QUANTITATIVE INVESTIGATION ON THE EFFECT OF …Moscona & Moscona (1963), when they had demonstrated...

J. Cell Set. a, 323-340 (1967) 323

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QUANTITATIVE INVESTIGATION ON THE

EFFECT OF PUROMYCIN ON THE

AGGREGATION OF TRYPSIN- AND

VERSENE-DISSOCIATED CHICK

FIBROBLAST CELLS

R. B. KEMP, B. M. JONES, I. CUNNINGHAM AND M. C. M. JAMESThe Department of Zoology, University of Wales, Aberystwyth

SUMMARY

Embryonic chick fibroblast cells were used to test the effects of puromycin on cell adhesionand aggregation. Single cell suspensions were prepared by dissociating the muscle tissues of5- and 9-day-old chick embryos with either trypsin or versene according to standard cell dis-sociation procedures.' Cell aggregation was quantitatively estimated by a turbidimetric method.

Quantitative analyses of the components in cell-free supernatants revealed that both trypsinand versene when used as cell dissociating agents removed substantial amounts of proteinfrom the surfaces of the cells and the intercellular regions. Trypsin removed larger quantitiesof protein and of iV-acetylneuraminic acid than did versene. Nucleic acids were also releasedduring trypsin and versene dissociation, and estimations of these nucleic acids gave a measureof the extent of cell lysis. The proteins and peptides were separated by gel filtration. Thesolubilized proteins removed by versene treatment were separated into three peaks on SephadexG.200. The tryptic digest gave one main peptide peak on Sephadex G.50.

Both trypsin- and versene-dissociated cells, on rotation in Hanks's balanced salts solutionwith or without serum, began to adhere to one another at the start of the test period, indicatingthat the cell dissociation procedures had not impaired the adhesive properties of the separatedcells.

When puromycin at a final concentration of 10 fig/ml was introduced at zero time into a cellsuspension with or without serum, it did not delay the adhesion of cells to one another, and didnot prevent the formation in the first hour of initial aggregates; it succeeded only in preventingprogress in aggregation beyond the initial stages. Adding puromycin at the 2nd or 3rd hourof the 4-h test period, when aggregation had already taken place, produced results of a similarpattern, progress in aggregation being brought to a halt an hour later. Puromycin at the aboveconcentration effectively blocked protein synthesis by more than 90 % as measured by theincorporation of L-[a-14C]leucine into the proteins of the rotated cells. These results impliedthat aggregation was not dependent on the synthesis of ' cell aggregating' material to replenishthe loss of such material from the surfaces of the cells owing to the trypsin treatment. It issignificant that 2,4-dinitrophenol, which is known to suppress cellular metabolism by in-hibiting the production of ATP, also brought cell aggregation to a halt only after a lag periodduring which initial aggregates were formed.

It is suggested, as evidenced in the results of our investigations, that puromycin may wellbe exerting its inhibitory effect by ultimately depressing cellular metabolism below the levelat which certain basic adhesive mechanisms at the cell surface can operate.

324 R. B. Kemp, B. M. Jones, I. Cunningham and M. C. M. James

INTRODUCTION

Puromycin, an inhibitor of amino acid transfer from s-RNA into protein (Yarmo-linsky & de la Haba, 1959), is capable of suppressing RNA-dependent protein syn-thesis in a variety of tissues and organs (Nathans & Neidle, 1963). Indeed, as measuredby the incorporation of radioactive leucine, puromycin has been shown to have animmediate and striking inhibitory effect on protein synthesis in dissociated cells(Humphreys, 1966).

Moscona & Moscona (1963), when they had demonstrated that puromycin exerteda reversible inhibitory effect on the aggregation of rotated trypsin-dissociated cells,attributed the ability of this inhibitor to halt cell aggregation to its effectiveness inblocking protein synthesis. On this assumption, they maintained that the trypsin-dissociated cells required to regenerate cell-binding constituents in order to aggregate.They predicted that the trypsin in the cell dissociation method removed cell-bindingconstituents from the cell surface, and that these constituents had to be replenishedbefore the cells could reaggregate.

Curtis & Greaves (1965) drew attention to the inhibitory effect that serum couldpresumably exert on the aggregation of cells, and they went as far as to argue that itwas the serum and not the puromycin in the medium used by Moscona & Moscona(1963) that inhibited aggregation. The validity of this argument rested on whetherpuromycin could inhibit aggregation in a serum-free medium. Curtis & Greaves(1965) claimed that it could not, whereas Moscona & Moscona (1966), after repeatingtheir earlier experiments and reaffirming their results, maintained that it did. However,these conflicting results and interpretations, as realized by Moscona & Moscona (1966),may have been derived from defining aggregation differently. The use of versene byCurtis & Greaves (1965), instead of trypsin as a cell dissociating agent, also invited thesuggestion that this may well have been a contributory cause of the conflicting results.

Although the hypothesis favoured by Moscona & Moscona (1963, 1966) has stimu-lated much useful discussion, it has lacked convincing support despite the isolation(Moscona, 1963) and identification (Margoliash et al. 1965; Humphreys, 1965) ofcell-surface components which participate in the aggregation of sponge cells. Suchsupport is needed from two directions: (1) the demonstration of a significant delaybefore trypsin-dissociated cells on rotation begin to adhere to one another; such adelay would reflect a need by the cells for time to replenish the loss, owing to degra-dation by trypsin, of the required cell-binding products; and (2) the demonstrationthat puromycin, which immediately and strikingly blocks protein synthesis (Hum-phreys, 1966), prevents dissociated cells recovering their adhesive properties andacquiring a capacity to produce even initial aggregates.

The purpose of the present work has therefore been: to try to determine whendissociated cells on rotation recover their adhesiveness after treatment with trypsin orversene; to analyse and quantify the materials removed by these respective celldissociating agents; and to estimate quantitatively by a turbidimetric method theadhesiveness of trypsin- and versene-dissociated cells and their ability to aggregatein the presence of puromycin in a medium with or without serum.

Effect of puromycin on cell aggregation 325

MATERIALS AND METHODS

The present studies were based on the use of fibroblast cells from embryonic chick muscletissue. Pieces of this tissue from embryos of the required age (5 or 9 days old according to thenature of the experiment) were washed in warm Hanks's balanced salts solution (BSS), beforebeing treated for iomin at 37 CC with 0-25 % trypsin (Difco 11250) in Hanks's BSS atpHv6.Dissociation was aided by flushing with a pipette. The dissociated cells were washed in Hanks'sBSS and finally resuspended in this same solution with or without 10 % calf serum dependingon the aim of the experiment.

Since Curtis & Greaves (1965) obtained their versene-dissociated cells from tissues of 5-day-old embryos, we used muscle tissue from embryos of the same age and dissociated it withversene according to their method. Pieces of the muscle tissue were washed and then treatedfor 10 min using 0-002 M ethylenediaminetetra-acetic acid (EDTA) buffered to pH 8 in calcium-and magnesium-free Hanks's solution. The dissociation of the tissue into separate cells by thismethod tended to be inferior to that involving trypsin treatment, because the suspensions con-tained fragments which could not be separated from the single cells. But by careful centri-fugation, a cell suspension sufficiently free from fragments to be usable as a test system in ourexperiments was obtained.

Aggregation

Aggregation was estimated quantititatively by a turbidimetric method, as used previouslyfor tissue cells (Jones, 1965; Knight, Jones & Jones, 1966). The details of this standardprocedure employed in our laboratory has been described by Cunningham & Hirst (1967).The density of cells in the test suspensions was adjusted to 7 x 10' cells/ml using a haemocyto-meter, and 2-ml aliquots were placed in 2-ml siliconized test tubes, each of which was insertedin an absorptiometer (EEL Quantitrator with Unigalvo 20 attached). The cell suspension wasrotated by a siliconized glass-covered rod stirrer which, while present at the bottom of the tube,was magnetically rotated at 450 rev/min. The cell suspension was kept at 37 °C, and the periodof the test was 4 h. Changes in the optical density of the rotated cell suspension were recordedat a wave-length of 600 m/i; the optical density decreases as cell aggregation progresses andincreases if aggregates disperse. However, in the case of suspensions of cells dissociated withtrypsin the initial phase of aggregation coincided with an increase in optical density to producean initial peak. This peak did not occur when versene-dissociated cell suspensions, whichcontained fragments and small groups of cells at the start of the 4-h period, were rotated.

Progress in aggregation could also be calculated by examining samples of a test cell suspen-sion and counting the single cells that remained. Cell viability was rapidly checked by adding1 drop of a 2 % aqueous solution of either trypan blue or lissamine green stain to 9 drops ofthe suspension; viable cells do not take up these stains whereas dead or damaged cells do soreadily. When necessary, cell viability was also calculated by transferring treated dissociatedcells to culture vessels and counting the cells which failed to display the appearance and changesin shape assumed by viable cells.

Puromycin was added to cell suspensions with or without serum to give a final concentrationof 10 /tg/ml. Fresh solutions of 1 x IO~4M 2,4-dinitrophenol (DNP) in Hanks's BSS were usedto test the effect of this compound on cell aggregation. The dissociated cells were resuspendedin this solution and the preparation was immediately inserted in the absorptiometer and rotated.

Radioisotope studies

In the radioisotope studies to determine protein synthesis, c 1 ml of a 20 /ic/ml solution ofL-[a-uC]leucine (specific activity 261 fic/mg) in Hanks's BSS was added at zero time (o-h)to single cells suspended in Hanks's BSS with or without serum to give a final concentration of1 /ic/ml/7 x io° cells. Puromycin was added to a parallel series of suspensions to give a finalconcentration of 10 /tg/ml. The cell suspensions contained in 2-ml test tubes were rotatedaccording to the procedure adopted in the turbidimetric method. The uptake of radioactiveleucine into proteins during the first 15 min was then determined according to Humphreys'procedure (1966). Samples to be plated for counting were transferred to chilled conical gradu-

21 Cell Sci. 2


ated centrifuge tubes with careful washing of the 2-ml test tubes, and the cells centrifuged at500 g for 5 min. The cells were then washed with excess cold Hanks's BSS, resuspended incold s % trichloroacetic acid (TCA) and stored overnight at 4 °C. The precipitate was centri-fuged and resuspended in 5 % TCA at 90 °C for 30 min. The preparation was cooled to roomtemperature, centrifuged and washed successively in 1 ml of 40 /*g/ml 'cold' L-leucine, 70%ethanol, 100% ethanol and diethyl ether. The precipitate was then dissolved in I N NaOH(2 h at 25 CC), neutralized with 0-3 N HC1 and made up to 5 % TCA by addition of 30 % TCA.The precipitate was plated on a Millipore membrane (27 mm diameter) using a Milliporefilter (Catalogue no. XX10/025/00). The membranes were placed in planchettes and retainedin a flattened position with nylon rings. The planchettes were stored in a desiccator but wereremoved and equilibrated with the atmosphere 12 h prior to counting. The samples were countedon a Nuclear Chicago Model 186A gas-flow radioactive counter to a standard error of 0-5 %.

Analytical methods

The procedures adopted for the analyses of the materials present in the cell-free supernatantsobtained after the preparation of suspensions of dissociated cells by trypsin- or versene-treatment of the tissues were as follows. Glucose was omitted from the Hanks's BSS whensupernatants of cell preparations were required for analysis. The supernatants, prior to analysis,were also centrifuged at 2500 g for 5 min to remove fine particles of tissue debris.

The supernatant obtained after using the versene-dissociation method was routinely scannedin the ultraviolet spectrum using a Unicam SP 800. Clarification was frequently necessary andwas attained by centrifugation at 30000 £ (MSE Superspeed 50) for 1 h, but during thisprocedure, it was estimated that about 10 % of the protein was lost.

Sialic acids were assayed as iV-acetylneuraminic acid by the 2-thiobarbituric acid methodof Warren (1959). In view of the recent objections of Eichberg & Karnovsky (1966), no attemptwas made to distinguish' free' sialic acid from' bound' sialic acid. The supernatant was simplyhydrolysed at 80 °C for 1 h using sulphuric acid at a final normality of O-IN (Svennerholm,1958). The yield of iV-acetylneuraminic acid (NAN) was estimated spectrophotometricallywith a Unicam SP 500 against a reagent blank, using NAN (Koch-Light) as a standard andemploying Warren's (1959) correction equation for sialic acid estimation in the presence ofsubstantial amounts of 2-deoxyribose, that is:

/wnole NAN = 0-138 x ODMt-ooog x 0£>Hl,

where ODKt and OZ)Mj are the optical densities at 562 and 532 m/i, respectively. This correc-tion was considered necessary owing to the presence of significant quantities of deoxypentosesugars as nucleic acids in the supernatants.

Total sugars were estimated using Dische's (1962) modifications of the Molisch a-naphtholtest. The presulphonated a-naphthol (Devor, 1950) was stabilized by allowing it to stand in thedark for 8 h at 18 CC (Devor, 1952). The solutions were assayed spectrophotometrically (UnicamSP 500) at 550 m/i against a reagent blank using glucose as a standard.

Total hexose was assayed by Dische's (1962) method using 2 % anthrone reagent.Nucleic acids as sugars were assayed by Dische's (1944) carbazole reagent using the procedure

described by Snell & Snell (1957). This reagent reacts with both pentose and deoxypentosenucleic acids (Gurin & Hood, 1939, 1941). Colour development was measured spectrophoto-metrically at 520 m/t against nucleic acid standards and reagent blank. Deoxyribose wasestimated by the Dische (1930) diphenylamine reaction using the Davidson & Waymouth (1944)procedure. In both cases the nucleic acids were extracted from protein and lipid in the standardway (Davidson, 1965), using trichloroacetic acid or perchloric acid.

Protein was estimated by the Lowry, Rosenburgh, Farr & Randall (1951) modifications of theFolin-Ciocalteu (1927) reaction. Total protein was estimated after alkaline hydrolysis with INNaOH for 10 min at 100 °C. In this case the 20 % (w/v) sodium carbonate reagent was notmade up in O-IN NaOH as in the estimation of soluble protein, which does not requirehydrolysis. The protein content of trypsin complicated the estimation of Folin-Ciocalteu-positive substances in the tryptic partial digest. Much of the trypsin was removed along withthe cells and tissue debris to which it was adsorbed, as shown by fluorescent methods using afluorescein isothiocyanate-trypsin conjugate, so simple difference methods could not be used


to estimate the tissue-derived compounds. Since trypsin elutes in the void volume of a Sepha-dex G.25 column, gel filtration was used to separate the trypsin from the peptides, which werethen concentrated by freeze-drying and estimated in the usual way. Trypsin-released proteinswere not detected by this procedure but any discrepancy as a result of this was minimized insome experiments by allowing digestion to continue to completion in the cell-free preparation.The digestion was monitored by recording the amount of O-IN NaOH consumed per unittime required to maintain the pH at 8-o (Bennich, 1961). The reaction was not arrested after acertain time by a drop in pH, since this precipitated a quantity of protein.

Ckromatography

The gel nitration studies (Porath & Flodin, 1959) involved the use of the cross-linkeddextran ' Sephadex' (A.B. Pharmacia, Uppsala, Sweden) at a bed volume (K() of 205 cm3 in a45 x 2-54 cm Pharmacia chromatography column, temperature-regulated to 4 °C. The bufferflow was regulated by a peristaltic pump. The effluent was monitored continuously in an LKBUvicord ultraviolet absorptiometer (254 m/t) and collected as 15-min fractions in an LKBfraction collector. The standard basic technique for gel-filtration column chromatography(Gelotte, 1964) was largely adhered to.

Sephadex G.25 (Wr. 2-5 ± 0-2 g/g, particle size 20-80/i) was used to isolate peptides fromtrypsin; the applied sample was 15 ml, the eluting buffer was 0-145 M NaCl, phosphate-buffered (o-oi M) to pH 7-1, and the flow rate was 90 ml/h. The peptides from the tryptdc digest(10-ml samples) were separated using either Sephadex G.50 (Wr. 5-0 ±0-3 g/g, particle size20-80 fi) or Sephadex G.75 (Wr. 7-5 ± 0-5 g/g, particle size 40-120 fi), both having a buffer-flowrate of 50 ml/h. If the peptides were not initially passed through Sephadex G.25, they weredeproteinized using trichloroacetic acid at a final concentration of 5 % (w/v) for 15 min at20 °C, and then centrifuged at 500 g for 2 min (Cook, Heard & Seaman, 1962). The trichloro-acetic acid-soluble peptides were dialysed (except in certain cases) overnight against runningtap water (Langley & Ambrose, 1964), during the course of which the 'free' sialic acid, 97 %of the hexose, and possibly (Ledvina, 1963) some of the mucopeptides were removed. The eluantwas either IM acetic acid, pH 2-6 (Bennich, 1961) or phenol:acetic acidrwater (1:1:1, w/v/v)(Carnegie, 1965). In the latter case a Pharmacia column could not be used owing to the corrosivenature of the eluant; a modified QVF glass tube of the same dimensions as the column was usedinstead. In another series of experiments the crude tryptic digest was either directly applied tothe column, or it was treated as follows: acidified to pH 4-6 with 1M acetic acid (pH 2-6) at25 °C, left for 30 min, and centrifuged at 1200 g for 20 min; then the supernatant was appliedto a G.50 column and eluted with 1M acetic acid, pH 2-6.

The supernatant obtained after versene dissociation was desalted using Sephadex G.25(Flodin, 1961) and lyophilized; 3-5 mg of protein from the lyophilate were redissolved in 10 mlof the eluant and applied to a Sephadex G.200 column (bed volume 205 cm3, Wr. 20 ± 2 g/g,particle size 140-400 mesh, Lot no. T0.4762). The sample was eluted at 2-o ml/cma/h usingO-IM TRIS-HC1 in IM NaCl, pH 8-o (Gelotte, Flodin & Killander, 1962), with 0-02% (w/v)sodium azide as a bacteriocide. In certain cases, lipoproteins were also removed from the re-dissolved lyophilate by increasing the non-protein density to 1-06 g/ml using sodium chlorideand centrifuging at 105 000 g (MSE Superspeed 50) for 19 h (Wallenius, Trantman, Kunkel& Franklin, 1957). The lipoproteins were removed from the top 3-5 ml by the technique of deLalla & Gofman (1954).

The void volume (Vo) of the columns was experimentally determined using haemoglobin inO-O2M phosphate buffer, pH 6-8 (Gelotte, 1964), for G.25, and using o-i% Blue Dextran2000 (A.B. Pharmacia, Uppsala, Sweden) in eluant buffer for G.50, G.75 and G.200. As across-check, Vo was calculated from the equations given by Gelotte (1964).

RESULTS

Adhesiveness of trypsin-dissociated cells

When a suspension of single trypsin-dissociated cells in Hanks's BSS with orwithout serum was rotated, the cells immediately began to adhere to one another.


Counts of single cells in samples taken at intervals confirmed this observation, sinceit was calculated that the number of cells decreased shortly after the start of theexperiment and continued to do so throughout the 4-h period (Fig. 1). Therefore,although trypsin removed a substantial amount of material from the treated tissues(see Table 1), it did not seem to impair the effectiveness of the cellular adhesivemechanism.

Table 1. Analysis of the major componentsin cell-free supernatants from preparations of cellsobtained by dissociating embryonic chick muscle tissue with trypsin and versene (EDTA)

Age of embryo, days. . .Tissue utilized, mg/embryoDissociating agent, ml/mg tissueCells dissociated/embryoCells dissociated/mg tissueN-acetylneuraminic acid, /imol/mg tissueTotal sugars, /ig/mg tissueHexose, /ig/mg tissueNucleic acids, /ig/mg tissueTotal protein, /ig/mg tissueSoluble protein, /ig/mg tissue

r

EDTA

5117

6-84 x io~3

5 00 x 10'4-27 x io4

1-75 x io"5

1-1303690-2932-191-49

Dissociating agentA

\

Trypsint

5142

4-65 x 10-3

480 x 10"3-38 x i o 4

138 x 10-4

——

0-6725-90

—

93 0 2

6-62 x io~3

2-25 X io'7-33 xio4

1-17 x io~*I-OI0-2720-5303-90

—

"b 6

£ 4.Sm

o J

V3

•

•

\

0 1 2 3 4Hours

Fig. 1. Shows progressive reduction in the number of single cells remaining in a sus-pension of trypsin-dissociated embryonic chick fibroblast cells rotated at 37 °C duringthe 4-h period, at the end of which the process of aggregation is completed. Notethat the number of single cells starts to decrease at the beginning of the period.

Effect ojpu.Tom.ycin on cell aggregation 329

The initial rise in optical density of the cell suspension coincided with the singlecells adhering to one another to form small initial aggregates; many single cells,however, remained. It has been suggested that this initial optical density peak isproduced only when suspensions at the start of rotation are comprised almost entirelyof single cell9 and that it is linked with progress from the single-cell state to the forma-tion of small aggregates which, in attaining a certain critical size, increase the overalllight scatter (M. C. M. James, B. M. Jones & R. B. Kemp, in preparation). This initialrise in optical density is followed by a decrease which continues as shown by thepronounced downward inflexion of the optical density curves of the control prepara-tions in Figs. 2, 3.

06

-a

•a

O

0 1 2 3 4 0 1 2 3 4Hours

Fig. 2. Effect of puromycin on the optical density curve of a rotated suspension oftrypsin-dissociated fibroblast cells from o-day-old chick embryos, when introducedato-h to give a final concentration of 10 /ig/ml. A, cell suspensions rotated in Hanks'sBSS at 37 CC without (c) and with puromycin (t). B, cell suspensions rotated inHanks's BSS containing 10 % calf serum at 37 CC without (c) and with puromycin (t).Initial aggregates were produced in the presence of puromycin with or without serum.

Effect of puromycin on the adhesiveness and aggregation of trypsin-dissociated cells

When puromycin was added at o-h to a suspension of trypsin-dissociated cells at afinal concentration of io/ig/ml, the optical density increased in the first half-hour toan initial peak. This rise occurred irrespective of whether or not serum was presentin the Hanks's BSS (Fig. 2). and it coincided with the production of small initialaggregates, but aggregation did not progress beyond this initial stage (Fig. 8). Theoptical density curves for such treated preparations levelled out after the initial rise(Fig. 2). The small aggregates, which were produced in the first hour, persisted; theywere still present in the puromycin-treated preparations at the end of the 4-h period.

Puromycin was also added to cell suspensions at a final concentration of 10/ig/mlafter the cells had been rotating and aggregating for either 1 or 2 h. Since o-i ml ofHanks's BSS containing the puromycin to give the required final concentration wasadded to the suspension, o-i ml of Hanks's BSS was simultaneously added to thecontrol preparations at the corresponding times.

It will be seen from the family of optical density curves in Fig. 3 that when puro-


mycin was added to a cell suspension after i h, the optical density curve continued toslope downward for a further hour before levelling off (Fig. 3.B). When added after2 h the optical density again decreased in the following hour at a rate comparablewith that of the control (Fig. 3 C) over the corresponding period, but from the thirdhour onwards it remained constant (Fig. 3D). The levelling out of the optical densitycurves of the treated preparation coincided with a halt in aggregation; aggregation inthese preparations was therefore incomplete in comparison with the controls.

Fig. 3. The effects of puromycin on the optical density curve of rotated suspensionsof trypsin-dissociated cells in Hanks's BSS at 37 °C, when introduced to give a finalconcentration of 10 ftg/ml at different times during the 4-h period. A, control pre-paration to which o-i ml of Hanks's BSS was added after 1 h (H). B, a preparation towhich o-i ml of Hanks's BSS containing the amount of puromycin (P) required togive 10 /ig/ml was added after 1 h. C, control preparation to which Hanks's BSS wasadded after 2 h. D, preparation to which puromycin was added after 2 h. Note thelevelling out of the optical density curves 1 h after the puromycin had been added.

These results, which were qualitatively reproducible, showed that even when thedissociated cells had been allowed adequate time to synthesize and replenish the lossof the supposed 'cell-binding constituents', the puromycin could still, after a lagperiod of about an hour, bring progress in aggregation to a halt.

Effect of puromycin on cell aggregation

Effect of puromycin on the aggregation of versene-dissociated cells

When suspensions of versene-dissociated cells in Hanks's BSS with or withoutserum were rotated at 37 °C the cells adhered to one another and produced aggregates.However, these were not as well-formed, or as compact as those produced in sus-pensions of trypsin-dissociated cells. The elongated fragments which could not beseparated from the single cells (Fig. 10) frequently became caught up in the cohesivecell masses (Fig. 11). Even so, it was evident that the versene-dissociated cells wereadhesive and capable of forming relatively large aggregates in the control preparationsas reflected by the downward slope of the optical density curve (Fig. 4). In contrast,the optical density of the puromycin-treated preparation decreased only slightly, sig-nifying inhibition of progress in aggregation.

0-5

aO

0-4

0 1 2 3 4Hours

Fig. 4. The effect of puromycin on the optical density of a rotated suspension ofversene-dissociated cells in Hanks's BSS at 37 °C, when introduced at o-h. Opticaldensity curves for treated (t) and untreated (c) preparations. Initial aggregates wereproduced in the presence of puromycin.

Under the standardized conditions of the turbidimetric method, preparations ofversene-dissociated cells on rotation, unlike those of trypsin-dissociated cells, did notproduce an initial optical density peak in the first hour. If an essential requirement toproduce such a peak is that at o-h, the suspension should be composed almost entirelyof single cells so that initial progress in aggregation can proceed from the single stateto the formation of small initial aggregates (M. C. M. James et al. in preparation),


then the failure of preparations of versene-dissociated cells to produce such a peakmay be attributed to fragments and small groups of cells already being present at thestart of an experiment (Fig. 10).

When puromycin was added to suspensions of versene-dissociated cells after theyhad been rotating and aggregating for either i or 2 h, the results were similar to thoseobtained when the same tests were conducted with suspensions of trypsin-dissociatedcells (see Fig. 3).

Effect of puromycin on protein synthesis of trypsin-dissociated cells

Puromycin introduced at o-h into cell suspensions with or without serum to give afinal concentration of io/^g/ml immediately and effectively inhibited cellular proteinsynthesis, according to determinations of incorporated radioactive leucine in the TCA-precipitated fraction. It was shown that puromycin inhibited protein synthesis within15 min by 92'53 % in cells rotated in Hanks's BSS without serum, and by 90-6 % incells in Hanks's BSS with serum. In these preparations containing both puromycinand radioactive leucine the cells continued to aggregate for 60 min to form initialaggregates corresponding to those already described (Fig. 8).

Effect of dinitrophenol on the aggregation of trypsin-dissociated cells

It was evident from the results of testing the effect of puromycin on the aggregationof dissociated cells, that there was a considerable time lag before the inhibitoryeffect of puromycin was expressed. This pattern occurred despite protein synthesisbeing blocked immediately by the puromycin.

Since it has been demonstrated that puromycin was capable of suppressing cellularmetabolism independently of its inhibition of protein synthesis (Hofert & Boutwell,1963; Giudice, 1965; Frankel, 1966; Gutman, Autor & Lynn, 1966), attention wasnext turned to testing the effect on aggregation of DNP, which suppresses cellularmetabolism by decreasing the production of ATP (Webb, 1963).

When DNP was introduced into the cell suspension at o-h, at a final sublethalconcentration of 1 x I O ^ M , the optical density increased initially to produce in thefirst hour a significant peak corresponding to that in the control. In the second hourthe optical density decreased less than it did in the control, and it remained constantin the second half of the period, as shown by the levelling out of the optical densitycurve (Fig. 5).

In the DNP-treated suspensions the cells produced initial aggregates, but aggrega-tion did not go beyond this stage. The pattern of results obtained by treating the cellsuspensions with DNP was therefore qualitatively the same as that obtained withpuromycin.

Materials removed by trypsin and versene in cell dissociation procedures

The quantitative estimations of components in the supernatants from preparationsof cells derived from the muscle tissue of 5-day-old chick embryos was based on theaverage from the results of a series of assays involving a total of 61 embryos, whenversene was used as the dissociating agent, and 15 embryos when trypsin was used


(see Table 1). The analysis of the tryptic digests, derived from the relatively smallnumber of embryos, was carried out to provide comparative results. A further seriesof six analyses of tryptic digests from twenty seven 9-day-old chick embryos was alsocarried out and the mean results are shown in the Table.

The determinations are expressed in units mass/mg of tissue since the analysedmaterial present in the supernatants is not only derived from the surfaces of thedissociated cells but also from tissue fragments, clumps of undissociated cells andintercellular tissue fluids. It will be seen in the Table that the number of dissociated

0-5

-o

•0

O

0-4

I

0 1 2 3 4Hours

Fig. 5. The effect of DNP on the optical density of a rotated suspension of trypsin-dissociated cells in Hanks's BSS at 37 °C, when present at o-h at a final concentrationof 1 x IO~*M. Optical density curves for control (c) and treated (t) preparations.Initial aggregates were produced in the treated preparations.

cells per mg of tissue is relative to the age of the embryo from which the tissue wasobtained, and not to whether trypsin or versene is used in the method of cell dis-sociation. Contrary to common belief, the method of cell dissociation using verseneremoved substantial quantities of protein, although when the two methods of dis-sociating 5-day-old embryo tissue are compared, relatively larger quantities of proteinwere removed when trypsin was used. If the nucleic acid levels are taken as a measureof lysis, then the relatively larger quantities of protein present after trypsin treatmentcould be partly accounted for by the release of intracellular proteins. However, theseintracellular proteins would be heterogeneous and in relatively small quantities, andso they could not account for the predominant peptides present in the digest of both

334 R- B- Kemp, B. M. Jones, I. Cunningham and M. C. M. James

5- and 9-day-old embryos. When the muscle tissue from the batch of 5-day-oldembryos was treated with trypsin, a gel-like material was produced. It contained85 % of the nucleic acids and accounted for half the protein present in the supernatant.The gel could be solvated by addition of physiological saline in a volume ratio of4:1, saline:gel.

A noteworthy result is that the quantity of iV-acetylneuraminic acid in the supernatantafter trypsin treatment was greater than it was in the supernatant after treatment withversene.

50 100 150 200Effluent volume, ml

250

Fig. 6. Gel filtration of trichloroacetic acid-soluble peptides in tryptic digest obtainedfrom muscle tissues of 9-day-old chick embryos by the method of cell dissociation.The gel used was Sephadex G.50, the eluant was 1 M acetic acid, pH 2-6, and theflow rate was 50 ml/h. The resolution of the peptides is shown before (broken line)and after (continuous line) overnight dialysis against running tap water.

The gel-filtration studies made in this investigation on the proteins and peptidesreleased after trypsin and versene treatment of the tissues represent an initial step inthe characterization of these constituents. So far, it has been shown that the proteinsin the supernatants produced after versene dissociation, which produced a peak at260 m/i in the ultraviolet spectrum, separated into 3 peaks on Sephadex G.200, thesecond two of which could not, however, be completely resolved by this straight-forward method but were resolved by recycling chromatography. Despite applicationof centrifugation procedures to remove possible lipoproteins, the pattern of the 3peaks remained unchanged.

After treatment with trichloroacetic acid, the tryptic partial digest from the dis-sociation of the tissue from 9-day-old embryos produced, on elution with 1 M aceticacid at pH 2-6 through a Sephadex G.50 column, one main peak with a distributionconstant (Kd) of 1-41, preceded by two very much smaller peaks (Fig. 6). The resolu-tion of the TCA-soluble substances into three fractions was not altered by continueddigestion of the cell-free preparation to completion, which usually took 3 h. Thequantity of peptide in the main peak was, however, significantly greater.


Since the above Kd indicated that adsorption had taken place, the theoreticalupper limit of Kd being i-o, phenol:acetic acid:water (1:1:1, w/v/v) was adopted aseluant for the rechromatography of the main peak on a G.50 column. The Kd ofthis sample was now approximately 0-47, although this could not be calculatedaccurately owing to difficulties in interpreting water regain in this eluant.

Resolution of the crude digest on Sephadex G.50 gave the same pattern of results,except that there was a large peak in the void volume, which consisted mostly oftrypsin. The acetic acid-treated digest produced a single peak with a Kd of 1-4 onelution through G.50 with 1 M acetic acid, pH 2-6.

The main peak, judging from its adsorption properties on Sephadex, would appearto contain aromatic amino acids. This peak is being analysed further.

DISCUSSION

The results of this investigation show that trypsin and versene treatment as used incell dissociation procedures did not impair the effectiveness of the adhesive mechan-isms of the treated cells. The dissociated cells began to adhere to one another shortlyafter they were rotated at the start of the test period.

Puromycin did not delay the adhesion of the cells to one another and did not inhibitthe formation of initial aggregates (Fig. 8) when it was added at o-h to suspensions ofeither trypsin- or versene-dissociated cells to give a final concentration of 10/ig/ml.This concentration of puromycin effectively blocked protein synthesis in the rotatedcells as shown by the 90 % inhibition within 15 min in the incorporation of L-[a-14C]leu-cine into the intracellular proteins of cells, which continued to aggregate for thefollowing 45 min. This recalls the finding by Humphreys (1966) that dissociatedsponge cells aggregated normally for 6 h after protein synthesis had been shown to beblocked by puromycin.

Initial aggregates were formed in the presence of puromycin irrespective of thepresence or absence of calf serum in the medium. Hence our curiosity over the reportthat versene-dissociated cells remained in the single state when horse serum waspresent together with puromycin in the medium, and that 3- to 4-cell aggregates wereformed when the horse serum was omitted (Curtis & Greaves, 1965). Our results arein agreement with their observation that dissociated cells were capable of adhering toone another in the presence of puromycin, but we did not find, under the conditionsof our turbidimetric method for estimating aggregation, that the cells failed to adhereto one another when both calf serum and puromycin were present, although, likehorse serum, calf serum is supposed to possess aggregation-inhibitory properties(Curtis, 1965).

Therefore, regardless of possible aggregation-inhibitory factors in horse serum,though certainly not in the calf serum we used, our results are not in agreement withthe implication in the argument of Curtis & Greaves (1965) that puromycin does notexert an inhibitory effect on cell aggregation, but, at the same time, they do notsupport the conclusion as broadly drawn by Moscona & Moscona (1966) that puro-mycin completely inhibits cell aggregation as they defined it. Our results show


that puromycin will bring progress in aggregation to a halt only after initial aggre-gates have already been formed, and it is in this context alone that we agree thatpuromycin is capable of inhibiting aggregation.

It also seems to us that if the initially formed ' loose clusters' of cells observed byMoscona & Moscona (1966) are comparable with the initial aggregates produced inour cell suspensions after puromycin had been introduced at o-h (Fig. 8), then theseauthors could equally have decided to emphasize that puromycin does not inhibit cellaggregation, and that it is capable only of halting progress in aggregation eventually,after a lag period, during which time the cells form initial aggregates.

Analysis of the materials in the supernatants derived from the trypsin- and versene-treated cell preparations showed that both trypsin and, contrary to general belief,versene removed substantial amounts of protein during the cell dissociation pro-cedures (Table 1). It is likely that in the application of the two cell-dissociating agentsto 5-day-old embryos, the greater amount of proteinaceous material in the super-natant after trypsin dissociation could be accounted for partly by more extensive lysis(as indicated by a higher nucleic acid content), which would result in a proportionallygreater release of intracellular proteins. Similarly, the proportionally less yield (unitmass per quantity of dissociated cells) of protein and nucleic acid from tissues from9-day-old embryos dissociated with trypsin would suggest that lysis of these cellswas less. This may reasonably be expected when one considers that the tissues arerelatively older and, presumably, more resistant to the trypsin-treatment. It is evidentthough that most of the Folin-Ciocalteu-positive material after trypsin-dissociationis derived from the surfaces of the cells, since only one peptide peak, which may beassociated with sialic acid (see Table 1), predominated in the gel-filtration experi-ments. This finding is in agreement with that obtained by Langley & Ambrose (1964),who used trypsin to isolate a mucopeptide from the surfaces of Ehrlich ascites tumourcells, and by Ohkuma & Ikemoto (1966), who isolated a sialoglycopeptide liberatedfrom red blood cells, when these cells were treated with trypsin. If the bulk of thepeptides in the tryptic digest had been derived from intracellular proteins, one wouldhave expected a more heterogeneous distribution after gel filtration, since it is mostunlikely that the whole range of intracellular proteins would be split into aromaticpeptides of similar molecular weight.

While it is fair to assume that the levels of nucleic acids may be a measure of celllysis, it is probable that these levels are abnormally high owing to other sugarsinterfering with the carbazole test. Nevertheless after trypsin-dissociation of 5-day-oldembryos, the production of the frequently reported (Cook et al. 1962; Moscona, 1962)gel-like material, which is destroyed by DNase (Steinburg, 1963), would seem tosupport the suggestion that lysis does occur. Similarly, the absence of a gel aftertrypsin dissociation of 9-day-old embryos probably reflects the earlier-postulatedmuch-lesser degree of lysis. In both cases, however, some of the nucleic acids de-tected could have been derived from cells damaged at the cut edges of the tissuefragments (Steinberg, 1963).

One of the remarkable features that emerges from the analyses of the supernatants(see Table 1) is the fact that there was a yield of iV-acetylneuraminic acid after


versene dissociation. It can be postulated that this acid was derived partly from glyco-proteins solubilized, either directly or indirectly, by the action of the chelating agent;and partly from the internal membranes (Wallach & Kamat, 1966) of cells rupturedduring the preparation of the cell suspension. The different levels of sialic acid in thesupernatants (see Table 1) obtained by the trypsin- and versene-dissociation methodsemployed in our cell aggregation experiments would not seem to support the suggestionthat sialic acid, by analogy to its postulated structural role in salivary mucoprotein(Gottschalk, i960), has an important function is some aspect of cell contacts (Weiss,1965) since, presumably, trypsin-dissociated cells had little surface sialic acid, whereasversene-dissociated cells had a great deal; yet they both aggregated equally efficientlyin the controls (Figs. 9, 11). This finding supports our view that quantitatively im-portant cell-surface components are unimportant in relation to the ability of thebasic adhesive mechanisms to initiate adhesion.

It will be apparent that the considerations up to this point already cast doubt on thevalidity of the hypothesis that protein synthesis is necessary for providing trypsin-dissociated cells with cell-binding constituents to restore their adhesive properties(Moscona & Moscona, 1963, 1966). The further finding that cells which had alreadybeen rotating and aggregating for 1 h or even 2 h at 37 °C before puromycin wasintroduced stopped aggregating 1 h later, strongly suggests that the puromycin couldbe producing its inhibitory effect on aggregation by some means other than by pre-venting the regeneration of cell-binding constituents. There would have been adequatetime, one would have thought, during the 2-h or even the i-h period prior to thepuromycin being introduced, for the production of enough cell-binding constituentsto allow aggregation to continue during the remaining part of the 4-h period.

As evidenced by the results of adding puromycin at different times during the 4-hperiod and of the radioactive studies, cell surface constituents removed during dis-sociation procedures do not function as an integral part of a basic adhesive mechanism,which is probably of similar constitution in most types of cells. One is therefore ledto consider evidence to suggest that puromycin may well be bringing progress in cellaggregation to a halt in rotated cell suspensions by indirectly affecting basic cellularadhesive mechanisms. A crucial fact supporting this possibility is that it is now welldocumented that the action of puromycin is not specific to its inhibition of proteinsynthesis. Quite independently of this function puromycin is known to depress carbo-hydrate metabolism; it causes glycogenolysis in mouse liver (Hofert & Boutwell, 1963)and affects the uptake and utilization of glucose in fat cells of the rat (Gutman et al.1966). Giudice (1965) found that puromycin suppressed respiratory metabolism ofdissociated sea-urchin cells by inhibiting their oxygen uptake and Frankel (1966)showed that puromycin could depress oxygen uptake by up to 20 % in Tetrahymenapyriformis, G.L. We suggest therefore that puromycin may well be exerting its in-hibitory effect on the aggregation of embryonic chick fibroblast cells by suppressingcellular metabolism to a level at which the adhesive mechanisms cannot operate. Thissuppression would explain the observation by Humphreys (1966) that sponge cells,which had been aggregating for 6 h after protein synthesis had been inhibited bypuromycin, died after 12 h.


Further evidence for our suggestion can be found in the action of DNP, since whenthis substance was introduced at o-h to a test suspension, aggregation proceedednormally for the first hour, as shown by the initial optical density peak, to produceinitial aggregates, but was then arrested. It is significant that the pattern of this resultresembles that obtained when puromycin was introduced at o-h. DNP is known toreduce the energy supply in the cell by uncoupling oxidative phosphorylation (Webb,1963), thus suppressing cellular metabolism. One may therefore conclude that DNPbrought progress in aggregation to a halt by directly suppressing cellular metabolism.It is likely that low suboptimal temperatures (Steinberg, 1962; Curtis, 1963; Mos-cona, 1963) also inhibit cell aggregation by suppressing cellular metabolism to a levelat which the cells round up and lose their adhesiveness. By rapidly raising or loweringthe temperature within the range of 8-37 °C at different times, it appears that themechanisms of adhesion, judged by the turbidimetric method, are extremely sensitiveto changes in temperature (M. C. M. James, B. M. Jones & R. B. Kemp, in prepara-tion).

If one accepts our suggestion that puromycin ultimately brings progress in cellaggregation to a halt by suppressing cellular metabolism, it is not too far fetched toassume that puromycin in lowering the rate of cellular metabolism is reducing theeffectiveness of certain basic, enzymically controlled, adhesive mechanisms at the cellsurface. This explanation is compatible with the hypothesis of cell adhesion (B. M.Jones, 1966, 1967) which stems from observations on the effects of exogenous nucleo-tide phosphates on cell adhesion and aggregation (Knight et al. 1966; B. M. Jones,1966; P. C. T. Jones, 1966) and from good evidence marshalled in support of theexistence of ATPases at the cell surface and of contractile proteins of the actomyosintype in non-muscular cells (B. M. Jones, 1966). The hypothesis is based on themechano-chemical principle involved in the contraction and relaxation at the cellsurface of a contractile actomyosin-like protein with ATPase activity, cellular ad-hesiveness depending on the relationship or balance existing between the contractingand relaxing mechanisms.

It is a matter of common observation that cells tend to assume a spherical shapereflecting contraction at low suboptimal temperatures. It was suggested, on the basisof the hypothesis, that the suppression of cellular metabolism in reducing the availableenergy supply inactivates the relaxing system (B. M. Jones, 1966). It is furthersuggested that this system then fails to perform its function of withdrawing calciumfrom the contractile elements. Accordingly, in the presence of excess calcium thecontractile elements assume a contracted state, which is unfavourable to adhesion.This conception of how a suppression of cellular metabolism could inhibit adhesionand aggregation by interfering with the operating of basic adhesive mechanisms atthe cell surface will be further developed in a subsequent publication.

We wish to thank Mrs Alise Howse for technical assistance and Mr R. Moore for preparingthe photographs. The work has been aided by generous grants provided by the British EmpireCancer Campaign for Research and by the Science Research Council.


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(Received 3 May 1967)

journal of Cell Science, Vol. 2, No. 3

For legends see next page

R. B. KEMP, B. M. JONES, I. CUNNINGHAM AND M. C. M. JAMES(Facing p. 340)

Fig. 7. Single cells in a sample taken from a test suspension of trypsin-dissociatedcells in Hanks's BSS at the beginning of the 4-h period, x 650.Fig. 8. Initial aggregates formed by trypsin-dissociated cells in Hanks's BSS at 30 °Cafter puromycin had been introduced at o-h to give a final concentration of 10 /ig/ml.x 650.Fig. 9. Relatively large aggregates produced by trypsin-dissociated cells at the end ofthe 4-h period after rotation in Hanks's BSS at 37 °C. x 650.

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Fig. 10. Single cells and fragments in a sample taken from a test suspension ofversene-dissociated cells at the beginning of the 4-h period, x 650.Fig. 11. Aggregates produced by versene-dissociated cells at the end of the 4-hperiod after rotation in Hanks's BSS at 37 °C. x 650.

R. B. KEMP, B. M. JONES, I. CUNNINGHAM AND M. C. M. JAMES

QUANTITATIVE INVESTIGATION ON THE EFFECT OF …Moscona & Moscona (1963), when they had demonstrated...

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