J. Cell Sci. 42, 169-182 (1980)Printed in Great Britain © Company of Biologists Limited igSo

ROLE OF CELL SURFACE RECEPTOR

MOBILITY IN CONCANAVALIN A-INDUCED

AGGLUTINATION OF NOVIKOFF HEPATOMA

AND NORMAL RAT LIVER CELLS

PAOLA MASTROMARINO, GIOVANNI NERI,* ANGELO SERRAAND EARL F. WALBORG, jRfDepartment of Human Genetics, Catholic University School of Medicine,00168 Rome, Italy, and fThe University of Texas System Cancer Center,Science Park-Research Division, Smithville, Texas 78957, U.S.A.

SUMMARYThe relationship between Con A-receptor mobility and Con A-induced agglutination of

Novikoff hepatoma and normal rat liver cells was investigated. Novikoff cells, incubated withfluorescein-labelled Con A at 3 °C displayed uniform, ring-like surface fluorescence. Increasingthe temperature of the cells to 37 °C caused capping of Con A receptors in approximately 65 %of the cells, a phenomenon that could be prevented by prefixing the cells with glutaraldehyde.In spite of these variations in Con A-receptor distribution, Con A-induced agglutination wasremarkably constant over a temperature range from 3 to 37 °C. In contrast to NovikofF cells,normal hepatocytes displayed a uniform, ringlike surface fluorescence at both 3 and 37 °C. Nocapping was observed. However, hepatocytes, similar to Novikoff cells, were agglutinable bylow concentrations of Con A. These findings indicate that, in this model system, Con A-inducedcytoagglutination is not dependent upon long-range lateral mobility of Con A receptors. Thequalitative differences in the lateral mobility of cell-surface Con A leceptors of normal andmalignant rat liver cells may represent a marker for neoplastic transformation during hepato-carcinogenesis, adaptation to growth in ascitic form, or progression of a tumour to a moremalignant state.

INTRODUCTION

Since the first report by Aub, Sanford & Cote (1965) and subsequently by others(Burger & Goldberg, 1967; Inbar & Sachs, 1969; Sela, Lis, Sharon & Sachs, 1970;Nicolson & Blaustein, 1972) that malignantly transformed cells are more readilyagglutinated by plant lectins than their normal counterparts, a large effort has beendirected toward elucidation of the factors responsible for preferential lectin-inducedcytoagglutination of malignant cells (reviewed by Nicolson, 1974). In a very simplifiedway, lectins can be viewed as acting like molecular bridges extending betweenneighbouring cells and causing agglutination by a mechanism analogous to thatresponsible for antibody-induced cell agglutination.

To determine whether alterations of cell-surface lectin receptors are responsible forthe increased agglutinability of tumour cells, investigations have focused mainly on

* To whom correspondence and reprint requests should be addressed at: Istituto diGenetica Umana, Universita Cattolica, Via Pineta Sacchetti, 644-00168 Rome, Italy.

12 CEL42

170 P. Mastromarino, G. Neri, A. Sena and E. F. Walborg, Jr

cell-surface saccharide-containing components, particularly glycoproteins. Severaldifferences have been reported between normal and malignant cells with respect tothe number, structure, topography and dynamics of cell-surface lectin receptors(reviewed by Smith & Walborg, 1977). Several hypotheses have been advanced toexplain the increased lectin-induced agglutination of tumour cells (Rapin & Burger,1974; Smith & Walborg, 1977). To date, none of these hypotheses has proved ofgeneral applicability, possibly because different mechanisms are operating in differentcell types. Moreover, the concept that increased agglutinability is a distinctiveproperty of cancer cells is no longer tenable. A number of studies on various mousetumours failed to show positive correlation between malignancy and lectin-inducedagglutinability (Gantt, Martin & Evans, 1969; Friberg, 1972; Dent & Hillcoat, 1972).More recently Starling, Capetillo, Neri & Walborg (1977) showed that Novikoffhepatoma cells and normal rat hepatocytes are both agglutinable by low concen-trations of concanavalin A (Con A) and wheat germ agglutinin. In this same modelsystem, it was found that glutaraldehyde-fixed Novikoff cells were still agglutinableby Con A, suggesting that Con A-induced agglutination of these cells is independentof cell-surface receptor mobility (Walborg et al. 1975). Moreover, microfilament-active drugs, such as cytochalasins b and d, proved capable of inhibiting Con A-induced agglutination of Novikoff cells, but did not have any apparent effect on thetopography of Con A receptors (Glenney, Hixson & Walborg, 1979). The presentstudy was undertaken to clarify the relationship between Con A-induced cyto-agglutination and the mobility of Con A receptors on Novikoff hepatoma and normalrat liver cells.

MATERIALS AND METHODS

Cells

Novikoff hepatoma cells (Novikoff, 1957) were maintained in 6- to 10-week-old femaleSprague-Dawley rats (Charles River Italia, Calco, Italy) by weekly intraperitoneal trans-plantation. Normal hepatocytes were obtained as a suspension of cells (largely single cells)from the liver of 6- to 10-week-old female Sprague-Dawley rats by the collagenase perfusionmethod of Starling et al. (1977). Papain digestion and control incubation of Novikoff hepatomacells were performed as previously described (Neri, Smith, Gilliam & Walborg, 1974). Cellviability was assessed on the basis of vital dye exclusion test, using nigrosine (Kaltenbach,Kaltenbach & Lyons, 1958).

Labelling of Con A with fluorescein

Con A (Grade III Sigma Chemical Co., St Louis, Missouri) was conjugated with fluoresceinisothiocyanate component A (Serva Feinbiochimica, Heidelberg, West Germany) by directmixing at pH 90, as described by Mallucci (1976). The conjugate was purified by affinitychromatography on a column of Sephadex G-50 (Pharmacia Fine Chemicals, Uppsala,Sweden). Material that did not bind to the column or that could be removed by elution with001 M glucose was discarded. The active fraction, eluted from the column with o-i M glucose,was concentrated by ultrafiltration on Amicon PM 10 membrane (Amicon B.V., Costerhout,Holland) to a volume of approximately 3-0 ml, and dialysed exhaustively against standardsalt solution, pH 7̂ 4 (Mallucci, 1976) to remove glucose. The ratio of the optical densities at495 and 280 nm, a measure of the degree of conjugation, was 0-5. The concentration of Con Ain the conjugate (53 mg/ml) was calculated from the extinction coefficient of the lectin

Con A receptor mobility and cytoagglutination 171

(E asciii'm = '3)> corrected for the absorption of fluorescein at 280 nm. A virtually identicalestimate of the concentration of Con A was obtained by an independent method, i.e. bymeasuiing the haemagglutinating activity of fluorescein-labelled Con A (Fl-Con A) using thehaemagglutination assay of Smith, Neri & Walborg (1973). This assay also showed thatFl-Con A had the same specific activity as freshly prepared, unlabelled Con A.

Assay of Con A receptor mobility

Untreated, control-incubated and papain-treated Novikoff cells or untreated normalhepatocytes were washed three times in Ca3+- and Mg2+-free phosphate-buffered saline(CMF-PBS) prepared according to Dulbecco & Vogt (1954), resuspended in the same bufferat a concentration of 5 x io6 cells/ml and submitted to the following procedure. To a 4-mlaliquot of cell suspension a specified amount of Fl-Con A was added, and the mixture allowedto incubate for 30 min (unless otherwise specified) at either 3 °C, over crushed ice, at 23 °C,in a temperature-regulated waterbath, or at 37 °C in a metabolic water incubator. Cellsuspensions were then chilled to 3 °C and washed 3 times with cold CMF-PBS in a refrigeratedcentrifuge. The cells were resuspended in 4 ml of buffer and fixed for 30 min at 3 °C byaddition of glutaraldehyde to a final concentration of 2 %. The cells were subsequently washed3 times in CMF-PBS, resuspended in 0-5 ml of the same buffer and observed under a coverslipwith a Leitz Orthoplan ultraviolet photomicroscope equipped with epi-illumination andspecific filters for fluorescein. In a few experiments the cells were fixed with glutaraldehydeprior to incubation with Fl-Con A. The specificity of binding of Fl-Con A to the cell surfacewas assessed by incubating the cells with Fl-Con A in presence of 0-5 M methyl-a-D-mannopyranoside (a-MM), a specific inhibitor of Con A (Poretz & Goldstein, 1970). Alter-natively, cells incubated with Fl-Con A were washed 3 times in CMF-PBS and post-incubatedtwice for 15 min at 37 °C in CMF-PBS containing of 02 M a-MM. Visual scoring of Fl-ConA-labelled cells allowed their assignment to 1 of 3 classes of cells with different patterns offluorescence: ringlike, caplike and patchlike. The proportions of cells belonging to the 3 classeswere then estimated for the various experimental conditions, and the trend of these proportionswas analysed.

Cytoagglutination assays

Untreated, control-incubated or papain-treated Novikoff cells were washed 3 times in CMF-PBS and assayed for Con A-induced agglutination following the procedure of Neri et al. (1974).Con A, purchased from Pharmacia Fine Chemicals, was dissolved in CMF-PBS. Proteinconcentration was determined spectrophotometrically using the extinction coefficient,E 28<tam = X3- The cytoagglutination assays were performed at 3, 23 and 37 °C, with differentconcentrations of Con A. For the assay at 3 °C, the cytoagglutination plates were set on a thinmetal sheet lying over a pan filled with crushed ice. After pipetting the cells and the lectin intothe wells, and mixing with a toothpick, the plates were transferred to the cold room, andremoved 30 min later for microscopic scoring. For the assay at 23 °C, all operations wereperformed at room temperature. For the assay at 37 °C, pipetting of the lectin and cells andmixing were performed at room temperature. The plates were then immediately transferredto an incubator at 37 °C, covered with Petri dishes to minimize evaporation, and removed30 min later for scoring. All agglutination assays were repeated on different days using freshreagents. The degree of agglutination was scored visually on a serological scale from o to 4(Wray & Walborg, 1971). Then, the least square regression lines of agglutination on the logof the Con A concentration at the 3 temperatures were fitted.

RESULTS

Con A receptor mobility of Novikoff cells

Incubation of Novikoff cells with Fl-Con A resulted in labelling of the cell surfacein a ringlike or in a caplike pattern. Caps could take the form of either a protrudingfluorescent mass at the uropod of the cell, or a condensation of fluorescence at one

172 P. Mastromarino, G. Neri, A. Serra and E. F. Walborg, Jr

Con A receptor mobility and cytoagglutination 173

pole of the cell. Cell-surface labelling was specific, and could be completely inhibitedby the presence of 0-5 M a-MM. Discrete patches of fluorescence were also observedin some of the cells scored. The location of these patches at the surface of the cellsseems unlikely since they could not be removed by postincubation of the cells witha-MM. Examples of these fluorescence patterns are illustrated in Fig. 1 (panelsA-E). The cells shown in the plate were incubated with Fl-Con A (66/tg/ml) for30 min at 37 °C (except for panel A, showing a cell incubated at 3 °C). These experi-mental conditions yielded maximal capping as shown in Figs. 2 and 3. Fig. 2, wherethe percentage of cells with cap is plotted against the concentration of Con A, showsthat at 66 /<g of Con A/ml the proportion of cells with cap was maximal. This con-centration of Con A is sufficient for maximal cell agglutination (Neri et al. 1974). Inreceptor-mobility experiments, however, cytoagglutination was largely prevented byusing conditions, such as low cell concentration and repeated pipetting of the cellsuspension, that would minimize cell-cell contacts. The incubation time (30 min)was chosen to render receptor mobility data directly comparable to cytoagglutinationdata, previously standardized on a 30-min incubation (Neri et al. 1974), and wasoptimal in terms of allowing maximal capping. This is shown in Fig. 3, where thepercentage of cells with cap is plotted against time.

50 75 100

[Con A ] , /jg/ml

125 150

Fig. 2. Cap formation in Novikoff cells incubated with various concentrations ofFl-Con A for 30 min at 37 °C.

Fig. 1. Patterns of fluorescence in Novikoff cells (A-E) and normal hepatocytes (F)treated with Fl-Con A (66 fig Con A/ml) for 30 min at 37 CC (except for A, showinga cell incubated with Fl-Con A at 3 °C). x 540. A, ringlike pattern; B, cap; c, uropodalcap; D, patchlike pattern; E, absence of cell-surface fluorescence in Novikoff cellstreated with Fl-Con A in presence of 0-5 M a-MM; F, ringlike fluorescence in normalhepatocyte.

174 P- Mastromarino, G. Neri, A. Serra and E. F. Walborg, Jr

The proportions of Novikoff cells showing ring, cap or patch fluorescence at the3 temperatures tested, and at a Con A concentration of 66/tg/ml, are reported inTable i. In untreated cells, incubation with Fl-Con A at 3 °C resulted in theappearance of ringlike fluorescence in 95 % of the cells, and of caps (all non-uropodal)

40

Incubation time, min

60

Fig. 3. Cap formation in Novikoff cells incubated with Fl-Con A (66 fig Con A/ml)for different times at 37 °C.

Table 1. Pattern of fluorescence'1 of Novikoff cells at different temperatures

Treatmentof cells

Untreated"Incubatedd

Papain treated"

N6

216223211

3A

R,%

958770

c,%53

19

P,%0

1011

Temperature,A

N"

227677825

23A

R,%

849079

c,%164

1 1

°C

P,/n

O

610

N"

4424964Si

37

R,%

312736

c,%656831

P,0//o

45

33

" The pattern of fluorescence was designated: R = ringlike; C = caplike; P = patchlike.6 Total no. of cells scored.c Cells washed 3 times in PBS.d Cells incubated for 40 min in Buffer II (Wray & Walborg, 1971) at 37 °C." Cells incubated as in d above, but in presence of 2 units of papain (Worthington Biochemical

Co., Freehold, NJ.) per ml of cell suspension containing io7 cells/ml.

in 5%. Increase in temperature was accompanied by a significant shift in the patternof surface fluorescence from ringlike to caplike. The increase in the percentage ofcapped cells from 3 to 37 °C was about 13-fold. The trend analysis of proportions ofcapped cells showed that the observed increase with the temperature is not linear.

Con A receptor mobility and cytoagglutination 175

The appropriate statistics (Armitage, 1971; Snedecor & Cochran, 1971) indicatedthat the deviation of proportions from their linear regression on the temperature ishighly significant (Xi = 23-485; P < o-ooi), suggesting that a sudden changeprobably occurs at 37 CC. Patchlike fluorescence was absent at 3 or 23 °C, and presentin only 6% of the cells incubated at 37 °C. Control-incubated cells behaved in anapparently identical way. Here also, the deviation from linearity was highly significantCd = J64"47o; P < o-ooi). Treatment of Novikoff cells with papain resulted in theappearance of a relevant proportion of cells with caplike fluorescence at 3 °C. Thepercentage of capped cells increased further upon raising the temperature to 37 °C,at which, however, their proportion was only 31%, significantly lower than thatobserved in the untreated or control-incubated cells. This effect is clearly correlatedwith a significant increase in the number of cells displaying patchlike fluorescence.Nevertheless, the deviation from linearity, suggesting a sudden change at 37 °C, wasevident also in this experimental condition (xt = 44'1 1 2! P < o-ooi). Fixation ofNovikoff cells with glutaraldehyde prior to incubation with Fl-Con A under any ofthe experimental conditions employed, resulted in a pattern of surface fluorescencecomparable to that observed at 3 °C under those same conditions.

Con A-receptor mobility of normal hepatocytes

Isolated normal rat hepatocytes were incubated with Fl-Con A under experimentalconditions identical to those described for Novikoff cells. Only untreated cells, i.e.cells that had not undergone papain treatment or control incubation, were tested.Surface fluorescence had a ringlike form in 100% of the viable cells at all 3 tempera-tures tested, and at Con A concentrations ranging from 1-3 to 66/tg/ml. The latterconcentration was routinely used, because it gave more distinct and visible rings. Anormal hepatocyte is illustrated in Fig. IF .

Effect of temperature upon cytoagglutination of Novikoff cells

The fitted least-square lines of regression of Con A-induced agglutination ofNovikoff cells upon Con A concentration at 3, 23 and 37 °C are reported in Fig. 4.It clearly appears, on inspection, that agglutination is dependent on the concentrationof Con A. The intercept of each agglutination line with the abscissa represents theconcentration of Con A required for threshold agglutination at that particulartemperature. The point on the abscissa corresponding to the score value of 2 on theordinate represents the concentration of Con A necessary for half-maximal aggluti-nation. Threshold and half-maximal values are reported in Table 2. The untreatedcells show at 37 °C a slight increase in agglutinability, since a smaller concentrationof Con A is required for threshold and half-maximal agglutination. Although aconstant enhancement of agglutinability at 37 °C is also suggested by the parallelismof the regression lines (Fig. 4A) the approximate standard error of the estimated line(sy_x = + 0-485) indicates, however, that such increase cannot be considered significant.The behaviour of the control-incubated cells was virtually superimposable on that ofthe untreated cells. Treatment of the cells with papain caused an increase in agglutin-ability by Con A, an effect that had been previously observed (Neri et al. 1974). At

176 P. Mastromarino, G. Neri, A. Serra and E. F. Walborg, Jr

the same mean concentration of Con A (log x = 1-32) the mean agglutinability at37 °C was 3-13 + 0-27 for papain-treated cells against 2-48 + 0-12 for incubated cellsor 2-27±o-io for untreated cells. A test for the difference of means shows that thisincrease is significant both with respect to untreated cells (t = 2-938; P < o-ooi)and to incubated cells (t = 2-667; P < o-ooi).

37° 3° 23°

5 25

[Con A],

Fig. 4A For legend see page 178.

100

DISCUSSION

Interest in lectin-induced cell agglutination was greatly stimulated by the obser-vation that neoplastic cells are usually agglutinated by lower concentrations of lectinsthan their normal counterpart. The molecular mechanisms responsible for thedifferential lectin-induced cytoagglutination of normal and malignant cells has beenthe subject of numerous investigations (Nicolson, 1974; Smith & Walborg, 1977).Much of this research focused on the role of lectin receptor mobility as a determinantof lectin-induced cytoagglutination. From work of Rosenblith, Ukena, Berlin &Karnowski (1973), of Nicolson (1973) and of Guerin et al. (1974) it might be con-

Con A receptor mobility and cytoagglutination 177

eluded that clustering of lectin receptors in discrete areas of the cell surface is aprerequisite for the increased agglutination of transformed cells. The evidence,however, is only circumstantial, since, as pointed out by Smith & Walborg in theircomprehensive review (1977), these authors did not investigate the agglutinabihty ofcells carrying clustered sites (e.g. transformed cells incubated with Con A at 37 °C)

37"

Z- 2

S1

/ I5 25 100

[Con A], /jg/ml

Fig. 4B For legend see page 178.

as compared with the same cells carrying uniformly distributed sites (transformedcells incubated with Con A at o °C). More direct evidence was provided by Inbaret al. (1973), who showed that aldehyde fixation of mouse lymphoma cells inhibitedagglutination by Con A. Similarly, Noonan & Burger (1973) reported that Py3T3 orSV3T3 cells, that were not agglutinable by Con A at o °C, became agglutinablewhen the temperature was raised to 22 °C. On the other hand, Schnebli et al. (1977),in a series of detailed studies utilizing human erythrocytes as well as cells from normaland malignant tissues, came to the conclusion that cell agglutination by Con A wasnot blocked at o °C, a temperature at which mobility of receptors in the plane of themembrane was inhibited. Rutishauser & Sachs (1974), in a study utilizing normal

178 P. Mastromarino, G. Neri, A. Serra and E. F. Walborg, Jr

mouse lymphocytes and lymphoma cells attached to nylon fibres, provided evidencethat cell-cell binding induced by Con A required short-range lateral movements ofcell-surface lectin receptors, and that clustering of the receptors was not necessary andseemed to hinder cell-cell binding.

The purpose of the present report was to clarify the relationship between the

I25

[Con A], jug/ml

100

Fig. 4. Estimated regression lines of the degree of agglutination upon Con A concen-tration at temperatures of 3, 23 and 37 °C respectively, A, untreated cells; B,control-incubated cells; C, papain-treated cells. Abscissa: Con A concentration ona logarithmic scale. Ordinate: agglutination scores. The dotted lines represent theapproximate standard error of the estimated regression line at 37 °C.

topography and dynamics of Con A receptors at the cell surface and Con A-inducedcytoagglutination of normal and malignant rat liver cells. These studies utilizedNovikoff hepatoma cells and normal rat hepatocytes, cells that are agglutinable bylow concentrations of Con A (Neri et al. 1974; Starling et al. 1977). We took advantageof the fact that lectin-induced receptor movement is a temperature-dependentphenomenon and can be blocked at 0-4 °C, when the membrane lipid bilayer is

Con A receptor mobility and cytoagglutination 179

'frozen' (Reinert & Steim, 1970). Con A receptor movements were monitored atvarious temperatures utilizing fluorescein-conjugated Con A, and were comparedwith Con A-induced agglutination, scored under the same experimental conditions,using a modification of a previously described method (Neri et al. 1974). We foundthat temperature variations had profound effects on the distribution pattern of Con A

Table 2. Con A concentration (/<g/ml)fcr threshold and half-maximal agglutinationof Novikoff cells at different temperatures

Temperature, °C

23 37*

Treatment of cells Thresh." i/2Max4 Thresh." 1/2M1K' Thresh." i/2Max6

Untreated" 45 250 4-0 250 28 i6'3Incubated" 31 190 30 220 2-1 125Papain-treated" 0-45 60 0-75 ic-6 014 36

" Threshold agglutination.b Half-maximal agglutination.<=•*•• As in Table 1.

receptors on Novikoff cells. At 3 °C the distribution was uniform over the entire cellsurface as indicated by the ringlike shape of the membrane fluorescence in 95 % ofthe cells examined. This uniform distribution probably represents the inherenttopographical pattern of the Con A receptors, since an identical pattern was observedwhen the cells were fixed with glutaraldehyde, prior to incubation with Fl-Con A.Upon shifting the temperature to 37 °C, the Con A receptors underwent a redistri-bution, as indicated by the appearance of caps in 65% of the cells scored. In a fewcells the fluorescence appeared clustered in discrete patches over the cell. Howeverappropriate controls in presence of a-MM, indicated that the fluorescence could notbe removed by post-incubating the cells with a-MM, suggesting that the fluorescentpatches were intracellular and probably represented pynocytosed material. Thesituation at 23 °C was similar to that observed at 3 °C. This accounts for the non-linearity of the increase of the proportion of capped cells with temperature, but seemsrather surprising, since the transition temperature of the membrane lipids is below23 °C (Reinert & Steim, 1970). At the latter temperature the lipid bilayer should bein a fluid state and should allow lateral diffusion of membrane macromolecules. Ifthis does not happen, the most likely explanation is that the process of cap formationrequires energy and is therefore partially inhibited at 23 °C. The previous observationof Glenney et al. (1979) that at 22 °C ferritin-labelled Con A was uniformly distributedover the surface of Novikoff cells, is in agreement with the present finding.

In spite of these variations in Con A-receptor distribution, Con A-induced cellagglutination was remarkably stable over the temperature range from 3 to 37 °C.This observation suggests that Con A-induced agglutination of Novikoff cells doesnot depend upon diffusion and capping of the lectin receptors. It was previously

180 P. Mastromarino, G. Neri, A. Sena and E. F. Walborg, Jr

demonstrated that Novikoff cells become more agglutinable by Con A after papaintreatment (Neri et al. 1974). This finding was quantitatively confirmed in the presentreport, and additional evidence was provided, showing that the papain effect was ofthe same magnitude at each of the 3 temperatures tested. On the other hand,temperature-dependent Con A-receptor diffusion could still be observed in thepapain-treated cells, indicating once more that capping of receptors and cytoaggluti-nation varied independently. The only significant effect of papain treatment uponCon A-receptor dynamics, especially apparent at 37 °C, was an enhanced internal-ization of Fl-Con A, as indicated by the increased percentage of cells with patchlikefluorescence, that could not be removed by post-treatment with a-MM. A lessmarked effect was the appearance of caps in a small percentage of cells incubated withFl-Con A at 3 °C.

Additional evidence that cytoagglutination is not dependent upon receptorclustering was provided by a study of normal rat hepatocytes. These cells are alsoagglutinable by low concentrations of Con A (Starling et al. 1977). Yet, when assayedfor receptor mobility using Fl-Con A, all of the viable cells scored invariably displayeda ring of surface fluorescence with no evidence of receptor diffusion and redistributioninto caps or patches, even at 37 °C. The molecular basis for the observed differencein Con A-receptor mobility between normal hepatocytes and hepatoma cells ispresently unclear. At any rate, it may be concluded that Con A-induced cytoaggluti-nation and long-range Con A-receptor mobility vary independently in normalhepatocytes and Novikoff hepatoma cells. Furthermore Con A-receptor mobilitymay represent another marker of neoplastic transformation during hepatocarcino-genesis, adaptation to growth in the ascitic form, or progression of a tumour to a moremalignant state.

This work was supported in part by grant NCT 75 00699.04 from the Consiglio Nazionaledelle Ricerche of Italy.

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(Received 17 July 1979)