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
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ABSTRACT |
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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.
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- 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.
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 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.
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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
, 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).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Properties of the enzyme preparations used in this study
The BioPORTER technique has been shown to successfully
introduce antibodies, dextran sulfates, phycobiliproteins, albumin, and
functional enzymes like -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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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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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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DISCUSSION |
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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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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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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.
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