Isolation and sequencing of a cDNA coding for the human DF3 breast carcinoma-associated antigen.

Abstract

The murine monoclonal antibody (mAb) DF3 reacts with a high molecular weight glycoprotein detectable in human breast carcinomas. DF3 antigen expression correlates with human breast tumor differentiation, and the detection of a cross-reactive species in human milk has suggested that this antigen might be useful as a marker of differentiated mammary epithelium. To further characterize DF3 antigen expression, we have isolated a cDNA clone from a Agtll library by screening with mAb DF3. The results demonstrate that this 309-base-pair cDNA, designated pDF9.3, codes for the DF3 epitope. Southern blot analyses of EcoRI-digested DNAs from six human tumor cell lines with 32P-labeled pDF9.3 have revealed a restriction fragment length polymorphism. Variations in size of the alleles detected by pDF9.3 were also identified in Pst I, but not in HindIII, DNA digests. Furthermore, hybridization of 32P-labeled pDF9.3 with total cellular RNA from each of these cell lines demonstrated either one or two transcripts that varied from 4.1 to 7.1 kilobases in size. The presence of differently sized transcripts detected by pDF9.3 was also found to correspond with the polymorphic expression of DF3 glycoproteins. Nucleotide sequence analysis of pDF9.3 has revealed a highly conserved (G+C)-rich 60-base-pair tandem repeat. These rmdings suggest that the variation in size of alleles coding for the polymorphic DF3 glycoprotein may represent different numbers of repeats.A human breast carcinoma-associated antigen has been identified by using a murine monoclonal antibody (mAb), designated DF3. mAb DF3 was prepared against a membrane-enriched fraction of a human breast carcinoma metastatic to liver (1). DF3 antigen is expressed on the apical borders of secretory mammary epithelial cells and in the cytosol of less differentiated malignant cells (1). DF3 antigen expression also correlates with the degree of breast tumor differentiation and estrogen receptor status (2). These findings and the detection of DF3 antigen in human milk (3) The present studies were performed to further examine genetic mechanisms responsible for the heterogeneity of DF3 antigen expression. We describe the isolation of a partial cDNA clone,t designated pDF9.3, coding for the DF3 antigen. This cDNA clone encompasses 309 nucleotides of DF3 mRNA and has tandemly repeated sequences. By using this clone, we also demonstrate that the heterogeneity of DF3 antigen is related to size variations in DF3 alleles and DF3 transcripts. MATERIALS AND METHODSLibrary Screening. An oligo(dT)-primed cDNA library prepared from human MCF-7 breast carcinoma cells in Agtll was kindly provided by P. Chambon (Institut de Chimie Biologique, Strasbourg, France) (8). Immunologic screening of the Agtll library was performed as described (9) by using affinity-purified mAb DF3 (0.25 ,ug/ml) and antimouse IgG conjugated with alkaline phosphatase (Promega Biotec, Madison, WI). Positive plaques were isolated and the phage was further purified to homogeneity by repeated antibody screening. DNA was i...

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