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Thioredoxin Expression and Localization in Human Cell Lines: Detection of Full-Length and Truncated Species
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
Department of Biosciences, Novum, Karolinska Institute, Huddinge, Sweden.
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
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1997 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 236, no 1, 181-192 p.Article in journal (Refereed) Published
Abstract [en]

Thioredoxin (Trx) is an intracellular multifunctional 12-kDa protein with a reduction/oxidation (redox) active disulfide constitutively expressed by most cells of the human body. Trx can also be released by cells such as lymphocytes upon activation or oxidative stress exposure and exert a cocytokine and cytoprotective activity. In addition, a truncated 10-kDa form of Trx has been reported. In order to better understand the function of full-length and truncated Trx, we have produced, for the first time, specific monoclonal antibodies, which can discriminate between the two forms. Using these novel antibodies, designated αTrx1 to αTrx4, a panel of cell lines derived from human B and T lymphocytes, monocytes, granulocytes, and melanomas was analyzed by immunochemical techniques. The cellular distribution differed between the two forms. All lines contained full-length Trx, also located to a minor extent on the cell surface. One exception was the melanoma cell line FM28.4, which did not show any Trx expression. Truncated Trx was present in most cells in minimal amounts only, whereas the monocytic cell lines THP-1 and U-937 expressed high amounts on the cell surface, as shown by flow cytometric analysis of living cells and confocal laser-scanning microscopy. The biological importance and function of the short versus long forms of Trx as detected by the antibodies are discussed.

Place, publisher, year, edition, pages
1997. Vol. 236, no 1, 181-192 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-13714DOI: 10.1006/excr.1997.3699OAI: oai:DiVA.org:liu-13714DiVA: diva2:21196
Available from: 2002-01-11 Created: 2002-01-11 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Immunological Studies in Malignant Melanoma: Importance of TNF and the Thioredoxin System
Open this publication in new window or tab >>Immunological Studies in Malignant Melanoma: Importance of TNF and the Thioredoxin System
2001 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Malignant melanoma is a tumor whose incidence is dramatically increasing in persons with light-coloured skin in all parts of the world. Due to its resistance against traditional chemo- and radiotherapy, melanoma has been a favourite target of alternative therapies, in particular those involving immunological mechanisms. Cytokines and particularly tumor necrosis factor (TNF) have been studied as possible antitumoral agents, but also as endogenous growth or differentiation factors. Previous studies showed that melanomas could express TNF in situ and that this expression correlated to decreased lymphocyte infiltration. On the other hand, redox reagents can modulate expression of cytokines, and the thioredoxin (Trx) system is particularly known to influence expression and secretion of TNF in vitro.

The overall aim of this research was to explore immunological aspects of melanoma, particularly the role of TNF both in vitro and in vivo, as well as its possible modulation by Trx.

In the in vitro studies first we developed a novel method for obtention of monoclonal antibodies against melanoma antigens, and generated and characterized specific monoclonal antibodies against both full-length and truncated Trx. We studied the cytokine expression of a panel of normal and transformed melanocytic cells by immunofluorescence, all of which presented TNF and Trx at levels comparable to monocytic cells, and TNF-receptors (TNFR) at low but detectable levels. Melanoma cells did not secrete TNF upon stimulation in spite of its presence in the Golgi apparatus. However, melanoma cells expressed the TNF-processing enzyme TACE and were capable of cleaving transfected GFP-tagged TNF. Imaging studies point to a possible cell-cell tranfer of endogenous TNF in melanoma cells.

On the other hand, TNF and Trx expression in melanoma cell lines correlated to resistance against exogenous TNF. We studied then the in situ expression of TNF and Trx by immunohistochemistry in a group of 44 cutaneous melanoma patients. Trx expression did not correlated to survival or other clinicalpathological parameters. TNF expression significantly correlated to better survival in tumors thicker than 0,8 mm, and constituted an independent prognostic factor.

These results point to a biological role of endogenous TNF in malignant melanoma, either by constituting an autocrine/paracrine differentiation factor or by modulating communication with other cell types, particularly of the host’s immune system.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2001. 79 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 703
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-5226 (URN)91-7373-142-0 (ISBN)
Public defence
2001-12-06, Berzeliussalen, Campus US, Linköpings universitet, Linköping, 09:30 (English)
Opponent
Supervisors
Note
Article 4 changed significantly during the publication process.Available from: 2002-01-11 Created: 2002-01-11 Last updated: 2017-09-22Bibliographically approved
2. Thioredoxin system in normal and transformed human cells
Open this publication in new window or tab >>Thioredoxin system in normal and transformed human cells
2000 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Thioredoxin (Trx) and thioredoxin reductase (TrxR), together with NADPH, constitute the Trx system, a major antioxidant entity that helps maintain a reducing environment within living cells. Trx designates a family of proteins that are related on the basis of structure and function. Human full-length Trx is a 12 kDa redox-active protein that contains the evolutionarily conserved active site sequence Cys-Gly Pro-Cys. Truncated Trx is a shorter, 10 kDa form of human Trx that shows complete homology to the N-terminal 80 or 84 amino acids of 12 kDa Trx. Truncated Trx displays no reducing activity, even though it contains an intact active site. Human TrxR is a homodimeric, FAD-containing, selenoprotein that reduces oxidized Trx back to the enzymatically active form by consuming NADPH. Expression of human Trx and TrxR is induced by a variety of stressors. The Trx system functions directly and indirectly in important biological features such as DNA synthesis, gene expression, co-cytokine activity, chemokine properties, scavenging reactive oxygen intermediates, apoptosis, and growth regulation.

Aims: The overall objective of this research was to examine the regulation and biological role of the Trx system in normal and transformed cells, and a possible connection with tumorigenesis. The specific aims were as follows: i) to study the presence and function of full-length and truncated Trx in normal and transformed human cells; ii) to assess the expression and function of TrxR; iii) to investigate the functional importance of truncated Trx; iv) to examine the regulatory mechanisms behind secretion of Trx, truncated Trx, and TrxR; v) to study full-length secreted Trx in human plasma.

Methods: Using Köhler and Milstein hybridoma technology, monoclonal antibodies against full-length and truncated Trx, as well as TrxR, were developed and applied in various immunological methods to detect the proteins in and secreted from normal and transformed human cells.

Results: Using selective monoclonal antibodies distinct intracellular localization of full-length and truncated Trx was shown. The latter was present in lower amounts, primarily associated with the plasma membrane; only small amounts of the 12 kDa were present on the plasma membrane. Moreover, we found that human TrxR was secreted from normal and transformed cells via a Golgi-dependent pathway. Using sensitive EUSPOT, TrxR release was quantified at the single-cell level and found to be inducible. TrxR was detected in plasma from healthy blood donors, and this protein was overexpressed in transformed cells, compared to their normal counterparts. Truncated 10 kDa Trx was present in and secreted from normal and transformed cells. Treatment of normal peripheral blood monocytes with LPS, IL-1, IFN-γ, PMA, ionomycin, and the thiol oxidant diarnide induced secretion of Trx. Trx was found within platelets, and liberation of this Trx was induced by diamide, implying a novel mechanism for release of Trx.

Conclusions: Monoclonal antibodies were generated against full-length and truncated Trx as well as TrxR and proved to be useful tools for investigating these proteins. Full-length and truncated Trx were found to have different cellular functions, suggesting that C-temrinal truncation regulates the activity of Trx. Secretion of TrxR and the presence of thls enzyme in plasma imply that the Trx system has both intracellular and extracellular functions. The presence of Trx in platelets indicates participation of the protein in coagulation and possibly also in the effects of platelets that maintain the reducing capacity of human plasma.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2000. 86 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 642
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25707 (URN)10083 (Local ID)91-7219-743-9 (ISBN)10083 (Archive number)10083 (OAI)
Public defence
2000-10-13, Berzeliussalen, Hälsouniversitetet, Linköping, 09:30 (Swedish)
Opponent
Supervisors
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-09-22Bibliographically approved

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Söderberg, AnitaSpyrou, GiannisBarral, Anna-MariaRosén, Anders

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