Rapid Induction of Cell Death by Selenium-compromised Thioredoxin Reductase 1 but Not by the Fully Active Enzyme Containing Selenocysteine*

Karin Anestål and Elias S. J. ArnérDagger

From the Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, 171 77 Stockholm, Sweden

Received for publication, October 21, 2002, and in revised form, February 5, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Three separate mammalian thioredoxin reductases are known. These include widely expressed cytosolic TrxR1,1 mitochondrial TrxR2, and a third isoform mainly located in testis (1-5). All of these enzymes are selenoproteins and carry a selenocysteine (Sec) residue in a C-terminal redox active motif having the amino acid sequence -Gly-Cys-Sec-Gly-COOH. The Sec residue in this motif is essential for catalytic activity (2, 6). Together with the neighboring cysteine, it forms a redox active selenenylsulfide/selenolthiol motif (7) that receives electrons from a redox active -Cys-Val-Asn-Val-Gly-Cys- motif present in the N-terminal domain of the other subunit in the dimeric holoenzyme (8). This N-terminal redox active motif is also present in other enzymes of the pyridine nucleotide disulfide oxidoreductase family, such as glutathione reductase and lipoamide dehydrogenase; as in all of these enzymes, this motif receives electrons from a flavin prosthetic group that in turn is reduced by NADPH (7-9).

Beside glutathione reductase (10), cytosolic TrxR1 is believed to be a most important enzyme for control of the cellular redox state, antioxidant defense, and redox regulation of cellular processes. These diverse functions mainly derive from cellular actions by its main substrate, thioredoxin 1 (Trx1), which has a remarkable range of cellular activities (11, 12). A main function of TrxR1 is hence to reduce Trx1, and many of the intracellular functions held by reduced Trx1 are known to promote cell viability. Among these functions are inhibition of apoptosis signal-regulating kinase 1 (13), regeneration of peroxiredoxins (14), and methionine sulfoxide reductase (15), with the latter enzymes playing central roles for cell (16) and organism (17) survival or aging. Moreover, TrxR1 is readily inhibited by a number of electrophilic agents used as anticancer agents in clinical use, including quinones (18, 19) and cisplatin (20, 21), and the drug auranofin that is used against rheumatic disorders (22). The potential importance of TrxR1 as a clinical drug target was recently reviewed (23).

In light of such studies as those referred to above, that all in essence identify the selenium-dependent TrxR1 and the thioredoxin system as important for normal cell function and viability, it is most interesting that TrxR1 has also been shown to function as a cell death-promoting factor. GRIM-12 (gene associated with retinoid interferon-induced mortality), subsequently shown to be TrxR1, was reported to mediate the cell death effects seen in tumor cell lines treated with a combination of interferon-beta and all-trans-retinoic acid (24). The identification of GRIM-12 was elegantly made by use of an antisense cDNA library expressed under control of an interferon-induced promoter in HeLa cells, screened for induced resistance to retinoid- and interferon-induced cell death, cloned in Escherichia coli and subsequently ascertained for protective effects when transfected into MCF-7 cells (24). Thus, antisense TrxR1 cDNA expression protected MCF-7 cells from cell death induced by the retinoid-interferon combination, suggesting that TrxR1 activity was a prerequisite for the cell death effect. Subsequent studies clearly indicated that the cell death was apoptotic, dependent upon wild type Trx1 and involved both caspase-8 and p53 (25-27). A link between Trx1 and p53 was known previously (28), and GRIM-12/TrxR1 was shown to be a prerequisite for retinoid/interferon-induced cell death but notably not for cell death induced by tumor necrosis factor alpha , etoposide, or vincristine (24). This reflects a need for a functional thioredoxin system in the induction of apoptosis through p53 upon treatment of cells specifically with the retinoid/interferon combination (24).

We were, however, intrigued by the fact that transient transfection with TrxR1 constructs was shown to directly induce cell death in MCF-7 cells, with 25% cell death 72 h post-transfection compared with 8% in vector controls (27) or, in a subsequent study, up to 40% cell death 40 h post-transfection with similar results in T47D, COS-7, and HeLa cells (29). How could this selenoprotein, hitherto considered essential for cell viability, display such potent cell death-promoting effects? Considering the well known inefficiency in the mammalian selenoprotein translation machinery, which also has been demonstrated using transfection constructs for TrxR1 (30), we hypothesized that these cell death-promoting effects of TrxR1 might have been due to the formation of truncated selenium-deficient protein species. This assumption was corroborated by the fact that the selenocysteine insertion sequence element in the 3'-untranslated region, necessary for TrxR1 selenoprotein production (2, 30), had not been included in the constructs directly promoting cell death (27, 29). Furthermore, selenocysteine in TrxR1 is as in all selenoproteins co-translationally inserted at a UGA codon, which normally confers termination of translation (1, 2, 30, 31). In case of GRIM-12, this UGA codon was initially interpreted as a termination codon and replaced with a nucleotide sequence encoding a c-myc tag (24). That construct has, nonetheless, subsequently been utilized for transfection studies with results interpreted to show that "wild type" thioredoxin reductase may induce cell death (27, 29, 32). In an attempt to scrutinize whether the direct deleterious effects by TrxR1 on cell viability could specifically be attributed to selenium-compromised forms of TrxR1, we have here made use of a recently developed methodology for lipid-mediated delivery of intact proteins into mammalian cells (33). This approach was chosen to avoid any production of a mixture of full-length and truncated TrxR1 that may result from inefficient selenoprotein synthesis using transfection experiments, even using constructs including the selenocysteine-encoding UGA codon and a correct mammalian selenocysteine insertion sequence element. As reported herein, we found that selenium-compromised TrxR1, either a selenium-deficient truncated form or an enzyme derivatized at the selenocysteine residue, indeed rapidly induced cell death, whereas such direct effect on viability could not be seen using the full-length selenocysteine-containing enzymatically active form of the enzyme.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chemicals and Reagents-- BioPORTER protein delivery reagent was obtained from Gene therapy systems, fetal calf serum came from Biotech Line AS, whereas Dulbecco's modified Eagle medium, L-glutamine, and phosphate-buffered saline (PBS) were from Invitrogen, and antibiotics came from BIO-Whittaker Belgium. GR (purified from yeast), GSSG, 1-chloro-2,4-dinitrobenzene (DNCB), CDDP, 2'-[4-ethoxyphenyl]-5-[4-methyl-1piperazinyl]-2,5'-bi-1H-benzimidazole (Hoechst 33342), 5-iodoacetamidofluorescein (5-IAF), and propidium iodide came from Sigma.

Preparation of Different Forms of Recombinant TrxR1-- Recombinant selenocysteine containing TrxR1 (rTrxR1) was purified from an overproducing E. coli system as described previously (34), and full-length TrxR1 was prepared from rTrxR1 using a recently developed technique based upon selective binding to phenyl arsine oxide-Sepharose.2 For fluorescence labeling, Alexa (Molecular Probes) or 5-IAF was conjugated to TrxR1 by prior reduction of rTrxR1 (1 µg/µl) in 50 µl of 50 mM Tris-Cl, pH 7.5, 2 mM EDTA with the addition of 11 µl of 40 mM dithiothreitol in PBS buffer, pH 7.4, and incubation at 4 °C for 1 h, followed by the addition of 1 µl of 80 mM 5-IAF and incubation for an additional 2 h at 20 °C. This reaction was stopped by addition of 5 µl of 100 mM GSH. For DNCB derivatization (35), rTrxR (100 µg approx  1.8 nmol subunit) was preincubated with NADPH (250 nmol) 30 min at 20 °C in 20 µl of 50 mM Tris-Cl, 2 mM EDTA, pH 7.5, whereupon 0.5 µl of 340 mM DNCB in ethanol was added, and derivatization was allowed for 20 min at 20 °C, hence producing dnp-TrxR1. Preparation of a DNCB control followed the same procedure as the preparation of dnp-TrxR1, except for excluding rTrxR from the reaction. CDDP-TrxR1 was prepared by incubating 20 µM rTrxR1 with 100 µM CDDP in Me2SO in the presence of 1 mM NADPH in 50 µl of 50 mM Tris-Cl, 2 mM EDTA, pH 7.5, for 20 min at 20 °C. The CDDP control was prepared by the same procedure without the addition of enzyme. To separate CDDP-TrxR1, fluorescent TrxR1, or dnp-TrxR1 from reductants, co-factor, and unbound electrophilic reactant, the derivatized enzyme samples were run on Fast desalting PC 3.2/10 columns equilibrated with 50 mM Tris-Cl, 2 mM EDTA, pH 7.5, using the SMART system (Amersham Biosciences). The protein concentrations were determined by the Bradford method according to protocol from Bio-Rad using bovine serum albumin as standard. The DNCB and CDDP controls were subjected to the same desalting procedures, with the fractions corresponding to the enzyme fractions used for the control cell experiments using equivalent volumes. To confirm labeling of TrxR1 with 5-IAF, it was also analyzed on a Tris-Cl SDS-PAGE gradient 8-16% gel, and fluorescent protein was detected by exposure of the gel to UV light.

Enzymatic Assays-- The TrxR1 activities of different preparations were determined using the standard DTNB assay (36). NADPH oxidase activity was determined as described previously (35) by following A340 in a reaction mixture with 500 µl of 50 mM Tris-Cl, pH 7.5, 2 mM EDTA, and 200 µM NADPH using 2.55 µM enzyme. The activity of GR was determined following the initial linear decrease of A340 in a 500-µl reaction mixture containing 50 mM Tris-Cl, pH 7.5, 2 mM EDTA, 200 µM NADPH, and 2 mM GSSG. The NADPH oxidase activity of GR was determined according to the same protocol as for TrxR1.

Cell Cultures and BioPORTER Experiments-- A549 (human lung carcinoma) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin in an humidified atmosphere of 5% CO2 and 95% air at 37 °C. The cells were seeded in LabTec II chamber slides (0.7 cm2/well) (Nunc, Denmark) 24 h before incubation with BioPORTER, which was prepared according to the manufacturer's protocol, briefly described as follows. Chloroform (250 µl) was used to dissolve the dried BioPORTER reagent present in one tube as delivered (catalog number BP502401; Gene Therapy Systems), which was vortexed 20 s and added in aliquots (2.5 µl) to the bottom of Eppendorf tubes, whereupon the tubes were left open in a hood to evaporate the chloroform. For our initial experiments, different quantities of each TrxR1 preparation (0.1-100 ng) were diluted in PBS to a final volume of 15 µl and were used to hydrate the aliquots with dry BioPORTER reagent by incubation for 5 min at 20 °C. In the experiments shown in Fig. 4, only 100 ng of the TrxR1 preparations and 350 ng of GR were used in each assay. Each tube was then briefly vortexed before the addition of serum free cell medium to a final volume of 250 µl. This medium preparation with BioPORTER/TrxR1 was added to the cells grown in chamber slides (10,000 cells/well), which had first been washed once with serum-free medium. The cells were subsequently incubated for 4 h at 37 °C before microscopy assessment.

Microscopy and Assessment of Cell Viability-- The cells were washed three times with PBS prior to microscopy with a power Leica DMRB microscope equipped with Fluotar PL 10X and Fluotar PL 20× objectives with filters for 4',6-diamidino-2-phenylindole (DAPI), rhodamine, and fluorescein isothiocyanate. A Hamamatsu digital camera 4742-95 was used to obtain the microscopic images. Cells treated with 5-IAF-TrxR1 or Alexa-labeled TrxR were subjected to bright field and fluorescein isothiocyanate-filtered images, whereas assessment of viability was performed with bright field, DAPI (for Hoechst staining), and rhodamine (for propidium iodide staining) filters. For staining, the cells washed in PBS were first incubated for 15 min 20 °C with Hoechst 33342 dye (5 µg/ml in PBS), whereupon propidium iodide was added (50 µg/ml from 5 mg/ml stock in PBS), and then the cells were incubated for an additional period of 5 min. Cell morphology was assessed as follows. Viable cells were considered to have Hoechst-stained normal-sized smooth nuclei without red propidium iodide staining, whereas cells having red-stained nuclei with either multiple bright specks of fragmented chromatin or one or more spheres of condensed chromatin with more compact nuclei than normal were judged to be apoptotic, and necrotic cells had red-stained smooth nuclei of roughly the same size as viable cells.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The aim of this study was to introduce different forms of TrxR1 into mammalian cells and study effects on cell viability. As mentioned in the Introduction, use of a novel lipid-mediated delivery system (33), commercially available under the brand name BioPORTER, would bypass inefficiencies in selenoprotein translation and thereby open the possibility to study isolated effects of the selenium-containing compared with selenium-compromised forms of TrxR1. We wished to introduce into cells (a) fully active selenocysteine-containing TrxR1, (b) truncated selenocysteine-deficient TrxR1, and (c) TrxR1 derivatized with DNCB. The latter yields an enzyme devoid of the Trx1 reducing capacity but with an increased NADPH oxidase activity, producing superoxide (35). Furthermore, we also utilized TrxR1 derivatized with the clinically used antitumor agent cisplatin, which inhibits TrxR1 activity but does not induce its inherent NADPH oxidase activity (20). To have well defined preparations of TrxR1, we made use of purified recombinant enzyme produced in E. coli either using a system tailored for heterologous production of the full-length selenoprotein or a construct without a selenocysteine insertion sequence element, thereby producing the two-amino acid truncated selenium-deficient form of the enzyme (34). However, the system for production of recombinant selenoprotein results in a mixture of truncated and full-length enzyme (34). Therefore we have further isolated the full-length recombinant TrxR1 from the purified preparation, as described under "Experimental Procedures." We also used the original preparation of rTrxR1 containing a mixture of full-length and truncated enzyme species. Furthermore, we analyzed the effects of GR, which is a non-selenoprotein enzyme closely related to mammalian TrxR1 in both structure (8) and primary sequence (2) but naturally lacking the C-terminal Sec-containing redox active center (2, 7). We also found GR to have a greater inherent NADPH oxidase activity than TrxR1. The properties of the enzyme preparations used in this study are summarized in Table I.


                              
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Table I
Properties of the enzyme preparations used in this study
The enzyme activities shown are the means ± S.D. of three or four separate determinations.

The BioPORTER technique has been shown to successfully introduce antibodies, dextran sulfates, phycobiliproteins, albumin, and functional enzymes like beta -galactosidase and caspases into living cells (33). However, it could not be used to introduce apoptosis-inducing cytochrome c (33), which contrasts the introduction into cells of cytochrome c using microinjection techniques (37). Therefore, we first used fluorescently labeled TrxR1 to probe whether the BioPORTER technique could be used for introduction of TrxR1 protein into living cells. Reportedly, the highest proportion of protein introduced into cells upon use of the BioPORTER technique was seen after 4 h of incubation (33). In agreement with this, we could detect fluorescent TrxR1 in most cells after a 4-h incubation period. A slight fluorescence at some cells also in the absence of BioPORTER seemed to derive from enzyme aggregating at the cell membrane, whereas the inclusion of BioPORTER clearly led to a stronger and intracellular fluorescence in nearly all cells (Fig. 1). We hence continued with our studies using the TrxR1 forms listed in Table I, with the aim to analyze possible effects on cell viability.


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Fig. 1.   Lipid-mediated delivery of TrxR1 into A549 cells. The figure shows light microscopy (A) and fluorescence microscopy (B) images of cells incubated with fluorescent Alexa-derivatized TrxR1 in the absence (left panels) or presence (right panels) of BioPORTER. Note that fluorescence of TrxR1 was seen in nearly all cells upon use of the BioPORTER reagent.

The ratio of protein to BioPORTER reagent may affect the dynamics of the delivery system (33). Therefore we used different amounts of each TrxR1 form (0.1, 0.5, 1, 10, and 100 ng) incubated with a fixed number of A549 cells, a constant amount of BioPORTER reagent, and a 4-h incubation time. We then assessed the effects on cell morphology and viability using Hoechst staining and propidium iodide staining after incubation of A549 cells, in the presence or absence of BioPORTER, with full-length TrxR1, truncated TrxR1, or the irreversibly inhibited dinitrophenyl-derivatized TrxR1. Apoptotic morphology was denoted as condensed nuclei being slightly propidium iodide-colored, and cells with large and intensely colored nuclei were considered necrotic, whereas cells lacking propidium iodide staining were considered live cells (Fig. 2). However, because this distinction between apoptotic and necrotic cells is somewhat capricious and because in all cases the clear majority of the dying cells had apoptotic and not necrotic characteristics, the statistic evaluation was performed on the effects on total cell death (apoptotic + necrotic). We could not find any clear dose response curve in the effects using any of the TrxR1 forms. Such lack of clear dose-response curves and a certain degree of interassay variability in effects using the BioPORTER reagent has also been seen by others, analyzing BioPORTER-mediated introduction of nucleoside kinases,3 likely to be due to a variability in the reconstitution and hydration of the original lipid reagent (see "Experimental Procedures" for the method) leading to a variability in the ratios of reagent to protein. Nonetheless, we found a striking difference in cell death-promoting capacity when comparing the effects of selenium-compromised TrxR1 derivatives with the enzymatically active selenium-containing form of the enzyme. Fig. 3 summarizes the results of these experiments, showing that full-length TrxR1 could not produce any significant increase in cell death as compared with controls, whereas introduction of truncated TrxR1 resulted in a cell death of 38 ± 29%, which was a highly significant increase as compared with controls (Fig. 3). Introduction of dinitrophenyl-derivatized TrxR1 resulted in an increase of cell death similar to that of truncated TrxR1. Control experiments with sole DNCB solution treated identically but without protein gave no effects on viability, which excluded that the cell death induction was due to traces of DNCB left in solution and should therefore be attributed to the derivatized enzyme. The mixture of full-length and truncated TrxR1, being the recombinant TrxR1 as produced, gave no significant induction of cell death, indicating that simultaneous addition of full-length TrxR1 may counteract the effect of the truncated enzyme.


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Fig. 2.   Assessment of cell death-promoting effects. For each experiment, summarized in the legends to Figs. 3 and 4, cells with Hoechst-stained nuclei were counted and judged as either viable (lack of propidium iodide staining), apoptotic (Hoechst-stained condensed nuclei with propidium iodide-stained spots), or necrotic (large nuclei intensely stained with Hoechst and propidium iodide-stained). This figure illustrates such morphology assessment, with some of the cells judged to be apoptotic indicated with arrows, some of the viable cells indicated with open arrowheads, and a necrotic cell indicated with the letter n. For the differences in induction of cell death by each form of TrxR1 or by GR, see Figs. 3 and 4.


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Fig. 3.   Cell death-promoting effects by different forms of TrxR1. A549 cells were incubated for 4 h without addition of protein (controls) or with the addition of fully active recombinant selenocysteine-containing TrxR1 (TrxR1), a less active rTrxR1 (rTrxR1), truncTrxR1 (truncTrxR1), and dnp-TrxR1 (dnp-TrxR1) in the absence (-BP, open bars) or presence (+BP, filled bars) of BioPORTER reagent. For each experiment, all of the attached cells with Hoechst-stained nuclei within a microscopical field were counted and judged as either viable, apoptotic, or necrotic as described in the legend to Fig. 2. In cases of extensive cell death, the cells also became more spherical and started to detach from the surface. Nonetheless, in each individual experiment 200-600 attached cells were evaluated. As described in the text, we found no clear dose-response curve using 0.1-100 ng of protein with a fixed number of cells and a fixed amount of BioPORTER reagent. This graph therefore summarizes the combined results from two or three experiments for each dose of TrxR1 derivative, resulting in a total of 14 separate experiments performed for each form of TrxR1 (i.e. in total 2800-8400 cells evaluated per treatment). All of the evaluations were performed by the same person, who at the time of assessment was unaware of the treatment to which the cells had been exposed. The figure summarizes the total cell death (error bars indicate mean ± S.D.) for each form of treatment. A two-tailed heteroscedastic Student's t test was used for a statistical evaluation in comparison with the cell death occurring in the relevant control cells (in the presence or absence of BioPORTER). There was no significant difference in cell death between the two groups of control cells (p = 0.056). Statistically relevant increases in cell death are indicated in the figure with asterisks. *, p < 0.01; **, p < 0.005. n = 14 for each treatment.

We next analyzed whether TrxR1 derivatized with cisplatin could induce cell death in a manner similar to truncated or dinitrophenyl-derivatized TrxR1, which indeed was the case (Fig. 4A). This suggested that the NADPH oxidase activity of the derivatized enzyme seemed not to be an essential factor for the cell death induction, because truncated TrxR1 or CDDP-TrxR1, in contrast to dnp-TrxR1, lacked a significant increase in the inherent NADPH oxidase activity (Table I). We also analyzed the effects of a glutathione reductase preparation that had higher inherent NADPH oxidase activity than the utilized TrxR1 preparations (Table I). We found no induction of cell death using this glutathione reductase (Fig. 4B).


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Fig. 4.   Effects of cisplatin-derivatized TrxR1 and glutathione reductase on cell viability. In A, A549 cells were incubated with rTrxR1, truncTrxR1, or cisplatin-derivatized TrxR1 in the presence or absence of BioPORTER and subsequently analyzed for viability and evaluated as described in the legends of Figs. 2 and 3. The CDDP control shows the effects of a control sample treated identically to CDDP-TrxR1, except that no enzyme had been added in the original sample. In B, an experiment comparing the effects of truncTrxR1 with fully active GR (see Table I) is shown. **, p < 0.005; ***, p < 0.001; n = 4 for each treatment in both A and B.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The studies presented here show that truncated selenium-deficient TrxR1, or forms of the enzyme compromised at the selenocysteine residue with either cisplatin or dinitrophenyl moieties, may rapidly induce cell death. The enzymatically active full-length enzyme did not hold this property. Remarkably, the only difference between truncated and full-length TrxR1 protein is the presence in the latter of the two final C-terminal amino acids (-Sec-Gly-COOH). In case of truncated TrxR1 and also CDDP-TrxR1, use of BioPORTER was needed for cell death to be provoked, which demonstrated that the cell death-promoting actions of these TrxR1 derivatives were intracellular (Figs. 3 and 4A). The dinitrophenyl-derivatized enzyme gave a similar induction of cell death in the presence of BioPORTER, but there was also a certain induction of cell death by dinitrophenyl-derivatized TrxR1 in the absence of lipid reagent (Fig. 3). This suggests that extracellularly dinitrophenyl-derivatized TrxR1 can have access to reducing equivalents, perhaps by interaction with other reductive proteins at the cell surface, so that its increased oxidase activity (Table I) producing superoxide (35) may also induce cell death when present on the outside of cells. Glutathione reductase with a higher NADPH oxidase activity (Table I) could not provoke cell death under similar conditions, neither extracellularly nor intracellularly (Fig. 4B). All of the other forms of NADPH oxidases can thereby not mimic the mechanism for induction of cell death by dnp-TrxR1 in the absence of BioPORTER; this could be indicative of specific functions of TrxR1 at the cell surface.

How many TrxR1 derivative proteins were introduced into the cells using BioPORTER in these studies, in comparison with the endogenous TrxR1 levels in A549 cells? Based on TrxR1 determinations of others (38, 39), it can be estimated that A549 cells contain approximately 100-200 ng of TrxR1 and 1.5 mg of total protein/106 A549 cells. It was reported that BioPORTER may introduce 10-50% of the added protein into treated cells under optimal conditions (33). Because we utilized BioPORTER with 0.1-100 ng of protein with 10,000 A549 cells, this results in the estimate that 0.01-50 ng of TrxR1 could have been introduced into the cells. Endogenously 10,000 A549 cells contain approximately 1-2 ng of TrxR1. Therefore the amount of TrxR1 derivatives introduced should have encompassed the endogenous TrxR1 levels with a wide margin both on the lower and upper end. Within the utilized range, however, we could see similar deleterious effects on viability using both the lower and higher amounts of selenium-compromised TrxR1, albeit with a certain interassay variability. We believe this variability probably depended upon variable efficiency of protein delivery caused by the BioPORTER technique, as further described under "Results." However, because substoichiometric amounts of TrxR1 derivatives, as compared with the endogenous TrxR1 levels, could also induce cell death, this suggests a rather specific signaling event.

The rapid induction of apoptotic cell death upon the introduction of selenium-compromised TrxR1 into the A549 cells within 4 h is reminiscent of the 2-4-h time frame of apoptotic induction upon microinjection (37, 40, 41) or pinocytic loading (42) of cells with cytochrome c. This time frame should be compared with the cell death-promoting effects using transient transfections with vector constructs for GRIM-12/TrxR1, where 35% cell death was seen 40 h post-transfection (29). Because direct introduction of selenium-compromised TrxR1 protein into cells bypasses the need for translation, it is natural that the cell death induction occurred faster compared with what is seen using transfection experiments.

What is the mechanism for cell death induced by different forms of TrxR1? From the published GRIM-12 studies, it seems clear that an intact thioredoxin system is a prerequisite for a functional apoptotic machinery upon treatment of cells with a retinoid-interferon combination (24). The importance for an intact functional TrxR1/Trx system in induction of apoptosis after the retinoid-interferon combination clearly involved p53 modulation and caspase-8 activation (26, 27, 29, 43). The necessity of intact TrxR1 for p53 maturation was recently also demonstrated in a separate study showing derivatization of the selenocysteine in TrxR1 by electrophilic lipid derivatives, resulting in impaired maturation and function of p53 (44). The direct cell death-promoting effects of wild type TrxR1 that were proposed (27), however, were questionable to us and were the reason for the design of the here presented study. Because the transient transfection experiments with wild type GRIM-12/TrxR1 shown to directly promote cell death (27, 29) in fact involved production of truncated TrxR1 (see the Introduction), some is already known about the death-promoting effects of truncated TrxR1. It seems that a preserved N-terminal redox active -Cys-Val-Asn-Val-Gly-Cys- motif is a prerequisite, because when this motif was replaced by a dipeptide -Gly-Ala- motif, the cell death-promoting effect was lost (29). Moreover, removal of the whole interface domain of TrxR1, i.e. the C-terminal third of the protein (2) governing subunit association (8), resulted in even further increased cell death-promoting effects as compared with solely truncated TrxR1 (called wild type in Refs. 27 and 29), whereas both the FAD- and NADPH-binding domains (2, 8) were necessary to maintain to have a direct cell death-promoting effect (43).

It could be hypothesized that selenium-compromised TrxR1 can interact with the endogenous selenium-dependent thioredoxin system by formation of mixed complexes with endogenous Trx1 or TrxR1 subunits. If the endogenous thioredoxin system indeed would be inhibited in a dominant manner, apoptosis may be provoked because of both a reduced general antioxidant defense and direct activation of apoptosis signal-regulating kinase 1, as proposed to be the possible downstream effects of general TrxR1 inhibition (12, 23). However, induction of cell death by substoichiometric amounts (0.01 to maximum 0.1 ng introduced into cells containing 1-2 ng of endogenous TrxR1, at the lowest amounts utilized; see above) suggests that selenium-compromised TrxR1 can directly provoke cell death with yet unknown mechanisms. One possibility is that selenium-compromised TrxR1 may function in a manner similar to (or possibly connected with) that of AIF. AIF is a flavoprotein that is normally localized to the mitochondrial intermembrane space and that, when translocated to the cytosol and nucleus, induces caspase-independent apoptosis (45, 46). Interestingly, both AIF (46) and TrxR1 (8) are 55-57-kDa flavoprotein oxidoreductases with a structural fold similar to glutathione reductase. Moreover, the importance of the native TrxR1 oxidoreductase activity for maintained cell viability (11, 12) is reminiscent of the fact that the redox activity of AIF also promotes cell viability as a property separate from the apoptosis induction capacity (45).

We conclude, based upon the findings presented here and a thorough analysis of the design and results of the prior GRIM-12/TrxR1 experiments, that we find no evidence that enzymatically fully active selenocysteine-containing TrxR1 can have direct cell death-promoting effects, as has been claimed (24, 25, 27, 29, 32, 43). In contrast, it is clear that truncated or derivatized selenium-compromised forms of TrxR1 may directly promote apoptosis. This fact is most interesting and may also have medical relevance. It should be noted that truncated TrxR1 could possibly be formed in vivo under conditions of limited selenium supply, as has been shown in cell lines (47). The direct cell death-promoting effects of truncated TrxR1 may thereby be linked to some of the pathophysiology seen at events of selenium deficiency. Moreover, because many chemotherapeutic drugs in clinical use inhibit TrxR1 (23), of which CDDP was used in the present study, the possibility that such inhibition in a dominant manner could directly induce cell death as a part of the therapeutic effect should be considered. The importance for a functional thioredoxin system in maturation and activity of p53 and p53-mediated cell death (28, 44) requires an additional stimulus activating p53 for cell death execution to occur and is hence an alternative pathway in which the thioredoxin system is involved in apoptosis. Furthermore, the inhibition of apoptosis signal-regulating kinase 1 and thereby inhibition of apoptosis by reduced thioredoxin (13) as well as antiapoptotic functions of peroxiredoxins being dependent on a functional thioredoxin system (16) result in a delicate balance between promotion of cell viability and induction of apoptosis, regarding effects mediated by the thioredoxin system. The different pathways for cell viability promotion or apoptosis induction that are affected by the status of the thioredoxin system are summarized in Fig. 5. Further studies on the direct cell death-promoting effects of selenium-compromised TrxR1 are certainly warranted and necessary to consider as an additional pathway for induction of apoptosis.


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Fig. 5.   Summary of pathways affecting cell viability or apoptosis that involve the mammalian thioredoxin system. An enzymatically fully active thioredoxin system composed of intact TrxR1 and Trx1 (circled) normally promotes cell viability (a). Moreover, it is important for maturation and activity of p53 mediated apoptosis, which also requires an additional stimulus activating p53 (b). If thioredoxin is oxidized, as in excessive oxidative stress, apoptosis signal-regulating kinase 1 may be activated and initiate apoptosis (c). As shown in the present study, selenium-compromised forms of TrxR1, formed either by derivatization at the Sec residue with electrophilic agents or possibly by formation of truncated enzyme at events of selenium deficiency, may also promote apoptosis in a direct and dominant manner (d). For references and further discussion, see the text.


    ACKNOWLEDGEMENTS

We are grateful to Drs. Mathias Lundberg and Magnus Johansson for initial assistance and suggesting use of the BioPORTER reagent. We are also thankful to Linda Johansson for providing preparations of full-length recombinant TrxR1 and Dr. Chunying Chen for fluorescence labeling.

    FOOTNOTES

* This work was supported by grants from the Karolinska Institute and the Swedish Society of Medicine and by Swedish Cancer Society Projects 3775 and 4056.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 46-8-728-69-83; Fax: 46-8-31-15-51; E-mail: Elias.Arner@mbb.ki.se.

Published, JBC Papers in Press, February 6, 2003, DOI 10.1074/jbc.M210733200

2 O. Rengby, L. Johansson, L. A. Carlson, E. Serini, P. Kårsnäs, and E. S. J. Arnér, manuscript in preparation.

3 M. Johansson, personal communication.

    ABBREVIATIONS

The abbreviations used are: TrxR1, full-length Sec-containing thioredoxin reductase 1; Sec, selenocysteine; truncTrxR1, Sec-deficient two-amino acid truncated TrxR1; rTrxR1, recombinant TrxR1 preparation; GR, glutathione reductase; AIF, apoptosis inducing factor; DNCB, 1-chloro-2,4,-dinitrobenzene; dnp-TrxR1, dinitrophenyl-derivatized rTrxR1 made by reaction with DNCB; CDDP or cisplatin, cis-diamminedichloroplatinum (II); CDDP-TrxR1, rTrxR1 derivatized by reaction with CDDP; PBS, phosphate-buffered saline; 5-IAF, 5- iodoacetamidofluorescein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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