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Thioredoxin system in normal and transformed human cells
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-25707Local ID: 10083ISBN: 91-7219-743-9 (print)OAI: oai:DiVA.org:liu-25707DiVA: diva2:246255
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
List of papers
1. Thioredoxin Expression and Localization in Human Cell Lines: Detection of Full-Length and Truncated Species
Open this publication in new window or tab >>Thioredoxin Expression and Localization in Human Cell Lines: Detection of Full-Length and Truncated Species
Show others...
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13714 (URN)10.1006/excr.1997.3699 (DOI)
Available from: 2002-01-11 Created: 2002-01-11 Last updated: 2017-12-13Bibliographically approved
2. Monoclonal Antibodies to Human Thioredoxin Reductase
Open this publication in new window or tab >>Monoclonal Antibodies to Human Thioredoxin Reductase
1998 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 249, no 1, 86-89 p.Article in journal (Refereed) Published
Abstract [en]

The thioredoxin system consisting of thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH is an electron donor for ribonucleotide reductase but has also been implicated in other cellular events, including secretion, growth promotion, regulation of transcription factors, protection against oxidative stress, and apoptosis. Mammalian TrxR is a dimeric flavoprotein with 58 kDa subunits each with a catalytically active selenocysteine residue. To study the function and expression of TrxR, we have produced and characterized, for the first time, monoclonal antibodies against human TrxR. Native placenta TrxR was used for immunization of BALB/c mice, followed by hybridization, cloning, and establishment of hybridomas producing specific antibodies against human TrxR. Three clones of IgG1,κ subclass, termed anti-TrxR1, anti-TrxR2, and anti-TrxR3, were studied in detail. The isoelectric points (pIs) of the mAbs were 6.5, 6.0, and 6.5, respectively. The affinities (Ka) of the mAbs were 2 × 108M−1.Inhibition ELISA using biotin-labeled versus nonconjugated mAb IgG revealed that all three mAbs recognized one immunodominant epitope. Western blot analysis showed that the antibodies specifically bound to a 58 kDa protein, representing the subunit of TrxR. A Trx-dependent insulin reduction assay was used for analysis of enzymatic activity and the antibodies neutralized the reductase activity.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79884 (URN)10.1006/bbrc.1998.9053 (DOI)
Available from: 2012-08-14 Created: 2012-08-14 Last updated: 2017-12-07Bibliographically approved
3. Thioredoxin Reductase, a Redox-active Selenoprotein, Is Secreted by Normal and Neoplastic Cells: Presence in Human Plasma
Open this publication in new window or tab >>Thioredoxin Reductase, a Redox-active Selenoprotein, Is Secreted by Normal and Neoplastic Cells: Presence in Human Plasma
2000 (English)In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 60, no 8, 2281-2289 p.Article in journal (Refereed) Published
Abstract [en]

Thioredoxin (Trx) and Trx reductase (TrxR) are redox-active proteins that participate in multiple cellular events, including growth promotion, apoptosis, and cytoprotection. Studies on overexpression of Trx and TrxR in human cancers have indicated a role of these proteins in tumor development. In this study, we analyzed the expression of TrxR in peripheral blood cells, tumor-transformed leukemia, and melanoma cells and found, in addition to abundant plasma membrane localization, that TrxR was released from these cells. Secretory cells were observed at the single cell level using a sensitive enzyme-linked immunospot assay. The release was inducible, and physiological stimulation of human monocytes by IFN-γ, lipopolysaccharide, and interleukin 1α significantly increased the number of TrxR-secreting cells (P = 0.004). Secretion of TrxR followed the classical Golgi pathway, and it was confirmed by metabolic labeling using [35S]methionine and[ 35S]cysteine. TrxR was also detected for the first time in fresh healthy blood donor plasma (n = 21; median concentration, 18.0 ng/ml), with biological activity as determined by insulin reduction assay.

These results highlight the role of extracellular Trx and TrxR during inflammation and tumor progression. Released Trx, with its active site motif containing amino acids Cys-X-X-Cys, was recently shown to have chemoattractant properties beside its previously described antioxidant and cocytokine activities. Regeneration of oxidized Trx requires available TrxR outside the cell, the presence and induction of which is described in this paper for normal and transformed cells.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25334 (URN)9776 (Local ID)9776 (Archive number)9776 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
4. Secretion of 10-kDa and 12-kDa Thioredoxin Species from Blood Monocytes and Transformed Leukocytes
Open this publication in new window or tab >>Secretion of 10-kDa and 12-kDa Thioredoxin Species from Blood Monocytes and Transformed Leukocytes
2000 (English)In: Antioxidants and Redox Signaling, ISSN 1523-0864, E-ISSN 1557-7716, Vol. 2, no 4, 717-726 p.Article in journal (Refereed) Published
Abstract [en]

Thioredoxins (TRX) are ubiquitous, small redox-active proteins with multiple functions, including antioxidant, cytoprotective, and chemoattractant activities. In addition to a 12-kDa intracellular form, extracellular 10-kDa and 12-kDa TRX have been defined. The biological activities of the 10-kDa TRX were previously measured as eosinophil cytotoxicity enhancing activity or B-cell stimulatory activity. Cytotrophoblastic cell lines also release a 10-kDa TRX form. To study the biological role of 10-kDa TRX, we established two highly sensitive enzyme-linked immuno-spot assays (ELISPOT), which detect secreted truncated 10-kDa and full-length 12-kDa TRX at the single cell level. TRX secretion was investigated in several cell lines including the T-helper cell hybridoma MP6, the Jurkat T-cell leukemia, the U-937 myelomonocytic leukemia, and the 3B6, EBV-transformed, lymphoblastoid B-cell line. The highest number of secreting cells was found in 3B6 cultures, median = 34 (quartiles, 27–39) per well (105 cells). Peripheral blood monocytes isolated from healthy donors secreted significantly more TRX after stimulation with ionomycin, phorbol myristate acetate (PMA), fMLP, and lipopolysaccharide (LPS), compared to unstimulated cells. Oxidative stress induced by thioloxidant diamide also induced the secretion of both truncated and full-length TRX measured in ELISPOT (p = 0.047 and p = 0.031, respectively). The biological activity of the truncated and full-length forms was tested in a cell migration assay. Truncated TRX was devoid of protein disulfide reductase activity, but retained strong chemoattractant activity for human monocytes, in the same range as full-length TRX, as previously reported.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25335 (URN)10.1089/ars.2000.2.4-717 (DOI)9777 (Local ID)9777 (Archive number)9777 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
5. Direct Evidence for Thioredoxin in Platelets: Studies on its release into human plasma
Open this publication in new window or tab >>Direct Evidence for Thioredoxin in Platelets: Studies on its release into human plasma
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Thioredoxin is a multifunctional protein involved in protecting cells against oxidative stress, and it acts as a co-cytokine and chemotactic factor during an immune response. Mechanisms behind thioredoxin release and transport in the blood are unknown, although the presence of thioredoxin in extracellular fluids is indisputable. We investigated the role of circulatory thioredoxin. We have previously found that release of selenoprotein thioredoxin reductase to the blood is induced by oxidative stress, and thioredoxin reductase is present in plasma (Söderberg, Sabaf, and Rosén, Cancer Res. 60, 2281; 2000). Both thioredoxin and protein disulfide isomerase are substrates for thioredoxin reductase, and both are exposed on the surface oflymphocytes and monocytes. Plasma thioredoxin level is elevated by oxidative stress, a condition often seen in burn patients and HIV carriers. Three of our present findings demonstrate that platelets contain thioredoxin and that circulatory thioredoxin is transported primarily in platelets in healthy blood donors. First, thioredoxin, but not thioredoxin reductase was detected in platelets by deconvolution fluorescence microscopy. Second, plasma thioredoxin concentration was sensitive to thrombocytolysis: a significant decrease in thioredoxin was seen in plasma samples (n = 20) pretreated with the platelet degranulation inhibitors theophylline, adenosine, dipyridamol, acetylsalicylic acid, and apyrase (thioredoxin decreased from 28 down to 8 ng/ml; p < 0.0001). Release of thioredoxin from platelets was induced by the thioloxidant diamide but not by platelet degranulation caused by thrombin, a thrombin peptide (SFLLRN) and collagen, ADP, or PMA-ionophore. Third, in thrombocytopenic and thrombocytemic patients, plasma levels of thioredoxin, but not ß- thromboglobulin, were strongly correlated with platelet numbers (correlation coefficient, r = 0.7). Summarizing, we found direct evidence that thioredoxin is present in platelets and is liberated by oxidative stress (diamide). This suggests that platelets are essential for prompt delivery of this cellular reducing agent to sites of injury, where it can activate coagulation factors and subsequently reduce or balance reactive oxygen species released by inflammatory macrophages.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-79886 (URN)
Available from: 2012-08-14 Created: 2012-08-14 Last updated: 2017-09-22Bibliographically approved

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