Evidence for long-range electrostatic repulsion between HeLa cells

Printed in Sweden Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved

Experimental Cell Research 89 (1974) 206-216

EVIDENCE

FOR LONG-RANGE

ELECTROSTATIC

REPULSION

BETWEEN HeLa CELLS J. J. DEMAN

and E. A. BRUYNEEL

Department of Experimental Cancerology, Clinic for Radiotherapy and Nuclear Medicine, Academic Clinic, State University, Ghent, Belgium

SUMMARY Agglutination curves obtained on addition of low molecular weight poly-L-lysines (mol. wt 4 000-23 000) to HeLa cells, show a deviation from linearity at low polymer concentration. This probably indicates the existence of a [-potential which has to be lowered before agglutination can take place. Experiments with dilysine support the assumption that cell surface charge is lowered on addition of low concentrations of short chain poly-L-lysines. Long poly-L-lysine molecules (mol. wts 70 000; 100 000) yield linear agglutination curves already at the lowest polymer concentrations. This might indicate that these polymers are able to bridge the original repulsion gap between HeLa cells. After removal of peripheral sialic acid by neuraminidase, linear agglutination curves are obtained with all poly-L-lysines irrespective of their chain lengths. This is interpreted as evidence for involvement of sialic acid residues in the charge organization responsible for electrostatic repulsion. The magnitude of the presumed repulsion effect is shown to vary with the cell density at the time the HeLa cells were harvested from the culture. The largest repulsion effect is obtained with cells from density inhibited cultures which also have the lowest tendency for mutual adhesion. With fast growing cells from low density cultures linear agglutination curves are obtained with short chain poly-L-lysines. This is interpreted as evidence for a strong diminishment or absence of long-range electrostatic repulsion between such cells.

Long-range electrostatic repulsion between cells presupposes the occurrence of a high charge density at the cell surface resulting in the building up of a c-potential. In the absence of electric field the structurally bound charges at the electrokinetic shear plane might be partly or wholly neutralized in situ by mobile counter ions. Hence it is obvious that electrokinetic data cannot be used as a proof for the existence of long-range repulsive forces in natural conditions. As far as we know the most direct proof for the existence of these forces has been obtained with erythrocytes [l, 21, a typical nonadhesive cell type. For adhesive cells such as Exptl Cell Res 89 (1974)

tissue cells, the evidence is less direct although there is a lot of indications which strongly suggest that electrostatic repulsion forces exist and might be a controlling factor in intercellular adhesion [3-51. Unfortunately the dextran-covering method used for the demonstration of those forces on erythrocytes did not appear to be applicable to adhesive cells. Dextran molecules are mutually adhesive and also when adsorbed to the cell surface will alter the original thickness and adhesivity of the cell coat. Our use of poly-L-lysine is based on the ability of these molecules to form polymeric cation bridges which cross-link opposed

Long-range repulsion between cells

membrane surfaces [6]. At neutral pH the shape of the polymer is that of a stiff rod due to mutual repulsion between its positively charged side chains [7]. Polylysines apparently do not penetrate into the cell [7] but selectively bind to the negative charges of the cell surface [8]. If it is assumedthat longrange repulsion counteracts mutual adhesion then there are two ways in which the tendency for cell aggregation could be increased by the polymers: (1) neutralization of the surface charge; (2) bridging of the intercellular repulsion gap. The agglutinative effect of long polylysine molecules may be based on both functions jointly [9]. Short chain length polycations may reduce the electrostatic forces of repulsion of each cell surface and in this way the surfaces might come sufficiently close for these polycations to crosslink them [3]. Other evidence for the double role of polylysines comes from the experiments of Katchalsky [6]. The erythrocyte surface potentials measured electrophoretically at the critical point at which agglutination set in were more close to the original potential with long polymers than with shorter ones, suggesting the existence of long-range repulsion forces together with the need for a prealable lowering of the C-potential in the case of the shorter polylysines. In the present investigation we compared the agglutinative effects on HeLa cells produced by poly-L-lysines of different chain lengths. We tried to discern their gap-bridging function from their effect on surface charge neutralization by studying the effect of dilysine, a molecule supposed to be too short for gap bridging. The effects obtained on HeLa cells were compared to those on erythrocytes, i.e. cells for which the existence of long-range repulsion has already been established.

201

MATERIALS AND METHODS Cell culture HeLa cells were grown in spinner cultures using modified [lo] Basal Medium Eagle supplemented with 10% calf’s serum. Cell growth was started by dilution of a high density suspension to ca 0.5 x lo6 cells/ml with fresh culture medium. Two different types of spinner vessels were used. One type had a volume of 10 1 and contained 3 1 of suspension, whereas the other type had a volume of 2 1 and contained 600 ml of suspension. It was observed that cell growth in the larger suspension proceeded at a slower rate than in the smaller suspension. However, in both cell growth became stationary at a density between 2.0 and 2.5 x 10s cells/ml. This high density level was usually reached after 72 h of culture in the smaller vessel and after 96 h in the larger one. Prolongation of the culture time after renewal of the medium did not result in a considerable increase in cell density since, as a rule, the cell concentration did not exceed 2.5 x lo6 cells/ml. Therefore it was assumed that cell density was the primary cause for the in-

hihltlon of cell growth.

Comparison of the agglutinative effects of long and short chain length polylysines were performed on cells harvested after 72 h from 3 1 suspensions. At harvest the cell concentration varied between 1.4 and 1.8 x lo8 cells/ml. In experiments designed to compare cells at different growth rates cells were used from the 600 ml suspensions because in these the growth rate was usually exponential in the first 24 h of culture. The cell concentration at the time of harvest was determined microscopically by repeated countings in a Btirker chamber. Because of the large quantities of cells needed, it was necessary in most cases to start several 600 ml suspensions simultaneously. Upon harvest the mean value of the cell densities was calculated and the experiments were performed on the combined cells.

Preparation of the suspensions The cells were washed once with NaC10.9 % and then were suspended to a concentration of ca 1.8 x lo6 cells/ml in a solution which contained 8.0 g NaCI, 0.3 g KCl, 0.05 g NaH,PO,.H,O, 0.025 g KH,POI, 1.0 g NaHCO, and 3.0 g Tris/l of distilled water. The pH of the solution was adjusted to pH 7.5 by Na-acetate buffer 0.5 M, pH 4.0. The suspensions were placed in a water bath at 37°C for 15 min with occasional shaking. After this time poly+lysine or dilysine was added to concentrations varying between 0.5 and 10.0 mg/l of suspension. The suspensions were made homogeneous by inverting the flasks several times. Ninety ml was transferred to the container for measurement of the agglutination rate. This procedure was followed for each individual suspension containing a given poly+lysine concentration. The cells not used directly were conserved at 4°C as pellets after the initial washing with NaC10.9 %. Human erythrocytes were obtained from fresh blood treated with EDTA. The erythrocyte concentration was determined using an electronic particle counter (celloscope 401, AB Lars Ljungberg & Exptl Cell Res 89 (1974)

208

Deman and Bruyneel

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Figs 1-2. Abscissa: poly-r.-lysine cont. @g/ml of suspension); ordinate: rel. cell cont. Fig. 1. Agglutination of HeLa cells with poly-L-lysines of mol. wts (a) 4000; (b) 11000; (c) 15000 and (d) 23 000. Effects of previous incubation withneuraminidase(Nase). Cell cultures were started at 0.5 x lo8 cells/ml and were harvested after 72 h at cell concentrations between 1.4 and 1.8 x lo8 cells/ml.

d

Co, Stockholm). Preparation of the cell suspensions proceeded as described for HeLa cells extent for the circumstance that three previous washings with NaCl 0.9 % were intercalated together with the higher erythrocyte concentration of 180 x lo6 cells/ml in the suspension.

strated [12] the enzyme splits of sialic acid from surface glycoproteins but not from cell surface bound glycolipids. The amounts of sialic acid liberated were the maximal amounts releasable by the enzymes. Details of the incubation procedure were the same as those described elsewhere [13].

Measurement of agglutination

Radioactive labeling of poly+lysine

The method is based on the measurement of the total cell concentration in the top layer of a slowly rotating suspension. The value obtained is divided by the cell concentration of the same suspension in which the cells were distributed homogeneously, i.e. before the cells were transferred to the container for measurement. The ratio, called the relative cell concentration, decreases with increasing agglutination rate. The method has been described in detail [ll].

Poly-L-lysine of mol. wt 23 000 was tritiated according to the method described by Rifkin & al. [14] except for the substitution of HEPES buffer by Moscona’s solution. Sodium borotritide was obtained from the Radiochemical Centre, Amersham (specific activity 221 mCi/mg, batch 34). After labeling, the mixture was dialysed against Moscona’s solution. Dialysis was stopped when the radioactivity in the dialysate after installment of equilibrium had become less than 1% of the radioactivity inside the bag. The tritium activity was measured with a liquid scintillation spectrometer (Packard, TRI-CARB, model 3380).

Cell viability The viability was assessed by the trypan blue exclusion test. At the time the cells were harvested from the spinner culture, the viability was above 90%. After measurement of the agglutination rate it ranged from 80.5 to 85.0 %.

Poly-L-lysines and dilysine Following poly-L-lysine. HBr polymers were obtained from Sigma Chem. Co., St. Louis, MO., USA: mol. wt 4000, lot 112C-5.510; mol. wt 11 000, lot 91C-5 000; mol. wt 15 000, lot 33C-5.060; mol. wt 23 000, lot 113C-5.250; mol. wt 70000, lot 113C5.150. Also L-lysyl-L-lysine. 2 HCI (dilysine) was a Sigma product. Poly-L-lysine. HBr mol. wt 100 000, lot 33 220 was obtained from Serva, Heidelberg.

Elektrokinetic

measurements

Cell electrophoresis was carried out at 25°C according to the method described by Bangham et al. [15] making use of a cylindrical microelectrophoresis apparatus (Rank Bros, Bottisham, UK). As suspension medium the same solution was used as that described above for the measurement of the agglutination rate. The position of the stationary layer inside the capillary tube was checked making use of the known mobility of human erythrocytes in NaCl 0.9 % [16]. The applied field strength was 4 V/cm.

RESULTS

Neuraminidase treatment

(1) Agglutination

c+Neuraminidase (3 : 2 : 1 : 18) (from Fibrio cholerue; Serva, Heidelberg) was used to remove sialic acids from the cell surface. As has been recently demon-

(a) Comparison between poly-L-lysines of different chain lengths. The relative cell con-

Expti Cell Res 89 (1974)

of HeLa cells

Long-range repulsion between cells

209

approximately linear increase. At ca 7.0 ,ug polylysine per ml of suspension, the agglut tination is complete. :\ Although the length of the polymer molet. cules varies, there are no marked differences 0.6 \ 0 Intact between the four curves presented. On the \ assumption that the polypeptide chains of the polylysines are fully stretched, the distance covered by an individual amino acid is 3.67 A. It follows that the length of poly-La lysine with molecular weights 4 000 and 23 000 a 6 2 4 are respectively ca 70 and 400 A. Becausewe do not know the average lengths of the poly0.8 lysine parts which are adsorbed unto the cell I surface, our experiments can give no precise information on the width of the presumed repulsion gap. The mutual adhesivity of the HeLa cells increases on removal of peripheral sialic acid as is easily seen on comparing the relative cell concentrations in the absenceof polymer. The agglutination rate of desialylated HeLa b cells increases linearly with increasing poly4 a 2 6 lysine concentration indicating that the polymer can bind to other charges than sialic Fig. 2. Agglutination of HeLa cells with poly-Llysines of mol. wt (a) 70000; (b) 100000. Culture acid. Despite the decreasein surface charge conditions as in fig. 1. by removal of sialic acid (see further), the extent of agglutination at high polymer concentrations at the surface of the suspensions centration is approximately the same as with are plotted against polylysine concentrations intact cells. expressed on a weight-per-volume basis. In fig. 2 the agglutination curves obtained This way of expression ensures that polymers with polylysines of mol. wts 70 000 and of different chain lengths are on the same 100 000 are presented. There is a linear inscale with respect to the charge concentra- crease in agglutination rate with intact cells tion they produce in solution. as well as with neuraminidase pretreated Fig. 1 shows the agglutination curves ob- cells. This suggeststhat the initial deviation tained with poly+lysines of mol. wts 4 000, from linearity, caused by the presence of 11000, 15 000 and 23 000. HeLa cells not peripheral sialic acid, is circumvented in this preincubated with neuraminidase display an case by the greater chain length of the polyirregularity in their curves at low polymer mer. concentration indicating that there the poly(b) Agglutination of cells harvested from lysines are somewhat less effective in produc- culture at different cell densities. The cell ing agglutination. On increasing the con- growth in our spinner cultures was usually centration, the rate of agglutination shows an started at a cell density of ca 0.5 x lo6 cells/ 0.8 I

Exptl Cell Res 89 (1974)

210 Deman and Bruyneel ml. During the first day of culture growth proceeded very rapidly, the cell density generally being found to have doubled after 24 h. During the following days the growth rate decreased until a stationary cell density between 2.0 and 2.5 x lo6 cells/ml was reached. HeLa cells harvested from the high density cultures were found to be less adhesive than cells from low density cultures. We examined whether this difference in adhesivenesshad an influence on the magnitude of the presumed electrostatic repulsion effect as observed in our agglutination curves. On agglutination of low adhesivity cells with poly-L-lysine mol. wt 11 000, a large !initial departure from linearity is observed -T (fig. 3). The effect is less pronounced with I I I I II 11 11 1 0 2 4 6 a 10 cells of intermediate adhesiveness and disappears completely with cells harvested after Fig. 4. Agglutination with poly-L-lysine mol. wt 23 Ooo of HeLa cells harvested at different cell densities. Cell growth was started at 0.5 x lOa cells/ml. Curve I, harvest at 2.00 x lo6 cells/ml after 72 h; curve 2, harvest at 1.74 x lo8 cells/ml after 48 h; curve 3, harvest at 0.94 x 10s cells/ml after 24 h.

-0

2

4

Figs 3-6. Abscissar poly-r.-lysine cont. bg/ml of suspension); ordinate: rel. cell cont. Fig. 3. Agglutination with poly-L-lysine mol. wt 11 000 of HeLa cells harvested at different cell densities. Cell growth was started at 0.5 x lo8 cells/ml. Curve 1, harvest at 1.82 x lo6 cells/ml after 48 h; curve 2, harvest at 1.56 x lo6 cells/ml after 72 h; curve 3, harvest at 1.07 x lo6 cells/ml after 24 h. Nase, harvest at 1.76 x lo6 cells/ml after 72 h and treated with neuraminidase. Expti Cell Res 89 (1974)

24 h at the lowest cell density. The latter cells have an adhesivenesswhich is quite comparable to that of high density cultures which had been treated with neuraminidase. Also with both types of cells the curves are linear at low binding values of polymer. Note that curve 2 and the neuraminidase curves are those already presented in fig. 1. Fig. 4 shows the same observation on agglutination with poly+-lysine mol. wt 23 000. Fig. 5 indicates that the chain length of the polymer is critical. With poly+-lysine, mol. wt 70 000, the curves are linear at low polymer concentration both with low adhesivity cells from high density cultures as well as with more adhesive cells harvested at low density. These results indicate that the magnitude of the departure from linearity at low polymer concentration is in some way related to

Long-range repulsion between cells

211

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Fig. 5. Agglutination with poly-L-lysine mol. wt 70 000 of HeLa cells harvested at different cell densities. Cell growth: curve I, 0.7 x 1CPcells/ml at start and 1.95 x lo@cells/ml at harvest after 48 h; curve 2, 0.5 x lOa cells/ml at start and 1.02 x lo8 cells/ml at harvest after 24 h.

cell adhesivenesswhich in its turn appears to be dependent on the cell density and/or growth rate of the cells at the time of harvest. If it is admitted that the diminished adhesiveness is caused by an enhancement of the strength of electrostatic repulsion, then from our results it is not apparent whether or not the diminishment in adhesivenessof high density cells is actually accompanied by an increase of the width of the repulsion gap. (2) Agglutination

of human erythrocytes

The results obtained with intact erythrocytes are quite analogous to those obtained with intact HeLa cells (fig. 6). Removal of sialic acid abolishes the capacity for agglutination by the lower molecular weight polylysines, whereas polylysine of mol. wt 100 000 is still agglutinative on such cells.

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Fig. 6. Agglutination of human erythrocytes with poly+lysine of mol. wts (a) 15 000; (b) 23 000; and (c) 100000. Effect of previous neuraminidase treatment (0).

Agglutination curves obtained with polylysines of mol. wts 4 000 and 11 000 on intact erythrocytes are not represented. Their general characteristics are those of the curves of intact cells in fig. 6 a and b. Polylysine of mol. wt 70 000 gives rise to an agglutination curve on intact cells quite similar of fig. 6 c.

to that

Exptl Cell Res 89 (1974

212 Deman and Bruyneel Table 1. Binding of poly+lysine

(mol. wt 23 000) to HeLa cells and human erythrocytes

not preincubated with neuraminidase Concentration of tritiated polylysine in pg/ml of suspension 1. HeLa cells cont. 1.8 x 10s cells/ml 2. Human erythrocytes cont. 160 x lo6 cells/ml Ratio. bound polylysind ’ added polylysine

N.D.b

0.333

0.293

0.280

0.270

0.257

0.360

0.354

0.317

0.542

0.523

0.476

0.490

0.453

0.444

N.D.

N.D.

0.426

0.50

1.0

2.0

3.0

4.0

5.0

6.0

7.5

10.0

a The radioactivity in each sample was measured for 30 min. In the sample containing unbound polylysine, the number of counts varied from 12.441 net cpm at 10 ,ug PL/ml to 455 net cpm at 1.0 pg PL/ml for HeLa cells. For erythrocytes the corresuonding values were 10 358 net cpm and 992 net cpm. The background level was ca 25 cpm. ’ N.D., not determined.

(3) Binding affinity

We examined the question whether the diminished tendency for agglutination at the lower polymer concentration was caused by a diminished binding tendency in this concentration range. Different cell suspensions were prepared to which tritiated polylysine of mol. wt 23 000 was added in the same concentrations as those of the previous experiments. After 30 min of incubation at 37°C with occasional shaking, the suspensions were made homogeneous by reversal of the test tubes and 1 ml was taken for measurement of the radioactivity. Thereafter the suspensions were centrifuged and 1 ml was taken from the supernatant. Substraction of the number of counts found in both samplesand subsequent division by the number of counts present in the homogeneous suspension yields the ratio of bound polylysine to the total added polylysine. Table 1 does not give evidence for a significant change in the binding efficiency with the lower polymer concentrations. The results confirm those reported earlier in which a constant binding efficiency was found with erythrocytes [7] and Ehrlich ascites tumor Exptl CeN Res 89 (1974)

cells [17] in the same concentration range of polylysine as that used in our experiments. (4) Treatment with dilysine

We used dilysine in an attempt to discriminate between the agglutinative (cross-linking) effect of the polylysines and their possible promoting effect on mutual adhesion due to alterations produced in the surface charge of the cells. Dilysine which has a length of ca 7.3 A was thought as being a molecule too short for gap bridging in the case long range electrostatic repulsion should be operative. Because of the similarity in chemical structure it was assumed that no essential difference exists between the binding characteristics of the dimer and those of the polymeric polylysines. (a) Addition of dilysine to cells harvested at different cell densities. From fig. 7 it is ob-

served that addition of dilysine to intact HeLa cells increases their rate of aggregation only to a value which is approximately the same as that obtained on neuraminidase treatment of the cells. No agglutination can be observed on visual inspection of the suspensions.HeLa cells cultured in Eagle’s medium without serum but supplemented with 10 pugdilysine

Long-range repulsion between cells

2 13

The increase in intercellular adhesiveness produced by dilysine can be ascribed to a reduction of the cell surface charge by the adsorption of the dimer. The curves obtained with dilysine ressemble the initial parts of the short chain length polylysine curves which suggeststhat the lower concentrations of those polymers increase cell adhesivenessby a similar process. Consequently it was investigated if cross-linking (agglutination) by short chain length polylysine would be promoted by addition of given amounts of dilysine previous to-or together with-the addition of polylysine. As is demonstrated in fig. 8, previous adwith short chain polylysine.

Fig. 7. Abscissa: dilysine cont. (,ug/ml of suspension); ordinate: rel. cell cont. Effect of dilysine on the aggregation of HeLa cells harvested at different cell densities. Cell growth: curue 1, 0.8 x 106 cells/ml at start and 2.11 x lo6 cells/ml at harvest after 48 h; curve 2, 0.5 x 10’ cells/ ml at start and 1.57 x 10s cells/ml at harvest after 48 h; curve 3, 0.5 x 1V cells/ml at start and 1.08 x lo6 cells/ml at harvest after 24 h. Nase: 0.5 x lo6 cells/ml at start and 1.54 x 106 cells/ml at harvest after 48 h and treated subsequently with neuraminidase.

per ml of suspension, display the same increased adhesiveness after 24 h as that observed after 15 min indicating that the dimer is not taken up by the cells. Therefore the increased aggregation rate with dilysine appears to be due to an enhanced mutual cell adhesiveness.The effect increases with lower adhesivenessof the original cells at harvest. Addition of dilysine to suspensions of desialylated cells or to cells harvested at low density produces no effect. The dimer also has no effect on the adhesivenessof intact or neuraminidase pretreated human erythrocytes. Actual knowledge on the influence of the surface charge on the adhesiveness of the latter cells is in line with this, because it has been established that removal of sialic acid which is responsible for the quasitotality of the electrokinetic surface charge of human erythrocytes [18] does not enhance their mutual adhesiveness[ 13, 191. (6) Effect of dilysine on the agglutination

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Fig. 8. Abscissa: poly+lysine cont. @g/ml of suspension); ordinate: rel. cell cont. Effect of previous dilysine addition on the agglutination of high density cells with poly-L-lysine mol. wt 11 000. Dilysine cont.: curve I, 0 pg/ml; curve 2, 4 ,ug/ml; curve 3, 8 pg/ml. Cell growth: curve 1, 0.5 x 1W cells/ml at start and 2.05 x 1V cells/ml at harvest after 72 h; curve 2, 0.5 x lo8 cells/ ml at start and 1.86 x log cells/ml at harvest after 72 h; curve 3, 0.7 x log cells/ml at start and 2.10 x lo8 cells/ml at harvest after 48 h. Exptl Cell Res 89 (1974)

214 Deman and Bruyneel dition of 4 ,ug dilysine per ml of cell suspension caused the agglutination to start at a lower concentration of poly+lysine mol. wt 11 000. With 8 ,ug dilysine per ml a linear curve was obtained already at the lowest polymer concentration. Fig. 8 also shows the increase in adhesiveness in the absence of polylysine and which should be ascribed to the effect of dilysine.

centration, indicating that agglutination might be absent or strongly diminished in that concentration range. The experiment with radioactive polylysine makes it unlikely that this phenomenon could be ascribed to a decreasedbinding efficiency of the polylysines in that concentration range. The initial irregularity in the agglutination curve disappears on previous desialylation of the cells or when cells are taken from rapidly (5) Elektrokinetic surface charge of growing cultures. Linear agglutination curves HeLa cells are also obtained with long chain polylyThe electrophoretic mobility of intact HeLa sines indicating that the length of the polycells measured at 25°C averaged 1.02 pm mer is of critical importance. The latter reset-lV-l cm (S.D.+O.lO). After removal of sult suggeststhe existence of an electrostatic all neuraminidase releasable sialic acid the repulsion gap which can only be bridged by mobility was reduced to 0.71 (S.D.kO.19). cationic polymers of sufficient length. Measurement of the mobilities in presence Binding of the positively charged polylyof 10 ,ug dilysine per ml yielded mobilities sine molecules to the cell surface diminishes equal to 1.05 (S.D.+O.ll) and 0.73 (SD. the [-potential [6, 71 and hence decreasesthe -t 0.16) for intact and neuraminidase-treated distance over which electrostatic repulsion is HeLa cells respectively. The reduction in operative. It follows that only the initial mobility caused by the enzyme is compa- shape of the agglutination curves at low polymer concentration bears information conrable to that reported [20]. The absence of a dilysine effect on the cerning the original distance over which remobility can be reconciled with the previous pulsion is felt by the cells. The difference befinding of a dilysine effect on the adhesivity tween the agglutination curves of the long by making allowance for the possibility that polylysines (mol. wts 70 000, 100 000) and the dilysine molecules are bound to sites those of the shorter ones (mol. wt G23 000) deeper than the electrokinetic shear plane. is most logically explained by the assumption Mayhew et al. [17] recently found that only that at low polymer concentration the chief a small fraction of polylysine bound to Ehr- effect of the short chain polylysines consists lich ascites tumor cells in the absence of in the gradual neutralization of the surface electric field was electrokinetically detectable. charge with an ensuing diminishment of the distance of the electrostatic repulsion gap. Long chain polylysines apparently do not DISCUSSION need previous neutralization but are able to The agglutination curves of intact HeLa cells span the original repulsion gap between HeLa harvested in conditions of moderate or sta- cells and to cross-link opposing cell surfaces. After removal of sialic acid, linear agtionary growth are shown to display different shapes according to the molecular weight of glutination curves are obtained with all polythe polylysine used. With short chain poly- lysines. This might indicate that sialic acids lysines (mol. wt <23 000) a departure from have an essential role in the building up of linearity is observed at low polymer con- the <-potential on HeLa cells. A similar conExptl Cell Res 89 (1974)

Long-range repulsion between cells

elusion was reached for human erythrocytes by Jan & Chien [2]. However, in this last case it must be realized that sialic acids constitute the quasi-totality of the electrokinetic cell surface charge [2, 181.With HeLa cells this is only about 30% and several other charged groups are known [21]. It is possible that these other groups do not partake in the building up of the c-potential. Alternatively, if they partake, an essential role must be attributed to the sialic acid residues in the maintenance of the charge organization which is the cause for electrostatic repulsion. The dimeric dilysine was used in an attempt to distinguish between the cross-linking effects and the effects of cell surface charge reduction, which will both be found with long chain length polylysines. Dilysine was supposed to be a molecule too short for gap bridging if a repulsion gap should really exist. It was found that dilysine did not produce agglutination but increased mutual adhesion between HeLa cells. Also after previous addition of dilysine a linear agglutination curve was obtained with short chain length polylysine. This, together with the fact that the initial parts of the curves obtained with short chain polylysines on intact cells are similar to those obtained with dilysine, corroborates the thesis that the initial effect of the short chain polylysines consists in the reduction of the cell surface charge and a shortening of the repulsion distance. The increasein mutual cell adhesivenessmust probably be attributed to a diminution of the electrostatic repulsive force. Dilysine produces an increase in mutual cell adhesiveness towards a value which is approximately the same as that obtained on desialylation of the HeLa cells. There is no ground for the belief that dilysine should specifically neutralize the carboxyl charges of sialic acid because polymeric polylysines, which have a similar chemical structure, exert

215

a pronounced agglutinating effect on desialylated HeLa cells. The result rather might confirm the previous finding of an involvement of sialic acids in the regulation of mutual cell adhesion [13, 22, 231 and might possibly indicate an exclusive role of these residues in the maintenance of the cell surface charge organization as already mentioned. The results obtained with human erythrocytes can be interpreted as a further confirmation of the existence of long-range repulsive forces between these cells [1, 21. Therefore they constitute an argument in favour of the soundness of our methodological approach. The contrasting effects of long and short polylysines on desialylated erythrocytes are probably related to considerable differences in the binding affinity in conditions of low cell surface charge density. The electrostatic repulsion effect was found to become more pronounced with HeLa cells from high density cultures. Also the aggregation rate in absence of polylysine which is a measure for mutual adhesiveness, decreaseswith higher cell density. It seems logical to relate both findings and to assume that the enhanced adhesivenessof fast growing cells at low density is caused by the diminishment or disappearance of the longrange repulsive forces. Also the increase in adhesiveness,caused by addition of dilysine, is most pronounced with cells with the lowest adhesiveness.This again is most readily explained by admitting that the dilysine effect on cell adhesion resides on its ability to reduce the strength of the repulsive forces, the largest effect being obtained with highly repulsive cells. The finding of a variation in the strength of electrostatic repulsion during different phases of asynchronous cell growth indicates the existence of a relation between growth and mutual cell adhesiveness. Summarizing we believe that the occurExptl Cell Res 89 (1974)

216 Deman and Bruyneel rence of long-range repulsion forces of HeLa cells is indicated by several strong arguments. (Z) The difference in the agglutination curves obtained with short and long chain polylysines indicates that long polymers are able to cross-link opposing cell surfaces directly, whereas short polymers first have to reduce the cell surface charge which lowers the distance of the repulsion gap. (2) The initial parts of the curves obtained with short polylysines resemble the curves obtained with dilysine, a molecule too short for cross-linking in the presence of longrange repulsion. In both casesthe increase in mutual cell adhesivenesscan be ascribed to a reduction of the cell surface change and a concomitant weakening of the electrostatic repulsive forces. (3) Short chain polylysines are able to cross-link cell surfaces already at the lowest polymer concentrations if dilysine has been added previously. This suggests that the width of the intercellular gap is controlled by the strength of the electrostatic repulsion forces. (4) Cells harvested in the stationary phase of growth have the lowest tendency for mutual adhesion and display the strongest long-range repulsion effect. This is very suggestive for the assumption that electrostatic repulsion should actually be the cause for the diminished adhesiveness of these cells. From this it follows that the strength of mutual adhesion should increase with decreasing repulsive force, which wholly conforms to the interpretation given to our other results.

Exptl Cell Res 89 (1974)

The authors are indebted to Mr G. Debruyne for radioactive labeling of poly+lysine, and to MS R. Van Cauwenberghe and MS F. De Wulf for technical assistance. This study was supported by a grant from the Fund for Medical Research (FGWO), Belgium.

REFERENCES 1. Brooks, D E & Seaman, G V F, Nature 238 (1972) 251. 2. Jan, K M & Chien, S, J gen physiol 61 (1973) 638. 3. Curtis, A S G, Progr biophys mol biol 27 (1973) 316. 4. Pethica, B A, Exptl cell res 8, suppl. (1961) 123. 5. Curtis, A S G, Biol rev Cambr 37 (1962) 82. 6. Katchalsky, A, Danon, D, Nevo, A & De Vries, A, Biochim biophys acta 33 (1959) 120. 7. Nevo, A, De Vries, A & Katchalsky, A, Biochim biophys acta 17 (1955) 536. 8. Mamelak, M, Wissig, S L, Bogoroch, R & Edelman, I S, J membr biol 1 (1969) 144. 9. Quinton, P M & Philpott, C W, J cell biol 56 (1973) 787. 10. Miller, A 0 A, Arch biochem biophys 122 (1967) 270. 11. Deman, J & Bruyneel, E, Exptl cell res 81 (1973) 151 ___.

12. Barton, N W & Rosenberg, A, J biol them 248 (1973) 7353. 13. Deman, J J, Bruyneel, E A & Mareel, M M, J cell biol 60 (1974) 641. 14. Rifkin, D B, Compans, R W & Reith, E, J biol them 247 (1972) 6432. 15. Bangham, A D; Pethica, B A & Seaman, G V F, Biochemistry 69 (1958) 12. 16. Seaman, G V F, J supramol struct 1 (1973) 437. 17. Mayhew, E, Harlos, J P & Juliano, R L, J membr biol 14 (1973) 213. 18. Eylar, E H, Madoff, M A, Brody, 0 V & Oncley, J L, J biol them 237 (1962) 1992. 19. Kemp, R B, J cell sci 6 (1970) 751. 20. Ruhenstroth-Bauer, G, Fuhrmann, G F, Kubler, W, Rueff. F & Munk. K. Z Krebsforsch 65 (1962) 37.’ 21. Sherbet, G V, Lakshmi, M S & Rao, K V, Exptl cell res 70 (1972) 113. 22. Vicker, M G & Edwards, J G, J cell sci 10 (1972) 759, 23. Sauter, C, Lindenmann, J & Gerber, A, Eur j cancer 8 (1972) 451. Received April 9, 1974 Revised version received June 18, 1974