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J Taylor-Papadimitriou

Publications and source records attributed to J Taylor-Papadimitriou.

At least 19 recordsLinked to original sources
PubMed2004

Bioprocess development for the production of a recombinant MUC1 fusion protein expressed by CHO-K1 cells in protein-free medium.

The mucin MUC1 is a candidate for use in specific immunotherapy against breast cancer, but this requires the large-scale production of a MUC1 antigen. In this study, a bioprocess for the expression of a recombinant MUC1 fusion protein with a cancer associated glycosylation in CHO-K1 cells has been developed. Cells permanently expressing parts of the extracellular portion of MUC1 fused to IgG Fc were directly transferred from adherent growth in serum-containing medium to suspension culture in the protein-free ProCHO4-CDM culture medium. Using the Cellferm-pro system, optimal culture parameter as pH and pO(2) were determined in parallel spinner flask batch cultures. A pH of 6.8-7.0 and a pO(2) of 40% of air saturation was found to give best cell growth and productivity of secreted recombinant protein. Specific productivity strongly depended the pO(2) and correlated with the online monitored oxygen uptake rate (OUR) of the cells, which indicates a positive influence of the rate of oxidative phosphorylation on productivity. The optimised conditions were applied to continuous perfusion culture which gave very high cell densities and space time yields of the recombinant MUC1 fusion protein, allowing production at gram scale. The product degradation was much lower in supernatants from continuous perfusion culture compared to batch mode. Antibodies reacting with cancer associated MUC1 glycoforms strongly bound to the fusion protein, indicating that the desired glycoforms were obtained and suggesting that the recombinant MUC1 protein could be tested for use in immunotherapy.

Animals↗
PubMed2001

Up-regulation of MUC1 in mammary tumors generated in a double-transgenic mouse expressing human MUC1 cDNA, under the control of 1.4-kb 5' MUC1 promoter sequence and the middle T oncogene, expressed from the MMTV promoter.

In this study we examined the regulation of expression of the human MUC1 gene in vivo, by developing MUC1 transgenic mice. The data showed that epithelial-specific expression of MUC1 can be directed by just 1.4 kb of 5' flanking sequence using MUC1 cDNA as a reporter gene in vivo. Furthermore, high levels of MUC1 expression were seen in the lactating mammary gland and in spontaneous mammary tumors generated by crossing the MUC1 transgenics with mice transgenic for the polyoma middle T oncogene under the control of the mouse mammary tumor virus promoter. This pattern of expression in epithelial tissues is comparable to the expression of MUC1 in humans and also to the expression pattern in another transgenic mouse line developed with a 10.6-kb genomic MUC1 fragment. This study confirmed that MUC1 is a compact gene and demonstrated that the 1.4-kb 5' sequence not only directs epithelial-specific expression of MUC1 in vivo but also contains the elements governing the up-regulation observed during lactation and in malignancy.

Animals↗
PubMed2001

Identification of three non-VNTR MUC1-derived HLA-A*0201-restricted T-cell epitopes that induce protective anti-tumor immunity in HLA-A2/K(b)-transgenic mice.

The human epithelial mucin MUC1 is over-expressed in more than 90% of carcinomas of the breast, ovary, and pancreas as well as in some other tumours, making it a potential target for tumour immunotherapy. We have identified several MUC1-derived peptides mapping outside the variable number tandem repeat region that comply with the peptide-binding motif for HLA-A*0201 and that become processed into stable major histocompatibility complex-peptide complexes as assessed by in vitro assays. In A2/K(b) transgenic mice, 3 peptides, namely MUC(79-87) (TLAPATEPA), MUC(167-175) (ALGSTAPPV) and MUC(264-272) (FLSFHISNL) elicit peptide-specific cytotoxic T lymphocyte (CTL) immunity, which protects these mice against a challenge with MUC1, A2/K(b)-expressing tumour cells. These peptides therefore represent naturally processed MUC1-derived CTL epitopes that could be used as components in peptide-based vaccines and for the analysis of anti-MUC1 CTL responses in A*0201-positive patients with MUC1-expressing tumours.

Animals↗
PubMed2001

O-linked glycosylation in the mammary gland: changes that occur during malignancy.

Glycosylation is a very important posttranslational modification of many biologically relevant molecules. A change in the structure of glycans added to glycoproteins and glycolipids is a common feature of the change to malignancy. With the cloning of many of the glycosyltransferases and the identification of specific target molecules, it is now possible to define these changes at the molecular level and to dissect the mechanisms involved. Within the mammary gland, mucin-type O-linked glycosylation has been studied most extensively. In normal resting, pregnant and lactating breast, mucin O-glycans are largely extended (core 2 type) structures. In contrast, mucin O-glycans found in breast carcinomas are often truncated (core 1 type). One mechanism that is responsible for this increase in core 1 structures is a change in the expression of glycosyltransferases, particularly an increase in the expression of the sialyltransferase, ST3Gal-1. The loss, at least to some degree, of core 2 based glycans is a consistent feature of MUC1 mucin when it is expressed by mammary tumours as demonstrated by the unmasking of the SM3 epitope in greater than 90% of breast carcinomas.

Animals↗
PubMed2001

Breast cancer and the immune system: opportunities and pitfalls.

The identification of tumor-associated antigens, and advances in our understanding of human immunology, have resulted in renewed interest in tumor immunology. A variety of approaches have been utilized in recent years against different tumor types. The results from some of these studies have been encouraging, but it is not yet clear whether they will be applicable to patients with breast cancer.

Adult↗
PubMed2001

The Sp1 transcription factor regulates cell type-specific transcription of MUC1.

Because the MUC1 mucin is highly expressed in breast and other carcinomas, interest is focused on the MUC1 promoter, particularly in the context of the delivery of genes to carcinomas. Earlier in vitro studies showed that the region between -152 and -66 of the MUC1 promoter is required for transcriptional activity in MUC1-expressing cells. Experiments reported here showed that sequences -119/-62 within this region are able to modulate transcription of the heterologous constitutively active herpes simplex virus thymidine kinase promoter in a pattern consistent with MUC1 expression. Band-shift experiments showed that although several factors (including Sp1 and Sp3) bind to these sequences, the element important in directing this MUC1 pattern of expression was an Sp1 GC box at -97. The data also show that the positioning or phase of the GC box was crucial for directing expression. The importance of the Sp1 transcription factor was confirmed by demonstrating that overexpression of Sp1 in MUC1-nonexpressing cells increased, not only the expression of a reporter gene driven by the 1.4-kb MUC1 promoter, but also the expression of MUC1 from the endogenous gene. Together, these data define an important role for Sp1 in the cell type-specific transcription of MUC1.

Base Sequence↗
PubMed2001

Regulation of MUC1 expression in human mammary cell lines by the c-ErbB2 and ras signaling pathways.

The MUC1 protein is a highly O-glycosylated transmembrane molecule that is expressed at the luminal surface of most glandular epithelial cells and is upregulated in carcinomas. Here, we report the effect of the activation of the c-ErbB2 --> Ras pathway on the expression of the MUC1 gene in the nontumorigenic mammary cell lines MTSV1-7 and HB2 and in the malignant cell lines T47D and ZR75. Endogenous levels of MUC1 mRNA and protein in HB2 clones permanently overexpressing c-ErbB2 or V12-H-Ras were markedly reduced compared with levels in the parental cell lines. Furthermore, in transient transfection assays, the transcription of a CAT reporter construct driven by the MUC1 promoter was inhibited when cotransfected with a c-ErbB2 or a V12-H-ras expressing vector. Transient transfections using mutant forms of the ras oncogene, and the inhibitor chemical wortmannin, indicated that the pathway activated by c-ErbB2 proceeds via activation of Ras and that the Raf and phosphoinositide 3-kinase pathways are involved. Finally, cotransfection assays using a reporter gene driven by the MUC1 promoter carrying abolishing mutations in some of the cis-acting elements showed that a GC box at -99/-91 is crucial for responsiveness to c-ErbB2 inhibition of transcription.

Androstadienes↗
PubMed2001

Mouse ST6Gal sialyltransferase gene expression during mammary gland lactation.

The sialyltransferase ST6Gal mediates the biosynthetic addition of sialic acid, via an alpha2,6 linkage, to the nonreducing end of terminal lactosamine structures. Transcription of the murine ST6Gal gene, Siat1, is regulated by the selective use of multiple promoters in a tissue- and development-specific manner. Here we report that Siat1 mRNA expression is dramatically elevated in lactating (relative to virgin) mouse mammary gland. The predominant ST6Gal mRNA species expressed in lactating mammary gland is a heretofore undocumented isoform containing a unique 5'-untranslated region originating from the mouse Siat1 genetic region, now defined as Exon L, residing 549-bp 5' of the previously characterized Exon X(2). Thus, the novel ST6Gal mRNA form initiates transcription from the region designated as p4 and incorporates the unique sequence from Exon L in 5'-juxtaposition to commonly shared sequences encoded on Exon I to Exon VI. In contrast, cells derived from virgin mammary tissue expressed only the housekeeping mRNA form derived from p3, with Exon O sequence preceding Exons I-VI. The Exon L-containing, p4 class of mRNA was also not detected in a survey of eight other mouse tissues. Previous reports have indicated a strong correlation between mammary cancers and elevated ST6Gal expression in rats and in human patients. However, we uncovered neither elevated expression of ST6Gal mRNA nor appearance of p4 class in mouse breast carcinomas experimentally induced by transformation with the polyoma-middle T oncogene. A number of established breast carcinoma cell lines were also examined, with ST6Gal mRNA and activity generally low. Moreover, with the exception of the Shionogi cell line, p4 class of ST6Gal mRNA was not expressed in any of the mouse breast carcinoma specimens examined. Taken together, our data indicate that murine ST6Gal induction during lactation is achieved by de novo recruitment of a normally silent promoter. Furthermore, the data provide no support for elevated Siat1 expression on the mRNA level in association with murine mammary gland carcinogenesis. With the single exception of the Shionogi cell line, the p3 class remains the predominant ST6Gal mRNA expressed in all other murine mammary carcinoma cells examined.

5' Untranslated Regions↗
PubMed2000

The relative activities of the C2GnT1 and ST3Gal-I glycosyltransferases determine O-glycan structure and expression of a tumor-associated epitope on MUC1.

In breast cancer, the O-glycans added to the MUC1 mucin are core 1- rather than core 2-based. We have analyzed whether competition by the glycosyltransferase, ST3Gal-I, which transfers sialic acid to galactose in the core 1 substrate, is key to this switch in MUC1 glycosylation that results in the expression of the cancer-associated SM3 epitope. Of the three enzymes known to convert core 1 to core 2, by the addition of GlcNAc to GalNAc in core1 C2GnT1 is the dominant enzyme expressed in normal breast tissue. Expression of C2GnT1 is low or absent in around 50% of breast cancers, whereas expression of ST3Gal-I is consistently increased. Mapping of ST3Gal-I and C2GnT1 within the Golgi pathway showed some overlap. To examine functional competition, the enzymes were overexpressed in T47D cells, which normally make core 1-based structures, have no detectable C2GnT1 activity and express the SM3 epitope. Overexpression of C2GnT1 resulted in loss of binding of SM3 to MUC1, accompanied by a decrease in the GalNAc/GlcNAc ratio, indicative of a switch to core 2 structures. Transfection of a C2GnT1 expressing line with ST3Gal-I restored SM3 binding and reduced GlcNAc incorporation into MUC1 O-glycans. Thus, even when C2GnT1 is expressed, the O-glycans added to MUC1 become core 1-dominated structures, provided expression of ST3Gal-I is increased as it is in breast cancer.

Breast Neoplasms↗
PubMed2000

The lectin domain of UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-T4 directs its glycopeptide specificities.

The initiation step of mucin-type O-glycosylation is controlled by a large family of homologous UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). Differences in kinetic properties, substrate specificities, and expression patterns of these isoenzymes provide for differential regulation of O-glycan attachment sites and density. Recently, it has emerged that some GalNAc-transferase isoforms in vitro selectively function with partially GalNAc O-glycosylated acceptor peptides rather than with the corresponding unglycosylated peptides. O-Glycan attachment to selected sites, most notably two sites in the MUC1 tandem repeat, is entirely dependent on the glycosylation-dependent function of GalNAc-T4. Here we present data that a putative lectin domain found in the C terminus of GalNAc-T4 functions as a GalNAc lectin and confers its glycopeptide specificity. A single amino acid substitution in the lectin domain of a secreted form of GalNAc-T4 selectively blocked GalNAc-glycopeptide activity, while the general activity to peptides exerted by this enzyme was unaffected. Furthermore, the GalNAc-glycopeptide activity of wild-type secreted GalNAc-T4 was selectively inhibited by free GalNAc, while the activity with peptides was unaffected.

Amino Acid Sequence↗
PubMed2000

Activation of the alpha2beta1 integrin prevents c-erbB2-induced scattering and apoptosis of human mammary epithelial cells in collagen.

Constitutive overexpression of c-erbB2 in the mammary epithelial cell line MTSV1-7 has been shown to result in epithelial-mesenchymal conversion, anchorage-independent growth and loss of organized morphogenesis in collagen. To elucidate the events leading to this drastic change, MTSV1-7 cells and its subclone HB2 (which shows a more strictly epithelial phenotype) were transfected with the hybrid trk-neu receptor consisting of the extracellular domain of the trkA nerve growth factor (NGF) receptor and the transmembrane and cytoplasmic domains of c-erbB2 (neu). In cells expressing this construct, c-erbB2 homodimerization can be mimicked by addition of NGF. In trk-neu transfectants of HB2 cells, modest expression led to increased cell proliferation upon NGF treatment. When clones with higher expression levels were grown in collagen, NGF instead induced cell scattering, diminished viability and dramatically increased apoptosis. Interestingly, both the dissociation of colonies and loss of cell viability could be completely reversed by treatment of the cells with antibodies that activate the adhesive capacity of the alpha2beta1 integrin. Long-term NGF treatment of high-expressing transfectants generated fibroblastic clones displaying a reduced expression of integrin alpha2 and E-cadherin, and extensive apoptosis in collagen. These results, which indicate that strong c-erbB2 signalling may lead to downregulation and/or inactivation of the alpha2beta1 integrin, promoting apoptosis in collagen, provide one possible explanation to the increased apoptosis frequently seen in early tumour development.

Antibodies, Monoclonal↗
PubMed2000

Targeted macrophage cytotoxicity using a nonreplicative live vector expressing a tumor-specific single-chain variable region fragment.

Antigen-specific recognition and subsequent destruction of tumor cells is the goal of vaccine-based immunotherapy of cancer. Often, however, tumor antigen-specific cytotoxic T lymphocytes (CTLs) are either not available or in a state of anergy. In addition, MHCI expression on tumor cells is often downregulated. Either or both of these situations can allow tumor growth to proceed unchecked by CTL control. We have shown previously that tumor antigen-specific monoclonal antibodies can be expressed in vaccinia virus and that activated macrophages infected with this virus acquire the ability to kill tumor cells expressing that antigen. Here we show that a membrane-anchored form of the scFv portion of the MUC1 tumor antigen-specific monoclonal antibody, SM3, can be expressed on activated macrophages with the highly attenuated poxvirus, modified vaccinia Ankara (MVA), as a gene transfer vector. Cells infected with the MVA-scFv construct were shown to express the membrane-bound scFv by Western blot and FACS analysis. That cells expressing the membrane-anchored scFv specifically bind antigen was shown by FACS and by BIAcore analysis. GM-CSF-activated macrophages were infected with the construct and shown to recognize specifically MUC1-expressing tumor cells as measured by IL-12 release. Furthermore, activated macrophages expressing the membrane-bound scFv specifically lyse target cells expressing the MUC1 antigen but not cells that do not express MUC1.

Animals↗
PubMed2000

Immunity to murine breast cancer cells modified to express MUC-1, a human breast cancer antigen, in transgenic mice tolerant to human MUC-1.

The high incidence of breast cancer in women and the severity of the disease have stimulated a need for improved and novel forms of therapy. The product of the MUC-1 gene has been identified as a breast cancer-associated antigen in breast cancer patients. The gene has been cloned and sequenced. Transgenic mice were prepared that express human mucin and are naturally tolerant to the molecule, providing a unique opportunity to investigate immunotherapeutic strategies in experimental animals that might eventually be applied to breast cancer patients. A cell line (410.4) derived from a mouse mammary adenocarcinoma that arose in a BALB/c mouse was transduced with a retroviral vector (R1-MUC1-pEMSVscribe) that encoded MUC-1. After confirmation of the expression of human mucin, the cells (E3) were further modified by transduction with retroviral vectors encoding interleukin (IL)-2, IL-4, IL-12, or IFN-gamma to evaluate the effect of cytokine-secretion on the immunogenic properties of the cells in the MUC-1 transgenic mice. The results indicated that modification of the breast cancer cells to secrete IL-12 reduced and at times eliminated the tumorigenic growth properties of the cells. Under similar circumstances, progressively growing tumors formed in MUC-1 transgenic mice that received injections of unmodified E3 cells or with E3 cells modified to secrete IL-2, IL-4, or IFN-gamma. Immunity to breast cancer developed in MUC-1 transgenic mice that had rejected IL-12-secreting E3 cells because the animals were resistant to challenge with (non-cytokine-secreting) E3 cells. In vitro analyses confirmed the presence of T cell-mediated cytotoxicity toward the breast cancer cells in MUC-1 transgenic mice immunized with the IL-12-secreting cells. Our data obtained in a unique animal model system point toward an analogous form of therapy for breast cancer patients.

Adenocarcinoma↗
PubMed2000

A transgenic mouse model for tumour immunotherapy: induction of an anti-idiotype response to human MUC1.

MUC1 is a membrane bound, polymorphic epithelial mucin expressed at the luminal surface of glandular epithelium. It is highly expressed in an underglycosylated form on carcinomas and metastatic lesions and is, therefore, a potential target for immunotherapy of cancer. The monoclonal antibody HMFG1 binds the linear core protein sequence, PDTR, contained within the immunodominant domain of the tandem repeat of MUC1. The efficacy of murine and humanized HMFG1 (Ab1) used as an anti-idiotypic vaccine was examined in mice transgenic for human MUC1 (MUC1.Tg) challenged with murine epithelial tumour cells transfected with human MUC1. Humoral idiotypic cascade through Ab2 and Ab3 antibodies was observed in MUC1.Tg mice following multiple antibody inoculations in the presence of adjuvant. Impaired tumour growth at day 35 and highest Ab3 levels were found in mice that had received mHMFG1 with RAS adjuvant. However, comparison of Ab3 levels in individual mice with tumour size in all treatment groups did not show a correlation between smaller tumours and increased levels of anti-idiotype antibody. This suggests that the anti-tumour effects of anti-idiotype vaccination are not solely related to the induction of idiotypic antibody cascades and probably involve other mechanisms.

Adjuvants, Immunologic↗
PubMed2000

Morphogenetic and proliferative responses to heregulin of mammary epithelial cells in vitro are dependent on HER2 and HER3 and differ from the responses to HER2 homodimerisation or hepatocyte growth factor.

The effects of heregulin on the cell line HB2, derived from immortalised human luminal mammary epithelial cells, have been examined. HB2 cells, which normally form smooth spherical colonies in collagen gels, exhibited a striking heregulin-induced morphological change to colonies projecting a large number of spiky branches. A mitogenic effect of heregulin on HB2 cells was also seen, which was more pronounced on collagen than on plastic, whereas cell motility was unaffected. HB2 cells were found to express the heregulin receptor subunits HER2 and HER3, but not HER4. Treatment of HB2 cells with heregulin also induced tyrosine phosphorylation of a band shown by immunoprecipitation to contain HER3. Using specific receptor-blocking antibodies, it was found that both the morphogenetic and proliferative responses of heregulin in HB2 cells were mediated by HER2 and HER3. To compare the effects of HER2 in heregulin signaling to heregulin-independent HER2 homodimerisation (thought to be a carcinoma-associated event), HB2 cells were transfected with the trk-neu hybrid receptor which could be induced to form homodimers by NGF. Although activated HER2 homodimers induced proliferation in the HB2 transfectants in collagen, a morphological response in collagen was not seen, suggesting that HER3 signaling is important for morphogenesis in this cell type.

Breast↗
PubMed1999

MUC1 and cancer.

The MUC1 membrane mucin was first identified as the molecule recognised by mouse monoclonal antibodies directed to epithelial cells, and the cancers which develop from them. Cloning the gene showed that the extracellular domain is made up of highly conserved repeats of 20 amino acids, the actual number varying between 25 and 100 depending on the allele. Each tandem repeat contains five potential glycosylation sites, and between doublets of threonines and serines lies an immunodominant region which contains the epitopes recognised by most of the mouse monoclonal antibodies. The O-glycans added to the mucin produced by the normal breast are core 2 based and can be complex, while the O-glycans added to the breast cancer mucin are mainly core 1 based. This means that some core protein epitopes in the tandem repeat which are masked in the normal mucin are exposed in the cancer associated mucin. Since novel carbohydrate epitopes are also carried on the breast cancer mucin, the molecule is antigenically distinct from the normal breast mucin. (Changes in glycosylation in other epithelial cancers have been observed but are not so well documented.) Immune responses to MUC1 have been seen in breast and ovarian cancer patients and clinical studies have been initiated to evaluate the use of antibodies to MUC1 and of immunogens based on MUC1 for immunotherapy of these patients. The role of the carbohydrates in the immune response and in other interactions with the effector cells of the immune system is of particular interest and is discussed.

Amino Acid Sequence↗
PubMed1999

Treatment of breast cancer with fibroblasts transfected with DNA from breast cancer cells.

This investigation was based on the hypothesis that weakly immunogenic, breast cancer-associated Ags, the products of mutant or dysregulated genes in the malignant cells, will be expressed in a highly immunogenic form by semiallogeneic IL-2-secreting fibroblasts transfected with DNA from breast cancer cells. (Classic studies indicate that transfection of genomic DNA can stably alter both the genotype and the phenotype of the cells that take up the exogenous DNA.) To investigate this question, we transfected LM mouse fibroblasts (H-2k) modified to secrete IL-2 with genomic DNA from a breast adenocarcinoma that arose spontaneously in a C3H/He mouse (H-2k). To increase their nonspecific immunogenic properties, the fibroblasts were also modified before transfection to express allogeneic MHC determinants (H-2Kb). Afterward, the IL-2-secreting semiallogeneic cells were cotransfected with DNA from the spontaneous breast neoplasm, along with a plasmid (pHyg) conferring resistance to hygromycin. Pooled colonies of hygromycin-resistant cells were then tested in C3H/He mice for their immunotherapeutic properties against the growth of the breast neoplasm. The results indicated that tumor-bearing mice immunized with the transfected cells survived significantly longer than mice in various control groups. Similar beneficial effects were seen in C57BL/6 mice injected with a syngeneic breast carcinoma cell line (EO771) and semiallogeneic, IL-2-secreting fibroblasts transfected with DNA from EO771 cells. The immunity was mediated by CD8+ T cells since immunized mice depleted of CD8+ cells failed to resist tumor growth.

Adenocarcinoma↗