Monoclonal Antibodies Against Native and Denatured Forms of Estrogen-Induced Breast Cancer Protein (BCEI/Ps2) Obtained by Expression in Escherã¬Chã¬Acoli1

(CANCER RESEARCH 50. 2390-2396, April 15, 1990] Monoclonal Antibodies against Native and Denatured Forms of Estrogen-induced Breast Cancer Protein (BCEI/pS2) Obtained by Expression in EscherÃ¬chÃ¬acoli1

Jean-FranÃ§ois Prud'homme, AndrÃ©Jolivet, Marie-France PichÃ³n,Jean-FranÃ§ois Savouret, and Edwin Milgrom2

Croupe de Recherches sur la Biochimie Endocrinienne et la Reproduction (INSERM U. 135), FacultÃ©deMÃ©decineParis-Sud, 94275 Le Kremlin-BicÃ©treCedex, France

ABSTRACT protein in tissue extracts, plasma, and urine and to analyze tissue biopsies by immunocytochemistry. Moreover, immu Several vectors Â»ereused to express the complementary DNA for breast cancer estrogen-induced protein BCEI (also called pS..) in Esche noaffinity chromatography should allow the purification of rÃ¬chÃ¬acoli.The best results were obtained by using the pUR 290 large amounts of the protein enabling the study of its function expression vector after deletion of the sequence encoding the signal and its possible growth factor properties. We report here the peptide of the protein. In these conditions, /3-galactosidase-BCEI/pS? expression of the cDNA in Escherichia coli and the develop fusion protein accounted for - 2(1'I of total proteins in bacterial extracts. ment from the recombinant protein of polyclonal and monoclo It was purified by Chromatograph) on DEAE-Trisacryl or by gel electro- nal antibodies. phoresis and electroelution. Polyclonal antibodies were obtained by immunization of rabbits and goats, and monoclonal antibodies were raised in mice. Two types of monoclonal antibodies were obtained: one class recognized the native MATERIALS AND METHODS protein and was very efficient for the immunoprecipitation and immuno- purification of the protein from breast cancer cells; a second class Cell Culture. MCF-7, ZR-75-1, T-47D, and SK-Br-3 cell lines (14, recognized the denatured protein and was especially effective for immu- 15) were maintained in Dulbecco's modified Eagle's medium or RPMI noblot studies. 1640 (Gibco, Madison, WI) supplemented with 10% FCS (Gibco) and M( I I |)S could be detected by immunocytochemistry in breast cancer 0.6 Mg/ml insulin. Twenty-four h after plating flasks (Nunc, Roskilde, biopsies using monoclonal antibodies on frozen or paraffin-embedded Denmark), the cells were refed for 48 h with fresh medium containing sections. One of the antibodies (mBCEIn) exhibited high affinity for the phenol red, insulin, and 3% charcoal-stripped fetal calf serum as de protein and could be used at 1.9 Mg/mlconcentration for immunolabeling scribed previously (7). Estradici (5 HM; Roussel-Uclaf, Romainville, of histolÃ³gica)sections. France) or tamoxifen (1 ^M; ICI, Macclesfield, England) were added to The mBCEIii antibody was used in immunoaffinity chromatography the medium. to purify the peptide in a single step from culture media of estrogen- In serum-free cultures, the medium was progressively (48 h) ex treated MCF-7 cells. changed for HBCa medium (Institut Jacques Boy, Reims, France) (16). It was then collected every 48 h and stored after centrifugation at â€”¿20"C INTRODUCTION until used. Expression of BCEI/pS2 cDNA in E. coli. The cDNA containing the Human breast cancer is a hormone-dependent disease (1). entire coding sequence of pre-BCEI/pS2 and a dG tail was excised by Extensive studies have been performed to understand the role Pstl from previously described clone A (7). The fragment was inserted of hormones in the occurrence, growth, and spread of this in the Pstl site of pUR 292 expression vector (17). A second construct cancer. The role of receptors for estrogen and progesterone has was obtained by excision with Ncol and Pstl of the insert and introduc tion into the corresponding sites of pKK 233-2 expression vector (18) been analyzed (2) and more recently hormonally regulated gene products have been characterized (3). By constructing a cDNA3 (Pharmacia, Uppsala, Sweden). A third construct involved deletion of the putative signal sequence of pre-BCEI/pS2 through Banl digestion library derived from mRNAs of MCF-7 breast cancer cells and (this deleted a fragment encoding the first 20 amino acids). The frag screening it with probes prepared from estrogen-stimulated and ment was blunted with Klenow polymerase as described (19) and nonstimulated cells, two groups independently isolated and introduced into the Pstl site of pUR 290 (the vector DNA was also sequenced clones corresponding to a 0.6-kilobase estrogen- blunted by Klenow polymerase to obtain the correct reading frame). induced mRNA (4, 5, 6, 7) (called either BCEI for breast cancer Plasmids were transfected in F'l 1 ree A strain for pUR 290-292 (17), estrogen-induced or pS2) (8). This mRNA was found to be RB 796 strain for pKK 233-2 (18), amplified (20), and identified by expressed in a subset of breast cancers (9) and in stomach (10) restriction enzyme analysis. but was absent in a large variety of other tissues including Purification of the Fusion Protein /3-Galactosidase-BCEI/pS?. Produc tion of hybrid protein (/3-galactosidase-BCEI/pSj) in Luria broth me normal breast tissue. The sequence of the protein deduced from dium was induced by 0.5 min isopropyl-/3-D-thiogalactopyranoside the cloned cDNA showed similarity with growth factors (11) (Boehringer Mannheim, Mannheim, Federal Republic of Germany) and immunological analogy with EGF has also been reported added during 5 h when the /J550of the culture had reached 0.5. The cell (12). The protein has a signal peptide which is cleaved upon paste was suspended in one-tenth of initial volume in 20 miviTris-HCl secretion (13). The availability of monoclonal antibodies should (pH 8), 150 irmi NaCl, 5 mM 0-mercaptoethanol, 1 mM MgCl2, and 1 allow one to establish immunoassay methods to measure the mM PMSF buffer. All subsequent steps were performed at 4'C. The cells were lysed by 6 successive 30-s sonifications. After centrifugation Received 8/8/89; revised 12/14/89. (20,000 x g for 30 min) the supernatant was precipitated by ammonium 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 in sulfate (40% saturation, pH 7) for 2 h. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The precipitate was redissolved in a minimal volume of 20 mM Tris- ' This work was supported by the Institut National de la SantÃ©et de la HCI-pH 7.4-50 mM NaCI-1 mM /J-mercaptoethanoI-1 mM MgCl2-l Recherche MÃ©dicale,thÃ©CentreNational de la Recherche Scientifique, thÃ©UnitÃ© d'Enseignement et de Recherches Kremlin-BicÃ©tre,thÃ©Association pour la Re mM PMSF and dialyzed against the same buffer or desalted by passing cherche sur le Cancer Transbio Company, and thÃ©Fondation pour la Recherche through a GF05 column (IBF, Villeneuve-la-Garenne, France). MÃ©dicaleFranÃ§aise. In some experiments the recombinant protein was purified by a 5% 2To whom requests for reprints should be addressed. polyacrylamide gel electrophoresis in denaturing conditions and dec 1The abbreviations used are: cDNA. complementary DNA; BCEI/pS2, breast cancer estrogen-induced protein; EGF, epidermal growth factor; FCS, fetal calf troeluted as described (21). scrum; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; In other experiments the supernatant was clarified by centrifugation ELISA, enzyme-linked immunosorbent assay; BSA, bovine serum albumin. and applied to a DEAE-Trisacryl column (IBF) and eluted by a linear 2390

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1990 American Association for Cancer Research. ESTROGEN-INDUCED BREAST CANCER PROTEIN gradient of 0.1-0.35 M NaCI. The main protein peak (0.15 M NaCl) IgG (Sigma) were added to the supernatant and precipitated for 2 h at 4Â°Cwithrabbit anti-mouse Â¡mmunoglobulins(Sigma). The precipitates was pooled and the protein was concentrated by precipitation with ammonium sulfate (50% saturation. pH 7). The precipitate was dis were washed twice with PBS-0.5% Nonidet P-40 (Sigma) and 0.1% BSA, dried, and resuspended in Laemmli's buffer (24). The samples solved in PBS and dialyzed against the same buffer. The resulting protein was subjected to a 5% polyacrylamide gel were submitted to a 15% acrylamide gel electrophoresis in denaturing electrophoresis and stained by Coomassie blue. The purity was esti conditions (24). Gels were fixed in formaldehyde according to the method of BÃ¼rket mated by scanning the gels. The concentration of the protein was al. (25) and then processed for fluorography with En!-Hance (New estimated by comparison with known amounts of /3-galactosidase (Boehringer) electrophoresed and stained in the same conditions. England Nuclear, Berkeley, CA). Polyclonal Antibodies. Three rabbits were given s.c. injections at Western Blot Experiments. These were performed according to the monthly intervals of 150 Â¿igofelectropurified fusion protein. The hybrid method of Van Eldik et al. (26) with two modifications: methanol was omitted from transfer medium and transfer was performed at 180 mA protein was emulsified with an equal volume (500 ti\) of complete Freund's adjuvant for the first injection and incomplete Freund's ad for 45 min instead of 30 min. Ami-BCEI/pS2 polyclonal and monoclo nal antibodies were used, respectively, at a 1/100 dilution and at a juvant for subsequent injections. Anti-BCEI/pS2 antibodies were de concentration of 5 Â¿ig/ml.Nitrocellulose paper (Bio-Rad, Richmond, tected by ELISA (described below) with a peroxidase-conjugated don CA), swine anti-rabbit IgG (Nordic, Tilberg, The Netherlands), rabbit key anti-rabbit antibody (Amersham, England) after saturating ;miÂ¡.; anti-mouse IgG (Sigma), IDA 10, a monoclonal antibody not related galactosidase antibodies by tf-galactosidase in large excess. to breast cancer proteins, and '"I-labeled protein A were used as Monoclonal Antibodies. Two mice were given injections of 500 Mgof described previously (23). Autoradiographies were performed on HS- DEAE-Trisacryl purified fusion protein with an equal volume (160 n\) 90 film (Orwo, German Democratic Republic) at -70Â°C. of Freund's adjuvant. The mice were given 4 s.c. injections over a period Immunocytochemistry of BCEI/pS2. Cryostat sections (5 Mm)of pri of 5 months. ELISA indicated the presence of anti-BCEI/pS2 antibodies mary breast cancers (stored in liquid nitrogen) were prepared and in the blood. Three days before fusion, the mouse with the highest titer mounted on poly-L-lysine-coated slides. Two sections (positive and of antibodies received a booster, by i.v. injection, of 500 Mgof the same control) were prepared for each tumor and fixed for 15 min at room antigen. Cell fusion was performed as described previously (22). Hy- temperature with 3.7% formalin in PBS, pH 7.4. bridomas supernatants were screened by ELISA. Microtiter plates Paraffin sections prepared after 18-24 h fixation in 3.7% formalin (Immulon, Nunc) were coated in 50 mM potassium phosphate buffer, were deparaffinized in xylene (3x5 min), absolute alcohol, and 95% pH 8.4, overnight at 4Â°Cwith one type of antigen per lane of wells. alcohol (2x5 min for each). Before immunolabeling the slides were The following antigens were used: 1 n\ of serum-free culture medium rehydrated for 2 x 5 min in PBS at room temperature. from estradiol-treated MCF-7 cells; 5 ng (~30 fmol/well) of electro- All incubations were performed at room temperature and separated purified and eluted /â€¢'.co//'-derived/3-galactosidase and the same amount by two 5-min PBS washes. The sections were incubated for l h with mBCEI,, (1.9 Mg/ml in PBS-1% BSA) or control serum (nonimmunized of electropurified and eluted E. coli derived fusion protein between ÃŸ- mouse serum diluted at 1.9 Mg/ml in PBS-1% BSA). Then, the sections galactosidase and BCEI/pS2. After blocking free binding sites by incu were successively incubated for 30 min with biotinylated anti-mouse bation with 1% BSA, 25-M>aliquots of each hybridoma culture super immunoglobulin (Amersham) diluted 1/200 in PBS-1% BSA and for natant were incubated in duplicate with the three antigens overnight at 4Â°C.After washing with PBS-0.1% Tween 20, horseradish peroxidase- 15 min with streptavidin-biotinylated horseradish peroxidase (Amer sham) diluted 1/100 in PBS-1% BSA. The sections were finally incu conjugated sheep anti-mouse IgG (Amersham) was added and incubated bated for 10 min with 0.05 M 3,3'-diaminobenzidine tetrahydrochloride for l h at room temperature and for 30 min at 4Â°C.After washing, o- (Sigma) in PBS containing 0.01% H2O2. After two washes in distilled phenylenediamine (2 mg/ml) and 0.06% H2O2 in 0.15 M phosphate water, the sections were weakly counterstained for 2 min with Harris' citrate buffer (pH 5.2) were added and incubated for 30 min. The hematoxylin (Ortho Diagnostics, New Brunshwick. NJ), diluted at 10% reaction was stopped by adding 3 N HCI and the absorbance was in distilled water, and rinsed twice in tap water. The slides were measured at 492 nm. The hybridomas were cloned by limiting dilution dehydrated in graded alcohol and xylene and mounted. using splenocytes as feeder layer. The isotype was determined by ELISA A control was performed with monoclonal antibody mBCEIH pre- with isotype specific rabbit antisera (Miles Scientific, Naperville, IL). saturated with the antigen. mBCEI,, (19 Mg/ml) was incubated for 6 h at room temperature and 80 h at 4Â°Cwith the hybrid protein ÃŸ- The development of ascites as well as the preparation and purification of immillinoli ilmlins have been described previously (23). galactosidase BCEI/pS2 (2 mg/ml) in PBS containing 1% BSA. The Cross-reactivity of antibodies with human EGF (Progen, Heidelberg, saturated antibody was diluted 10-fold before incubation with the Federal Republic of Germany), mouse EGF (Sigma Chemical Co., St. sections, and the immunocytochemical reaction was performed as Louis, MO), and human transforming growth factor Â«(Biomedicai above. Technologies, Stoughton, MA) was analyzed by ELISA (10 pmol/well). Immunoaffinity Purification of BCEI/pS2. Estradiol (5 nM) was added Cell Labeling. MCF-7 cells (10s) were plated in T25 flasks in RPMI to MCF-7 cells in culture. Two days later the culture medium was collected ( 150-800 ml) and chromatographed on 3 successive columns: containing 3% FCS, 0.6 Mg/ml insulin, and 5 n\i estradici. deactivated Affi-Gel 10 (Bio-Rad) (5 ml); nonrelated IDA-10 antibody On day 4, cells were washed and incubated for 2 h in RPMI lacking (23) coupled to Affi-Gel 10 (1 ml containing 15 mg of antibody); and cysteine, supplemented with 1% charcoal-stripped and dialyzed FCS, mBCEI i, antibody coupled to Affi-Gel 10 (1 ml containing 15 mg of plus insulin and estradiol. Labeling was then performed for 9 h in the presence of 50 nCi/ml of ["Sjcysteine (specific activity, 1365 Ci/mmol; antibody). After the last column was disconnected, its content was washed with Amersham). The medium (1.5 ml) was collected by centrifugaron (5 the following buffers: phosphate-buffered saline (equal volume to that min at 1200 x g) and supplemented with 1 mM PMSF and 10 mM cold of culture medium); 10 mM Tris-0.3 M NaCl, pH 7.4; 10 mM Tris-I M cysteine. The cells were washed twice with cold PBS and centrifugea. NaCI, pH 7.4; 10 mM Tris, pH 9; 50 mM glycine, pH 6.2; 50 mM The pellet was resuspended in 200 n\ of 20 mM Tris (pH 7.4)-1.5 mivi glycine, pH 5 (in all cases, except for the first wash, 50 ml of buffer EDTA and lysed by five freeze-thaw cycles. Media and cell extracts were used). were stored at -20Â°C until used. To assess ("Sjcysteine incorporation Elution was performed with 50 mM glycine buffer, pH 2.2 (7 ml). into newly synthesized proteins. 10 n\ of medium or 1.5 M!of cellular Eluted fractions were rapidly neutralized with 1 M Tris, pH 9, or with extract were trichloroacetic acid precipitated. l M 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 8.3. Immunoprecipitation and Gel Electrophoresis. Culture medium from estrogen-treated MCF-7 cells (500 n\) was incubated with hybridoma supernatant (500 M')overnight at 4Â°Cin the presence of 1 ITIMPMSF. For 35S-Iabeled proteins, cell culture medium (100 M') or cell extract RESULTS (25 M') supplemented up to 100 n\ with PBS was incubated overnight Production of Recombinant Protein. Several prokaryotic vec at 4"C with 900 Â¿ilof hybridoma culture medium containing 1 mM tors were used encoding either /3-galactosidase fusion protein PMSF and 1 mM cold cysteine. After centrifugation, 2 Mgof mouse or the native protein. 2391

The pUR 292 expression vector contains a Tac promoter. The cDNA encoding the pre-BCEI/pS2 was inserted into the 66â€” Pstl cloning site yielding a fusion protein with ÃŸ-galactosidase. 45â€” Several clones were isolated. Upon induction by isopropyl-/3-D- 36â€” thiogalactopyranoside, it was observed by gel electrophoresis 29â€” that the hybrid protein accounted for about 1% of total protein 24â€” whereas a band of the size of d-galactosidase accounted for ~20% of total protein. Several bacterial strains were tested 20â€” (F'l 1 ree A, CMK 603, TGI) as well as different experimental conditions (medium, temperature, etc.) without improvement (data not shown). 14 â€”¿ We suspected that the signal peptide of pre-BCEI/pS2 was 6.5â€” recognized by the bacterial membranes thus leading to a pro- teolysis of the protein. A second construction was therefore prepared in which the signal peptide was deleted and the cDNA encoding BCEI/pS2 was inserted into pUR 290 vector. This markedly improved the yield of fusion protein which accounted for 20% of total protein. Excision of BCEI/pS2 seemed limited since the band corresponding to Â£?-galactosidaseon polyacryl- 1234 5678 amide gel electrophoresis accounted for only 2% of proteins Fig. 2. Immunoblot of BCEI/pS2 with rabbit polyclonal antibody. Lanes 1 and 5. cell culture medium taken prior to contact with cells. Lanes 2 and 6. (Fig. 1). medium taken from MCF-7 cells cultured in the absence of steroid (charcoal- Finally, we also tried to produce BCEI/pS2 without fusion to stripped serum), but in the presence of phenol red: Lanes 3 and 7. medium taken /3-galactosidase. The cDNA encoding pre-BCEI/pS2 was di from MCF-7 cells cultured in the presence of 5 nM estradici; Lanes 4 and A, medium taken from MCF-7 cells cultured in the presence of I ^M tamoxifen. In rectly inserted behind the Tac promoter of pKK 233-2 expres Lanes 1-4, rabbit polyclonal antiserum raised against the fusion protein (ÃŸ- sion vector. However, analysis of the proteins produced by galactosidase pre-BCEl/pS2) was used. In Lanes 5-8. rabbit preimmune serum was used. bacteria transformed with this construct did not show any protein of the size of BCEI/pS2 in either total or periplasmic 2.0i1.5-1.0-0.5-ngoloctosidoseÂ¿l\'/'/'/\t%^if/'.'tyYSAl 1 ÃŸâ€” space extract. The mammalian protein may have been unstable Â£ protein! Fugion when produced under these conditions. For immunization pro C mediu!ri//;/i^^Ã•^iv/^â€¢1EH MCF7 culture CM cedures, the protein was purified by two methods. Extracts OÃ• from cells transformed with the pUR 290-BCEI/pS2 plasmid DNA were chromatographed on DEAE-Trisacryl yielding a Ãœ fusion protein of about ~30% purity. Extracts from cells trans z OD formed with pUR 292-pre-BCEI/pS2 DNA were electropho- o: O (fi m n.-ra 205- C D Fig. 3. Characterization of monoclonal antibodies using ELISAs against var ious antigens. A mouse was immunized with ff-galaclosidase-BCEI/pS2 fusion protein (30C; pure) yielding different types of monoclonal antibodies reacting with antigens. I. typical result obtained with antM-galactosidase antibodies. B, typical pattern obtained with antibodies against native BCEI/pS2 (antibody mBCEI,,-IgGl). Similar results were obtained with mBCEI,2(IgGl), mBCEI,3 (IgG2a). mBCEInUgGl), and mBCEI,,(IgGI). C, typical pattern obtained with antibodies against denatured BCEI/pS2 (antibody mBCEIi-IgGl). Similar results were obtained with mBCEl:(IgGl). D, typical pattern obtained with antibodies nonrelated to breast cancer proteins (IDA 10-IgGI). The following antigens were used: /j-galactosidase purified through polyacrylamide gel electrophoresis in denaturing conditions (Lane I); pre-BCEI/pS2 prepared in the same conditions (Lane 2); culture medium from estrogen-treated 66â€” MCF-7 cells (Lane 3).

resed on polyacrylamide gels and the fusion protein was electroeluted. Preparation of Polyclonal Antibodies. Three rabbits were given injections of 150 ng of the /i-galactosidase-pre-BCEI/pS2 fusion protein purified by polyacrylamide gel electrophoresis from 1 2 3 bacterial extracts. After three injections, antibodies were de Fig. I. Synthesis of /3-galactosidase-BCEI/pS2 fusion protein in E. coli trans tected by ELISA and were further boosted by successive injec formed with recombinant pUR-290 vectors. Lane 1, size marker proteins: Lane tions of antigen. As expected, the antibodies recognized an 2, E. coli transformed with a nonrecombinant pUR-290 (encoding /i-galactosid- estrogen-stimulated M, ~6000 protein in MCF-7 cell culture ase); Lane 3, E. coli transformed with recombinant pUR-290 vector (encoding ÃŸ- galactosidase-BCEI/pS2 fusion protein). Tac promoter activity was induced with medium as shown by immunoblot experiment (Fig. 2). The isopropyl-rf-D-thiogalactopyranoside. In each case 1.5 ml of bacteria medium were antibodies were specific, recognizing no other antigen in the centrifuged and the pellet was resuspended in Laemmli buffer. An aliquot (one- fifth) was run on 5% polyacrylamide gel (see "Materials and Methods") and MCF-7 cell extract. Polyclonal antibodies were also prepared stained with Coomassie blue. in goat (not shown). 2392

66â€” 66â€”

45â€” 45â€” 36â€” 36â€” 29â€” 24â€” 29â€” 24- 20â€” 20â€”

14â€”

6.5â€” 14â€” 6.5â€”

1234 5678 Fig. 4. Immunoblot with a monoclonal antibody recognizing denatured BCEI/ pS2. Lanes I and 5, culture medium from MCF-7 cells grown in charcoal-stripped serum (absence of steroid) but in the presence of phenol red; Lanes 2 and 6, 1234 culture medium from MCF-7 cells grown in the presence of 5 nM estradici; Lanes Fig. 6. Immunoprecipitation of native BCEI/pS2 followed by immunoblot 3 and 7, culture medium from MCF-7 cells grown in the presence of I MM with antibodies detecting the denatured form of the protein. MCF-7 cells were tamoxifen; Lanes 4 and 8. culture medium prior to contact with cells. In Lanes grown in the presence of estrogen (see "Materials and Methods"). The proteins 1-4, antibody mBCEl, was used. In Lanes 5-8. antibody IDA 10, nonrelated to in the culture medium were immunoprecipitated with a nonspecific antibody (IDA breast cancer proteins, was used. The M, ~50,000 band corresponds to serum 10) (Lane /). with antibody mBCEIn recognizing the native protein (Lane 2), and IgG present in culture medium and revealed by its binding of '"I-labeled protein with antibody mBCEl, recognizing the denaturated protein (Lane 3). The im- A. munoprecipitates were analyzed in duplicate by immunoblot with mlÂ«T I,. Lane 4, immunoblot of the culture medium without any immunoprecipitation. The MT 50,000 and 25,000 bands correspond to the binding of '"l-labeled protein A to immunoglobulins. 92.5â€” 69â€” plates coated either with ^-galactosidase-BCEI/pS2 fusion pro tein purified by polyacrylamide gel electrophoresis, or 0-galac- 46â€” tosidase, or estrogen-treated MCF-7 cell culture medium. The 2250 wells which were screened yielded 3 different types of 30â€” results. Thirty-four hybridomas clearly secreted anti-/3-galactosidase antibodies (reacting strongly both with the latter antigen and the fusion protein). Thirty-one hybridomas secreted anti bodies which reacted against the culture medium from estrogen- stimulated MCF-7 cells, but not against ÃŸ-galactosidaseor the 14.3â€” ( electroeluted fusion protein. Four other hybridomas secreted antibodies which reacted strongly against the fusion protein, 3.4/2.3â€” did not react against 0-galactosidase, but did react slightly against culture medium from estrogen-treated MCF-7 cells (Fig. 3). These results were compatible with two different classes of anti-BCEI/pS2 antibodies: one class recognizing mostly the native form of the protein; and a second class recognizing its 1 2345 67 8 denatured form. The following experiments were performed to Fig. 5. Immunoprecipitation of 3'S-labeled BCEI/pS2 by monoclonal antibod test this hypothesis using mainly antibody mBCEl,, (supposed ies. MCF-7 cells were grown in the presence of estrogen and [35S]cysteine.Aliquots to recognize the native protein) and antibody mBCEl, (sup (100 fÂ¿l)ofthe culture medium were immunoprecipitated with monoclonal anti posed to recognize the denatured protein). bodies (see "Materials and Methods") (Lanes 2-4). The same experiment was Monoclonal Antibodies against Native and Denatured Forms repeated after denaturation of proteins in culture medium by heating for 3 min at 100'C (Lanes 5-7) prior to immunoprecipitation. Lane I, molecular weights of BCEI/pS2. Culture media from MCF-7 cells treated by estra- (xIO~3) of marker proteins (Amersham); Lanes 2 and 5, immunoprecipitation diol or the antiestrogen tamoxifen were used for immunoblot with control monoclonal antibody IDA 10 (nonrelated to breast cancer proteins). experiments (the protein is denatured during the sodium do- Lanes 3 and 6. immunoprecipitation with mlÂ«T I,, monoclonal antibody; Lanes 4 and 7, immunoprecipitation with mlÂ«TI, monoclonal antibody; Lane 8, decyl sulfate-polyacrylamide gel electrophoresis). As shown in trichloroacetic acid precipitation of the same aliquot of the culture medium. Fig. 4, antibody mBCEl, (IgGl) recognized an antigen of the expected size corresponding to the protein BCEI/pS2, which Monoclonal Antibody Preparation. Two mice were given 4 s.c. was induced by estrogen and suppressed by tamoxifen. On the injections of fusion /i-galactosidase-BCEI/pS2 protein (30% contrary antibody mBCEl,, (IgGl) did not react with this purity). The mouse giving the strongest plasma antibody re protein (not shown). Opposite results were obtained when sponse as evaluated by ELISA was used for preparation of studying the ability of the different antibodies to precipitate hybridomas. BCEI/pS2 (the protein is in its native state). MCF-7 cells were These hybridomas were screened by 3 different ELISAs using labeled by [35S]cysteine and cultured in presence of estradiol. 2393

-Â£â€¢->*f\- .â€¢â€¢>:,-A â€¢¿B

C-

Fig. 7. Immunocytochemical detection of BCEI/pS2 in human breast cancer. Frozen (A, C, D. and E) or paraffin (fi) breast cancer sections were prepared and incubated (see "Materials and Methods") with mBCEIn antibody (A, fi. and E) or the same antibody saturated with 100-fold excess antigen (C) or control preimmune serum i/'i All the tumors shown are invasive ductal carcinomas. A, the immunolabeling is mainly located on the plasmic membrane of epithelial cells. /, carcinoma without detectable BCEl/pS2. Bar = 100 firn (E). 200 firn (A, C. and O), 520 >,m (fi).

Culture media were treated by antibodies mBCEI, and estrogen-treated MCF-7 cells was immunoprecipitated using mBCEI,,. Only the mBCEIn antibody precipitated the labeled antibody mBCEIn and the precipitate was analyzed by immu BCEI/pS2 (Fig. 5, Lane 3). However, when the protein was noblot with antibody mBCEI, (Fig. 6). In this experiment partially denatured by heating, it could be immunoprecipitated antibody mBCEI,, immunoprecipitates the native protein and by antibody mBCEI, (Fig. 5, Lane 6). antibody mBCEI, reveals it by immunoblotting after it has been Finally, immunoprecipitation and immunoblot analysis were denatured during the run on a sodium dodecyl sulfate-polyacryl- combined in the following experiment. Culture medium from amide gel. 2394

ies by preimmune serum. The mBCEIn antibody had a high E e apparent affinity for BCEI/pS2 since it could be used for im CM munostaining at a very low concentration (0.8 ng/m\ on cryostat s * Washes en sections). UJ O Immunoaffinity Purification of BCEI/pS2. Antibody mBCEI,, was coupled to Affi-Gel 10. Cell culture medium from estrogen- ce *; 09 treated MCF-7 cells was chromatographed on this immuno O 1 g 5 matrix, preceded by two other columns containing gels devised to decrease the nonspecific binding of proteins (deactivated P Affi-Gel 10 and an immunomatrix with nonrelated IDA 10 iec. antibody coupled to Affi-Gel 10). The specific immunomatrix o. retained ~85% of the antigen and very little of the applied 200 400 0246 proteins. After extensive washings proteins were eluted from F1LTRATE( ml ) ELUATE ( ml ) the mBCEI,,-Affi-Gel column. ELISAs followed BCEI/pS2 Fig. 8. Immunoaffinity purification of BCEI/pS2. Cell culture medium (130 purification (Fig. 8). The peptide has been extensively purified ml) from estrogen-treated MCF-7 cells was chromatographed on mBCEIM-Affi- Gel 10 column. After 6 extensive washings (arrows, see "Materials and Methods") as shown by gel electrophoresis (Fig. 9). It was possible to elution was performed with 50 mM glycine buffer, pH 2.2. Neutralization was obtain 75 Â¿tgofpurified BCEI/pS2 from 1 liter of cell culture rapidly performed with I M Tris buffer, pH 9. medium. Aliquots (2 /il) of each fraction were diluted 100-fold and coated on Nunc plates, and ELISAs were performed as described with mBCEI,, antibody. DISCUSSION 66- Hormone dependency of breast cancers is presently assessed 45. mainly by their content of steroid hormone receptors. BCEI/ pS2 is a potentially interesting clinical marker of estrogen 29- dependency. Its presence in a series of 180 tumors has been analyzed through immunocytochemistry and detection of 20.1- mRNA by Northern blot analysis (9). Subsets of patients were defined according to the presence of estrogen and progesterone receptors and BCEI/pS2. However, availability of monoclonal 14.2- antibodies would allow to devise extensive clinical trials for 6- testing the importance of this protein as a marker of breast cancer prognosis and hormone dependency. The monoclonal antibodies described in this paper can now be used for radio- immunoassay and immunometric or immunoenzymatic assay of BCEI/pSi.4 The protein has also been assayed in the serum and thus it will be possible to determine if it is a circulating marker of the presence and/or the extension of breast cancers. The monoclonal antibodies allow the detection of BCEI/pS2 1 2 3 by immunohistological methods in both frozen and paraffin- Fig. 9. Polyacrylamide gel electrophoresis of immunoaffinity purified BCEI/ embedded sections. These properties are of interest since steroid pS2. Proteins were electrophoresed on a 15% polyacrylamide gel in the presence of sodium dodecyl sulfate (24). The gel was fixed as described (25) and silver hormone receptors are usually detected in frozen sections (28, stained. Lane I, culture medium from estrogen-treated MCF-7 cells (IO ni); Lane 29) whereas retrospective studies can be done in most cases 2, flowthrough of immunoaffinity chromatography (14 ^1);Lane 3, protein eluted only on paraffin sections (30). BCEI/pS2 being only expressed from the immunomatrix (see Fig. 8) (1 /jg of protein). in a fraction of cancers, it may be used, like other specifically expressed antigens, to try to define subclasses of breast cancers. ELISAs were also performed using antibodies against the Studies should be undertaken to try to correlate the phenotypic native BCEI/pS2 protein to analyze culture media from estro gen-treated MCF-7, ZR-75-1, T-47D, and SK-Br-3 cells. BCEI/ differences between individual cancers and their response to various types of chemotherapy. pS2 was detected in media from the first two cell types, but not The biological role of BCEI/pS2 is not yet understood. with the last two (data not shown), confirming previous results Expression of the gene has been detected only in breast cancer showing the presence or the absence of BCEI/pS2 mRNA in and gastric epithelium (10). Various structural features of the these various breast cancer cell lines (27). Moreover, the anti bodies did not cross-react with human EGF, mouse EGF, and polypeptide such as small size and number of putative disulfide bonds have led to comparisons with growth factors (11). These human transforming growth factor a (data not shown). characteristics may also determine a very constrained tertiary Immunocytochemical Detection of BCEI/pS2 in Breast Cancer. Monoclonal antibodies could be used for the immunocytochem- structure and explain the existence of the two classes of anti bodies recognizing either the native or the denatured protein. ical detection of BCEI/pS2 on either frozen sections or paraffin BCEI/pS2 was found to be homologous with spasmolytic sections of breast cancer biopsies (Fig. 7). In most cases the polypeptide, a protein that has hormone-like actions (11). It plasma membrane of the epithelial cells was labeled. When the tumor cells contained a high concentration of BCEI/pS2, cyto- was suggested (11) that BCEI/pS2 might have actions analogous to EGF, since EGF-like spasmolytic protein is stable in gastric plasmic and perinuclear staining could also be observed. The juice and can act on gastric epithelium. This hypothesis was specificity of the reaction was shown by signal extinction when supported by the finding that BCEI/pS2 was secreted by stom- the antibodies were preincubated with excess BCEI/pS2 and by lack of immunostaining when replacing anti-BCEI/pS2 antibod 4J. Predine and J. F. Prud'homme, unpublished observations. 2395

10. Rio, M. C., Bellocq. J. P., Daniel. J. Y.. Tomasetto, C., Lathe, R., Chenard, ach mucosa (IO). Mori et al. (12) concluded that BCEI/pS2 M. P., Batzenschlager, A., and ChambÃ³n, P. Breast cancer-associated pS2 copurified with a small amount of EGF that was recognized by protein: synthesis and secretion by normal stomach mucosa. Science (Wash. their antibody to EGF. When EGF was removed from the DC), 241: 705-708, 1988. 11. Baker, M. E. Estrogen-induced pS2 protein is similar to pancreatic spasmo BCEI/pS2 preparation, then there was no recognition by this lytic polypeptide and the kringle domain. Biochem. J., 253: 307-309, 1988. anti-EGF antibody. Thus, BCEI/pS2 appears to be distinct from 12. Mori, K., Fujii, R., Kida. N.. Ohta, M.. and Hayashi. K. Identification of a EGF, although it may have some similar actions. polypeptide secreted by human breast cancer cells (MCF-7) as the human estrogen-responsive gene (pS2) product. Biochem. Biophys. Res. Commun., In order to study the function of BCEI/pS2 it was necessary 755:366-372. 1988. to prepare pure protein in relatively large amounts. This could 13. Nunez, A-M., Jakowlew, S., Briand. J-P.. Gaire, M., Krust, A., Rio, M-C., be obtained either by purification of the protein from the MCF- and ChambÃ³n. P. Characterization of the estrogen-induced pS2 protein secreted by the human breast cancer cell line MCF-7. Endocrinology. 121: 7 cell culture medium or by expression in bacterial systems. 1759-1765. 1987. This last alternative could bring about some difficulties con 14. Engel. L. W., and Young. N. A. Human breast carcinoma cells in continuous culture: a review. Cancer Res., 38: 4327-4339, 1978. cerning the correct secondary and tertiary structures of the 15. Keydar, I., Chou, L., Karby, S.. Weiss, F. R., Delasea, J., Radu, M., Chaitcik, expressed protein. Using immunoaffinity chromatography we S., and Brenner. H. J. Establishment and characterization of a cell line of have purified in a single step large amounts of the protein from human breast carcinoma origin. Eur. J. Cancer, 15:659-670, 1979. 16. Calvo, F., Brower, M., and Carney, D. N. Continuous culture and soft conditioned culture media of estrogen-treated cancer cells. Fi agarose cloning of multiple human breast carcinoma cell lines in serum-free nally the cDNA as well as the gene coding for the BCEI/pS2 medium. Cancer Res., â€¢¿Â«.â€¢4553-4559,1984. 17. RÃ¼ther,U.. and MÃ¼ller-Hill.B. Easy identification of cDNA clones. EMBO protein have been cloned (7, 31). The gene was localized to J..2: 1791-1794, 1983. chromosome 21 (32). The regulation of BCEI/pS2 expression 18. Amann, E., and Brosius, J. "ATG vectors" for regulated high-level expression by estrogens could be an interesting model of steroid action in of cloned genes in Escherichia coli. Gene, 40: 183-190, 1985. 19. Maniatis, T., Fritsch, E. F.. and Sambrook, J. Molecular Cloning: a Labo humans. The availability of well defined and high affinity mono ratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, clonal antibodies will facilitate these studies. 1982. 20. Birnboim, H. C., and Doly, J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res., 7: 1513-1523, ACKNOWLEDGMENTS 1979. 21. Hunkapiller. M-W., Lujan, E., Ostrander, F., and Hood, L. E. Isolation of We thank Dr. O. Mercereau-Puijalon (Institut Pasteur-Paris) and microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol.. 91: 227-236, 1983. Pr. Muller-Hill (Cologne, Federal Republic of Germany) for the gift of 22. LogeÃ¢t,F.,Vu Hai, M. T., Fournier, A.. Legrain. P., Buttin, G., and Milgrom, pUR 290-292 vector system. The manuscript was typed by V. FranÃ§ois. E. Monoclonal antibodies to rabbit progesterone receptor: crossreaction with other mammalian progesterone receptors. Proc. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1990 American Association for Cancer Research. Monoclonal Antibodies against Native and Denatured Forms of Estrogen-induced Breast Cancer Protein (BCEI/pS 2) Obtained by Expression in Escherichia coli

Jean-François Prud'homme, André Jolivet, Marie-France Pichon, et al.

Cancer Res 1990;50:2390-2396.

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