| 1. |
- Bäckström, Malin, 1967, et al.
(författare)
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Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells.
- 2003
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Ingår i: The Biochemical journal. - 1470-8728. ; 376:Pt 3, s. 677-86
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Tidskriftsartikel (refereegranskat)abstract
- We have developed an expression system for the production of large quantities of recombinant MUC1 mucin in CHO-K1 (Chinese-hamster ovary K1) cells. The extracellular part of human MUC1, including 16 MUC1 tandem repeats, was produced as a fusion protein with murine IgG Fc, with an intervening enterokinase cleavage site for the removal of the Fc tail. Stable MUC1-IgG-producing CHO-K1 clones were generated and were found to secrete MUC1-IgG into the culture medium. After adaptation to suspension culture in protein-free medium in a bioreactor, the fusion protein was secreted in large quantities (100 mg/l per day) into the culture supernatant. From there, MUC1 could be purified to homogeneity using a two-step procedure including enterokinase cleavage and ion-exchange chromatography. Capillary liquid chromatography MS of released oligosaccharides from CHO-K1-produced MUC1 identified the main O-glycans as Galbeta1-3GalNAc (core 1) and mono- and di-sialylated core 1. The glycans occupied on average 4.3 of the five potential O-glycosylation sites in the tandem repeats, as determined by nano-liquid chromatography MS of partially deglycosylated Clostripain-digested protein. A very similar O-glycan profile and site occupancy was found in MUC1-IgG produced in the breast carcinoma cell line T47D, which has O-glycosylation typical for breast cancer. In contrast, MUC1-IgG produced in another breast cancer cell line, MCF-7, showed a more complex pattern with both core 1- and core 2-based O-glycans. This is the first reported production of large quantities of recombinant MUC1 with a breast cancer-like O-glycosylation that could be used for the immunotherapy of breast cancer.
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| 2. |
- Jenndahl, Lachmi E, et al.
(författare)
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Characterization of integrin and anchorage dependence in mammary epithelial cells following c-erbB2-induced epithelial-mesenchymal transition.
- 2006
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Ingår i: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. - : Springer Science and Business Media LLC. - 1010-4283. ; 27:1, s. 50-8
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Tidskriftsartikel (refereegranskat)abstract
- Signalling from the proto-oncogene c-erbB2 in mammary epithelial cells has earlier been shown to result in epithelial-mesenchymal transition (EMT) giving rise to fibroblast-like cells, and acquisition of anchorage-independent growth (AIG) usually determined by growth capacity in soft agar. In this study, we have analysed AIG associated with c-erbB2-induced EMT in a human mammary epithelial cell line. Intriguingly, cells capable of growth in soft agar were shown to be dependent on the function of beta(1) integrin extracellular matrix receptors for growth in collagen. We therefore tested the hypothesis that apparent AIG was due to deposition of extracellular matrix in the agar. Although the fibroblastic cells had strongly upregulated expression of the fibronectin receptor subunit integrin alpha(5) andabundant fibronectin fibrils, these properties did not have a positive correlation with AIG. Furthermore, antibody blocking of integrin alpha(5) and beta(1) failed to inhibit AIG. These results indicate that the anchorage-independent cells are not dependent on connection to extracellular matrix, but instead may be subject to a growth-inhibitory effect from the collagen in the absence of integrin signalling. This notion was supported by the finding that integrin blocking of the fibroblastic cells in fibrin was without effect on proliferation.
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| 3. |
- Link, Thomas, et al.
(författare)
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Bioprocess development for the production of a recombinant MUC1 fusion protein expressed by CHO-K1 cells in protein-free medium
- 2004
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Ingår i: J Biotechnol. ; 110, s. 51-62
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Tidskriftsartikel (refereegranskat)abstract
- 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 pO2 were determined in parallel spinner flask batch cultures. A pH of 6.8–7.0 and a pO2 of 40% of air saturation was found to give best cell growth and productivity of secreted recombinant protein. Specific productivity strongly depended the pO2 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.
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| 4. |
- Pinto, Rita, et al.
(författare)
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Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ proximity ligation
- 2012
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Ingår i: Journal of Cellular and Molecular Medicine (Print). - : Wiley. - 1582-1838 .- 1582-4934. ; 16:7, s. 1474-1484
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Tidskriftsartikel (refereegranskat)abstract
- Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification of these biomarkers has been based on the detection of either the protein or the O-glycan modifications. We therefore aimed to identify the combined mucin and O-glycan features, i.e. specific glycoforms, in an attempt to increase specificity of these cancer biomarkers. Using in situ proximity ligation assays (PLA) based on existing monoclonal antibodies directed to MUC1, MUC2, MUC5AC and MUC6 mucins and to cancer-associated carbohydrate antigens Tn, Sialyl-Tn (STn), T, Sialyl-Lea (SLea) and Sialyl-Lex (SLex) we screened a series of 28 mucinous adenocarcinomas from different locations (stomach, ampulla of Vater, colon, lung, breast and ovary) to detect specific mucin glycoforms. We detected Tn/STn/SLea/SLex-MUC1 and STn/SLea/SLex-MUC2 glycoforms in ≥50% of the cases, with a variable distribution among organs. Some new glycoforms – T/SLea-MUC2, STn/T/SLea/SLex-MUC5AC and STn/T/SLea/SLex-MUC6 – were identified for the first time in the present study in a variable percentage of cases from different organs. In conclusion, application of the PLA technique allowed sensitive detection of specific aberrant mucin glycoforms in cancer, increasing specificity to the use of antibodies either to the mucin protein backbone or the O-glycan haptens alone.
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| 5. |
- Rughetti, Aurelia, et al.
(författare)
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Recombinant tumor-associated MUC1 glycoprotein impairs the differentiation and function of dendritic cells
- 2005
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Ingår i: J Immunol. ; 174:12, s. 7764-7772
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Tidskriftsartikel (refereegranskat)abstract
- Tumors exploit several strategies to evade immune recognition, including the production of a large number of immunosuppressive factors, which leads to reduced numbers and impaired functions of dendritic cells (DCs) in the vicinity of tumors. We have investigated whether a mucin released by tumor cells could be involved in causing these immunomodulating effects on DCs. We used a recombinant purified form of the MUC1 glycoprotein, an epithelial associated mucin that is overexpressed, aberrantly glycosylated, and shed during cancer transformation. The O-glycosylation profile of the recombinant MUC1 glycoprotein (ST-MUC1) resembled that expressed by epithelial tumors in vivo, consisting of large numbers of sialylated core 1 (sialyl-T, ST) oligosaccharides. When cultured in the presence of ST-MUC1, human monocyte-derived DCs displayed a modified phenotype with decreased expression of costimulatory molecules (CD86, CD40), Ag-presenting molecules (DR and CD1d), and differentiation markers (CD83). In contrast, markers associated with an immature phenotype, CD1a and CD206 (mannose receptor), were increased. This effect was already evident at day 4 of DC culture and was dose dependent. The modified phenotype of DCs corresponded to an altered balance in IL-12/IL-10 cytokine production, with DC expressing an IL-10highIL-12low phenotype after exposure to ST-MUC1. These DCs were defective in their ability to induce immune responses in both allogeneic and autologous settings, as detected in proliferation and ELISPOT assays. The altered DC differentiation and Ag presentation function induced by the soluble sialylated tumor-associated mucin may represent a mechanism by which epithelial tumors can escape immunosurveillance.
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| 6. |
- Sewell, Robert, et al.
(författare)
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The ST6GalNAc-I sialyltransferase localizes throughout the Golgi and is responsible for the synthesis of the tumor-associated sialyl-Tn O-glycan in humna breast cancer
- 2006
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Ingår i: J Biol Chem. ; 281:6, s. 3586-3594
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Tidskriftsartikel (refereegranskat)abstract
- The functional properties of glycoproteins are strongly influenced by their profile of glycosylation, and changes in this profile are seen in malignancy. In mucin-type O-linked glycosylation these changes can result in the production of mucins such as MUC1, carrying shorter sialylated O-glycans, and with different site occupancy. Of the tumor-associated sialylated O-glycans, the disaccharide, sialyl-Tn (sialic acid {alpha}2,6GalNAc), is expressed by 30% of breast carcinomas and is the most tumor-specific. The ST6GalNAc-I glycosyltransferase, which can catalyze the transfer of sialic acid to GalNAc, shows a highly restricted pattern of expression in normal adult tissues, being largely limited to the gastrointestinal tract and absent in mammary gland. In breast carcinomas, however, a complete correlation between the expression of RNA-encoding ST6GalNAc-I and the expression of sialyl-Tn is evident, demonstrating that the expression of sialyl-Tn results from switching on expression of hST6GalNAc-I. Endogenous or exogenous expression of hST6GalNAc-I (but not ST6GalNAc-II) always results in the expression of sialyl-Tn. This ability to override core 1/core 2 pathways of O- linked glycosylation is explained by the localization of ST6GalNAc-I, which is found throughout the Golgi stacks. The development of a Chinese hamster ovary (CHO) cell line expressing MUC1 and ST6GalNAc-I allowed the large scale production of MUC1 carrying 83% sialyl-Tn O-glycans. The presence of ST6GalNAc-I in the CHO cells reduced the number of O-glycosylation sites occupied in MUC1, from an average of 4.3 to 3.8 per tandem repeat. The availability of large quantities of this MUC1 glycoform will allow the evaluation of its efficacy as an immunogen for immunotherapy of MUC1/STn-expressing tumors.
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