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

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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 Escherìchìacoli1 Jean-François Prud'homme, AndréJolivet, Marie-France Pichón,Jean-François Savouret, and Edwin Milgrom2 Croupe de Recherches sur la Biochimie Endocrinienne et la Reproduction (INSERM U. 135), FacultédeMédecineParis-Sud, 94275 Le Kremlin-BicétreCedex, France ABSTRACT protein in tissue extracts, plasma, and urine and to analyze tissue biopsies by immunocytochemistry. Moreover, immu Several vectors »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 rìchìacoli.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 histológica)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 —¿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 Santéet de la HCI-pH 7.4-50 mM NaCI-1 mM /J-mercaptoethanoI-1 mM MgCl2-l Recherche Médicale,théCentreNational de la Recherche Scientifique, théUnité d'Enseignement et de Recherches Kremlin-Bicétre,théAssociation pour la Re mM PMSF and dialyzed against the same buffer or desalted by passing cherche sur le Cancer Transbio Company, and théFondation pour la Recherche through a GF05 column (IBF, Villeneuve-la-Garenne, France). MédicaleFrançaise. 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 4°Cwithrabbit anti-mouse ¡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 Bürket 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 ¿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 ¿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 ;mi¡.; 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.
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