SURFACE MODIFICATION AND ELECTRO- PHORESIS OF NORMAL AND TRANSFORMED BHK21 CELLS

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1 J. CM Sci. 14, (1974) 3 Printed in Great Britain SURFCE MODIFICTION ND ELECTRO- PHORESIS OF NORML ND TRNSFORMED BHK21 CELLS. L. LTNER ND G.. TURNER Cancer Research Unit, University Department f Clinical Bichemistry, Ryal Victria Infirmary, Newcastle upn Tyne, England SUMMRY The mean electrphretic mbilities at ph 7-5 f virus-transfrmed (Py6) and nrmal BHK21 cells were very similar, whether they were harvested mechanically r by the use f trypsin. fter frmaldehyde treatment, there was significantly increased mbility in bth cell types; the transfrmed cells shwed significantly the greater change. fter neuraminidase treatment, the mean electrphretic mbility was decreased t the same extent in bth types f cell. Treatment with neuraminidase and frmaldehyde had n effect n the mean electrphretic mbility f the nrmal cells but slwed the transfrmed variety. The mbility in histne slutin had n relatinship t histne cncentratin but was statistically crrelated with the amunt f histne per cell, calculated frm ttal histne present divided by the ttal number f cells; a linear relatinship being btained with the nrmal cells but an initial plateau was demnstrated with the transfrmed cells. The nrmal cell line shwed a similar plateau after neuraminidase treatment. The pssible significance f these results is discussed. INTRODUCTION Many studies have been carried ut with the bjective f elucidating surface prperties unique t malignant cells. Recent reprts (Burger & Gldberg, 1967; Inbar & Sachs, 1969) n the binding f sme plant-derived lectins t the cell surface have illustrated differences in the surface prperties f nrmal and neplastic transfrmed cells. Earlier wrk tended t indicate that the malignant prperties f the cell were assciated with a higher net negative surface charge (Lwick, Purdm, James & mbrse, 1961; Ruhenstrth-Bauer, Fuhrmann, Granzer, Kiibler & Rueff, 1962; Saki, 1967) but cnflicting results have appeared regarding this crrelatin (Vassar, 1963; Ck & Jacbsn, 1968) and the situatin is by n means defined. Studies n ascites tumur cells (Ck, Heard & Seaman, 1962; Ward & mbrse, 1969) have emphasized that bth negative and psitive cmpnents cntribute t the verall electrical prperties f the cell surface, and it is cnsidered pssible that variatins in bth f these cmpnents culd explain the diversity f the results btained in the past. With this in mind, the present investigatins were undertaken. It was decided t cmpare the electrphretic mbility f a line f baby hamster kidney cells (BHK21/C13) with that f its malignant viral-transfrmed derivative (BHK2i/Ci3/Py6). These cell lines are subsequently referred t as BHK21 and Py6 respectively. With bth lines, attempts were made t study cntributin t cell

2 4. L. Latner and G.. Turner charge by varius grupings. Fr this purpse, frmaldehyde was used in relatin t the amin grups (Ck et al. 1962; Ward & mbrse, 1969); and neuraminidase in relatin t sialic acid grups (Ck et al. 1962; Frrester, mbrse & Stker, 1964; Ward & mbrse, 1969). s wrk in this labratry had demnstrated that histnes culd prduce changes clsely resembling malignant transfrmatin in BHK21 cells (Latner & Lngstaff, 1971), it was als decided t study the effect f histnes n the electrphretic behaviur f these and the Py6 cells, since it was pssible that the BHK21 transfrmatin bserved was a result f a membrane phenmenn. MTERILS ND METHODS Tissue culture Using 2-z. (56-ml) glass medical flats, BHK21 and Py6 cells were grwn t cnfluence in 10 ml grwth medium cntaining 80 % (v/v) Eagle's Medium - Dulbecc's Mdificatin (Flw Labratries), % (v/v) calf serum (Flw Labratries), 0-47 mg ml" 1 glutamine, 2-95 mg ml" 1 NaHCOj, 500 units ml" 1 penicillin G, 0-25 mg ml" 1 streptmycin sulphate and 60 units ml" 1 mycstatin. The whle was equilibrated with an atmsphere f % CO, in air and placed in an incubatr at 37 C. Prir t harvesting cells, the cnfluent mnlayer was washed with 10 ml f phsphate-buffered saline, ph 7-4 (PBS) (Dulbecc & Vgt, 1954). Passage f the cells was carried ut by harvesting with 5 ml f 0125 % (w/v) trypsin (Flw Labratries 1:250) in PBS, and subcultures were set up at 1:10 dilutin. Cells used fr experimental purpses were harvested under mre clsely cntrlled cnditins t ensure minimum disruptin f the cell surface. The cells were incubated with 5 ml f 004 % (w/v) crystalline trypsin (Behringer) in PBS at 37 C fr 1 min. This was pured ff and the mnlayer with its adhering film f trypsin incubated at 37 C fr a further 3 min. Three millilitres f PBS cntaining 2 % (v/v) calf serum were added t each bttle f cells, and suspensin btained by gently pipetting t and fr. Each bttle prvided apprximately i 7 cells. Cells were separated mechanically by remving the medium with a hypdermic syringe and squirting the fluid against the wall f the culture bttle. fter several repetitins, a sufficient number f cells had been separated fr subsequent bservatin. Cell cncentratins were measured using an electrnic cell cunter (Cellscpe, Grant Instruments, Cambridge, U.K.). Cell electrphresis knwn number f cells were taken and washed nce with 10 ml istnic sucrse (SPB) buffered t ph 75 (0-25 M sucrse, M NaH,PO,,, M NajHPO 4 ); a further 2 washes with this buffer did nt change the mbility f the cells, which demnstrated that the single wash was sufficient. The cells were then suspended in 6 ml f SPB and transferred t the rectangular chamber f a particle electrphresis apparatus (Rank Brthers, Bttisham, Cambridgeshire, U.K.). cnstant vltage, in the range f V cm" 1, was applied between the platinized platinum electrdes and the velcity f a single cell, mving in the statinary layer, was measured acrss the graticule. The plarity was reversed and the velcity f the same cell was measured in the ppsite directin. The cell velcity adpted was the average value f these 2 measurements. The velcities f at least 10 cells were measured fr each preparatin. ll measurements were perfrmed at 25-0 ±01 C and human erythrcytes frm heparinized r defibrinated bld were used as cntrls t check the peratin f the instrument. The packed cells frm 1 ml f whle bld were washed 4 times with 10 ml PBS and then suspended in 100 times their vlume f this slutin; 1 ml f the suspensin was sedimented and washed nce with 10 ml SPB befre resuspending in the 6 ml f this slutin fr electrphresis. The mbility f the washed human erythrcytes in SPB was fund t be 2-57 ±i4/im s" 1 V" 1 cm. Since standard cnditins were used thrughut and the temperature was kept cnstant at 25-0 ±-i C, it was nt cnsidered necessary t crrect the mbility values t the viscsity f water at 25 C.

3 Electrphresis f surface-mdified cells 5 In rder t ensure that trypsin did nt affect the electrkinetic surfaces f the cells, a limited number f electrphresis bservatins at different ph were carried ut n cells harvested mechanically, and cmpared with similar measurements n trypsinized cells. The ph f the SPB was altered with istnic HC1 r NaOH. The mbilities at different ph levels f cells separated by the 2 methds were fund t be identical within experimental errr. Therefre, all future electrphresis experiments discussed in this paper were carried ut with cells separated by the use f trypsin. Frmaldehyde treatment suspensin f i 7 cells in PBS cntaining 2 % calf serum was taken and resuspended in 10 ml PBS, after which 0-4 ml f 40% nalar frmaldehyde slutin were added with cntinuus stirring. fter a minimum perid f 30 days fixatin at rm temperature (Ward & mbrse, 1969), the cells were centrifuged dwn and the frmaldehyde discarded. The cells were then mixed with 10 ml PBS and again centrifuged dwn and the PBS discarded. This washing prcess was repeated 3 mre times befre the cells were prepared fr electrphretic measurements as described in the previus sectin. Micrscpically, the final suspensins cntained rund intact cells with very few clumps. fter fixatin fr 30 days, n decrease in cell number was detected in 6 bservatins with Py6 cells and 4 with BHK21 cells. Mrever, investigatins using frmaldehyde prepared frm parafrmaldehyde (Ward & mbrse, 1969) gave similar electrphresis readings t thse btained with nalar frmaldehyde (BHK ±0- fim s" 1 V" 1 cm, 29 bservatins; Py6 2'62 ±0-19 /im s" 1 V" 1 cm, 22 bservatins). It was therefre felt justified in cncluding that the nalar frmaldehyde did nt prduce cell lysis and the measurements btained were nt affected by crss-linking prducts f such lysis t the cell surface. It is interesting t nte that in this respect the cell lines investigated were far mre stable than red cells (Heard & Seaman, 1961). It shuld als be pinted ut that the ph f the suspensin in frmaldehyde slutin was peridically checked and fell by n mre than 0-25 ph unit ver the whle perid. Neuraminidase treatment Neuraminidase was btained cmmercially (Behringwerke G Hechst Pharmaceuticals) and was stated t give negative tests fr prteases, aldlases and lecithinase C. One neuraminidase unit was defined by the manufacturers as the amunt f enzyme required t release 1 fig f N-acetylneuraminic acid frm ai-acid glycprtein in 15 min at 37 C, at ph 55. The tissue culture cell suspensins were washed nce with 10 ml PBS prir t incubatin with 10 units f neuraminidase per 10' cells in 1 ml PBS at 37 C fr 30 min. fter incubatin, the cells were washed nce with 10 ml SPB r PBS befre recnstituting the apprpriate suspensin fr electrphretic measurements r fixatin in frmaldehyde respectively. Histne preparatin Histnes were prepared frm bullck thymus chrmatin btained by the technique described by Marushige & Bnner (1966). The methd invlved extractin f the basic nuclear prteins frm the chrmatin with ice-cld 0-25 N HC1 and subsequent precipitatin in 90 % (v/v) aqueus acetne at 4 C. The resultant material was washed with acetne, vacuum-dried and stred in a vacuum dessicatr at 4 C. ll preparatins were characterized by their ultravilet absrptin spectra and by the patterns btained after electrphresis n plyacrylamide gel (Jhns, 1967). The electrphretic patterns gave 4 distinct bands which were clsely similar t thse reprted fr calf thymus histnes (Jhns, 1967). ls, analysis f the preparatins fr nucleic acids prved negative, and amin acid analysis was clsely similar t that published by Jhns (1971) fr calf thymus histnes.

4 2O6. L. Latner and G.. Turner Histne treatment knwn number f cells were sedimented at lw speed n a bench centrifuge and washed nce with 10 ml SPB. The histnes, i- mg ml" 1 SPB, were then added in calculated amunts t a suspensin f the cells in SPB and the final vlume adjusted t 6 ml with SPB. Mbilities were measured in the presence f the histnes after incubatin at 25 C fr 30 ± 15 min. Variatins in time f incubatin f the rder given did nt appear t affect the resultant mbilities. RESULTS Untreated cells. The results with BHK21 cells are shwn in Fig. 1 and thse with Py6 in Fig. 2. The BHK21 cells shwed a mean electrphretic mbility f 2-04/tm s" 1 V~ x cm (s.d ; 89 bservatins). The Py6 cells shwed a mean mbility f 2-08/«n s~ x V -1 cm (s.d. ±0-27; 148 bservatins). lthugh there is a greater spread f values fr the transfrmed cells, especially in the upper limits f the distributin (variance rati, P < -i), the difference between the 2 means was nt significant (P > -i). 40 = t 40r _ Electrphretic mbility, /;m s" 1 V" 1 cm Fig. 1. Histgrams shwing the percentage distributin f electrphretic mbilities f BHK21 cells;, untreated; B, c, D, treated with frmaldehyde, neuraminidase and neuraminidase fllwed by frmaldehyde, respectively. Mean values + s.d. fr the ppulatins f cells measured, and the ttal numbers f bservatins fr, B, C, D were, respectively: 2-04 ±0-12, 89; ±-18, 76; i'47 ±-14,44; and 8 ± 022, 52.

5 Electrphresis f surface-mdified cells 7 It is a matter f sme interest that when electrphretic mbilities were determined under identical cnditins t thse described by Frrester, mbrse & Macphersn (1962), the mbility fr BHK21 cells was i-i6±-i2/tm s" 1 V" 1 cm (33 bservatins) and that fr the Py6 cells was /n s" 1 V" 1 cm (33 bservatins). Frmaldehyde treatment. The results with BHK21 cells are shwn in Fig. IB and 40 g S -40 V E r Electrphrectic mbility, ftm s" 1 V" 1 cm Fig. 2. Histgrams shwing the percentage distributin f electrphretic mbilities f Py6 cells:, untreated; B, C, D, treated with frmaldehyde, neuraminidase, and neuraminidase fllwed by frmaldehyde respectively. Mean values ± s.d. fr the ppulatins f cells measured and the ttal numbers f bservatins fr, B, C, D were, respectively: , 148; , 66; I-47±O-I8, 43; and -i-77± 0-18, 38. thse with Py6 in Fig. 2B. fter frmaldehyde treatment, the BHK21 cells shwed a mean electrphretic mbility f 2-13 i-18/im s -1 V" 1 cm (76 bservatins). This represented a small but significant increase f 4% ver the value fr the untreated cells (P < -i). The Py6 cells, hwever, shwed a dramatic increase f 22% after treatment with a mean value f fim s -1 V" 1 cm (66 bservatins). s was the case in the untreated cells, the spread f mbilities was slightly greater fr Py6 than fr BHK21 (variance rati, 0-05 > P > -i). Neuraminidase treatment. The results with BHK21 cells are shwn in Fig. ic and thse with Py6 in Fig. 2 c. It is quite bvius that neuraminidase significantly re-

6 8. L. Latner and G.. Turner duced the mbility in bth instances, as wuld be expected (Frrester et al. 1964). The mean mbilities fr bth ppulatins f treated cells were identical. The BHK21 cells having a mean f ±0-14 fim s -1 V" 1 cm (44 bservatins) and the Py6 cells having a mean f fim s~ 1 V" 1 cm (43 bservatins) r T -'- ~ 1 ' «-08.c CL O t t * ft t munt f histne present, //g ml"' Fig. 3. bsence f any relatinship when the cncentratin f histne in the suspending medium is pltted against the electrphretic mbility f BHK21 cells. In this and subsequent figures, each pint is the mean value f at least 10 bservatins. 1 In additin t these electrphresis measurements, bservatins were als made f the amunt f sialic acid (Warren, 1959) released by the neuraminidase treatment. N significant difference was fund between the BHK21 and Py6 cells. The frmer yielded an average amunt f 40-6 fig sialic acid/10 8 cells (range ver 3 bservatins 35*2~45"7 fig) and the latter an average amunt f 37-6/ig/i 8 cells (range ver 3 bservatins /^). Treatment with neuraminidase fllwed by frmaldehyde. The results with BHK21 cells are shwn in Fig. ID and thse with Py6 in Fig. 2D. BHK21 cells after the cmbined treatment shwed a mean electrphretic mbility f fim s~ x V" 1 cm (52 bservatins) which was nt significantly different frm the mbility f the untreated BHK21 cells. In marked cntrast, hwever, the Py6 cells shwed a mean mbility f 1-77 ± 0-18 fim s" 1 V" 1 cm (38 bservatins) which was a reductin f abut 15 % cmpared with untreated Py6 cells. Behaviur f cells in histne slutin. Cell suspensins were added t histne slutins f cncentratins ranging frm 8 t 50 fig ml" 1. Initially, the number f cells used in each suspensin varied frm experiment t experiment ver the range 3-4 x i 6 t 16-3 x i 6 and the final ttal vlume after adding the cell suspensin was

7 Electrphresis f surface-mdified cells 9 6 ml. In later experiments, the cell number used was always between 4-0 x i 6 and 60x i 8. With the nrmal BHK21 cells, as can be seen in Fig. 3, there was n crrelatin between the electrphretic mbility and the cncentratin f histne present. On the ther hand, as can be seen in Fig. 4, ver the range f histne cncentratins we emplyed, there was a high degree f negative crrelatin (crrelatin cefficient 0-91) when the amunt f histne per cell, calculated frm ttal histne present -2-4 T E S -0-8 Q. g munt f histne present, pg/cell 60 Fig. 4. The relatinship between the cncentratin f histne and the electrphretic mbilities f BHK21 ( ) and Py6 (O--O) cells. divided by the ttal number f cells, was pltted against electrphretic mbility. Fr this reasn, it was felt best, ver the range f histne cncentratins we emplyed, t express ur histne experiments in terms f cncentratin per cell. The linear relatinship demnstrated in Fig. 4 is quite striking. It is, hwever, apparent that the slpe f the line calculated is such that it wuld nt g thrugh the zer pint. It can nly be inferred, therefre, that at histne cncentratins between and 10 pg cell" 1 there is n significant change in electrphretic mbility. On the ther hand, as can be seen in Fig. 4, the behaviur f the Py6 cells in histne slutin shwed quite a striking difference. Over the range 0-35 pg cell" 1, there was virtually n change in electrphretic mbility f the cells. The slight slpe f the calculated line is nt significantly different frm zer. t cncentratins abve 40 pg cell" 1, the calculated slpe shwed a rapid fall-ff in electrphretic mbility and eventually at cncentratins rund abut 50 pg cell" 1, the mbility f the Py6 cells was the same as that f the BHK21 cells. Behaviur f neuraminidase-treated cells in histne slutins. fter cells had been treated with neuraminidase, their electrphretic mbility in histne slutin was such that there was a clser degree f resemblance between the BHK21 cells and the 14 C E L 14

8 210. L. Latner and G.. Turner Py6 cells (Fig. 5). Bth types f cells shwed n significant change in electrphretic mbility ver the range 0-25 pg celh 1, but then mbility began t fall. This fall, initially, ccurred mre rapidly with BHK21 cells but the pints then levelled ut s that nce again at the cncentratin f apprximately 50 pg cell" 1, bth lines met. It is interesting t nte that at this cncentratin, electrphretic mbilities were virtually the same as thse shwn in Fig. 4. E - r I >> ' O I munt f histne present, pg/cell Fig. 5. The relatinship between the cncentratin f histne and the electrphretic mbilities f BHK21 ( ) and Py6 (O O) cells after treatment with neuraminidase. DISCUSSION The results presented in this paper shw that we culd nt demnstrate any difference in the mean electrphretic mbilities f untreated baby hamster kidney cells and their virus-transfrmed derivatives. In ther wrds, the net surface charge was the same in each cell type. The Py6 cells shwed a slightly larger spread abut the mean than the BHK21 cells but, even emplying their methd f bservatin, this was by n means as large as that reprted by Frrester et al. (1962), wh fund that the mbilities f their plyma-virus-transfrmed cells gave a cefficient f variance times greater than that f the BHK21 cells. Later results frm this grup f wrkers (Frrester et al. 1964) shwed that increased electrphretic mbility f freshly transfrmed cells was assciated with 2 types f clnal mrphlgy, s-called Types 1 and 2, the basic difference being the release f large numbers f runded cells int the medium by Type 2. Type 2 eventually reverted t Type 1. Increased mbilities were in general seen nly with Type 1. It was prpsed that the hetergeneity f earlier preparatins resulted frm the accumulatin f spntaneus variants in lng-term culture. The Py6 cells used in ur experiments have nt been typed, and it is pssible that they were f Type 2, althugh n extensive release f rund cells int the medium was ever bserved. In additin, it wuld have been expected frm the length f time they had spent in culture that they had reverted t

9 Electrphresis f surface-mdified cells 211 Type 1. We d nt, therefre, cnsider that the difference in ur findings frm thse f Frrester et al. (1962) can be explained in terms f these 2 types. Indicatin that the transfrmed cells d have mre negatively charged grups n their cell surface than d nrmal cells was given by the results btained after treatment f the cells with frmaldehyde. The mbility f the Py6 cells rse by 22 % cmpared t nly 4% fr the BHK21 cells. study f the chemistry f the reactins f frmaldehyde with prteins has shwn that the charged grup which reacts at neutral ph is the amin grup NHj~ (French & Edsall, 1945) and that in this way the psitive charge is lst. Thus, it wuld appear that the Py6 cells have a higher number f negatively charged grupings at the cell surface than the BHK21 cells but that these are masked by the presence f an equally increased number f amin grups. Psitively charged amin grups have been previusly shwn t exist in significant amunt n the surfaces f leukemia (Ward & mbrse, 1969) and ascites cells (Ck et al. 1962; Mehrishi, 1970). detailed study by Ck & Jacbsn (1968) demnstrated a basic grup present n the surface f acute lymphblastic leukemia cells which was absent n the surface f nrmal lymph nde cells. fter neuraminidase treatment, there was still n significant difference in the electrphretic mbility f transfrmed and untransfrmed cells and the diminutin f mbility as cmpared with untreated cells was virtually the same in each instance. It has been shwn that the neuraminidase treatment f each type f cell remved amunts f sialic acid nt significantly different frm each ther. It is pssible, hwever, that remval f sialic acid results in rearrangement f cell membrane (Ward & mbrse, 1969). When neuraminidase-treated cells were subsequently subjected t frmaldehyde, there was a highly significant difference in the respnse f each type f cell. In each instance, the frmaldehyde treatment increased significantly the electrphretic mbility but this increase was much greater with the BHK21 cells than with the Py6. n increase wuld, f curse, be expected in bth cases, since psitively charged amin grups are being masked. It is, hwever, f great interest that mre such grups were masked when BHK21 cells were treated with neuraminidase than when Py6 cells were s treated. This must mean that in sme way mre amin grups had been expsed t the actin f frmaldehyde and wuld pint t the pssibility that remval f sialic acid frm the BHK21 cell surface had resulted in a rearrangement f that surface (Ward & mbrse, 1969). It wuld be interesting t speculate that this rearrangement pssibly results in the expsure f a greater area f subsurface. It culd pssibly be pstulated that ur results were in sme way due t fixatin artifacts as a result f neuraminidase treatment. We have, hwever, fund n difference in ur bservatins after repeated washing prir t frmaldehyde treatment. ls, frmaldehyde treatment f cells incubated with heat-inactivated neuraminidase yielded mbilities very similar t thse btained with cells treated with frmaldehyde alne (BHK ±0-21 /im s -1 V" 1 cm, 13 bservatins; Py fim s" 1 V" 1 cm, 12 bservatins). Finally, treatment with neuraminidase after prir fixatin with frmaldehyde gave 14-2

10 212. L. Latner and G.. Turner similar mbilities t thse btained by treatment with frmaldehyde after prir treatment with neuraminidase (BHK fim s~ 1 V -1 cm, 24 bservatins; Py fim s" 1 V" 1 cm, 24 bservatins). We therefre cnsider that it is highly unlikely that ur findings are explicable in terms f ' fixatin' artifacts. It is a matter f great interest that within the range f histne cncentratins we emplyed, there was a highly significant linear relatinship between the amunt f histne, when expressed per cell, and electrphretic mbility, whereas n such relatinship ccurred with regard t the actual cncentratin f histne in the slutin. This must mean that under ur defined experimental cnditins there was sme srt f equilibrium situatin between cells and histne in slutin and that the amunt f histne taken up per cell was determined by this equilibrium. Hwever, with transfrmed cells, this appears t have ccurred nly in the higher range f cncentratins. This wuld supprt the current cncept that there is a definite change in the structure f the surface f the transfrmed cells (Burger & Gldberg, 1967; Inbar & Sachs, 1969). If ne assumes that in the cell surface there are negatively charged grupings with varying affinities fr psitively charged macrmlecules like the histnes, it then fllws that the transfrmatin prcess may render the higher affinity znes less available and that the histnes d nt cmbine with the surface until the cncentratin is such as t be cmpatible with cmbinatin with negatively charged grups f smewhat lwer affinity. We think that negatively charged grups must be invlved with histne binding and that the latter cmbine with the frmer by virtue f their basic amin acids, since we have btained similar results with ther experiments in which we have used plylysine r plyarginine. It has already been pinted ut that ur results indicate that the net surface charge is the same in each cell type and that in the Py6 cells the increased amunt f amin grups must be accmpanied by an increased amunt f negatively charged grupings. Since the histne effect n electrphretic mbility results frm cmbinatin with negatively charged grups, these psitively charged macrmlecules must bviusly be in cmpetitin with thse psitively charged grupings which neutralize negative charges in the cell surface. It is pssible that the histne mlecules cmbine nly with the negatively charged grupings present in excess; hwever, this seems unlikely, since this excess is the same in transfrmed and untransfrmed cells but the behaviur with histnes is different. If ne returns t the cmpetitin cncept, it is nt difficult t envisage a situatin where fr varius reasns such as spatial relatinship, the psitive grupings riginally in the cell surface are cmbined mre r less firmly with thse which are negatively charged. In ther wrds, in sme cases it wuld be difficult fr histnes t replace the psitive grupings (lw histne affinity sites) and in thers relatively easy (high histne affinity sites). The difference in behaviur between the BHK21 cells and the Py6 cells in the presence f histnes culd then be explained by the fact that the surface rearrangement in the latter type f cells is such as t make less available the high-affinity histne sites. Recent evidence has indicated that neuraminidase treatment f a cnfluent mnlayer f BHK21 cells results in the release f cells frm the density-dependent inhibitin f grwth (Vaheri, Ruslahti & Nrdling, 1972). Thus, the behaviur f the

11 Electrphresis f surface-mdified cells 213 BHK21 cells, in shwing lss f cntact inhibitin, resembles that f transfrmed cells. Cnsequently, it is a matter f interest that after neuraminidase treatment, the mbility f the BHK21 cells in the presence f histnes gave a very similar plateau t that btained with neuraminidase-treated Py6 cells. This wuld seem t indicate that the remval f sialic acid frm the BHK21 cell surface resulted in a mlecular rearrangement which rendered unavailable the sites with high affinity fr histne. Hence, ne might assume that the surface f the BHK21 cells was nw very similar t that f the Py6 cells. Hwever, the slpe subsequent t the plateau was steeper with the BHK21 cells, and this suggests that any pssible surface rearrangements still left differences between the 2 types f cell. Our results appear t indicate a difference between the surface membranes f nrmal and transfrmed BHK21 cells. This difference, hwever, des nt affect electrphretic mbility but depends n a different distributin f charged grups in the surface. The findings in the experiments in which we emplyed bth neuraminidase and frmaldehyde are f great interest, since, regardless f the rder in which these reagents were emplyed, the final electrphretic mbilities were the same. We culd cnclude that sialic acid mlecules d nt cntribute significantly t the surface charge f the BHK21 cells, since treatment with frmaldehyde after neuraminidase restred the mbility t the riginal level. In ther wrds, a net increase in psitive charge was cmpletely neutralized by frmaldehyde, which wuld have n effect n a phenmenn dependent n the remval f negatively charged sialic acid mlecules. When the bservatins were carried ut using frmaldehyde initially, there were nly slight changes in the electrphretic mbility f the BHK21 cells at any stage. This must mean that there are, at mst, nly a few free NH 3 + grupings expsed at the surface and that such negative charge as is present at the surface f the BHK21 cells des nt arise frm sialic acid mlecules. lternatively, it culd be argued that remval f these latter mlecules results in the expsure f a crrespnding negative charge, alng with the additinal psitive gruping expsed. The results with Py6, hwever, lead us t the cnclusin that the sialic acid mlecules d cntribute significantly t surface charge r, alternatively, that their remval des nt expse an equal negative charge. In either case, this is further evidence t supprt the cncept that there are differences in the surface mlecular arrangement between BHK21 and Py6 cells. We gratefully acknwledge much valuable technical assistance by Mr C. Crnell, and we als wish t thank Dr M. Lunn fr carrying ut the amin acid and nucleic acid analyses f the histne preparatins. Prfessr M. Stker, Imperial Cancer Research Fund, Lndn, kindly supplied the riginal cultures f BHK21 and Py6 cells.

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