Pericellularmatrix and Cell Surface Glycoproteinsof Virus Transformedmouse Epithelialcells'

[CANCERRESEARCH42,1147-1153,March1982] 0008-5472/82/0042-0000$02.00 PericellularMatrix and Cell Surface Glycoproteinsof Virus transformedMouse EpithelialCells'

Jorma Keski-Oja,2Carl G. Gahmberg,and Kari Alitalo3

Departments of Virology (J. K-O., K. A.) and Biochemistry (C. G. G.j, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 29, Finland

ABSTRACT disturbances of the regulation of epithelial cell growth and maturation. Mouse embryo Mus musculus castaneous epithelial cells, Transformation of fibroblastic cells with viruses have been transformed with Moloney murine sarcoma virus (MSV) or with reported to induce changes both in the pericellular matrix and ecotropic murine leukemia virus (MuLV), were analyzed for in the cell surface glycoproteins (38, 42, 43, 46). Particularly, production of pericellular matrix glycoproteins. The nontrans the loss of pericellular fibronectin has been a characteristic formed, MSV-transformed, and MuLV-transformed cells pro feature in almost all virally transformed cell systems studied duced fibronectin, laminin, type I collagen, and small amounts (42, 43). Loss of another noncollagenous matrix glycoprotein, of type III collagen when studied by immunofluorescence using laminin, from virally transformed cells has recently been dem specificantibodies.Thevirus-transformedepithelialcellspro onstrated (14). It has also been reported that viral transforma duced enhanced amounts of fibronectin into their growth me tion of chick fibroblasts leads to a decrease in their collagen dia. Nontransformed M. muscu!us castaneaus epithelial cells synthesis (11, 18, 28, 37, 38). Both chemical and viral trans mainly produced type I collagen, as shown by metabolic label formation can change the collagen phenotype of cultured fibro ing and polypeptide analysis. A significant increase in the blastic mouse cells (13). glycoprotein production was seen by the MuLV-transformed In the present study, we used cultures of mouse embryo cells,whereassmallchangesinthecollagenproductionwere epithelial cells, MMC-E4 (34), to study changes in the pericel apparent after MSV transformation. MuLV-transformed cells lular matrix and cell surface glycoproteins of epithelial cells produced increased amounts of type I collagen and also some after viral transformation. MMC-E cells were infected with Mo collagenous polypeptides that comigrated with procollagen loney MSV or ecotropic MuLV.5 Infection of the cells with the type IV chains. The ratio of the procollagen type I chains viruses resulted in the growth of morphologically transformed deposited in the matrix was altered in transformed cells. Radio cell colonies that were isolated using soft-agar techniques. active surface labeling of the cells revealed changes of the With the use of metabolic and cell surface labeling techniques high-molecular-weight glycoproteins in both the MSV- and the and tests for collagen isotypes and the noncollagenous poly MuLV-transformed cells. Unlike virus-transformed fibroblastic peptides, fibronectin and laminin, we found that viral transfor cells, these transformed epithelial cells deposited and retained mation modulates the pericellular matrix and the surface gly connective tissue glycoproteins in their pericellular matrices. coproteins of mouse epithelial cells. Unlike the situation with The results indicate that viral transformation modulates the virus-transformed fibroblasts, a loss of the pericellular matrix pericellular matrix and surface glycoproteins of cultured mouse did not take place. epithelial cells. The ability of virus-transformed epithelial cells to deposit pericellular matrices is a major difference between them and virus-transformed fibroblastic cells. MATERIALS AND METHODS Cell Cultures.Stockculturesof MMC-Emouseepithelialcells (34) INTRODUCTION and their viral transformants were subcultuned once a week at a 1:5 ratio. Syngeneic fibroblastic cells, MMC-F, were cultivated as do Studies on the biochemical alterations that occur after viral scnibed(34). The cells weregrownin Eagle'sbasalmediumsupple transformation of epithelial cells have been hampered by the mented with 10% fetal calf serum plus penicillin (1 00 lU/mI) and lack of pure cultures of nontransformed epithelial cells. Most streptomycin (50 jig/mI). studies carried out thus far have been pertormed with cultured Transformation of MMC-ECells with MuLV and with Moloney rat liver epithelial cells (5, 23, 45) or cell lines established from MSV. MMC-E cells were seededon plastictissueculturedishesat a tumors(12,25,26).Carcinomasarethemostcommonneo density of 1 x 1O@cells/60-mm diameter plate. Eighteen hr after seeding, the cells were infected with a high multiplicity of Moloney plasmsofhumans,andthebiologyofcarcinomacellsdiffers MSV or ecotnopicC3H MuLV. The cells were maintainedon culture considerably from that of sarcoma cells (cf. Refs. 7 and 45). dishes until foci of morphologically transformed cells could be seen. Identification of changes in transformed epithelial cells may Thecellswerethentrypsinizedandseededin soft agar.Cellcolonies provide a tool for elucidating aspects concerned with the that grew in soft agar were isolated, and the MSV-transfonmed cell clonestudiedherewasfoundtobeabletoproducepoorlydifferentiated 1ThisworkwassupportedbygrantsfromtheFinnishCancerFoundation,the carcinomasin nudemice(34). The MuLV-transformedcellclonewas ScienceFoundationofLÃ Ã¤keCorporation(Turku,Finland),theAssociationof alsoisolatedusingthe soft-agartechnique.5ControlMMC-Ecellsand Finnish Life Assurance Companies, the Academy of Finland and NCI grant 5R01 CA26294-02.Presentedinpartat theVIIEuropeanCongressofPathology,

Helsinki, 1981 (19). 4 The abbreviations used are: MMC-E cells, Mus musculus castaneous epithe 2Towhomrequestsforreprintsshouldbeaddressed. hal cell line (33); MSV, mouse sarcoma virus; PAuLV,murineleukemia virus. 3 Present address: Department of Biochemistry, University of Washington, 5 U. R. Rapp and J. Keski-Oja. Transformation of a mouse epithelial cell line, Seattle,Wash.98195. MPAC-E by IudR-induced C3H/PAuLV enhanced by a tumor promoter, submitted Received June 10, 1981 ; accepted November 5, 1981. for publication.

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MSV-tnansfonmed cells were negative, but the MuLV-transfonmed cells NaB3H4labeling,the cells were incubatedwith 5 units of galactose were positivein assaysfor reversetranscniptasecarriedout as do oxidase(Kabi,Stockholm,Sweden)and12.5 unitsof VibrioCholerae scnibed(35, 36). neuraminidase (Behningwerke, Marburg-Lahn, Federal Republic of Immunofluorescenceof the PericellularMatrices. Confluentcul Germany)for 30 mmat 37Â°.Thecells were washedand incubated tunesof cells grownon glasscovenslipswereextracted3 timeswith with 2 mCiof NaB3H4(8Ci/mmol; NewEnglandNuclear)in 0.5 ml of sodium deoxycholate in hypotonic buffer (0.5% deoxycholate:1 m@ phosphate-bufferedsaline.After 30 mm of reduction,the label was phenylmethylsulfonyl fluonide:0.14 MTnis-HCIbuffer, pH 8.0) on an ice chasedwith 1 mMNaBH4for10 mm,and the cells werewashedand bathasdescribedindetail(16).Thematriceswerethenrapidlywashed extracted with gel sample buffer. The samples were analyzed in sodium 3 times with 2 mM Tnis-HCIbuffer (pH 8.0) containing 1 mM phenyl dodecylsulfate:polyacrylamidegelsfollowedby fluorography(4). methylsulfonylfluoride.After a washing,the matriceswerefixed with 3.3% paraformaldehyde, as described in detail (1 , 16). RESULTS Indirect immunofluonescence for fibronectin, Iaminin, and pnocolla gen isotypes was performed according to the method of Alitalo et a!. Immunofluorescence Analysis of the Pericellular MatrIces. (2). Rabbitantilamininantibodieswerea gift fromDr.GeorgeS. Martin MMC-E cells and their viral transformants were studied for their (National Institute of Dental Research, NIH, Bethesda, Md.). Dilution 1: ability to deposit pericellular matrices. Surface staining of 200 was used for the staining which could be blocked by absorption of the antiserum with purified laminin (2). No staining was seen by normal confluent cultures of MMC-E cells had demonstrated only low rabbit serum. amounts of fibronectin on their surfaces (34). Confluent cul Metabolic Labeling and PolypeptideAnalysis. Cellcultureswere tures of cells grown on glass coverslips were then extracted labeledwith[14C]glycine(5mCi/liter; 113 Ci/mol; TheRadiochemical with sodium deoxycholate in hypotonic buffer to remove the Centre, Amersham, United Kingdom) on with both [2-3H]glycine (20 cells as described in detail (16), and the pericellular matrices mCi/liter; 23 Ci/mmol)andL-[5-3H]proline(20mCi/liter; 15 Ci/mmol; were visualized by indirect immunofluorescence using specific the Radiochemical Centre) in media containing sodium ascorbate (50 antibodies. pg/mI) and $-aminopropionitnile fumarate (50 @g/ml).Both the cell Analysis of the matrices with antifibronectin antibodies mdi layers and the culture media were analyzed for the presence of radio cated that both the MSV-transformed and the MuLV-trans activepolypeptidesinsodiumdodecylsulfate:polyacrylamidegels(24). formed cells could retain a pericellular matrix around them The collagenous polypeptides in culture media were identified by their susceptibilityto digestionby bacterialcollagenase(FormIll; 30 selves as did the control MMC-E cells (Fig. 1; data of the units/mI, 60 mm at 37Â°;Advance Biofactures Corp., Lynbrook, N. V.) MuLV-transformed cells are not shown). The matrices were and by their electrophoretic mobility when compared to the previously also studied using specific antibodies against procollagen identified collagen polypeptides produced by 3T3 cells (see Fig. 4),Â° types I, Ill, and IV, and against laminin (2). Matrices of all cells The collagenasepreparationdid not contain contaminatingprotein contained procollagen type I. The matrices contained also ases. For pepsin digestion, acetic acid was added to the medium at a small amounts of procollagen type Ill (Fig. 1, d and h) but final concentration of 0.5 M (final pH 3.0), and pepsin was added to a procollagen type IV could not be demonstrated (data not concentration of 100 @g/ml(3 times crystallized, dissolved in 0.5 M shown). The matrices contained also some laminin that was acetic acid; Sigma Chemical Co., St. Louis, Mo.). The medium was distributed with a characteristic punctate pattern (Fig. 1, b and then incubated at 15Â°for 18 hr. Digestion was terminated by neutral f). No differences were observed between the MMC-E cells izing the aliquots of the samples with equimolar amounts of 1 M NaOH. Proteinaseinhibitors were then added [2 mM phenylmethylsulfonyl and their viral transformants. Unlike virus-transformed fibro fluoride (Sigma) and 200 units aprotinin per ml (Trasylol; Bayer, Lev blastic cells, both the MSV- and MuLV-transformed epithelial orkusen, Federal Republic of Germany]. The radioactive polypeptides coIls were able to retain a pericellular matrix. produced by the cells were concentrated by precipitation with ammo PolypeptidesProducedbyVIrus-transformedMouseEpi nium sulfate (1 76 mg/mI) in the presence of proteinase inhibitors and thelial Cells. To analyze the collagenous proteins produced by using gelatin as a carrier protein (2). As another test for collagenous the cells, confluent cultures of MMC-E cells and their MuLV- or proteins, the specific hydroxylations of proline and lysine were inhibited MSV-transformed counterparts were radiolabeled metabol by chelationof Fe2@bya,a'-dipynidyl;this is knownto slowdownthe ically, and the cell layers were analyzed by gel electrophoresis secretion of underhydroxylated collagen (17, 32). A change in mobility followed by fluorography. Analysis of the cell layers after a and increased specific radioactivity of a polypeptide band in material short labeling indicated that the major labeled polypeptides from the treated cell layer then identified the accumulated collagenous comigrated with fibronectin and procollagen chains. In addition, polypeptide. Cell SurfaceLabeling.Surfacelabelingof the cellswasperformed there were other radiolabeled polypeptides that were not fur eitherby thepeniodate:NaB3H4labelingmethod(8)or bythegalactose ther characterized (Fig. 2). The major difference in the poly oxidase:NaB3H4method(9, 10). Cells grownon plastic disheswere peptide patterns between the MSV-transformed cells and the washed3 timeswithphosphate-bufferedsaline(0.14 MNaCIin 10 mM nontransformed cells was an apparent decrease in radioactivity phosphate buffer, pH 8.0) and incubated with 1 m@NalO4for 10 mm of the procollagen a2(l) chain (Fig. 2, Lane 2). In MuLV-trans at 0Â°,washedwith phosphate-bufferedsaline,scrapedwith a rubber formed cell layers, the procollagen a2(l) chain was detectable, policeman, and resuspended in 0.5 ml of phosphate-buffered saline. but its amount appeared lower than that of the control cells. Thereafter,the cells wereincubatedwith NaB3H4(2mCi;8 Ci/mmol; To study the possibility of altered production or deposition NewEnglandNuclear,Boston,Mass.).After30 mmat roomtempera of procollagen a2(I) chains in the transformed cell cultures, we tune,the cells were washedwith phosphate-bufferedsaline,and the cells weredissolvedin gel samplebuffer.For the galactoseoxidase studied the processing and accumulation in the cell layers of newly synthesized collagen in the presence of a,a'-dipyridyl, an inhibitor of the hydroxylation of lysine and proline. As a 6 K. Alitalo, E. Kuismanen, R. MyllylÃ¤, U. Kiistala, S. Asko-Seljavaara, and A. result of this treatment, we found a characteristic change in Vaheri.Humanepidermalkeratinocytessynthesizeanddepositnovelcollagen chains and secrete together with feeder layer 3T3 cells firbronectin, type IV the mobility of the procollagen chains in the cell layers (Fig. 3). collagen and laminin, submitted for publication. The accumulation of procollagen a2(l) chains in MMC-E cell

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@1h@ f: 9_@__@t@f:L@L@- Fig.1. Immunotluorescenceanalysisof the matrixproteinsof PAPAC-Ecellsand their MSVtransformants.Confluentcell layerswere extractedwith sodium deoxycholate In hypotonic buffer (16) and fixed with paraformaldehyde for immunofluorescence. Indirect immunofluorescence for fibronectin and laminin was performedasdescribed(2).a,e,phase-contrastphotomicrographsofcontrolMMC-EcellsandPASV-transformedcells;b,f, lamininstainingofthematrices;c,g, fibronectinstainingofthematrices;d,h, procollagentypeIll stainingofthematrices,respectively.x 180

1 2 3 M MOMSVMMC.EMULV ir@ @@ FN P' 200 @ procti(I) 0 @ proa2(l)' r â€˜1'S i@ -proct@,RI B , 1@@ -@ . -proc1@2[i] @ â€˜lOO

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Fig. 2. Radlelabellng of PAPAC-Ecellsand their viral transformants with [â€˜4C]glycine.Thecells were grown to confluency and labeled in culture for 3 hr with 5 mCi of[â€•C@lycineperlIter. After labeling, the cell layers were washed 3 times with phosphate-buffered saline and extracted with the gel sample buffer. An autofluorogram ofan8%pelyacrylamldegelisshown.Lane1,MMC-Ecells;Lane2,MSV-transformedcells;Lane3,PAuLv-transformedcells.M,molecularweightmarkers(myosin, phoephorylaseb,bovineserumalbumin,andovalbumin);FN,positionoffibronectin.proa1(I)andproa2(I),mobilityofprocollagentype1 chains. Fig.3. InhIbitionofhydroxylation.Confluentcultureswerelabeledfor2 hrwith[â€˜4C]glycinewithor without0.3 [email protected],a'-dipyridyl.Analysisofthesampleswasas in Fig. 2. MOMSV, PAoloneyPASV-transformedPAMC-Ecells. MMC-E, control MPAC-Ecells; MuLV, PAuLV-transformedPAPAC-Ecells.The underhydroxylated, intracellularly accumulated chains of procollagen type I are indicated left, 1â€˜and2' corresponding to the procollagen proal(l); right, proa2(l) chains. An autofluorogram of a 6% polyacrylamide gel is shown.

MARCH 1982 1149

J. Keski-Oja et a!. cultures was clearly seen. The analysis of both the MSV- and digestion experiments of the culture media of the cells sug MuLV-transformed cells indicated that there was a negligible gested that the MSV-transformed cells could also produce accumulation of procollagen a2(l) chain in the cell layers, normal type I procollagen molecules (gel not shown). All cells whereas control MMC-E cells contained both chains of type I including the MSV-transformed cells produced collagen type I procollagen (Fig. 3). These results strongly suggest that polypeptides into their media, and both procollagen chains type I procollagen in MSV-transformed cells is mainly depos could be identified in the gels as shown by densitometric ited in cell layers in altered form, probably as homotrimers scanning (Chart 1; Fig. 5). The major difference was that the [pro-si (I)]3 and that procollagen a2(I) chain is also somehow transformed cells also produced significant amounts of fibro modified after MuLV transformation. nectin, whereas the control MMC-E cells produced, under DigestionwithCollagenase.Thecollagenouspolypeptides these conditions, only small amounts of fibronectin. The total produced by the cells were also identified by collagenase production of polypeptides, as measured by incorporation of digestion after labeling of the cells with radioactive amino radioactivity in polypeptides produced by the cells, was much acids. MMC-E cells produced collagenous polypeptides that enhanced in the MuLV-transformed cells (see legend to Chart comigrated with procollagen type I and type III chains, but the 1 for details). syngeneic fibroblastic cells MMC-E also produced type IV When the labeling was carried out for 18 hr, collagenous collagen as did the fibroblastic 3T3 control cells (Fig. 4). Pepsin polypeptides that comigrated with the procollagen type IV

I4@L@i cv)oo MuLV MoMSV 3T3

. â€œ â€˜4â€• proal(IVL ;@ @I4 4 proa@2(lV)= e@ â€”proa[ffl@ :1 proc@1(l)@ â€˜I. procE2(l)â€” - a2(I)' @.

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Fig. 4. Comparison of the polypeptides produced by 3T3 cells with the polypeptides produced by MMC-E epithelial cells and the syngeneic PAMC-Ffibroblastic cells. The cells were labeled for 4 hr with radioactive glycine and proline, and the radiolabeled polypeptides were analyzed in 5% gels followed by autofluorography. Left.collagenasesensitivityofthepolypeptidesproducedby3T3cells.Someprocessedformsof collagensareseenin additiontothedifferentprocollagenchains indicated. Both MMC-E and MMC-F cells produced polypeptides that comigrated with type I and type Ill procollagen chains. Fig. 5. Production of type Iv collagen-like peptides by virus-transformed MMC-E cells. The cells were labeled for 18 hr with radioactive glycine in the presence @ of ascorbate, as described in Materials and Methods.â€•Thesupernatant fluids were collected, clarified by centrifugation, digested with collagenase for 1 hr and processed for 5% polyacrylamide gel electrophoresis (see â€˜â€˜MaterialsandMethodsâ€•andChart 1). The migration of the proa 1 and proa2 chains of mouse procollagen type IV and of collagen type I a2-chain are shown. MuLV, supernatant fluid from MuLV-transformed cells; M0MSV, supematant fluid from PAoloneyPASV-transformed cells. Fibronectin migrates in these gels close to the proa 1(W) chain.

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trimer, as shown previously in cultures of virally and chemically A transformed murine fibroblasts (1 3). However, the MSV-trans @c@J @:H@;j @nj formed cells were able to produce normal procollagen type I S 5@ @a @s into their culture media. Surface Glycoprotein Analysis of the Cells. The cells were radiolabeled in vitro using the periodate:NaB3H4 labeling method. Fibronectin was heavily labeled both in the nontrans formed MMC-E cells and in their viral transformants, in accord 1'@'@:@L;Jv@&J%@@JkLE ance with the immunofluorescence results of isolated pericel lular matrices. Since the cells were cultivated without $-ami

BC nopropionitrile fumarate, collagen type I was found in the processed form. Collagen type I chains, sized and collagenase sensitive, were labeled in all cell layers (Fig. 7A, the doublet above the M.W. 100,000 marker). However, the label was slightly decreased in the a2(l) chain of both the MSV- and the MuLV-transformed cells further suggesting a change in the phenotype of collagen type I deposited in the cell layers. In both clones of virus-transformed cells, a M.W. 95,000 glyco protein was found (Fig. 7A, Lanes 2 and 3). In addition, a novel glycoprotein (M.W. 68,000) was seen in the MuLV-transformed Chart1. Productionof collagenousproteinsby virus-transformedPAPAC-E cells (Fig. 7A, Lane 3; see also Fig. 7B, Lane 3). cells.Thecellsgrownon 100-mm-diametertissueculturedisheswerelabeled When the galactose oxidase:NaB3H4 labeling method was for 2 hr with 20 mCi of [3H]glycine per liter and 20 mCi of [3H]proline per liter in the presence of sodium ascorbate. The supematant fluids were clarified by used, the patterns of radioactive polypeptides differed from centrifugation and incubated with collagenase for 1 hr. The cell layers were those obtained by the periodate labeling method. Fibronectin trypsinized, and their cell number was determined in an electric cell counter. was not as heavily labeled as by the periodate:NaB3H4method. Results of duplicate determinations are shown here. Approximately equal amounts of radioactivity was analyzed in gels followed by densitometric scanning. Thus,theamountofeachpolypeptideisnotdirectlycomparabletothecorre sponding peak In the untreated materiel but indicates the collagenase-sensitive MoMSV MMC-E MuLV and-insensitIvepolypeptidesintherespectivemedium. radioactivity in the medium before collagen 10@)MMC-E6.770.6MSV6.884.6PAuLv7.0346.1CellsCell no. (x 10_6)Protein-boundase treatment (cpm x

@ FN@ 0- A, B, MMC.Ecells;C,D, MuLV-transformedcells;E,F, PASV-transformedcells. proa,(I) @4 B,D,F,collagenasedigestions.Thepositionsoffibronectin(FN)andprocollagen type I chaise are shown. proci@2(I) C chains of control 3T3 cells were clearly seen in the autoradi ogramsoftheculturemediaoftheMuLV-transformedcells, faintly in the media of MSV-transformed cells, but not at all in the media of control cells (Fig. 5; data of MMC-E cells are not . @11 shown).Fibronectinpolypeptidesthatmigratecloseto the procollagen al(IV) chains were produced by both of these virus-transformed cell clones and were insensitive to collagen ase as expected (Fig. 5). The collagen a2(l) chain was also clearly seen in the culture media of the MuLV-transformed cells but was only barely visible in the media of MSV-transformed cells. Transformation by MuLV may thus affect the production of type IV collagen by mouse epithelial cells. The polypeptides of MMC-E cells and their viral transform ants were also subjected to collagenase. Procollagen type I chains were readily removed from MMC-E cells by collagenase, whereastheamountofprocollagena2(l)chainwasnegligible Collagenase â€” + - _+ - + In the MSV-transformed cells and also slightly different from controls in the MuLV-transformed cells (Fig. 6). Procollagen Â© Fig. 6. Collagenase treatment of the cell layers. Confluent cultures were ccl(lll) chains and their processed forms were also identified in labeledwithradioactiveglycine,andthecellswereextractedanddigestedwith MMC-E cells by their susceptibility to collagenase. The depo collagenase as described in (Materials and Methods.â€•Analysiswas as in Fig. 2. sitionoftypeI collagenintothematrixthusseemstobe MoMSV,MoloneyMSV-transformedPAMC-Ecells;MMC-E,controlMPAC-Ecells; MuLv,MuLV-transformedcells.Left.migrationoffibronectin(FN)andprocolla affected by transformation. Collagen type I is evidently depos gen type I chains. An autofluorogram of a 6% gel is shown. All gel tracks have ited in the matrix of MSV-transformed cells partly as a homo been taken from the same gel.

MARCH 1982 1151

J. Keski-Oja et a!.

1 2 3 Â® M I 2 3 M Â® MMC-E cell clones,5 we now find that transformation can bring about changes in the glycoprotein production and cell surface glycoproteins of these cells. Both of these virus transformed epithelial cell colonies also contain keratin, mdi @ , - .@ â€˜200 cating that they are of epithelial origin.1 The pericellular matrix @@@@ FN- -(@tin â€” â€”â€˜1002 in these epithelial cells resembles the matrix produced by @, cultured endothelial cells which deposit fibronectin underneath @@ 100- $ -@2L'I â€˜68x the cell layer (3). Endothelial cells, however, do not contain 68- keratin (27). C.) It has been reported that viral transformation of fibroblastic 43- . . â€” â€” chicken cells brings about a decrease in their collagen produc @@ .43 -fo@'med tion (11, 18, 28, 37, 39). Unlike the situation in the virus 30-transformed fibroblastic cells, the collagen production of nei theâ€˜30 ther MSV- nor MuLV-transformed cells was decreased. On after transformation,the cells began to produce Â© slightly enhanced amounts of type I procollagen sooner. Some type IV collagen was also produced by the MuLV-transformed Fig. 7. Surface carbohydrate labeling and glycoprotein polypeptide analysis ofcollagenlabeled mouse epithelial cells and their viral transformants. Confluent cultures werecontrary,cells. This might be due to a general stimulation of either using the periodate:NaB3H4method (A) or the galactose oxidase and not because of an induction of type IV collagen NaB3H4method(B).A, autofluorogramof5 to 16%gradientpolyacrylamidegel. Left,molecularweightmarkers(x 10@).FN,fibronectin.a1(l)anda2(l),colla synthesis by transformation. It is tempting to speculate that the genase-sensitivecellsMW. processed chains of type I collagen. Arrows, PAW.95,000 andsynthesisincrease in collagen synthesis in the MuLV-transformed 68.000proteinsseeninvirus-transformedcells.B,autofluorogramofan due to the presence of presumably several provirus copies 8% gel. Lane 1, MMC-E cells; Lane 2, PASV-transformedcells; Lane 3, MuLV transformed cells; M, marker proteins myosin, phosphorylase b, bovine serum including viral promoter regions in these cells. It has been stimulatepolypeptidealbumin, and carbonic anhydrase. Arrowheads, prominent alterations in theis shown in other cases that these promoters may patterns of the transformed cell clones (see text), Lane 2; M.W. of flanking cellular genes in certain cells (15, 30, 115,000 protein, Lane 3; MW. I 60,000 protein (top) and MW. 68,000 protein (bottom).transcription 31). In addition to the enhanced production of collagenous pro In the MSV-transformed cells, a glycoprotein with a molecularteins, the virus-transformed epithelial cells also produced en weight of 115,000 was found with a concomitant decrease ofhanced amounts of fibronectin into their media. Virus-trans a(14,respond M.W. 130,000 protein (Fig. 7B, Lane 2). These bands cor formed fibroblastic cells are known to produce fibronectin nontransformedcellto the migration of the collagen type I chains in these44), although in some cases less than their infectedcultures. The glycosylation of collagen may thus be af counterparts (44). Epithelial cells also produce fibronectin theMW.by transformation. In the MuLV-transformed cells, aculture (2, 33, 40). In the nontransformed MMC-E cells, aftercrease160,000proteinwasobservedwitha concomitantde production of fibronectin was low, but it was enhanced onis of a M.W. 175,000 protein (Fig. 7B, Lane 3). Its identitytransformation as shown in the present study. Studies thatobservednot known. A novel polypeptide (M.W. 68,000) was alsometastatic human carcinoma cell lines in culture showed (Fig. 7B, Lane 3), probably corresponding to thethey deposited only low levels of fibronectin into their pericel polypeptide found using the periodate labeling method (seelular matrices, whereas cell lines derived from primary carci above).nomasusedDISCUSSIONin had bundles of matrix fibronectin (40). The cells the presentstudy seemto correspondbetter to the latter cases.Mouse situation, since pericellular matrix was found in all embryo epithelial cells, MMC-E, may prove a useful of the glycoproteins produced by cell lines estab model in studies involved in the malignant transformation of Iish&I from tumors has suggested that identification of certain epithelial cells. MMC-E cells have been classified as epithelial matrix glycoproteins may serve as tools in the histopathological by their morphology and several other criteria (34). Recently, classification of tumors (1). Mesenchymal and epithelial cells we have found that they also contain keratin-type intermediate have characteristic profiles of glycoproteins, and analysis of filaments typical of epithelial cells (22). Transformation of these human solid tumors has indicated that the distribution of fibro cells by MSV (34) or by polycyclic aromatic hydrocarbons (29) nectin in sarcomas differs from that in carcinomas (41). Trans resulted in cell colonies capable of inducing poorly differen formation is a complex phenomenon which may affect several tiated carcinomas in nude mice. steps in the metabolism of cells. Understanding of the molec The cells possess a large number of cell surface receptors ular events and their disturbances involved in the formation of for epidermal growth factor. The treatment of MMC-E with the pericellular matrix would expand our knowledge of the role epidermal growth factor induced membrane changes in asso of the matrix for different types of cells. The ability of virus ciation with rapid changes in the distribution of their keratin transformed epithelial cells to deposit pericellular matrix gly filaments (21 , 22). Sarcoma growth factors produced by MSV coproteins is a major difference between them and the virus transformedculture.MMC-E fibroblastic cells (6) can reversibly transformAnalysistransformed fibroblastic cells in cells, suggesting that the transformation of both fibro blastic and epithelial cells may involve similar mechanisms J. Keski-Oja, V-P. Lehto, T. Vartio, R. A. Badley, and I. Virtanen. PAicrofila (20).7cells,Using ments and intermediate filaments in virus-transformed mouse epithelial nontransformed, MuLV-transformed, and MSV-trans submitted for publication.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research. PeriCe!Iular Matrix of Transformed Epithe!ial Ce!!s ACKNOWLEDGMENTS 22. Keski-Oja,J., Lehto,V-P., and Virtanen,I. Keratinfilamentsof cultured mouse epithelial cells are rapidly affected by epidermal growth factor. J. We thankOre.A. Vahenand U. R. Rappfor discussions,T.Jelvefor fine Cell Biol., 90: 537â€”541,1981. assistance, and P. Arminen for secretarial work. 23. Knowles, PA.A., and Franks, L. PA.Stages in neoplastic transformation of adult epithelial cells by 7,12-dimethylbenz(a)-anthracene in vitro. Cancer Res., 37: 3917â€”3924,1977. 24. Laemmli, U. K. Cleavage of structural proteins during the assembly of the REFERENCES head of bacteriophage T4. Nature (Lond.), 227: 680â€”685,1970. 25. Lasfargues, E. Y., and Moore, D. H. A method for the continuous cultivation 1. Alitalo, K., Keski-Oja, J., and Vaherl, A. Extracellular matrix proteins char of mammary epithelium. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1982 American Association for Cancer Research. Pericellular Matrix and Cell Surface Glycoproteins of Virustransformed Mouse Epithelial Cells

Jorma Keski-Oja, Carl G. Gahmberg and Kari Alitalo

Cancer Res 1982;42:1147-1153.

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