Cisplatin and oxaliplatin are toxic to cochlear outer hair cells and both target thioredoxin reductase in organ of Corti cultures.

CONCLUSION: Inhibition of thioredoxin reductase (TrxR) may be a contributing factor in cisplatin-induced ototoxicity. Direct exposure of organ of Corti to cisplatin and oxaliplatin gives equal loss of hair cells. OBJECTIVES: Platinum-containing drugs are known to target the anti-oxidant selenoprotein TrxR in cancer cells. Two such anti-cancer, platinum-containing drugs, cisplatin and oxaliplatin, have different side effects. Only cisplatin induces hearing loss, i.e. has an ototoxic side effect that is not seen after treatment with oxaliplatin. The objective of this study was to evaluate if TrxR is a target in the cochlea. Loss of outer hair cells was also compared when cisplatin and oxaliplatin were administered directly to the organ of Corti. METHODS: Organ of Corti cell culture was used for direct exposure to cisplatin and oxaliplatin. Hair cells were evaluated and the level of TrxR was assessed. Immunohistochemical staining for TrxR was performed. An animal model was used to evaluate the effect on TrxR after treatment with cisplatin and oxaliplatin in vivo. RESULTS: Direct exposure of cochlear organotypic cultures to either cisplatin or oxaliplatin induced comparable levels of outer hair cell loss and inhibition of TrxR, demonstrating that both drugs are similarly ototoxic provided that the cochlea becomes directly exposed.

Human Protein Atlas of redox systems - what can be learnt?

BACKGROUND: High-throughput screening projects are popular approaches to yield a vast amount of information amenable for database mining and "hypothesis generation". The keys to success for these approaches depend upon the quality of primary data, choice of algorithms for data analyses, solidity in data annotations and the general usefulness of the results. A large initiative aimed at mapping the expression of all human proteins is the Human Protein Atlas (www.proteinatlas.org), encompassing immunohistochemical analyses of human tissues utilizing antibodies raised against a large number of human proteins. Here, we wished to probe what could be learnt from this atlas using a manual in-depth analysis of the results regarding the expression of key proteins in the human glutathione and thioredoxin systems. METHODS: The freely available on-line data of immunohistochemical analyses for selected human redox proteins within the Human Protein Atlas were here analyzed, provided that reasonably solid data existed for the antibodies that were employed. This included tissue expression data for thioredoxin 1 (Trx1), Trx2, thioredoxin reductase 1 (TrxR1), TrxR2, glutathione reductase (GR), glucose 6-phosphate dehydrogenase (G6PD), γ-glutamyl cysteinyl synthase (gGCS) and the six peroxiredoxins Prx1 to Prx6. The data were further complemented with a screen using a polyclonal peptide antibody raised against the unique glutaredoxin domain of TXNRD1_v3 ("v3"). The results from fifteen major tissues and organs are presented (lung, kidney, liver, lymph node, testis, prostate, ovary, breast, pancreas, cerebellum, hippocampus, cerebral cortex, skin, skeletal muscle and heart muscle) and discussed considering earlier findings described in the literature. RESULTS: Staining patterns proved to be highly variable and often unexpected both in terms of tissues analyzed and the individual target proteins. Among the analyzed tissues, only macrophages of the lung, tubular cells of the kidney, lymphoid cells of lymph nodes, Leydig cells in the testis, glandular cells of the prostate and exocrine glandular cells of the pancreas, showed positive staining with all of the fourteen antibodies that were analyzed. Among these antibodies, those against Trx1, TrxR2 and G6PD showed the most restricted staining across different tissues, while others including the antibodies against Trx2, TrxR1, GR, Prx3, Prx4 and Prx6 gave strong staining in most tissues. Staining for v3 was strong in many cells and tissues, which was unexpected considering previous results mapping transcripts for this protein. No obvious co-variation in staining across tissues could be noted when comparing any two of the analyzed antibodies. Staining for G6PD was weak in most tissues, except for cells of the seminiferous ducts in testis and follicular cells of the ovary, where G6PD staining was strong. CONCLUSIONS: Results from high-throughput screening projects such as the Human Protein Atlas must be taken with caution and need to be duly confirmed by thorough in-depth follow-up studies. The varying staining intensities comparing tissues as seen here for most of the analyzed antibodies nonetheless suggest that the overall profile of the human redox systems may vary significantly between different cell types and between different tissues. GENERAL SIGNIFICANCE: The Human Protein Atlas data suggest that the individual proteins of the human thioredoxin and glutathione systems may be strikingly tissue- and cell type-specific in terms of expression levels, but we also conclude that these type of high-throughput results should be taken with significant caution and must be duly verified using subsequent focused and detailed hypothesis-guided follow-up studies. This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.

Induction of cell membrane protrusions by the N-terminal glutaredoxin domain of a rare splice variant of human thioredoxin reductase 1.

The human thioredoxin system has a wide range of functions in cells including regulation of cell proliferation and differentiation, immune system modulation, antioxidant defense, redox control of transcription factor activity, and promotion of cancer development. A key component of this enzymatic system is the selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the TXNRD1 gene. Transcription of TXNRD1 involves alternative splicing, leading to a number of transcripts also encoding isoforms of TrxR1 that differ from each other at their N-terminal domains. Here we have studied the TXNRD1_v3 isoform containing an atypical N-terminal glutaredoxin (Grx) domain. Expression of the transcript of this isoform was found predominantly in testis but was also detected in ovary, spleen, heart, liver, kidney, and pancreas. By immunohistochemical analysis in human testis with antibodies specific for the Grx domain of TXNRD1_v3, the protein was found to be predominantly expressed in the Leydig cells. Expression of the TXNRD1_v3 transcript was also found in several cancer cell lines (HCC1937, H23, A549, U1810, or H157), and in HeLa cells, it was induced by estradiol or testosterone treatments. Surprisingly, green fluorescent protein fusions with the complete TXNRD1_v3 protein or with only its Grx domain localized to distinct cellular sites in proximity to actin, and furthermore, had a potent capacity to rapidly induce cell membrane protrusions. Analyses of these structures suggested that the Grx domain of TXNRD1_v3 localizes first in the emerging protrusion and is then followed into the protrusions by actin and subsequently by tubulin. The results presented thus reveal that TXNRD1_v3 has a unique and distinct expression pattern in human cells and suggest that the protein can guide actin polymerization in relation to cell membrane restructuring.

Heat-inducible amplifier vector for high-level expression of granulocyte-macrophage colony-stimulating factor.

PURPOSE: In cytokine immunotherapy of cancer it is critical to deliver sufficiently high local cytokine concentrations in order to reach the therapeutic threshold needed for clinical efficacy. Simultaneously, for optimal clinical safety adverse effects caused by high systemic cytokine levels must be minimized. One of the most promising anti-cancer therapeutic cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF), has elicited anti-tumour immune responses in animal studies and clinical trials. However, the clinical efficacy has been limited, with local GM-CSF levels being therapeutically insufficient and systemic toxicity being a limiting factor. METHODS: To address these problems we have developed a novel GM-CSF expression vector, pAD-HotAmp-GM-CSF, which can provide high levels of GM-CSF expression, and induction of cytokine expression to limited tissue areas. This expression system combines inducible and amplifying elements in a single multi-genic construct. The first transcriptional unit contains the inducible element, the heat shock protein 70B (HSP70B) promoter that regulates expression of the transcription-activating factor tat. RESULTS: Upon the binding of tat to the second promoter, the HIV2 long terminal repeat amplifies downstream gene expression of the therapeutic cytokine GM-CSF. Moderate hyperthermia at 42 degrees C for 30 min induced GM-CSF expression in pAD-HotAmp-GM-CSF that was over 2.5- and 2.8-fold higher than levels reached with HSP70B promoter alone and the prototypical human cytomegalovirus promoter. CONCLUSIONS: Thus, the inducible amplifier vector, pAD-HotAmp-GM-CSF, represents a novel system for regulated and enhanced GM-CSF expression, which enables both greater efficacy and safety in cytokine immunotherapy of cancer.