Human mammary cells present on the cell surface a polymorphic epithelial mucin (PEM) which is developmentally regulated and aberrantly expressed in tumors. PEM carries tumor-associated epitopes recognized by monoclonal antibodies HMFG-1, HMFG-2, SM-3. Previously isolated partial cDNA clones revealed that core protein contained large domain consisting of variable numbers 20-amino acid repeat units. We now report full sequence for PEM, as deduced from sequences. The encoded consists three...

Three hybridomas producing monoclonal antibodies (IgG), reacting with components of the human mammary milk fat globule have been isolated. When tested for binding to a wide range cell lines and strains, all three show negative reactions fibroblasts, lymphoblastoid cells, large number epithelial non-breast origin. Two (1.10.F3 3.14.A3) reacted seven out eight breast cancer tested, cells cultured from milk. The other antibody (3.15.C3) only two lines.

The nucleotide sequences of partial cDNA clones coding for the core protein a human polymorphic epithelial mucin were determined, and large domain was found to consist 60-base pair tandem repeat sequence. originally selected (Gendler, S. J., Burchell, J. M., Duhig, T., Lamport, D., White, R., Parker, Taylor-Papadimitriou, (1987) Proc. Natl. Acad. Sci. U. A. 84, 6060-6064) using three monoclonal antibodies which show differential reactivity with produced by normal malignant breast. Two...

Abstract The monoclonal antibody (MAb) SM‐3, which was raised to chemically deglycosylated milk mucin, reacts with an epitope present on the core protein of this mucin we have referred as PEM (polymorphic epithelial mucin). Although is abundantly expressed by both lactating breast and carcinomas, SM‐3 shows very little or no reactivity former but does react 92% carcinomas. Furthermore, stains primary carcinomas lung, colon ovary, corresponding normal tissue at a much reduced level not all....

Abstract Introduction The identification of potential breast cancer stem cells is importance as the characteristics suggest that they are resistant to conventional forms therapy. Several techniques have been proposed isolate or enrich for tumorigenic cells, including (a) culture in non-adherent non-differentiating conditions form mammospheres and (b) sorting by their surface phenotype (expression CD24 CD44). Methods We cultured metastatic found pleural effusions from patients without serum...

Abstract MUC1 is a highly attractive immunotherapeutic target owing to increased expression, altered glycosylation, and loss of polarity in >80% human cancers. To exploit this, we have constructed panel chimeric Ag receptors (CAR) that bind selectively tumor-associated MUC1. Two parameters proved crucial optimizing the CAR ectodomain. First, observed binding CAR-grafted T cells anchored subject steric hindrance, independent glycosylation status. This was overcome by insertion flexible...

MUC-1 mucin is considered to be aberrantly glycosylated in breast, ovary, and other carcinomas comparison with from corresponding normal tissues. In order clarify these differences glycosylation, we have compared the O-linked carbohydrate chains immunoprecipitated [3H]GlcN-labeled breast epithelial cell lines (MMSV1-1, MTSV1-7, HB-2) derived cells cultured human milk, three cancer (MCF-7, BT-20, T47D). Analysis by high pH anion chromatography showed that had a higher ratio of GlcN/GalN more...

The product of the MUC1 gene, polymorphic epithelial mucin (PEM) is aberrantly glycosylated in breast and other carcinomas, resulting exposure normally cryptic peptide epitopes. PEM expressed by cancer cells contains more sialylated O‐glycans has a lower GlcNAc content than that normal cells. epitopes thus thought to be due sugar side chains being shorter on tumour‐associated mucin. To investigate possible mechanisms underlying different pattern glycosylation cells, we analysed pathways...

Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptideN-acetylgalactosaminyltransferases (GalNAc-transferases). The role each GalNAc-transferase plays in unclear. In this report we characterized the specificity and kinetic properties of three purified recombinant GalNAc-transferases. GalNAc-T1, -T2, -T3 were expressed as soluble proteins insect cells to near homogeneity. enzymes have distinct but partly overlapping specificities with short peptide acceptor...

Human mammary epithelial cells secrete and express on their cell surfaces complex mucin glycoproteins (Mr greater than 250,000) that are developmentally regulated, tumor-associated, highly immunogenic. Studies using monoclonal antibodies directed to these suggest molecular structures can vary with differentiation stages in the normal gland malignancy. To analyze nature of glycoproteins, milk was affinity-purified deglycosylated hydrogen fluoride, yielding bands at 68 72 kDa silver-stained...

The MUC1 mucin represents a prime target antigen for cancer immunotherapy because it is abundantly expressed and aberrantly glycosylated in carcinomas. Attempts to generate strong humoral immunity by immunization with peptides have generally failed partly of tolerance. In this study, we developed chemoenzymatic synthesis extended TR glycopeptides cancer-associated O-glycosylation using panel recombinant human glycosyltransferases. different densities Tn STn glycoforms conjugated KLH were...

The cell membrane mucin MUC1 is over-expressed and aberrantly glycosylated in many cancers, cancer-associated glycoforms represent potential targets for immunodiagnostic therapeutic measures. We have recently shown that with GalNAcα1-O-Ser/Thr (Tn) NeuAcα2-6GalNAcα1-O-Ser/Thr (STn) O-glycosylation a cancer-specific glycoform, Tn/STn-MUC1 glycopeptide-based vaccines can override tolerance human transgenic mice induce humoral immunity high specificity (Sorensen AL, Reis CA, Tarp MA, Mandel U,...

Autoantibodies to cancer antigens hold promise as biomarkers for early detection of cancer. Proteins that are aberrantly processed in cells likely present autoantibody targets. The extracellular mucin MUC1 is overexpressed and glycosylated many cancers; thus, we evaluated whether autoantibodies generated aberrant O-glycoforms might serve sensitive diagnostic Using an antibody-based glycoprofiling ELISA assay, documented truncated glycoforms were not detected sera patients. An O-glycopeptide...

Abstract The polymorphic epithelial mucin (PEM) appears to be the target molecule for many monoclonal antibodies (MAbs) which react with tumour‐associated and epithelium‐specific antigens. PEM contains a large domain made up of 20 aminoacid tandem repeats are highly immunodominant as reactive this recognize epitopes within area. Using overlapping peptide octamers, we have precisely mapped 4 MAbs including one, SM‐3, shows enhanced tumour specificity. We report that core SM‐3 epitope...

The functional properties of glycoproteins are strongly influenced by their profile glycosylation, and changes in this seen malignancy. In mucin-type O-linked glycosylation these can result the production mucins such as MUC1, carrying shorter sialylated O-glycans, with different site occupancy. Of tumor-associated disaccharide, sialyl-Tn (sialic acid α2,6GalNAc), is expressed 30% breast carcinomas most tumor-specific. ST6GalNAc-I glycosyltransferase, which catalyze transfer sialic to GalNAc,...

A fourth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T4, was cloned and expressed. The genomic organization of GalNAc-T4 is distinct from GalNAc-T1, -T2, -T3, which contain multiple coding exons, in that the region contained a single exon. placed at chromosome 12q21.3-q22 by situ hybridization linkage analysis. expressed Sf9 cells or stably transfected Chinese hamster ovary cell line exhibited unique acceptor substrate specificity. transferred GalNAc to...

Detection of serum biomarkers for early diagnosis breast cancer remains an important goal. Changes in the structure O-linked glycans occur all cancers resulting expression glycoproteins that are antigenically distinct. Indeed, assay widely used monitoring disease progression (CA15.3), detects a glycoprotein (MUC1), but elevated levels antigen cannot be detected stage patients. However, since immune system acts to amplify antigenic signal, antibodies can sera long before antigen. We have...