Tomas Nordman

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.

Loss of nicotinic receptors induced by beta-amyloid peptides in PC12 cells: Possible mechanism involving lipid peroxidation

The mechanisms involved in the loss of nicotinic acetylcholine receptors (nAChRs), seen in brains of patients with Alzheimers disease (AD) and in cultured cells treated by β‐amyloid peptides (Aβs), remain elusive. We give results to show that lipid peroxidation induced directly by Aβ might be involved in the deficits of nAChRs. In the study, PC12 cells were treated by addition of 5 μM of Aβ25–35 and Aβ1–40, respectively, with or without a antioxidant, vitamin E. Besides significantly decreased MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5,diphenyltetrazolium bromide) reduction, an increased lipid peroxidation was detected in the cells, but no protein oxidation. Significant reductions in [3H]epibatidine and [125I]α‐bungarotoxin binding sites and in the protein levels of the α3 and α7 nAChR subunits were observed in the cells treated with Aβs. Furthermore, Aβ25–35 decreased the level of ubiquinone‐9 in PC12 cells, but did not change the amount of cholesterol, providing further evidence for lipid peroxidation. Interestingly, when PC12 cells were pretreated by antioxidant before the addition of Aβs, the lipid peroxidation and the decreased ubiquinone resulted from Aβs were prohibited. The decreases of nAChR binding sites and subunit proteins resulted from Aβs were mostly prevented by the pretreatment with antioxidant. These findings suggest that lipid peroxidation stimulated by Aβs might be a mechanism for the loss of nAChRs associated with the pathogenesis of AD.

Lovastatin stimulates up‐regulation of α7 nicotinic receptors in cultured neurons without cholesterol dependency, a mechanism involving production of the α‐form of secreted amyloid precursor protein

The cholesterol‐lowering drug lovastatin enhances the secretion of the α‐secretase cleavage product of amyloid precursor protein (APP). To investigate whether this effect is mediated via activation of α7 nicotinic acetylcholine receptors (nAChRs), we treated SH‐SY5Y cells and PC12 cells with lovastatin and measured the levels of α7 nAChRs, the α‐form of secreted APP (αAPPs), and lovastatin‐related lipids, including cholesterol and ubiquinone. The results showed that low concentrations of lovastatin significantly induced up‐regulation of α7 nAChRs. No effects of lovastatin were observed on α3‐containing nAChRs, muscarinic receptors, or N‐methyl‐D‐aspartate receptors. αAPPs levels increased in the culture medium of cells treated with lovastatin, whereas no change in whole APP was observed. The increase in αAPPs was inhibited by prior exposure of these cells to α‐bungarotoxin, an antagonist of α7 nAChRs. The concentrations of lovastatin used in the study did not change the cholesterol content, but high doses can decrease the levels of ubiquinone and cell viability. These results indicate that lovastatin may play a neuronal role that is cholesterol independent. We also show that the up‐regulation of α7 nAChRs stimulated by lovastatin is involved in a mechanism that enhances production of αAPPs during APP processing.

Extramitochondrial reduction of ubiquinone by flavoenzymes.

Publisher Summary This chapter summarizes the current methods for extramitochondrial regeneration of the reduced antioxidant active form of ubiquinone, ubiquinol, by flavoenzymes belonging to a family of pyridine nucleotide oxidoreductases, and studies of these reductions in homogenates and cytosols isolated from cell lines. For the antioxidant function of ubiquinol and because it is widely spread in all membranes, it is of great importance that the reduced form can be regenerated at all these locations. It describes the regeneration of the reduced form of ubiquinone, ubiquinol, by the three well-known enzymes lipoamide dehydrogenase (LipDH), mammalian thioredoxin reductase (TrxR-1), and glutathione reductase (GR), and by homogenate and cytosol isolated from cells. As these enzymes are located at overlapping and at various different intracellular locations and in vitro studies show different enzymatic reaction characteristics in the reduction of ubiquinone, it is believed that these reactions are important to protect the cell under various cellular conditions caused by, for example, oxidative stress. These protecting mechanisms are correlated with other functions in the cell crucial for cell survival. Many investigators have so far studied the regeneration of ubiquinone, and different quinone reductases are proposed as reduction enzymes.

Adriamycin cytotoxicity may stimulate growth of hepatocellular tumours in an experimental model for adjuvant systemic chemotherapy in liver transplantation

Adjuvant treatment with adriamycin has been suggested to improve results after liver transplantation for hepatocellular cancer. Here we have applied an animal model for evaluation of treatment with adriamycin and/or cyclosporine A on liver tumour growth. Three chemically induced rat liver tumours with various degree of differentiation were transferred to the spleens of syngenic rats. Each recipient group was divided into four subgroups, treated with adriamycin and/or cyclosporine A or none of the drugs. When the tumour was well differentiated no proliferation was found in any of the subgroups. When the tumour exhibited a more pronounced dysplasia, adriamycin stimulated tumour growth. This effect was further increased by cyclosporine. In the animals transplanted with the most aggressive tumour, adriamycin inhibited tumour growth. When given together with cyclosporine this inhibition was counteracted. These data suggest that adriamycin, especially when given together with cyclosporine, may have a stimulatory effect on liver tumour cell growth.