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Studies on cellular adhesion in tissue culture
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Experimental Cell Research83 (1974) 311-318
STUDIES ON CELLULAR
ADHESION
IN TISSUE CULTURE
XIV. Positively Charged Surface Groups and the Rate of Cell Adhesion L. WEISS Department
of Experimental Pathology, Roswell Park Memorial Buffalo, N. Y. 14203, USA
Institute,
SUMMARY Attempts were made to identify positively charged groups at the surfaces of Ehrlich ascites tumour (EAT) cells, and particles of polystyrene polymer which had adsorbed proteins after incubation in serum-containing culture medium. The cells and particles were treated with 2,4,6-trinitrobenzene sulphonic acid (TNBS) or 2,3-dimethylmaleic anhydride (DMA), which react with amino and other cationic groups. The increases in cell and particle anodic electrophoretic mobility were consistent with approx. 5 % of the total surface charge of each, being due to positively charged groups. The effects of DMA or TNBS treatment of the cells and/or polystyrene surfaces, on the rates of cell adhesion to these surfaces were then determined. The significantly slower rates of adhesion after some modes of treatment suggest that positively charged groups at the surfaces of EAT cells play a part in their initial contact with and adhesion to, protein-coated plastic surfaces. However, quantitatively the role of cationic groups is a minor one in this part of the adhesion process.
Many electrokinetic studies have revealed that a very wide variety of cells from vertebrates carry a net negative charge at their surfaces, which is thought to partially regulate many different types of cell contact interactions [ 11, 121.Purely electrostatic interactions between cell and other surfaces separated by distances greater than approx. 2 nm will be largely determined by net surface charge, and will result in mutual repulsion. However, as the separation between the surfaces decreases, electrostatic interactions between discrete units of ionized species are expected to become increasingly important, and the possibility arises that coulombic attractions between negatively and positively charged sites at opposing cell surfaces could promote cell contact [17].
The studies described here fall into two parts. First, in an attempt to demonstrate surface amino groups, and other anionic sites, the electrophoretic mobilities of Ehrlich ascites tumour cells treated with reagents for these groups were measured. Mandatory [16] associated observations were made on cell integrity and leakage as a consequence of this treatment. The reaction of these reagents with serum proteins adsorbed to the surfaces of polymeric styrene spheres was also followed by electrokinetic methods. Second, the rates of cell adhesion to polystyrene microtest plate surfaces in the presence of serum-containing medium or phosphate buffered saline were determined, after reacting the cells and/ or plastic surfaces with the reagents. Exptl Cell Res 83 (1974)
312 L. Weiss MATERIALS AND METHODS Cells. Ehrlich-Lettrk hyperdiploid ascites carcinoma (EAT) cells were grown at 37°C in suspension culture at densities of 2 to 6 x 105/ml, in medium RPM1 1630 [8] supplemented with 5 “/: foetal calf serum (FCS). Viability, assessedby trypan blue exclusion tests, was in excess of 97 %. Plusticpariicles. Styrene divinylbenzene latex particles (Dow Diagnostics, Ind.; Lot. No. 2F2B) having a mean diameter of 5.7 pm (t 1.5 S.D.), were obtained as a suspension in distilled water containing 10% solids. In order to approximate the reactions occurring in culture at the surfaces of the microtest plates, which are fabricated from styrene polymer, the particles were incubated in culture media or PBS, with and without 5% foetal calf serum for 30 min at 37°C. They were then treated in a similar manner to the cells.
ReaPent.7. EAT ceils and the stvrene spheres were in-
cubated with either 2,4,6-trinitrobeniene sulphonic acid (TNBS) or 2.3-dimethvlmaleic anhvdride (DMA) which, undkr defined co
This followed the description given by Mehrishi [7]. Cells (or particles) were washed twice in 0.145 M NaCl adjusted to pH 7.2 with 0.5 N NaHCO,, and resuspended in 0.145 M NaCl (pH 6.5 to 6.8), to give a concentration of 2 y 10” cells/ml. To 10 ml of the ‘experimental’ group, was added 5 /cl DMA in ethanol (10e2 M), and 5 ~1 of ethanol alone was added to the controls. The mixtures were incubated at 25°C for 15 min. Culture medium or PBS (&FCS) was added to the suspensions used for adhesion tests, whereas those used for electrophoresis measurements were diluted with saline. The reaction of DMA with amino groups is shown in fig. 1b, which indicates the substitution of an amino group by a negatively charged species. DMA-treatment.
Cell zkbility. Routine checks of cell viability were made, by means of trypan blue exclusion tests during every experiment. In addition, the cultures on which rates of adhesion were determined, were reincubated and re-examined after 24 and 48 h for morphologic and reproductive integrity.
Cell leakage. NazjlCrO, having 25 ,&i activity was added to 1.5 x 10’ EAT cells suspended in 2 ml of serum-containing medium. After 30 min incubation Exptl Cell Res 83 (1974)
at 37’C, the cells were washed once and resuspended in medium and kept at 4°C for 30 min. They were then washed twice in medium and resuspended in either 0. I45 M NaCl (pH 6.5) or PBS (pH 8). The cells were next treated with DMA, or TNBS, with appropriate controls, as described above. Following centrifugation (300 g) at 4°C for 20 min, the deposits and supernatant fluids were counted in a gamma-spectrometer, and the counts on the fluids (leakage) expressed as a percentage of deposit plus supernate. Partide electrophoresis. The electrophoretic mobilities of cells and particles in specified suspending fluids, were measured in a cylindrical cell apparatus of the type described by Bangham et al. [I]. All measurements were made at 37”C, with reversal of current, in a known voltage gradient applied between grey, sintered platinum electrodes, within 5-10 min of completion of the described reactions.
Rate of cell adhesion. This was determined using polystyrene microtest plates (Falcon Plastics, Los Angeles, Calif.), as described in detail elsewhere [13]. One drop (0.0068 ml) of cell suspensions, containing 2.5 IO4 cells/ml in specified solutions, was placed into each well through 25 gauge needles attached to 1 ml (tuberculin) syringes. After incubation for periods of up to 2 h in moist 5 % CO, in air, the plates were inverted for 1 h and the residual adherent cells then counted. The numbers of adherent cells in individual wells are expressed as percentages of the mean of the controls for each plate. In some experiments, the microtest plate surfaces were modified. in a similar manner to those of the styrene particles. The plates were first filled with RPM1 1630 1 5 I:~,FCS. incubated for 30 min at 37°C and then reacted with’TNBS or DMA as described above. In the first group of experiments, cells reacted with either TNBS or DMA. and their appropriate controls, were suspended in KPMI 1630~j-546 FCS and allowed to adhere to unmodified microtest plates. In the second group of experiments, rates of adhesion were determined for unmodified cells, suspended in PBS and 5 :; FCS, adhering to modified microtest plate surfaces. In the third group of experiments, reacted and non-reacted cells, suspended in PBS= FCS, were allowed to adhere to the modified microtest plate surfaces.
RESULTS Cell uiabilities No significant changes in cell viability were demonstrably associated with the treatments associated with the electrokinetic and adhesion experiments described below.
Leakage experiments In experiments with tubes were used for
EAT cells, in which 28 each group, the PBS
Cell adhesionand positive charge 313 Table 1. Electrophoretic mobilities of polyJtyreneparticlesfirst incubated with serum-containing medium and then reacted with TNBS or DMA together with appropriate controls, measuredat 37”C, in PBS + 5 % foetal calf serum Serum-treated particles reacted with
Mean mobility pm set-l volt-’ cm +S.E. (No. of obs.)
% of control
f-test (vs control)
105 102 107
0.1 :‘p z-o.05 0.6;p>O.5 0.01 -‘p :~o.ool
106 109 111
0.01 :‘p 0.001 p dO.001 p : 0.001
108
p ‘-0.001
106
0.05 ‘p -, 0.02
Measured in PBS
PBS (control) TNBS 3.4 i: lo-” M TNBS 3.4 x 1O-5M TNBS 3.4 ): 1O-aM Ethanol (control) DMA 5 i lo-” M DMA 5 ,,:1O-5M DMA 2.5 ): IO-& M
~ 1.08 kO.02 -1.13F0.02 - 1.10~0.02 - 1.15 kO.02 ~l.lOkO.02 -1.17-tO.02 -1.19kO.02 -- 1.22f0.02
(165) (151) (149) (149) (116) (142) (118) (120)
--0.98kO.02 ~ 1.06t0.01 -0.88~0.01 -0.93f0.02
(118) (154) (100) (112)
Measured in PBS i 5 :b FCS
PBS (control) TNBS Ethanol (control) DMA
controls exhibited a mean of 16.0+0.91 $4 leakage of Wr, the ethanol controls showed 17.4+ 2.0 Y/o and the DMA-treated cells 15.2F 0.81 ~6. These differences are not significant. Similar results with TNBS have been reported previously [ 151.
Electrophoretic mobiliries Cells: The mobilities of TNBS-treated EAT cells were -1.25 (kO.04 SE., n=lOO) pm se& volt-l cm, compared with - 1.19 (+ 0.03; 100) for the PBS controls. This 5% increase is not significant (0.3 >p >0.2). Incubation with DMA resulted in a mean mobility of -1.28 (20.03; 100) compared with -ml.15 (kO.03; 100) for the ethanol control. The increased surface negativity of the DMAtreated cells over both the ethanol (11 %) and PBS (8 :b) controls is statistically significant (p -0.01). In 5 other separate experiments, PBS-, ethanol- and DMA-treated EAT cells were washed twice and resuspended in saline before their electrophoretic mobilities were de-
termined. Compared with saline-treated cells, increases of 5 and 11 % were detected in the ethanol and DMA-treated cells respectively. The DMA-treated cells showed a mean increase of 5.8 y0 in their net surface negativity over their ethanol controls (~~0.05). Therefore, in the later adhesion experiments, the cells were not washed after treatment with the various reagents. In other experiments, after treatment with the reagents, EAT cells were centrifuged and fresh EAT cells were incubated with the supernatant fluids. No significant changes in mobilities (i.e. -0.4 to -1.5 o/o) were observed in the ‘fresh’ cells. Therefore, the omission of washing after treatment of EAT cells with DMA or TNBS, does not leave amounts of these reagents in the medium which are significant in the present context.
Particles The mean electrophoretic mobilities of the particles suspended in RPM1 1630 was -~ 1.94 (f0.02; 172), compared with -0.93 (kO.01; Exptl Cell Res 83 (1974)
314 L. Weiss 122) for particles suspended and measured in RPM1 1630 + 5 % FCS. On washing and resuspending these particles in RPM1 1630, their mobilities showed little change ( - 1.05 i 0.02: 64). That the reduction in anodal mobility following incubation and measurement in serum-containing medium was due to adsorption of serum constituents, as distinct from viscosity differences is indicated by the observation that the mean mobilities of human erythrocytes in medium with and without serum were -1.39 (10.02; 100) and -~ 1.41 (lO.01; 100) respectively. The tabulated results therefore refer to experiments made on ‘coated’ particles. The results given in table 1 show that when coated styrene particles, were treated with the ‘usual’ concentrations of the two reagents, small increases in anodal mobility were observed when the particles were suspended in PBS. These increases are statistically significant after DMA-treatment, and insignificant after TNBS-treatment. It is also seen that increasing the final concentrations of TNBS IOO-fold, and of DMA 50-fold, only produced increases of net negativity of 2 and 5 96 respectively, over the effect of the usual concentrations. As shown in table 1, the increases in the negativity of the serum-treated particles produced by DMA and TNBS are manifest when the particles are electrophoresed in PBS + 5 % FCS.
Rates of cell adhesion The numerical results of the experiments with the ‘unmodified’ microtest plates are summarized in table 2. In RPM1 1630+5 % FCS, apart from the statistically insignificant (0.2 >p >O. 1) reduction in the rate of adhesion of EAT cells after 30 min initial exposure to TNBS, both modes of treatment produced significant (p
of adhesion of the EAT cell compared with their PBS or ethanol controls. The effects of reacting positively charged groups on microtest plate surfaces after prior incubation with serum-containing medium, on the rates of adhesion of unmodified cells (after 2 h), are summarized in table 3. Neither DMA nor TNBS produced a significant change in rate. The rates of adhesion of modified and unmodified cells, to modified and unmodified, serum-treated microtest plates, in PBS + 5 % FCS are shown in table 4. By comparison of the results of experiments 1 and 2 it can be seen that in the absence of serum in the suspending medium, treatment of both the cells and the microtest plate surfaces with DMA produced a significant decrease in adhesion rate, which was greater than that produced by treating the cells alone. This latter treatment also decreased the rate to 85 % of that of the ‘untreated’ cells. These rate decreases were not observed when the cells were suspended in PBS in 5 % FCS. The results of experiments 5 and 6 show significant decreases in the adhesion rates of cells suspended in PBS, following treatment of both them and the microtest plates with TNBS, compared with either untreated cells and surfaces (expt 5) or TNBStreated cells and untreated surfaces (expt 6). Comparison of experiments 5 and 6, shows no significant decrease in adhesion rate due to TNBS treatment of the cells alone. These effects following TNBS-treatment were also observed when the cells were suspended in PBS containing serum (expts 7, 8). DISCUSSION All current models of the molecular architecture of the cell periphery depict proteins at the cell surface, and some postulate the presence of the hydrophilic heads of phospholi-
Cell adhesion and positive charge pids [lo]. Therefore, among the positively charged groups possibly present at the cell surface are the side chain amino-groups of lysine and hydroxylysine; terminal protein CIamino-groups; imidazolic groups of histidine residues; guanidino-groups of arginine and phospholipid and glycolipid amines. In summary, the electrokinetic experiments indicate the existence of bound cationit sites, accounting for approx. 5% of the surface charge of both the EAT cells and styrene polymer particles, after the latter were exposed to serum-containing culture medium. Analogous changes to those observed at the surfaces of the plastic particles are thought to occur at the surfaces of the microtest plates, where adsorption of protein from similar culture medium has been detected by attenuated total reflectance spectroscopy in the infra-red [13]. The results also show that these positively charged groups react with TNBS and DMA under the conditions used here. lOO- and 50-fold increases respectively, in the concentrations of these reagents produced comparatively little change in the electrophoretic mobilities of the particles over those produced by the ‘usual’ levels. The effectiveness of TNBS and DMA is therefore unlikely to be limited by concentration, although it may be limited by the physicochemical nature of the reacting cationit sites [9]. It should be noted that TNBS reacts with unprotonated amino groups, and that reaction of the CI- and a-amino groups is second order. Although in simple amino acid solutions, the SH-group of N-acetylcysteine was found by Freedman & Radda [3] to be more reactive to TNBS than many amino groups, the resulting -S-TNP group is much more unstable at alkaline pH than the -HN-TNP group [5]. Our failure to detect a reaction of cationic groups at cell surfaces with TNBS by electrophoresis therefore, probably reflects
3 15
Table 2. Rates of adhesion of TNBS- and D MA-treated cells suspended in serum-containing medium, to microtest plate surfaces, expressed as percentages of appropriate controls “; adhent cells ri SE. 30 min TNBS PBS Controls 60 min TNBS PBS Controls 120 min TNBS PBS Controls 30 min DMA Ethanol Controls 60 min DMA Ethanol Controls 120 min DMA Ethanol Controls
96.8 + 1.7 (249) 100 + 1.7 (291) 90.4 + 1.7 (279) 100 + 1.6 (285) s1.5+1.4 (373) 100 + 1.3 (360) 85.0 k2.2 (85) 100 t-2.3 (85) 84.6 & 1.9 (86) 100 +2.1 (81) 92.5 & I .4 (146) 100 & 1.5 (143)
their low density at the cell surface, rather than simple competititve inhibition for TNBS by sulphydryl groups. The interpretation of adhesion experiments following TNBS treatment is further complicated because reaction of cells with sulphydryl-binding reagents results in a diminution of their rates of adhesion to a variety of surfaces [4]. Some indication of the extent of the reaction of DMA with sulphydryl groups comes from the work of Dickens & Cooke [2]. Under similar reaction conditions to those used in the present studies, namely at 25°C in an aqueous environment buffered at pH 8, when DMA reacts with cysteine, SH-groups are lost at a rate of 0.08 I/mole-l min-l compared with 70 for maleic anhydride. Acid production additional to hydrolysis, due to the formation of carboxyl groups was undetectable (i.e. ~0.01 1 mole-l min-l), indicating little or no acylation of the SH-groups, Exptl Cell Res 83 (1974)
316 L. Weiss Table 3. Adhesion of ‘untreated’ cells to microtest plate surfaces treated as indicated Adhesion is expressed as % of appropriate controls after 2 h incubation Coated’ microtest plate incubated with
Suspending fluid
% adherent cel1sIS.E. (No. of obs)
DMA Ethanol DMA Ethanol TNBS PBS TNBS PBS
PBS v 5 % FCS PBS I 5 “b FCS PBS PBS + 5 :b FCS 5 “6 FCS PBS PBS
97.3 F 2.9 (60) 100.0+2.2 (60) 100.6i 3.0 (60) 100.0+2.5 (60) 106.9+ 2.3 (60) 100.0& 1.9 (59) 106.8& 3.3 (60) 100.0& 2.6 (59)
although there may be some addition to the double-bond. The increase in surface negativity following DMA-treatment, was demonstrated in the absence of viability changes and detectable leakage of intracellular contents, and a similar lack of toxic effects has been previously reported for TNBS [14, 151.The electrophoretic mobility data indicate that approx. 5 “/b of the total charges at the electrokinetic surfaces of EAT cells are cationic, since following their removal with TNBS there was an approx. 5 y0 increase in surface negativity, and following their removal and replacement with negative charges after DMA-treatment, there was an approx. 10% increase. If the hypothetical cationic surface groups are concentrated in clusters of the type demonstrated for some anionic groups at the surfaces of EAT cells [17, 181, then the importance and effectiveness of small total numbers (per cell) of positively charged groups in promoting localized regions of cell contact with negatively charged regions of an opposing surface would be disproportionate to their total number. Cationic groups may play a role in determining the rate of cell adhesion in the model system studied here, and possibly in cell-cell adhesion, in 3 non-exclusive ways, namely: Exptl Cell Res 83 (1974)
t-tests 0.5 ‘p -,0.4 0.9 p-,0.8 0.05:~p-.o.o2 0.2:,p-,O.l
(1) Positively charged groups on cells may bind to negatively charged groups on the microtest plate surfaces. (2) Positively charged groups adsorbed to microtest plate surfaces from the serumcontaining medium, may react with negatively charged groups at the cell surfaces. (3) Positively charged groups on macromolecules in the medium may bind to negatively charged groups on the cell and microtest plate surfaces, thereby ‘gluing’ them together. Some indication of the quantitive importance of these possible mechanisms is expected to be reflected in decreases in the rates of cell adhesion to microtest plates, compared with appropriate controls, in simple media with and without macromolecules, after reacting cationic sites at the cell and/or microtest plate surfaces. The experiments summarized in table 2 indicate that in serum-containing culture medium in which the cells ‘normally’ grow, prior treatment of the cells with either TNBS or DMA, produced small but statistically significant changes in their rate of adhesion to microtest plates. Within interpretative limitations to be discussed later, this suggests that positively charged groups at the surface of EAT cells play a demonstrable part in deter-
Cell adhesion and positive charge
3 17
Table 4. Adhesion of cells to microtest plates after 2 h, when both were reacted with DMA or TNBS Adhesion is expressedas a :: of appropriate controls
Expt
Cells
Microtest plates
I
Ethanol DMA DMA DMA DMA DMA Ethanol DMA PBS TNBS TNBS TNBS PBS TNBS TNBS TNBS
Ethanol DMA Ethanol DMA Ethanol DMA Ethanol DMA PBS TNBS PBS TNBS PBS TNBS PBS TNBS
2 3 4 5 6 7 8
Suspending fluid
% adherent cells& S.E. (No. Obs)
PBS PBS PBS PBS PBS+ 5 56FCS PBS+ 5 % FCS PBS+5% FCS PBS+ 5 % FCS PBS PBS PBS BPS PBS+ 5 % FCS PBS i 5% FCS PBS : 5% FCS PBS in5 % FCS
loo.O& 1.4 (179) 76.4* 1.2 (180) lOO.O+lS (180) 90.3i I.9 (180) 100.0~1.9 (115) 99.6-tl.8 (120) 100.0~1.9 (117) 101.8k2.1 (119) 100.0+1.9 (116) 88.8* 1.7 (120) 100.0kl.9 (118) 86.1i2.0 (116) 100.0~1.9 (117) 90.7kl.8 (117) 100.0~1.6 (178) 90.5* 1.5 (179)
mining their rate of adhesion. This role is not limiting, because the majority of cells adhere in spite of the treatment. The experiments summarized in table 3 indicate that in the presence or absence of serum, when no other macromolecules were deliberately added to the medium, positively charged groups on the substratum play no demonstrable role in determining the rates of cell adhesion. In these and later experiments, PBS was used instead of regular culture medium to avoid the presence of amino acids with reactive amino groups. It should be noted however that PBS, with or without serum is an inadequate growth medium. Therefore, as metabolic processes play some part in the mode of establishment of cell adhesion, it is not surprising that the results obtained with PBS were not numerically similar to those obtained in culture medium. In the absence of serum, after reacting both cell and substratum surfaces, the effects of absence
f-tests p .‘O.OOl pe 0.001 0.9 -.p .0.8 0.6 “p c 0.5 p e. 0.001
p.~o.OOl P.‘O.OOl p ,’ 0.001
or major removal of positively charged adhesion-promoters were expected to be maximal. The data given in table 4 show that in fact, in the case of DMA-treated cells, there was a maximal reduction in adhesion rate compared with untreated cells and microtest plates (expt 1) and a lesser effect compared with treated cells and untreated microtest plates (expt 2). The greater reduction seen in expt 1 than expt 2, suggests the synergistic effect expected if positively charged groups on both surfaces contribute to their adhesion. Although reductions in adhesion-rate were obsevered in analogous experiments with TNBStreatment (expts 5, 6), no evidence of synergism was observed. This latter finding is more consistent with the data given in table 3. The observation that in the presence of serum, prior TNBS-treatment of both surfaces produced significant reductions in adhesion-rate (expts 7, 8) whereas DMA-treatment did not (expts 3, 4), suggests more complex secondExptl Cell Res 83 (1974)
318 L. Wiess
-CONH
R
Fig. I. Reaction of amino-groups with (a) TNBS and (b) DMA, under conditions specified in text.
order interactions of the cells than can be currently interpreted. In common with many other reagents, it might be argued that the effects of the present reagents on the rate of EAT cell adhesion were due to steric hindrance as indicated in fig. 1, rather than loss of positively charged surface groups per se. Steric hindrance of this type could only occur if other sites involved in this part of the contact process were close enough to the reacted sites to be masked by the comparatively small TNBS or DMA molecules. At present there is no supportive evidence for this possibility. Unfortunately, direct experimental tests involving the use of reagents of smaller molecular size, such as formaldehyde are impractical since these enter cells, modify their mechanical properties and are toxic; and even with these reagents, the basic question of the effects of steric hindrance and/or conformational change would remain unanswered. It could also be argued in the case of the DMA-treated cells, that the reduction in adhesion rate is due to the acquisition of additional negative charges (fig. 1) rather than the loss of positive charges. However, this argument is clearly not applicable to the reduction
in adhesion
TNBS-treatment. Exptl Cell Res 83 (1974)
rate
observed
after
The present experimental results in general, support the concept at least in the system studied here, that DMA and TNBS- ‘sensitive’ cationic groups at the cell and substratum surfaces influence the initial rates of adhesion, and that this parameter is sensitive to the presence of serum in the culture medium. However, as cell adhesion occurs after both surfaces have been reacted with either TNBS or DMA, without the addition of the hypothetical serum ‘glues’, and allowing for the reaction of some of the TNBS with sulphydryl groups as discussed above, it would appear that cationic surface sites play at most a minor rather than an all-or-none role in this type of cell contact reaction. I am grateful to Dr Geoffrey Seaman for his constructive criticism of this work and Dr Jake Belle for his advice on relevant organic chemistry. My sincere thanks are also due to MS T. L. Cudney, MS C. Maver and Mr D. Huber for their assistance. This work was partially supported by Grant BC-87F from the ACS.
REFERENCES I. Bangham, A D, Flemans, R, Heard, D H & Seaman, G V F, Nature (Lond) 182 (1958) 642. 2. Dickens, F & Cooke, J, Brit j cancer 19 (1965) 404. 3. Freedman. R B & Radda. G K. Biochem i 108 (1968) 383’. 4. Grinnell, F, Milam, M & Srere, P A, Arch biothem biophys 153 (1972) 193. 5. Kotaki, A, Harada, M & Yagi, K, J biochem Tokyo 55 (1964) 553. 6. Mehrishi, J N, Eur j cancer 6 (I 970) 127. - Progr biophys mol biol 25 (1972) I. s7: Moore,-G E,.Sandberg, A A & Ulrich, K, J natl cancer inst 36 (1966) 405. 9. Papahadjopoulos, D &Weiss, L, Biochim biophys acta 183 (1969) 417. 10. Weiss, L, Int rev cytol 26 (1969) 63. Il. -The chemistry of biosurfaces (ed M L Hair) vol. 2, p. 377. Dekker, New York (1972). 12. - Exptl cell res 74 (1972) 21. 13. - Ibid 81 (1973) 57. 14. Weiss, L, Belle, J & Cudney, T L, Int j cancer 3 /
I
11968) 795.
15. tiei&, L & Cudney, T L, Int j cancer 4 (I 969) 776. 16. Weiss. L & Harlos. J P, Progr surface sci I (1971) 355. 17. Weiss, L, Jung, 0 S & Zeigel, R, Int j cancer 9 (1972) 48. 18. Weiss, L & Zeigel, R, J theor biol 34 (1972) 21.
Received June 4, 1973 Revised version received August 28, 1973
Experimental Cell Research83 (1974) 311-318
STUDIES ON CELLULAR
ADHESION
IN TISSUE CULTURE
XIV. Positively Charged Surface Groups and the Rate of Cell Adhesion L. WEISS Department
of Experimental Pathology, Roswell Park Memorial Buffalo, N. Y. 14203, USA
Institute,
SUMMARY Attempts were made to identify positively charged groups at the surfaces of Ehrlich ascites tumour (EAT) cells, and particles of polystyrene polymer which had adsorbed proteins after incubation in serum-containing culture medium. The cells and particles were treated with 2,4,6-trinitrobenzene sulphonic acid (TNBS) or 2,3-dimethylmaleic anhydride (DMA), which react with amino and other cationic groups. The increases in cell and particle anodic electrophoretic mobility were consistent with approx. 5 % of the total surface charge of each, being due to positively charged groups. The effects of DMA or TNBS treatment of the cells and/or polystyrene surfaces, on the rates of cell adhesion to these surfaces were then determined. The significantly slower rates of adhesion after some modes of treatment suggest that positively charged groups at the surfaces of EAT cells play a part in their initial contact with and adhesion to, protein-coated plastic surfaces. However, quantitatively the role of cationic groups is a minor one in this part of the adhesion process.
Many electrokinetic studies have revealed that a very wide variety of cells from vertebrates carry a net negative charge at their surfaces, which is thought to partially regulate many different types of cell contact interactions [ 11, 121.Purely electrostatic interactions between cell and other surfaces separated by distances greater than approx. 2 nm will be largely determined by net surface charge, and will result in mutual repulsion. However, as the separation between the surfaces decreases, electrostatic interactions between discrete units of ionized species are expected to become increasingly important, and the possibility arises that coulombic attractions between negatively and positively charged sites at opposing cell surfaces could promote cell contact [17].
The studies described here fall into two parts. First, in an attempt to demonstrate surface amino groups, and other anionic sites, the electrophoretic mobilities of Ehrlich ascites tumour cells treated with reagents for these groups were measured. Mandatory [16] associated observations were made on cell integrity and leakage as a consequence of this treatment. The reaction of these reagents with serum proteins adsorbed to the surfaces of polymeric styrene spheres was also followed by electrokinetic methods. Second, the rates of cell adhesion to polystyrene microtest plate surfaces in the presence of serum-containing medium or phosphate buffered saline were determined, after reacting the cells and/ or plastic surfaces with the reagents. Exptl Cell Res 83 (1974)
312 L. Weiss MATERIALS AND METHODS Cells. Ehrlich-Lettrk hyperdiploid ascites carcinoma (EAT) cells were grown at 37°C in suspension culture at densities of 2 to 6 x 105/ml, in medium RPM1 1630 [8] supplemented with 5 “/: foetal calf serum (FCS). Viability, assessedby trypan blue exclusion tests, was in excess of 97 %. Plusticpariicles. Styrene divinylbenzene latex particles (Dow Diagnostics, Ind.; Lot. No. 2F2B) having a mean diameter of 5.7 pm (t 1.5 S.D.), were obtained as a suspension in distilled water containing 10% solids. In order to approximate the reactions occurring in culture at the surfaces of the microtest plates, which are fabricated from styrene polymer, the particles were incubated in culture media or PBS, with and without 5% foetal calf serum for 30 min at 37°C. They were then treated in a similar manner to the cells.
ReaPent.7. EAT ceils and the stvrene spheres were in-
cubated with either 2,4,6-trinitrobeniene sulphonic acid (TNBS) or 2.3-dimethvlmaleic anhvdride (DMA) which, undkr defined co
This followed the description given by Mehrishi [7]. Cells (or particles) were washed twice in 0.145 M NaCl adjusted to pH 7.2 with 0.5 N NaHCO,, and resuspended in 0.145 M NaCl (pH 6.5 to 6.8), to give a concentration of 2 y 10” cells/ml. To 10 ml of the ‘experimental’ group, was added 5 /cl DMA in ethanol (10e2 M), and 5 ~1 of ethanol alone was added to the controls. The mixtures were incubated at 25°C for 15 min. Culture medium or PBS (&FCS) was added to the suspensions used for adhesion tests, whereas those used for electrophoresis measurements were diluted with saline. The reaction of DMA with amino groups is shown in fig. 1b, which indicates the substitution of an amino group by a negatively charged species. DMA-treatment.
Cell zkbility. Routine checks of cell viability were made, by means of trypan blue exclusion tests during every experiment. In addition, the cultures on which rates of adhesion were determined, were reincubated and re-examined after 24 and 48 h for morphologic and reproductive integrity.
Cell leakage. NazjlCrO, having 25 ,&i activity was added to 1.5 x 10’ EAT cells suspended in 2 ml of serum-containing medium. After 30 min incubation Exptl Cell Res 83 (1974)
at 37’C, the cells were washed once and resuspended in medium and kept at 4°C for 30 min. They were then washed twice in medium and resuspended in either 0. I45 M NaCl (pH 6.5) or PBS (pH 8). The cells were next treated with DMA, or TNBS, with appropriate controls, as described above. Following centrifugation (300 g) at 4°C for 20 min, the deposits and supernatant fluids were counted in a gamma-spectrometer, and the counts on the fluids (leakage) expressed as a percentage of deposit plus supernate. Partide electrophoresis. The electrophoretic mobilities of cells and particles in specified suspending fluids, were measured in a cylindrical cell apparatus of the type described by Bangham et al. [I]. All measurements were made at 37”C, with reversal of current, in a known voltage gradient applied between grey, sintered platinum electrodes, within 5-10 min of completion of the described reactions.
Rate of cell adhesion. This was determined using polystyrene microtest plates (Falcon Plastics, Los Angeles, Calif.), as described in detail elsewhere [13]. One drop (0.0068 ml) of cell suspensions, containing 2.5 IO4 cells/ml in specified solutions, was placed into each well through 25 gauge needles attached to 1 ml (tuberculin) syringes. After incubation for periods of up to 2 h in moist 5 % CO, in air, the plates were inverted for 1 h and the residual adherent cells then counted. The numbers of adherent cells in individual wells are expressed as percentages of the mean of the controls for each plate. In some experiments, the microtest plate surfaces were modified. in a similar manner to those of the styrene particles. The plates were first filled with RPM1 1630 1 5 I:~,FCS. incubated for 30 min at 37°C and then reacted with’TNBS or DMA as described above. In the first group of experiments, cells reacted with either TNBS or DMA. and their appropriate controls, were suspended in KPMI 1630~j-546 FCS and allowed to adhere to unmodified microtest plates. In the second group of experiments, rates of adhesion were determined for unmodified cells, suspended in PBS and 5 :; FCS, adhering to modified microtest plate surfaces. In the third group of experiments, reacted and non-reacted cells, suspended in PBS= FCS, were allowed to adhere to the modified microtest plate surfaces.
RESULTS Cell uiabilities No significant changes in cell viability were demonstrably associated with the treatments associated with the electrokinetic and adhesion experiments described below.
Leakage experiments In experiments with tubes were used for
EAT cells, in which 28 each group, the PBS
Cell adhesionand positive charge 313 Table 1. Electrophoretic mobilities of polyJtyreneparticlesfirst incubated with serum-containing medium and then reacted with TNBS or DMA together with appropriate controls, measuredat 37”C, in PBS + 5 % foetal calf serum Serum-treated particles reacted with
Mean mobility pm set-l volt-’ cm +S.E. (No. of obs.)
% of control
f-test (vs control)
105 102 107
0.1 :‘p z-o.05 0.6;p>O.5 0.01 -‘p :~o.ool
106 109 111
0.01 :‘p 0.001 p dO.001 p : 0.001
108
p ‘-0.001
106
0.05 ‘p -, 0.02
Measured in PBS
PBS (control) TNBS 3.4 i: lo-” M TNBS 3.4 x 1O-5M TNBS 3.4 ): 1O-aM Ethanol (control) DMA 5 i lo-” M DMA 5 ,,:1O-5M DMA 2.5 ): IO-& M
~ 1.08 kO.02 -1.13F0.02 - 1.10~0.02 - 1.15 kO.02 ~l.lOkO.02 -1.17-tO.02 -1.19kO.02 -- 1.22f0.02
(165) (151) (149) (149) (116) (142) (118) (120)
--0.98kO.02 ~ 1.06t0.01 -0.88~0.01 -0.93f0.02
(118) (154) (100) (112)
Measured in PBS i 5 :b FCS
PBS (control) TNBS Ethanol (control) DMA
controls exhibited a mean of 16.0+0.91 $4 leakage of Wr, the ethanol controls showed 17.4+ 2.0 Y/o and the DMA-treated cells 15.2F 0.81 ~6. These differences are not significant. Similar results with TNBS have been reported previously [ 151.
Electrophoretic mobiliries Cells: The mobilities of TNBS-treated EAT cells were -1.25 (kO.04 SE., n=lOO) pm se& volt-l cm, compared with - 1.19 (+ 0.03; 100) for the PBS controls. This 5% increase is not significant (0.3 >p >0.2). Incubation with DMA resulted in a mean mobility of -1.28 (20.03; 100) compared with -ml.15 (kO.03; 100) for the ethanol control. The increased surface negativity of the DMAtreated cells over both the ethanol (11 %) and PBS (8 :b) controls is statistically significant (p -0.01). In 5 other separate experiments, PBS-, ethanol- and DMA-treated EAT cells were washed twice and resuspended in saline before their electrophoretic mobilities were de-
termined. Compared with saline-treated cells, increases of 5 and 11 % were detected in the ethanol and DMA-treated cells respectively. The DMA-treated cells showed a mean increase of 5.8 y0 in their net surface negativity over their ethanol controls (~~0.05). Therefore, in the later adhesion experiments, the cells were not washed after treatment with the various reagents. In other experiments, after treatment with the reagents, EAT cells were centrifuged and fresh EAT cells were incubated with the supernatant fluids. No significant changes in mobilities (i.e. -0.4 to -1.5 o/o) were observed in the ‘fresh’ cells. Therefore, the omission of washing after treatment of EAT cells with DMA or TNBS, does not leave amounts of these reagents in the medium which are significant in the present context.
Particles The mean electrophoretic mobilities of the particles suspended in RPM1 1630 was -~ 1.94 (f0.02; 172), compared with -0.93 (kO.01; Exptl Cell Res 83 (1974)
314 L. Weiss 122) for particles suspended and measured in RPM1 1630 + 5 % FCS. On washing and resuspending these particles in RPM1 1630, their mobilities showed little change ( - 1.05 i 0.02: 64). That the reduction in anodal mobility following incubation and measurement in serum-containing medium was due to adsorption of serum constituents, as distinct from viscosity differences is indicated by the observation that the mean mobilities of human erythrocytes in medium with and without serum were -1.39 (10.02; 100) and -~ 1.41 (lO.01; 100) respectively. The tabulated results therefore refer to experiments made on ‘coated’ particles. The results given in table 1 show that when coated styrene particles, were treated with the ‘usual’ concentrations of the two reagents, small increases in anodal mobility were observed when the particles were suspended in PBS. These increases are statistically significant after DMA-treatment, and insignificant after TNBS-treatment. It is also seen that increasing the final concentrations of TNBS IOO-fold, and of DMA 50-fold, only produced increases of net negativity of 2 and 5 96 respectively, over the effect of the usual concentrations. As shown in table 1, the increases in the negativity of the serum-treated particles produced by DMA and TNBS are manifest when the particles are electrophoresed in PBS + 5 % FCS.
Rates of cell adhesion The numerical results of the experiments with the ‘unmodified’ microtest plates are summarized in table 2. In RPM1 1630+5 % FCS, apart from the statistically insignificant (0.2 >p >O. 1) reduction in the rate of adhesion of EAT cells after 30 min initial exposure to TNBS, both modes of treatment produced significant (p
of adhesion of the EAT cell compared with their PBS or ethanol controls. The effects of reacting positively charged groups on microtest plate surfaces after prior incubation with serum-containing medium, on the rates of adhesion of unmodified cells (after 2 h), are summarized in table 3. Neither DMA nor TNBS produced a significant change in rate. The rates of adhesion of modified and unmodified cells, to modified and unmodified, serum-treated microtest plates, in PBS + 5 % FCS are shown in table 4. By comparison of the results of experiments 1 and 2 it can be seen that in the absence of serum in the suspending medium, treatment of both the cells and the microtest plate surfaces with DMA produced a significant decrease in adhesion rate, which was greater than that produced by treating the cells alone. This latter treatment also decreased the rate to 85 % of that of the ‘untreated’ cells. These rate decreases were not observed when the cells were suspended in PBS in 5 % FCS. The results of experiments 5 and 6 show significant decreases in the adhesion rates of cells suspended in PBS, following treatment of both them and the microtest plates with TNBS, compared with either untreated cells and surfaces (expt 5) or TNBStreated cells and untreated surfaces (expt 6). Comparison of experiments 5 and 6, shows no significant decrease in adhesion rate due to TNBS treatment of the cells alone. These effects following TNBS-treatment were also observed when the cells were suspended in PBS containing serum (expts 7, 8). DISCUSSION All current models of the molecular architecture of the cell periphery depict proteins at the cell surface, and some postulate the presence of the hydrophilic heads of phospholi-
Cell adhesion and positive charge pids [lo]. Therefore, among the positively charged groups possibly present at the cell surface are the side chain amino-groups of lysine and hydroxylysine; terminal protein CIamino-groups; imidazolic groups of histidine residues; guanidino-groups of arginine and phospholipid and glycolipid amines. In summary, the electrokinetic experiments indicate the existence of bound cationit sites, accounting for approx. 5% of the surface charge of both the EAT cells and styrene polymer particles, after the latter were exposed to serum-containing culture medium. Analogous changes to those observed at the surfaces of the plastic particles are thought to occur at the surfaces of the microtest plates, where adsorption of protein from similar culture medium has been detected by attenuated total reflectance spectroscopy in the infra-red [13]. The results also show that these positively charged groups react with TNBS and DMA under the conditions used here. lOO- and 50-fold increases respectively, in the concentrations of these reagents produced comparatively little change in the electrophoretic mobilities of the particles over those produced by the ‘usual’ levels. The effectiveness of TNBS and DMA is therefore unlikely to be limited by concentration, although it may be limited by the physicochemical nature of the reacting cationit sites [9]. It should be noted that TNBS reacts with unprotonated amino groups, and that reaction of the CI- and a-amino groups is second order. Although in simple amino acid solutions, the SH-group of N-acetylcysteine was found by Freedman & Radda [3] to be more reactive to TNBS than many amino groups, the resulting -S-TNP group is much more unstable at alkaline pH than the -HN-TNP group [5]. Our failure to detect a reaction of cationic groups at cell surfaces with TNBS by electrophoresis therefore, probably reflects
3 15
Table 2. Rates of adhesion of TNBS- and D MA-treated cells suspended in serum-containing medium, to microtest plate surfaces, expressed as percentages of appropriate controls “; adhent cells ri SE. 30 min TNBS PBS Controls 60 min TNBS PBS Controls 120 min TNBS PBS Controls 30 min DMA Ethanol Controls 60 min DMA Ethanol Controls 120 min DMA Ethanol Controls
96.8 + 1.7 (249) 100 + 1.7 (291) 90.4 + 1.7 (279) 100 + 1.6 (285) s1.5+1.4 (373) 100 + 1.3 (360) 85.0 k2.2 (85) 100 t-2.3 (85) 84.6 & 1.9 (86) 100 +2.1 (81) 92.5 & I .4 (146) 100 & 1.5 (143)
their low density at the cell surface, rather than simple competititve inhibition for TNBS by sulphydryl groups. The interpretation of adhesion experiments following TNBS treatment is further complicated because reaction of cells with sulphydryl-binding reagents results in a diminution of their rates of adhesion to a variety of surfaces [4]. Some indication of the extent of the reaction of DMA with sulphydryl groups comes from the work of Dickens & Cooke [2]. Under similar reaction conditions to those used in the present studies, namely at 25°C in an aqueous environment buffered at pH 8, when DMA reacts with cysteine, SH-groups are lost at a rate of 0.08 I/mole-l min-l compared with 70 for maleic anhydride. Acid production additional to hydrolysis, due to the formation of carboxyl groups was undetectable (i.e. ~0.01 1 mole-l min-l), indicating little or no acylation of the SH-groups, Exptl Cell Res 83 (1974)
316 L. Weiss Table 3. Adhesion of ‘untreated’ cells to microtest plate surfaces treated as indicated Adhesion is expressed as % of appropriate controls after 2 h incubation Coated’ microtest plate incubated with
Suspending fluid
% adherent cel1sIS.E. (No. of obs)
DMA Ethanol DMA Ethanol TNBS PBS TNBS PBS
PBS v 5 % FCS PBS I 5 “b FCS PBS PBS + 5 :b FCS 5 “6 FCS PBS PBS
97.3 F 2.9 (60) 100.0+2.2 (60) 100.6i 3.0 (60) 100.0+2.5 (60) 106.9+ 2.3 (60) 100.0& 1.9 (59) 106.8& 3.3 (60) 100.0& 2.6 (59)
although there may be some addition to the double-bond. The increase in surface negativity following DMA-treatment, was demonstrated in the absence of viability changes and detectable leakage of intracellular contents, and a similar lack of toxic effects has been previously reported for TNBS [14, 151.The electrophoretic mobility data indicate that approx. 5 “/b of the total charges at the electrokinetic surfaces of EAT cells are cationic, since following their removal with TNBS there was an approx. 5 y0 increase in surface negativity, and following their removal and replacement with negative charges after DMA-treatment, there was an approx. 10% increase. If the hypothetical cationic surface groups are concentrated in clusters of the type demonstrated for some anionic groups at the surfaces of EAT cells [17, 181, then the importance and effectiveness of small total numbers (per cell) of positively charged groups in promoting localized regions of cell contact with negatively charged regions of an opposing surface would be disproportionate to their total number. Cationic groups may play a role in determining the rate of cell adhesion in the model system studied here, and possibly in cell-cell adhesion, in 3 non-exclusive ways, namely: Exptl Cell Res 83 (1974)
t-tests 0.5 ‘p -,0.4 0.9 p-,0.8 0.05:~p-.o.o2 0.2:,p-,O.l
(1) Positively charged groups on cells may bind to negatively charged groups on the microtest plate surfaces. (2) Positively charged groups adsorbed to microtest plate surfaces from the serumcontaining medium, may react with negatively charged groups at the cell surfaces. (3) Positively charged groups on macromolecules in the medium may bind to negatively charged groups on the cell and microtest plate surfaces, thereby ‘gluing’ them together. Some indication of the quantitive importance of these possible mechanisms is expected to be reflected in decreases in the rates of cell adhesion to microtest plates, compared with appropriate controls, in simple media with and without macromolecules, after reacting cationic sites at the cell and/or microtest plate surfaces. The experiments summarized in table 2 indicate that in serum-containing culture medium in which the cells ‘normally’ grow, prior treatment of the cells with either TNBS or DMA, produced small but statistically significant changes in their rate of adhesion to microtest plates. Within interpretative limitations to be discussed later, this suggests that positively charged groups at the surface of EAT cells play a demonstrable part in deter-
Cell adhesion and positive charge
3 17
Table 4. Adhesion of cells to microtest plates after 2 h, when both were reacted with DMA or TNBS Adhesion is expressedas a :: of appropriate controls
Expt
Cells
Microtest plates
I
Ethanol DMA DMA DMA DMA DMA Ethanol DMA PBS TNBS TNBS TNBS PBS TNBS TNBS TNBS
Ethanol DMA Ethanol DMA Ethanol DMA Ethanol DMA PBS TNBS PBS TNBS PBS TNBS PBS TNBS
2 3 4 5 6 7 8
Suspending fluid
% adherent cells& S.E. (No. Obs)
PBS PBS PBS PBS PBS+ 5 56FCS PBS+ 5 % FCS PBS+5% FCS PBS+ 5 % FCS PBS PBS PBS BPS PBS+ 5 % FCS PBS i 5% FCS PBS : 5% FCS PBS in5 % FCS
loo.O& 1.4 (179) 76.4* 1.2 (180) lOO.O+lS (180) 90.3i I.9 (180) 100.0~1.9 (115) 99.6-tl.8 (120) 100.0~1.9 (117) 101.8k2.1 (119) 100.0+1.9 (116) 88.8* 1.7 (120) 100.0kl.9 (118) 86.1i2.0 (116) 100.0~1.9 (117) 90.7kl.8 (117) 100.0~1.6 (178) 90.5* 1.5 (179)
mining their rate of adhesion. This role is not limiting, because the majority of cells adhere in spite of the treatment. The experiments summarized in table 3 indicate that in the presence or absence of serum, when no other macromolecules were deliberately added to the medium, positively charged groups on the substratum play no demonstrable role in determining the rates of cell adhesion. In these and later experiments, PBS was used instead of regular culture medium to avoid the presence of amino acids with reactive amino groups. It should be noted however that PBS, with or without serum is an inadequate growth medium. Therefore, as metabolic processes play some part in the mode of establishment of cell adhesion, it is not surprising that the results obtained with PBS were not numerically similar to those obtained in culture medium. In the absence of serum, after reacting both cell and substratum surfaces, the effects of absence
f-tests p .‘O.OOl pe 0.001 0.9 -.p .0.8 0.6 “p c 0.5 p e. 0.001
p.~o.OOl P.‘O.OOl p ,’ 0.001
or major removal of positively charged adhesion-promoters were expected to be maximal. The data given in table 4 show that in fact, in the case of DMA-treated cells, there was a maximal reduction in adhesion rate compared with untreated cells and microtest plates (expt 1) and a lesser effect compared with treated cells and untreated microtest plates (expt 2). The greater reduction seen in expt 1 than expt 2, suggests the synergistic effect expected if positively charged groups on both surfaces contribute to their adhesion. Although reductions in adhesion-rate were obsevered in analogous experiments with TNBStreatment (expts 5, 6), no evidence of synergism was observed. This latter finding is more consistent with the data given in table 3. The observation that in the presence of serum, prior TNBS-treatment of both surfaces produced significant reductions in adhesion-rate (expts 7, 8) whereas DMA-treatment did not (expts 3, 4), suggests more complex secondExptl Cell Res 83 (1974)
318 L. Wiess
-CONH
R
Fig. I. Reaction of amino-groups with (a) TNBS and (b) DMA, under conditions specified in text.
order interactions of the cells than can be currently interpreted. In common with many other reagents, it might be argued that the effects of the present reagents on the rate of EAT cell adhesion were due to steric hindrance as indicated in fig. 1, rather than loss of positively charged surface groups per se. Steric hindrance of this type could only occur if other sites involved in this part of the contact process were close enough to the reacted sites to be masked by the comparatively small TNBS or DMA molecules. At present there is no supportive evidence for this possibility. Unfortunately, direct experimental tests involving the use of reagents of smaller molecular size, such as formaldehyde are impractical since these enter cells, modify their mechanical properties and are toxic; and even with these reagents, the basic question of the effects of steric hindrance and/or conformational change would remain unanswered. It could also be argued in the case of the DMA-treated cells, that the reduction in adhesion rate is due to the acquisition of additional negative charges (fig. 1) rather than the loss of positive charges. However, this argument is clearly not applicable to the reduction
in adhesion
TNBS-treatment. Exptl Cell Res 83 (1974)
rate
observed
after
The present experimental results in general, support the concept at least in the system studied here, that DMA and TNBS- ‘sensitive’ cationic groups at the cell and substratum surfaces influence the initial rates of adhesion, and that this parameter is sensitive to the presence of serum in the culture medium. However, as cell adhesion occurs after both surfaces have been reacted with either TNBS or DMA, without the addition of the hypothetical serum ‘glues’, and allowing for the reaction of some of the TNBS with sulphydryl groups as discussed above, it would appear that cationic surface sites play at most a minor rather than an all-or-none role in this type of cell contact reaction. I am grateful to Dr Geoffrey Seaman for his constructive criticism of this work and Dr Jake Belle for his advice on relevant organic chemistry. My sincere thanks are also due to MS T. L. Cudney, MS C. Maver and Mr D. Huber for their assistance. This work was partially supported by Grant BC-87F from the ACS.
REFERENCES I. Bangham, A D, Flemans, R, Heard, D H & Seaman, G V F, Nature (Lond) 182 (1958) 642. 2. Dickens, F & Cooke, J, Brit j cancer 19 (1965) 404. 3. Freedman. R B & Radda. G K. Biochem i 108 (1968) 383’. 4. Grinnell, F, Milam, M & Srere, P A, Arch biothem biophys 153 (1972) 193. 5. Kotaki, A, Harada, M & Yagi, K, J biochem Tokyo 55 (1964) 553. 6. Mehrishi, J N, Eur j cancer 6 (I 970) 127. - Progr biophys mol biol 25 (1972) I. s7: Moore,-G E,.Sandberg, A A & Ulrich, K, J natl cancer inst 36 (1966) 405. 9. Papahadjopoulos, D &Weiss, L, Biochim biophys acta 183 (1969) 417. 10. Weiss, L, Int rev cytol 26 (1969) 63. Il. -The chemistry of biosurfaces (ed M L Hair) vol. 2, p. 377. Dekker, New York (1972). 12. - Exptl cell res 74 (1972) 21. 13. - Ibid 81 (1973) 57. 14. Weiss, L, Belle, J & Cudney, T L, Int j cancer 3 /
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15. tiei&, L & Cudney, T L, Int j cancer 4 (I 969) 776. 16. Weiss. L & Harlos. J P, Progr surface sci I (1971) 355. 17. Weiss, L, Jung, 0 S & Zeigel, R, Int j cancer 9 (1972) 48. 18. Weiss, L & Zeigel, R, J theor biol 34 (1972) 21.
Received June 4, 1973 Revised version received August 28, 1973