Joy Burchell

Comparison of O-Linked Carbohydrate Chains in MUC-1 Mucin from Normal Breast Epithelial Cell Lines and Breast Carcinoma Cell Lines: DEMONSTRATION OF SIMPLER AND FEWER GLYCAN CHAINS IN TUMOR CELLS

MUC-1 mucin is considered to be aberrantly glycosylated in breast, ovary, and other carcinomas in comparison with mucin from corresponding normal tissues. In order to clarify these differences in glycosylation, we have compared the O-linked carbohydrate chains from MUC-1 immunoprecipitated from [3H]GlcN-labeled breast epithelial cell lines (MMSV1-1, MTSV1-7, and HB-2) derived from cells cultured from human milk, with three breast cancer cell lines (MCF-7, BT-20, and T47D). Analysis by high pH anion chromatography showed that the normal cell lines had a higher ratio of GlcN/GalN and more complex oligosaccharide profiles than the cancer cell lines. Structural analyses were carried out on the oligosaccharides from MTSV1-7 and T47D MUC-1, and the following structures were proposed. MUC-1 from T47D had rather a simple glycosylation pattern, with NeuAcα2-3Galβ1-3GalNAc-ol, Galβ1-3GalNAc-ol, and GalNAc-ol predominating; in contrast, MUC-1 from MTSV1-7 had more complex structures, including a number of disialo, core 2 species, i.e. NeuAcα2-3Galβ1-4GlcNAcβ1-6[NeuAcα2-3Galβ1-3]GalNAc-ol and NeuAcα2-3Galβ1-4GlcNAcβ1-6[NeuAcα2-3Galβ1-4GlcNAcβ1-3Galβ1-3]GalNAc-ol. Double-labeling experiments with [3H]GlcN and 14C-aminoacids and analysis of GalNAc or GalNAc-ol:protein ratios in MUC-1 showed that there was also a significant difference in the degree of glycosylation of the mucin between the two cell types. We conclude that MUC-1 from breast cancer cell lines has simpler, and fewer, carbohydrate chains than MUC-1 from normal breast epithelial cells, and that these differences, combined or separately, explain the differential tumor specificity of some MUC-1 antibodies and T cells.

Androgen-dependent synthesis of basic secretory proteins by the rat seminal vesicle.

1. Two basic proteins were purified from secretions of rat seminal vesicles by using Sephadex G-200 chromatography and polyacrylamide-gel electrophoresis under denaturing conditions. 2. It is not certain that these two proteins are distinct species and not subunits of a larger protein, but their properties are similar. Highly basic (pI = 9.7), they migrate to the cathode at high pH and their amino acid composition shows them to be rich in basic residues and serine. Threonine and hydrophobic residues are few. Both proteins are glycoproteins and have mol.wts. of 17000 and 18500. 3. Together these two proteins account for 25-30% of the protein synthesized by the vesicles, but they are absent from other tissues. 4. Changes in androgen status of the animal markedly affect these proteins. After castration, a progressive decrease in the basic proteins is observed and the synthesis of the two proteins as measured by [35S]methionine incorporation in vitro is is decreased. Testosterone administration in vivo rapidly restores their rates of synthesis. 5. These effects on specific protein synthesis are also observed for total cellular protein, and it is suggested that testosterone acts generally on the total protein-synthetic capacity of the cell and not specifically on individual proteins. Proliferative responses in the secretory epithelium may also be involved. 6. The extreme steroid specificity of the induction process suggests that the synthesis of these basic proteins is mediated by the androgen-receptor system. 7. The biological function of these proteins is not clear, but they do not appear to be involved in the formation of the copulatory plug.

The polymorphic epithelial mucin : potential as an immunogen for a cancer vaccine

Abstractu2002The identification and cloning of several tumour antigens together with an improvement in the understanding of the mechanisms involved in antigen presentation and immune recognition has opened up the possibility of using active specific immunotherapy as a treatment for certain cancers. This review discusses the tumour-associated MUC1 gene product of the polymorphic epithelial mucin (PEM), as a potential target molecule for cancer treatment. PEM is both over-expressed and aberrantly glycosylated in many carcinomas resulting in an antigenically distinct molecule. Furthermore, immune responses specific for PEM have been detected in cancer patients. Both syngeneic and transgenic murine model systems have been developed in order to compare the efficacy and toxicity of various PEM-based immunogens in tumour rejection studies, and to further improve the understanding of antigen presentation and the mechanisms underlying tumour rejection. Such models also allow the examination of MUC1-based immunogens as a treatment for existing tumours. Clinical trials in progress using immunogens based on the MUC1 gene product are briefly discussed.

The product of the human MUC1 gene when secreted by mouse cells transfected with the full-length cDNA lacks the cytoplasmic tail

The polymorphic epithelial mucin (PEM) is found as a cell associated transmembrane protein with an extracellular domain made up largely of tandem repeats and also as a soluble form in some body fluids and culture supernatants. To determine whether the soluble form can arise without the mechanism of alternative splicing mouse cells have been transfected with an expression construct containing the full-length cDNA, and the supernatants of the transfectants analyzed for the presence of the mucin. The presence of mucin in the supernatants could indeed be detected in a radioimmunoassay and by immunoprecipitation using monoclonal antibodies to the tandem repeat region of the core protein, indicating that release of the soluble form can occur without alternative splicing. The soluble form was not however precipitated with a polyclonal antiserum to the cytoplasmic tail, suggesting that it was released from the membrane by the action of a protease.

Antibodies to Human Milk Fat Globule Molecules

AbstractSince the development of the first polyclonal antiserum to human milk fat globule (HMFG) membranes (1), this immunogen has been used by numerous workers to produce polyclonal and monoclonal antibodies. These reagents have been extremely useful in identifying markers of breast epithelial differentiation and have had a number of clinical applications. In addition, monoclonal antibodies to HMFG and to many tumour-associated antigens have brought into focus a group of highly immunogenic glycoproteins which have the characteristics of mucins and are expressed by many carcinomas. In this review we will discuss mainly work which has been done with the human milk fat globule, but it should be borne in mind that the same membranes and their components have been extensively studied in rodents and in the cow.

Lack of β-Casein production by human breast tumours revealed by monoclonal antibodies

An immunohistochemical study with four monoclonal antibodies to human beta-casein was carried out to examine the expression of this milk protein in a wide range of normal tissues, in 127 breast tumours and in a heterogeneous panel of 42 malignancies of other histogenesis. The only normal tissue stained positively by the antibodies was the mammary gland in late pregnancy, during lactation and in the post-lactational regression period. None of the tumours of non-mammary origin showed any staining. Furthermore, only two of 40 benign breast lesions and one anaplastic primary carcinoma with its metastasis (among 87 breast carcinomas) showed any reactivity. The immunohistochemical results were supported by immunoblotting data and suggested beta-casein expression has no role to play as a marker in the diagnosis or monitoring of human breast cancer.

Molecular and Immunological Analysis of Mucin Molecules Produced by Normal and Malignant Human Mammary Epithelial Cells

The mammary gland is a complex tissue containing several lineages, including the luminal or secretory epithelial cells and the basal or myoepithelial cells which line the ducts and alveoli. These two cell types can be distinguished in tissue sections by immunohistochemical staining with monoclonal antibodies to structural components and to functional products characteristic of each cell type. Such studies have focused attention on the luminal or secretory epithelial cell lineage, since the dominant cell in breast cancers expresses both the simple epithelial keratins and the mucin molecules expressed by the luminal epithelial cells in the normal gland1. In these particulars the breast cancer cell resembles other adenocarcinomas, such as those from the colon, lung and ovary.

Epithelial Mucin Antibodies and Their Epitopes: Core Protein Epitopes of a Polymorphic Epithelial Mucin (PEM)

Largely because of their complexity, the detailed structure of the mucins has been difficult to analyze. This group of compounds is categorized mainly by the fact that they contain a high level of carbohydrate which is attached in O-linkage to serine and/or threonine via the linkage sugar N-acetylgalactosamine. The mucous secretions produced by some epithelial cells, particularly those lining the gastro-intestinal tract and the lungs, contain mucins along with other products and these components have been studied for some time at the biochemical level. However, other glandular epithelial cells, such as the salivary gland, breast, ovary, endometrium, and sweat glands, also produce mucins, and some of these simpler mucins have recently received much attention. This is because many antibodies selected for epithelial or tumor specificity have been found to react with high molecular weight glycoproteins which are produced by simple epithelial cells and have the properties of mucins.1–9

41 The muci gene product, PEM as a target antigen in carcinomas

Most glandular epithelial cells lining ducts express on their apical surface a transmembrane glycoprotein the polymorphic epithelial mucin (PEM) which is heavily O-glycosylated. PEM is overexpressed in more than 90% of breast and ovarian carcinomas and in some lung and colon tumours. Although the sequence of the MUC1 gene coding for the core protein is not altered in the cancer associated mucin, the O-glycans which are added are shorter and more heavily sialylated. This results in the appearance of novel carbohydrate epitopes and the unmasking of core protein epitopes which lie between the O-glycosylation sites. The increased expression of an antigenically distinct molecule, which is no longer restricted in its expression to the apical surface appears to induce both Band T cell responses in some breast, ovarian and pancreatic cancer patients. To compare immunogens based on the MUC1 gene or its product, and to optimise antigen presentation, syngeneic and transgenic mouse models have been developed. In the syngeneic model, intramuscular injection of cDNA coding for the MUC1 gene (driven by the actin promoter) given before injection of MUC1-expressing mouse tumour cells can inhibit tumour growth. In the animals rejecting the tumour, cytotoxic T cells can be isolated and their development correlates to some degree with effective tumor immunity, while the appearance of circulating antibodies does not. In a mouse transgenic for the human MUC1 gene, PEM expressing tumours are rejected when antigen presentation is enhanced by expression ofthe B7 molecule.

Characterization and Evolution of an Expressed Hypervariable Gene for a Tumor-Associated Mucin, MUC-1

Mucins, present on highly polarized, secretory epithelial cells, have gained prominence in recent years as many monoclonal antibodies selected for their reactivity on differentiated or normal tissues react with epitopes present on these molecules. Although difficult to analyze biochemically because of their large size and large amount of O-linked carbohydrate, recent cloning studies from a number of different laboratories have produced structural information of the core protein. In most cases cDNA clones were obtained from λgt11 expression libraries following the development of antibodies to the stripped core protein. Thus far, three human mucin partial or full-length cDNA clones have been characterized as well as the porcine submaxillary mucin and a Xenopus integumentary mucin. In each case a domain of the core protein was found to consist of tandem repeats of a defined length. Although no homology exists between the tandem repeats of the various mucin genes at either the DNA or protein level, the repeated sequences in each case code for molecules which could be highly O-glycosylated. One characteristic feature of mucins is the presence of between 50 and 90% carbohydrate which is linked to serines or threonines via an O-glycosidic linkage to N-acetylgalactosamine. A second characteristic is the presence of prolines which along with glycosylation help to provide the extended core protein structure characteristic of mucins. All of the predicted proteins coded for by the mucin clones contain these features.