[CANCER RESEARCH 49, 2928-2934, June 1, 1989]

Differential Induction by Interferons of Major Histocompatibility Complex-encodedand Non-Major Histocompatibility Complex-encoded Antigens in HumanBreast and Ovarian Carcinoma Cell Lines1

Cinda M. Boyer,2 Deborah V. Dawson, Sharon E. Neal, Lisa F. Winchell, David S. Leslie, David Ring, and

Robert C. Bast, Jr.Departments of Medicine [C. M. B., S. E. N., L. F. W., D. S. L., R. C. B.], Community and Family Medicine and Biometry [D. V. D.], and Microbiology and Immunology[R. C. B.¡,Duke University Medical Center, Durham, North Carolina 27710 and The Cetus Corporation [D. RJ, Emeryville, California 94608

ABSTRACT

Treatment of cancer cells with interferons can modulate expression ofcell surface antigens, particularly those of the major histocompatibilitycomplex (Ml 1C). To examine the effect of recombinant 7- and «-interféronson expression of non-MHC antigens, murine monoclonal antibodies have been used to quantitate 14 distinct tumor-associated cell surfaceantigens from five breast cancer cell lines and five ovarian cancer celllines using a live cell radioimmunoassay. Both Class I and Class II MHCantigens could be augmented or induced with •y-interferon.Significantly

increased expression of MHC antigens was observed in nine of 10 celllines with induction indices as high as 11-fold. When 17 non-MHCepitopes were measured on 10 cell lines, minimal (1.3-2.7-fold) inductionwas observed in 10 of the 170 instances evaluated. Expression of onlytwo epitopes, 2G3 and 735B11, was increased on more than one cell Une.On six cell lines expression of non-MHC epitopes could not be increased.Consequently, among many different cell surface determinants, interferons produced a highly selective augmentation or induction of MHCantigens, whereas augmentation or induction of other tumor-associatedantigens was apparently restricted to a few epitopes.

INTRODUCTION

Interferons modulate expression of MHC3 antigens at thesurface of both normal and malignant cells (1-6). MHC antigenexpression in human cells is regulated by interferon at the levelof gene transcription (7). Recent data indicate that modulationof antigen expression by a- and 7-interferons may not be limitedto MHC antigens, but can also extend to additional tumor-associated cell surface antigens expressed by melanomas orcarcinomas (8-11).

Monoclonal antibodies reactive with a variety of independently expressed and chemically distinct tumor-associated antigens have been raised against breast carcinomas (12). Many ofthese epitopes are also expressed by ovarian cancers. Conversely, some epitopes expressed primarily by ovarian carcinomas, such as CA 125, can also be found on a fraction of breastcancers. Using monoclonal reagents, at least 14 different cellsurface antigens have been detected in breast and ovarian neoplasms. Given probes which recognize a substantial number ofdistinct antigens, it has been possible to ask whether interferonsproduce a selective induction of MHC antigens or a moregeneral modulation of many different antigen families. Coordinate changes in expression among apparently distinct antigens might point to mechanisms by which interferons affectgene expression. If expression of other tumor-associated surface

Received 5/30/88; revised 11/3/88, 2/17/89; accepted 2/28/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Funded in part by Grant 5-RO1CA39930.7To whom requests for reprints should be addressed, at Box 3843, Duke

University Medical Center, Durham, North Carolina 27710.3The abbreviations used are: MHC, major histocompatibility complex; BSA,

bovine serum albumin; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid; PBS, phosphate buffered saline; RMFI, relative mean fluorescence intensity;MFI, mean fluorescence intensity; CEA, carcinoembryonic antigen.

antigens can be enhanced by an interferon, treatment withinterferon in vivo might facilitate serotherapy using monoclonalantibodies and their conjugates with drugs, radionuclides, andtoxins.

MATERIALS AND METHODS

Cell Lines. The CAM Al (13), SKBR3 (14), BT483 (15), and MCF7(16) breast carcinoma cell lines were obtained from Cetus Corporation(Emeryville, CA). The BT20 breast cancer cell line was obtained fromDr. Arthur Bogden (17). Five ovarian carcinoma cell lines were developed from ascites obtained from ovarian carcinoma patients. Four(OVCA 420, OCVA 429, OVCA 432, OVCA 433) were established incollaboration with Dr. Herbert Lazarus (18) and one (DOV 13) wasderived in our laboratory at Duke.4 Culture media were obtained from

Hazelton Research Products (Lanexa, KA). Culture conditions wereoptimized for each cell line used. Cells were cultured in medium RPMI1640 (SKBR3, BT483, and BT20), Dulbecco's modified minimal essential medium (CAMA 1and MCF7) or Eagle's minimal essential medium(ovarian cell lines) supplemented with 5-15% fetal bovine serum(GIBCO Laboratories, Inc., Grand Island, NY) and glutamine (Hazel-ton). Media for the ovarian cancer cell lines were also supplementedwith nonessential amino acids, sodium pyruvate. penicillin, and streptomycin (Hazelton). Insulin (Sigma Chemical Co., St. Louis, MO) wasadded to the medium for three cell lines (BT20, MCF7, and DOV 13).Each cell line was tested bimonthly for mycoplasma and remained freefrom contamination (18).

Antibodies (Table 1). Hybridomas producing monoclonal antibodiesreactive with human Class I MHC (W6/32) (20) or Class II MHC(L243) (21) antigens were obtained from the American Type CultureCollection. Hybridoma tissue culture supernatant* were collected andused undiluted (L243) or at a 1:5 dilution (W6/32) in radioimmuno-assays. Monoclonal antibodies reactive with non-MHC tumor-associated antigens included 10 purified antibodies obtained from CetusCorporation (Emeryville, CA) (11); 19-9 (22), B72.3 (23), and 5E91E9(24) were obtained from Centocor, Inc. (Malvern, PA); DU-PAN-2(25) was obtained from Dr. Richard Met/gar at Duke University; Mov8 (26) was obtained from Dr. Maria Colnaghi at Instituto Nazionaleper lo Studio e la Cura dei Tumori (Milan); 225 (27) from Dr. HideoMasui at Sloan Kettering Memorial Institute (New York, NY); andOC 125 (18) was generated in our laboratory. An ascites preparation ofA2C6, an IgGl antihepatitis B monoclonal antibody obtained fromCentocor, was used as a negative control. Ascites preparations ofantibodies were diluted 1:1000 in radioimmunoassays and 1:100 inindirect immunofluorescence assays. Purified antibodies were dilutedto 5 A'K/rnlin both radioimmunoassays and immunofluorescence assays.

Interferons. Recombinant 7-interferon was kindly provided by BiogenInc. (Cambridge, MA) and recombinant a-interferon consensus] sequence was obtained from Amgen (Thousand Oaks, CA). Lyophilizedinterferon preparations were reconstituted according to the manufacturer's instructions and stored in frozen aliquots at —70°C.

Live Cell Radioimmunoassay. The live cell radioimmunoassay wasmodified slightly from the method of Greiner (9). In brief, 2x10* cellswere plated in flat-bottomed RemovaCell microtiter plates (DynatechLaboratories, Inc., Alexandria, VA). Interferon was added in doses

4 Unpublished observation.

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Table 1 Tumor-associated cell surface antigens tested for Interferon induction

MonoclonalantibodyW6/32L2432G3OC125B72.3Mov8Du-PAN-219-9520C9113F15E91E9225454A129C633F8735B11260F9317G5140A7Antigenrecognized"Class

IHLAClassII HLA(DR)HMW

mucinglycoproteinHMWmucinglycoproteinHMWmucinglycoproteinHMWmucinglycoproteinHMWmucinglycoproteinHMWmucinglycoproteinM,

200,000proteinM,200,000/100,000760,000/40,000

glycoproteinM,

180,000glycoprotein(CEA)M,

175,000 EGFreceptorglycoproteinM,

95,000transferrinreceptorglycoproteinM,

70,000proteinM,66,000proteinM,61,000/65,000proteinA/r55,000glycoproteinM,42,000glycoproteinGlycolipid

tal interferon-treated cells compared to the mean fluorescence intensityof untreated control cells.

RMFI = experimental mean channel for fluorescence intensitycontrol mean channel for fluorescence intensity

" HMW, high molecular weight.

ranging from 0 to 1000 units/ml and plates were incubated at 37°Cin

an atmosphere of 5% CO2 and 95% humidified air. After 24 to 72 h,assay wells were washed once with 200 u\ of RPMI 1640 medium.Aliquots (200 n\) of RPMI 1640 supplemented with 10% BSA (Sigma),12.5 HIM HEPES buffer (Hazelton), and 0.08% sodium azide wereadded to each well and incubated for 1 h. Plates were washed once with200 ¿i'of the same medium. Fifty n\ of mouse monoclonal antibodydiluted in assay medium (RPMI 1640 medium supplemented with 1%BSA, 12.5 mM HEPES and 0.08% sodium azide) was added and plateswere incubated for 1 h at 37°C.Following three washes with assaymedium, 125I-labeled sheep antimouse F(ab')2 fragments (10s cpm)

(New England Nuclear, Boston, MA) were added in 50 /il of assaymedium and plates were incubated for 1 h at 37°C.Plates were washed

three times with assay medium and RemovaCell wells were separatedand counted for 1 min in a gamma counter (Auto-Gamma 5780;

Packard Instruments, Sterling, VA). The induction index is a ratio usedto describe changes in antibody binding following interferon treatmentof cells.

Induction index_ mean antibody binding (cpm) with optimal dose of interferon

mean antibody binding (cpm) without interferon

Indirect Immunofluorescence and Flow Cytometry. For indirect im-

munofluorescence tumor cell lines were trypsinized and cell suspensionswere washed once in cold assay buffer (PBS, pH 7.2, supplementedwith 1% fetal bovine serum and 0.02% sodium azide) and distributedinto microtubes at 5 x 10s cells/tube. Mouse monoclonal antibodies

were diluted in assay buffer and added in a 50-^1 volume. A mouse IgGpreparation (Coulter Immunology, Hialeah, FL) was used as a negativecontrol. Tubes were incubated for 30 min on ice followed by threewashes with assay buffer. Fifty n\ of fluorescein-conjugated goat anti-

mouse IgG (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD)diluted 1:50 in assay buffer was added and tubes were incubated for 30min on ice. Following three washes in assay buffer, the final cell pelletwas resuspended in PBS (500 ¡A)and 25 p\ of formaldehyde to fix cellsfor subsequent analysis. Cells were analyzed with an EPICS V flowcytometer (Coulter Electronics, Hialeah, FL). Fluorescent signals weregated on the basis of forward and right angle light scattering to eliminatedead cells and aggregates from analysis. Gated signals (IO4) were

analyzed on a linear scale and mean fluorescence intensity determinedusing the software of a Coulter MDADS. The index RMFI is a ratioused to describe the mean fluorescence intensity changes of experimen-

Cellular Proliferation Assay. Cellular proliferation was assayed bytritiated thymidine incorporation. Cells were plated at 2 x 104/well in

96-well flat-bottom plates (Costar, Cambridge, MA) and interferonswere added at doses ranging from 0 to 1000 units/ml. Plates wereincubated at 37°Cin a 5% CO2 atmosphere for 72 h. During the final8 h of incubation plates were pulsed with 1 ¿iCi/well[3H]thymidine (6.7

Ci/mmol; New England Nuclear, Boston, MA). Wells were washedthree times with Hanks balanced salt solution (Hazelton) and 50 p\ of2 M NaOH was added to solubilize cells. Samples were absorbed intocellulose acetate harvesting elements (Skatron, Inc., Sterling, VA),dried, transferred to vials, covered with Betafluor (National Diagnostics, Somerville, NJ), and counted for 1 min in a scintillation counter(Tri-Carb 4640, Packard Instruments).

Statistics. Radioimmunoassays and proliferation were analyzed bythe Spearman rank correlation test (28) to evaluate the possibility ofan increasing dose-response relationship once optimal experimentalconditions had been established. Adjustment for multiple comparisonswas made by the Bonferroni method (29). For example, the predetermined overall level of significance (0.05) was divided by the number ofcomparisons made (e.g., 20 antibodies) to calculate the corrected levelof significance (0.05 divided by 20 = 0.0025). Descriptive levels ofsignificance (P values) less than 0.05 but greater than 0.0025 may beregarded as suggestive. The P values for each antibody for two or threereplicate experiments were combined (Table 3) according to the methodof Fisher (30). Since we were looking for a systematic increase inantigen expression which correlated with increasing interferon dose,combined P values derived from correlations which departed from thesecriteria were not considered meaningful. This approach is slightly moreconservative than the use of one-sided tests for the detection of increasing dose responses. For this analysis values for all concentrations ofinterferons are considered and precise correlation with the inductionindex at the optimal interferon concentration as calculated above is notnecessarily observed.

RESULTS

Tumor-associated Surface Antigen Expression of Breast andOvarian Carcinoma Cell Lines. MHC class I antigens wereexpressed by all of the breast and ovarian cancer cell linestested, but only two of the ovarian cell lines expressed Class II(DR) MHC antigen (Fig. 1). Several of the non-MHC tumor-associated antigens (Table 1) were expressed by all cell linesincluding M, 42,000 and 55,000 antigens, 95,000 transferrinreceptor, and the high molecular weight glycoprotein detectedby the 2G3 monoclonal antibody. The epitope on the highmolecular weight glycoprotein complex defined by OC 125 wasdetected on all five ovarian carcinoma cell lines but on only onebreast carcinoma cell line. The prevalence of different cellsurface antigens was otherwise remarkably similar betweenbreast and ovarian cancer cell lines, consistent with their derivation from hormonally responsive epithelial tissues (Fig. 1).Antigens were heterogeneously assorted among the cell linessuggesting an unlinked pattern of expression for these chemically distinct antigen families.

Effect of 7-Interferon on MHC and Non-MHC Tumor-associated Antigen Expression and Cellular Proliferation. To determine optimal conditions for interferon induction of surfaceantigens, cell lines were treated with 0, 10, 100, or 1000 units/ml -y-interferon for 24, 48, or 72 h and assayed for binding of

monoclonal antibodies to hepatitis B antigen (irrelevant control), Class I and Class II MHC antigens. As early as 24 h aftertreatment of SKBR3 breast cancer cells with 1000 units/ml 7-

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BreastCell Line

Ovarian

I

M «S "i

Class I MHCClass II MHC

mucin

mucin

mucin

200 Kd

200/100/60/40 Kd

180 Kd175 Kd

95 Kd

72 Kd

66 Kd

61/65 Kd

55 Kd

42 Kd

glycolipid

Fig. 1. Phenotype of breast and ovarian cancer cell lines by live cell radio-immunoassay. Cell lines were considered positive if antibody binding exceededthe mean binding for the negative control antibody (A2C6) by > two standarddeviations. •,antigen-positive; D, antigen-negative.

H

hU6

i

3000 -

2000

1000 -

3000

2000 -

1000

1.243

e 3000 -

2000 -

1000 -

A 24 hi

B 48 hr

72 hr

Interferon ( units/ml )Fig. 2. Induction of MHC antigens on SKBR3 breast cancer cell line with y-

¡nterferon.Duplicate wells of SKBR3 cells were treated with O P), 10 (•).100(Sí),or 1000 (:;:'•)units/m 7-interferon for 24 (A), 48 (B), or 72 (C) h. Resultsare expressed as mean cpm ±SD. P values were determined by Spearman's rankcorrelation test. *, significant at the overall 0.05 level after adjustment for multiplecomparisons.

interferon, Class I MHC antigen expression increased 3.8-fold(induction index = 3.8) compared to the diluent controls (Fig.2A). Induction indices of 2.7 and 2.5 were observed with 100units/ml and 10 units/ml doses of interferon, respectively. Nosignificant induction of Class II antigens was observed at 24 h.After treatment for 48 h, however, induction indices of 2.0 and3.7 were observed at 100 units/ml and 1000 units/ml respectively (Fig. 2B). Both Class I and Class II MHC antigensincreased about 5-fold after treatment for 72 h with 1000 units/ml interferon (Fig. 2C). Each cell line was evaluated for MHCantigen expression in the absence of interferon (Table 2). Insome cases, induction of MHC antigen was observed (Table 3)where constitutive expression of antigen could not be detectedwithin the limits of live cell radioimmunoassay (Fig. 1, Table

2). When cells are assayed by this technique, however, it isdifficult to exclude the presence of a subpopulation which mightexpress low levels of MHC antigen in the absence of interferon.If MHC antigen is constitutively expressed in the absence ofinterferon, increases in expression of antigen in the presence ofinterferon may more accurately be referred to as augmentationrather than induction.

Optimal lengths of incubation with interferon were determined for each cell line in analogous experiments (data notshown). Maximal effects for most cell lines occurred aftertreatment for 72 h with interferon, with the exception of MCF7and DOV 13 where 48 h was optimal. Cell lines differed withregard to whether both Class I and II MHC antigens wereincreased following interferon treatment (Table 3). Both ClassI and II MHC antigens were significantly increased following7-interferon treatment of breast cancer cell lines with an averageof more than a twofold increase of both antigens on each cellline. Lower levels of MHC antigen induction were observedwith the ovarian cancer cell lines. Class I or II MHC antigenswere significantly increased in four of five ovarian cancer celllines. Only one cell line (OVCA 420) failed to show significantlyincreased expression of either Class I or II MHC antigens, andone cell line (DOV 13) had significantly increased expressionof Class I MHC antigen only.

After the optimal interval required for 7-interferon to induceMHC antigens was determined for each cell line, the effect ofinterferon was evaluated for 12 chemically distinct non-MHC

cell surface antigens ranging in size from MT42,000 to 300,000.In six of the 10 cell lines tested, 7-interferon did not affect theexpression of non-MHC antigens. Treatment of SKBR3,CAMA1, BT483, or BT20 cells with 7-interferon for 72 hincreased expression of selected non-MHC antigens (Table 4).A representative experiment with SKBR3 cells is shown in Fig.3. When results were combined from replicate experiments, the2G3 epitope on high molecular weight mucin was the onlyepitope which was significantly increased on three cell lines(Table 4). The M, 61,000/65,000 protein recognized by 735B11antibody was induced on two cell lines. CEA was induced onlyon the SKBR3 cell line. Epitopes recognized by OC 125,113F1,317G5, and 140A7 antibodies were induced only on the BT20cell line. In summary of the 17 non-MHC epitopes evaluatedon 10 cell lines, induction or augmentation was observed inonly 10 of 170 possible instances.

Since the live cell radioimmunoassay measures antigenexpression on a per well basis rather than a per cell basis, itwas important to determine whether there were comparablenumbers of cells in interferon-treated wells and untreated wells.Cohort cultures of SKBR3 cells assayed for antigen expressionwere pulsed with [3H]thymidine to assess cellular proliferation.Although interferon induction of antigen expression was max-

Table 2 Expression of MHC antigens on breast and ovarian cancer cell linesAntibody binding"

CelllineSKBR3CAM

AlMCF7BT483BT20OVCA

420OCVA429OVCA432OVCA433DOV

13A2C6955

±12*375

±152392±35997±327376±63875±407417

±64360±178963±190138±55W6/322,260

±1781,209±341,380

±351,350±921,560±1084,445±3093,903±872,188±9411,

934±4401,391±158L243980

±120403±66450±88582±185474±263975±4352,463±80440±471,400±141234±40

°Live cell radioimmunoassay.* Mean cpm ±SD of triplicate wells.

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Table 3 y-interferon induction of Class 1 and IIMHC antigens on breast and ovarian carcinoma cell linesMean induction index0

Class I MHC Class II MHC

Rank correlation6 Combined P value'

CelllineBreastSKBR3CAMA1MCF7BT483BT20OvarianOVCA

420OVCA429OVCA432OCVA433DOV

13No.

of experiments3322322322Mean4.02.82.63.83.51.61.51.71.21.2(Range)(3.6-4.5)(2.4-3.2)(2.1,3.0)(3.0,4.6)(2.0-5.1)(1.4,1.7)(1.4,1.6)(1.5-1.9)(1.2,1.2)(1.2,1.2)Mean4.56.94.46.34.41.22.55.71.72.2(Range)(1.6-8.5)(3.2-10.9)(3.4,5.3)(5.3,7.3)(3.0-6.3)(0.7,1.7)(2.0,2.9)(1.9-11.1)(1.0,2.4)(0.9,3.5)Class

Imean0.82730.90700.76650.97200.82730.36700.73400.63330.12950.7725Class

IImean0.89270.85670.70200.97200.9140-0.66450.93950.93600.51850.2915Class

Imean«cO.OOOl<0.00010.0001<0.0001<0.0001NS"0.00010.0001NS0.0001Class

IImean<0.0001•CO.OOOl0.0002«cO.OOOl<0.0001NS«cO.OOOl<0.00010.0009NS

" Induction index was calculated as described in "Materials and Methods."b Rank correlations were determined by the method of Spearman as described in "Materials and Methods."c The P values for the replicates of each result were combined by the method of Fisher as described in "Materials and Methods.'d NS, not significant.

Table 4 Summary ofy-interferon induction of tumor-associated antigens AJCt Wt/U LUJ ICI OCUf tnj Ho.. DU-MN-1

CelllineSKBR3CAM

AlBT483BT20IrNo.

of experiments Antibodyty3323auction

index"can2G3

1.55E91E92.72G3735B112G3OC125113F1735B11.3.3.7.7.7.5317G5

1.8140A72.2(Range)(1.3-1.8)(1.5-3.9)(1.2-1.5)(1.0,1.6)(1.2-2.0)(1.7-1.9)(1.2-2.0)(1.0-2.3)(1.3-2.2)(1.0-3.0)Rank

correlation*

mean0.65530.58300.64070.47500.66200.71970.67670.41770.51100.6190Combined Pvalue'0.00020.00130.00070.00090.00010.00010.00020.00020.0009<0.0001

" Induction index was calculated as described in "Materials and Methods."* Rank correlations were determined by the method of Spearman as described

in "Materials and Methods."c The P value for the replicates of each result were combined by the method of

Fisher as described in "Materials and Methods."

imal at 100 or 1000 units/ml, [3H]thymidine incorporated

varied less than twofold at interferon doses of 0, 10, 100, or1000 units/ml (not significant; Spearman rank correlation)(Fig. 4). In separate experiments, cell counts of SKBR3 after72 h of 1,000 units/ml 7-interferon were 50% lower than inuntreated cultures (data not shown). A 50% decrease in cellnumber would result in an underestimate of induction of cellsurface antigens by interferon in the live cell radioimmunoassay.It seemed unlikely that increased antigen expression could beaccounted for by increased numbers of cells in wells exposed tointerferon.

Increased Density of Antigen Expression in SKBR3 Cells afterTreatment with 7-Interferon. Increased antigen expression couldbe due to an increased fraction of antigen positive cells, anincreased antigen density per cell, or both. To distinguishbetween these possibilities, binding of different monoclonalantibodies to SKBR3 cells was assayed by indirect immunoflu-orescence and analyzed by flow cytometry following treatmentwith 7-interferon or diluent. Both the percentage of positivecells and their MFI were determined (Fig. 5). RMFI wascalculated as described in "Materials and Methods" to compare

the MFI of untreated and 7-interferon-treated cells stained withthe same monoclonal antibody. Both the percentage and MFIof cells expressing MHC antigens increased after treatmentwith 7-interferon. MHC Class I antigen-positive cells increasedfrom 74 to 98%. MHC Class II antigen-positive cells increasedto 71% from an undetectable level (7%) which was equivalentto background staining with the mouse IgG negative control

5200» 113F1 3E91E9 US 454A11 fCt UFI 733B11 »Of« JÕ7C5 140A7

Gamma Interferon ( anils/ml )Fig. 3. Induction of tumor-associated antigens on SKBR1 breast cancer cell

line with >-interferon. Triplicate wells of SKBR3 cells were treated with 0 CO),10 (•).100 (iifl or 1000 (•'•:'••)units/ml i-interferon for 72 h. Results are expressedas mean cpm ±SD. /' values were determined by Spearman's rank correlationtest. P values for W6/32, L243, 2G3, and DU-PAN-2 antibodies were significantat the overall 0.05 level after adjustment for multiple comparisons.

(9%). RMFI for Class I and Class II MHC antigens were 7.1(Fig. 5, A and B) and 6.7 (Fig. 5, C and D), respectively,indicating a 7-fold increase in the average antigen density ofMHC antigen after 7-interferon treatment. Although the per

centage of cells expressing the 2G3 determinant was similarbefore (86%) and after (79%) interferon treatment the MFI ofthe 2G3 determinant on interferon-treated cells was approximately double that for untreated cells (Fig. 5, E and F; RMFI =1.8). The expression of theMr 200,000 glycoprotein recognizedby 520C9 antibody remained unchanged after 7-interferontreatment. In addition, no size difference could be detected byforward angle light scatter when untreated SKBR3 cells werecompared to interferon treated SKBR3 cells. These data indi-

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10 100

Gamma Interferon ( units/ml )

1000

Fig. 4. ['HjThymidine incorporation of untreated and -y-interferon-treated

SKBR3 cells. Triplicate wells of SKBR3 cells were treated with 0, 10, 100, or1000 units/ml -y-interferon for (•)72 h. Wells were pulsed with 1 ¿iCi[3H]thymidine for 8 h. Results are expressed as average cpm ±SD.

UNTREATED GAMMA INTERFERONTREATED

A W6/32MFI=28

C L243MFI =3

DL243MFI=20

E 2G3MFI=32

O 520C9MFI=140

H520C9MFI =I34

64 128 192 256 64 128 192RELATIVE FLUORESCENCE INTENSITY

296

Fig. 5. Increased density of antigen expression on 7-interferon treated SKBR3cells. SKBR3 cells were treated with 1000 units/ml i-interferon for 24 h, stainedwith monoclonal antibodies by indirect immunofluorescence and analyzed by flowcytometry. Linear fluorescence intensity histograms illustrate binding of monoclonal antibodies: W6/32 to untreated and treated cells (A, B), L243 to untreatedand treated cells (C, D), 2G3 to untreated and treated cells (£,f), and 520C9 tountreated and treated cells (G, H). MFI, mean fluorescence intensity (expressedas channel number).

cate that interferon exposure increased both the number of cellsexpressing MHC antigen and the average density of MHCantigens. Since the percentage of 2G3-positive cells was similarwith or without interferon exposure, the increase in 2G3 expression was primarily due to increased antigen density per cell.

Comparison of a- and 7-Interferons for Induction of AntigenExpression. Since a-interferon has been reported to increaseexpression of tumor-associated antigens, we compared the effects of a- and 7-interferon treatment on antigen expression bySKBR3 cells. Treatment with 7-interferon significantly increased Class I MHC antigens (induction index = 5.8) and the2G3 determinant on HMW mucin (induction index = 1.9; Fig.6A). Only Class I MHC antigen (induction index = 4.4; Fig.6B) was significantly increased after treatment with a-inter-feron. The induction of Class II MHC antigens by 7-interferon

did not retain significance after correction for multiple comparisons (Fig. 6A). In separate experiments, a-interferon failed

to affect expression of CEA detected by 5E91E9 antibody.

QM+1

5uB

A GammaA2C6 W6/32 L243 2G3

p = 0.0001* p = 0.0373 p =0.0001*S20C9

B Alpha

p = 0.0001*

r»n l0000 0000

WOO WOO»HO WO

0 0 O OWOO

wo

OOOO 0 O 0 OWOO WOO

wo wo

Interferon ( units/ml )Fig. 6. Comparison of consensusi sequence a- and -y-interferons for induction

of tumor-associated antigen expression. Triolicate wells of SKBR3 cells weretreated with 0 P), 10 (•),100 pi ) or 1000 (: :•)units/ml 7- (A) or consensus,sequence o-interferon (B) for 48 h. Results are expressed as mean CPM ±SD. Pvalues were determined by Spearman's rank correlation test. *, significant at the

overall 0.05 level after adjustment for multiple comparisons. Other quoted Pvalues may be regarded as suggestive.

DISCUSSION

We have evaluated the ability of recombinant y- and a-interferons to modulate expression of a large number of chemically distinct tumor-associated cell surface antigens. The effectof the interferons on surface antigen expression was remarkablyselective for MHC antigens. When 17 epitopes on 12 non-MHC antigens were studied on 10 cell lines, only four breastcancer cell lines exhibited a modest increase in the expressionof seven determinants judged by whole cell radioimmunoassay.Six cell lines showed no induction or augmentation of non-MHC epitopes. The increase in antibody binding could not beaccounted for by increased numbers of cells in interferon treatedwells. Using flow cytometry, the density of 2G3 antigen but notthe number of antigen bearing cells increased after interferontreatment. In contrast MHC Class I or II antigens were augmented or induced on nine of 10 breast and ovarian cancer celllines.

Optimal induction or augmentation of MHC Class I and IIantigens was obtained after 48 or 72 h of treatment with 7-interferon. Both Class I and Class II MHC antigens wereincreased on breast carcinoma cell lines. 7-Interferon increasedMHC antigens on four of five ovarian cancer cell lines, butproduced smaller increases than were observed with the breastcancer cell lines. The reason for this apparent difference between tumor types is not known. As a group, however, theovarian cancer cell lines tended to have a higher initial level ofClass I MHC antigen than did the breast cancer cell lines.Apparent induction of Class II MHC antigen was observed forsome cancer cell lines since constitutive levels were not detectable in the live cell radioimmunoassay. This may reflect thethreshold of detection in our assay. Since we cannot rule outthe existence of positive subpopulations of cells, our resultscannot definitively distinguish between Je novo induction ofantigen expression versus augmentation of preexisting antigenexpression.

Induction of MHC antigen by 7-interferon is known to beaccompanied by induction of MHC antigen-specific messenger

RNA (7). Selective induction of MHC antigens could reflectdifferent regulatory pathways for synthesis of MHC and non-MHC cell surface antigens. Although expression of the 2G3

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and OC 125 epitopes were increased following 7-interferontreatment of SKBR3, CAMA1, or BT20 cells, the amounts ofother determinants found on high molecular weight mucinswere unchanged. Mucin determinants recognized by monoclonal antibodies are often carbohydrate or a combination ofpeptide and carbohydrate (31, 32) rather than purely peptidedeterminants. Different mucin determinants tested may becoexpressed on the same molecules (33). The selective augmentation or induction of only two among the several mucin epitopes evaluated raises the possibility that interferon might affectglycosylation rather than transcription of the mucins.

We were unable to confirm reports by Greiner et al. (9, 11)that interferon augments expression of two non-MHC antigensassociated with MCF7 cells, determinants recognized by theB72.3 and anti-CEA antibodies. Such differences may be dueto the fact that the MCF7 cell line employed by Greiner et al.in their studies (9,11) and the MCF7 cell line used in our studywere from different sources. MCF7 cells from different sourcesmay vary in a number of antigenic and biological characteristics.The MCF7 cell line utilized by Greiner et al. (9, 11) expresseddeterminants recognized by B72.3 and anti-CEA monoclonalantibodies. Clones of MCF7 cells are known to vary in theirexpression of determinants recognized by anti-CEA and B72.3antibodies (34). Since our MCF7 cell line does not constitu-tively express the determinant recognized by B72.3 we couldnot test whether interferon augmented its expression. OurMCF7 cells did constitutively express the CEA antigen, however, in our study interferon did not augment its expression.Several factors may account for this discrepancy. Our MCF7cells may differ in their responsiveness to interferons, althoughMHC Class I antigen expression was augmented on MCF7cells in both our study and the studies of Greiner et al. Inaddition, we used a different anti-CEA monoclonal antibody inour study (5E91E9), whereas Greiner et al. used the B1.1 anti-CEA monoclonal antibody. In our studies, induction of CEAwas observed on SKBR3 cells and increases occurred in expression of the TAG72 antigen recognized by the B72.3 antibodybut these did not reach statistical significance. Our own dataindicate that cell lines vary in their responsiveness to inductionor augmentation of cell surface antigens by interferons. Although the 2G3 determinant was expressed by all 10 cell lines,7-interferon augmented expression only on the SKBR3,CAMA1, and BT20 cell lines. The type of interferon may alsohave been important. In previous reports, induction of theB72.3, B6.1, and B 1.1 determinants was obtained by Type Aor Type B a-interferon while several other types were unreactive(11). a-Interferon is a family of at least 13 different molecularspecies which may vary in as much as 20% of their amino acidsequence. In the present study we used recombinant aconsensus, sequence, which is an analogue designed as a consensus of the known human a-interferon subtypes based oncomparison of their primary structures. Our own data suggestthat the type of interferon is an important variable since wewere able to induce or augment MHC antigens with both a-and 7-interferons, but the 2G3 determinant was only augmented with 7-interferon. A recent study reported two- tothreefold increases in TAG72 and CEA antigens following ai-or 7-interferon treatment of carcinoma cells derived from effusions (35). Since our results were obtained with cell lines, wecannot rule out the possibility that antigen levels of primaryeffusion cells might be more responsive to interferons.

Augmentation of tumor-associated antigens with interferonsis an appealing strategy for increasing the expression of antigenswhich might be used as targets for diagnosis or therapy of

tumors with monoclonal antibodies. Increased antigen expression could improve the ability of monoclonal antibodies todetect or destroy tumor cells. This strategy would be mosthelpful if a wide variety of antigens could be augmented and ifmost tumor cells were susceptible to modulation. Aside fromMHC antigens, however, only a limited set of cell surfaceepitopes are increased following interferon exposure. In addition, since cell lines vary in their responsiveness to interferon's

ability to augment non-MHC antigens, it is possible that tumorsfrom different patients or cells within a single tumor might alsovary in responsiveness to interferon. Alternative approachessuch as exposure to hormones, growth factors or differentiatingagents might modulate a larger set of cell surface tumor associated antigens which could serve as targets for diagnosis ortherapy.

ACKNOWLEDGMENTS

The authors thank Ms. N. Holmes for outstanding assistance inmanuscript preparation.

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1989;49:2928-2934. Cancer Res   Cinda M. Boyer, Deborah V. Dawson, Sharon E. Neal, et al.   Carcinoma Cell LinesComplex-encoded Antigens in Human Breast and OvarianComplex-encoded and Non-Major Histocompatibility Differential Induction by Interferons of Major Histocompatibility

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