[CANCER RESEARCH 39. 4744-4748, November 1979]0008-5472/79/0039-OOOOS02.00

Relationship between Rous Sarcoma Virus-induced Expression of MembraneAntigen and Phenotypic Transformation1

Paolo M. Comoglio,2 Bianca Pani, Maria Prat, Guido Tarone, and Carlo Monti-Bragadin

Cattedra di Istologìaed Embriologia Generale ¡P.M. C.. M. P. G. TJ and Istituto di Microbiologia ¡B.P.. C. M-B.]. University of Trieste School of Medicine. Trieste.

Italy

ABSTRACT

Rous sarcoma virus-transformed hamster BHK fibroblastsexpress a virus-induced cell surface antigen undetectable incells either transformed by unrelated viruses or infected bytransformation-defective strains of Rous sarcoma virus. Toclarify whether this antigen plays any role in the process ofmalignant transformation or is expressed at the cell surfaceonly as a consequence of the acquisition of the transformedphenotype, we have investigated its expression at the cellsurface of Rous sarcoma virus-transformed BHK cells treatedwith dibutyryl cyclic adenosine 3':5'-monophosphate and at

the surface of parental BHK cells transiently transformed bythe tumor promoter phorbol myristate acetate. In the dibutyrylcyclic adenosine 3':5'-monophosphate-treated cells, in which

most of the parameters of the transformed phenotype arereverted to normality, but the product of the transforming geneis still present, virus-induced cell surface antigen is expressed.

In the mirror experiment, this antigen is not expressed byphenotypically transformed but genetically normal phorbol myristate acetate-treated cells. It is concluded that the tumormembrane antigen studied is intimately associated with theexpression of the function(s) controlled by the transforminggene.

INTRODUCTION

Several lines of evidence suggest that plasma membranealterations may play a fundamental role in malignant transformation. Antibodies are among the most sensitive tools to identify such alterations when these lead to the appearance of newantigenic specificities at the cell surface. RSV3-transformed

hamster fibroblasts express at their surface 2 tumor membraneantigens undetectable in control cells, either uninfected orinfected by transformation-defective strains of virus (7, 8). One

(EA) is common to hamster cells transformed by unrelated DNAor RNA oncoviruses and to hamster embryo fibroblasts4; the

second one (VCSA) is a virus-induced antigen different from

known virion structural proteins. It has been previously shown(7, 8) that the expression of VCSA is controlled by the srcgene, the RSV nucleotide sequence the sole function of which

1 Supported by the Progetto Finalizzato Virus. Sottoprogetto Virus Oncogeni,

of the Italian National Research Council.2 To whom requests for reprints should be addressed, at Istituto di Anatomia

Umana Normale, Università di Torino, Corso Massimo d'Azeglio, 52, 10126

Torino, Italy. Paolo M. Comoglio holds a joint appointment with the Istituto diAnatomia Umana, University of Torino School of Medicine.

3 The abbreviations used are: RSV, Rous sarcoma virus; VCSA. virus-inducedcell surface antigen; cAMP. cyclic adenosine 3':5'-monophosphate; dBcAMP,dibutyryl cyclic adenosine 3':5'-monophosphate; PMA, phorbol-12-myristate-13-

acetate; Con A, concanavalin A.4 M. Prat et al., manuscript in preparation.

Received March 12, 1979; accepted June 19, 1979.

is the induction and maintenance of the neoplastic state (30).However, a variety of other functional and structural propertiesof the plasma membrane are altered as a consequence ofneoplastic transformation. These include altered metabolitetransport (11); activation of surface proteases (29); abnormalagglutinability by plant lectins (19); and quantitative variationsof membrane glycolipids (10), glycopeptides (31 ), and a limitednumber of surface proteins (13). This group of alterations,indicated by the general term transformed phenotype, belongsto a self-amplifying cascade of secondary events triggered by

the product of the src gene. Many of these alterations haveproved to be merely a consequence of transformation since (a)they may revert to normality in genetically transformed cells bytreatment with various compounds, such as exogenous cAMP(21), or, conversely, (b) they may be altered in geneticallynormal cells treated with drugs able to induce reversible transformation only at the phenotypic level (9).

In this paper, we have examined the relationship betweenthe expression of the tumor-specific membrane antigen VCSA

and some parameters of the transformed phenotype. We haveobserved that the appearance of VCSA at the cell surface isindependent of the acquisition of the transformed phenotype.This suggests that this membrane antigen is intimately associated with some early function(s) controlled by the transforminggene.

MATERIALS AND METHODS

Cells and Viruses. The B46 line is a clone of BHK C13/21hamster fibroblasts transformed in vitro by the env~ Bryan

strain of RSV, a strain lacking genetic information for virusenvelope glycoproteins. The clone was selected in this laboratory for its high expression of VCSA (7). The C13/SN cellsare a clone of the parental BHK C13/21 hamster fibroblastline, designated "supernormal' ' after selection for lack of ability

to grow in semisolid agar, as described by Bouck and Di-Mayorca (4). All cells were grown in Dulbecco-modified Eagle's

medium supplemented with 10% calf serum, antibiotics, andFungizone.

Antisera. The anti-VCSA serum was prepared in rabbits, as

previously described in detail (7). Briefly, rabbits were primedby an i.m. injection of 2.5 x 107 live B46 cells washed carefully

to minimize the bound amount of calf serum from the culturemedium and boosted by 2 identical doses of cells at 20-dayintervals. The antiserum was adsorbed for 30 min in ice withan equal volume of packed sheep erythrocytes, to removenonspecific cytophilic immunoglobulins and antibodies againstForssman antigen (1 ). It was then chromatographed through acolumn of calf serum insolubilized by covalent binding to Seph-arose 4B to remove contaminant anti-calf serum antibodies(28). Finally, 1-ml aliquots were exhaustively adsorbed with

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Expression of RSV-induced Tumor Membrane Antigen

control parental C13/SN fibroblasts by incubation 3 times with2 x 108 live cells at 0°for 60 min. After a further adsorption

under the same conditions with BHK fibroblasts transformedby polyoma virus, the antiserum, called anti-VCSA, had nocytotoxic activity against hamster cells, either controls or cellstransformed by oncogenic viruses other than RSV (7, 8). Thecytotoxic activity of the antiserum was titrated by the 51Cr

release assay as described elsewhere (7). Freeze-dried guineapig or frozen rabbit sera were used as a source of complementwith similar results; controls included cells incubated withantiserum alone or complement alone.

Other Analytical Procedures. Agglutinability by Con A wastested as described in detail by Becker ef al. (2) on subcon-fluent cells suspended in 0.02% (w/v) EDTA in 0.01 M phosphate-buffered (pH 7.2) 0.15 M NaCI and washed twice inserum-free medium. Lyophilized Con A (Miles-Yeda Ltd., Re-hovot, Israel) dissolved in 10 /¿Iof phosphate-buffered NaCIsolution was added in triplicate 2-fold scalar dilutions to Falconmicroplate wells containing 2 x 104 cells suspended in 10 /¿I.

Agglutination was scored by an inverted microscope afterincubation at room temperature for 1 hr.

Sugar uptake was determined by measuring the rate ofincorporation of D-glucosamine as described by Hatanaka (11 ).

Cell monolayers, in triplicate, were carefully washed and incubated in glucose-free Hanks' solution containing 2 x 10~5 MD-[3H]glucosamine. After washing, the radioactivity incorpo

rated was measured by liquid scintillation counting of cellsamples lysed by distilled water. The sugar uptake, calculatedas cpm per mg protein, was linear up to 20 min; the uptakerate was measured within 10 min.

Growth in soft agar was tested as described by Macphersonand Montagnier (18) using Dulbecco's medium supplemented

with 10% tryptose phosphate broth and 10% fetal calf serum.Cells (3x10" in growth medium partially solidified by 0.33%

agar) were plated in 5-cm Retri dishes over a basal feeder layer

of 0.5% agar. Formation of colonies was scored after 10 daysof incubation at 37°.

RESULTS

Subconfluent monolayers of B46 cells were treated, 12 to24 hr after plating, with dBcAMP at the final concentration of1 x 10~3 M. Forty-eight hr after addition of dBcAMP, the plates

were inspected by an inverted phase-contrast microscope. The

cells displayed a slight flattened morphology, and at confluencythe number of round cells was markedly reduced compared tothe untreated controls (data not shown). Saturation densitywas measured by plating, in triplicate, identical numbers ofdBcAMP-treated or control cells and by counting the numberof cells per sq cm 2 days after the cells had reached con-fluency. The culture medium was supplemented with 10% calfserum and replaced, with addition of fresh dBcAMP whereappropriate, every second day. Table 1 shows that in thepresence of dBcAMP the saturation density of B46 cells waslowered by a factor of 2. A toxic effect of the compound wasruled out by testing the plating efficiency and the spontaneousrelease of 51Cr (a sensitive test for cell viability) of the treated

cells.Phenotypic reversion was also monitored by measuring cell

agglutinability in the presence of various concentrations of thelectin Con A. B46 cells were agglutinated by Con A at concen

trations as low as 15 mg/ml, whereas dBcAMP-treated B46cells were unaffected by Con A concentrations as high as 250mg/ml (Table 1).

On the contrary, treatment with dBcAMP exerted no effecton the rate of D-[3H]glucosamine transport, the amount of sugar

taken up by treated and untreated cells being the same (Table1). Similarly, dBcAMP did not affect the amount of RSV group-specific core proteins synthesized by B46 cells, this amountbeing low, as previously shown by a radioimmunological assay(7), in both control and treated cells (data not shown).

The expression at the cell surface of the tumor-specificantigen VCSA was monitored by the cytotoxic activity of thespecific adsorbed rabbit antiserum against control anddBcAMP-treated cells labeled with 51Cr. The assay, lasting 1hr, was performed in the presence of 1 x 10~3 M dBcAMP, to

prevent possible rapid reversion of the treated cells to thetransformed phenotype as a consequence of the removal ofthe cyclic nucleotide. Both control and treated B46 cells werelysed by anti-VCSA serum. Chart 1 shows that the treated cells

were lysed with a slightly higher efficiency. The expression ofVCSA in dBcAMP-treated cells was confirmed by competition

experiments performed by adding increasing amounts of coldtreated cells to 51Cr-labeled B46 cells used as target cells

(Chart 2). In these experiments, it was observed that dBcAMPtreatment reduces to about one-half the number of cells re

quired to obtain 50% inhibition of the cytotoxic reaction. Thisis indicative of a higher amount of VCSA accessible to antibodies at the cell surface.

The mirror experiment was performed by measuring VCSAexpression on normal C13-SN cells phenotypically transformed

Table 1dBcAMP-induced phenotypic reversion of RSV-transformed fibroblasts

CellB46

B46/dBcAMPSaturation

density"(x 105)2.51.2Con

A agglutinabil

ity6

(/ig/ml)15

500o-Glucosamine

transport01

000 ±150°

1200 ±50

Number of cells: sq cm at confluency.0 Con A required to agglutinate 30% of the cells tested.0 cpm of r>{3H]glucosamineincorporated:mg of total cell proteins.d Mean ±S.D.

80

eo

20

1O 2O 4Oserum dilution" 80

Chart 1. Complement-dependent cytotoxic activity of rabbit anti-VCSA serumtested against 51Cr-labeledRSV-transformed B46 cells (•).B46 cells treatedwith 1 x 10 3MdBcAMP for 48 hr (O). and C13-SN cells grown in the presenceof 5 x 10 ' M PMA (A).

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P. M. Comoglio et al.

80

60

40

20

7.5 3.7 1.8 1competitor: target cell ratio

Chart 2. Inhibition of the cytotoxic activity of anti-VCSA serum. The antiserum,at a dilution giving 50% maximum lysis, was tested against "''Cr-labeled B46 cells

in the presence of increasing amounts of unlabeled competitor cells. Representative experiment in which B46 cells, either untreated (•)or treated with 1 X10~3 M dBcAMP (O), were tested. C13-SN cells phenotypically transformed inthe presence of 5 x 10"' M PMA (A) were ineffective.

by the tumor promoter phorbol myristate acetate. C13-SN

fibroblasts were grown for a minimum of 4 days in the presenceof 5 X 10~7 M PMA. The culture medium was changed every

other day, and fresh PMA was added.In the presence of the drug, the fibroblast-like cell morphol

ogy and agglutinability by Con A were almost unaffected (datanot shown). On the contrary, growth properties were markedlyaltered. The treated cells reached a saturation density higherthan that of the untreated controls (Table 2). Moreover, atconfluency the PMA-treated cells displayed several foci of

abnormally crowded and overlapping cells. These foci werenever observed in C13-SN control cultures. Moreover, whenplated in semisolid agar medium in the presence of PMA, C13-

SN cells were able to grow and to develop small colonies.These were smaller in size and developed with a plating efficiency 5 times lower than those developed by B46 cells (Table2; Fig. 1). As described in "Materialsand Methods," the clone

C13-SN did not form colonies in soft agar.In C13-SN cells, the rate of glucosamine transport was

unaffected by treatment with PMA, being already as high asthat of transformed B46 cells (Table 2).

The expression of the tumor antigen VCSA at the surface ofPMA-treated C13-SN cells was tested in the direct cytotoxicityassay with anti-VCSA serum and PMA-treated cells labeledwith 51Cr. PMA was added to the assay medium to prevent

possible rapid reversion to normality. As shown in Chart 1, theanti-VCSA serum did not have significant cytotoxic activity onPMA-treated cells. To rule out the possibility of expression of

low amounts of VCSA undetectable by direct lysis, competitionexperiments were performed by adding cold treated cells to51Cr-labeled VCSA-positive B46 cells used as targets. No com

petition was observed, showing that phenotypically transformed C13-SN cells were completely negative with respect to

VCSA expression (Chart 2).

DISCUSSION

Following RSV transformation in vitro, cells change theirshape, become less adherent to substratum, grow rapidly, losedensity-dependent inhibition of growth, and become able to

grow in soft agar (22). Many of these processes are controlledby the intracellular concentration of cAMP. Transformed cellstreated with an active cAMP derivative, dBcAMP, or agentsthat elevate endogenous cAMP levels increase their adhesiveness to substratum (14, 16), slow their growth rate (15), andflatten and in some cases may acquire an elongated morphology similar to that of normal cells (15). This phenotypic reversion to normality, which may be easily monitored by measuringagglutinability toward plant lectins (12, 25), has also beenobserved in dBcAMP-treated cells of various species trans

formed by RSV (17, 21).The dBcAMP-induced reversion of the RSV-transformed

hamster fibroblast line used in these experiments was measured by changes in growth properties and by loss of agglutinability by the lectin Con A. Cell morphology was only slightlyaffected, and the rate of glucose uptake was unchanged. Theseobservations are in agreement with previous studies reviewedby Pastan et al. (21 ) and Hatanaka (11 ).

In these RSV-transformed cells, phenotypic reversion is not

followed by disappearance of VCSA from the cell surface,since dBcAMP-treated cells were lysed by the specific antise-

rum. Paradoxically, the cyclic nucleotide even increased theexpression of the antigen. This unexpected observation is inagreement with similar results obtained by Kurth and Bauer(17) with an uncharacterized syngeneic antiserum againstRSV-induced mouse sarcoma.

This finding suggests that VCSA expression is a stableproperty of the tumor cell, independent of the acquisition ofthe phenotypic characteristics controlled by the intracellularlevel of cAMP. The mirror experiment performed on phenotypically transformed but genetically normal cells gives furthersupport to this hypothesis, since VCSA was not expressed byC13-SN fibroblasts treated with the drug PMA.

PMA is a phorbol derivative belonging to a class of tumorpromoters of plant origin, able to induce a constellation ofchanges reminiscent of the phenotype of cells transformed byoncogenic viruses or chemical carcinogens. These includetransient increase in intracellular cyclic guanosine 3':5'-mono-

phosphate levels (9), decrease in intracellular cAMP (3), andalterations of growth properties (6, 26, 27), as observed alsoin the cells used for these experiments. Moreover, extremelylow concentrations of PMA induce a marked increase in cellsurface plasminogen activator in a variety of cells of differentorigin, including hamster fibroblasts. Since good correlationhas been demonstrated between production of plasminogenactivator and the loss of anchorage-dependent growth (20,23), the observed growth in soft agar of PMA-treated C13-SN

cells is also explained and has been taken to be a marker ofphenotypic transformation.

Table 2

Phenotypic transformation of normal fibroblasts in the presence of the tumorpromoter PMA

CellC1

3-SNC13-SN/PMASatura

tion density"

(x 105)3.35.7Growthin soft

agar65

±3156 ±35D-Glucosamine

transport01

200 ±180"

980 ±105a Number of cells: sq cm at confluency." Number of colonies developed 10 days after plating 3 x 103 cells.0 cpm of D-[3H]glucosamine incorporated:mg of total cell proteins.dMean ±S.D.

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Expression of RSV-induced Tumor Membrane Antigen

Table 3

Relationship between VCSA expression and phenotypic transformation

CellB46B46/dBcAMPC13-SN/PMACia-SNCon

Aag-glutinabil-src*

ity+

High+Low—Low—

LowSaturation

densityReduced"Increased"Growthin

softagar+NTC+—GlucosaminetransportUnaffected"Unaffected"VCSA++-—

a Presence of an active src sequence integrated into the host cell genome.6 Compared to untreated B46 cells.c NT, not tested.d Compared to untreated C13-SN cells.

An overall picture of the phenotypic alterations that we havemeasured in hamster fibroblasts transformed by RSV, revertedby dBcAMP, or sham-transformed by PMA is summarized in

Table 3. Among the 5 biological parameters altered by transformation, namely, Con A agglutinability, saturation density,growth in soft agar, glucosamine transport, and expression ofVCSA at the cell surface, the only one that strictly correlateswith the presence of the transforming (src) gene is the latter.In fact, Con A agglutinability and saturation density revert tonormality in genetically transformed dBcAMP-reverted B46

cells, and abnormal growth and loss of anchorage dependenceare observed in untransformed C13-SN cells treated with PMA

as well as in cells truly transformed by RSV. Finally, glucosamine transport rate is similarly elevated in both RSV-trans-

formed and control cells. The tumor membrane antigen VCSA,on the contrary, only appears at the cell surface of RSV-

transformed cells, is insensitive to treatment with dBcAMP, andis not induced by the tumor promoter PMA.

It is concluded that VCSA expression is intimately associatedwith the expression of the function(s) directly controlled by thetransforming gene. It has been shown that this gene codes fora protein with a molecular weight of 60,000 with phosphoki-

nase activity (5, 24). The elucidation of the relationship between this protein and VCSA will contribute to the understanding of the role played by the plasma membrane in malignanttransformation.

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oC

Fig. 1. Growth in soft agar of hamster fibroblasts. a. RSV-transformed B46 cells showing large colonies after 10-day incubation. As shown in b, control C13-SNcells, under the same conditions, may undergo a few divisions (arrow) but do not form colonies. In c. in the presence ot 5 x 10 ' PMA, C13-SN cells develop asignificant number of colonies with 2 different appearances, "tight" (lower left) or "spread" (upper right). Arrow, an abortive colony, x. 100.

4748

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1979;39:4744-4748. Cancer Res   Paolo M. Comoglio, Bianca Pani, Maria Prat, et al.   of Membrane Antigen and Phenotypic TransformationRelationship between Rous Sarcoma Virus-induced Expression

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