Cloning of Partial cDNA Encoding Differentiation and Tumor-Associated Mucin Glycoproteins Expressed by Human Mammary Epithelium

• Published in: Proceedings of the National Academy of Sciences - PNAS 1987-09, Vol.84 (17), p.6060-6064
• Main Authors: Gendler, S. J., Burchell, J. M., Duhig, T., Lamport, D., White, R., Parker, M., Taylor-Papadimitriou, J.
• Format: Article
• Language: English
• Subjects: Antibodies; Antibodies, Monoclonal; Antigens, Surface - genetics; Antigens, Surface - immunology; Biological and medical sciences; Biotechnology; Breast - cytology; Breast - immunology; Breast - metabolism; Breast cancer; Breast Neoplasms - genetics; Breast Neoplasms - immunology; Breast Neoplasms - pathology; Cell Differentiation; Cell Line; Cell lines; Cloning, Molecular; DNA; DNA - genetics; Epithelial Cells; Epithelium - immunology; Epithelium - metabolism; Female; Fundamental and applied biological sciences. Psychology; Gels; Gene expression; Genetic engineering; Genetic technics; Glycoproteins; Humans; Messenger RNA; Methods. Procedures. Technologies; Molecular and cellular biology; Molecular cloning; Molecular genetics; Mucins; Mucins - genetics; Mucins - immunology; Polymorphism, Restriction Fragment Length; RNA; RNA, Messenger - genetics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.84.17.6060

Human mammary epithelial cells secrete and express on their cell surfaces complex mucin glycoproteins (Mr > 25,000) that are developmentally regulated, tumor-associated, and highly immunogenic. Studies using monoclonal antibodies directed to these glycoproteins suggest that their molecular structures can vary with differentiation stages in the normal gland and in malignancy. To analyze the molecular nature of these glycoproteins, milk mucin was affinity-purified and deglycosylated with hydrogen fluoride, yielding bands at 68 and 72 kDa on silver-stained gels. Polyclonal and monoclonal antibodies to the stripped core protein were developed and used to screen a λ gt11 expression library of cDNA made from mRNA of the mammary tumor cell line MCF-7. Seven cross-reacting clones were isolated, with inserts 0.1-1.8 kilobases long. RNA blot analysis, using as a probe the 1.8-kilobase insert subcloned in plasmid pUC8 (pMUC10), revealed transcripts of 4.7 and 6.4 kilobases in MCF-7 and T47D mammary tumor cells, whereas normal mammary epithelial cells from pooled milks have additional transcripts. The expression of mRNA correlates with antigen expression as determined by binding of two previously characterized anti-mucin monoclonal antibodies (HMFG-1 and HMFG-2) to seven cell lines. Restriction enzyme analysis detected a restriction fragment length polymorphism when human genomic DNA was digested with EcoRI or HinfI.