Interspecific DNA-mediated Transfer and Amplification of a Gene Specifying a Mr 100,000 Human Melanoma-associated Cell Surface Glycoprotein1

The gene that encodes the membrane-bound M, 100,000 human mel anoma-associated antigen (M VV) defined by mouse mAb 376.96, a leukocyte and fibroblast interferon-modulated glycoprotein having pref erential distribution on melanoma and carcinoma cells, has been transfected into the mouse melanoma cell line B78H1 as a step toward molecular cloning and characterization of the MAA. Primary, secondary, and tertiary B78H1 transfectants expressing the M, 100,000 MAA gene were generated by treatment with coprecipitated DNA from M, 100,000 MAA+ human or transfectant mouse cells and they were detected by an indirect RBC resetting assay. The M, 100,000 MAA gene was also transferred into K-1735 mouse melanoma cells and into nonmalignant and malignant mouse fibroblast lines. The species immunoprecipitated by mAb 376.96 from human melanoma cells (M, 100,000) and from mouse melanoma transfectant cells (M, 97,000-100,000) were both converted to molecule(s) having an M, of approximately 70,000 by enzymatic removal of asparagine-linked carbohydrate residues. Two in dependent secondary transformant clones of B78H1 cells express M, 100,000 MAA antigenicity at levels significantly higher than those observed when one or two copies of the gene are present. Clone M, 100,000 secondary-A spontaneously overexpresses M, 100,000 MAA at least 5-fold and has >10 times elevated levels of putatively M, 100,000 MAA gene-associated human alii family repeat element (A-a/u)-positive restriction fragments relative to "single" copy secondary transfectant cells. Clone M, 100,000 secondary-B has increased copy number and expression of M, 100,000 MAA as a consequence of a selective coamplification procedure which is targeted to a mouse wild type dihydrofolate reducÃ-ase(dhfr) gene expression vector. This vector was cointroduced into B78H1 cells in addition to the DNA of M, 100,000 MAA* primary transfectant cells and the initially selected aminoglycoside phosphotransferase (neo) gene vector. Stepwise selections of a secondary M, 100,000 MAA* transfectant clone with increasing concen trations of the dihydrofolate reductase-inhibitory antimetabolite methotrexate led to progressive increases in copy numbers of the introduced dhfr gene and to parallel increases in h-alii sequences, in cellular levels of dihydrofolate reducÃ-aseprotein, and in cellular mAb 376.96 reactivity. Levels of these entities ultimately reached 50-fold, relative to levels expressed prior to amplification. The array of h-alu* restriction fragments amplified in M, 100,000 secondary-B cell DNA is very similar to that observed in M, 100,000 secondary-A cell DNA. Co-amplified transfectant cells represent both an abundant source of the M, 100,000 MAA molecule and an enriched source of the V7,100,000 MAA genomic gene and of the corresponding mRNA; the latter are currently being used to facilitate isolation of recombinant clones related to this gene. INTRODUCTION Application of mAb3 technology for topographical analysis of human melanoma cells has led to the detection and the Received 7/7/89; revised 11/17/89: accepted 11/29/89. 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. 1This work has been supported by Grants CA 31937. CA 44107 (L. H. G.). CA 37959 (S. F.), and BRSG SS07RRO5396-24, awarded by the NIH. and by The Cancer Research League of New York City (L. H. G.). 2To whom requests for reprints should be addressed, at University of Illinois at Chicago, Center for Research in Periodontal Diseases and Oral Molecular Biology. 801 South Paulina Street. Chicago, IL 60612. 3The abbreviations used are: mAb, monoclonal antibody: HMW-MAA. high molecular weight melanoma-associated antigen: MAA, melanoma-associated an tigen; MTX, methotrexate; MEM. minimum essential medium; PCS, fetal calf serum; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; DHFR. dihydrofolate reducÃ-ase:pi. isoelectric PH. immunochemical characterization of a number of MAAs (1). Certain MAAs appear to have significance as indicators of the onset or progression of neoplasia, as markers for stages of melanocytic differentiation, or as possible targets for antibodymediated diagnosis and/or therapy (for review, see Ref. 2). The recombinant cloning of genes that encode MAAs promises to facilitate the molecular and functional characterization of these antigens, to elucidate the mechanisms that dictate cell or tissue specificity of their expression, and to provide genetic ap proaches to active and passive immunotherapy of melanoma (3, 4). Strategies based on interspecific DNA-mediated gene transfer have well documented usefulness for cloning genes that specify proteinaceous cell surface molecules (5-8); accordingly, we have carried out tumor cell DNA-mediated transfections of genes that encode several human tumor-associated antigens into the highly competent mouse melanoma cell line B78H1 (9-11). A human melanomaand carcinoma-associated cell mem brane-bound glycoprotein, the M, 100,000 MAA identified by mAb 376.96, is of interest for its preferential expression by melanomas, carcinomas, and neuroblastomas (as opposed to lymphoblastoid cells) among tumors and its restriction to endothelial cells and gastric epithelia among normal tissues (12,13). In addition, expression and shedding of M, 100,000 MAA are susceptible to modulation by leukocyte and fibroblast interfer ons (14). This communication describes the transfer of the human M, 100,000 MAA gene into B78H1 cells. Evidence is also pre sented for amplification of the M, 100,000 MAA gene with concomitant overexpression of M, 100,000 MAA antigen, both as a spontaneous phenomenon and in response to a stepwise biochemical selection. The latter co-amplification procedure has resulted in isolation of clonal mouse transfectant cell pop ulations that synthesize amounts of M, 100.000 MAA that are estimated by different criteria as being elevated 15to more than 50-fold over the amount synthesized by human melanoma cells. Thus, the transfection-co-amplification procedure makes feasible the detailed characterization of the M, 100,000 MAA as it is overproduced by the murine host cells, and it potentially simplifies the use of a variety of methods for the isolation of genomic and/or complementary DNA clones corresponding to the M, 100,000 MAA gene. MATERIALS AND METHODS Cell Lines. All cell lines were cultured in a humidified 5% CO2 atmosphere at 37°C.B78H1 mouse melanoma cells, LMTK" mouse malignant fibroblasts (15) and SK-MEL-37 and A-875 human mela noma cells were cultured in Earle's buffered MEM containing 10% heat inactivated PCS (GIBCO Laboratories, Grand Island. NY), 50 ¿ig/ ml gentamycin sulfate (GIBCO), and 1.25 Mg/ml fungizone (ER Squibb and Sons, Princeton, NJ). The human melanoma cell line Colo 38 was cultured in RPMI 1640 medium (GIBCO) supplemented with 15% PCS (GIBCO), 50 ^g/ml gentamicin sulfate (GIBCO), and 1.25 ug/m\ fungizone (Squibb). The embryo fibroblast line NIH 3T3 (16) was cultured in Dulbecco's modified Eagle's medium containing 10% heat-