Identification of a human osteosarcoma-associatedglycoprotein with monoclonal antibodies: relationship with alkaline phosphatase TAKASHI NAKAMURA' AND MICHAEL GROSS^ Department of Surgery, Faculty of Health Sciences, McMaster University, Hamilton, Ont., Canada and

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Ontario Cancer Treatment and Research Foundation, Hamilton Regional Cancer Center, Hamilton, Ont., Canada

TAKAO YAMAMURO Department of Orthopedic Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan AND

SHUEN-KUEI~ 1 ~ 0 ~ Department of Pathology, Faculty of Health Sciences, McMaster University, Hamilton, Ont., Canada and Ontario Cancer Treatment and Research Foundation, Hamilton Regional Cancer Center, Hamilton, Ont., Canada

Received February 13, 1987

NAKAMURA, T., GROSS,M., YAMAMURO, T., and LIAO,S.-K. 1987. Identification of a human osteosarcoma-associated glycoprotein with monoclonal antibodies: relationship with alkaline phosphatase. Biochem. Cell Biol. 65: 1091-1 100. The molecular nature of an osteosarcoma-associated antigen was investigated with the three monoclonal antibodies Ost6 (immunoglobulin (IgGl), Ost7 (IgGl), and Ost15 (IgG2a), which selectively react with frozen sections of osteosarcoma and chondrosarcoma tissues. When tested with a panel of 41 human cell lines in the mixed hemadsorption assay, the antibodies reacted similarly with three of six osteosarcomas, one choriocarcinoma, one teratoma, and one osteoblast-like culture, but failed to react with 32 lines of normal and other tumor cell types. Immunoprecipitation plus sodium dodec I sulfate (SDS) polyacrylamide gel electrophoresis and sequential immunoprecipitation studies revealed that in [15Slmethionine- or ['4~]glucosamine-labelledosteosarcoma cells the three antibodies detected a single glycoprotein, with an apparent molecular mass of 86 kilodaltons (kDa), which was not affected by reducing conditions. Tunicarnycin treatment and pulse-chase experiments showed glycosylation of this molecule to be N-linked; it arose from a 54-kDa polypeptide precursor. Alkaline phosphatase activity was detected in the material rich in 86-kDa molecules that was immunoprecipitated from serologically reactive cell lines with each antibody. These antibodies also cross-reacted with two isoenzymes of alkaline phosphatase (strongly with the liver and bone, and moderately with the placental isoenzyme), but not with the intestinal form. NAKAMURA, T., GROSS,M., YAMAMURO, T., et LIAO, S.-K. 1987. Identification of a human osteosarcoma-associated glycoprotein with monoclonal antibodies: relationship with alkaline phosphatase. Biochem. Cell Biol. 65 : 1091-1 100. Nous avons rechercht la nature moltculaire d'un antigene associC ?t un ostCosarcome avec trois anticorps monoclonaux, Ost6 (immunoglobuline (Ig) GI), Ost7 (IgG 1) et,Ost 15 (IgG2a), qui rkagissent stlectivement avec des coupes congelkes des tissus des ost6osarcomes et des chondrosarcomes. A l'aide d'un essai d'hemadsorption mixte, nous avons test6 la rCactivitC avec un ensemble de 41 lignCes cellulaires humaines. Les anticorps ont rCagi de la m&mefagon avec trois des six ostCosarcomes, un choriocarcinome, un tCratome et une culture de type osttoblaste, mais ils n'ont pas rCagi avec 32 lignCes de types de cellules normales et de cellules d'autres tumeurs. Les Ctudes d'immunoprCcipitation avec Clectrophorese sur gel de dodCcylsulfate de sodium (SDS) - polyacrylamide et d'immunoprtcipitation skquentielle rkvklent que les trois anticorps dCtectent une seule glycoprotCine des cellules ostCosarcomateuses marquCes avec la [35S]m~thionine ou la ['4~]glucosamine.Elle a une masse molCculaire de 86 kilodaltons (kDa) et elle n'est pas affectke par les conductions rkductrices. Le traitement avec la tunicamycine et les expCriences de marquage rapide avec chasse montrent que la glycosylation de cette molCcule est N-like; elle provient d'un

ABBREVIATIONS: Ig, immunoglobulin; SDS, sodium dodecyl sulfate; kDa, kilodalton(s); MABs, monoclonal antibodies; MEM, minimumessential medium; FCS, fetal calf serum; RBC, red blood cell; PBS, phosphate-buffered saline; sp.act., specific activity; PAGE, polyacrylamide gel electrophoresis. 'Author from whom reprints should be requested. Present address: Department of Orthopedic Surgery, Faculty of Medicine, Kyoto University, Kyoto, Japan. 2Present address: Division of Orthopaedic Surgery, Victoria General Hospital, Halifax, N.S., Canada B3H 2Y9. 'besent address: Biotherapeutics, 357 Riverside Drive, Franklin, TN, U.S.A. 37064.

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polypeptide prkcurseur de 54 kDa. Nous avons dCtectC une activitk phosphatasique alcaline dans le matkriel immunoprCcipit6 riche en molCcules de 86 kDa que nous avons obtenu des lignCes cellulaires qui rkagissent ~Crologiquementavec chaque anticorps. Ces anticorps rCagissent aussi avec deux isoenzymes de la phosphatse alacaline (fortement avec les isoenzymes du foie et des os et modCrCment avec I'isoenzyme placentaire), mais non avec la forme intestinale. [Traduit par la revue]

Introduction treatment and have made some progress the years, the prognosis for patients with osteosarcoma is still poor: 80% are found to have micmmetastases to the lung during surgical procedures (Goorin et al. 1985). Accordingly, control of micrometastases appears to be one of the most important steps needed to improve the prognosis for patients with osteosarcoma. Hybridoma technology provides an efficient means of producing many MABs against tumor-associated antigens. Some of these antibodies have been used for diagnosis and therapy, as well as in basic research on malignant tumors. Encouraging results for improving treatment of patients with malignant tumors have recently been reported (Miller et al. 1982; Houghton et al. 1985; Larson 1985). The use of MABs against osteosarcoma-associated antigens may allow earlier diagnosis and more specific treatment of pulmonary metastatic lesions. We have previously reported that the three murine MABs Ost6, Ost7, and Ost15, raised against a human osteosarcoma, have selective reactivity with osteosarcomas (Hosoi et al. 1982). These antibodies reacted in an irnmunofluorescence assay with sections from five of five osteosarcomas, a chondrosarcoma, and a few layers of chondrocytes adjacent to the subchondral bone. Osteosarcoma xenografts in athymic nude mice have been radioimaged with these antibodies (Nakamura et al. 1984). To increase our understanding of the nature of the osteosarcoma-associated antigens recognized by these antibodies, we first looked for appropriate target cell lines by testing these three antibodies for reactivity against a panel of tumor and normal human cell lines, using a mixed hemadsorption assay. The reactive target cell lines were used for characterizing the antigens, using combined immunochemical and biochemical means. Materials and methods Cell lines and culture methods The 41 cell lines used in this study are listed in Table 1. The four osteosarcoma cell lines (Saos-2, U-20S, MG63, and TE85) were obtained from American Type Culture Collection (Rockville, MD); the fetal osteoblast-like cell line (6A3) was from Dr. P. Grant, University of Toronto (Toronto, Ont.); and the three fibroblast cell lines (S6000, D, and A l ) were from Drs. D. Tompkins and M. Coughlin, McMaster University (Hamilton, Ont.). The embryonic intestine CCL-6 cell line

was obtained from Dr. M. PagC, UniversitCLaval (Sainte-Foy, Que). The sources of other cell lines have been reported (NAamura er al, 1984; Liao et al. 1982, 1985). The cells were grown as a monolayer in Eagle's MEM or RPMI-1640 medium containing 10% heat-inactivated FCS, loo u penicillinim~,and 100 , . ~ gstreptornycinJmL. The cultures were incubated at 37OC in a humidified atmosphere of 95% air and 5% C02. MABs and serum Ost6, Ost7, and Ost15 were purified from ascitic fluids of hybridoma-bearing BALBIc mice (Nakamura et al. 1984); Ost6 and Ost7 are of the IgGl isotype and Ost15 is of the IgG2a isotype. Normal mouse serum was obtained from 8-week BALBIc mice by heart puncture, and rabbit anti-mouse Ig antiserum was purchased from Jackson Immunoresearch Laboratories (Avandale, PA). Mouse anti-sheep RBC antiserum was prepared in our laboratory by immunizing BALBIc mice with sheep RBCs. Mixed hemadsorption assay We used the mixed hemadsorption assay to detect antibodies binding to target cells grown in wells of microtiter plates (Liao etal. 1982). The sheep RBC that were sensitized with mouse anti-sheep RBC antiserum, followed by a second sensitization with goat anti-mouse antiserum, were used as indicators to detect the murine MABs binding to the target cells. The endpoint titer was the highest dilution at which at least 10% of the target cells was positive. Target cells were scored positive if five or more RBCS were adherent to the cell surface: Metabolic labelling of cells Cells were grown in a 75-cm2 culture flask until near confluency and washed twice with a 30-mL volume of sterile PBS (pH 7.4). They were metabolically labelled with 200 pCi of L-[35~]methionine(New England Nuclear, Boston, MA; sp.act., 1200 Cilmmol; 1 Ci = 37 GBq) in 10 mL of methionine-free MEM supplemented with 5% dialyzed FCS (Khosravi et al. 1985). Some cultures were treated with tunicamycin, for studying glycosylation. The antibiotic was dissolved in PBS at a concentration of 100 ,.~g/mLas a stock solution; various concentrations of tunicamycin (1, 10, 50 pg/mL) were added to the medium 2 h before the cultures were metabolically labelled with [35S]methionine in the medium containing cbrresponding concentrations of tunicamycin. In pulse-chase experiments, cells were first pulse labelled for the indicated time with [35~]methionine, washed twice with 30 mL of sterile PBS, and then chased with 10 mL of complete medium containing 10% FCS and 30 mg methionine for various durations. Metabolic incorporation of ~ - l [ l - ' ~ C ] glucosamine hydrochloride (New England Nuclear; sp.act., 54.2 mCiImmol) was performed by adding 200 pCi to each culture containing complete MEM plus 10% FCS. The cultures were incubated for 18 h at 37OC. Labelled cells were washed twice with 30 mL of PBS con-

NAKAMURA ET AL.

taining cold L-methionine (2 mg/mL), scraped from the culture flask with a rubber policeman, pelleted by centrifugation at 800 x g for 20 min, and washed three times with PBS containing L-methionine. After each washing the supernatant was removed by centrifugation as described above. The cell pellet was extracted with 2 mL of lysing buffer (0.5% NP-40 in PBS containing 0.1 mM phenylmethylsulfonyl fluoride and 0.1 m M benzamidine hydrochloride) for 1 h at 4"C, and cellular debris was removed by centrifugation (100 000 x g , 1 h).

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Kodak XRP- 1 Film (Eastman Kodak Company, Rochester, NY). I4c-labelled protein markers (New England Nuclear, Boston, MA) were included in each gel. -

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Competitive cell-binding assay '25~-labelledOst6 and 1251-labelled Ost7 were used. Semiconfluent cells scraped from culture flasks were pelleted and washed in PBS containing 0.2% gelatin by centrifugation at 1000 x g for 10 min. Aliquots of the cell suspension at a concentration of 5 x 105/(100 p,L.microfuge tube) were prepared; each was mixed with an indicated amount of asdites Chloroglycouril-mediatediodination Cultured cells were surface labelled with 12'1 (Markwell and which contained unlabelled MAB (3 mg IgGImL). Fifty micFox 1978). Cell cultures at near confluency were washed three rolitres of iodinated antibody (having radioactivity of 12 900 times with PBS, scraped from each flask, and pelleted by cpm and containing 0.01 pg IgG for '251-labelledOst6, or 10 centrifugation. The cell pellet was suspended in 0.5 mL PBS (4 498 cpm and 0.01 pg IgG for '25~-labelledOst7) was added. x lo6 cells) and placed in a test tube (13 x 100 rnm) coated The mixtures were incubated for 90 min at 4°C with occasional with 100 pg 2,3,4,6-tetrachloro-3a,6a-diphenylglycoud agitation. Cells were pelleted and washed five times with PBS (IODO-GEN, Pierce Chemical, Rocheford, IL); 1 mCi '251 containing 0.2% gelatin. The cell suspensions were introduced (New England Nuclear) was added, and the mixture was incu- to and pelleted in new tubes. The radioactivity of iodinated bated at room temperature for 10 min. The cells were removed antibodies bound to the cells was counted in a gamma counter to a new tube, washed three times with PBS to remove free (model 300, Beckman, Palo Alto, CA). Results, expressed as iodine, and pelleted by centrifugation; the pellet was extracted means + SD of four determinations, were analyzed by the ANOVA and Newman-Keuls multiple range test for statistical with 2 mL of lysing buffer as above. For competitive cell-binding assays, purified MABs were analysis. P < 0.05 was considered significant. labelled with '251(IODO-GEN), as described by Nakamura et Assay of alkaline phosphatase (EC 3.1.3.1) al. (1984). The fetal osteoblast-like cell line 6A3 and all of the osteosarImmunoprecipitation coma cell lines were lysed as above, except that the buffer used For indirect immunoprecipitation, 50 p L of MABs or nor- for extraction was 10 mM Tris-HC1 (pH 7.5) containing 0.5 mal mouse serum was mixed with 300 p L of cell lysate for 90 rnM MgC12 and 0.5% NP-40. min at 4°C; 10 p L of rabbit anti-mouse Ig antiserum was added The activity of alkaline phosphatase was assayed by deterand mixed for 60 min at 4OC; and finally, 200 pL of 10% mining the release of p-nitrophenol from p-nitrophenyl phosprotein A - Sepharose C1-4B beads (Pharmacia, Uppsala, phate using spectrophotometry at 405 nm. The substrate soluSweden) was added and mixed for 40 min. The beads were tion contained 5 mg p-nitrophenyl phosphate in 1.0 M diethpelleted and washed five times with lysing buffer by centri- anolamine buffer (100 mL, pH 10.3) and 0.5 rnM MgC12. The fugation at 800 x g for 3 min. Immunocomplexes were reaction was stopped by adding the same volume of 1N NaOH. dissociated by heating the beads at 100°C for 2 min in 50 pL of A standard solution of p-nitrophenol (Sigma, St. Louis, MO) double-strength SDS-polyacrylamide gel sample buffer was used for assay of released p-nitrophenol. One unit of (Laemmli 1970). activity corresponds to the release of 1 pmol p-nitrophenoll For sequential immunoprecipitation, immunodepletion was min. The protein content of the samples was determined by first performed (Liao et al. 1987). Then, 300-pL aliquots of Bio-Rad protein assay using bovine serum albumin as a stanlysate, from cultured cells metabolically labelled with dard (Bio-Rad Laboratories, Richmond, CA). [3SS]methioninefor 18 h, were incubated with 50 p L of one of the MABs. The immunocomplexes were removed by protein Testfor cross-reactivity of antibody with puriJied human alkalinephosphatasesfrom various tissue types A - Sepharose beads as described above and the supernatant Purified alkaline phosphatases of human bone, liver, fluid (immunodepleted material) was immunoprecipitated with each of the three MABs as described above. In reciprocal placental, and fetal intestinal origins were kindly provided by fashion, experiments were performed in which the second or Professor R. A. Stinson of the University of Alberta (Edmonthird MAB was used to imrnunodeplete the antigen from ton, Alta.) (Stinson and Seargeant 1981). Their activities are radiolabelled lysate, and the immunodepleted material was 870, 990, 940, and 1040 UIL, respectively. The crossreactivity of antibodies with these isoenzymes was determined precipitated with each of the MABs. by the method of Meyer et al. (1982). Briefly, antibodies were SDS-polyacrylamide gel electrophoresis and autoradiogra- dissolved in 50 mM carbonate buffer (pH 9.8) and adjusted to a concentration of 10 p g IgGImL. Fifty microlitres of each P ~ Y SDS-polyacrylamide slab gels (1.5 mm thick) were pre- antibody was pipetted into the wells of a Immulon I flat-bottom pared according to the method of Laemmli (1970). Samples plate (Dynatech Laboratories Inc., Alexandria, VA) and incuwere analyzed under reducing and nonreducing conditions by bated for 3 h at a room temperature. The wells were washed six electrophoresis on 10 or 7.5-15% gradient polyacrylamide at times with PBS containing 1% bovine serum albumin. Thirty 140 V for 16 h. Enhancers (Autofluor, National Diagnostics, microlitres of alkaline phosphatase (1:50 dilution) was pipetSomewille, NJ) were used in processing the gels by scintilla- ted into the wells and incubated for 1 h at room temperature. tion autoradiography and radiolabelled bands were detected on The wells were washed three times with PBS containing 1%

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TABLE1. Reactivity of Ost6, Ost7, and Ost15 to human cell lines as tested by mixed hemadsorption assay Reciprocal titer Cell line

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-

Osteosarcoma Saos-2 KT005 TE85 MG63 u-20s R970 Neuroblastoma IMR-6 SKNSH SHSY-SY Glioblastoma LN 18 LN 40 LN 135 LN 229 Teratoma Tera-2 Fetal osteoblast-like cell 6A3 Fibrosarcoma HT 1080 Melanoma RPMI 5966 CaCL 73-36 CaCL 78-1 Carcinoma HT-29 (colon cancer) HCT 8R (colon cancer) MGHUI (bladder cancer) KB (oral cancer) BT-20 (breast cancer) MCF-7 (breast cancer) A549 (lung cancer) CaLu 3 (lung cancer) SK-Lu- 1 (lung cancer) HS0229T (lung cancer) CaLu 1 (lung cancer) HeLa (cervical cancer) JAR (Choriocarcinoma) Fibloblast S6000 D HF172 MRC-5 A1 5004-42 MCHF-2 Amnion epithelium HAEI70 Embryonic intestine CCL-6

FIG. I. Fluororadiographs showing SDS-polyacrylamide gel electrophoresis profiles of [35S]methionine (A) and 125~labelled (B) cell lysates of Saos-2 cells immunoprecipitated with Ost6, Ost7, and Ost15. (A) 10% gel; (B) 7.5-15% gradient gel. Lanes 1 and 5,Ost6; lanes 2 and 6,Ost7; lanes 3 and 7, Ost15; lane 4, normal mouse serum; lane 8, positive control ([35~]methionine-labelledcell lysate immunoprecipitated with Ost6); lane 9, [rnethyl-14~]methylated protein markers (myosin, 200 kDa; y-globulin, 52 kDa (heavy chain) and 22.5 kDa (light chain); phosphorylase b, 97.4 kDa; ovalbumin , 4 6 m a ) . bovine serum albumin. One hundred microlitres of alkaline phosphatase substrate solution was added to the wells and incubated for 30 min at room temperature. The reaction was stopped by the addition of 100 FL of 1 N NaOH. The samples were read for OD at 405 nm in a Multiscan apparatus. The results were expressed as a ratio of captured alkaline phosphatase activity to total alkaline phosphatase activity.

Results Reactivity of MABs with human cell lines The results of the mixed hemadsorption assay are summarized in Table 1. Ost6, Ost7, and Ost15 reacted strongly with two of six osteosarcoma cells lines (Saos-2 and KT005) and with the teratoma cell line (Tera-2). They reacted weakly with an osteosarcoma cell line (TE85), a fetal osteoblast-like cell (6A3), and a choriocarcinoma (JAR). The patterns of selective reactivity exhibited by the three MABs were very similar. However, they did not react with the other 36 cell lines tested. Molecular profile of antigenfs)detected by MABs Metabolic labelling and irnrnunoprecipitation were

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mass as the [35S]methionine-labelledantigens precipitated from the same cell lines (Fig. 1).

FIG.2. Sequential immunoprecipitation.Fluororadiographs of SDS-polyacrylamide gel electrophoresis profiles of im-

munoprecipitates resulting from imrnunodepleted or nonimmunodeple%edmaterials. fmm~node~letion was performed by incubating Saos-2 cell lvsates with - 135Slmethionine-labelled - protein A - beads that were bound with a before irnmunoprecipitationwas conducted with the corresponding antibody or other antibodies. Antibodies used for immunodepletion and immunoprecipitation are indicated at the bottom of the figure(lanes 1-9). Lane 10, molecular mass protein markers. performed with eight cell lines, including serologically reactive and nonreactive cell lines (Saos-2, KT005, TE85, MG63, Tera-2, 6A3, CaCL 78-1, and HT-29). All three MABs precipitated the diffuse bands of 86 kDa from [35S]methionine-labelledSaos-2 and KT005 lines, as visualized by SDS-polyacrylamide electrophoresis under reducing conditions (Fig. I); this suggests that the antigens were the same or extremely similar glycoprotein(s). The position of the antigen in the gel electrophoresis was not affected under nonreducing conditions (results not shown). These MABs did not immunoprecipitate any specific proteins from the cell lines that showed negative reactivity in the mixed hemadsorption assay (CaCL 78- 1, HT29, and MG63). Similarly, no specific band could be detected from the cell lines TE85 and 6A3, which were weakly reactive to antibodies by the mixed hemadsorption assay. From the Tera-2 line we detected a very faint band consistent with a molecular mass of 86 kDa. Surface iodination of the Saos-2 osteosarcoma cell line was performed. The three MABs immunoprecipitated '25~-labelledproteins with the same molecular

Sequential immunoprecipitation To determine whether the three MABs recognized the same antigenic molecule, sequential immunoprecipitation experiments were performed. The radiolabelled lysate of the Saos-2 cells was depleted of antigens, by incubation with protein A - beads bound with one of the three MABs before immunoprecipitation with each antibody. The results, shown in Fig. 2, revealed that none of the three MABs precipitated the 86-kDa molecule in the lysate depleted by noncorresponding MAB, suggesting that they recognized a same molecular entity. Glycosylation and tunicamycin studies Because of the diffuse nature of the resulting bands precipitated by the three MABs from osteosarcoma cells, we sought to determine if the antigen was a glycoprotein. The Saos-2 cells were metabolically labelled with ['4C]glucosamine for 18 h. Immunoprecipitation and SDS-PAGE of the cell lysate with Ost6 showed that a radioactive band had the same molecular mass as the [35~]methionine-labelledantigen and established the glycoprotein nature of the antigen (Fig. 3A). To further characterize the antigen as a glycoprotein, the Saos-2 cells were treated with tunicamycin at concentrations of 1, 10, and 50 pg/mL before metabolic labelling with [35S]methionine.With concentrations of 10 and 50 ~ g / m L of tunicamycin, there was a new band with an estimated molecular mass of 54 kDa in SDSPAGE and a concomitant disappearance of the 86-kDa protein (results not shown). At a concentration of 1 ~g/mL of tunicamycin, two distinct bands of 54 and 86 kDa were detected (Fig. 3A). We performed pulsechase labelling of osteosarcoma cells with [ 3 5 ~ ] methionine in the absence of tunicamycin for 1 h and a chase for 0-3 h (Fig. 3A). At 0 h of chase, four to five multiple bands in a range of 55-67 kDa were detected. At 3 h of chase, label was evident only in a 86-kDa band. The results taken together suggest that the glycosylation of the unglycosylated polypeptide precursor (54 kDa) occurs within a short period of time (