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J Taylor-Papadimitriou

Publications and source records attributed to J Taylor-Papadimitriou.

At least 19 recordsLinked to original sources
PubMed1995

Mechanisms underlying aberrant glycosylation of MUC1 mucin in breast cancer cells.

The product of the MUC1 gene, the polymorphic epithelial mucin (PEM) is aberrantly glycosylated in breast and other carcinomas, resulting in exposure of normally cryptic peptide epitopes. PEM expressed by breast cancer cells contains more sialylated O-glycans and has a lower GlcNAc content than that expressed by normal cells. The exposure of peptide epitopes is thus thought to be due to the sugar side chains being shorter on the tumour-associated mucin. To investigate possible mechanisms underlying the different pattern of glycosylation in breast cancer cells, we analysed the pathways involved in the biosynthesis of O-glycan chains of mucins in normal and cancerous mammary epithelial cells. An immortalized mammary epithelial cells line originating from normal human milk. MTSV1-7, and three human breast cancer cell lines, BT20, MCF-7 and T47D, were studied. Glycosyltransferase activities assembling, elongating and terminating O-glycan core-1 [Gal beta 1-3GalNAc alpha-R] and core-2 [GlcNac beta 1-6 (Gal beta 1-3) GalNAc alpha-R] were present in the normal mammary cell line. Many of the glycosyltransferase activities were also expressed at variable levels in breast cancer cells. However, a sialyltransferase activity (CMP-sialic acid Gal beta 1-3GalNAc alpha 3-sialyltransferase) was increased several fold in all three cancer cell lines. Moreover, mammary cancer cell lines BT20 and T47D have lost the ability to synthesize core-2, as shown by the lack of UDP-GlcNAc: Gal beta 1-3GalNAc (GlcNAc to GalNAc) beta 6-GlcNAc-transferase activity, which corresponded to the absence of the mRNA transcript. However, MCF-7 breast cancer cells expressed this enzyme. Thus, the mechanism for the exposure of peptide epitopes in BT20 and T47D cells is proposed to be the loss of core-2 branching leading to shorter, sialylated O-glycan chains. A different mechanism is proposed for MCF-7 breast cancer cells.

Amino Acid Sequence↗
PubMed1994

Branching morphogenesis of human mammary epithelial cells in collagen gels.

To study the morphogenesis of human epithelial cells in vitro we have used a three-dimensional collagen matrix and a newly developed mammary epithelial cell line, 1-7 HB2. In standard medium 1-7 HB2 cells formed compact balls/spheres inside collagen type I gels, while cocultivation with various fibroblast cell lines or growth in fibroblast-conditioned media resulted in the appearance of branching structures. At least two different soluble factors secreted by fibroblasts were found to be implicated in the branching morphogenesis. Firstly, hepatocyte growth factor/scatter factor could induce branching in a concentration-dependent manner. Moreover, a polyclonal serum against hepatocyte growth factor/scatter factor completely inhibited the branching morphogenesis induced by medium conditioned by MRC-5 fibroblast cells. In contrast, a morphogenetic activity secreted by human foreskin fibroblasts was identified that appears to be different from hepatocyte growth factor/scatter factor and from a number of other well-characterized growth factors or cytokines. This model system has been used to examine the role of integrins in mammary morphogenesis. The expression of the alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 4 integrins was decreased when cells were plated on collagen gels. The addition of specific blocking monoclonal antibodies directed to the alpha 2- and beta 1-integrin subunits to growth media impaired cell-cell interactions and interfered with the formation of compact structures inside collagen gels, suggesting that the alpha 2 beta 1 integrin can control intercellular adhesion in mammary morphogenesis. In contrast one of the blocking monoclonal antibodies against the alpha 3-integrin subunit (P1B5) mimicked the effect of soluble 'morphogens'. Our results suggest that the modulation of alpha 3 beta 1 activity may represent an important event in the induction of branching morphogenesis of human mammary epithelial cells.

Animals↗
PubMed1993

Effect of modification of carbohydrate side chains on the reactivity of antibodies with core-protein epitopes of the MUC1 gene product.

The product of the MUC1 gene, the polymorphic epithelial mucin (PEM), contains a large domain consisting of tandem repeats of 20 amino acids. Each repeat contains five potential sites for O-glycosylation, suggesting that this region forms a scaffold for the attachment of the O-linked carbohydrate which makes up more than 50% of the molecule. A number of monoclonal antibodies have been shown to recognize core-protein epitopes within the tandem repeat domain. One such antibody, SM3, reacts with the mucin expressed by breast carcinomas but shows little or no reaction with normal resting or lactating breast. Using primary mammary epithelial cells (HuME), an immortalized cell line derived from HuME (MTSV1-7) and a breast carcinoma cell line (BT20) the influence of the carbohydrate side chains on the binding of antibodies to core-protein epitopes has been investigated. We unequivocally show that the masking of the SM3 epitopes in normal breast epithelial cells is due to the carbohydrate side chains. In addition we demonstrate that the binding of two other antibodies (HMFG1, HMFG2) to core-protein epitopes is influenced by the carbohydrate side chains. The binding of HMFG-1 is particularly affected by sialic acid whereas the binding of HMFG2 is influenced by the length of the oligosaccharide side chains. Furthermore, inhibited elongation of O-linked carbohydrate side chains does not seem to interfere with the cell trafficking of the mucin to the cell surface.

Antibodies, Monoclonal↗
PubMed1993

Recombinant vaccinia mucin vector: in vitro analysis of expression of tumor-associated epitopes for antibody and human cytotoxic T-cell recognition.

We have constructed a recombinant vaccinia virus vector that contains human mucin MUC-1 cDNA. Analysis of the recombinant virus isolates showed the tendency of the vaccinia to delete large portions of the mucin tandem repeat region. Epstein-Barr virus (EBV)-immortalized B cell lines from humans and chimpanzees were infected and analyzed for expression of the mucin on the cell surface and the presence of specific epitopes in the tandem repeat region previously shown to be preferentially expressed on tumor cells and recognized by tumor-specific mouse monoclonal antibodies and human cytotoxic T lymphocytes (CTL). We found that this recombinant vector encodes expression of mucin that contains all the epitopes recognized by the antibodies. The tumor-specific epitopes can be further exposed by inhibition of O-linked glycosylation in infected cells. Lack of multiple tandem repeats, however, prevents major histocompatibility complex (MHC)-unrestricted recognition by the CTL of the majority of infected cell lines. Still, we show two examples of an apparent MHC-restricted recognition of vaccinia-encoded mucin that may depend on one or very few rare human lymphocyte antigen (HLA) types capable of presenting the mucin peptides.

Amino Acid Sequence↗
PubMed1992

Studies of clonal cell lines developed from primary breast cancers indicate that the ability to undergo morphogenesis in vitro is lost early in malignancy.

In an attempt to obtain cell lines from the different components found in primary breast cancers, we used a low-calcium medium to culture epithelial cells of mixed phenotype and a recombinant T antigen containing retrovirus to immortalize cells found in these cultures. In each case, the histology of the sample used for culture was examined in detail and the best growth was obtained from samples associated with a substantial in situ or benign component and from lobular rather than ductal carcinomas. Clonal cell lines were developed from each of 4 tumours: 1 infiltrating ductal (tumour number 2), 2 infiltrating lobular (tumours 3 and 5) and 1 mucoid (tumour 6). To try to identify the phenotype and origin of the cell lines, immunohistochemical markers, histological analysis of tissue sections and behavioural markers were used. All the cell lines expressed mainly luminal epithelial cell markers, but the basal epithelial keratin, keratin 14, was also expressed homogeneously or heterogeneously. Growth in agar was seen with some but not all cell lines derived from only 1 tumour (tumour 5) and tumour development in nude mice was observed (with low efficiency) with cell lines from only 1 tumour (tumour 6). The data suggest that the cell lines obtained from the infiltrating ductal carcinoma (tumour 2) developed from cells cultured from the associated benign component, while the cell lines from tumours 3, 5 and 6 may each have developed from a cell in an early stage of malignancy. When tested for their ability to undergo morphogenesis on extracellular matrix components, cell lines from tumour 2 made well-developed ductal-alveolar-like structures, while those from the other tumours did not.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

Antibodies to the cytoplasmic domain of the MUC1 mucin show conservation throughout mammals.

An antiserum against the carboxy-terminal seventeen amino acids of the human MUC1 mucin has been raised and extensively characterized. This antiserum, CT1, immunoprecipitates two high molecular weight polymorphic bands (greater than 200 kDa) from a metabolically labelled breast cancer cell line corresponding to the two alleles which have previously been shown to contain different numbers of a twenty amino acid repeat. The CT1 antiserum reacted with tissues from many mammalian species and immunoprecipitated large polymorphic proteins, suggesting that the cytoplasmic portion of the molecule is well conserved. The cell and tissue distribution of Muc-1 mucin in the mouse has been studied by immunocytochemistry. This protein is abundant at the apical surfaces of epithelial tissues and is found expressed in the stomach, kidney, mammary gland, pancreas, salivary gland, lung, trachea, uterus, cervix and vagina.

Amino Acid Sequence↗
PubMed1992

Tissue-specific expression of a human polymorphic epithelial mucin (MUC1) in transgenic mice.

The human MUC1 gene codes for the core protein of a mucin which is expressed by glandular epithelia and the carcinomas which develop from these tissues. The core protein is aberrantly glycosylated in cancers, and some antibodies show specificity in their reactions with the cancer-associated mucin, which also contains epitopes recognized by T-cells from breast and pancreatic cancer patients. For evaluating the potential use of mucin-reactive antibodies and mucin-based immunogens in cancer patients, a mouse model, expressing the MUC1 gene product PEM (polymorphic epithelial mucin) as a self antigen, would be extremely useful. To this end, we have developed transgenic mouse strains expressing the human MUC1 gene product in a tissue-specific manner. The TG4 mouse strain was established using a 40-kilobase fragment containing 4.5 kilobases of 5' and 27 kilobases of 3' flanking sequence. The TG18 strain was developed using a 10.6-kilobase SacII fragment from the 40-kilobase fragment; this fragment contained 1.6 kilobases of 5' sequence and 1.9 kilobases of 3' flanking sequence. Both strains showed tissue specificity of expression of the MUC1 gene, which was very similar to the profile of expression seen in human tissues. The antibody SM-3 is directed to a core protein epitope, which is selectively exposed in breast cancers and which shows a more restricted distribution on normal human tissues. It was established that the distribution of the SM-3 epitope of PEM in the tissues of the transgenic mice is similar to that seen in humans. The transgenic mouse strains described here should form the basis for the development of a preclinical model for the evaluation of PEM-based antigens and of antibodies directed to PEM in cancer therapy.

Animals↗
PubMed1992

Collagen-induced rapid morphogenesis of human mammary epithelial cells: the role of the alpha 2 beta 1 integrin.

The cell line MTSV1-7, originally derived by immortalizing mammary epithelial cells cultured from human milk was able to form three-dimensional structures in collagen gel. We have now found that these cells, cultured as a monolayer, are able to undergo rapid morphogenesis forming ridges and balls around collagen fibres, when soluble collagen type I is added to the medium. Monoclonal antibodies to the alpha 2 (P1E6) and beta 1- (mAB13) subunits of VLA-2, but not to the alpha 3-subunit (P1B5) of VLA-3, could block this collagen-induced rapid morphogenesis (CIRM). The effect of the antibodies on cell attachment, spreading, and migration on collagen gels was analyzed to identify alpha 2 beta 1 dependent steps which might be involved in CIRM. The results suggest that while other proteins, besides alpha 2 beta 1, are also involved in cell attachment and migration, cell spreading was specifically blocked by antibodies to the VLA-2, but not to the VLA-3 integrin. The results demonstrate that the alpha 2 beta 1 integrin plays a crucial role in the collagen-induced morphogenesis of human mammary epithelial cells and implicate the process of VLA-2-dependent cell spreading as an important step in this morphogenesis.

Antibodies, Monoclonal↗
PubMed1992

Isolation of azatyrosine-induced revertants from ras-transformed human mammary epithelial cells.

Non-transformed revertant clones were isolated from the ras-transformed MTSV1-7 (ras) cell line, after treatment with the antibiotic azatyrosine. Azatyrosine significantly inhibited the growth of the ras-transformed cells but not of the normal MTSV1-7. After 7 days of azatyrosine treatment, approximately 30% of MTSV1-7 (ras) cells survived, and revertant cell lines were selected by random cloning. The azatyrosine-induced revertants (six clones) were considered non-transformed on the basis of (a) their substantially reduced ability to form colonies in soft agar, and (b) their inability to produce tumours in nude mice. Molecular analysis of the revertants revealed that each contains multiple copies of the v-H-ras gene and expresses high levels of v-H-ras mRNA, and all revertants sustain elevated levels of p21ras protein. Thus, the revertant phenotype induced by azatyrosine does not result from inactivation of v-H-ras oncogene or inhibition of its expression. In vivo guanine nucleotide binding to p21ras in the revertant cell lines demonstrated binding of both GTP and GDP, indicating that reversion to the non-transformed phenotype was not due to inability of p21ras to bind GTP. The expression of the human K-rev-1 gene, a known tumour-suppressor gene in ras-transformed NIH3T3 cells, was studied in the isolated azatyrosine revertants. All six revertants showed a significant increase in the K-rev-1 transcript levels compared with the ras-transformed MTSV1-7 cells. These results suggest that tumorigenic transformation of human mammary epithelial cells by v-H-ras may be influenced by the level of expression of the tumour-suppressor gene, K-rev-1.

Alanine↗
PubMed1991

Expression of the gene coding for a human mucin in mouse mammary tumor cells can affect their tumorigenicity.

The human epithelial mucin which is the product of the MUC1 gene is expressed by many carcinomas, including those of breast, ovary, colon, and lung. The core protein is aberrantly glycosylated in the tumors resulting in the exposure or appearance of novel epitopes. To examine the possibility of using the MUC1 gene and its products in active immunization against breast and other carcinomas, we have developed a syngeneic mouse model, by transfecting the gene into the mouse mammary epithelial tumor cell 410.4. An 8.3-kilobase EcoRI fragment of the gene was transfected using the expression vector pEMSV scribe alpha 2. Transcripts of the correct size, initiating from the transcriptional start site seen in human cells, were observed in the transfectants. The mucin was expressed in the cytoplasm and in the membrane, and the glycosylation pattern appeared to be similar to that seen in human tumor cells, since the core protein epitopes recognized by antibodies HMFG-1, HMFG-2, and SM-3 were exposed. The 410.4 transfectants expressing the human mucin showed a reduction in tumor incidence at low inocula and a delay in tumor growth at higher inocula. Pretreatment with 10(4) transfected cells could inhibit the development of tumors from a subsequent inoculum of 10(6) transfectants, but had no effect on the tumor development of the untransfected 410.4 cells. Our results suggest that the human mucin expressed by the 410.4 cells may mobilize an immune response which inhibits tumor development. They also indicate that the mouse model will be useful for evaluation of efficacy of immunogens based on the MUC1 gene and its product.

Animals↗
PubMed1991

Cytotoxic T-lymphocytes derived from patients with breast adenocarcinoma recognize an epitope present on the protein core of a mucin molecule preferentially expressed by malignant cells.

A population of tumor-reactive cytotoxic T-cells can be propagated from tumor-draining lymph nodes of patients with breast adenocarcinoma. These T-cells specifically recognize breast and pancreatic tumor cells in a major histocompatibility complex (MHC)-unrestricted fashion but not other tumors of epithelial origin or the natural killer target K562. The tumor-specific but MHC-unrestricted lytic activity of these cytotoxic T-lymphocytes (CTLs) is mediated through the alpha/beta T-cell receptor. The molecule recognized by these CTLs is ductal epithelial mucin produced by breast and pancreatic adenocarcinomas. The protein core of the mucin consists of multiple tandem repeats of a 20-amino acid sequence. Antibody SM3, directed against a determinant on the mucin protein core preferentially expressed on malignant cells is able to significantly inhibit lysis of tumor cells by the CTL, while other antibodies binding to different core epitopes are not. Normal breast epithelial lines, which also express mucin but not the SM3 epitope, are not lysed by these tumor-reactive CTLs or act as cold target inhibitors of lysis of tumor lines. The data suggest that the highly repetitive nature of the mucin allows cross-linking of the T-cell receptor on mucin-specific T-cells and therefore accounts for the lack of MHC restriction seen in this system. They further suggest that the mucin core epitope recognized on tumor cells is not expressed on normal epithelial cells in a manner that can be recognized by tumor-reactive CTLs. These findings support the role of mucins as important tumor-associated antigens mediating the cellular response to certain human cancers and suggest that epithelial mucin core sequences might form the basis for an effective vaccine to augment the antitumor immune response.

Adenocarcinoma↗
PubMed1991

Efficient immortalization of luminal epithelial cells from human mammary gland by introduction of simian virus 40 large tumor antigen with a recombinant retrovirus.

When defined in terms of markers for normal cell lineages, most invasive breast cancer cells correspond to the phenotype of the common luminal epithelial cell found in the terminal ductal lobular units. Luminal epithelial cells cultured from milk, which have limited proliferative potential, have now been immortalized by introducing the gene encoding simian virus 40 large tumor (T) antigen. Infection with a recombinant retrovirus proved to be 50-100 times more efficient than calcium phosphate transfection, and of the 17 cell lines isolated, only 5 passed through a crisis period as characterized by cessation of growth. When characterized by immunohistochemical staining with monoclonal antibodies, 14 lines showed features of luminal epithelial cells and of these, 7 resembled the common luminal epithelial cell type in the profile of keratins expressed. These cells express keratins 7, 8, 18, and 19 homogeneously and do not express keratin 14 or vimentin; a polymorphic epithelial mucin produced in vivo by luminal cells is expressed heterogeneously and the pattern of fibronectin staining is punctate. Although the cell lines have a reduced requirement for added growth factors, they do not grow in agar or produce tumors in the nude mouse. When the v-Ha-ras oncogene was introduced into two of the cell lines by using a recombinant retrovirus, most of the selected clones senesced, but one entered crisis and emerged after 3 months as a tumorigenic cell line.

Animals↗
PubMed1991

Morphological differentiation of hybrids of human mammary epithelial cell lines is dominant and correlates with the pattern of expression of intermediate filaments.

Hybrids have been developed from two cell lines originally derived from human mammary epithelial cells. MTSV1-7 (hygro), developed from cultured milk epithelial cells, shows a cuboidal morphology, expresses high levels of keratins (but no vimentin), and forms ball-like three-dimensional structures in collagen gels. 5.3.1E (neo), derived from a cell cultured from tissue taken from a primary breast cancer, shows an elongated morphology, expresses high levels of vimentin and low levels of keratins, and does not form structures on collagen gels. An examination of the hybrids formed by fusion of MTSV1-7 cells and 5.3.1E cells showed that while all could form three-dimensional structures in collagen gels, the type of structures formed resembled either ductal or alveolar-ball-like structures depending on the individual hybrid. Nine hybrids were examined and a clear correlation was observed between cell shape as seen on plastic, intermediate filament expression, and the form of the structures as seen in collagen gels. All the hybrids resembling the parent MTSV1-7 cells by showing a cuboidal morphology and high level of keratin expression formed ball-like structures; on the other hand, hybrids resembling 5.3.1E by showing an elongated morphology and a high level of vimentin expression formed duct-like structures in collagen gels. The results show that the ability to form structures in collagen gels was inherited in a dominant fashion from the MTSV1-7 parent. They also suggest that the profile of intermediate filament expression in the hybrids may influence both cell shape on plastic and the morphology of the three-dimensional structures formed in collagen gels.

Blotting, Southern↗
PubMed1991

Structure and biology of a carcinoma-associated mucin, MUC1.

Although mucins have been studied at the biochemical and biophysical level for some time, attempts to define their structures in detail were only partially successful because of their size and complexity. The advent of monoclonal antibodies reactive with these molecules introduced a new approach to structural studies by defining antigenic epitopes, by allowing purification of the mucin molecules by affinity chromatography, and by providing a means to clone genes coding for the core proteins. By their profile of reactivity with the normal and cancer-associated mucin in a particular tissue, the antibodies also defined a difference in the mucin derived from the two sources. It is now clear that this difference lies in the carbohydrate side chains, as the core proteins are identical. Because the mucins are tumor-associated antigens and the cancer mucins can express epitopes that are relatively tumor specific, this family of molecules is now being intensively studied. There is also considerable interest in elucidating the normal function of the mucin and in determining whether, through an altered structure, this function is subverted in malignancy. In the next few years we should expect that the structure of other mucins will be defined in the same detail as the product of the MUC1 gene. We should also expect to see the continued application of mucin-reactive antibodies in the clinic and the investigation of mucins as agents for immunotherapy of some cancers. As to the function(s) of these molecules, perhaps we will learn enough in the future to make a critical reappraisal of the name.

Amino Acid Sequence↗
PubMed1990

Structure and expression of the human polymorphic epithelial mucin gene: an expressed VNTR unit.

The human polymorphic epithelial mucin (PEM) is expressed apically by glandular epithelium and by the carcinomas that develop from these tissues. Previously isolated cDNA clones revealed that the core protein contained a large domain consisting of variable numbers of 60 bp tandem repeats (TR), making it an expressed minisatellite. We now report the full genomic sequence of the PEM gene, including 803 bp of 5' flanking sequence. The gene is composed of 7 exons and varies in size from approximately 4 to approximately 7 kb, depending on the number of tandem repeats in exon 2. Expression of PEM was obtained from a genomic clone in an Epstein-Barr virus based vector, after transfection into a human epithelial cell line, indicating the presence of effective regulatory sequences in this clone.

Amino Acid Sequence↗