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2-Chlorodeoxyadenosine (CdA) is an antileukaemic agent used in treatment of hairy cell leukaemia (HCL) and chronic lymphocytic leukaemia of B- and T-cell type (B-CLL and T-CLL). The aim of this study was to elucidate the interpatient variability of CdA phosphorylation and its relation to response to CdA treatment. In extracts of peripheral blood mononuclear cells of patients with B-CLL (n = 39), CdA phosphorylation was significantly higher than in HCL (n = 19) when calculated per protein (391 +/- 155 pmol CdA phosphorylated/mg protein/min versus 288 +/- 166 pmol/mg/min, P < 0.001), but was the same when calculated per cell (12 +/- 5.9 pmol/10(6) cells/min versus 14 +/- 5.9 pmol/10(6) cells/min) due to a larger cell volume in HCL. In T-CLL (n = 6), CdA phosphorylation was significantly lower than in B-CLL, both when calculated per protein (128 +/- 68 pmol/mg/min, P < 0.001) or per cell (5.7 +/- 2.7 pmol/10(6) cells/min, P < 0.05). This low CdA phosphorylation in T-CLL was unexpected because normal B- and T-lymphocytes contain equal amounts of CdA phosphorylation. With B-CLL, 21 patients who responded (complete and partial response) to CdA treatment showed a significantly higher CdA phosphorylation than 13 patients not responding to CdA treatment (456 +/- 170 pmol/mg/min versus 309 +/- 97 pmol/mg/min, P < 0.01). We conclude that the level of CdA phosphorylation is correlated with the response of leukaemias to CdA treatment.

Deoxynucleoside kinases are key enzymes in deoxyribonucleoside salvage, activating several clinically important chemotherapeutic drugs. The four known kinases, cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK) and the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK), have been purified and characterized as to the subunit structure as well as specificity with a large number of analogs. These results are summarized and used to establish selective assays for the four enzymes in crude extracts of normal and malignant human peripheral blood mononuclear cells, gastrointestinal tissues and sarcomas. TK2 and dGK activities were found at low levels in all tissues, possibly correlated to the content of mitochondria. TK1 activity was detected only in samples containing a significant number of S phase cells. We have measured dCK activity as well as dCK polypeptide level by immuno blotting in these extracts. High levels of dCK were found in normal mononuclear leukocytes (91-145 ng dCK/mg protein) and in B-cell chronic lymphocytic leukemia (80 +/- 30 ng/mg, n = 23). Hairy cell leukemia contained lower levels (28 +/- 23 ng/mg, n = 7), as did unexpectedly three samples of T-cell chronic lymphocytic leukemia (18 +/- 14 ng/mg). Phytohemaglutinine stimulation of normal lymphocytes did not lead to any substantial increase in either dCK activity or expression (less than 2.5-fold). In colon adenocarcinomas, the dCK content was significantly higher (21 +/- 9.3 ng/mg, n = 20) than in normal colon mucosa (8.2 +/- 3.7 ng/mg, n = 19, p < 0.05). A similar pattern of dCK expression was found in gastric adenocarcinomas (21 +/- 13 ng/mg, n = 5) and normal ventricular mucosa (6.2 +/- 5.4 ng/mg, n = 5, p < 0.15). One leiomyosarcoma and one extra-skeletal osteosarcoma showed a dCK levels comparable to those found in normal lymphocytes (84 +/- 6 and 109 +/- 4 ng/mg), while other sarcoma samples contained levels comparable to the gastrointestinal adenocarcinomas (20 +/- 7 ng/mg, n = 12). We confirm that dCK is expressed constitutively and predominantly in lymphoid cells, but conclude that a significant expression may be found in non-lymphoid tissues as well, with increased levels in the corresponding tumor tissue. 2-Chlorodeoxyadenosine (CdA), an antileukemic agent used in treatment of hairy cell leukemia and chronic lymphocytic leukemias (B-CLL), is phosphorylated by dCK which was used as the selective substrate for this enzyme. A study was performed to investigate if there was a correlation between the dCK levels and the response to CdA treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

The selenoprotein thioredoxin reductase 1 (TrxR1) has in recent years been identified as a promising anticancer drug target. A high-throughput assay for discovery of novel compounds targeting the enzyme is therefore warranted. Herein, we describe a single-enzyme, dual-purpose assay for simultaneous identification of inhibitors and substrates of TrxR1. Using this assay to screen the LOPAC1280 compound collection we identified several known inhibitors of TrxR1, thus validating the assay, as well as several compounds hitherto unknown to target the enzyme. These included rottlerin (previously reported as a PKCδ inhibitor and mitochondrial uncoupler) and the heme precursor protoporphyrin IX (PpIX). We found that PpIX was a potent competitive inhibitor of TrxR1, with a Ki = 2.7 μM with regard to Trx1, and in the absence of Trx1 displayed time-dependent irreversible inhibition with an apparent second-order rate constant (kinact) of (0.73 ± 0.07) × 10− 3 μM− 1 min− 1. Exogenously delivered PpIX was cytotoxic, inhibited A549 cell proliferation, and was found to also inhibit cellular TrxR activity. Hemin and the ferrochelatase inhibitor NMPP also inhibited TrxR1 and showed cytotoxicity, but less potently compared to PpIX. We conclude that rottlerin-induced cellular effects may involve targeting of TrxR1. The unexpected finding of PpIX as a TrxR1 inhibitor suggests that such inhibition may contribute to symptoms associated with conditions of abnormally high PpIX levels, such as reduced ferrochelatase activity seen in erythropoietic protoporphyria. Finally, additional inhibitors of TrxR1 may be discovered and further characterized based upon the new high-throughput TrxR1 assay presented here.

6 páginas, 5 figuras.-- et al., Selenoproteins, in particular thioredoxin reductase, have been implicated in countering oxidative damage occurring during aging but the molecular functions of these proteins have not been extensively investigated in different animal models. Here we demonstrate that TRXR-1 thioredoxin reductase, the sole selenoprotein in Caenorhabditis elegans, does not protect against acute oxidative stress but functions instead together with GSR-1 glutathione reductase to promote the removal of old cuticle during molting. We show that the oxidation state of disulfide groups in the cuticle is tightly regulated during the molting cycle, and that when trxr-1 and gsr-1 function is reduced, disulfide groups in the cuticle remain oxidized. A selenocysteine-to-cysteine TRXR-1 mutant fails to rescue molting defects. Furthermore, worms lacking SELB-1, the C. elegans homolog of Escherichia coli SelB or mammalian EFsec, a translation elongation factor known to be specific for selenocysteine in E. coli, fail to incorporate selenocysteine, and display the same phenotype as those lacking trxr-1. Thus, TRXR-1 function in the reduction of old cuticle is strictly selenocysteine dependent in the nematode. Exogenously supplied reduced glutathione reduces disulfide groups in the cuticle and induces apolysis, the separation of old and new cuticle, strongly suggesting that molting involves the regulated reduction of cuticle components driven by TRXR-1 and GSR-1. Using dauer larvae, we demonstrate that aged worms have a decreased capacity to molt, and decreased expression of GSR-1. Together, our results establish a function for the selenoprotein TRXR-1 and GSR-1 in the removal of old cuticle from the surface of epidermal cells., The work was supported by grants from Cancerfonden (09 0507) and Vetenskapsrådet (K2009-67X-20441-03-3) (to S.T.), grants from The Swedish Foundation for Strategic Research, Vetenskapsrådet, and the NordForsk Nordic C. elegans Network (to P.S.), a grant from The Carl Trygger Foundation and Vetenskapsrådet (B650-19981122/2000) (to O.P.P.), grants from the Instituto de Salud Carlos III (Projects PI050065 and PI080557, cofinanced with the Fondo Social Europedeo) and Junta de Andalucía (Projects P07-CVI-02697 and P08-CVI-03629) (to A.M.-V.), and a predoctoral fellowship from CONACYT, Mexico (to B.C.-V.).

Selenium research must be said to have began in 1817, when Berzelius discovered this element. The first genuine publication describing this research was published by Berzelius in 1818, in a paper where he also named the element as Selenium. Here, in this chapter on the history of selenium research, an attempt is made to take a “bird’s-eye” view at the development of this research field since 1817 until today. The tool chosen is an analysis of the scientific literature on selenium research, thereby attempting to give an unbiased assessment of this research field. Finally, as in all assessments of historic trends, we should also ask where the future of selenium research might take us. By necessity, the answer to that question is uncertain. However, we can conclude that never before has selenium research been as vigorous and expanding as it is today, which also holds major promise for the future.

Resting and stimulated human peripheral blood lymphocytes and monocyte-derived macrophages were incubated with tritiated deoxycytidine labeled at the 5-position. Release of tritiated water into the medium was thereupon detected utilizing its lack of binding to active charcoal, which is an established technique to measure in situ thymidylate synthase activity. It was found that tritiated dihydrouracil, a deoxycytidine catabolite, was formed during incubation with tritiated deoxycytidine. Like water, dihydrouracil does not bind to active charcoal, and its presence in the cell medium can result in an overestimation of the in situ thymidylate synthase activity. The catabolism of dCyd was highest in macrophages where 25% of the added dCyd (0.5 μM, 0.5 nmol/million cells) had been converted to dihydrouracil after 30 min, and 90% after 12 h. The in situ thymidylate synthase activity was found to be the highest in stimulated lymphocytes. If the interference of dihydrouracil had not been considered, the activity in macrophages would have been greatly overestimated and would have appeared to be higher than that of stimulated lymphocytes.

Palladium (Pd), platinum (Pt), and gold (Au) are noble metals, two of which have established medical use. Pt has anticancer efficacy, predominantly as cisplatin, whereas the gold compound auranofin is used against arthritis. Both compounds inhibit the selenoprotein thioredoxin reductase (TrxR), but Pd has not been studied in this regard. Using salts of Pd, Pt, and Au as well as cisplatin and auranofin we found that Pd and Au were strikingly more potent inhibitors of recombinant TrxR1 than Pt. The TrxR-related nonselenoprotein glutathione reductase in pure form (but less so in a cellular context), as well as cellular thioredoxin (Trx) activities, were inhibited by the gold salt KAuCl(4) but were little affected by auranofin or the other compounds. In an analysis of three cancer cell lines, the extent of inhibition of TrxR activity and decrease in cell viability depended upon the choice of both noble metal and ligand and also seemed independent of p53 status. During treatment of cells with cisplatin, covalent complexes of TrxR1 with either Trx1 or TRP14 (Trx-related protein of 14kDa) were formed, as verified by Western blot analyses and mass spectrometry. These results reveal that Au and Pd are strong inhibitors of TrxR, but Pt and cisplatin trigger highly specific cellular effects on the Trx system, including covalent cross-linking of TrxR1 with Trx1 and TRP14.

Schistosomiasis is a chronic parasitic disease affecting hundreds of millions of individuals worldwide. Current treatment depends on a single agent, praziquantel, raising concerns of emergence of resistant parasites. Here, we continue our explorations of an oxadiazole-2-oxide class of compounds we recently identified as inhibitors of thioredoxin glutathione reductase (TGR), a selenocysteine-containing flavoenzyme required by the parasite to maintain proper cellular redox balance. Through systematic evaluation of the core molecular structure of this chemotype we define the essential pharmacophore, establish a link between the nitric oxide donation and TGR inhibition, determine the selectivity for this chemotype versus related reductase enzymes and present evidence that these agents can be modified to possess appropriate drug metabolism and pharmacokinetic properties. The mechanistic link between exogenous NO donation and parasite injury is expanded and better defined. The results of these studies verify the utility of oxadiazole-2-oxides as novel inhibitors of TGR and as efficacious anti-schistosomal reagents.

24 páginas, 6 figuras. Material suplementario 2 figuras., Thioredoxin reductases are important selenoproteins maintaining cellular redox balance and regulating several redox dependent processes in apoptosis, cell proliferation and differentiation. Specific functions of dedicated splice variants may add further complexity to the functions of these proteins. We show here that a splice variant of human thioredoxin reductase 1, TXNRD1_v3, forms both dynamic cytoplasmic filaments and provokes instantaneous formation of dynamic cell membrane protrusions identified as filopodia. Using truncated versions of the protein we found that both the cytoplasmic filaments and the filopodia formation were exclusively dependent on the glutaredoxin domain of the protein. Interestingly, actin polymerization was required for filopodia formation triggered by TXNRD1_v3, but not for generation of cytoplasmic filaments. We conclude that the glutaredoxin domain of TXNRD1_v3 is an atypical regulator of the cell cytoskeleton that potently induces formation of highly ordered cytoplasmic filaments and cell membrane filopodia., This work was supported by the Swedish Cancer Society, the Swedish Research Council ProjectK99-13X-10370, the Knut and Alice Wallenberg Foundations and the Karolinska Institutet. A.M.-V. was supported by a “Ramón y Cajal” contract from the Spanish Ministry of Science.

Selenoproteins contain a highly reactive 21st amino acid selenocysteine (Sec) encoded by recoding of a predefined UGA codon. Because of a lack of selenoprotein supply, high chemical reactivity of Sec, and intricate translation machineries, selenoprotein crystal structures are difficult to obtain. Structural prerequisites for Sec involvement in enzyme catalysis are therefore sparsely known. Here we present the crystal structure of catalytically active rat thioredoxin reductase 1 (TrxR1), revealing surprises at the C-terminal Sec-containing active site in view of previous literature. The oxidized enzyme presents a selenenylsulfide motif in trans-configuration, with the selenium atom of Sec-498 positioned beneath the side chain of Tyr-116, thereby located far from the redox active moieties proposed to be involved in electron transport to the Sec-containing active site. Upon reduction to a selenolthiol motif, the Sec residue moved toward solvent exposure, consistent with its presumed role in reduction of TrxR1 substrates or as target of electrophilic agents inhibiting the enzyme. A Y116I mutation lowered catalytic efficiency in reduction of thioredoxin, but surprisingly increased turnover using 5-hydroxy-1,4-naphthoquinone (juglone) as substrate. The same mutation also decreased sensitivity to inhibition by cisplatin. The results suggest that Tyr-116 plays an important role for catalysis of TrxR1 by interacting with the selenenylsulfide of oxidized TrxR1, thereby facilitating its reduction in the reductive half-reaction of the enzyme. The interaction of a selenenylsulfide with the phenyl ring of a tyrosine, affecting turnover, switch of substrate specificity, and modulation of sensitivity to electrophilic agents, gives important clues into the mechanism of TrxR1, which is a selenoprotein that plays a major role for mammalian cell fate and function. The results also demonstrate that a recombinant selenoprotein TrxR can be produced in high amount and sufficient purity to enable crystal structure determination, which suggests that additional structural studies of these types of proteins are feasible.

Thioredoxin systems, involving redox active thioredoxins and thioredoxin reductases, sustain a number of important thioredoxin-dependent pathways. These redox active proteins support several processes crucial for cell function, cell proliferation, antioxidant defense and redox-regulated signaling cascades. Mammalian thioredoxin reductases are selenium-containing flavoprotein oxidoreductases, dependent upon a selenocysteine residue for reduction of the active site disulfide in thioredoxins. Their activity is required for normal thioredoxin function. The mammalian thioredoxin reductases also display surprisingly multifaceted properties and functions beyond thioredoxin reduction. Expressed from three separate genes (in human named TXNRD1, TXNRD2 and TXNRD3), the thioredoxin reductases can each reduce a number of different types of substrates in different cellular compartments. Their expression patterns involve intriguingly complex transcriptional mechanisms resulting in several splice variants, encoding a number of protein variants likely to have specialized functions in a cell- and tissue-type restricted manner. The thioredoxin reductases are also targeted by a number of drugs and compounds having an impact on cell function and promoting oxidative stress, some of which are used in treatment of rheumatoid arthritis, cancer or other diseases. However, potential specific or essential roles for different forms of human or mouse thioredoxin reductases in health or disease are still rather unclear, although it is known that at least the murine Txnrd1 and Txnrd2 genes are essential for normal development during embryogenesis. This review is a survey of current knowledge of mammalian thioredoxin reductase function and expression, with a focus on human and mouse and a discussion of the striking complexity of these proteins. Several yet open questions regarding their regulation and roles in different cells or tissues are emphasized. It is concluded that the intriguingly complex regulation and function of mammalian thioredoxin reductases within the cellular context and in intact mammals strongly suggests that their functions are highly fi ne-tuned with the many pathways involving thioredoxins and thioredoxin-related proteins. These selenoproteins furthermore propagate many functions beyond a reduction of thioredoxins. Aberrant regulation of thioredoxin reductases, or a particular dependence upon these enzymes in diseased cells, may underlie their presumed therapeutic importance as enzymatic targets using electrophilic drugs. These reductases are also likely to mediate several of the effects on health and disease that are linked to different levels of nutritional selenium intake. The thioredoxin reductases and their splice variants may be pivotal components of diverse cellular signaling pathways, having importance in several redox-related aspects of health and disease. Clearly, a detailed understanding of mammalian thioredoxin reductases is necessary for a full comprehension of the thioredoxin system and of selenium dependent processes in mammals.

Thioredoxin reductase (TrxR) is a key selenoprotein antioxidant enzyme and a potential target for anti-cancer drugs. One potent inhibitor of TrxR is the gold (I) compound auranofin, which can trigger mitochondrial-dependent apoptosis pathways. The exact mechanism of apoptosis induction by auranofin is not yet clear, but there are indications that mitochondrial oxidative stress is a central event. We assessed the redox state of the peroxiredoxins (Prxs) in Jurkat T-lymphoma cells treated with auranofin, and found that mitochondrial Prx3 was considerably more sensitive to oxidation than the cytosolic Prx1 and 2, indicating selective mitochondrial stress. Prx3 oxidation was detected at apoptotic doses of auranofin in several cell types, and occurred before other mitochondrial events including cytochrome c release and mitochondrial depolarisation. Auranofin was also able to sensitise U937 cells to TNF-alpha-mediated apoptosis. Auranofin-induced apoptosis was effectively blocked by the overexpression of Bcl-2, and Bax/Bak deficient mouse embryonic fibroblasts were also resistant to apoptosis, indicating a central role for the pro-apoptotic proteins of this family in auranofin-triggered apoptosis. Auranofin exposure inhibited the proliferation of apoptosis-resistant cells, and at higher doses of auranofin could cause cell death through necrosis. We conclude that auranofin induces apoptosis in cells through a Bax/Bak-dependent mechanism associated with selective disruption of mitochondrial redox homeostasis in conjunction with oxidation of Prx3.

Isothiocyanates are phytochemicals with anti-cancer properties that include the ability to trigger apoptosis. A substantial body of evidence suggests that reaction of the electrophilic isothiocyanate moiety with cysteine residues in cellular proteins and glutathione accounts for their biological activity. In this study we investigated the effect of several different isothiocyanates on the redox states of the cysteine-dependent peroxiredoxins (Prx) in Jurkat T lymphoma cells, and compared this to known effects on the selenoprotein thioredoxin reductase, glutathione reductase and intracellular GSH levels. Interestingly, oxidation of mitochondrial Prx3 could be detected as early as 5 min after exposure of cells to phenethyl isothiocyanate, with complete oxidation occurring at doses that only had small inhibitory effects on total cellular thioredoxin reductase and glutathione reductase activities. Peroxiredoxin oxidation was specific to the mitochondrial isoform with cytoplasmic Prx1 and Prx2 maintained in their reduced forms at all analyzed time points and concentrations of isothiocyanate. Phenethyl isothiocyanate could react with purified Prx3 directly, but it did not oxidize Prx3 or promote its oxidation by hydrogen peroxide. A selection of aromatic and alkyl isothiocyanates were tested and while all lowered cellular GSH levels, only the isothiocyanates that caused Prx3 oxidation were able to trigger cell death. We propose that pro-apoptotic isothiocyanates selectively disrupt mitochondrial redox homeostasis, as indicated by Prx3 oxidation, and that this contributes to their pro-apoptotic activity.

BackgroundSecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins) can be formed from the selenoprotein thioredoxin reductase (TrxR) by targeting of its selenocysteine (Sec) residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function.Principal findingsBoth human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS) and consequently antioxidants could protect against the cell killing by SecTRAPs.ConclusionsWe conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity.

18 páginas, 5 figuras, 1 esquema, 1 película., 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., This study was supported by grants from the Swedish Cancer Society, the Swedish Research Council (Medicine), the Åke Wibergs Foundation and Karolinska Institutet.

In this chapter we describe strategies to produce synthetic selenoproteins, with a focus on recombinant selenoprotein production in E. coll. We further discuss the possible use of selenocysteine (Sec) in proteins for biotechnological applications. Such applications are based upon either the introduction of a selenium isotope, with specific characteristics such as high-energy radioactivity (as for 75Se and 73Se) or nuclear spin (77Se), or on the high reactivity of the nucleophilic Sec residue enabling site-specific conjugation-based applications with electrophilic ligands. Utilization of Sec insertion for protein purification or detection purposes has recently been demonstrated in a number of different experimental systems and we envision that further such applications will be developed in the near future.

Thioredoxin (Trx), NADPH and thioredoxin reductase (TrxR) comprise a thioredoxin system which exists in nearly all living cells. It functions in thiol-dependent thiol-disulfide exchange reactions crucial to control of the reduced intracellular redox environment, cellular growth, defense against oxidative stress or control of apoptosis and has multi-facetted roles in mammalian cells including implications in cancer. Eg reduced Trx activates DNA binding of transcription factors and is involved in antioxidant defense through repair of oxidatively damaged proteins or as an electron donor to peroxiredoxins. The Trx system functions in synthesis of deoxyribonucleotides for DNA synthesis, both replication and repair, by ribonucleotide reductase. Trx and truncated Trx (Trx80) act in modulation of immune cell function. TrxR isoforms in the cytosol and the mitochondria are essential selenoenzymes with a selenocysteine in the active site. These enzymes display a remarkably broad substrate specificity but are also targets for existing chemotherapeutic drugs. Mammalian TrxR enzymes are linked to selenium metabolism as a result of being selenoproteins, but can also directly reduce low molecular selenium compounds like selenite and have been implicated in the chemoprevention effects of selenium against cancer. Numerous scientific reports describe higher expression of Trx and TrxR in some, but not all tumors. Some data suggest that high Trx could be linked to resistance to chemotherapies while others suggest that high Trx and TrxR may induce apoptosis and reduce the mitotic index of certain tumors linked to the p53 dependent cell death. Recent data suggest that TrxR is essential for the carcinogenic process and invasive phenotype of cancer. Both Trx and TrxR have been regarded as interesting targets for chemotherapy.

The thioredoxin system is ubiquitous, providing reducing equivalents to essential biosynthetic enzymes like ribonucleotide reductase. It is essential for cellular redox regulation, control of oxidative stress, and protection against oxidative damage. This unit includes protocols for measuring thioredoxin or thioredoxin reductase in biological preparations or as purified enzymes.

Selenocysteine (Sec), the 21st amino acid, exists naturally in all kingdoms of life as the defining entity of selenoproteins. Sec is a cysteine (Cys) residue analogue with a selenium-containing selenol group in place of the sulfur-containing thiol group in Cys. The selenium atom gives Sec quite different properties from Cys. The most obvious difference is the lower pK(a) of Sec, and Sec is also a stronger nucleophile than Cys. Proteins naturally containing Sec are often enzymes, employing the reactivity of the Sec residue during the catalytic cycle and therefore Sec is normally essential for their catalytic efficiencies. Other unique features of Sec, not shared by any of the other 20 common amino acids, derive from the atomic weight and chemical properties of selenium and the particular occurrence and properties of its stable and radioactive isotopes. Sec is, moreover, incorporated into proteins by an expansion of the genetic code as the translation of selenoproteins involves the decoding of a UGA codon, otherwise being a termination codon. In this review, we will describe the different unique properties of Sec and we will discuss the prerequisites for selenoprotein production as well as the possible use of Sec introduction into proteins for biotechnological applications. These include residue-specific radiolabeling with gamma or positron emitters, the use of Sec as a reactive handle for electophilic probes introducing fluorescence or other peptide conjugates, as the basis for affinity purification of recombinant proteins, the trapping of folding intermediates, improved phasing in X-ray crystallography, introduction of 77Se for NMR spectroscopy, or, finally, the analysis or tailoring of enzymatic reactions involving thiol or oxidoreductase (redox) selenolate chemistry.

Mammalian thioredoxin reductase (TrxR) is important for cell proliferation, antioxidant defense, and redox signaling. Together with glutathione reductase (GR) it is the main enzyme providing reducing equivalents to many cellular processes. GR and TrxR are flavoproteins of the same enzyme family, but only the latter is a selenoprotein. With the active site containing selenocysteine, TrxR may catalyze reduction of a wide range of substrates, but can at the same time easily be targeted by electrophilic compounds due to the extraordinarily high reactivity of a selenolate moiety. Here we addressed the inhibition of the enzyme by major anticancer alkylating agents and platinum-containing compounds and we compared it to that of GR. We confirmed prior studies suggesting that the nitrosourea carmustine can inhibit both GR and TrxR. We next found, however, that nitrogen mustards (chlorambucil and melphalan) and alkyl sulfonates (busulfan) efficiently inhibited TrxR while these compounds, surprisingly, did not inhibit GR. Inhibitions were concentration and time dependent and apparently irreversible. Anticancer anthracyclines (daunorubicin and doxorubicin) were, in contrast to the alkylating agents, not inhibitors but poor substrates of TrxR. We also found that TrxR, but not GR, was efficiently inhibited by both cisplatin, its monohydrated complex, and oxaliplatin. Carboplatin, in contrast, could not inhibit any of the two enzymes. These findings lead us to conclude that representative compounds of the major classes of clinically used anticancer alkylating agents and most platinum compounds may easily target TrxR, but not GR. The TrxR inhibition should thereby be considered as a factor that may contribute to the cytotoxicity seen upon clinical use of these drugs.

Reactive oxygen species (ROS) are generated as toxic by-products of aerobic metabolism, but are also essential biomolecules in cell signaling. The thioredoxin (Trx) system is a major enzymatic system modulating ROS levels and is important for redox regulation of cellular function. It consists of Trx and thioredoxin reductase (TrxR), which reduces Trx using NADPH. Most, if not all, of the functions of Trx depend on the activity of TrxR. Mammalian TrxR enzymes are selenoproteins with broad substrate specificities, and alteration of cytosolic TrxR1 expression and activity is likely to be an important determinant for the control of cellular redox regulation. TrxR1 activity in cells seems to be modulated by an intricate interplay, involving a housekeeping type promoter in combination with alternative splice variants and transcriptional start sites, posttranscriptional regulation through AU-rich elements, inactivation by electrophilic agents and by itself modulating the effects of several key signaling molecules. TrxR1 activity is also intimately linked with several aspects of selenium metabolism, and hence selenoprotein function in general. Here, we summarize the current knowledge of these different levels of TrxR1 regulation in diverse cell types and in response to growth and signaling events.

29 páginas, 4 figuras, 2 tablas., Human thioredoxin reductase 1 (TrxR1, the TXNRD1 gene product) is a ubiquitously expressed selenoprotein with many important redox regulatory functions. In this study, we have further characterized the recently identified core promoter region of TXNRD1. One critical Sp1/Sp3 site was found to be important in A549 and HeLa cells, whereas another Sp1/Sp3 site and one Oct1 site bound transcription factors but were, nonetheless, dispensable for transcription. We also experimentally identified several 5′-region TXNRD1 transcript variants using 5′-RACE with cDNA derived from different tissues, and we analyzed all available TXNRD1-derived EST sequences. The results show that the core promoter governs transcription of the clear majority of TXNRD1 transcripts but also that alternative promoters may be activated under rare conditions or in specific cell types. Furthermore, extensive alternative splicing occured in the 5′ region of TXNRD1. In total, 21 different transcripts were identified, potentially encoding five isoforms of TrxR1 carrying alternative N-terminal domains. One isoform encompassed a glutaredoxin domain, whereas another encoded a predicted mitochondrial localization signal. These results reveal that the human thioredoxin system is intriguingly complex. Cell-specific transcription of the TXNRD1 gene encoding different isoforms of TrxR1 must be taken into account to fully understand the functions of the human thioredoxin system., This study was supported by grants from The Swedish Society for Medical Research (to A.-K. R.), the Swedish Medical Society (to E. S. J. A.), The Karolinska Institute (to E. S. J. A. and A. M.-V.), the Swedish Cancer Society (Projects 3775 and 4056 to E. S. J. A.), the Swedish Medical Research Council (Projects 03P-14096-01A and 03X-14041-01A to A. M.-V.), and the Åke Wibergs Stiftelse (A. M.-V.).

Uptake of modified low-density lipoproteins (LDLs) by macrophages in the arterial wall is an important event in atherogenesis. Indeed, oxidatively modified LDLs (oxLDLs) are known to affect various cellular processes by modulating oxidation-sensitive signaling pathways. Here we found that the ubiquitous 55 kDa selenoprotein thioredoxin reductase 1 (TrxR1), which is a key enzyme for cellular redox control and antioxidant defense, was upregulated in human atherosclerotic plaques and expressed in foam cells. Using reverse transcription polymerase chain reaction analysis, we also found that oxLDLs, but not native LDLs (nLDLs), dose-dependently increased TrxR1 mRNA in human monocyte-derived macrophages (HMDMs). This stimulating effect was specific for oxLDLs, as pro-inflammatory factors, such as lipopolysaccharides (LPSs), interleukin-1beta (IL-1beta), interleukin-6 (Il-6), and tumor necrosis factor alpha (TNFalpha), under the same conditions, failed to induce TrxR1 mRNA levels to the same extent. Moreover, phorbol ester-differentiated THP-1 cells or HMDMs transiently transfected with TrxR1 promoter fragments linked to a luciferase reporter gene allowed identification of a defined promoter region as specifically responding to the phospholipid component of oxLDLs (p.05 vs. phospholipid component of nLDLs). Gel mobility shift analyses identified a short 40-nucleotide stretch of the promoter carrying AP-1 and HoxA5 consensus motifs that responded with an altered shift pattern in THP-1 cells treated with oxLDLs, however, without evident involvement of either the Fos, Jun, Nrf2 or HoxA5 transcription factors.

Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.

Mammalian thioredoxin reductases are selenoproteins. For native catalytic activity, these enzymes utilize a C-terminal -Gly-Cys-Sec-Gly-COOH sequence (where Sec is selenocysteine) forming a redox active selenenylsulfide/selenolthiol motif. A range of cellular systems depend upon or are regulated by thioredoxin reductase and its major protein substrate thioredoxin, including apoptosis signal-regulating kinase 1, peroxiredoxins, methionine sulfoxide reductase, and several transcription factors. Cytosolic thioredoxin reductase 1 (TrxR1) is moreover inhibited by various electrophilic anticancer compounds. TrxR1 is hence generally considered to promote cell viability. However, several recent studies have suggested that TrxR1 may promote apoptosis, and the enzyme was identified as GRIM-12 (gene associated with retinoid interferon-induced mortality 12). Transient transfection with GRIM-12/TrxR1 was also shown to directly induce cell death. To further analyze such effects, we have here employed lipid-mediated delivery of recombinant TrxR1 preparations into human A549 cells, thereby bypassing selenoprotein translation to facilitate assessment of the protein-related effects on cell viability. We found that selenium-deficient TrxR1, having a two-amino acid-truncated C-terminal -Gly-Cys-COOH motif, rapidly induced cell death (38 +/- 29% apoptotic cells after 4 h; p < 0.005 compared with controls). Cell death induction was also promoted by selenium-compromised TrxR1 derivatized with either cis-diamminedichloroplatinum (II) (cisplatin) or dinitrophenyl moieties but not by the structurally related non-selenoprotein glutathione reductase. In contrast, TrxR1 with intact selenocysteine could not promote cell death. The direct cellular effects of selenium-compromised forms of TrxR1 may be important for the pathophysiology of selenium deficiency as well as for the efficacy of antiproliferative drugs targeting the selenocysteine moiety of this enzyme.

Mammalian thioredoxin reductases (TrxR) are homodimers, homologous to glutathione reductase (GR), with an essential selenocysteine (SeCys) residue in an extension containing the conserved C-terminal sequence -Gly-Cys-SeCys-Gly. In the oxidized enzyme, we demonstrated two nonflavin redox centers by chemical modification and peptide sequencing: one was a disulfide within the sequence -Cys 59 -Val-Asn-Val-Gly-Cys 64 , identical to the active site of GR; the other was a selenenylsulfide formed from Cys 497 -SeCys 498 and confirmed by mass spectrometry. In the NADPH reduced enzyme, these centers were present as a dithiol and a selenolthiol, respectively. Based on the structure of GR, we propose that in TrxR, the C-terminal Cys 497 -SeCys 498 residues of one monomer are adjacent to the Cys 59 and Cys 64 residues of the second monomer. The reductive half-reaction of TrxR is similar to that of GR followed by exchange from the nascent Cys 59 and Cys 64 dithiol to the selenenylsulfide of the other subunit to generate the active-site selenolthiol. Characterization of recombinant mutant rat TrxR with SeCys 498 replaced by Cys having a 100-fold lower k cat for Trx reduction revealed the C-terminal redox center was present as a dithiol when the Cys 59 -Cys 64 was a disulfide, demonstrating that the selenium atom with its larger radius is critical for formation of the unique selenenylsulfide. Spectroscopic redox titrations with dithionite or NADPH were consistent with the structure model. Mechanisms of TrxR in reduction of Trx and hydroperoxides have been postulated and are compatible with known enzyme activities and the effects of inhibitors, like goldthioglucose and 1-chloro-2,4-dinitrobenzene.

Mammalian thioredoxin reductase catalyzes NADPH dependent reduction of a wide variety of substrates and plays a central role in redox regulation and antioxidant defence. Recently the enzyme was discovered to be a selenoprotein with a catalytically active penultimate selenocysteine residue. Dinitrohalobenzenes irreversibly inhibit the enzyme with a concomitant induction of an NADPH oxidase activity, producing superoxide. A model explaining the reactivity of dinitrohalobenzenes with thioredoxin reductase is presented, involving dinitrophenyl-derivatization of both the selenocysteine residue and its neighboring cysteine residue, reduction by NADPH of the enzyme-bound flavin in dinitrophenyl-alkylated enzyme (dnp-TrxR), followed by two consecutive one-electron transfers from the flavin to nitro groups of the dnp-moieties in dnp-TrxR, forming nitro anion radicals. The nitro radicals react with oxygen to form superoxide, again generating dnp-TrxR with an oxidized flavin, which may then follow another cycle of NADPH-dependent superoxide production. Dinitrohalobenzene compounds are well known for their immunostimulatory properties. Here it is proposed that the inflammatory components of this immunostimulation can be mediated by interaction with the thioredoxin system, via effects on cell function by superoxide production, oxidative stress and increased extracellular levels of thioredoxin.

Publisher Summary This chapter describes the preparation and assay of mammalian thioredoxin and thioredoxin reductase (TrxR). The amino acid sequences of mammalian TrxR revealed a strikingly high homology to glutathione reductase. 14,19 The conserved features of all the structural components of glutathione reductase are preserved in mammalian TrxR, including a redox active disulfide motif in the N-terminal FAD region, the NADPH binding region, and the carboxyterminal interface region that governs the association of the two subunits in the homodimeric holoenzyme. Mammalian thioredoxin reductase has gained increased interest due to its wide reductive capacity, the discovery of selenium in the enzyme, and its lipid hydroperoxide reductase activity. As thioredoxin shows a growing number of new roles in redox regulation of cellular processes and as an extracellular cytokine, the interest in TrxR in these contexts naturally follows. Future studies of mammalian thioredoxin systems should be an exciting area of research, yielding results required for a deeper understanding of thiol redox control and mechanisms protecting against oxidative stress.

The immunostimulatory dinitrohalobenzene compound 1-chloro-2, 4-dinitrobenzene (DNCB) irreversibly inhibits mammalian thioredoxin reductase (TrxR) in the presence of NADPH, inducing an NADPH oxidase activity in the modified enzyme (Arnér, E. S. J., Björnstedt, M., and Holmgren, A. (1995) J. Biol. Chem. 270, 3479-3482). Here we have further analyzed the reactivity with the enzyme of DNCB and analogues with varying immunomodulatory properties. We have also identified the reactive residues in bovine thioredoxin reductase, recently discovered to be a selenoprotein. We found that 4-vinylpyridine competed with DNCB for inactivation of TrxR, with DNCB being about 10 times more efficient, and only alkylation with DNCB but not with 4-vinylpyridine induced an NADPH oxidase activity. A number of nonsensitizing DNCB analogues neither inactivated the enzyme nor induced any NADPH oxidase activity. The NADPH oxidase activity of TrxR induced by dinitrohalobenzenes generated superoxide, as detected by reaction with epinephrine (the adrenochrome method). Addition of superoxide dismutase quenched this reaction and also stimulated the NADPH oxidase activity. By peptide analysis using mass spectrometry and Edman degradation, both the cysteine and the selenocysteine in the conserved carboxyl-terminal sequence Gly-Cys-Sec-Gly (where Sec indicates selenocysteine) were determined to be dinitrophenyl-alkylated upon incubation of native TrxR with NADPH and DNCB. A model for the interaction between TrxR and dinitrohalobenzenes is proposed, involving a functional FAD in the alkylated TrxR generating an anion nitroradical in a dinitrophenyl group, which in turn reacts with oxygen to generate superoxide. Production of reactive oxygen species and inhibited reduction of thioredoxin by the modified thioredoxin reductase after reaction with dinitrohalobenzenes may play a major role in the inflammatory reactions provoked by these compounds.

The low-molecular-weight compound APR-246 (PRIMA-1(MET)) restores wild-type conformation and function to mutant p53, and triggers apoptosis in tumor cells. We show here that APR-246 also targets the selenoprotein thioredoxin reductase 1 (TrxR1), a key regulator of cellular redox balance. APR-246 inhibited both recombinant TrxR1 in vitro and TrxR1 in cells. A Sec-to-Cys mutant of TrxR1 was not inhibited by APR-246, suggesting targeting of the selenocysteine residue in wild-type TrxR1. Preheated APR-246 and its conversion product methylene quinuclidinone (MQ) were much more efficient TrxR1 inhibitors than APR-246 itself, indicating that MQ is the active compound responsible for TrxR1 enzyme inhibition. TrxR1 inhibited by MQ was still functional as a pro-oxidant NADPH oxidase. Knockdown of TrxR1 caused a partial and reproducible attenuation of APR-246-induced tumor cell death independently of p53 status. Cellular TrxR1 activity was also inhibited by APR-246 irrespective of p53 status. We show that APR-246 can directly affect cellular redox status via targeting of TrxR1. Our findings provide an explanation for the previously observed effects of APR-246 on tumor cells lacking mutant p53.

The nucleoside analog 2-chlorodeoxyadenosine (CdA, Cladribine) is a chemotherapeutic agent for treatment of leukemias and lymphomas, most successfully used in hairy cell leukemia and B-cell chronic lymphocytic leukemia. CdA is phosphorylated intracellularly to its monophosphate derivative by the enzymes deoxycytidine kinase and deoxyguanosine kinase. Cell lines deficient in deoxycytidine kinase were shown to be resistant to CdA and a high deoxycytidine kinase level in combination with low 5'-nucleotidase has been proposed to partly explain the selectivity in CdA toxicity for lymphoid cells. In this report biochemical properties in CdA phosphorylation mediated by deoxycytidine kinase and deoxyguanosine kinase are reviewed and discussed in relation to the further metabolism of CdA 5'-monophosphate, the different possible mechanisms of action and the correlation with clinical response. It is concluded that much is known about the metabolism and mechanisms of action of CdA, but that the remarkable therapeutic effect in hairy cell leukemia has yet to be explicitly explained.

Deoxycytidine kinase (dCK) activates several clinically important drugs, including the recently developed antileukaemic compound 2-chlorodeoxyadenosine (CdA). The distribution of dCK in cells and tissues has previously been determined by activity measurements, which may be unreliable because of the presence of other enzymes with overlapping substrate specificities. Therefore we have measured dCK polypeptide levels in extracts of normal and malignant human peripheral blood mononuclear cells, gastrointestinal tissues and sarcomas, using a specific immunoblotting technique, as well as the phosphorylation of CdA in the same extracts. High levels of dCK were found in all major subpopulations of normal mononuclear leucocytes (120 +/- 19 ng dCK/mg protein) and in B-cell chronic lymphocytic leukaemia (81 +/- 30 ng/mg, n = 23). Hairy-cell leukaemia contained lower levels (28 +/- 23 ng/mg, n = 7), as did three samples of T-cell chronic lymphocytic leukaemia (18 +/- 14 ng/mg). Phytohaemagglutinin stimulation of normal lymphocytes did not lead to any substantial increase in either dCK activity or protein expression (less than 2.5-fold). The human CEM wt T-lymphoblastoid cell line contained 56 +/- 1 ng/dCK/mg protein, while in the CEM ddC50 and AraC8D mutants that lack dCK activity, no dCK polypeptide could be detected. In colon adenocarcinomas, the dCK content was significantly higher (20 +/- 9 ng/mg, n = 20) than in normal colon mucosa (8 +/- 3.5 ng/mg, n = 19, P0.05). A similar pattern of dCK expression was found in gastric adenocarcinomas (21 +/- 13 ng/mg, n = 5) and normal stomach mucosa (6 +/- 5 ng/mg, n = 5, P0.15). One leiomyosarcoma and one extra-skeletal osteosarcoma showed dCK levels comparable with those found in normal lymphocytes (84 +/- 6 and 109 +/- 4 ng/mg, respectively), while other sarcoma samples contained lower levels, comparable to the gastrointestinal adenocarcinomas (20 +/- 7 ng/mg, n = 12). Thus, dCK is expressed constitutively and predominantly in lymphoid cells, but it is also found in solid non-lymphoid tissues, with increased levels in malignant cells. The phosphorylation of CdA in crude extracts showed a close correlation to the dCK polypeptide level.

The four major mammalian anabolic enzymes in the salvage pathway of deoxyribonucleosides are deoxy-cytidine kinase (dCK), the two thymidine kinase isoenzymes (TK1 and TK2) and deoxyguanosine kinase (dGK). These enzymes were recently cloned and/or homogeneously purified and thereby thoroughly characterized (reviewed by Arner, 1993; Arner and Eriksson, 1994). dCK and TK1 are both considered to be cytosolic, whereas dGK is regarded to have a mitochondrial location. The subcellular location of TK2, however, is not fully clarified, as discussed below.

Background In vivo imaging using Annexin A5-based radioligands is a powerful technique for visualizing massive cell death, but has been less successful in monitoring the modest cell death typically seen in solid tumors after chemotherapy. Here we combined dynamic positron emission tomography (PET) imaging using Annexin A5 with a serum-based apoptosis marker, for improved sensitivity and specificity in assessment of chemotherapy-induced cell death in a solid tumor model. Methodology/principal findings Modest cell death was induced by doxorubicin in a mouse xenograft model with human FaDu head and neck cancer cells. PET imaging was based on (11)C-labeled Sel-tagged Annexin A5 ([(11)C]-AnxA5-ST) and a size-matched control. 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]-FDG) was utilized as a tracer of tissue metabolism. Serum biomarkers for cell death were ccK18 and K18 (M30 Apoptosense® and M65). Apoptosis in tissue sections was verified ex vivo for validation. Both PET imaging using [(11)C]-AnxA5-ST and serum ccK18/K18 levels revealed treatment-induced cell death, with ccK18 displaying the highest detection sensitivity. [(18)F]-FDG uptake was not affected by this treatment in this tumor model. [(11)C]-AnxA5-ST gave robust imaging readouts at one hour and its short half-life made it possible to perform paired scans in the same animal in one imaging session. Conclusions/significance The combined use of dynamic PET with [(11)C]-AnxA5-ST, showing specific increases in tumor binding potential upon therapy, with ccK18/K18 serum measurements, as highly sensitive markers for cell death, enabled effective assessment of modest therapy-induced cell death in this mouse xenograft model of solid human tumors.

In the present study we have explored the catabolism of dCyd and 2',3'-dideoxycytidine (ddC) in mature monocyte-derived macrophages (M/M) and focused on its relation to the accumulation of intracellular anabolites. We found that dCyd catabolism in M/M was significant in 1-week old cultures (15% of 0.5 microM dCyd, 0.5 nmole/million cells, catabolized within one hour of incubation) and further increased in more mature 3-week and 5-week old cultures (> 85% catabolized within one hour). Dihydrouracil (DHU) was irreversibly formed as end product of the dCyd catabolism, with dUrd and Ura as intermediate metabolites. Intracellularly, dCyd nucleotides were detected up to three hours of incubation, with dCTP maintained at steady levels between one and three hours. However, after twelve hours of incubation DHU was the only detectable intracellular as well as extracellular metabolite. In contrast to what was observed with dCyd, we found that ddC was resistant to catabolism, with no detectable catabolites formed within twenty-four hours of incubation. Therefore ddC anabolism was unopposed by catabolic pathways and consequently intracellular anabolites of ddC accumulated throughout twenty-four hours of incubation. Based on these results, we propose that lack (or inhibition) of catabolism is at least as important as efficient anabolic phosphorylation for obtaining therapeutic effects of pyrimidine nucleoside analogs.

Human cells salvage pyrimidine deoxyribonucleosides via 5′-phosphorylation which is also the route of activation of many chemotherapeutically used nucleoside analogs. Key enzymes in this metabolism are the cytosolic thymidine kinase (TK1), the mitochondrial thymidine kinase (TK2) and the cytosolic deoxycytidine kinase (dCK). These enzymes are expressed differently in different tissues and cell cycle phases, and they display overlapping substrate specificities. Thymidine is phosphorylated by both thymidine kinases, and deoxycytidine is phosphorylated by both dCK and TK2. The enzymes also phosphorylate nucleoside analogs with very different efficiencies. Here we present specific radiochemical assays for the three kinase activities utilizing analogs as substrates that are by more than 90 percent phosphorylated solely by one of the kinases; i.e. 3′-azido-2′,3′-dideoxythymidine (AZT) as substrate for TK1, 1-β-D-arabinofura-anosylthymidine (AraT) for TK2 and 2-chlorodeoxyadenosine (CdA) for dCK. We determined the fraction of the total deoxycytidine and thymidine phosphorylating activity that was provided by each of the three enzymes in different human cells and tissues, such as resting and proliferating lymphocytes, lymphocytic cells of leukemia patients (chronic lymphocytic, chronic myeloic and hairy cell leukemia), muscle, brain and gastrointestinal tissue. The detailed knowledge of the pyrimidine deoxyribonucleoside kinase activities and substrate specificities are of importance for studies on chemotherapeutically active nucleoside analogs, and the assays and data presented here should be valuable tools in that research.

3'-Azido-3'-deoxythymidine (AZT) is HIV-inhibitory in human macrophages, which is surprising in view of the low AZT phosphorylation reported in macrophage extracts. To elucidate the mechanism of AZT activation, we studied AZT anabolism as well as catabolism in human lymphocytes and macrophages, and compared it to that of thymidine. Thymidine kinase (TK)-specific activity in mitogen-stimulated lymphocytes was 15 times higher than in macrophages. However, the TK activity per cell was only 1.3 times higher, because of the large macrophage cell volume. Total cellular TK activity, but not specific activity, matched the level of intracellular AZT anabolism. The substrate specificity of TK in macrophages strongly suggests that mitochondrial TK2 was the enzyme phosphorylating thymidine and AZT in these cells, whereas it was cytosolic TK1 in stimulated lymphocytes. In vivo thymidine catabolism was extensive, forming thymine and dihydrothymine. In macrophages more than 95% of the added thymidine (0.5 microM) was degraded within 60 min. AZT, in contrast, was not catabolized, which explains the high AZT nucleotide accumulation, a process opposed only by AZTMP excretion. The lack of catabolism together with phosphorylation by TK2 clarifies how AZT can inhibit human immunodeficiency virus in macrophages. The fact that TK2 and not TK1 phosphorylates AZT in macrophages should have important implications for combination chemotherapy.

Deoxynucleoside kinases catalyze 5′-phosphorylation of deoxynucleosides and deoxynucleoside analogs used in chemotherapy. Cytosolic thymidine kinase (TK1), mitochondrial thymidine kinase (TK2) and cytosolic deoxycytidine kinase (dCK) are key enzymes in this metabolism. They have been isolated from many sources and diverging results have been reported on their biochemical properties (reviwed in 1,2).

Background Schistosomiasis—infection with helminth parasites in the genus Schistosoma, including S. mansoni—is a widespread, devastating tropical disease affecting more than 200 million people. No vaccine is available, and praziquantel, the only drug extensively utilized, is currently administered more than 100 million people yearly. Because praziquantel resistance may develop it is essential to identify novel drug targets. Our goal was to investigate the potential of a unique, selenium-containing parasite enzyme thioredoxin glutathione reductase (TGR) as a drug target. Methods and Findings Using RNA interference we found that TGR is essential for parasite survival; after silencing of TGR expression, in vitro parasites died within 4 d. We also found that auranofin is an efficient inhibitor of pure TGR (K i = 10 nM), able to kill parasites rapidly in culture at physiological concentrations (5 μM), and able to partially cure infected mice (worm burden reductions of ~60%). Furthermore, two previously used antischistosomal compounds inhibited TGR activity, suggesting that TGR is a key target during therapy with those compounds. Conclusions Collectively, our results indicate that parasite TGR meets all the major criteria to be a key target for antischistosomal chemotherapy. To our knowledge this is the first validation of a Schistosoma drug target using a convergence of both genetic and biochemical approaches., Using both genetic and biochemical approaches, David Williams and colleagues show that the parasite thioredoxin glutathione reductase meets all the major criteria to be a key target for antischistosomal chemotherapy., Editors' Summary Background. More than 200 million people are infected with schistosomes, a type of parasitic worm. Schistosomes have a complex life cycle that starts with them reproducing in freshwater snails. The snails release free-swimming, infectious parasites that burrow into the skin of people who swim in the contaminated water. Once in the human host, the parasites turn into larvae and migrate to the liver where they become juvenile worms. These mature into 10- to 20-mm-long adult worms and take up long-term residence in the veins draining the gut (Schistosoma mansoni and S. japonicum) or bladder (S. haematobium). Here, the worms mate and release eggs, some of which pass into the feces and so back into water where they hatch and infect fresh snails. Schistosomiasis causes serious health problems (including chronic liver, gut, bladder, and spleen damage) in about 20 million people, making it a disease of great public-health and socioeconomic importance in the developing countries in which it mainly occurs. Why Was This Study Done? The only drug available to treat schistosomiasis is praziquantel. Although it is very effective, people regularly get reinfected and need to be retreated once or twice a year. All told, 100 million people are currently being treated with praziquantel. Reliance on a single drug, however, is problematic, as the parasites are likely to develop resistance to the drug over time. The identification of new drug targets in schistosomes is therefore an urgent goal. In this study, the researchers have investigated whether thioredoxin glutathione reductase (TGR), a parasitic enzyme with several functions, might be a key target for antischistosomal chemotherapy. They chose this enzyme because adult worms need to make antioxidants (chemicals that prevent oxygen from damaging cells) to protect themselves against the human immune response. Antioxidant production in these worms depends on TGR; in mammalian cells, two specialized enzymes do its job. The researchers reasoned, therefore, that TGR might be an essential parasite protein and a potentially important drug target. What Did the Researchers Do and Find? The researchers made large quantities of pure TGR and tested its activity against various substrates. The enzymatic properties and substrate preferences of TGR, they found, differed somewhat from those of its mammalian counterparts. They then screened different types of compounds for their ability to inhibit TGR. Praziquantel had no effect on TGR activity, but two antischistosomal compounds that are no longer used, potassium antimonyl tartrate and oltipraz, inhibited the enzyme. The most potent inhibitor of TGR, however, was a gold-containing complex called auranofin, low levels of which inhibited TGR in test tubes, completely killed larval, juvenile, and adult parasites living in laboratory dishes within hours, and more than halved the worm burden in infected mice. Finally, the researchers used a technique called RNA silencing to test the importance of TGR for worm survival. Fragments of double-stranded RNA (dsRNA) stop proteins being made from messenger RNA that contains an identical sequence. The addition of TGR dsRNA to larval parasites in a dish greatly reduced TGR enzyme activity and killed nearly all the parasites within days. What Do These Findings Mean? These findings suggest TGR as a key target for antischistosomal drug development. Indeed, the discovery that two previously used antischistosomal compounds inhibit TGR suggests that the enzyme has already served as a target protein. The RNA silencing experiment shows that TGR is essential for parasite survival, and the biochemical analyses indicate that TGR and its mammalian counterparts have different substrate specificities. Thus, it should be possible to find compounds that inhibit TGR but have much less effect on the mammalian enzymes. This is certainly true for auranofin, a drug used to treat rheumatoid arthritis. Whether auranofin will be an effective treatment for schistosomiasis remains to be seen—an agent that completely kills schistosomes in animals would be preferable. However, even a 50% reduction in worm burden would decrease the human health problems caused by schistosomiasis, and a combination of auranofin (or another TGR inhibitor) with an agent that works by a different mechanism might be more effective and would also reduce the chances of the parasite developing drug resistance. Additional Information. Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040206. World Health Organization provides information on schistosomiasis, including a fact sheet in English, Spanish, French, Arabic, Chinese, and Russian US Centers for Disease Control and Prevention provide information for the public and for professionals on schistosomiasis MedlinePlus encyclopedia includes an entry on schistosomiasis (in English and Spanish) The Schistosomiasis Control Initiative has information on the disease and its control Wikipedia has a page on schistosomiasis that is available in several languages (note: Wikipedia is a free online encyclopedia that anyone can edit)

Deoxycytidine kinase specific activity was high in human peripheral lymphocytes and increased less than 2-fold when the lymphocytes were stimulated by phytohemagglutinin A. Ion-exchange chromatography showed the same profile of deoxycytidine kinase activity in resting and proliferating cells. This enzyme could also efficiently phosphorylate deoxyadenosine and deoxyguanosine. In contrast, the thymidine kinase activity was very low in resting peripheral lymphocytes and increased more than 40-fold upon stimulation. Similar relative changes in the activities of the two enzymes were observed in human T-lymphoblast cells (CCRF-CEM) separated by centrifugal elutriation into cells of different cell cycle phases. The ratio of deoxycytidine to thymidine kinase activities is 20:1 in extracts from resting human lymphocytes and 1:2 in PHA-stimulated cells. This drastic change in deoxyribonucleoside phosphorylating activities during the cell cycle in human lymphocytes is of importance for studies on unscheduled DNA synthesis, for the design of therapies to interfere with viral DNA metabolism, and for a correct interpretation of the compartmentation effects observed in DNA precursor metabolism.

Antidiuretic hormone (ADH) increases transepithelial flux of water and particular solutes across the amphibian urinary bladder and mammalian collecting duct by increasing the permeability of the apical surface. We find that if each challenge with ADH is ended by replacing the medium bathing both the mucosal and serosal surfaces of the toad bladder, then rechallenge with the same supramaximal dose of ADH 36-100 min later produces flux equivalent to or greater than the original response, but rechallenge after 15 min produces only 68% of the original response. If the medium bathing the mucosal surface is neither replaced nor returned to its original volume, complete recovery of the osmotic flux response to ADH does not occur. Maximal restimulation by ADH occurs with transepithelial osmotic gradients between 119 and 180 mosmol/kg during both challenges (the serosal bath is always isotonic amphibian Ringers). In addition, ADH-containing serosal baths that have maximally activated transport across bladders for 30-60 min can be reused and again produce maximal activation of ADH responses in fresh bladders or in the original bladders after washing. These results are in contradistinction to reports of desensitization of transepithelial flux upon rechallenge with ADH after an initial stimulation under many conditions. Our findings suggest that desensitization in vitro may result from experimental design rather than intrinsic biological characteristics of the system.

Thioredoxin reductase 1 (TrxR1) is a key regulator in many redox-dependent cellular pathways, and is often overexpressed in cancer. Several studies have identified TrxR1 as a potentially important target for anticancer therapy. The low molecular weight compound RITA (NSC 652287) binds p53 and induces p53-dependent apoptosis. Here we found that RITA also targets TrxR1 by non-covalent binding, followed by inhibition of its activity in vitro and by inhibition of TrxR activity in cancer cells. Interestingly, a novel approximately 130 kDa form of TrxR1, presumably representing a stable covalently linked dimer, and an increased generation of reactive oxygen species (ROS) were induced by RITA in cancer cells in a p53-dependent manner. Similarly, the gold-based TrxR inhibitor auranofin induced apoptosis related to oxidative stress, but independently of p53 and without apparent induction of the approximately 130 kDa form of TrxR1. In contrast to the effects observed in cancer cells, RITA did not inhibit TrxR or ROS formation in normal fibroblasts (NHDF). The inhibition of TrxR1 can sensitize tumor cells to agents that induce oxidative stress and may directly trigger cell death. Thus, our results suggest that a unique p53-dependent effect of RITA on TrxR1 in cancer cells might synergize with p53-dependent induction of pro-apoptotic genes and oxidative stress, thereby leading to a robust induction of cancer cell death, without affecting non-transformed cells.