69.2%) of ACC. These findings suggest that NCAM is associated not only with a cell-to-cell adhesion mechanism, but also with tumorigenesis, including growth, development and perineural invasion in human salivary gland tumors.

Modulation of Peroxisome Proliferator-Activated Receptor δ Activity Affects Neural Cell Adhesion Molecule and Polysialyltransferase ST8SiaIV Induction by Teratogenic Valproic Acid Analogs in F9 Cell Differentiation

It has been suggested that the teratogenic effects of the antiepileptic drug valproic acid (VPA) is reflected in vitro by the differentiation of F9 cells, activation of peroxisome proliferator-activated receptor delta (PPARdelta), and inhibition of histone deacetylases (HDACs). The aim of this study was to identify genes involved in the differentiation of F9 cells induced by VPA, teratogenic VPA derivatives, or the HDAC inhibitor trichostatin A (TSA) and to characterize the role of PPARdelta. Treatment of the cells with teratogenic VPA derivatives or TSA induced differentiation of F9 cells, mRNA, and protein expression of the neural cell adhesion molecule (NCAM) as well as activated the 5'-flanking region of the NCAM promoter, whereas nonteratogenic VPA derivatives had no effect at all. The polysialyltransferases [ST8SiaIV (PST1) and ST8SiaII] are responsible for the addition of polysialic acid (PSA) to NCAM. The mRNA expression of PST1 was highly induced by only teratogenic VPA derivatives and TSA. As shown by fluorescence-activated cell sorting analysis the level of PSA was higher after treatment of F9 cells with teratogenic VPA derivatives. It is interesting that overexpression of the PPARdelta but not PPARalpha or PPARgamma in F9 cells resulted in higher induction of NCAM mRNA and protein expression and of PST1 mRNA expression (and a higher PSA level) than in mock-transfected F9 cells. Furthermore, repression of PPARdelta activity in F9 cells inhibited these effects. We conclude that NCAM and PST1 are molecular markers in F9 cell differentiation caused by treatment with teratogenic VPA compounds or TSA and suggest that in addition to HDAC inhibition PPARdelta is involved in the signaling pathway.

唾液腺腺様嚢胞癌の増殖・進展メカニズムに関する研究―ＮＣＡＭの発現とその機能について―

Neural cell adhesion molecule (NCAM) is one cell surface glycoprotein and a member of the immunoglobulin superfamily. It has been reported that NCAM may be associated with perineural invasion by malignant glandular tumors, such as bile duct carcinoma or gallbladder carcinoma. However, it remains unclear whether NCAM is really associated with the development of salivary gland tumors. Previous studies have demonstrated that NCAM is constitutively expressed in the human salivary gland tumor cell line HSG in vitro. In this study, molecular mechanisms of perineural invasion were examined using HSG cells in which NCAM-mediated inhibition of salivary gland tumor proliferation is caused by homophilic binding. In addition, the localization of NCAM in adenoid cystic carcinomas (ACCs) was examined immunohistochemically. The levels of NCAM mRNA and protein expression were found to decrease in a dose-dependent manner on treatment with anti-NCAM antibody. The MTT assay revealed a significant reduction in the number of viable HSG cells. Confocal laser microscopy showed that HSG cells undergo apoptosis in response to treatment with anti-NCAM antibody. The activation of caspases 3, 7, and 9 was observed in HSG cells after treatment with anti-NCAM antibody, thus confirming the induction of activated caspase-mediated apoptosis. The up-regulation of TGF-β1-mediated NCAM expression seemed to lead to the activation of homophilic NCAM binding, further accelerating HSG cell proliferation. Immunohistochemical examinations revealed that NCAM was slightly to moderately positive in 9 of 13 cases (69.2%) of ACC.These findings suggest that NCAM is associated not only with a cell-to-cell adhesion mechanism but also with tumorigenesis, including growth, development, and perineural invasion in human ACCs.

N-CAM expression and localization in PC12 cells modulated by extracellular peptides

The neural cell adhesion molecules (N-CAMs) play an important role in mediating cell–cell interactions in the nervous system. Different isoforms of these membrane proteins are involved in the formation of the neuronal network and in the dynamic phases of neuronal plasticity. We studied the early stages of the pseudo neuronal differentiation of PC12 cells induced by a class of small acidic peptides capable of modulating gene expression in these cells. The data presented here indicate that peptides with specific sequences induce an increase in N-CAM mRNA expression and protein translocation to the plasma membrane to a comparable degree as NGF.