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Maj N Enevoldsen,
Artur Kochoyan,
Monika Jurgenson,
Külli Jaako,
Oksana Dmytriyeva, Peter S Walmod,
Jesper D Nielsen,
Janne Nielsen,
Shizhong Li,
Irina Korshunova,
Boris Klementiev,
Tatiana Novikova,
Alexander Zharkovsky,
Vladimir Berezin,
Elisabeth Bock
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ABSTRACT: The fibroblast growth factor receptor (FGFR) plays a vital role in the development of the nervous system regulating a multitude of cellular processes. One of the interaction partners of the FGFR is the neural cell adhesion molecule (NCAM), which is known to play an important role in neuronal development, regeneration and synaptic plasticity. Thus, simultaneous activation of FGFR- and NCAM-mediated signaling pathways may be expected to affect processes underlying neurodegenerative diseases. We here report the identification of a peptide compound, Enreptin, capable of interacting with both FGFR and NCAM. We demonstrate that this dual specificity agonist induces phosphorylation of FGFR and differentiation and survival of primary neurons in vitro, and that these effects are inhibited by abrogation of both NCAM and FGFR signaling pathways. Furthermore, Enreptin crosses the blood-brain barrier after subcutaneous administration, enhances long-term memory in normal mice and ameliorates memory deficit in mice with induced brain inflammation. Moreover, Enreptin reduces cognitive impairment and neuronal death induced by Aβ(25-35) in a rat model of Alzheimer's disease, and reduces the mortality rate and clinical signs of experimental autoimmune encephalomyelitis in rats. Thus, Enreptin is an attractive candidate for the treatment of neurological diseases.
Neurobiology of Disease 07/2012; 48(3):533-45. · 5.40 Impact Factor
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ABSTRACT: Valproic acid (VPA) is a known teratogen. In the present study, the effects of VPA and seven VPA derivatives with different teratogenic potencies (isobutyl-, 5-methyl-, ethyl-, propyl-, butyl-, pentyl- and hexyl-4-yn-VPA) were investigated in L929 cells in vitro. Evaluated end-points included changes in cell proliferation, growth, cell cycle distribution, morphology, speed, glycogen synthase kinase-3β (GSK-3β) and Erk1/2 phosphorylation, and histone H3 acetylation. Changes in proliferation, growth, speed, Erk1/2 and GSK-3β-Tyr216 phosphorylation, and H3 acetylation were significantly associated with the teratogenic potencies of the VPA derivatives. However, in contrast to changes in Erk1/2 phosphorylation and H3 acetylation, significant changes in GSK-3β phosphorylation could only be obtained in response to prolonged incubation at high drug concentration. There was an association between changes in H3 acetylation and GSK-3β-Tyr216 phosphorylation, whereas none of these end-points were associated with changes in Erk1/2 phosphorylation. These results suggest that the teratogenic potencies of VPA and VPA derivatives are related to effects on both Erk1/2 and histone deacetylase activities, whereas changes in GSK-3β activity are possibly a secondary effect.
Basic & Clinical Pharmacology & Toxicology 03/2011; 109(3):164-74. · 2.18 Impact Factor
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Nikolaj Kulahin,
Ole Kristensen,
Kim K Rasmussen,
Lars Olsen,
Patrik Rydberg,
Bente Vestergaard,
Jette S Kastrup,
Vladimir Berezin,
Elisabeth Bock, Peter S Walmod,
Michael Gajhede
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ABSTRACT: The ectodomain of olfactory cell adhesion molecule (OCAM/NCAM2/RNCAM) consists of five immunoglobulin (Ig) domains (IgI-V), followed by two fibronectin-type 3 (Fn3) domains (Fn3I-II). A complete structural model of the entire ectodomain of human OCAM has been assembled from crystal structures of six recombinant proteins corresponding to different regions of the ectodomain. The model is the longest experimentally based composite structural model of an entire IgCAM ectodomain. It displays an essentially linear arrangement of IgI-V, followed by bends between IgV and Fn3I and between Fn3I and Fn3II. Proteins containing IgI-IgII domains formed stable homodimers in solution and in crystals. Dimerization could be disrupted in vitro by mutations in the dimer interface region. In conjunction with the bent ectodomain conformation, which can position IgI-V parallel with the cell surface, the IgI-IgII dimerization enables OCAM-mediated trans-interactions with an intercellular distance of about 20 nm, which is consistent with that observed in synapses.
Structure 02/2011; 19(2):203-11. · 6.35 Impact Factor
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ABSTRACT: The anti-epileptic drug valproic acid (VPA) has attracted attention as an anti-cancer agent.
The present study investigated effects of VPA exposure on histone deacetylase (HDAC) inhibition, cell growth, cell speed, and the degree of Erk1/2 phosphorylation in 10 cell lines (BT4C, BT4Cn, U87MG, N2a, PC12-E2, CSML0, CSML100, HeLa, L929, Swiss 3T3).
VPA induced significant histone deacetylase (HDAC) inhibition in most of the cell lines, but the degree of inhibition was highly cell type-specific. Moreover, cell growth, motility and the degree of Erk1/2 phosphorylation were inhibited, activated, or unaffected by VPA in a cell type-specific manner. Importantly, no relationship was found between the effects of VPA on HDAC inhibition and changes in the degree of Erk1/2 phosphorylation, cell growth, or motility. In contrast, VPA-induced modulation of the MAPK pathway downstream of Ras but upstream of MEK (i.e., at the level of Raf) was important for changes in cell speed.
These results suggest that VPA can modulate the degree of Erk1/2 phosphorylation in a manner unrelated to HDAC inhibition and emphasize that changes in the degree of Erk1/2 phosphorylation are also important for the anti-cancer properties of VPA.
BMC Cancer 01/2010; 10:383. · 3.01 Impact Factor
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Advances in experimental medicine and biology 01/2010; 663:23-53. · 1.09 Impact Factor
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Advances in experimental medicine and biology 01/2010; 663:403-20. · 1.09 Impact Factor
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[show abstract]
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ABSTRACT: Cell adhesion molecules (CAMs) mediate cell-to-cell interactions and interactions between cells and the extracellular matrix
(ECM). The neural cell adhesion molecule (NCAM), a prototypic member of the immunoglobulin (Ig) superfamily of CAMs, mediates
adhesion through homophilic and heterophilic interactions, thereby modulating a range of biological processes. This review
summarizes interactions between NCAM and other CAMs and ECM proteins. Additionally, the role of NCAM as a receptor for rabies
virus, and its implications in rabies infections is briefly described. Interactions between NCAM and its heterophilic partners
involve most of the NCAM extracellular modules and are mediated via amino acids and carbohydrates. The interactions promote
cell adhesion and trigger signal transduction and alterations in cytoskeletal dynamics and organization. Heterophilic NCAM
interactions may modulate, or be modulated by, homophilic NCAM interactions. Furthermore, some of the interactions are mutually
exclusive, whereas others might lead to the formation of multimeric protein complexes. Consequently, biological processes
affected by NCAM interactions are regulated in a complex manner involving many extracellular protein interactions.
KeywordsCell adhesion molecule-Extracellular matrix-Proteoglycan-Prion-Rabies virus
12/2009: pages 23-53;
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ABSTRACT: Cell adhesion molecules (CAMs) constitute a large class of plasma membrane-anchored proteins that mediate attachment between
neighboring cells and between cells and the surrounding extracellular matrix (ECM). However, CAMs are more than simple mediators
of cell adhesion. The neural cell adhesion molecule (NCAM) is a well characterized, ubiquitously expressed CAM that is highly
expressed in the nervous system. In addition to mediating cell adhesion, NCAM participates in a multitude of cellular events,
including survival, migration, and differentiation of cells, outgrowth of neurites, and formation and plasticity of synapses.
NCAM shares an overall sequence identity of ∼44% with the neural cell adhesion molecule 2 (NCAM2), a protein also known as
olfactory cell adhesion molecule (OCAM) and Rb-8 neural cell adhesion molecule (RNCAM), and the region-for-region sequence
homology between the two proteins suggests that they are transcribed from paralogous genes. However, very little is known
about the function of NCAM2, although it originally was described more than 20 years ago. In this review, we summarize the
known properties and functions of NCAM2 and describe some of the differences and similarities between NCAM and NCAM2.
KeywordsNCAM-NCAM2-Neural cell adhesion molecule-OCAM-Olfactory system-RNCAM
12/2009: pages 403-420;
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ABSTRACT: NCAM-type proteins modulate multiple neuronal functions, including the outgrowth and guidance of neurites, the formation,
maturation, and plasticity of synapses, and the induction of both long-term potentiation and long-term depression. The ectodomains
of NCAM proteins have a basic structure of five amino-terminal immunoglobulin (Ig), followed by two fibronectin type III (FnIII)
modules. As a result of alternative splicing, many NCAM-type proteins exist in several isoforms, including both transmembrane
and glycosylphosphatidylinositol (GPI)-anchored versions. Extracellularly, NCAM proteins mediate cell–cell adhesion through
homophilic interactions and bind to growth factors, growth factor receptors, glutamate receptors, other CAMs, and components
of the extracellular matrix. Intracellularly, NCAM-type proteins interact with various cytoskeletal proteins and regulators
of intracellular signal transduction. A central feature of the synaptic function of NCAM proteins is the regulation of their
extracellular interactions by adhesion-modulating glycoepitopes, their removal from the cell surface by endocytosis, and the
elimination of their adhesion-mediating interactions by the proteolytic cleavage of their ectodomains. Although specific aspects
of NCAM proteins have changed through evolution, core structural and functional features are conserved between NCAM-type proteins
in vertebrates and invertebrates, demonstrating that the functions of this class of adhesive proteins are of general importance
during nervous system formation.
KeywordsapCAM-ATP-Fasciclin II-Immunoglobulin-NCAM-PSA
06/2009: pages 265-299;
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ABSTRACT: The brain acid-soluble protein BASP1 (CAP-23, NAP-22) belongs to the family of growth-associated proteins, which also includes GAP-43, a protein recently shown to regulate neural cell adhesion molecule (NCAM)-mediated neurite outgrowth. Here, the effects of BASP1 overexpression were investigated in PC12E2 cells and primary hippocampal neurons. BASP1 overexpression stimulated neurite outgrowth in both cell types. The effects of BASP1 and trans-homophilic NCAM interactions were additive, and BASP1-induced neurite outgrowth was not inhibited by ectopic expression of cytoplasmic NCAM domains. Furthermore, inhibition of signaling via the fibroblast growth factor receptor, Src-family nonreceptor tyrosine kinases, protein kinase C, or GSK3beta, and expression of constructs of the cytoskeletal proteins spectrin and tau inhibited NCAM- but not BASP1-induced neurite outgrowth. Expression of BASP1 mutated at the serine-5 phosphorylation site stimulated neurite outgrowth to a degree comparable to that observed in response to overexpression of wild-type BASP1, whereas expression of BASP1 mutated at the myristoylation site at glycine-1 completely abrogated the stimulatory effects of the protein on neurite outgrowth. Finally, coexpression experiments with dominant negative and wild-type versions of GAP-43 and BASP1 demonstrated that the two proteins could substitute for each other with respect to induction of NCAM-independent neurite outgrowth, whereas BASP1 was unable to replace the stimulatory effect of GAP-43 on NCAM-mediated neurite outgrowth. These observations demonstrate that BASP1 and GAP-43 have overlapping, but not identical, functions in relation to neurite outgrowth and indicate that the main function of BASP1 is to regulate the organization and morphology of the plasma membrane.
Journal of Neuroscience Research 09/2008; 86(10):2201-13. · 2.74 Impact Factor
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Raino Kristian Hansen,
Claus Christensen,
Irina Korshunova,
Martin Kriebel,
Nadine Burkarth,
Vladislav V Kiselyov,
Marianne Olsen,
Søren Ostergaard,
Arne Holm,
Hansjürgen Volkmer, Peter S Walmod,
Vladimir Berezin,
Elisabeth Bock
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ABSTRACT: A combinatorial library of undecapeptides was produced and utilized for the isolation of peptide binding to the fibronectin type 3 modules (F3I-F3II) of the neural cell adhesion molecule (NCAM). The isolated peptides were sequenced and produced as dendrimers. Two of the peptides (denoted ENFIN2 and ENFIN11) were confirmed to bind to F3I-F3II of NCAM by surface plasmon resonance. The peptides induced neurite outgrowth in primary cerebellar neurons and PC12E2 cells, but had no apparent neuroprotective properties. NCAM is known to activate different intracellular pathways, including signaling through the fibroblast growth factor receptor, the Src-related non-receptor tyrosine kinase Fyn, and heterotrimeric G-proteins. Interestingly, neurite outgrowth stimulated by ENFIN2 and ENFIN11 was independent of signaling through fibroblast growth factor receptor and Fyn, but could be inhibited with pertussis toxin, an inhibitor of certain heterotrimeric G-proteins. Neurite outgrowth induced by trans-homophilic NCAM was unaffected by the peptides, whereas knockdown of NCAM completely abrogated ENFIN2- and ENFIN11-induced neuritogenesis. These observations suggest that ENFIN2 and ENFIN11 induce neurite outgrowth in an NCAM-dependent manner through G-protein-coupled signal transduction pathways. Thus, ENFIN2 and ENFIN11 may be valuable for exploring this particular type of NCAM-mediated signaling.
Journal of Neurochemistry 12/2007; 103(4):1396-407. · 4.06 Impact Factor
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ABSTRACT: 2-n-Pentyl-4-pentynoic acid (PE-4-yn-VPA) is a derivative of the antiepileptic and mood-stabilizing drug valproic acid (VPA). PE-4-yn-VPA exists as R- and S-enantiomers, the latter being more teratogenic. PE-4-yn-VPA also possesses antiepileptic, antiproliferative, and cell-differentiating properties. Moreover, the less teratogenic enantiomer, R-PE-4-yn-VPA, was recently shown to improve learning and memory. We here present a detailed investigation of the enantioselective properties of PE-4-yn-VPA using a range of in vitro and in vivo assays including measurements of cellular growth and migration, neuronal differentiation and survival, intracellular signal transduction, synaptic plasticity and maturation, and short-term memory as determined by the social recognition test. The results show that the enantiomers of PE-4-yn-VPA largely had similar effects in vitro. However, in all in vitro experiments the more teratogenic enantiomer, S-PE-4-yn-VPA, exhibited a stronger potency than R-PE-4-yn-VPA, and only S-PE-4-yn-VPA had a detrimental effect on cell survival. Interestingly, both the R- and S-enantiomer improved learning and memory. In contrast, the beneficial effect of S-PE-4-yn-VPA on memory was lost by time, whereas the effect of R-PE-4-yn-VPA administration was longer lasting, suggesting that the beneficial effect of the S-enantiomer on memory formation may be counteracted by its detrimental effect on neuronal cell survival.
Neuropharmacology 04/2007; 52(3):764-78. · 4.81 Impact Factor
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ABSTRACT: The neural cell adhesion molecule, NCAM, is involved in multiple cis- and trans-homophilic interactions (NCAM binding to NCAM) thereby facilitating cell-cell adhesion through the formation of zipper-like NCAM-complexes. NCAM is also involved in heterophilic interactions with a number of proteins and extracellular matrix molecules. Some of these heterophilic interactions are mutually exclusive, and some interfere with or are dependent on homophilic NCAM interactions. Furthermore, both homo- and heterophilic interactions are modulated by posttranslational modifications of NCAM. Heterophilic NCAM-interactions initiate several intracellular signal transduction pathways ultimately leading to biological responses involving cellular differentiation, proliferation, migration and survival. Both homo- and heterophilic NCAM-interactions can be mimicked by synthetic peptides, which can induce NCAM-like signalling, and in vitro and in vivo studies suggest that such NCAM mimetics may be used for the treatment of neurodegenerative disorders.
Neurochemical Research 12/2004; 29(11):2015-35. · 2.24 Impact Factor
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ABSTRACT: Screening for potential teratogenicity of 20 test compounds was performed using a computerised microscope workstation for determination of cytotoxicity, proliferation and morphology of fibroblastoid murine L929-cells. The test compounds, which were divided into four classes according to teratogenicity were: 5-bromo-2(')-deoxyuridine, 6-aminonicotinamide, acrylamide, boric acid, D-(+)-camphor, dimethadione, dimethyl phthalate, diphenhydramine, hydroxyurea, isobutyl-ethyl-valproic acid, lithium chloride, methyl mercury chloride, methotrexate, methoxyacetic acid, penicillin G, all-trans-retinoic acid, pentyl-4-yn-valproic acid, saccharin, salicylic acid and valproic acid. All compounds, with the exception of dimethadione inhibited proliferation in a linear dose-dependent manner, and there were statistically significant compound class-dependent differences between the IC(50)-values for the compounds (p<0.0374), the strongest teratogens being the most potent. Furthermore, the average efficacies (maximum relative change) for 10 parameters describing cell morphology exhibited statistically significant compound class-dependent differences (p<0.0001), the class I and II compounds exhibiting significantly lower efficacies than the class III and IV compounds (p<0.01). Thus, test compounds affected both the proliferation and morphology of L-cells in manner demonstrating a general relationship with the teratogenic potency of the compounds. However, the moderate teratogens dimethadione and lithium chloride only had minor effects on the morphology and proliferation of the cells whereas the non-teratogen diphenhydramine had effects on both proliferation and morphology comparable to the strong teratogens.
Toxicology in Vitro 09/2004; 18(4):511-25. · 2.78 Impact Factor
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ABSTRACT: The neural cell adhesion molecule (NCAM) stimulates neurite outgrowth by activating intracellular signaling cascades. We investigated the role of the transcriptional repressor HES-1 in NCAM-dependent neurite outgrowth by estimating neurite extension from PC12-E2 cells grown in coculture with NCAM-negative or NCAM-positive fibroblasts. PC12-E2 cells were transiently transfected with an expression plasmid encoding HES-1. We found that expression of HES-1 inhibited NCAM-dependent neurite outgrowth. Treatment with arachidonic acid (an important messenger in NCAM-dependent signaling) restored NCAM-induced neurite outgrowth inhibited by HES-1. These results suggest that HES-1 is a regulator of intracellular signal transduction stimulated by cell adhesion molecules involved in neurite outgrowth.
Journal of Neuroscience Research 02/2003; 71(1):1-6. · 2.74 Impact Factor
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Peter S Walmod,
Anton Berezin,
Helen C Gallagher,
Ute Gravemann,
Eugene A Lepekhin,
Vadym Belman,
Christopher L Bacon,
Heinz Nau,
Ciaran M Regan,
Vladimir Berezin,
Elisabeth Bock
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ABSTRACT: We present a new in vitro assay for screening of potential teratogens, based on staining of cultured mouse fibroblastoid L929 cells for the determination of number of live and dead cells and of cell morphology, employing automatic video recording, followed by detection of the stained specimen and calculation of endpoint values by the use of a computerized microscope workstation. Ten different parameters were combined empirically into a single index describing general alterations in cell morphology, and, subsequently, measurements of alterations in morphology and proliferation were combined to produce a single empirical index aimed at predicting teratogenic potency. The assay was employed in two different laboratories on 10 coded compounds; 7 compounds that have demonstrated in vivo teratogenic potentials: valproic acid (VPA), pentyl-4-yn-VPA, retinoic acid (RA), 13-cis-RA, AM580, thalidomide, and alpha-EM12 and 3 compounds for which no teratogenic potential has been demonstrated: isobutyl-4-yn-VPA, phytanic acid, and beta-EM12. Within each of the three groups of compounds the nonteratogens generally caused smaller alterations in cell morphology than the teratogens, although the effects of thalidomide and related compounds generally were minor or insignificant. The data support the hypothesis that cell morphology and proliferation in combination with other endpoints may be employed for in vitro screenings of potential teratogens, although studies of additional compounds are needed in order to establish the general validity of the procedure.
Toxicology and Applied Pharmacology 06/2002; 181(1):1-15. · 4.45 Impact Factor
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Søren Prag,
Eugene A Lepekhin,
Kateryna Kolkova,
Rasmus Hartmann-Petersen,
Anna Kawa, Peter S Walmod,
Vadym Belman,
Helen C Gallagher,
Vladimir Berezin,
Elisabeth Bock,
Nina Pedersen
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ABSTRACT: Cell migration is required during development of the nervous system. The regulatory mechanisms for this process, however, are poorly elucidated. We show here that expression of or exposure to the neural cell adhesion molecule (NCAM) strongly affected the motile behaviour of glioma cells independently of homophilic NCAM interactions. Expression of the transmembrane 140 kDa isoform of NCAM (NCAM-140) caused a significant reduction in cellular motility, probably through interference with factors regulating cellular attachment, as NCAM-140-expressing cells exhibited a decreased attachment to a fibronectin substratum compared with NCAM-negative cells. Ectopic expression of the cytoplasmic part of NCAM-140 also inhibited cell motility, presumably via the non-receptor tyrosine kinase p59(fyn) with which NCAM-140 interacts. Furthermore, we showed that the extracellular part of NCAM acted as a paracrine inhibitor of NCAM-negative cell locomotion through a heterophilic interaction with a cell-surface receptor. As we showed that the two N-terminal immunoglobulin modules of NCAM, which are known to bind to heparin, were responsible for this inhibition, we presume that this receptor is a heparan sulfate proteoglycan. A model for the inhibitory effect of NCAM is proposed, which involves competition between NCAM and extracellular components for the binding to membrane-associated heparan sulfate proteoglycan.
Journal of Cell Science 02/2002; 115(Pt 2):283-92. · 6.11 Impact Factor
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ABSTRACT: The shape of a cell reflects cell-substratum and cell-cell attachment and organization of the cytoskeleton (1–4). Many studies on cell morphology have until recently been purely qualitative for the simple reason that morphological changes
resulting from e.g., modulation of cell attachment or perturbation of cytoskeletal components often are so pronounced that
quantification may seem unnecessary. However, quantitative determination of cellular morphology greatly expands the possibility
of characterizing the role and regulation of macromolecules involved, e.g., in cytoskeletal functions and cellular differentiation.
12/2000: pages 85-100;
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ABSTRACT: The coordinated displacement of a cell is a highly complex process which relies on the integration of a series of signaling
pathways and on the function of a large number of molecular components including all main structures of the cytoskeleton (1,2). Thus, a detailed knowledge of the regulation and function of the cytoskeleton is of central importance for the understanding
of cell motility, and conversely, investigations of cell motility may shed light on the function of cytoskeletal components.
12/2000: pages 59-83;
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ABSTRACT: Valproic acid (VPA) is an established human teratogen that causes neural tube defects in 1–2 % of human foetuses exposed to the drug during early pregnancy. In this study, individual cell motility was evaluated using short- and long-term time-lapse video-recording and computer assisted image analysis, and it was found that VPA and selected VPA-analogues inhibited individual cell motility of L-cells in a dose-dependent manner. The compounds caused a decrease in the root-mean-square speed, S, and in the rate of diffusion, R, but an increase in the time of persistence in direction, P. Using short-term recordings and measurements of mean-cell speed, the reduction in the motile behaviour was shown to correlate with the teratogenic potency of the tested compounds. The observed effects of VPA on cell motility was independent of the employed L-cell clone, and could be reproduced in cells containing the neuronal marker NCAM and in the neuronal cell line N2a. Furthermore, the observed effect was independent of culture substratum, being observed for L-cells grown on fibronectin as well as on plastic. Immunofluorescence microscopy revealed that VPA-treatment of mouse L-cells caused a redistribution of F-actin and of a series of focal adhesion proteins, indicating that the effect of VPA on cell motility may be causally related to increased cell-substratum interactions or to alterations in the organisation or dynamics of the actin cytoskeleton. Cell Motil. Cytoskeleton 40:220–237, 1998. © 1998 Wiley-Liss, Inc.
Cell Motility and the Cytoskeleton 12/1998; 40(3):220 - 237. · 4.19 Impact Factor
