[CANCER RESEARCH 49, 2615-2620, May 15, 1989] Ligand-induced Phosphorylation of a Murine Tumor Surface Protein (TSP-180) Associated with Metastatic Phenotype1 A. Sacchi,2 R. Falcioni, G. Piaggio, M. A. Gianfelice, N. Perrotti, and S. J. Kennel Laboratorio di Oncogenesi Molecolare Istituto Regina Elena per lo studio e la cura dei tumori, Roma, Italia [A. S., R. F., G. P., M. A. G.J; Istituto di Oncologia Sperimentale e ClÃ-nica,Facoltà di Medicina, Calamaro, Italia ¡N.P.]; and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 [S. J. K.J ABSTRACT A tumor surface protein (TSP-180) has been identified on murine lung carcinomas using two monoclonal antibodies (MoAbs) (135-13C and 346- 11A). Quantitative analysis of TSP-180 on 3LL variants maintained either in vitro or in vivo indicates that TSP-180 is highly expressed in highly malignant metastatic cells. In reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns of TSP-180 obtained with MoAb 135-13C from cell lysates of 3LL metastatic cells show three proteins migrating to M, 204,000, 134,000, and 116,000. In the same experimental conditions MoAb 135-13C precipitates from low metastasizing ones only one band, corresponding to the lower molecular weight (M, 116,000). All bands of TSP-180 observed in 3LL variants are labeled by lactoperoxidase-catalyzed radioiodination of viable cells, incorporate 1!1'().,, and contain carbohydrates, as judged by binding to wheat germ agglutinin. These results indicate that all proteins have external exposure on the cell surface and that at least some of TSP-180 proteins could be differentially regulated in different tumor cells (highly metastatic versus low metastatic). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis banding patterns and immunoblots obtained from cell lysates of 3LL variants by using a monoclonal antibody to phosphotyro- sine (IG-2) indicate that this MoAb recognizes proteins migrating with molecular weights identical to those reported for TSP-180. Moreover, the immunoblots of solubili/od immunocomplex, obtained from cell ly sates of 3LL variants by using MoAb 135-13C, demonstrate that MoAb IG-2 specifically reacts with TSP-180 proteins. Experiments undertaken in order to assess if some or all of TSP-180 proteins have tyrosine kinase activity demonstrate that MoAb 135-13C binding to the cell surface induces specific phosphorylation of the M, 204,000 protein of TSP-180. Phosphoaminoacid analysis of the ligand- induced phosphorylated protein (pp204) demonstrates that this protein is phosphorylated at serine and tyrosine. Results reported lead us to hypothesize that TSP-180 is involved in growth-regulation mechanisms and that its high expression on cells with more malignant phenotype could be responsible for a proliferative advan tage of such tumor clones. INTRODUCTION The growth of malignant tumors is a complex process due to genetic changes and host selection: together these two events can result in the development of specific characteristics of tumor cells inducing increased biological malignancy and tumor progression. Both processes could endow tumor cells with the ability to adapt themselves to unfavorable microenvironments allowing them to acquire new characteristics with increased probability of expanding clones with selective advantages. De spite extensive investigations, identification of specific cellular alterations responsible for the proliferation advantage of such metastatic clones has still remain elusive. Changes in the amplification of c-onc genes have attracted Received 6/10/88; revised 11/29/88; accepted 1/25/89. 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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' Partially supported by Consiglio Nazionale delle Ricerche Progetto Finaliz zato 'Oncologia' Grant 870158344, by Associazione Italiana per la Ricerca sul Cancro, and by Oak Ridge National Laboratory . 2To whom requests for reprints should be addressed, at Laboratorio di On- cogenesi Molecolare, Istituto Regina Elena per lo studio e la cura dei tumori. Viale Regina Elena, 291-00161 Rome, Italy. considerable attention and have been proposed as important determinants in tumor progression (1-3), although recent stud ies on animal cells indicate that a direct relationship between one gene expression and tumor progression cannot be estab lished with certainty (4, 5). one genes have been identified by their expression as viral proteins or by transfection experiments (6, 7) and therefore, it is possible, that there is a large number of expressed one genes which are not easily identified by these techniques. Polyclonal or monoclonal antibodies have been used to iden tify numerous tumor markers which are preferentially expressed on tumor versus normal cells or on metastatic versus primary tumor cells, and, therefore, they could be helpful tools to detect products of one genes not previously identified. Recently, we have reported that expression of TSP-180,3 correlates with metastatic potential of Lewis lung carcinoma (3LL) clones (8, 9) and that monoclonal antibody (MoAb 135-13C) to TSP-180 stimulates tumor cell growth in vitro (10). In this paper further biochemical characterization of TSP- 180 on highly malignant metastatic cells demonstrate that this tumor surface protein is glycosylated, phosphorylated, and rec ognized by a monoclonal antibody to phosphotyrosine. More over, it is reported that addition of MoAb 135-13C to intact cells in the presence of 32PO4 induces specific phosphorylation of a Mr 204,000 protein of the TSP-180. Phosphoaminoacid analysis of this protein indicates that serine and tyrosine are both phosphorylated. MATERIALS AND METHODS Tumor Lines. In vivo tumor cell lines M1087 and BM 21548 (11), and the parental line (OL) of 3LL were passaged in 2-3-month-old male C57B1/6 mice. Each mouse was injected in the leg muscle with 0.1 ml of a suspension containing 2.5 x IO5 viable tumor cell. Lung métastaseswere evaluated 21 days after tumor implantation. Line 1 is a cell line established from a murine type 2 alveolar carcinoma that arose spontaneously in a female BALB/c mouse (12). This line is highly malignant and metastatic. In vitro C87 and BC215 clones were derived from spontaneous métastases of 3LL parental line and were maintained in culture according to previously reported methods (13). Spontaneous and artificial métastaseswere evaluated in C57BI/6 mice injected i.m. or i.V.,respectively, with 2.5 x IO5viable cells. Tumor-bearing animals were sacrificed 19 (artificial) or 24 (spontaneous) days after injections and lungs removed and fixed in Bouin's solution. Lung nodules and métastases were counted by the aid of a dissecting microscope (14). Monoclonal Antibodies. The generation and the characterization of MoAb 135-13C (IgG2a) have been previously described (12). MoAb 346-11 A, a monoclonal antibody to a second epitope of TSP-180 protein complex, was produced by hybridomas formed from spleen cells of F344 rat, immunized with TSP-180 protein purified by immu- noaffinity chromatography (15). Rat MoAb 133-13A, showing same isotype as MoAb 135-13C (IgG2a) and used as positive control, was from a hybridoma isolated from the same fusion as 135-13C and binds 'The abbreviations used are: TSP-180, tumor surface protein (M, 180,000); MoAb, monoclonal antibody; 3LL, Lewis lung carcinoma; WGA, wheat germ agglutinin; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electropho resis; BSA, bovine serum albumin; NP-40, Nonidet P-40; PBS, phosphate buff ered saline; PMSF, phenylmethylsulphonyl fluoride. 2615 on June 12, 2015. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from