Further characterization of the antigen defined by the monoclonal antibody

M27

MICHAEL E. BRAMWELL and SUSAN M. HUMM

Cancer Research Campaign, Cell Biology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford 0X1 3RE, England

Summary

Using immunoblotting techniques, the antigen thatbinds the monoclonal antibody M27 has beenclearly defined in terms of apparent molecularmass and distribution. In reducing conditions it hasan apparent mass of 178K (K= 103 Mr) and is presentin the cytoplasm and membranes of all mammaliantissue culture cells so far examined. It is absentfrom lines derived from avian, piscine and amphib-ian sources. It is also absent from foetal liver ofboth rat and mouse, but subsequently appears after

cultivation in vitro. Similarly, it can be detected onrat lymphocytes only after mitogenic stimulation.However, it is found on both hepatoma and lym-phoma cells in vitro, and on in vivo tumours frommurine sources. It thus appears to be associatedwith cell proliferation.

Key words: antibody, antigen, cell proliferation.

Introduction

The monoclonal antibody code-named M27 was isolatedsome years ago from a fusion using the spleen of miceinoculated with antigen derived from the human cervicalcarcinoma cell line HeLa (Gingrich et al. 1981a). Theantigen used in that study was prepared from SDS-PAGE gels and had an apparent unreduced molecularmass of 200K (K=103Mr). This molecule, on reduction,is a glycoprotein of apparent molecular mass 100K andwas shown to be present in increased amounts in themalignant segregants of suppressed hybrid cells (Bram-well and Harris, 1978). In the event, the antibody thatwas produced and code-named M27 reacted with anantigen that at that time could not be satisfactorilydefined, but was not the 100/200K glycoprotein. How-ever, the antibody did have interesting properties(Gingrich et al. 19816) and it was shown that theexpression of the antigen on the cell surface variedinversely with the level of glucose in the medium.Moreover, it was demonstrated that expression peakedearly in S phase.

However, the main interest in the properties of theantibody lay in the fact that, although the majority of cellsin tissue culture possessed the antigen on the cell surface,absorption studies on normal murine in vivo tissuerevealed little or no antigen present. Clearly, therefore,the antigen, although not tumour-specific, may play arole in cell proliferation both in vivo and in vitro.

Using immunoblotting techniques, the antigen hasnow been clearly defined in terms of its apparent molecu-lar mass and distribution.

Journal of Cell Science 94, 725-731 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

Materials and methods

Cell cultureCells were routinely cultured at 37 °C as monolayers in minimalessential medium (MEM) containing 10% foetal calf serum(FCS) except where indicated.

CellsH.Ep.2: a human cell line derived from a carcinoma of thelarynx (Moore et al. 1955). MRC5: human embryonic lungfibroblast cells (Jacobs et al. 1970). DAUDI: a human lym-phoma (Klein et al. 1967). RBC: human red blood cells werecollected by sedimentation in Dextraven 110 (Fisons Pic,Loughborough, UK) washed twice in PBS (phosphate-buffered saline, pH7.4) and recentrifuged at 100g. The pelletwas then extracted as for whole cells. PG19: a derivative of aspontaneous melanoma in a C57 black mouse (Jonasson et al.1977). 707: mouse erythroleukaemia (Friend et al. 1971).TA3H: mouse mammary carcinoma (Hauschka, 1953). HTC:rat hepatoma (Thompson et al. 1966). BRL-3A: a buffalo ratliver cell line grown in Dulbeccos modified Eagle's medium(DMEM) + 10% FCS (Dulak and Temin, 1973). FRL: foetalrat liver prepared from 17-day embryos and propagated inDMEM+10% FCS. The foetal liver was chopped finely andrinsed in digestion medium (DM) [L-15 without serum mixedwith an equal volume of Trypsin-EDTA (0.125%, w/v, and0.02%, w/v, respectively and 0.25 mgml"1 of collagenase(Worthington Biochemical Corp.)]. After washing the liver,,pieces were incubated in 10 ml DM at 37°C for lOmin. Thesingle cell suspension which resulted was removed and foetalcalf serum added to 10 % (v/v). The process was repeated twiceand the suspensions pooled and centrifuged at 500 £ for 30 s.The cloudy supernatants were discarded and the pellets washedtwice in DMEM+10% FCS before plating out densely in the

725

same medium. NRK-TG3: rat kidney cell line andNRK.TG3.B77.Sc4: rat kidney cell line transformed by Roussarcoma virus strain B77 (Marshall, 1980).

Rat lymphocytes: after killing, two cervical lymph nodeswere removed and teased apart with forceps in PBS containing5% FCS. The cells were spun down at 70 g, washed once inPBS+5% FCS and resuspended in RPMI + 10% FCS withConcanavalin A (ConA) at 5/ igmP1 and mercaptoethanol(2X 10~ M) for mitogenic stimulation. A portion of the cells wasalso stimulated in the absence of serum by ConA at 0.3 /fginl" .(The cell suspension was kindly provided by M. Puklavec of theMRC Cellular Immunology Research Unit in this department).CEF: chick embryo fibroblast cultured in DMEM and 10%FCS. 16Q: Japanese quail fibroblast cell line, Rous sarcomavirus-transformed, grown in DMEM+10% FCS (Murphy,1977). AS: Atlantic salmon; an epithelial-like cell obtained fromFlow Laboratories Ltd, Irvine, Scotland grown at 20°C. BFA:Bovine foetal aorta endothelium grown in Ham's F12 and 20%heat-inactivated FCS, obtained from Dr E. Levine, WistarInstitute, Philadelphia, USA. FEL: feline fibroblast-like linedeveloped by Flow Laboratories Ltd. COS 7: a monkey kidneyfibroblast cell line transformed with SV40 and grown inDMEM+10% FCS (Gluzman, 1981). MDCK: a canine kid-ney fibroblast cell line obtained from Gibco Ltd, Paisley,Scotland. A2H: a Chinese hamster fibroblast cell line (Ash-wood-Smith et al. 1967). PSY: a porcine cell line derived fromepithelia of the synovial membrane, obtained from the Instituteof Research into Animal Diseases, Compton, Newbury, Berks,UK. The cells were grown in L-1S (Leibowitz medium) + l %FCS+5% TPB (tryptone phosphate broth). EF2: vole fibro-blasts were prepared from embryos by Dr P. R. Cook. XTC-2:Xenopus laevis cell line grown at 27°C (Pudney et al. 1973).

Resident mouse macrophages were prepared from the perito-neal cavity and separated from other cell types by allowing themto adhere to a plastic surface.

Elicited macrophages were prepared similarly after injectioninto the peritoneum of lml of sodium thioglycolate (0.4%,w/v) for 4 days prior to removal.

HarvestingCells were released from monolayers using PBS containingEDTA (0.02%, w/v) at pH7.4.

Extraction of cellular componentsCells were washed in PBS and collected by centrifugation at2000 £ for 2min. An equal volume of a solution containing0.2% (w/v) sodium deoxycholate and lOmM-Tris-HCl,pH8.0, at 20°C was added to the pellet and the cells resus-pended. The cell membranes dissolved rapidly and nuclei werepelleted by centrifugation in an Eppendorf microcentrifuge at9000 £ for 2min. The cytoplasmic fraction containing thedissolved membranes was removed and stored at —20°C untiluse. Occasionally the protease inhibitors Aprotinin at 1 |Ugml~'and phenyl methyl sulphonyl fluoride, 2mM, were included inthe extraction procedure, but they made no difference to theresults obtained in short-term experiments. However, long-term storage at — 20 °C did result in some breakdown if noinhibitors were present.

Extraction of tissue samplesTissues were finely minced in distilled water and extracted with0.2% sodium deoxycholate in 10mM-Tris-HCl, pH8.0, forS min at 20°C. Insoluble material was spun off in a microcentri-fuge at 9000 £• for 2 min and 25 jil of the supernatant taken foranalysis.

Enzyme treatmentsNeuraminidase. 50 f.t\ of a cell extract, prepared as above,

was mixed with 50f.l\ of a buffer containing 0.05M-sodiumacetate and 2mM-calcium acetate at pH5.6. 0.02U of neuram-inidase {Vibrio cholerae, Boehringer Mannheim, FDR) wasadded and incubation performed at 37°C for 60min.

O-Glycanase. Cells were extracted as described above exceptthat a Tris-maleate buffer, pH.8.0, was used instead ofTris-HCL, since chloride inhibits O-glycanase (Genzyme,Koch-Light Ltd, Haverhill, Suffolk, UK). Prior to reactionwith O-glycanase the extract was treated with neuraminidase asdescribed, followed by the addition of 2mU of O-glycanase for3 ha t 37 °C.

N-Glyca»ase. 50,ul of a cell extract was added to 100 fd ofsolution containing 0.2M-sodium phosphate, pH8.6, 10mM-1,10-phenanthroline hydrate and 1 U of iV-glycanase (Gen-zyme, Koch-Light Ltd, Haverhill, Suffolk, UK). The mixturewas incubated for 24 h at 37°C.

Proteolytic enzymes. 50 fd of cell extract prepared as abovewas mixed with 10 fd of a solution containing either pro-nase (O.SmgmP1), thermolysin (0.5 mgml"1), trypsin(0.5mgml~ ) or subtilisin (0.5mgml~ ) and incubated for 1 hat 37°C.

InhibitorsTunicamycin. Cells grown as a monolayer were incubated

for 24 h at 37°C in normal growth medium containing tunica-mycin (Sigma Chemical Co. Ltd, Poole, Dorset, UK) at2 jig ml"1 (White etal. 1984).

Cycloheximide. Cells grown as a monolayer were incubatedwith cycloheximide (Sigma Chemical Co. Ltd, Poole, Dorset,UK) at a concentration of 1 mM in normal growth medium forperiods up to 6h.

ElectrophoresisSDS-PAGE was performed in 4-12% gradient slab gels usingthe LKB vertical gel apparatus (Laemmli, 1970; Studier, 1973),run at 20 mA and 4 V cm"1 for 16 h. Molecular weight standardsused were myosin (200K), /3-galactosidase (116K), phosphory-lase (94K), bovine albumin (67K) and ovalbumin (43K),obtained from Bio-Rad Laboratories, Richmond, California,USA.

ImmiinoblottingSDS-PAGE gels were subjected to electrophoretic transfer inTris-glycine buffer, 25 mM and 192 mM, respectively, pH8.3,containing 20% methanol, using the Transblot system (Bio-Rad Laboratories). Proteins and glycoproteins were transferredto nitrocellulose membrane filters (Schleicher and Schuell,GmbH, Dassel, FDR), washed in PBS containing in addition0.4M-NaCl and 0.2% Tween-20 (PNT) and blocked in 1%bovine serum albumin in PNT for 30 min. After rinsing indistilled water the membrane was incubated in the primaryantibody solution (monoclonal antibody M27 culture super-natant) for 30 min. After being washed three or four times inPNT buffer the membrane was incubated in radio-iodinatedsecond antibody (12SI-labelled rabbit anti-mouse IgG) for30 min. Excess radioactivity was washed off with 0 .1%, w/v,SDS in PBS. After staining with Amido Black (Sebia), themembrane was dried and exposed to X-ray film (Fuji PhotoFilm Co. Ltd, Japan) at -70°C for 24 h.

Measurement of surface M27 antigen on ratlymphocytesThis was carried out as described previously (Gingrich et al.1981a) for a saturation binding radio-immunoassay. The results

726 M. E. Bramwell and S. M. Humm

are expressed in the number of counts bound for a givennumber of cells.

M, X 13

DensitometryScanning of gels and autoradiographs was performed on a LKBUltrascan XL laser densitometer (LKB, Bromma, Sweden),coupled to an Olivetti M24 PC using the LKB 2400 Gelscan XLsoftware. 2C

Results

Identification of the M27 antigenFig. 1 shows the result of immunoblotting with the M27monoclonal antibody on a series of in vivo cell extractsand in vitro cell line extracts. The molecule to which it isbound has an apparent molecular mass of 178K and ispresent in varying amounts in the samples examined.Thus H.Ep.2, NRK and embryonic fibroblasts showsubstantial binding; total embryo, spleen and lung showmuch less, and kidney, heart and liver none at all. Thereis also evidence of a molecule of about half the size of themajor band, which may be a degradation product, sincelong term storage of samples increases the proportion ofthis component.

Presence of the M27 antigen in foetal rat liverThe absence of the M27 antigen in the liver of theembryonic mouse prompted the question as to whether itwould appear on cultivation of hepatocytes in vitro. Forthis experiment foetal rat liver proved more suitable sinceconditions for culture were available and a population ofhepatocytes was established as described in Materials

HXKT 3

20O

116 »

94 >'

6 7 *•

45 r

t)Fig. 1. Autoradiograph of an immunoblot of various cellextracts probed with monoclonal antibody M27.Approximately 50 fig of protein were loaded on each track.Lanes: a, H.Ep.2; b, mouse embryo - total extract; c, NRK;d, embryo fibroblasts (murine); e, kidney (niurine); f, spleen(murine); g, lung (murine); h, heart (murine), and i, liver(niurine).

1f

b c

Fig. 2. Autoradiograph of an immunoblot of foetal rathepatocytes probed with monoclonal antibody M27.Approximately 50 jug of protein were loaded on each track.Lanes: a, H.Ep.2; b, foetal rat hepatocytes, day 7; c, foetalrat hepatocytes, day 20; d, BRL-3A; e, foetal rat liverextract, and f, HTC.

and methods. Cultures were grown for several weeks andextracts of the cells revealed the presence of the M27antigen after 7 days, increasing in amount at 20 days.(Fig. 2). It is also clear that an established line of rat livercells (BRL-3A) and rat hepatoma line (HTC) bothexpressed the antigen.

Presence of the M27 antigen on stimulated ratlymphocytesAnother test for the presence of the M27 antigen on non-dividing as opposed to dividing cells was provided bystimulating rat lymphocytes with concanavalin A. Apopulation of largely quiescent lymphocytes was pre-pared as described in Materials and methods. These wereresuspended in 40 ml RPMI medium in two 75 cm2

flasks, one containing FCS and one without serum.Concanavalin A was added at S^ugmP1 and 0.3 ^g ml"1,respectively. Samples of cells (10 ml) were taken at dailyintervals to five days, counted, extracted and analysed onSDS-PAGE for the relative amount of M27 antigen.

Fig. 3 shows the increase in viable cell number aftertreatment with Concanavalin A and growth in 10 % FCScompared with growth in the absence of serum. Clearly,there is a rapid increase beginning at day 3 in the presenceof serum, whilst flasks without serum barely manage tomaintain a viable population.

Fig. 4 shows the increase in the total M27 antigen/unitof cellular protein over 5 days in both cell populations. Itis clear that there is a rapid rise in the amount of the M27antigen on stimulation with concanavalin A, both in thepresence or absence of serum, of 50-fold and 10-foldrespectively, of the initial value.

Characterization of the M27 antigen 727

Fig. 3. Increase in viable cells after stimulation of ratlymphocytes with Con A.with 10% FCS; (•FCS.

( • • ) , Con A at 5 fig mlCon A at 0.3 ̂ (g ml without

Mrx 10~3Mrx 10~3

200*

200

94

67

94 •

6 7 *

45 •

Fig. 4. Histogram showing the increase in binding of theM27 antibody to total extracts of mitogenically-stimulatedlymphocytes in the presence (rilled blocks) and absence(shaded blocks) of 10% FCS.

Days5 5

(methanol-fixed)

Fig. 5. Histogram showing the increase in the binding of theM27 antibody to the surface of mitogenically-stimulatedlymphocytes and to methanol-permeabilized cells aftermaximum stimulation (day 5).

If a similar experiment is performed, but measuringsurface M27 binding instead of total, then the resultshown in Fig. 5 is obtained. In this case only cells in FCSare considered due to the complications of cell death thatoccur in the absence of serum. There is a similar rapidrise in surface M27 antigen on mitogenic stimulation.

45 •

Fig. 6. Autoradiograph of an immunoblot of cell extracts ofthe murine mammary carcinoma TA3H grown in vivo (lanea), in vitro (lane b), and of an immunoblot of a cell extract ofH.Ep.2 cells electrophoresed with DTT (lane c) and withoutDDT (lane e), probed with the monoclonal antibody M27.Lane d contained molecular weight standards. About 50,ug ofprotein was loaded on each track.

The increase in the surface area of stimulated lympho-cytes cannot account for the dramatic rise in antigenlevels, since this is of the order of three- or fourfoldcompared with about 20-fold for the amount of antibodybound.

When cells are permeabilised with 70 % methanol inwater prior to assay then total M27 antigen should beexposed. Fig. 5 shows that in the stimulated lymphocyteabout 85 % is located on the surface. This compares withH.Ep.2 cells, where only 25 % was found on the exteriormembrane (Gingrich etal. 19816). However, in thelatter case the cells were lysed with detergent prior toassay and may have released more antigen.

The M27 antigen is part of a disulphide-linked multimerIf an extract of H.Ep.2 cells is run on SDS-PAGEwithout prior reduction with mercaptoethanol or dithio-threitol (DTT) and then electroblotted, no antigen of178K can be detected (Fig. 6, lane e). The presumptivehalf molecule is, however, still present. However, if it iselectrophoresed in the presence of dithiothreitol, then theantigen is present at 178K (Fig. 6, lane c). This suggeststhat the intact antigen is present as a disulphide-linkedaggregate or multimer of greater than a trimer, sincemolecules of 400-500K will normally electroblot. Ma-terial was recovered from the top of the gel, reduced withdithiothreitol and re-electrophoresed, to yield a bandbinding the M27 antibody at 178K (unpublished result).The 105K molecule must therefore contain few if anysulphhydryl groups, since it does not appear to aggregatein the same way.

728 M. E. Bramwell and S. M. Hutmn

Table 1.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15Time post-mitosis (h)

Fig. 7. The amount of M27 antigen in synchronized cells(arbitrary units). Cells were sampled at hourly intervals post-synchronization and analysed for relative M27 antigencontent.

The antigen is expressed in murine tumours grown invivoExperiments involving metabolic labelling of H.Ep.2cells with radioactive amino acid precursors failed toshow any incorporation into immunoprecipitates pro-duced using M27, even though such precipitates showedthe presence of the antigen by immunoblotting (unpub-lished results). In addition, the treatment of cells withprotein synthesis inhibitors such as cycloheximide failedto reduce the level of antigen found in the cell extracts.

One possibility was that the antigen was a serumcomponent taken up by the cell in vitro. This wasexamined by M27 probing of immunoblots of serumsamples and by M27 probing of immunoblots of extractsof tumours grown in vivo for several months.

Samples of serum failed to show the presence of theM27 antigen (unpublished results) but tumours extracteddirectly had considerable amounts (Fig. 6, lanes a and b).

The expression of the M27 antigen peaks in early Sphase of the cell cycleHeLa cells growing in suspension culture were synchro-nised using nitrous oxide and a thymidine block asdescribed previously (Rao, 1968; Warren and Cook,1978). Cells were collected at intervals of one hour,extracted and analysed by SDS-PAGE followed byimmunoblotting with M27. The autoradiographs werescanned and the binding of the antibody quantified.Fig. 7 shows a histogram of the results. There is a clearincrease in the expression of the M27 antigen in early Sphase. This is total cellular content and it correlates withthat found on the cell surface previously (Gingrich et al.19816).

The M27 antigen is widespread in tissue culture celllinesSince it was shown that the M27 monoclonal antibodyrecognised an antigen present on the surface of humanand rodent cells it was of interest to check cells of otherspecies for its expression.

Table 1 lists the cells examined and indicates whichbind M27 and which do not. It can be seen that all the

Cell Species

Reactivity of totalcell extract withM27 monoclonal

antibody

H.Ep.2MRC 5LymphocyteActivated lymphocyteDAUDIRBCPG19707707/DMSO-treatedTA3HHTCBRL-3AFRLFRL in vitroNRKTG33.B77Sc4CEF16QASBFAFELCos7MDCKA2HPSYEF2XTC-2

TissuesLungSpleenHeartLiverKidneyRBCResident macrophagesElicited macrophages

HumanHumanHumanHumanHumanHumanMouseMouseMouseMouseRatRatRatRatRatRatAvianAvianPiscineBovineFelineMonkeyCanineChinese hamsterPorcineVoleAmphibian

MouseMouseMouseMouseMouseMouseMouseMouse

The amount of M27 binding was estimated relative to H.Ep.2 cellsand is based on similar amounts of protein applied to each well forelectrophoresis.

mammalian cell lines express the antigen and those ofavian, piscine and amphibian origin do not.

It is also noteworthy that the murine erythroleukaemialine 707 expresses the antigen at more or less the samelevel after induction of haemoglobin synthesis by DMSOand consequent inhibition of cell division.

The M27 antigen is a proteinAlthough it has not proved possible to label satisfactorilythe M27 antigen with radioactive amino acids, it issensitive to proteolytic degradation with trypsin, Pro-nase, thermolysin and subtilisin (data not shown). Inaddition it does not appear to contain any carbohydratechains sensitive to either O-glycanase, Ar-glycanase orneuraminidase, and the inhibitor of N-linked glycosyl-ation, tunicamycin, has no effect on the antibody bindingor mobility on SDS-PAGE (data not shown). Finally,the antigen does not bind to affinity columns usingconcanavalin A or wheat germ agglutinin linked toSepharose 4B Pharmacia, thus precluding the presence ofexposed mannose or iV-acetylglucosamine/sialic acid

Characterization of the M27 antigen 729

residues, respectively (unpublished results). There is,therefore, no evidence for the presence of carbohydrateresidues.

Discussion

The monoclonal antibody M27 binds to an antigen that ispresent both on the surface and in the cytoplasm of allmammalian tissue-culture cells tested. It has an apparentmolecular mass of 178K in the reduced form, but itappears to be a complex of disulphide-linked molecules inits native state with a molecular mass of >500K. Its widedistribution in tissue-culture cells of different species ledto consideration of the possibility that it was beingadsorbed and internalized from the medium, as is a-2-macroglobulin (Pastanef al. 1977). However, none couldbe detected in foetal calf serum, and its absence in avian,piscine and amphibian cells grown in similar mediummade that explanation unlikely.

The conserving of both the antigenic site and apparentsize over a diverse range of mammals suggest that themolecule may have an important biological role. Itsappearance in foetal rat liver explants after several daysand in rat lymphocytes after mitogenic stimulation inculture support this idea. Of the in vivo normal tissuesexamined, only the spleen and lung of the mouse boundthe antibody, and then only weakly. This may be due tothe presence of activated macrophages in these organs,since they are strongly positive for M27 binding, whereasresident ones react very weakly. On the other hand,tumour tissues extracted directly had considerable anti-body-binding capacity.

Some indication of the antigen's role in the cell may beindicated by the fact that a peak of expression occurs earlyin S phase. This confirms earlier results on studies on thecell surface expression (Gingrich et al. 19816; Banyardand White, 1984) and suggests a proliferative function.Many molecules of similar size have been reported andmany of them are receptors for various growth factors:epidermal growth factor, 190K (Das et al. 1977); trans-forming growth factor type /?+, 180K (Fanger et al.1986); nerve growth factor, 158K (Radeche et al. 1987);platelet-derived growth factor, 185K (Heldin et al. 1983;Glenn etal. 1982); granulocyte-macrophage colony-Stimulating factor, 180K (Park et al. 1986; type IIinsulin-like growth factor, 240K (Kiessei al. 1987). BothO'-2-macroglobulin and clathrin in their reduced formshave molecular masses of 185K and 180K, respectively(Swensson and Howard, 1979; Pearce, 1975), and bothform larger complexes in the native state and thusresemble the M27 antigen. However, immunoprecipi-tation using antisera to both a'-2-macroglobulin andclathrin fail to precipitate the M27 antigen, which canthus be distinguished (unpublished observation).

It is of course too early to ascribe a receptor function tothe M27 antigen and any further speculation must await asequence analysis following complete purification.Finally, no association with the glucose transporter canbe demonstrated by immunoprecipitation studies (Bram-well and Baldwin, unpublished). This latter point is

important because an apparent inverse relationship wasobserved between the level of glucose in the medium andthe binding of the M27 (Gingrich etal. 19816). It wasalso shown that those cells with greatest uptake of glucoseshowed the strongest binding to M27 affinity columns(Banyard and White, 1984). There was thus circumstan-tial evidence that the M27 antibody was binding to a siteassociated with the glucose transporter. However, theobserved correlation can now be better interpreted as onein which proliferating cells with high levels of the M27antigen expressed on their surface will be the ones thatalso rapidly take up glucose from the medium, thusreflecting greater metabolic activity.

The authors thank Professor Henry Harris for advice andsupport. M. E. Bramwell is the James Hanson Researgh Fellowof the Cancer Research Campaign.

References

ASH WOOD-SMITH, M. J., ROBINSON, D. M., BARNES, J. H. ANDBRIDGES, B. A. (1967). Radiosensitization of bacterial and mammaliancells by substituted glycoxals. Nature, Loud. 216, 137-139.

BANYARD, M. R. C. AND WHITE, M. K. (1984). Association of aninternal membrane protein with glucose transport and with aniontransport. J. Cell Sci. 67, 45-62.

BRAMWELL, M. E. AND BALDWIN, S. A. (1989). in preparation.BRAMWELL, M. E. AND HARRIS, H. (1978). An abnormal membrane

glycoprotein associated with malignancy in a wide range ofdifferent tumours. Pmc. R. Soc. B 201, 87-106.

DAS, M., MIYAKAWA, T., FOX, C. F., PRUSS, R. M., AHARONOV, A.AND HERSCHMAN, H. R. (1977). Specific radiolabelling of a cellsurface receptor for epidermal growth factor. Pmc. natn. Acad.Sci. U.S.A. 74, 2790-2794.

DULAK, N. C. AND TEMIN, H. M. (1973). A partially purifiedpolypeptide fraction from rat-liver cell conditioned medium withmultiplication-stimulating activity for embryo fibroblasts. J. cell.Physiol. 81, 153-160.

FANGER, B. O., WAKEFIELD, L. M. AND SPORN, M. B. (1986).Structure and properties of the cellular receptor for transforminggrowth factor type /3. Biochemistry, Pliilad. 25, 3083-3091.

FRIEND, C , SCHER, W., HOLLAND, J. G. AND SATO, T. (1971).Haemoglobin synthesis in murine virus induced leukemic cells invitm,: stimulation of erythroid differentiation by DMSO. Pmc.natn. Acad. Sci. U.S.A. 68, 378-382.

GINGRICH, R. D., WOUTERS, M., BRAMWELL, M. E. AND HARRIS, H.(1981a). Immunological delineation in normal and malignant cellsof a membrane protein involved in glucose transport. 1.Preparation and properties of the antibody. J. Cell Sci. 52, 99-120.

GINGRICH, R. D., WOUTERS, M., BRAMWELL, M. E. AND HARRIS, H.(19816). Immunological delineation in normal and malignant cellsof a membrane protein involved in glucose transport. II. Functionof the antigen. J. Cell Sci. 52, 121-135.

GLENN, K., BOWEN-POPE, D. F. AND ROSS, R. (1982). Platelet-derived growth factor II. Identification of a platelet-derived growthfactor receptor by affinity labelling. J . biol. Chew. 257, 5172-5176.

GLUZMAN, Y. (1981). SV40-transformed simian cells support thereplication of early SV40 mutants. Cell 23, 175-182.

HAUSCHKA, T. S. (1953). Cell population studies of mouse ascitestumors. Trans. N.Y.Acad. Sci. II 16, 64-73.

HELDIN, C. H., EK, B. AND RONNSTRAND, L. (1983).

Characterization of the receptor for platelet-derived growth factoron human fibroblasts. Demonstration of an intimate relationshipwith a 185 000 dalton substrate for the platelet-derived growthfactor-stimulated kinase. J. Biol. Chem. 258, 10054-10061.

JACOBS, J. B., JONES, C. M. AND BAILLE, J. P. (1970).

Characteristics of a human diploid cell designated MRC-5. Nature,Land. 227, 168-170.

JONASSON, J., POVEY, S. AND HARRIS, H. (1977). The analysis ofmalignancy by cell fusion. VII. Cytogenetic analysis of hybrids

730 M. E. Bramwell and S. M. Humm

between malignant and diploid cells and of tumours derived fromthem. J. Cell Sci. 24, 217-254.

KEISS, W., GREENSTEIN, L. A., WHITE, R. M., LEE, L., RECHLER,

M. M. AND NlSSLEY, S. P. (1987). Type II insulin-like growthfactor receptor is present in rat serum. Proc. natn. Acad. Sci.U.S.A. 84, 7720-7724.

KLEIN, E., KLEIN, G., NADKARNI, J. S., NADKARNI, J. J., WIGZELL,

H. AND CLIFFORD, P. (1967). Surface IgM specificity on cellsderived from a Burkitt's lymphoma. Lancet 2, 1068-1070.

LAEMMLI, U. K. (1970). Cleavage of structural proteins during theassembly of the bead of bacteriophage T4. Nature, band. Zll,680-685.

MARSHALL, C. J. (1980). Suppression of the transformed phenotypewith retention of the viral 'SRC gene in cell hybrids between Roussarcoma virus-transformed rat cells and in untransformed mousecells. Expl Cell Res. 127, 373-384.

MOORE, A. E., SABACHEWSKY, L. AND TOOLAN, H. W. (1955).

Culture characteristics of four permanent lines of human cancercells. Cancer Res. 15, 598-602.

MURPHY, H. M. (1977). A new replication-defective variant of theBryan high-titer strain Rous sarcoma virus. Virology 77, 705-721.

PARK, L. S., FRIEND, D., GILLIS, S. AND URDAL, D. L. (1986).

Characterisation of the cell surface receptor for granulocyte-macrophage colony-stimulating factor. J. biol. Chem. 261,4177-4183.

PASTAN, I. P. , WlLLINGHAM, M. , ANDERSON, W. AND G A L L O , M.

(1977). Localization of serum-derived cv-2-macroglobulin incultured cells and decrease after Moloney virus transformation.Cell 12, 609-617.

PEARSE, B. M. F. (1975). Coated vesicles from pig brain:

purification and biochemical characterization. J. molec. Biol. 97,93-98.

PUDNEY, M., VARMA, M. G. R. AND LEAKE, C. J. (1973).Establishment of a cell line (XTC-2) from the South AfricanClawed Toad, Xenopus laevis. Experimentia 29, 466-467.

RADEKE, M. J., MISKO, T. P., HSU, C , HERZENBURG, L. A. AND

SHOOTER, E. M. (1987). Gene transfer and molecular cloning ofthe rat nerve growth factor receptor. Nature, bond. 325, 593-597.

RAO, D. N. (1968). Mitotic synchrony in mammalian cells treatedwith nitrous oxide at high pressure. Science 160, 774-776.

STUDIER, F. W. (1973). Analysis of bacteriophage T7 early RNA andproteins on slab gels.J. molec. Biol. 79, 237-248.

SWENSON, R. P. AND HOWARD, J. B. (1979). Structuralcharacterization of human cv-2-macroglobulin subunits. J. biol.Chem. 254, 4452-4456.

THOMPSON, E. B., TOMPKIN, G. M. AND CURRAN, J. F. (1966).

Induction of tyrosine a ketoglutarate transaminase by steroidhormones in a newly established tissue culture cell line. Proc. natn.Acad. Sci. U.S.A. 56, 296-303.

WARREN, A. C. AND COOK, P. R. (1978). Supercoiling of DNA andnuclear conformation during the cell-cycle. ,7. Cell Sci. 30,211-226.

WHITE, M. K., BRAMWELL, M. E. AND HARRIS, H. (1984).

Alterations induced by glucose deprivation and tunicamycin in thekinetic parameters of hexose transport in hybrid cells. J. Cell Sci.68, 257-270.

(Received 31 March 1989 — Accepted, in revised form, 30 August1989)

Characterization of the M27 antigen 731