STUDIES ON THE SURFACE PROPERTIES OF HYBRID CELLS · 2005. 8. 21. · (a) Sialic aci -d the sample was mad toe 1 N HC1 and then heate idn a sealed tube to 100 °C for 90 s. (b) Hexosamine

J. Cell Set. 48, 147-170 (198O 147Printed in Great Britain © Company of Biologists Limited 1981

STUDIES ON THE SURFACE PROPERTIES OF

HYBRID CELLS

III. A MEMBRANE GLYCOPROTEIN FOUND ON THESURFACE OF A WIDE RANGE OF MALIGNANT CELLS

M. A. L. ATKINSON* AND M. E. BRAMWELLSir William Dunn School of Pathology, University of Oxford,Oxford OXi 3RE, England

SUMMARY

We report here the presence of a glycoprotein of apparent molecular mass 90000 daltons onthe surface of membranes of malignant cells, which is absent or very much reduced on thesurface of non-malignant cells. This glycoprotein is rich in sialic acid and appears to be sensitiveto the concentration of cAMP under certain conditions.

Analysis of the labelled sugars present in the glycoproteins of cells metabolically labelledwith [14C]gluco8amine suggests that all the enzymes necessary for the conversion of the tracerprecursor into the sugars normally found to be labelled are present in both the malignant andthe non-malignant cells.

INTRODUCTION

It is a widely held belief that malignancy, here defined as the ability of a cell togrow progressively and kill its host, results from a loss of responsiveness to thenormal cell growth control mechanisms. If these mechanisms were well understoodit would be a formidable task to define the lesion or lesions which occur in malignantcells and result in the malignant state. As it is, the mechanics of normal growthcontrol are only just beginning to be elucidated (Holley, 1975; Skehan, 1976; Holley,1976).

There has been no lack of reported differences associated with malignancy sinceWarburg's observation that cancer cells show a decreased rate of respiration andhigh levels of glycolysis (Warburg, 1926). Chromosomal and mitotic abnormalities,the pathological symptoms of anaplastic growth, changes in the nucleus/cytoplasmratio, pleiomorphism, invasiveness, the ability to metastasize and alterations in thetumour cell glycocalyx (Robbins, 1974) are all well documented phenomena. Evidencefor tumour-specific antigens both on the cell surface and in the plasma is plentiful(Old & Boyse, 1964; Klein, 1966).

The plasma membrane is a selective barrier between the cell interior and theexternal environment and so any growth control signal, whatever its form may be,must either interact with a receptor on the cell surface or pass through it to some

• Present address: Department of Pathology, Yale University School of Medicine,310 Cedar Street, New Haven, CT 06510, U.S.A.

148 M. A. L. Atkinson and M. E. Bramwell

internal receptor. It seems logical therefore, that the changes in growth controlassociated with malignancy may well be reflected in changes in the structure andcomposition of the surface membrane, especially as the interaction of certain sub-stances with the cell surface, for example, trypsin (Burger, 1970), phytohaemag-glutinin (Hadden, Hadden, Haddox & Goldberg, 1972), Concanavalin A (Naspity &Richter, 1968), lipopolysaccharide endotoxins (Janossey & Greaves, 1971), sodiumperiodate oxidation (Novogrodsky & Katchalski, 1971) and neuraminidase followed bygalactose oxidase treatment (Novogrodsky & Katchalski, 1973) all appear to stimulatecell division. The common denominator in these various interactions seems to becell-surface receptors which seem likely to be glycoproteins.

The power of the cell fusion technique as a test of linkage between any phenotypicmarker and malignancy, especially when the linkage is also tested by the differentialcytotoxic selection procedure in wheat germ agglutinin (Bramwell & Harris, 1978a),has already been discussed (Atkinson & Bramwell, 1980a). It is perhaps significantthat the only markers which have so far been reported to survive these tests of linkageare the abnormal membrane glycoprotein of molecular mass 100000 daltonsdescribed by Bramwell & Harris (19780,6) and the apparent increase in activityof the enzyme sialyl-transferase (Atkinson & Bramwell, 1980a, b). The 100 Kglycoprotein has been reported to have an isoelectric point of 4-0, to bind Con Astrongly and wheat germ agglutinin (WGA) weakly, and to exist in a dimer form inthe membrane. On the basis of these properties and the observation that glucoseenhances the binding of fluoro-dinitro [14C]benzene to the dimer form the authorshave suggested that the 100 K/200 K glycoprotein may be involved in glucosetransport. The conclusion is drawn that since no other abnormal glycoproteins areseen in this system it is likely that this is the only glycoprotein alteration associatedwith malignancy and therefore may represent an altered protein which as a con-sequence of its altered structure is abnormally glycosylated.

Con A is known to bind specifically to glucose and mannose residues (Lis & Sharon,1973) and it would seem likely on this basis that the abnormal 100 K glycoproteinprobably contains large amounts of exposed mannose residues. It is believed thatthe biosynthesis of asparagine-linked oligosaccharides proceeds via a mannose-richcore oligosaccharide which is transferred en bloc from a lipid-bound oligosaccharidederivative to the nascent polypeptide during synthesis (Robbins, Hubbard, Turco &Wirth, 1977; Li & Kornfeld, 1979). This core is subsequently matured by the removalof some of the mannose residues and the addition of other sugars from nucleotidesugar donors, amongst these are iV-acetyl-glucosamine and sialic acid, one of thesugars found in the terminal position oligosaccharides. Thus it is conceivable thatthe 100 K glycoprotein represents an incomplete glycoprotein.

If the primary lesion of malignancy is unique and the same lesion is present in allmalignant cells (Wiener, Klein & Harris, 1974) then one would not expect to findany other surface marker which survives the cell fusion test if the defect is involvedin the synthesis of the protein portion of the 100 K glycoprotein, but if the defectlies in the glycosylation of the 100 K or in some unidentified control factor then onewould expect that a number of other altered glycoproteins should be evident in

Surface properties of hybrid cells, III 149

malignant cells. Bramwell & Harris (1978a) have already reported that they foundno altered Coomassie blue-staining protein, in either their SDS-polyacrylamide gelsor in their 2-dimensional gel system, which consistently segregated with malignancyand that the 100 K abnormal glycoprotein was the only Con A staining band whichsegregated with malignancy. It seemed worth while to look for changes in glycosylationof membrane glycoproteins in systems in which glycoproteinswere labelled by differentmethods. The metabolic patterns of surface glycoproteins in cells fed a variety ofdifferent radioactive sugars were investigated and labelling with glycosamine seemedthe most promising approach.

MATERIALS AND METHODS

Cells and cell culture

The cells and the method of cell culture have been described in our two previous papers(Atkinson & Bramwell, 1980a, b).

Metabolic labelling with radioactive sugars

Cells were allowed to attach to large (75-mm1) Falcon tissue culture flasks and to grow toconfljency in the presence of 10/fCi of D-[U-I4C]glucosamine hydrochloride (250 mCi/mmolupprox.) from the Radiochemical Centre, Amersham, England, for at least 24 h.

Extraction and solubilization of glycoproteins

Glycoproteins were extracted from suspensions of packed cells with an equal volume of°'S % Triton X-100 in 10 mM Tris-HCl, pH 80, for 20 min at room temperature (Butters &Hughes, 1974). The suspension was then centrifuged at 2000 rev/min for 15 min and theextract carefully aspirated. The detergent extract was then dispersed in an equal volume of50 mM Tris-HCl, pH 84 containing 10 % glycerol, 2 % SDS, o-i M dithiothreitol and 001 %bromophenol blue. The solution was heated in a 100 CC waterbath for 2 min.

Electrophoresis

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in slab gels (Studier,1970). JV.iV'-diallyltartardiamide (DATD) was used as a cross-linker and 75-20% gradientgels were cast from a 50:2 acrylamide:DATD mixture. In some cases a 4% stacker gel wasused to enhance resolution. The running buffer system employed was that of Laemmli (1970)and consisted of 12 g of Tris base, 58 g of glycine in 2 1. of distilled water and a final con-centration of o-i % SDS.

Samples were run for at least 800 h together with protein molecular weight markers,collagenase (120K, 110K), rabbit muscle glycogen phosphorylase (97K), [14C]phosphorylase A(94K), [uC]bovine serum albumin (68K), katalase subunits (60K) and [14C]ovalbumin (45K).

Gels were stained for protein by constant rocking in a o-i % solution of Coomassie brilliantblue in a 5:5:1 mixture of methanol: distilled water: acetic acid and destained in the samesolution without Coomassie blue.

Fluorography

The stained gels were impregnated with 2,5-diphenyloxazole (PPO, Fisons Ltd) in dimethylsulphoxide (DMSO) (Bonner & Laskey, 1974), washed in distilled water, dried under vacuumon to Whatman No. 1 filter paper and exposed to Kodak X-OMat XHi film at — 70 °C for5-10 days. Labelling patterns revealed in the fluorographs were analysed with a Joyce-Loeblmicrodensitometer.


Affinity labelling of separated glycoproteins with [12&F\lectins

The method of Tanner & Anstee (1976) and Burridge (1976) as adapted by Bramwell &Harris (1978a) was used. Two lectins were used in these studies (both from Pharmacia,Uppsala) wheatgerm agglutinin (WGA) which binds principally to iV-acetyl-glucosamine(Lis & Sharon, 1973) and sialic acid (Bhavanandan, Umemoto, Banks & Davidson, 1977) andCon A which binds principally to mannose and glucose residues (Lis & Sharon, 1973).

The lectins were labelled with Nal u l and the gels affinity labelled as described by Bramwell& Harris (1978 a). The gels were dried and exposed to Kodirex X-ray film at least 24 h andthe binding patterns revealed in the autoradiographs analysed with the Joyce-Loeblmicrodensitometer.

Thin-layer chromatography

Glycoproteins extracted from io7 cells were dialysed against distilled water overnight at4°C and were then divided into 3 aliquots which were hydrolysed as follows to maximizethe liberation of various sugars:

(a) Sialic acid - the sample was made to 1 N HC1 and then heated in a sealed tube to 100 °Cfor 90 s.

(b) Hexosamines - the sample was made to 4 N HC1 and heated in a sealed tube at 100 °Cfor 4 h.

(c) Neutral sugars - the sample was made to 2 N H,SO4 and heated in a sealed tube at 100 °Cfor 4 h.

The samples were cooled, centrifuged at 2000 rev/min and the supernatant carefullyaspirated. The hydrolysate was diluted to 1 ml with distilled water and vacuum-desiccatedto dryness over CaCl, and NaOH. 150 fi\ of distilled water were then added to each sampleand they were vacuum desiccated to dryness again. Finally 25 fi\ of distilled water were addedto each sample and they were stored at — 20 °C until required. 3 fi\ aliquots were spotted on too-25-mm-thick cellulose MN300 thin layer chromatography plates (CamLab Ltd). Twobuffer-systems were used: (A) the solvent was a 6:4:3 mixture by volume of n-butanol:pyridine:distilled water; (B) the atmosphere was saturated with a 11:40:6 mixture by volumeof pyridine: ethyl acetate: distilled water and the solvent was a 5:5:1:3 mixture by volumeof ethyl acetate: pyridine: acetic acid: distilled water.

The plates were allowed to air dry in a fume cupboard after chromatography and were theneither stained by the silver nitrate method (Trevelyan, Proctor & Harrison, 1950) or sectionedand the radioactivity eluted from the plates and counted as outlined in the text.

RESULTS

Standardization of the labelling conditions

Before an interpretation can be placed on data obtained from the SDS-PAGEseparation of metabolically labelled glycoproteins it is essential to standardize theconditions under which the labelling is carried out and to ascertain: (1) the formsin which the labelled precursor is found in the final product; (2) the period of growthin the presence of the radioactive precursor which results in a maximal incorporationof label; (3) how the pattern of incorporation of label into the various glycoproteinsvaries with the period of growth in the presence of the precursor, and with the stateof growth of the cells; and (4) how the overall rates of incorporation and loss of labelcompare with that in individual glycoproteins.

Fig. 1A shows that the incorporation of D-[U-14C]glucosamine into Triton-extractable and non-extractable material, and the sum of these 2 forms, in a cultureof HeLa spinner cells on a per 5 ml of culture basis, while Fig. 1B shows the same

Surface properties of hybrid cells, HI

1

8•- v~>o•I *

II 150

Time of growth, h

Fig. 1. Incorporation of [14C]glucosamine into HeLa cells. 30 /*Ci of D-[U-"C]glucos-amine hydrochloride (254 mCi/mmol) were added to 200 ml of growing HeLa spinnercells at a cell density of 8 x io6 cells/ml. At intervals a 5-ml sample was removed, thecells counted (see inset) and the viability estimated. The cells were then pelletedgently, washed with PBS and the glycoproteins extracted with 50 fi\ of 05 % TritonX-100 in 10 min Tris-HCl pH 8-o for 20 min at room temperature. The cellulardebris was pelleted by centrifugation at 3000 rev/min for 10 min and the supernatantaspirated. The pellet was dissolved in 05 ml of Protosol and the radioactivity countedin 3 ml of Unisolve I. An aliquot of the extracted material was also counted in 3 ml ofUnisolve I and the remainder used for polyacrylamide gel electrophoresis (PAGE,Fig. 2). Twenty-six hours after the addition of label the culture was diluted (arrow A)with an equal volume of fresh growth medium and further samples taken as indicated.96 h after the initial addition of radioactive sugar the cells were spun down andresuspended (arrow B) in the same volume of fresh medium and allowed to continuegrowth with occasional samples being removed, until 121 h after the start of theexperiment when a further 15 (iCi of D-[U-14C]glucosamine hydrochloride wereadded (arrow C) to the culture and further samples taken.

A. The amounts of radioactivity in the various fractions and whole cells expressed onthe basis of cpm per 10' cells.

B. The cpm associated with the Triton-extracted material, the cellular debris andwhole cells (debris and extract) derived from the cells spun down from 5 ml of culture.

O, Triton-extract; • , total; • , remainder.

152 M. A. L. Atkimon and M. E. Bramwell

data on a per io6 cells basis. It is immediately clear that the pattern of incorporationis the same in both cases. This indicates that all the available precursor is takeninto the cells very rapidly. The results suggest that the peak of Triton non-extractablematerial may precede the peak of extractable material and this may indicate a flowof labelled material from the non-extractable to the extractable form. The

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Fig. 2. Densitometer tracings of fluorographs obtained from SDS-PAGE of samplesof Triton-extracted glycoproteins from the experiment described in the legend toFig. i prepared as described in Materials and methods. Peak values in this and allsubsequent figures are in K daltons.

incorporation curve indicates that the amount of label increases in an almost linearway for 5-6 h before beginning to level off. The amount of bound label does notbegin to decline until after 15-20 h.

If samples of the Triton-extracted material are analysed by SDS-PAGE followedby fluorography (Fig. 2) it appears that the relatively small increase in incorporatedlabel between 8 and 24 h after the addition of the labelled precursor is paralleledby a much greater relative increase in the amount of label associated with glyco-proteins. This result is indicative of a maturation process too, with the label beingpresent first in a low-molecular-weight form which would not be retained on thegels and which is eventually converted to the mature surface glycoproteins.

Surface properties of hybrid cells, 111

6 c; E m m ? m a UAC;U-.A L U U L U U P P P u 0 0

154 M. A. L. Atkinson and M. E. Bramtvell

When the rate of cell growth begins to slow 26 h after the addition of the precursora very rapid loss of label occurs. However, the addition of fresh medium stimulatesthe cells to continue growth. When cell growth begins to slow again the rate of lossof label is at a lower level and comparison of the labelling patterns of the majorglycoproteins suggests that these tend to turnover at a similar rate. The addition offresh precursor causes a further peak of label in both the extractable and non-extractable forms and in the specific activity of the major glycoproteins.

It is obvious that the metabolic labelling of surface glycoproteins with radioactivesugars may be affected by a number of factors beyond the rate of synthesis of theindividual glycoproteins: (1) the pool size of the sugar within the cell; (2) the rateof degradation of the added sugar to non-utilizable products; (3) the rate of conversionof added sugar to another utilizable sugar; and (4) the turnover rate of the finalglycoprotein in relation to the length of the labelling period.

If comparisons are to be made between different cell types in the labelling patternsof their glycoproteins it is essential to ensure that any differences which are observedare not merely reflections of differences in intermediary metabolism. To this end,the glycoproteins of PG19, a spontaneous melanoma of the C57 Black mouse,PG19 x T13H hybrid Clone 8, a hybrid clone formed between the PG19 cell line anda secondary culture of mouse embryonic fibroblasts of the T13HT13H mouse, whichshowed a suppression of malignancy, and the segregant rumour derived from thishybrid, were labelled with D-[U-14C]glucosamine and extracted with Triton X-100as described in Materials and methods. The extract was hydrolysed and analysedby thin layer chromatography (TLC).

The amount of radioactivity associated with various sections of the plate wasascertained as described in the legend to Table 1. The peaks of radioactivity areassigned sugar identities on the basis of their RF values compared with those ofknown sugar standards chromatographed under identical conditions and stained bythe silver nitrate method (Trevelyan et al. 1950). The amount of each sugar expressedas a percentage of the total label is given in Table 1.

Hydrolysate a shows that the malignant cells (PG19 and Clone 8 Turn 1) containless radioactive sialic acid in their glycoproteins than the non-malignant one. Thismay result from a decreased incorporation of sialic acid into glycoproteins, a decreasedconversion of the precursor to CMP-sialic acid, or an increased pool of unlabelledsialic acid in the malignant cells. It is not possible to draw any conclusions about therelative proportions of the other sugars since this hydrolysate is only a partialhydrolysate to maximize the release of sialic acid.

Hydrolysate b is designed to maximize the release of hexosamines. Unfortunately theconditions of hydrolysis also serve to deacetylate the iV-acetyl-glucosamine and theiV-acetyl-galactosamine residues; also glucosamine and galaciosamine migrate underthese conditions with a similar RF value to that of sialic acid. The solvent system B gavemaximum resolution of these sugars and the results suggest that there is more glucosa-mine associated with the malignant cells than with the suppressed hybrid while the levelsof galactosamine are similar in all 3 cell lines. Since very little radioactivity remainsassociated with the origin in this hydrolysate it-probably reflects a complete hydrolysis.

Surface properties of hybrid cells, HI 155

Hydrolysate c is designed to maximize the release of neutral sugars. Analysis of theprofile of this hydrolysate suggests that there are no detectable labelled neutral sugars.

Thus glucosamine is an ideal precursor for the metabolic labelling of glycoproteinssince it is converted into sugars found in glycoproteins (galactosamine, glucosamineand sialic acid). Moreover, the enzymes necessary for the interconversion of thevarious sugars appear to be present in both the malignant and the non-malignantcells. Notwithstanding, there does seem to be a greater amount of sialic acid anda commensurately lower amount of glucosamine in the non-malignant hybrid, inagreement with the higher levels of sialic acid seen in the glycoproteins of non-malignant cells (Atkinson & Bramwell, 1980a) and may indicate some abnormalityin the synthesis of CMP-sialic acid in malignant cells.

Comparison of the D-[U-uC]glucosamine labelling patterns of the surface glycoproteinsof malignant and non-malignant cells

It proved difficult to prepare membranes from labelled cells with sufficientradioactivity to be detectable by the technique of fluorography. However, densi-tometer scans of fluorographs prepared from the SDS-electrophoretograms ofsolubilized whole cells and Triton extracts appeared to be essentially the same. Inaddition the autoradiographs prepared from [126I]WGA affinity labelled SDS-PAGEgels of plasma membrane and Triton extracts were similar suggesting that the Tritonextracts are representative samples of the plasma membrane glycoproteins.

The fluorographs of labelled Triton extracts of the malignant PG19 (Fig. 3) werecompared with those of 2 non-malignant fibroblast cell lines and those of thePG19XT13H Clone 8 hybrid which showed a suppression of malignancy and thesegregant tumour derived from it (Fig. 4). It is immediately clear that a stronglylabelled glycoprotein with an apparent molecular mass of 90 K daltons is found only inthe malignant PG19 and PG19 x T13H Clone 8 tumour 1 cell lines. This is the onlymajor variation which is consistently linked to malignancy in these 5 profiles.Comparison of the Coomassie blue-staining bands in this region gave little informationas a large amount of Coomassie blue-staining material migrated just behind this band.

Extracts from a wide range of malignant and non-malignant cell types were analysedfor the presence of this particular glycoprotein band in fluorographs (Figs. 6, 7) pre-pared from SDS-electrophoretograms (Fig. 5). An analysis of the amount of the 90 Kglycoprotein in these various cells is given in Table 2. It is clear that there is a strongcorrelation between the presence of the glycoprotein and the ability of the cell to growprogressively in vivo and kill the host. All the cell? derived from tumours show a strongpositive band, while those derived from normal non-malignant tissues do not showit at all. The wheat germ agglutinin-resistant cells show a reduction in the level ofthe material compared with the amount present on the surface of the malignantparental PG19 cells while the tumours derived from the B3 resistant line showelevated amounts. It is interesting that the band appears to increase in amount withthe length of time that the B3 cell line is grown in culture. This supports thecontention that this line may represent a mixed population of malignant and non-malignant cells in which the malignant ones selectively overgrow the non-malignant

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M. A. L. Atkinson and M. E. Bramwell

9790 68

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Fig- 3 Fig. 4Fig. 3. Densitometer tracings of fluorographs obtained from SDS-PAGE of theTriton-extracts of: A, MRC 5 (human fibroblast); B, C57 (mouse fibroblast); andc, PG19 (mouse melanoma). Each cell line was metabolically labelled with ["C]-glucosamine as described in Materials and methods.Fig. 4. Densitometer tracings of fluorographs obtained from SDS-PAGE of theTriton-extracts of PG19 x mouse fibroblast hybrid cells, Cl. 8 (lower) (suppressed)and Cl. 8 Tx (upper) (malignant revertant), metabolically labelled with [

14C]-glucosamine.


ones. The results with other hybrid-cell pairs of non-malignant hybrid and segreganttumour confirm this pattern, although in most of the suppressed hybrids the band isonly reduced in amount and not totally lost. This is indicative, in genetic terms, ofa complementation of the malignant parental genome by the diploid parental one.

m

90 K

Fig. 5. Autoradiograph of gel from which microdensitometer tracings shown in Figs. 3and 4 were taken. 20-/*l and 10-fil samples were loaded on the gel. 1, C57 (mousefibroblast; 2, PG19 (mouse melanoma); 3, Cl. 8 (suppressed hybrid); 4, Cl. 8 Tj(malignant revertant).

In all cases the absence of dithiothreitol from the solubilizing buffer did not affectthe apparent mobility of the 90 K glycoprotein indicating that it is not present inthe membrane as a disulphide-linked oligomer and serving to distinguish it from the100 K glycoprotein of Bramwell and Harris. Moreover it was found in malignantcells harvested at different cell densities although it did appear to be present inmaximum amounts in cells harvested at confluency (Fig. 8).


Affinity labelling with radioactive lectins

Affinity labelling of separated glycoproteins on polyacrylamide gels with[mI]lectins is a powerful tool since it enables the researcher to probe the composition

Table 2. Analysis of PAGE fluorograph profiles in various malignant and non-malignantcell lines

Cell lineMalignant/

non-malignant 90 K Cell lineMalignant/

non-malignant 90 K

PG19CBAT.T.FibsMRC5 fibsC6,B1. Mel.PGA9\VGAB4PGI9WGAB/T!P G I 9 W G A B 4 T JPGi9\VGAC,PGi9\VGAD4NRK3B77SC4 W/TTA3

MNNMM/NMMNNNMM

HeLaDAUDINS/MSWBSHTCSEWAPG19XT13HCI. 8PG19XT13HCI. 8Tumi

PG19 x HumanlymphocyteCl. i9Si6TGCl. i9Sx6TGTum 1

MMMMMMNM

NM

NRK (normal rat kidney), and 3B77SC4W/T ,(Rous sarcoma transformed rat kidney) celllines were kindly supplied by Dr C. J. Marshall.

of the surface glycoproteins as they actually appear in the membrane without anyinterfering effects resulting from dilution of the label in the intracellular pool, fromdifferential rates of synthesis between various glycoproteins, or from a differentialrate of degradation between the various labelled molecular species. The responseof the film to varying amounts of labelled material is linear (Bramwell & Harris,1978 a) and so a microdensitometer scan of the autoradiograph represents the varyingamounts of [126I]lectin bound. Thus the lectin-binding pattern gives an instantaneouspicture of the amount and type of sugar residues bound to individual glycoproteins.

Fig. 9 shows the scans of autoradiographs of Triton-extracted glycoproteinsaffinity labelled with [126I]WGA compared with ["CJglucosamine metabolic labelling.It is apparent that the [mI]WGA-binding pattern is very similar to theD-[U-14C]glucosamine-labelling pattern of metabolically labelled cells adding furtherproof that the metabolic labelling pattern is not artifactual.

The Con A-labelling patterns shown in Fig. 10 are very different from those seenwith [12SI]WGA and D- [U-14C]glucosamine. The increased [mI]Con A binding tothe extracted malignant cell glycoproteins in the 100 K region, as reported byBramwell & Harris (1978 a, b), is immediately apparent. There does not seem to besignificant binding of Con A to the 90 K region, thus distinguishing the2 glycoproteins.


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Fig. 6. Densitometer tracings of fluorographs obtained from SDS-PAGE of Tritonextracts of [14C]glucosamine labelled cells: A, PG19 WGA-resistant clones, Ct (lower)and D4 (upper); B, PG19 WGA-resistant Cl. B4 (top) after growth in vitro (middleand lower); c, HeLa (human uterine carcinoma); D, P3NSi-Ag4-i (mouse myeloma).

i6o M. A. L. Atkinson and M. E. Bramwell

The form in which the label is present in the 90 K material

The analysis of the labelled sugars found in Triton-extracted glycoproteinsfollowing growth in the presence of D-[U-MC]glucosamine by TLC has already beendescribed. The results indicate that the principal labelled sugars were sialic acid,galactosamine and glucosamine. Our earlier results (Atkinson & Bramwell, 1980a, b)

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Fig. 7. Densitometer tracings of fluorographs obtained from SDS-PAGE of Tritonextracts of [14C]glucosamine labelled cells: A, Daudi (human lymphoma); B, NRK(rat kidney cell line) - lower; RSV-transformed NRK - upper; C, MSWBS (methylcholanthrene-induced sarcoma); D, HTC (rat hepatoma).

suggested that while malignant cells show elevated levels of sialyl transferase activity,the amount of bound sialic acid found in malignant cells is generally less than thatfound in non-malignant ones. The appearance of a Con A-binding abnormal glyco-


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Fig. 8. Densitometer tracings of fluorographs obtained from SDS-PAGE of Tritonextracts of PGio cells labelled for 24 h with ["CJglucosamine at different celldensities: A, confluent; B, near confluent; c, semiconfluent; D, sparse.

protein of 100 K molecular mass indicates a high mannose content glycoproteinwhich may be indicative of incomplete glycosylation. This could result from eitheran abnormality in glycosylation per se or could be the result of the synthesis of anabnormal protein which prevents normal glycosylation. The 90 K band does notbind high amounts of Con A and so is unlikely to result from a similar defect. Theheavy labelling in this region with D- [U-14C]glucosamine and [^IJWGA could,however, indicate the presence of elevated amounts of either glucosamine or sialic


acid. Since the TLC results which have already been discussed suggest that theremay be more glucosamine and less sialic acid in the labelled material from malignantcells as compared to that from non-malignant ones grown in the presence ofD- [U-14C]glucosamine it seemed worthwhile to investigate which of these 2 sugarswas responsible for labelling in the 90 K glycoprotein.

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Fig. 9. Densitometer tracings of fluorographs obtained from SDS-PAGE, of Tritonextract of confluent PG19 cells, affinity labelled with [mI]WGA (lower) comparedwith metabolic labelling using [MC]glucosamine.

To do this the labelling pattern of Triton-extracted glycoproteins was comparedbefore and after treatment with neuraminidase. Fig. 11 shows the densitometer scansof the fluorographs of PG19 glycoproteins before and after treatment with neura-minidase. It is clear that there is a marked reduction in the labelling of the 90 K bandand of several other bands following the neuraminidase treatment. This suggeststhat there is a substantial incorporation of the labelled precursor into sialic acid ofthese bands, in the face of an overall decrease in the amount of bound sialic acid.


The role of cAMP in the regulation of the level of the 90 K glycoprotein

The role of cAMP in the regulation of cell growth has been extensively reviewed(Pastan & Johnson, 1974; Pastan, Johnson & Anderson, 1975) and derived fromexperiments in which the intracellular level of cAMP was elevated by inhibitingthe cAMP phosphodiesterase with theophylline or caffeine, by activating the adenylatecyclase with prostaglandin E or by feeding cAMP analogues such as iVa-Oa-dibutyryladenosine 3',5'-monophosphate (Bt2cAMP).

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Fig. 10. Densitometer tracings of autoradiographs obtained from SDS-PAGE, ofTriton-extracted confluent PG19 x mouse fibroblast Cl. 8 (lower) and Cl. 8 Tx (upper),affinity labelled with ["MJCon A.

Elevated levels of cAMP have been associated in certain cells with the inhibitionof growth at confluency (Otten, Johnson & Pastan, 1971, 1972), and transformed cellswhich do not stop growing at confluency have been shown not to elevate theirintracellular cAMP levels. Pardee (1974) has suggested that the Bt2cAMP growth-

164 M. A. L. Atkinson and M. E. Bramtcell

restriction point is in the Gx phase of the cell cycle and that the inhibition of growthresulting from serum, isoleucine and glutamine starvation all act through this commonpoint. Serum starvation has been reported to elevate cAMP levels (Seifert & Paul,1972; Kiam, Mamont & Tomkins, 1973) and the addition of amino acids to glutamine-or histidine-starved cells results in a drop in cAMP levels (Seifert & Rudland, 1974).Cells showing normal growth control are often observed to cease growth in Gl (Gu)

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Distance moved, cm

Fig. 11. Densitometer of fluorographs obtained from SDS-PAGE of Triton-extractedconfluent PG19 cells after treatment with and without neuramidase. Two ioo-/ilaliquots of Triton extract were incubated with (lower) and without 10 units ofneuraminidase at 37 °C for 1 h. The reaction was stopped by the addition ofsolubilizing buffer prior to electrophoresis.

correlating with the observations that cAMP levels are elevated in G^-arrested cellsand that Bt2cAMP arrests cells in Gv It was therefore, of interest to observe theeffect of exogenously supplied Bt2cAMP on PG19 cells and the results are shown inFig. 12. A reduction in the 90 K band was seen as the concentration of Bt2cAMP wasincreased from o to 0-5 mM but above this concentration there was no furtherreduction in the amount of the material. Interestingly this reduction is not seen whenconfluent cells are treated with Bt2cAMP, suggesting that cell growth is required.


16

8 10

Distance moved, cm

Fig. 12. Densitometer tracings of fluorographs obtained from SDS-PAGE of Triton-extracted PG19 cells after treatment with dibutryl cAMP. Lower tracing, 0-5 ITIM;upper tracing, i-o mM. As compared with Fig. 11 (upper tracing) the 90 K band issubstantially reduced.

DISCUSSIONIn this investigation glycoproteins have been principally labelled through inter-

mediary metabolism by feeding D-[U-14C]glucosamine. We have shown this to beincorporated into glycoproteins as iV-acetyl-glucosamine, A^-acetyl-galactosamine andsialic acid (Table 1). The kinetics of labelling have been studied (Fig. 1) and therate of appearance of label in the various glycoproteins compared (Fig. 2). In additionthe glycoprotein sugar compositions have been investigated by affinity labelling thegels with [1MI]lectins (Figs. 9, 10) and by neuraminidase treatment of the Triton-extracted glycoprotein material (Fig. 11). These studies have suggested that thepattern of labelling seen in fluorographs and autoradiographs is not an artifact ofdifferent rates of synthesis or degradation of the various glycoproteins. The labelledcells were routinely harvested at confluency at which the amount of the 90 Kglycoprotein in malignant cells appeared to be maximal. Glycoproteins were routinelyextracted from whole cells with non-ionic detergents at low ionic strength as describedin Materials and methods, however, there was no significant difference between theprofiles of the extracts obtained in this manner from those of plasma membranesor of whole cells solubilized in ionic detergents.


Scrutiny of a wide range of cell types (Figs. 3-7, Table 2) demonstrated thata glycoprotein migrating with an apparent molecular mass of 90000 daltons ispresent in the membranes of the malignant cells examined and is absent or verymuch reduced in those of non-malignant ones. (The band is broad and may becomposed of several related glycoproteins perhaps differing in the degree ofsialylation.) Moreover, when these malignant cells were compared with normaltissues derived from the same species of syngeneic animal (PG19 and C57 Blackmelanoma with C57 Black mouse trypsinized embryo fibroblasts; HeLa and Daudihuman tumour lines with the normal MRC5 human fiibroblast line) the 90 Kglycoprotein was not seen in any of these non-malignant lines. The PG19X humanlymphocyte hybrid clone i9S16TG and its segregant tumour was the first hybridpair examined to test the linkage between the presence of the glycoprotein and theability of a cell to grow progressively in vivo and to kill its host. This hybrid was usedas it is a common phenomenon in mouse-human hybrids that there is a very rapidloss of human chromosomes. In this case the original hybrid contained 1 human Xchromosome as the only visible human contribution to the karyotype of the hybrid.This was subsequently segregated by back selection in 6-thioguanine producing a cellline which had no visible chromosomal contribution from the non-malignant parentand yet showed a marked suppression of malignancy when compared to the malignantparent. Thus it was hoped that this hybrid would be a particularly strong test of thelinkage. The 90 K band appears to be very much reduced in the suppressed hybridbut is present in the tumour derived from the hybrid in amounts comparable withthat seen in the malignant PG19 parental cell. This pattern is repeated in the intra-specific hybrid between the mouse PG19 and the normal T13H mouse embryofibroblast hybrid pair. Equally significantly the amount of the 90 K band in theWGA-selected cells correlates closely with the take-incidence of these cells in vivo.

The [14C]glucosamine 90 K glycoprotein seems to contain a large amount of sialicacid which can be removed by treating the extracted glycoproteins with neuraminidase.The band also appears to be sensitive to the concentration of cAMP; the administrationof O'5-i-omM BtjcAMP causes a marked reduction within 24 b. This effect is notsimple and the state of the cells is crucial in the response, thus in confluent cellsno change is seen.

If the genetic lesion which causes malignancy is a single mutational event as suggestedby Wiener et al. (1974) then we are faced with the problem of how to reconcile this withthe identification of at least three phenotypic markers, the 100 K glycoprotein, the90 K glycoprotein and the elevation of sialyl transferase (Atkinson & Bramwell,1980a, b) all of which seem to be strongly linked to this lesion. Models could be con-structed to account for these parameters on an individual basis but we feel that the answerto the problem of understanding malignancy probably lies in trying to understand thesevarious peripheral changes, which may well be segregable from the cells' ability to growprogressively in vivo when enough hybrids are examined, but which are linked closelyin either a functional or a genetic relationship with the primary lesion of malignancy.

We have previously reported (Atkinson & Bramwell, 1980a, b) that malignantcells have elevated levels of sialyl transferase and we have shown here that malignant


cells are able to convert exogenously supplied tracer glucosamine to sialic acid andyet the amount of sialic acid bound per mg protein is much less in malignant cellsthan it is in non-malignant ones (Atkinson & Bramwell, 1980a). The assay of surfacesialyl transferase in mixtures of malignant and non-malignant cells compared withthat in the separate suspensions (Atkinson & Bramwell, 1980 ft) indicates thatincomplete glycoproteins may be present on the surface of malignant cells.

Since the core sugars of asparagine-linked oligosaccharides are believed to betransferred en bloc from a lipid intermediate then one might expect to find someglycoprotein with large amounts of mannose and this may be what the 100 Kglycoprotein is. On the other hand, the presence of a strongly sialylated glycoproteinneed not be contradictory, if the affinity of the sialyl-transferase varies with respectto different proto-glycoproteins, if the levels of the enzyme producing a2~3 and thatproducing az—6 ketosidic linkages differ or if there is merely much more of the 90 Kpolypeptide chain available for glycosylation.

A survey of the literature does not suggest that the carbohydrate components ofglycoproteins are important in controlling the activity of the molecules. Changes inthe oligosaccharide components of RNase, lipase, glucoamylase and chloroperoxidasehave little effect on their activity and very few of the enzymes central to metabolismcarry functionally significant carbohydrate groups (Warren, Buck & Tuszynski, 1978).The purpose of bound carbohydrate seems to be in functions such as lubrication,protection of surfaces, anti-desiccation (Tuszynski et al. 1978), antifreeze in arcticfish (Komatsu, DeVries & Feeney, 1970), cell recognition and aggregation (Pouyssegur,Willingham & Pastan, 1977; Balsalmo & Lilien, 1975; Hausman & Moscona, 1975),intercellular adhesiveness (Roseman, 1971; Yamada, Yamada & Pastan, 1976),adhesion to substratum, blood clotting (Blomback, 1972) or as structural elements, infor example, collagen (Spiro, 1972). Even while the antigenicity of some surfaceglycoproteins such as those responsible for the blood groupings of red blood cellsare clearly dependent on the carbohydrate component, when antibodies are raised toglycoproteins they are frequently found to react with the polypeptide component(Gottschalk, Bhargava & Murty, 1972). While there is no apparent role for thecarbohydrate units of the histocompatibility antigens (Nathenson & Cullen, 1974)those of the immunoglobulin molecules seem to be important in complement-inducedcytotoxicity (Kolde, Nose & Muramatu, 1977). Bound sialic acid also has a rolein the uptake of circulating glycoproteins in the liver (Pricer & Ashwell, 1971).

While precedent does not preclude the correlation of a functional alteration withthe appearance of an alteration in glycosylation it would seem more likely that theeffect of an alteration on glycosylation is on the insertion, configuration or amountof a glycoprotein on the cell surface and that it is this which is functionally significant.

At the present time the assignment of any function to the 90 K glycoprotein mustbe largely speculative. There is a good deal of evidence in the literature to supportthe hypothesis that the 100 K glycoprotein is involved in glucose transport (Bramwell& Harris, 1978a, b). Bramwell & Harris (19786) also note that glucose enhances thebinding of the erythrocyte glucose-transport inhibitor, i-fluoro-2,4-dinitrobenzene,to 3 bands in addition to the 100 K dimer, in the 50-55, 75-80 and the 90-95 K


regions, although to a lesser extent. The 90-95 K band may correspond to the glyco-protein described here. If the 100 and the 90 K proteins are associated in themembrane one envisages a multimeric protein complex in the cell membrane forthe binding and transport of glucose possibly with sites for the binding of hormonesand other growth control factors.

It is impossible to say whether glycoprotein changes are the cause or merely oneof the effects of malignancy. It is likely that the primary lesion of malignancy causesa variety of alterations, peripheral to the central biochemical pathways such that thecell's ability to function is not impaired but that its responses to the normal growthcontrol factors are altered. Pursuit of studies designed to segregate phenotypicmarkers from malignancy may eventually lead to the product of the primary lesion.In the meantime the raising of a specific antibody to one of these 'peripheral'glycoproteins, while not necessarily solving the riddle of malignancy, could be ofconsiderable significance.

During the course of this work M.A.L.A. was the recipient of an MRC studentship fortraining in Research Methods and of the Kate Erin research scholarship. M. E.B. is the JamesHanson Research Fellow of the Cancer Research Campaign.

REFERENCES

ATKINSON, M. A. L. & BRAMWELL, M. E. (1980a). Studies on the surface properties of hybridcells. I. Sialyl-transferase activity in homogenates of malignant and non-malignant cells.jf. Cell. Set. 46, 187-201.

ATKINSON, M. A. L. & BRAMWELL, M. E. (19806). Studies on the surface properties of hybridcells. II. Sialyl-transferase activity on the surface of malignant and non-malign.vit cells.y. Cell Set. 46, 203-220.

BALSALMO, J. & LILIEN, J. (1975). The binding of tissue specific adhesive molecules 10 thecell surface. A molecular basis for specificity. Biochemistry, N.Y. 14, 167-171.

BHAVANANDAN, V. P., UMEMOTO, J., BANKS, J. R. & DAVIDSON, E. A. (1977). Isolation and

partial characterization of sialoglycopeptides produced by a murine melanoma. Biocliemistry,N. Y. 16, 4426-4437.

BLOMBACK, B. E. G. (1972). Carbohydrates in blood clotting proteins. In Glycoproteins,Part B (ed. A. Gottschalk), 2nd edn, pp. 1069-1081. Amsterdam, London and New York:Elsevier.

BONNER, W. M. & LASKEV, R. A. (1974). A film detection method for tritium labelled proteinsand nucleic acids in polyacrylamide gels. Eur. y. Biochem. 46, 83-88.

BRAMWELL, M. E. & HARRIS, H. (1978a). An abnormal membrane glycoprotein associatedwith malignancy in a wide range of different tumours. Proc. R. Soc. B 201, 87-106.

BRAMWELL, M. E. & HARRIS, H. (19786). Some further information about the abnormalmembrane glycoprotein associated with malignancy. Proc. R. Soc. B 203, 93-99.

BURGER, M. M. (1970). Proteolytic enzymes initiating cell division and escape from contactinhibition of growth. Nature, Lond. 227, 170-171.

BURRIDGE, K. (1976). Changes in cellular glycoproteins after transformation: identificationof specific glycoproteins and antigens in sodium dodecyl sulphate gels. Proc. natn. Acad.Set. U.S.A. 73, 4457-4461.

BUTTERS, T. D. & HUGHES, R. C. (1974). Solubilization and fractionation of glycoproteinsand glycolipids of KB cell membranes. Biochem. y. 140, 469-478.

GOTTSCHALK, A., BHARCAVA, A. S. & MURTY, V. L. N. (1972). Submaxillary gland glyco-proteins. In Glycoproteins, Part B (ed. A. Gottschalk), 2nd edn, pp. 810-829. Amsterdam,London and New York: Elsevier.


HADDEN, J. W., HADDEN, E. M., HADDOX, M. K. & GOLDBERG, N. D. (1972). Guanosine3',5'-cyclic monopho8phate. A possible intracellular mediator of mitogenic influences in lym-phocytes. Proc. vatn. Acad. Sci. U.S.A. 69, 3024-3027.

HAUSMAN, R. E. & MOSCONA, A. A. (1975). Purification and characterization of the retina-specific cell aggregating factor. Proc. natn. Acad. Sci. U.S.A. 72, 916—920.

HOLLEY, R. W. (1975). Control of growth of mammalian cells in cell culture. Nature, Lond.258, 487-490.

HOLLEY, R. W. (1976). Mammalian cell growth regulation - Matters arising, reply. Nature,Lond. 263, 532.

JANNOSSBY, G. & GREAVES, M. F. (1971). Lymphocyte activation. I. Response of T and Blymphocytes to phytomitogens. Clin. exp. Immun. 9, 483-498.

KIAM, R., MAMONT, P. & TOMKINS, G. M. (1973). Pleiotypic control by adenosine 3',s'-cyclicmonophosphatc. A model for growth control in animal cells. Proc. natn. Acad. Sci. U.S.A.70, 1432-1436.

KLEIN, G. (1966). Tumour antigens. A. Rev. Microbiol. 20, 223-252.KOLDE, N., NOSE, N. & MURAMATU, T. (1977). Recognition of IgG by Fc receptor and

complement. Effect of glycosidase digestion. Biochem. biophys. Res. Commttn. 75, 838-844.KOMATSU, S. K., DEVRIES, A. L. & FEENEY, R. E. (1970). Studies of the structure of freezing

point depressing glycoproteins from an Antarctic fish. J. biol. Chem. 245, 2909-2913.LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head

of bacteriophage T4. Nature, Lond. 227, 680-685.Li, E. & KORNFELD, S. (1979). Structural studies of the major high mannose oligosaccharide

units from Chinese hamster ovary cell glycoproteins. J. biol. Chem. 254, 1600-1605.Lis, H. & SHARON, N. (1973). The biochemistry of plant lectins. A. Rev. Biochem. 42, 541-574.NASPITY, C. K. & RICHTER, M. (1968). The action of phytohaemagglutinin in vivo and in vitro,

a review. Progr. Allergy 12, 1-85.NATHENSON, S. G. & CULLEN, S. E. (1974). Biochemical properties and immunochemical-

genetic relationships of mouse H,-alloantigens. Biochbn. biophys. Ada 344, 1-25.NOVOGRODSKY, A. & KATCHALSKI, E. (1971). Induction of lymphocyte transformation by

periodate. FEBS Letters, Amsterdam 12, 297-300.NOVOGRODSKY, A. & KATCHALSKI, E. (1973). Induction of lymphocyte transformation by

sequential treatment with neuraminidase and galactose oxidase. Proc. natn. Acad. Sci. U.S.A.70, 1824-1827.

OLD, L. J. & BOYSE, E. A. (1964). Immunology of experimental tumours. A. Rev. Med. 15,167-186.

OTTEN, J., JOHNSON, G. S. & PASTAN, I. (1971). Cyclic AMP levels in fibroblasts: relationshipto growth rate and contact inhibition of growth. Biochem. biophys. Res. Commitn. 44,1192-1198.

OTTEN, J., JOHNSON, G. S. & PASTAN, I. (1972). Regulation of cell growth by cyclic adenosine3'-5'-monophosphate. J. biol. Chem. 247, 7082-7087.

PARDEE, A. B. (1974). A restriction point for control of normal animal cell proliferation.Proc. natn. Acad. Sci. U.S.A. 71, 1286-1290.

PASTAN, I. & JOHNSON, G. S. (1974). In Advances in Cancer Res. (ed. G. Klein, G. Weinhouse& A. Haddow), p. 303. New York and London: Academic Press.

PASTAN, I., JOHNSON, G. S. & ANDERSON, W. B. (1975). Role of cyclic nucleotides in growthcontrol. A. Rev. Biochem. 44, 491-522.

POUYSSECUR, I., WILLINGHAM, M. & PASTAN, I. (1977). Role of cell surface carbohydrates andproteins in cell behaviour: Studies on the biochemical reversion of an N-acetyl glucosamine-deficient fibroblast mutant. Proc. natn. Acad. Sci. U.S.A. 74, 243-247.

PRICER, W. E. & ASHWELL, G. (1971). The binding of desialylated glycoprotein by plasmamembranes of rat liver. J. biol. Chem. 246, 4825-4833.

ROBBINS, P. W., HUBBARD, S. C, TURCO, S. J. & WIRTH, D. F. (1977). Proposal for a commonoligosaccharide intermediate in the synthesis of membrane glycoproteins. Cell 12, 893-900.

ROBBINS, S. L. (1974). Pathological Basis of Disease, chapter 4. Washington: Arlington Press.ROSEMAN, S. (1971). The synthesis of complex carbohydrates by multi-glycosyl-transferase

systems and their potential function in intercellular adhesion. Chem. Phys. Lipids 5, 270-297.


SEIFERT, W. & PAUL, D. (1972). Levels of cyclic AMP in sparse and dense cultures of growingand quiescent 3T3 cells. Nature, New Biol. 240, 281-283.

SEIFERT, W. & RUDLAND, P. S. (1974). Cyclic nucleotides and growth control in culturedmouse cells; correlation of changes in intracellular 3'-5' cyclic GMP concentration witha specific phase of the cell cycle. Proc. natn. Acad. Sci. U.S.A. 71, 4920-4924.

SKEHAN, P. (1976). Mammalian cell growth regulation - matters arising. Nature, Lond. 263,531-532.

SPIRO, R. G. (1972). In GlycopToteins (ed. A. Gottschalk), 2nd edn, p. 964. Amsterdam,London and New York: Elsevier.

STUDIER, F. W. (1970). Analysis of bacteriophage T7 early RNAs and proteins in slab gels.J. molec. Biol. 79, 237-248.

TANNER, R. M. J. & ANSTEE, D. J. (1976). A method for the direct demonstration of thelectin binding components of the human erythrocyte membrane. Biochem. J. 153, 265-270.

TREVELYAN, W. E., PROCTOR, D. P. & HARRISON, J. S. (1950). Detection of sugars on paperchromatograms. Nature, Lond. 166, 444-445.

TUSZYNSKI, G. P., BAKER, S. R., FUHRER, J. P., BUCK, C. A. & WARREN, L. (1978). Glyco-peptides derived from individual membrane glycoproteins from control and Rous sarcomavirus-transformed hamster fibroblasts. J. biol. Chem. 253, 6092-6099.

WARBURG, O. H. (1926). Stofftvechsel der Tumoren. Berlin: Springer.WARREN, L., BUCK, C. A. & TUSZYNSKI, G.. P. (1978). Glycopeptide changes and malignant

transformation. A possible role for carbohydrate in malignant behaviour. Biochxm. biophys.Acta 516, 97-127.

WIENER, F., KLEIN, G. & HARRIS, H. (1974). The analysis of malignancy by cell fusion.VI. Hybrids between different tumour cells. J. Cell Sci. 16, 189-198.

YAMADA, K., YAMADA, S. & PASTAN, I. (1976). Cell surface protein partially restoresmorphology, adhesiveness and contact inhibition of movement to transformed fibroblasts.Proc. natn. Acad. Sci. U.S.A. 73, 1217-1221.

(Received 19 September 1980)

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STUDIES ON THE SURFACE PROPERTIES OF HYBRID CELLS · 2005. 8. 21. · (a) Sialic aci -d the sample was mad toe 1 N HC1 and then heate idn a sealed tube to 100 °C for 90 s. (b) Hexosamine