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Immunological Studies in Malignant Melanoma: Importance of TNF and the Thioredoxin System
Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-5226ISBN: 91-7373-142-0 (print)OAI: oai:DiVA.org:liu-5226DiVA: diva2:21200
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
List of papers
1. Cell–cell adherence as a selection method for the generation of anti-melanoma monoclonal antibodies
Open this publication in new window or tab >>Cell–cell adherence as a selection method for the generation of anti-melanoma monoclonal antibodies
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1997 (English)In: Journal of Immunological Methods, ISSN 0022-1759, Vol. 203, no 1, 103-109 p.Article in journal (Refereed) Published
Abstract [en]

The aim of the present study was to obtain monoclonal antibodies (mAbs) recognising human melanoma-associated antigens after immunisation of BALB/c mice with a 70–150 kDa membrane fraction from melanoma tumour tissues. Screening of specific antibody- producing hybridomas was performed using a novel cell–cell adherence method with the melanoma cell line M-14. Three mAbs of IgG1 isotype were selected: Mel-1, Mel-2 and Mel-3 which recognised the immunogen by ELISA and stained several melanoma cell lines positive in immunofluorescence. The molecular weight of the antigen was studied by different methods; a 170-kDa band was identified following immunoblotting of tumour lysate and a 72-kDa band was observed following immunoaffinity purification. Cell–cell adherence appears to be a reliable procedure for the generation of mAbs against native cellular antigens.

Keyword
Malignant melanoma; Hybridoma selection; Monoclonal antibody
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13713 (URN)10.1016/S0022-1759(97)00025-2 (DOI)
Available from: 2002-01-11 Created: 2002-01-11 Last updated: 2017-09-22
2. 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
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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.

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: 2017-09-22Bibliographically approved
3. Thioredoxin, thioredoxin reductase and tumour necrosis factor-alpha expression in melanoma cells: correlation to resistance against cytotoxic attack
Open this publication in new window or tab >>Thioredoxin, thioredoxin reductase and tumour necrosis factor-alpha expression in melanoma cells: correlation to resistance against cytotoxic attack
2000 (English)In: Melanoma research, ISSN 0960-8931, Vol. 10, no 4, 331-343 p.Article in journal (Refereed) Published
Abstract [en]

Although malignant melanomas are often associated with cytotoxic lymphocyte infiltration, these cells are largely ineffective in inducing tumour cell kill, indicating that the melanoma cells have protective mechanisms. These mechanisms are not fully understood, but cytokines and redox-active antioxidant proteins such as catalase, superoxide dismutase, thioredoxin (Trx) and Trx reductase (TrxR) present in the tumour cells constitute part of this protection. In this study firstly we investigated the constitutive intracellular expression of Trx, TrxR, the cytokines interleukin (IL)-1alpha, IL1beta, IL2, IL4, IL6, IL8, IL10, tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) in normal melanocytes and ten primary and metastatic malignant melanoma cell lines. Secondly, we analysed whether redox stimulation by Trx alone or in combination with the phorbol ester PMA affected the expression and release of TNFalpha. Thirdly, we explored the possible correlation between Trx/TrxR expression and resistance to exogenous TNFalpha. All the cultured cells showed intracellular overexpression of Trx and TrxR, which was not always the case for melanoma cells in vivo (tissue sections). The predominant intracellular cytokines found were TNFalpha, IL1alpha and IL1beta. In spite of its presence in the Golgi apparatus, none of the cell lines secreted TNFalpha constitutively, and only one melanoma, FM3, released detectable amounts after stimulation. In contrast, U-937 monocyte control cells released high amounts of TNFalpha on identical stimulation. All the melanoma cell lines were relatively resistant against exogenous TNFalpha, and there was a significant correlation (P < 0.01) between intracellular Trx/TrxR expression and TNFalpha resistance (IC50). In conclusion, Trx and TrxR, as well as TNFalpha, IL1alpha and IL1beta, were highly expressed in cultured normal skin melanocytes and malignant melanoma cell lines. In contrast to U-937 monocytic cells, TNFalpha showed a secretory block in these cells, suggesting a cytoprotective and possible autocrine role for TNFalpha. The intracellular expression of Trx and TrxR together with endogenous TNFalpha was correlated with the resistance to TNFalpha-induced cytotoxicity.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13715 (URN)10.1097/00008390-200008000-00004 (DOI)
Available from: 2002-01-11 Created: 2002-01-11 Last updated: 2017-09-22
4. Redox-signaling transmitted in trans to neighboring cells by melanoma-derived TNF-containing exosomes
Open this publication in new window or tab >>Redox-signaling transmitted in trans to neighboring cells by melanoma-derived TNF-containing exosomes
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2007 (English)In: Free Radical Biology and Medicine, ISSN 0891-5849, Vol. 43, no 1, 90-99 p.Article in journal (Refereed) Published
Abstract [en]

Hydrogen peroxide is known to be involved in redox signaling pathways that regulate normal processes and disease progression, including cytokine signaling, oxidative stress, and cancer. In studies on immune surveillance against cancer, hydrogen peroxide was found to disrupt cytotoxic T-cell function, thus contributing to tumor escape. In this study, secretion of TNF-containing vesicles of rab9+ endosomal origin, termed exosomes, was investigated using GFP-TNF constructs. We observed a polarized intracellular trafficking and apical secretion of TNF-positive nanovesicles. Cell-to-cell transfer of TNF was observed in exosomes in real-time microscopy, occurring separate from the melanin/melanosome compartment. Exosomes were prepared by ultracentrifugation or immunoisolation on anti-β2-microglobulin magnetic beads. TNF as well as TNF receptors 1 and 2 were present in the exosomes as determined by Western blot, flow cytometry, and deconvolution microscopy. The functional significance of melanoma-derived exosomes was established by their signaling competence with ability to generate significantly higher ROS levels in T cells compared with sham exosomes (P = 0.0006). In conclusion, we report here, for the first time, that TNF is found in tumor cell-derived exosomes and that these exosomes transmit redox signaling in trans to neighboring cells. The results are of importance for a better understanding of tumor escape mechanisms.

Keyword
TNF, Exosomes, Melanoma, Hydrogen peroxide, Redox signaling, Tumor escape mechanisms
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-13716 (URN)10.1016/j.freeradbiomed.2007.03.026 (DOI)000247395000011 ()
Available from: 2002-01-11 Created: 2002-01-11 Last updated: 2017-09-22
5. TNFα expression predicts a better outcome in thick malignant melanoma
Open this publication in new window or tab >>TNFα expression predicts a better outcome in thick malignant melanoma
Manuscript (Other academic)
Identifiers
urn:nbn:se:liu:diva-13717 (URN)
Available from: 2002-01-11 Created: 2002-01-11 Last updated: 2010-01-13

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