Int. J. Devl Neuroscience 18 (2000) 781–790

Cholinesterases modulate cell adhesion in human neuroblastomacells in vitro

Glynis Johnson *, Samuel W. MooreDepartments of Paediatric Surgery and Medical Biochemistry, Faculty of Medicine, Uni6ersity of Stellenbosch, PO Box 19063,

Tygerberg 7505, South Africa

Received 25 January 2000; received in revised form 23 June 2000; accepted 27 June 2000

Abstract

Cholinesterases are expressed non-synaptically during embryonic development, neoplasia and neurodegeneration. We haveinvestigated the effects of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) and, conversely, anti-AChE and -BChEantibodies and inhibitors on cell adhesion and neurite outgrowth in human neuroblastoma cells. Analysis of cholinesterase levelsand isoforms in undifferentiated and differentiated cells indicated a significant rise in AChE levels on differentiation. This increasewas related to both cell-associated and secreted enzyme, and was predominantly the G4 isoform. BChE levels and isoforms, onthe other hand, showed no significant variation. Coating the tissue culture plate with AChE stimulated neurite outgrowth, whileBChE had an anti-adhesive effect. Cell adhesion was affected by the BChE inhibitor, ethopropazine, and the AChE peripheral siteinhibitor, BW284c51, but not by eserine which binds to the active site. This indicates that the adhesion function is non-cholinergic,a finding supported by the lack of effect of AE-2, a monoclonal antibody that inhibits AChE, on cell adhesion. Four out of apanel of nine anti-AChE antibodies inhibited adhesion to varying degrees. Of these antibodies, two are catalytic, with epitopesassociated with the peripheral anionic site of AChE, and the remaining two have epitopes overlapping this site. Neither of the twoanti-BChE antibodies used had any effect on adhesion. These results indicate the importance of AChE in neuroblastoma celladhesion and neurite outgrowth, and suggest that the peripheral anionic site may be involved in these processes. © 2000 ISDN.Published by Elsevier Science Ltd. All rights reserved.

Keywords: Cholinesterases; Cell adhesion; Human neuroblastoma cells

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1. Introduction

Acetylcholinesterase (AChE; EC 3.1.1.7) is a compo-nent of the synapse and neuromuscular junction whereit is responsible for the hydrolysis of the neurotransmit-ter acetylcholine. AChE also has significant non-synap-tic expression, related to a secondary, non-cholinergic,function or functions. This non-synaptic expression isseen in developing neural and haematopoietic cells, inregenerating nerves and neoplastic cells, and also inneurodegenerative disorders such as Alzheimer’s disease[1]. Butyrylcholinesterase (BChE; EC 3.1.1.8) is ho-mologous to AChE, found in numerous tissues, and is

thought to function as a scavenger in the adult organ-ism [2,3]. In the embryo, BChE is associated withproliferation, and AChE with differentiation [4].

A number of reports have appeared in the last 6years providing evidence of secondary, non-cholinergicfunctions for AChE. Layer et al. [5] and Small et al. [6],both working on embryonic chick cells, found thatAChE was capable of stimulating neurite outgrowth ina non-cholinergic manner. Jones et al. [7] observedsimilar effects in rat dopaminergic neurons, whileKarpel et al. [8] found that cultured glioma cells trans-fected with different AChE transcripts were stimulatedto extend neurites. Koenigsberger et al. [9] describedsimilar findings in neuroblastoma cells. Andres et al.[10] have shown that overexpression of AChE results insignificantly enlarged motoneurons and later neuromo-tor impairment. AChE has also been shown to beinvolved in nerve regeneration in Aplysia [11], and it

Abbre6iations: AChE, acetylcholinesterase; BChE, butyryl-cholinesterase.

* Corresponding author. Tel.: +27-21-9389428; fax: +27-21-9337999.

E-mail address: gjo@gerga.sun.ac.za (G. Johnson).

0736-5748/00/$20.00 © 2000 ISDN. Published by Elsevier Science Ltd. All rights reserved.PII: S 0 7 3 6 -5748 (00 )00049 -6

G. Johnson, S.W. Moore / Int. J. De6l Neuroscience 18 (2000) 781–790782

appears to be promote in vitro aggregation of b-amy-loid fibrils associated with Alzheimer’s disease [12].

Abnormal expression of both AChE and BChE andin vivo amplification of their genes have been observedin megakaryocytopoietic disorders and leukemias [13]and brain and ovarian tumours [14]. The AChE gene islocated at 7q22 [15,16] and that for BChE at 3q26ter[17]. Both are within regions subject to non-randomchromosomal abnormalities observed in acute myeloidleukemia and the myelodysplastic syndromes. Stephen-son et al. [18] have shown that mutations at the AChElocus are indeed linked to both these disorders. Inaddition to the identification of abnormal expression ofcholinergic genes, antisense blocking of BChE expres-sion has been shown to result in an inhibition ofmegakaryocytopoiesis [13]. Application of similar tech-niques in embryonic chick retinal cells resulted in aninhibition of proliferation [19]. Antisense blocking ofAChE in haematopoietic cells resulted in an inhibitionof differentiation [20].

Although AChE is encoded by a single gene, amultiplicity of isoforms is generated through alternativemRNA processing and the association of oligomerswith structural subunits [21]. Alternative splicing givesrise to, firstly, soluble monomers without the capabilityof disulphide linkage to other oligomers, secondly, gly-cophosphotidylinositol-linked monomers and dimers,and, thirdly, amphiphilic monomers, dimers and te-tramers. Tetramers may be associated by disulphidelinkage with collagen-like or hydrophobic subunits,used for anchorage in the basal lamina or extracellularmembrane, respectively [22]. In the neuromuscularjunction, the predominant isoform is the asymmetricA12, in contrast to the only globular forms expressedearlier during development [1]. Although much less isknown about BChE, it also appears to have a similarmultiplicity of molecular forms [22].

The migration and aggregation of neural crest cells isdependent on the interaction of cell surface adhesionmolecules and molecules of the extracellular matrix asis axon growth and neurite extension [23–25]. Culturedneuroblastoma cells are adherent, growing as a mono-layer. Disruption of adhesion frequently results in celldeath [26–29]. The cholinesterase family includes anumber of proteins that function as cell adhesionmolecules, in particular, neurotactin and glutactin inDrosophila [30,31], and the vertebrate gliotactin [32]and neuroligin [33]. These molecules have an extracellu-lar domain homologous to AChE, but lack the catalyticfunction. Darboux et al. [34] replaced the extracellulardomain of neurotactin with the homologous domainfrom Drosophila AChE, with no subsequent diminutionin the chimaera’s adhesive capability, indicating thepresence of adhesive functions in AChE.

The active site of AChE, located at the bottom of a20 A deep gorge, consists of two ‘subsites’: the catalytic

triad responsible for the ester hydrolysis and an anionicsubsite that binds and positions the quaternary cholinemoiety of the substrate [35]. At the rim of the gorge,about 14 A away, lies a second anionic site, the periph-eral anionic site. BChE lacks the peripheral site. Thissite has been shown to be significant in the non-synap-tic functions of AChE [36].

In this study, we investigated, firstly, the effects ofAChE and BChE on adhesion and morphological dif-ferentiation of neuroblastoma cells, and, secondly,whether blocking the function of the cholinesteraseswith specific inhibitors and antibodies affected adhesionand cell viability.

2. Materials and methods

2.1. Reagents

Human erythrocyte AChE, human serum BChE, all-trans retinoic acid, dithiobisbenzoic acid, ethopro-pazine, BW284c51 (1.5 bis (4-allyldimethyl ammoniumphenyl) pentan-3-one dibromide), edrophonium and es-erine (physostigmine) were obtained from Sigma.

Tissue culture medium (Dulbecco’s MEM) and fetalbovine serum were obtained from Highveld Biological,Johannesburg, South Africa.

2.2. Culture and differentiation of neuroblastoma cells

The human neuroblastoma cell line N2a was ob-tained from Highveld Biological, Johannesburg, SouthAfrica. Two other cell lines, GIMEN and Kelly, wereobtained from the Department of Human Genetics,University of Stellenbosch. The Kelly cell line is origi-nally from Dr Fred Gilbert, Mount Sinai School ofMedicine, New York, NY. Cells were cultured in Dul-becco’s MEM with 10% fetal bovine serum, at 5% CO2

in 250 ml tissue culture flasks (Nunc, Denmark).Differentiation was induced by the culture of cells

with all-trans retinoic acid (Sigma) for 7 days. Theoptimal concentration was found to be 50 mM. Differ-entiation was assessed morphologically as the degree ofneurite outgrowth [37]: cells were grown on coverslipsin six well plates until confluent, fixed (50% methanol,50% acetone, 5 min at room temperature, and washedthree times in phosphate buffered saline) and examinedby phase contrast microscopy. Images of cells andneurites were recorded, and at least 100 cells assessedfor each experiment. Processes extending from the cellswere measured, and cells were considered to bedifferentiated or ‘with neurites’ if the processes wereequal to at least twice the diameter of the cell body[38]. The increase in AChE over baseline levels inundifferentiated cells was also used as a differentiationmarker.

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2.3. Density gradient fractionation of cell homogenates

Undifferentiated and differentiated cells were re-moved from four confluent flasks (total cell count�4×107 cells) with Versene, washed three times inphosphate buffered saline and homogenised in 50 mMTris/HCl (pH 7.4) containing 0.5% Triton X-100 and 1M NaCl. The final volume was 1 ml. Of this 0.5 ml waslayered onto each of two 5–20% (w/v) sucrose densitygradients in the above buffer (volume 10 ml) and thegradients centrifuged at 28 000 rpm (96 750×g) for 24h at 4°C and fractionated by upward displacement.Fractions were assayed for cholinesterase by themethod of Ellman et al. [39]. AChE activity was mea-sured on the same samples using 0.85 mM etho-propazine, a specific BChE inhibitor. BChE levels werecalculated as the difference between the totalcholinesterase and AChE values. The comparability ofBChE measurement using acetylthiocholine or bu-tyrylthiocholine as substrates was assessed by assay ofpurified standards using both substrates.

Sequential extracts in low salt and detergent-contain-ing buffers, respectively, were performed as follows: thecells were initially homogenised in 50 mM Tris/HCl,pH 7.4, and the homogenates centrifuged at 13 000 rpmfor 5 min in a microfuge. The supernatant from thiswas described as the low salt soluble fraction, and wasretained and fractionated on density gradients as de-scribed above. The pellet was again washed three timesin the same buffer, and then extracted in buffer con-taining 1% Triton X-100, and centrifuged as above. Thesupernatant from this extraction was described as thedetergent-soluble fraction, as was fractionated as de-scribed above.

2.4. Cholinergic inhibitors

Ethopropazine (Sigma) was used at concentrations of47.5, 23.8, 11.9 and 6.0 mg/ml medium. BW284c51 (1,5bis (4-allyldimethyl ammonium phenyl) pentan-3-onedibromide (Sigma) was used at concentrations of 17.9,8.96, 4.48 and 2.24 mg/ml medium. Eserine (physostig-mine; Sigma) was used at concentrations of 3.20, 1.60,8.0 and 4.0 mg/ml. All these concentrations were chosenafter preliminary experiments.

2.5. Antibodies

The polyclonal antibodies, PCB and PCW, wereraised against human erythrocyte AChE (Sigma) inrabbits, according to standard protocols [40]. Care andhandling of all animals was in accordance with theguidelines laid down by the Animal Research ReviewCommittee of the University.

The monoclonal antibodies, E8 and G9, were raisedagainst the same antigen in Balb/c mice, followed by

fusion of spleen lymphocytes with the mouse myelomaline SP2/Ag0, and cloned by limiting dilution, againaccording to standard protocols [40]. Four additionalanti-AChE monoclonals, raised against fetal bovineserum AChE and Torpedo AChE (antibodies E413D8,E62A1 and E65E8) as well as the anti-human erythro-cyte AChE monoclonal AE-2 [41] were a kind donationfrom B.P. Doctor and M.K. Gentry.

An anti-BChE polyclonal antibody was donated byA.S. Balasubramanian and an anti-BChE monoclonalwas a gift from S. Brimijoin. IgG from ascites fluid,tissue culture medium or serum was purified onProtein-A Sepharose (Pharmacia) according to stan-dard protocols [40]. Cells were plated at 2×104 cells/ml, and antibodies added at concentrations of 32, 16, 8and 4 mg IgG/ml. These concentrations were chosenafter preliminary experiments. Where azide had beenincluded in the antibody solution, an equivalent con-centration of azide was added to control wells.

2.6. Assessment of adhesion

Unattached cells were removed from triplicate wellsby pipetting, and retained. Each well was then rinsedwith 0.2 ml phosphate buffered saline, and the rinsingsolution combined with the unattached cells andcounted in a haemocytometer (Improved Neubauer).The remaining, attached, cells were removed withVersene and counted. These two values give the totalnumbers of unattached and attached cells in the well,respectively. The number of attached cells was ex-pressed as a percentage of the total (attached+unattached) and described as the ‘percentage adherent’.

The viability of both attached and unattached cellswas assessed at the time of counting by their ability toexclude 0.25% trypan blue. This was then expressed asa mean value for the total number of cells.

2.7. Coating the tissue culture plates

96 well tissue culture plates were pre-coated for 1.5 hat 37°C with 10, 20, 30 or 40 mg/ml AChE or BChE inserum-free medium. These values were chosen afterpreliminary experiments. After incubation, the solutionwas suctioned off, the plates washed twice with phos-phate buffered saline, and cells plated at 2×104/ml. Inseparate experiments, anti-AChE and anti-BChE anti-bodies were added at concentrations of 16 mg IgG tothe above AChE and BChE solutions, respectively.

2.8. Statistics

Statistical significance was calculated with Student’st-test, for two-tailed samples.

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3. Results

3.1. Induction of differentiation

Culture of the cells with 50 mM retinoic acid for 7 daysstimulated differentiation and resulted in the extensionof neurites from the cells. Similar effects were obtainedby culturing the cells in spent medium. This differenti-ated morphology could be reversed by removing theretinoic acid or by addition of fresh medium and serum.All three cell lines showed similar results, both ininduction of differentiation and in the experiments thatare described below; to avoid confusion and duplicationof data, however, the results that follow are thoseobtained with the N2a cell line only.

3.2. AChE and BChE le6els and isoforms indifferentiated and undiferentiated cells

The levels of AChE and BChE in undifferentiated anddifferentiated N2a cells, together with AChE values formedium at the time of plating and after 4 days cultureare shown in Table 1. It can be seen that, while BChElevels remain essentially constant (183.00922.05 mIU/ml for undifferentiated cells, and 161.47911.42 mIU/mlfor differentiated cells; the difference is non-significant),those for AChE rise on differentiation (164.01928.14mIU/ml for undifferentiated cells, to 243.3498.99 mIU/ml for differentiated cells; this increase is significant,PB0.05). There is also an increase in the amount ofAChE present in the medium (from 75.23910.23 to219.4299.24 mIU/ml; significant, PB0.01).

The density gradient fractionation of cell ho-mogenates from undifferentiated and differentiated cellsis shown in Fig. 1. AChE is present in three isoforms: themonomer G1 (fractions 2–7; 4S), a relatively smallamount of G2 (fractions 2–7; 7S) and the tetramer G4

(fractions 18–21; 11S). G1 and G2 levels appear approx-imately equal in both undifferentiated and differentiatedcells, while the levels of G4 increase approximatelyfourfold in the latter (Fig. 1a). Sequential extracts ofAChE from cell homogenates in low salt and detergent-containing buffers showed that while G1 was purelyhydrophilic, G4 was largely present in the detergent-sol-uble form (data not shown). In contrast, there appearsto be little difference in the distribution of the BChEisoforms in undifferentiated and differentiated cells (Fig.1b).

3.3. The effects of cholinergic inhibitors

The addition of ethopropazine, a specific BChE in-hibitor, reduced adhesion to 12.495.00% after 24 h(Table 2). Effects were visible after 1 h as a reduction inthe percentage of adherent cells (29.694.2%) althoughnot of viability. After 24 h, the viability had dropped to15%.

BW284c51, a specific AChE inhibitor, showed littleeffect after 24 h. However, by day 3, the cells incubatedwith the highest dose (17.9 mg/ml) began to show effectssimilar to those with ethopropazine. By day 4, thepercentage of adherent cells had dropped to 88% (Table2). These effects became more noticeable as time pro-gressed, and by day 7, the percentage of adherent cellshad decreased to 9% and the viability to only 5% (datanot shown). The reduction in adherent cells for bothethopropazine and BW284c51 is significant (PB0.001).Eserine (physostigmine), which inhibits both AChE andBChE, had no significant effect on the adhesion of thecells (Table 2).

3.4. The effects of anti-AChE antibodies on cells

Some, but not all, of the anti-AChE antibodies, whenadded directly to the culture medium of the cells,inhibited cell–substrate adhesion (Table 3). Results areexpressed for the highest concentration of antibody (40mg/ml); a dose-dependent effect was noted at lowerconcentrations. The cells remained rounded and insuspension; if the antibodies were added after the cellswere plated, they rounded up. The cell count remainedthe same, and although viability was initially high, itdropped rapidly. Addition of antibodies to cells thatwere already differentiating resulted in retraction ofneurites and a reversal of the differentiated morphology.Antibody effects were visible after 24 h incubation.

The antibody most effective in inhibiting these pro-cesses was the polyclonal, PCW, which prevented anycell–substrate adhesion. The monoclonal antibody, E8,was also an effective inhibitor, resulting in a markeddecrease in adhesion (8%). Both these results are highlysignificant (PB0.001). Again, dose-dependent effects,starting at 4 mg IgG/ml, were observed. Addition of the

Table 1AChE and BChE levels in undifferentiated and differentiated N2acells, and tissue culture medium (Dulbecco’s MEM with 10% FBS) atthe time of plating and after 4 days in culturea

BChE (mIU/ml)AChE (mIU/ml)

164928.14Undifferentiated cells 183922.05161.47911.42Differentiated cells 243.0598.99*

75.23910.23 N.D.Medium: day 0N.D.Medium: day 4 219.4299.24*

a Values are given as mIU/ml, where 1 IU is defined as the amountof enzyme capable of hydrolysing 1 mg of substrate (acetylthiocholine)per minute at 25°C, and 1 ml is the volume of homogenate from 107

cells. Results are given as means and standard deviations (n=4).N.D., not done.

* The difference between AChE in undifferentiated and differenti-ated cells is significant (P, 0.05), as is the increase in secreted AChEin the medium (PB0.01). Significant results are marked with aster-isks.

G. Johnson, S.W. Moore / Int. J. De6l Neuroscience 18 (2000) 781–790 785

Fig. 1. Density gradient fractionation of neuroblastoma cell homogenates. Gradients were 5–20% (w/v) sucrose in 50 mM Tris/HCl, pH 7.4,containing 0.1% Triton X-100 and 1 M NaCl. Fractionation was by upward displacement, hence the fractions on the left-hand side of the graphare those of lowest density. The positions of the sedimentation markers bovine serum albumin (4.6S), fibrinogen (7.63S) and catalase (11.2S) areshown by the arrows, reading from left to right. (a) AChE activity: undifferentiated cells ("); differentiated cells ( ). (b) BChE activity:undifferentiated cells ("); differentiated cells ( ).

Table 2The effects of cholinergic inhibitors on neuroblastoma (N2a) cells in vitroa

1 hInhibitor 4 h

Viability (%) Adherent cells (%) Viability (%)Adherent cells (%)

\95 18.593.2* 54*29.695.7*Ethopropazine\9593.392.1BW284c51 96.892.9 \95

95.791.894.694.6 \95\95Eserine\95 97.291.9 \95Control 96.392.5

a Results are shown as means and standard deviations (n=3).* Significant (PB0.01) results are marked with asterisks.

monoclonal antibodies, E62A1, E65E8 and the poly-clonal PCB resulted in a partial reduction of adhesion,while others, G9, E413D8 and the enzyme-inhibiting

antibody AE-2, had no effect at all.No cross-reaction between AChE and BChE was

observed for any of the antibodies (data not shown).

G. Johnson, S.W. Moore / Int. J. De6l Neuroscience 18 (2000) 781–790786

3.5. The effects of anti-BChE antibodies on cells

Addition of the two anti-BChE antibodies to theculture medium had no effect on adhesion (data notshown).

3.6. The effects of coating the tissue culture plate withAChE

Cells of all three neuroblastoma cell lines characteris-tically show a rounded or elongated shape in vitro andadhere to the surface of the tissue culture flask or plate(Fig. 2a). Coating the tissue culture plate with varyingconcentrations of AChE produced a clear dose-depen-dent stimulation of neurite extension (Fig. 2b; Table 4).Addition of AChE itself to the medium did not producethe same degree of stimulation (data not shown). Theaddition of the antibodies PCW and E8 (in stoichiomet-ric amounts) to the coating AChE solution preventedthe stimulation of neurite extension.

Conversely, inactivation of plated AChE with theinhibitory antibody, AE-2, again in stoichiometricamounts [41] had no effect on the stimulatory capabil-ity (Table 4).

3.7. The effects of coating the tissue culture plate withBChE

BChE, when used as a plate-coating, interfered withcell–substrate adhesion in a dose-dependent manner(Fig. 2c and Table 5). These differences were alsosignificant (PB0.001). As with AChE, reduced effectswere noted when the BChE was added directly to theculture medium (data not shown).

Addition of anti-BChE antibodies to the coatingsolution reduced the adhesion-inhibitory effect ofBChE in a dose-dependent manner (Table 5). Bothantibodies, the polyclonal and the monoclonal, pro-duced similar effects. Similarly, addition of the specificBChE inhibitor, ethopropazine, to the coating solution,also reduced the anti-adhesion effect.

4. Discussion

AChE is present in undifferentiated cells chiefly inmonomeric and dimeric forms. On induction of differ-entiation, either by retinoic acid or by culture in spentmedium, the level of total cell-associated AChE wasobserved to rise, and expression of the tetrameric, G4,isoform to increase. This tetrameric form is membrane-associated, as shown by sequential extraction in lowsalt and detergent-containing buffers, and presumablyhas the attached hydrophobic domain observed in otheramphiphilic tetrameric forms. There is also a secreted,hydrophilic, fraction. These results confirm other obser-vations on neuroblastoma cells. Cellular BChE levelsremain essentially the same, before and after differenti-ation, and there is also no variation in the relativedistribution of the isoforms. The values for AChE andBChE concentrations associated with the cells or in themedium are included as an indication of the relativedistribution of the cholinesterases in the different frac-tions and are not intended as absolute measurements.AChE activity is expressed as mIU/ml of cell ho-mogenate of 1×107 cells, as estimated in a haemocy-tometer. The values for BChE were calculated usingacetylthiocholine, an AChE substrate. That these arecomparable to using butyrylthiocholine, a BChE sub-strate, was shown by a lack of any significant differencein the hydrolysis of the substrates by purified standards.This is supported by the similarity in turnover numbers(Kcat) of BChE for both substrates: 24 000 min−1 [42]and 98 000930 000 min−1 [43] for butyrylthiocholine,and 40 000 min−1 for acetylthiocholine [44]. There isno chemical or structural basis for a difference insubstrate affinity: butyrylcholine is simply a largermolecule that cannot access the more constrained activesite of AChE. These results on neuroblastoma AChEand BChE levels, both cell-associated and in themedium, confirm those of other workers [45–47].

The AChE used in the experiments was a purified,rather than a recombinant, enzyme. We have observed

Table 3The effects of anti-AChE antibodies on neuroblastoma (N2a) cells in vitroa

Antibody Adherent cells (%) Cell count (×104/ml) Viability after 1 day (%) Viability after 4 days (%)

\9593.492.1E413D8 7.2 \9595.692.7 7.2AE-2 \95 \9541.298.5* 3.2E62A1 \95 5

8.093.6* 2.7E8 91.6 B5\95\957.494.192.7G9

4.6E65E8 80\9554.397.9*PCW 2.00.090* 88.5 B5

71.894.9 6.5 \95PCB 9096.592.3 7.5Control \95 \95

a Results are shown as means and standard deviations (n=9). The antibody concentration was 40 mg IgG/ml; cells were plated at 2×104/ml.Results shown are after 4 days incubation. The antibodies PCW and PCB are polyclonal, and the remainder monoclonal antibodies to AChE.

* Significant (PB0.01) decreases in percentages of adherent cells are indicated by asterisks.

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Fig. 2. Phase-contrast micrographs of N2a cells after 4 days culture.(a) Control (no treatment). (b) Cultured on a coating of 40 mg/mlAChE. (c) Cultured on a coating of 40 mg/ml BChE. Similar effects tothose in (c) were identified in cells cultured with cholinergic inhibitorsand antibodies. Bar=100 mm.

other antibodies do not. In the presence of the poly-clonal antibody, PCW and the monoclonal, E8, theneuroblastoma cells became morphologically roundedand detached from the substratum. Other antibodies, inparticular, E62A1 and E65E8, had a partial effect.Viability measurements using trypan blue exclusion,indicated that, at this point, the cells were still viable,with cell death occurring only subsequently. Loss ofadhesion in normally adherent cells is known to resultin apoptosis [26–29]. This suggests that the primaryeffect of the antibodies and inhibitors is an interference

Table 4The effects on adhesion and neurite outgrowth of growing cells (N2a)on a coating of AChEa

Cells with neurites (%)Adherent cells (%)AChE (mg/ml)

85.692.10 2.790.584.291.710 23.495.4*

23.392.9*92.993.42093.891.930 30.093.6*

40 38.592.7*95.594.540+PCWb 0.09025.497.9*

38.692.0*40+AE-2b 96.091.840+BWc 94.491.7 25.991.7*40+Eth.c 37.793.4*93.992.1

a The results shown are means and standard deviations, after 4days incubation (n=8).

b Addition of antibodies PCW and AE-2 to the AChE coatingsolution.

c Addition of the inhibitors BW284c51 and ethopropazine to theAChE coating solution.

* The difference between the number of cells with neurites, grownon a coating of 40 mg/ml AChE and that of the control, is significant(PB0.001). Significant (PB0.01) differences are marked with aster-isks.

Table 5The effects on adhesion and neurite extension of growing cells (N2a)on a coating of BChEa

Cells with neurites (%)Adherent cells (%)BChE (mg/ml)

85.692.9 2.790.905.692.1* 0.010

0.020.795.6*2030 6.091.9* 0.0

4.792.0* 0.04020.592.5* 0.040+Eth.b

0.040.096.1*40+MAbc

54.793.2*40+PAbd 0.190.05

a Results shown are means and standard deviations (n=3), after 4days incubation.

b Addition of the BChE-specific inhibitor, ethopropazine.c Addition of the anti-BChE-monoclonal antibody.d Addition of the anti-BChE polyclonal antibody to the BChE

coating solution.* The difference between the percentage of cells adherent, in those

grown on a coating of 40 mg/ml BChE and control, is significant(PB0.001). Significant differences (PB0.05) are marked with aster-isks.

the presence of several minor impurities in the prepara-tion on electrophoresis; it is, thus, possible for thesecontaminants to have influenced the results on neuriteoutgrowth. However, the close agreement of our resultswith those of other workers using recombinant [9] ortransfected [8] AChE suggests that the effects observedare AChE-specific. We have, furthermore, demon-strated clearly opposite effects with highly specific anti-AChE inhibitors and antibodies.

We have demonstrated that certain anti-AChE anti-bodies interfere with cell–substrate adhesion, whereas

G. Johnson, S.W. Moore / Int. J. De6l Neuroscience 18 (2000) 781–790788

with cell adhesion, and indicates that AChE is signifi-cantly involved in these processes. Conversely, neuriteand axon outgrowth is known to be supported andstimulated by cell adhesion molecules, e.g. NCAM, L1,and the integrins and extracellular matrix proteins suchas laminin [23–25].

BChE, when added to cells, exerts an anti-adhesiveeffect. In the embryo, high levels of BChE are associ-ated with proliferating, as opposed to aggregating anddifferentiating cells [1]. It has been proposed that BChEmay reciprocally regulate AChE expression [48,49]. Wewere, however, unable to confirm this effect in neurob-lastoma cells (data not shown). Cholinesterase in-hibitors have also been shown to affect AChE geneexpression [50]; these effects are related to AChE’scholinergic activity and are mediated by acetylcholineand the acetylcholine receptor and may not, therefore,apply to the non-cholinergic cell adhesion function. Afurther point that should be mentioned is that theamounts of AChE and BChE adhering to the tissueculture plate after coating are not necessarily the samedue to differences in glycosylation between the twoproteins: human BChE has seven occupied N-glycosyla-tion sites, whereas AChE has only three [51].

Because only some of the antibody panel were effec-tive, a specific epitope or epitopes, would appear to beinvolved. The findings of Darboux et al. [34] on chi-maeric AChE-neurotactin molecules indicated the N-terminal domain as the site of the adhesive function.This domain contains the active site gorge. The lack ofeffect, however, of the active site inhibitors, eserine (acarbamate acting on the active site serine residue) andedrophonium (a quaternary ammonium compoundbinding to the anionic subsite within the gorge) indi-cates that the adhesive function is non-cholinergic. Themonoclonal antibody AE-2, which was raised againsthuman erythrocyte AChE inhibits AChE activity bybinding to residues associated with the anionic site [41].We found that this antibody had no effect on neurob-lastoma cell adhesion, further evidence active site struc-tures are not involved. These findings on thenon-cholinergic nature of the adhesive function confirmthose of numerous other workers [5–7,11].

On the other hand, the peripheral site inhibitor,BW284c51, does interfere with adhesion. This confirmsother observations on chick embryonic neural cells [6]and nerve regeneration in Aplysia [11]. BW284c51 alsointerferes with the interaction of AChE with the b-amy-loid peptide in Alzheimer’s disease [12]. We have subse-quently demonstrated competition between theperipheral site inhibitors, BW284c51 and propidium,and antibody E8, localising the adhesion function tothe peripheral anionic site [36]. Supporting evidencecame from the fact that both E8 and PCW, the anti-bodies that inhibited adhesion most effectively, arecatalytic [52] and have epitopes associated with the

peripheral anionic site [53]. Interestingly, the two mon-oclonal antibodies that showed partial inhibition ofadhesion, E62A1 and E65E8, raised in B.P. Doctor’slaboratory in Washington, DC, have subsequently beenshown [54] to have epitopes partially overlapping theperipheral anionic site. Botti et al. [55] observed charac-teristic electrostatic motifs in the peripheral site regionsof the adhesive cholinesterases (AChE and neurotactin)but not in BChE. A recent publication [56] on the 3Dstructure of mouse AChE describes structural similari-ties between the peripheral site region of AChE andboth the b-amyloid peptide and the prion proteins, allof which have aggregation characteristics.

The question remains as to how AChE could func-tion as a cell adhesion molecule, as it has no intracellu-lar domain and therefore presumably no means oftransducing the signal to cell’s interior. Grifman et al.[57] have recently demonstrated functional redundancybetween AChE and neuroligin, a cholinesterase that is aligand for the b-neurexins. Neurexins are expressedthroughout the nervous system and are integral mem-brane proteins, having extracellular, transmembraneand cytoplasmic domains, capable of signal transduc-tion. Secreted AChE would thus seem to be important,perhaps as a ligand for such a cell-associated receptor.Secreted AChE could therefore feasibly account for thedifferentiation-inducing effect of spent medium, whichcontains significant levels of this fraction.

In conclusion, therefore, we have demonstrated thatAChE is capable of inducing morphological differentia-tion in neuroblastoma cells. BChE has an opposite,anti-differentiation, effect. AChE appears to functionas a cell adhesion molecule, the effects of which can beblocked by antibodies and inhibitors. The specificity ofthe latter allow us to identify the adhesion site on themolecule. These results may be of potential value indeveloping a therapy for neuroblastoma and other dis-eases, such as Alzheimer’s disease, where AChE isinvolved.

Acknowledgements

We acknowledge financial support from the MedicalResearch Council, the Cancer Association of SouthAfrica, and the Harry and Doris Crossley Foundation,University of Stellenbosch. We would particularly liketo thank Dr Mary K. Gentry of the Walter Reed ArmyInstitute, Washington, DC for donation of antibodiesE62A1, E65E8, E413D8 and AE-2, Professor StephenBrimijoin of the Mayo Foundation, Rochester, MN,for the anti-BChE monoclonal, and Professor A.S.Balasubramanian of the Christian Medical College andHospital, Vellore, for the anti-BChE polyclonal. Ourthanks also to Professor P. van Helden for laboratoryfacilities.

G. Johnson, S.W. Moore / Int. J. De6l Neuroscience 18 (2000) 781–790 789

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