From the Greenebaum Cancer Center, Department of
Microbiology and Immunology, Molecular and Cellular Biology Program,
University of Maryland School of Medicine, Baltimore, Maryland 21201 and
Department of Environmental Sciences, The Johns Hopkins
University School of Public Health, Baltimore, Maryland
Received for publication, January 16, 2001, and in revised form, April 30, 2001
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ABSTRACT |
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Interferons (IFNs) and retinoids are potent
biological response modifiers. The IFN- IFNs are a group of multifunctional cytokines that
stimulates antiviral, antitumor, and immunoregulatory activities. Using the Janus tyrosine kinases-signal transducing activator of
transcription (STAT)1 pathway
(1), IFNs induce the expression of a number of cellular genes that
mediates their diverse actions. The importance of IFNs in tumor cell
growth suppression is underscored by an increased incidence of
carcinogen-induced tumors in the IFN- Retinoids are vitamin A derivatives that bind to specific nuclear
receptors and induce the expression of genes involved in differentiation, growth control, and metabolism (16). Deprivation of
vitamin A in experimental animals results in an increased incidence of
various carcinomas and leukemias (17) and an abnormal rise in
myelopoiesis caused by a loss of spontaneous apoptosis (18). RA, a
prototypic natural metabolite of vitamin A, can either suppress or
reverse these effects in vivo. RA inhibits the growth of
certain neuroblastomas, promyelocytic leukemias, and teratocarcinomas in vitro (17). RA binds to specific nuclear retinoic acid
receptors (RARs), which in association with a structurally similar but
genetically distinct dimerizing partner, the retinoid X receptor (RXR),
and other co-activators induce gene transcription (19), leading to
growth suppression. However, the nature of these retinoid-regulated inhibitors of cell growth is unknown. The effects of RA are thought to
be mediated by a perplexing number of RAR and RXR isoforms present in
mammalian cells. Several synthetic and natural retinoids that can
differentially activate these receptors have been identified. Some of
these suppress the cell growth of certain primary skin dysplasias and
tumor cell lines (20). The growth-suppressive effects of some synthetic
retinoids are mediated in RAR-RXR-dependent and
-independent manners (20-22). For example, RA converts transcription factor E2F into a suppressor (23). Some synthetic retinoids can induce
cell death in p53-dependent or
caspase-dependent manners (24, 25). The
retinoid-dependent translocation of orphan nuclear receptor
TR3 from nucleus to the mitochondrion and the consequent release of
cytochrome c and activation of apoptosis have been shown
recently (26). Lastly, the antiapoptotic protein BAG-1 inhibits
RAR-RXR-dependent transcription by directly interacting with these nuclear receptors, which has been suggested as a mechanism of resistance to retinoid-induced growth suppression (27).
Clinical and experimental models have shown that the IFN/RA combination
is a more potent inhibitor of cell growth than either agent alone (15,
28). A number of studies have shown cross-talk between IFN- and
RA-regulated growth-suppressive pathways, although these ligands exert
their effects via disparate signaling mechanisms. Promyelocytic
leukemia protein-RAR We have shown earlier that the IFN/RA combination, but not the single
agents, causes cell death in vitro and suppresses tumor growth in vivo (28). Furthermore, no detectable activation
of IFN-stimulated protein kinase R or RNase L and no change in p53 and
pRb occurred under these conditions (38). These data suggested the
existence of novel IFN/RA-regulated mechanisms of cell death. To
identify the genes responsible for cell death and define their mechanism of action, we employed an antisense technical knockout approach (39). In this approach, specific cell death-associated genes
are identified by their ability to confer a growth advantage in the
presence of death inducers when expressed in an antisense orientation.
We have identified several genes associated with retinoid-IFN-induced
mortality that might participate in IFN/RA activated cell death. One of
the genes associated with retinoid-IFN-induced mortality was identical
to human thioredoxin reductase (TR), an intracellular redox regulatory
enzyme (38). We showed that suppression of TR by its antisense mRNA
inhibited IFN/RA-stimulated cell death. However, those studies have not
identified the molecular targets for TR-induced cell death. Here, we
show that the activation of certain members of the caspase family is
regulated by IFN/RA combination through thioredoxin reductase. Using
selected TR mutants we show that the wild-type, but not catalytically
inactive, mutant TR enzyme promotes cell death by stimulating caspase 8 activation. Such activation seems involved an up-regulation of death
receptor gene expression.
Reagents--
Restriction and DNA-modifying enzymes (New England
Biolabs); G418 sulfate,
isopropyl-1-thio- Cell Culture--
MCF-7 cells were cultured in phenol red-free
Eagle's minimal essential medium supplemented with 5%
charcoal-stripped fetal bovine serum and 10 Plasmids--
The mammalian expression vector pCXN2-Myc contains
a Myc epitope tag in its multiple cloning site. The chicken actin
promoter and the rabbit Generation and Expression of Mutant Proteins--
The open
reading frame of human TR1 cDNA was PCR-amplified using the
following primers. The 5' primer (forward)
(5'-CGGAATTCGCCACCATGAACGGCCCTGAAGAT-3') included an EcoRI restriction site (bold face), a Kozak
consensus sequence (italics) in the context of first AUG codon, and the coding region (underlined). The 3' primer (reverse)
(5'-GGCCATGGGCAGCCAGCCAGCCTGGAGGAT-3') consisted
of a coding sequence and a KpnI site. This primer lacked the
stop codon. After amplification (14 cycles) with AmpliTaq Gold
(PerkinElmer Life Sciences), the products were purified, digested with
EcoRI and KpnI, and subcloned into the modified mammalian expression vector pCXN2-Myc. The Myc-epitope tag was added to
the carboxyl terminus of the expressed protein.
Mutants with a replacement of specific amino acids were generated by a
two-step polymerase chain reaction method. In the case of Cys-mut,
PCR-directed mutagenesis has resulted in the replacement of the
Cys-Val-Asn-Val-Gly-Cys sequence with Gly-Ala. As a result, four amino
acids were lost in this mutant. The primers used for generating the
Cys-mut were primer A
(5'CCGGCGCCATACCTAAAAAACTGATGC-3') and primer B
(CCGGCGCCTGTTTCCTCCGAGACCCC-3'). The
NarI restriction sites in these oligonucleotides are shown
in bold face. In the first round of amplification, two PCR products
were generated. In one reaction the 5' (forward) primer and primer B
were used, and in the other reaction primer A and 3' (reverse) primer were used to generate PCR products, using the wild-type TR as a
template. These PCR products were purified and digested with
EcoRI + NarI and NarI + KpnI, respectively. The two digested products were ligated
to EcoRI and KpnI-digested pCXN2-Myc in a
three-way ligation mix. Protein domain analysis suggested the presence
of potential tyrosine kinase-induced phosphorylation sites at amino
acid positions 125, 129, and 150.
To generate a mutant domain lacking the critical tyrosines, four mutant
primers and four different PCRs were performed. Base changes in these
oligonucleotides are indicated in bold face with an underline. In the
first step, two separate PCR amplifications (12 cycles) were performed.
In one reaction, the 5' primer (forward) (5'-CGGAATTCGCCACCATGAACGGCCCTGAAGAT-3')
and TK primer 2 (5'-CATAAGCATTCTCAAAGACGAC-3') were
used, and in the other reaction, the 3'reverse primer
(5'-GGCCATGGGCAGCCAGCCAGCCTGGAGGAT-3') and TK
primer 1 (5'-GTCGTCTTTGAGAATGCTTATG-3') were
used for amplification with wild-type TR as the template. Products of
the first PCR were purified, mixed, denatured and renatured. They were
then used as a template for the second PCR reaction with the 5' forward
and 3'reverse primers. The resultant product was purified, digested
with EcoRI and KpnI, and subcloned into pGEM7Zf.
This step-1 mutant served as template for the second mutation with the
following primers. Again two PCRs were performed with complementary
oligonucleotides bearing the same mutation. In one reaction, the 5'
primer (forward)
(5'CGGAATTCGCCACCATGAACGGCCCTGAAGAT-3') and TK primer 3 (5'CTCTGCTGAAAAAATTTTTTCTTTGCC-3')
were used, and in the other reaction
5'-GGCCATGGGCAGCCAGCCAGCCTGGAGGAT-3' and TK
primer 4 (5'-GGCAAGAAAAAATTTTTTCAGCAGAG-3') were used to generate the PCR products. These products were purified, mixed,
denatured, and amplified with the 5' forward primer and the 3' reverse
primer. This final product lacking all the potential tyrosine kinase
target residues was subcloned as an EcoRI-KpnI fragment into the pCXN2-Myc vector. This mutant has been named TK-pm. All mutants were confirmed by sequencing, and
expression of the predicted protein was determined by transient
transfection assays in Cos-7 cells followed by Western blotting of the
extracts with Myc-epitope tag-specific antibodies prior to their use in the experiments.
Cell Growth Assay--
Cells (2000/well) were plated in 96-well
plates. Various inhibitory agents were added, and growth was monitored
using a colorimetric assay that quantifies cell numbers (41). Each
group of treatments had eight replicates. The cells were fixed with
trichloroacetic acid (final concentration 10%) at 4 °C for 1 h
at the end of the experiment and stained with 0.4% sulforhodamine B
(Sigma). The bound dye was eluted with 100 µl of Tris-HCl, pH 10.5, and the absorbance was monitored at 570 nm. One plate was fixed with
trichloroacetic acid 10 h after plating the cells. Absorbance
obtained with this plate was considered as 0% growth. Absorbance
obtained with wells containing untreated cells was considered as 100%
growth. An increase and decrease of A570 values
in the experimental wells relative to the initial value indicate cell
growth and death, respectively. When plotted as a percentage of
untreated control growth and death, values appear on the positive and
negative scales of the y axis, respectively.
Death Assays--
Cell death was also determined using an
alternate method, the annexin V binding assays. After treatment with
the indicated agents, cells were stained using a commercially available
kit (Trevigen, Inc.) per manufacturer recommendations. The cells were incubated with FITC-tagged annexin V and propidium iodide. Cells with
double staining were considered late apoptotic. FITC-stained cells were considered as apoptotic and quantified using a
Becton-Dickinson fluorescence-activated cell sorter (FACS). Annexin
V-positive cells (dead) were scored and expressed as a percentage of
the total number of cells.
Transient Transfection Assays--
Cell death was also monitored
in a transient transfection assay. Because the transfection efficiency
is only ~20%, it is important to score for the transfected cells.
Therefore, a Subcellular Fractionation--
Mitochondrial and cytosolic
fractions were prepared after homogenization of cells in ice-cold 20 mM HEPES buffer, pH 7.4, containing 250 mM
sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, and a commercially available protease
inhibitor mixture (Sigma) as described previously (42). After
centrifugation at 750 × g for 10 min, the supernatant
was centrifuged at 10,000 × g for 25 min. The pellet
suspended in the above buffer was considered to be the mitochondrial
fraction. The supernatant was further centrifuged at 100,000 × g for 1 h, and the supernatant was collected and represented the cytosolic fraction. An aliquot from the initial lysate
was saved for determining the total cytochrome c content.
Enzymatic Assay--
Thioredoxin reductase activity was
determined as described (43). Cell extracts were prepared after IFN/RA
treatment by freeze-thaw lysis. Twenty micrograms of extract was
incubated with insulin, NADPH, and thioredoxin in 0.2 M
HEPES, pH 7.6, for 20 min at 37 °C. Reactions were terminated after
the addition of 6 M guanidinium hydrochloride and 0.4 mg/ml
dithiobis(2-nitrobenzoic acid) prepared in 0.2 M Tris, pH
8.0. Absorbance at 412 nm was measured. In each case, a corresponding
control without Trx was used to determine the basal level of TR
activity (caused by endogenous Trx and NADPH). Absorbance values
obtained from these controls were subtracted from those obtained with
the reactions that contained Trx and NADPH. A negative control reaction
without cell extracts but with all the reaction components was also
used. Triplicate samples were measured for enzymatic activity. Pure TR
was used as a positive control.
Caspase Activity Assays--
The enzymatic activity of
individual caspases was determined using a commercially available kit
(BIOSOURCE). Briefly, cell lysates were prepared
after various treatments, and a comparable quantity of lysate (~25
µg) from each sample was incubated with the synthetic substrates
IETD-p-nitroanilide (pNA) (for caspase 8)
and LEHD-pNA (for caspase 9) at 37 °C for 3 h. The release of chromophore, pNA, from these substrates
was quantified by monitoring the absorbance at 405 nm in a microplate
reader. The assay was performed as recommended by the manufacturer.
Western Blot Analyses--
Equal quantities of cell extracts
were separated on 10-12% SDS-polyacrylamide gel electrophoresis and
Western blotted onto nylon membranes. Specific first antibodies were
incubated with the blots after blocking as described in our previous
publication (38). These blots were washed and incubated with an
appropriate second antibody tagged with horseradish peroxidase. Protein
bands were visualized using a commercially available enhanced
chemiluminescence (ECL) kit (Amersham Pharmacia Biotech).
Ribonulcease Protection Assays--
To determine the expression
of death-associated gene expression, cells were treated with various
agents for 3 days, and total RNA was extracted. An equal amount of RNA
(100 µg) from each sample was hybridized to 32P-labeled
multiple apoptosis-associated gene probes using a commercially available ribonuclease protection assay kit (PharMingen). The protected
RNAs were separated on a polyacrylamide gel. The gel was dried and
autoradiographed to detect specific bands. A control hybridization
reaction with yeast tRNA did not protect these bands (data not shown).
Generation of Mutant TR1 Proteins--
We have shown earlier that
down-regulation of cellular TR gene expression by its antisense
mRNA inhibits IFN- TR Is Critical for IFN/RA-induced Death--
To demonstrate a
critical role for TR in IFN/RA-induced cell death, MCF-7 cells were
stably transfected with the wild-type or mutant TR expression vectors.
Two sets of plates were set up in each case. In one case, the number of
G418-resistant colonies formed after transfection was determined by
staining the transfected plates with sulforhodamine B. In the other,
the resultant colonies were pooled and used in the experiments.
Transfection of the pCXN2-Myc vector alone into MCF-7 cells resulted in
the formation of ~230 G418-resistant colonies. A significantly
reduced number of colonies (~28% fewer) formed after transfection of
cells with wild-type TR or TK-pm. In contrast, plates
transfected with the Cys-mut yielded a significantly higher number of
colonies (35% more) than in the control (Fig.
2). There was a slight difference between the wild type and TK-pm in their ability to inhibit colony
formation, but it was not statistically significant. These data suggest
that overexpression of wild-type TR is cytotoxic, whereas the mutant is
protective. Cells that survived after selection with G418 expressed the
wild-type TR, Cys-mut, or TK-pm (Fig. 2B).
However, the wild type- and TK-pm-expressing cells grew
significantly slower than the vector- or Cys-mut-transfected cells.
These data indicate that moderate (tolerable) levels of TR impede cell
growth, and the Cys-mut dominantly interferes with the death
induction.
Overexpression of TR Promotes Cell Death--
To determine whether
cells expressing the wild-type or TK-pm mutant of TR were
more sensitive to the death-inductive effects of IFN/RA combination
than those expressing the vector alone, we have determined cell growth
in the presence of IFN/RA. In the first set, an equal number of cells
expressing the vector (wild type, Cys-mut, and TK-pm) was
exposed to the IFN-
Although the above data indicated the sensitivity and resistance of the
cells expressing wild type and Cys-mut, respectively, they did not
reveal the relative differences between the death sensitivities of wild
type-, TK-pm-, and vector-expressing cells. Therefore, MCF-7
cells stably transfected with the vector, wild type, Cys-mut, and
TK-pm were exposed to the IFN/RA combination, and growth was
monitored using the sulforhodamine B assay. Cell growth was measured on
3 different days, and the data were expressed as a percentage of the
untreated control. IFN/RA induced a time-dependent increase
in cell death (Fig. 3B). Importantly, the cells transfected with the wild type and TK-pm were 2-3-fold more sensitive
to IFN/RA-induced death compared with the vector-transfected ones. In
contrast, the cells transfected with the Cys-mut did not die, and their number increased slightly after IFN/RA treatment. To demonstrate these
differences more clearly, the percentage of cell death was plotted in
each case and presented in Fig. 3C. Although no death was
observed in Cys-mut-transfected cells, robust death activation occurred
in the vector-, wild type-, and TK-pm-transfected cells with
IFN/RA treatment.
The apoptotic nature of IFN/RA-induced cell death was confirmed using
the annexin V binding assays. Cells were exposed to the IFN/RA
combination for 3 days, fixed, and stained with a staining mixture
consisting of biotinylated annexin V and propidium iodide. Subsequently, they were stained with FITC-labeled annexin V. Annexin-positive cells were identified by FACS and expressed as a
percentage of the total number of cells in each case. Consistent with
the data shown in Fig. 3, A-C, the highest percentage of
annexin-positive cells was detected in wild type- and
TK-pm-expressing cells compared with the vector-transfected
cells after IFN/RA treatment (Fig. 3D). A minimal annexin V
staining was observed in Cys-mut-expressing cells. This number was
significantly smaller than the vector-transfected cells. These data
indicate that the Cys-mut interferes with IFN/RA-induced apoptosis.
We next examined the relationship between cell death activation and the
enzymatic activity of TR1. A comparable quantity of protein from cells
expressing the vector, wild type, TK-pm, and Cys-mut was
used for determining the TR activity (Fig. 3E). The lysates
from wild type- and TK-pm-expressing cells had 3-4-fold more enzyme activity than those from vector-expressing cells. In
contrast, the Cys-mut-expressing cells had a significantly lower
enzymatic activity. Together these data indicate that the enzymatic
activity of TR1 is critical for promoting death in response to the
IFN/RA combination.
IFN/RA-induced Cell Death Depends on Caspase
Activation--
Because most known cell death pathways culminate in
caspase activation, we next examined whether IFN/RA induces caspase
activities during cell death. If caspases were necessary for
IFN/RA-stimulated cell death to occur, their inhibition should result
in the prevention of death. For this purpose, a chemical caspase
inhibitor, Z-VAD-fmk, and two biological caspase inhibitors,
c-FLIP and v-FLIP, were employed in the
subsequent experiments (Fig. 4).
Treatment of MCF-7 cells with Z-VAD-fmk inhibited IFN/RA-induced cell
death in a concentration-dependent manner (Fig. 4A).
At a 50 µM dose, this inhibitor conferred the greatest
cell protection against IFN/RA-induced death.
Because Z-VAD-fmk is a general caspase inhibitor, we have examined the
influence of c-FLIP or v-FLIP (E8-FLIP) (44, 45) on IFN/RA-induced cell death. These two inhibitors suppress death receptor-induced apoptosis by interfering with caspase 8 functions. They are structural homologues of caspase 8 but lack the critical cysteine at the active site. For this purpose, three MCF-7 cell lines
stably transfected with the expression vector or the cDNAs for
c-FLIP or v-FLIP were generated. A FLAG epitope
was added to these cDNAs to permit their detection. In each case, a
pool of stably transfected pooled clones (~70 each) was employed in growth assays. Treatment of these cells with either IFN-
The inhibition of cell death by Z-VAD-fmk and FLIPs was also confirmed
by annexin V binding assays. Cells were exposed to the IFN/RA
combination for 5 days and stained with annexin V (Fig. 4D).
Z-VAD-fmk inhibited IFN/RA-induced cell death in vector and wild-type
TR-expressing cells. A smaller basal level of cell death was observed
in Cys-mut-expressing cells, which was not significantly affected by
the inhibitor. In a similar manner, annexin V staining was dramatically
lower in the c-FLIP- and v-FLIP-expressing cells than in those expressing vector alone (Fig. 4E) after IFN/RA treatment.
TR Activates Cell Death--
Because the above experiments
indicated that overexpression of TR causes a reduction in the number of
G418-resistant colonies formed (Fig. 2) and indirectly indicated a loss
of cell viability, the effect of TR overexpression on cell survival was
determined using a transient assay. MCF-7 cells were transfected with
CMV-
We next examined the influence of caspase inhibitors on TR-induced cell
death. In the first round of experiments, c-FLIP and a
catalytically inactive mutant of caspase 8 were employed. MCF-7 cells
were transiently transfected with a CMV- TR Modulates Caspase-induced Cell Death--
Because TR-activated
cell death was suppressed in the presence of the caspase 8 mutant or
caspase inhibitors, it was of interest to examine whether TR targets
specific caspases during the induction of cell death. We have chosen
caspases 8 and 9 for this study because they are activated in response
to a number of extracellular death activators (46, 47). Cells were
transfected with Rous sarcoma virus-
The inhibitory effect of Cys-mut on wild-type caspase 8-induced cell
death was also tested using various concentrations of the mutant
plasmid (Fig. 6B). Cells were transfected with Rous sarcoma
virus-
We next examined the influence of TR on another caspase, caspase 9, in
a similar assay (Fig. 6C). MCF-7 cells were transfected with
a caspase 9 expression vector along with the expression vectors for
wild-type TR or Cys-mut. Caspase 9-dependent cell death was slightly enhanced by the wild-type TR. In this case an additive effect
of TR was observed. However, unlike the other caspases, caspase
9-induced cell death was not inhibited in the presence of Cys-mut.
Together, these data suggest that TR controls the activity of only some caspases.
Activation of Caspase 8 and the Release of Cytochrome c during
IFN/RA-induced Cell Death--
To test directly whether caspase 8 was
activated during IFN/RA-induced cell death, Western blot analyses were
performed using specific antibodies. MCF-7 cells expressing various TR
mutants were treated with the IFN/RA combination for 72 h. Cell
lysates were prepared and used in Western blot analyses. As shown in
Fig. 7, caspase 8 was activated in
vector-transfected MCF-7 cells after IFN/RA treatment. In the cells
expressing wild-type TR, a higher magnitude of caspase 8 activation
occurred as evidenced by the conversion of most of the procaspase 8 into active form. In contrast, no activation of caspase 8 occurred in
cells expressing the Cys-mut. A similar pattern of caspase 8 activation
was noted in HeLa cells (data not shown). We also determined the
cleavage of PARP, a marker of caspase-mediated apoptosis (Fig. 7,
bottom panel). The IFN/RA combination induced the cleavage
of PARP in the vector-transfected cells. A similar but enhanced
cleavage of PARP was noted in cells expressing wild-type TR. In
contrast, PARP cleavage was not detected in cells expressing
Cys-mut.
Because TR promoted caspase-dependent death, it was of
interest to determine whether cytochrome c release occurred
in response to the IFN/RA combination. MCF-7 cells expressing various
mutants of TR were treated with the IFN/RA combination, and the
post-mitochondrial cytosolic fraction was examined for differences in
the amount of cytochrome c released. A basal level of
cytochrome c was detected in all the cell lines. However, it
rose significantly in the vector- or wild-type TR-expressing cells
(Fig. 8A). However, no such
enhancement of cytochrome c release occurred in
Cys-mut-expressing cells treated with IFN/RA. Significantly, a higher
amount of cytochrome c was released in response to the
IFN/RA combination in the wild-type TR-expressing cells compared with
the vector-transfected ones (Fig. 8C). The total amount of
cytochrome c in the cells, however, did not change whether
or not the TR gene was transfected into the cells (Fig.
8B).
Low Caspase Activities in Cells Expressing the Mutant TR--
The
death activation in response to the IFN/RA combination was a function
of caspase enzymatic activity. Differences in the caspase enzymatic
activity of MCF-7 cells expressing Cys-mut or wild-type TR were
compared in their capacity to stimulate caspase functions. Cell
extracts from IFN/RA-treated cells were incubated with synthetic
substrates specific for each of these caspases. In each case, the
substrate was tagged with a chromophore, pNA. These included
IETD-pNA (for caspase 8) and
LEHD-pNA (for caspase 9). The release of
pNA from these substrates by the respective caspases yields
a yellow-colored product, the level of which can be quantified. In each
case, pure enzyme was used as control (data not shown). As shown in
Fig. 9, the activities of caspases 8 and 9 are stimulated by the IFN/RA combination in the vector- or wild-type TR-expressing cells but not in the Cys-mut-expressing cells. More importantly, higher caspase activities were detected in the lysates prepared from cells expressing the wild-type TR than those expressing vector alone. There were no differences in the amount of total caspase
8 and 9 proteins expressing in these cells (Fig. 7 and data not
presented). Similar activity profiles were observed with caspases 8 and
9 in the HeLa cells expressing various TR mutants (data not shown).
IFN/RA Promotes the Expression of Death Receptor Gene
Expression--
Because caspase 8 activation requires death receptors,
and IFN/RA-induced death occurs with delayed kinetics, we examined whether the death induction involves the death receptor gene
expression. For MCF-7 cells treated with RA (for 3 days), IFN-
To identify the reasons for the differential death sensitivities of
wild-type- and mutant TR-expressing cells to IFN/RA, we next determined
the expression of death-related mRNAs in MCF-7 cells expressing
vector, wild type, and Cys-mut. A comparable quantity (100 µg) of RNA
from each sample was subjected to RPA (Fig. 10B). There were
no significant differences in the expression of caspase 8 mRNA
between the cells expressing various TR constructs. As shown in Fig.
10B, the expression of death-activating ligands, Fas ligand
and TRAIL, and the death receptors Fas, DR3, DR4, and DR5 (48) were
induced by the IFN/RA combination in the vector- or wild-type
TR-expressing cells. More importantly, the basal expression of Fas,
TRAIL, DR3, and DR4 was enhanced several -fold in cells expressing
wild-type TR. IFN/RA treatment further increased the expression of
these mRNAs. Notably, the Fas ligand expression was enhanced only
in the presence of the IFN/RA combination. In contrast, the Fas ligand,
TRAIL, DR3, DR4, and DR5 mRNAs were extremely low in
Cys-mut-expressing cells and were not increased after IFN/RA treatment.
Based on the above results, the expression of death-associated proteins
was examined by Western blotting. As representatives, death receptors
and death ligands Fas and TRAIL were chosen for this analysis, because
the expression of these mRNAs was up-regulated after IFN/RA
treatment. Cells were stimulated with IFN/RA for 72 h, and a
comparable amount of total protein from each sample was Western
blotted. The blots were probed with Fas- and TRAIL-specific monoclonal
antibodies. Consistent with the RPA, the expression of Fas and TRAIL
protein was induced upon IFN/RA treatment in the vector- and wild-type
TR-transfected cells but not in Cys-mut-expressing cells. The
expression of TRAIL and Fas in the cells expressing various TR mutants
was quantified using laser densitometry (Fig. 10D). IFN/RA
treatment caused a 4- and 3.5-fold increase in the expression of Fas
and TRAIL proteins, respectively, in the vector-transfected cells. More
importantly, in the presence of TR, basal expression of Fas (4-fold)
and TRAIL (5-fold) was higher than the untreated vector-transfected
cells, and it was further induced by IFN/RA treatment. Together these
data suggest that the differential sensitivity to cell death in the
presence of TR is in part caused by the expression of death receptors
and their ligands in the presence of the IFN/RA combination.
Caspase 8-dependent Death Activation by TR Is Mediated
through Trx1--
We next examined the mechanism through which TR
promotes caspase 8-dependent cell death. Two sets of transient
transfection assays were performed for this purpose. In the first, we
examined the role of its substrate Trx1 in death induction. MCF-7 cells were transfected with wild type or the mutant forms TR and Trx. The
critical cysteines at positions 32 and 35 were replaced by serines in
the Trx mutant. Along with these plasmids, a CMV-
To test the activity relevance of TR and Trx to caspase 8, we have performed a similar cotransfection assay (as in Fig.
11A), in which a
wild-type or mutant caspase 8 was cotransfected with the TR and Trx
expression vectors (Fig.
11C). In these experiments, suboptimal levels of expression vectors were transfected into MCF-7
cells to distinguish the differences between the effects of various
gene combinations. Transfection of wild-type TR, Trx, and caspase 8 resulted in 17, 11, and 20% death, respectively. As expected, no
significant death was noticed with mutant forms of TR (Cys-mut), Trx,
and caspase 8 lacking the critical cysteine residues. Cotransfection of
wild-type constructs of TR, Trx, and caspase 8 resulted in 69%
transfected cell death. Under these conditions, the presence of the Trx
mutant strongly suppressed the cell death. Similarly, in the presence
of a Cys-mut, wild-type Trx did not support the cell death stimulatory
effects of caspase 8. In addition, wild-type TR and wild-type Trx were
incapable of supporting death in the presence of caspase 8 mutant.
Thus, TR-mediated death effects require Trx,
which promotes the functional activity
of caspase 8. In all three cases the active site cysteines are
critical, because the inactivation of any one them significantly suppresses death, indicating the importance of the redox status for
their function.
Mammalian cells have evolved several antioxidative
deterrents including superoxide dismutase, catalase, glutathione,
thioredoxin, selenoproteins, and the thiol-specific antioxidant (49,
50). Thioredoxins are ubiquitously distributed proteins. Mammalian thioredoxins control cell growth and transcriptional and immune functions. The conserved cysteine residues of the sequence
Cys-Gly-Pro-Cys, are critical for their redox function (51). Several
thioredoxins or thioredoxin-like proteins exist in mammalian cells
(52-54), the physiologic roles of which have not been clearly
understood. The most well characterized member of these thioredoxins,
Trx1, is critical for mammalian development because a homozygous
deletion of this gene causes embryonic lethality in mice (55). However, the precise role of Trx or its modifying enzyme, TR, in the cell growth
control has been less well understood, although they have been
suggested to be important for a number of regulatory processes in cell
growth based on in vitro or chemical inhibition studies. For
example, Trx has been shown to play a regulatory role in yeast, Drosophila, and Xenopus cell division. The Trx
homologue of Drosophila, deadhead, is
required for female meiosis and early embryonic development (56).
Thioredoxin has also been shown to inhibit DNA synthesis in the
fertilized Xenopus eggs (57). In yeast, Trx gene mutation increases the frequency of the mitotic cell cycle (58). Loss of the TR
gene in fission and budding yeasts relieves p53-dependent growth suppression (59, 60). Although these studies highlighted the importance of redox enzymes in cell growth suppression, the exact targets of TR and Trx in these processes have not been defined clearly. Progress to this end has been relatively lagging, because of
the fact that these enzymes modify their substrates in an extremely transient manner, and no stable prosthetic groups are involved in this
process (51). Therefore, application of genetic methods will be useful
in identifying the downstream effectors.
The redox status of Trx is modified by an upstream regulatory enzyme,
thioredoxin reductase. This gene is now known as TR1 and is
expressed ubiquitously in mammalian tissues (51). Recently, two other
TR homologues, TR2 and TR3, have been identified
through data base searches or by biochemical purification.
TR3/TrxR2 is abundant only in the heart, liver,
kidney, and adrenal gland (61). The expression of TR2 is
limited to testis (62). More importantly, while TR1 is located
primarily in the cytoplasm, TR3 is present in the mitochondrion. The
specific roles of these enzymes in their respective environments are
unclear at this stage. Mammalian TR has broader substrate specificity,
unlike the prokaryotic or yeast enzymes (51). For example, it can
reduce unrelated compounds such as selenite, alloxan, and
5,5'-dithiobis(2-nitrobenzoic acid) in addition to Trx. It is likely
that each TR exhibits a narrow substrate specificity and acts in a
localized manner.
Using a genetic approach we demonstrated earlier that
TR1 is essential for the cell death regulatory processes
controlled by IFN/RA (38). TR1 was isolated in an antisense
knockout approach, wherein death genes are identified based on a
functional inactivation of their target genes (38). The IFN/RA
combination enhances the TR protein expression at a translational level
to promote cell death. In this study we have shown that the
overexpression of TR causes a significant decrease in the viable cell
colony formation. Although the cells expressing moderate levels of TR were viable, they grew slower than the vector-transfected cells and
were hypersensitive to death induction by IFN/RA (Figs. 2 and 3). That
TR1 exerts growth-suppressive functions via apoptosis in the presence
of exogenous ligands is also consistent with other studies that showed
that Trx1, when inactivated in a similar approach, suppresses
IFN- Although overexpression of wild-type TR suppressed the formation of
G418-resistant colonies, the Cys-mut enhanced them relative to the
vector-transfected control (Fig. 2). The Cys-mut strongly interfered
with the enzymatic activity and cell death mediated by TR1 exerting a
dominant negative effect. Although these observations indicate the
cytotoxic nature of TR, they do not prove that property clearly.
Transient transfection assays clearly revealed that the reduced number
of colonies formed might, indeed, be caused by cell death (Fig. 4). TR1
promotes the activation of caspases (Figs. 4-6). Several evidences
testify to this property of TR: 1) Transient transfection of TR
promoted cell death, which was blocked by the caspase inhibitor
Z-VAD-fmk and the biological inhibitors c-FLIP and
v-FLIP (Fig. 4), 2) wild-type TR promoted caspase-induced cell death, whereas a mutant inhibited it (Fig. 6), and 3) IFN/RA induced caspase activation and cell death was blocked upon
overexpression of a mutant (Fig. 7).
Proteins belonging to the BCL2 and caspase families
constitute the central core of the mammalian cell death machinery. The former alter mitochondrial permeability functions leading to the release of apoptogenic factors, and the latter degrade the proteins necessary for cell viability (46, 47). The post-translational modifications of these proteins by pro- and antideath signals regulate
their functions. For example, phosphorylation of caspase 9 inhibits its
biological activity (66). Phosphorylation of BAD, a member of
BCL2 family, by Akt/Protein kinase B inhibits its death-promoting
activity (67-69). Little is known about other potential
post-translational mechanisms of caspase regulation. Because death
signals originate from disparate receptors at the cell surface or
intracellular sources, it is crucial to define these signals. One
post-translational mechanism that can modulate caspase activity is
their catalytic site. The cysteine residue in the active site is
critical for these enzymes to execute cells in response to exogenous
death signals (46, 47). Indeed, C Although in transient transfection assays caspase 9 activity was not
ablated by the Cys-mut, in stable transfected cells it was able to
inhibit its function. This is because its activity depends on upstream
activators. For example, the release of cytochrome c from
mitochondrion was absent in the cells expressing the Cys-mut. Furthermore, caspase 8, an apical enzyme to mitochondrial damage (47),
is not activated in IFN/RA-treated cells (Fig. 7). Therefore, TR and
Trx seem to provide reducing power to caspase 8 under the control of
IFN/RA. Indeed, TR (38) and Trx levels (Fig. 11B) are
increased after IFN/RA treatment, and their presence correlates with
the time of cell death. In addition, only the IFN/RA combination, but
not the single agents, enhances the expression of death receptors and
their ligands. These death receptor and ligand complexes can recruit
caspase 8 to promote death (48). More importantly, the presence of
catalytically inactive TR suppresses the death receptor gene expression
(Fig. 10). Thus, a common factor(s) involved in death receptor gene
expression may also be a target of redox control. Indeed, a number of
stress-responsive transcription factors such as p53 (75),
hypoxia-inducing factor-1 In summary, we have shown that TR1 using Trx1 as substrate maintain the
redox status of caspase 8. The IFN/RA combination elevates the physical
levels of TR1 and Trx1 and the expression of death receptors and their
ligands, leading to the activation of a death pathway (Fig. 12).
and
all-trans-retinoic acid combination, but not these single
agents individually, induces death in several tumor cell lines.
To elucidate the molecular basis for these actions, we have employed an
antisense knockout approach to identify the gene products that mediate
cell death and isolated several genes associated with
retinoid-IFN-induced mortality (GRIMs). One of the GRIM
cDNAs, GRIM-12, was identical to human thioredoxin reductase (TR).
To define the functional relevance of TR to cell death and to define
its mechanism of death-modulating functions, we generated mutants of TR
and studied their influence on the IFN/RA-induced death regulatory
functions of caspases. Wild-type TR activates cell death that was
inhibited in the presence of caspase inhibitors or catalytically
inactive caspases. A mutant TR, lacking the active site cysteines,
inhibits the cell death induced by caspase 8. IFN/all-trans-retinoic acid-induced cytochrome
c release from the mitochondrion was promoted in the
presence of wild type and was inhibited in the presence of mutant TR.
We find that TR modulates the activity of caspase 8 to promote death.
This effect is in part caused by the stimulation of death receptor gene
expression. These studies identify a new mechanism of cell death
regulation by the IFN/all-trans-retinoic acid combination
involving redox enzymes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
receptor
/
and
STAT1
/
mice and a failure to reject the STAT1 null
tumors by the immune system. Consistent with their direct roles in the
control of apoptosis, defects in the expression of certain members of
the caspase family (2) and a loss of IFN-
-dependent
apoptosis (3) in STAT1
/
cells have been reported. Some
members of the IFN gene regulatory factor family such as IRF-1 and the
IFN consensus sequence-binding protein act as tumor growth suppressors
of certain human myeloid leukemias (4, 5). Because STAT1 and IFN gene
regulatory factors are IFN-regulated transcription factors, the
IFN-induced growth-suppressive effects ultimately depends on different
gene products regulated by them. Indeed, two IFN-induced enzymes, the protein kinase R and RNase L, have been implicated in stress-induced and spontaneous apoptosis (6-8). However, the protein kinase R- and
RNase L-independent growth-suppressive actions of IFNs have also been
described (9, 10). In addition to these, IFNs directly activate
growth-suppressive proteins such as pRb (11) and down-regulate
c-Myc, E2F, and cyclin D3 in certain lymphoid tumor cell lines
(12, 13). Despite their strong therapeutic activity as single agents in
a number of leukemias, IFNs are less effective in the therapy of solid
tumors (14). To overcome such resistance, a number of combination
therapies have been developed. Among these, the combination of IFNs
with retinoids is highly effective against several tumors (15).
However, the molecular basis for enhanced growth suppression of
IFN/all-trans-retinoic acid (RA) combination is unclear.
, a mutant retinoic acid receptor found
in certain acute promyelocytic leukemias, is generated by gene
translocation (29). Interestingly, this mutant receptor is induced by
IFNs and has been reported to participate in the anticellular actions
of IFN-
(30, 31). Furthermore, IFN can suppress the cell growth in
RA-resistant acute promyelocytic leukemias (32). The promyelocytic
leukemia protein forms a nuclear body consisting of several
IFN-inducible gene products (32, 33). Promyelocytic leukemia protein is
induced by IFNs, and its promoter contains STAT1 binding sites (34). We
and others have reported earlier that in certain IFN-resistant cells,
RA induces STAT1 levels, which leads to an enhancement of IFN responses
(35-37). RA has also been shown to induce some IFN-stimulated genes
directly (34).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside, and LipofectAMINE plus (Life Technologies, Inc.); nitrocellulose membranes, ECL reagents,
and horseradish peroxidase coupled to anti-rabbit or anti-mouse
antibodies (Amersham Pharmacia Biotech); human IFN-
ser (Berlex, Inc.), mouse monoclonal antibodies against actin and FLAG
epitope (Sigma), Myc epitope (Zymed Laboratories
Inc.), and thioredoxin (Serotec, Inc.); Fas and TNF-related
apoptosis-inducing ligand (TRAIL) (Santa Cruz Biotechnology); rabbit
polyclonal antibodies against caspase 8; caspase 9; poly(ADP-ribose)
polymerase (PARP); and cytochrome c (Santa Cruz
Biotechnology) were used in these studies. Fresh stocks of
all-trans-retinoic acid (Sigma-Aldrich) were prepared in
ethanol and added to cultures under subdued light.
11
M estradiol during treatment with IFN-
and RA. The cells
were grown in phenol red-free medium for 24 h before treatments
were initiated.
-globin polyadenylation sequences permit the
expression of cloned insert in high levels. The presence of a
neomycin-resistant marker allows the selection of transfected cells.
The c-FLIP and v-FLIP expression vectors
were provided by Jurge Tschopp (University of Laussane, Switzerland).
Mammalian expression vectors carrying caspase 8, caspase 9, and the
corresponding mutants were provided by Emad Alnemri (Thomas Jefferson
University, Philadelphia, PA) (40). Expression vectors carrying the
wild-type and cysteine mutant of thioredoxin (65, 77) were a gift from
Junji Yodoi (Institute for Virus Research, Shogin, Japan).
-galactosidase reporter gene driven by the enhancers of
either the Rous sarcoma virus long terminal repeat or the
cytomegalovirus (CMV) was cotransfected along with the effector
plasmids. This plasmid permits the detection of transfected cells after
staining with X-gal. Briefly, the cells were transfected with the
indicated plasmids (~1 µg) using the LipofectAMINE plus reagent.
Wherever multiple plasmids were transfected the total amount of DNA
transfected was kept to a maximum of 2-4 µg. At the end of the
experiment they were stained with a commercially available
in situ
-galactosidase detection kit containing X-gal (Stratagene), and the number of cells stained blue was determined microscopically. Cells with flat attached epithelial morphology were
considered alive, and those with a rounded and detached appearance were
considered dead. Multiple fields were scanned, and a total of 300-400
cells were counted to obtain statistically significant numbers.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/RA-induced cell death (38). To understand the
mechanism of TR-regulated cell death we first generated TR mutants that
were defective in various functional domains. Two mutants, Cys-mut
(lacking the critical cysteine residues at 58 and 61 of the active
site) and TK-pm (lacking the putative tyrosine
phosphorylation domain), were subcloned into a mammalian expression
vector, pCXN2-MycA (Fig. 1A).
The enhancer of the chicken actin gene controls the expression of
the cloned cDNA in this vector. A Myc tag was added to the
carboxyl terminus of each mutant after subcloning. This vector also
contains a neomycin resistance marker for selecting the stably
transfected cells. To confirm the expression of transgene, cell
extracts were Western blotted, and the blots were probed with Myc
tag-specific antibody. In all cases, proteins of the appropriate
Mr were expressed (Fig. 1B). These
mutants were used in the experiments described below. Throughout these
studies human IFN-
and all-trans-retinoic acid were used.
The term IFN/RA refers to the combination of these two agents. The term
TR refers to human thioredoxin reductase 1 (TR1).
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Fig. 1.
Structure of the mutant TR gene
products. A, various putative domains are indicated.
FAD, FAD binding domain; NBD, NADPH binding
domain; ID, interface domain. The cysteine residues of the
active site are indicated as circles. In addition, putative
tyrosine phosphorylation domains present in this protein are indicated
with small squares. B, the mutant
cDNAs were generated by PCR, cloned into mammalian expression
vector pCXN2-Myc, stably transfected into MCF-7 cells, and selected
with G418. Surviving cell clones were pooled, and Western blot
analysis was performed using a Myc tag-specific monoclonal antibody.
The bottom panel shows the same blot probed with
actin-specific antibody.
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Fig. 2.
Death-promoting effect of TR.
A, the overexpression of TR results in a loss of cell
viability. MCF-7 cells were transfected with equimolar amounts of
mammalian expression vector pCXN2-Myc or the same vector carrying
various TR mutants. After 3 weeks of selection with G418 (1 mg/ml), the
surviving colonies were counted. Each error bar represents
the mean ± S.E. of triplicates. WT, wild-type TR.
B, TR-expressing cells grow slower than the
vector-transfected ones. An equal number of cells (2000/well) stably
expressing various TR mutants were plated, and cell growth was
monitored after 5 days using the sulforhodamine B binding assay as
described under "Materials and Methods." Each data point represents
mean ± S.E. of six replicates.
/RA combination for 3 weeks in the presence of
G418 (for plasmid selection). The cell colonies were visualized after
fixation and staining with sulforhodamine B (Fig.
3A). As expected, the vector-,
wild-type TR-, and TK-pm-expressing cells were killed by the
combination. The cells carrying Cys-mut were resistant to cell death.
These data are consistent with our earlier results obtained with TR1 antisense constructs (38).
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Fig. 3.
MCF-7 cells transfected with mutant TR become
resistant to IFN/RA-induced death. A, MCF-7 cells
(105) expressing various plasmids were selected with G418
(1 mg/ml), IFN- (500 units/ml), and RA (1 µM) for 3 weeks and then with G418 alone for 1 week. Colonies surviving on the
plates were fixed and stained with sulforhodamine B as described above.
WT, wild-type TR. B, cell death induction by the
IFN-
and retinoic acid combination. Cell growth was measured using
sulforhodamine B as described under "Materials and Methods" (41).
Each data point represents mean ± S.E. of six replicates. The
absorbance value for 0% growth is 0.158, 0.164, 0.152, and 0.171. The
100% growth values for days 3, 5, and 7 are: 0.564, 0.407, 0.868, and
0.437; 0.756, 1.1612, 0.678, and 0.835; and 1.397, 0.812, 1.878, and
0.936, respectively. Values on negative scale indicate the death of
initially plated cells. Each data point is mean ± S.E. of six
replicates. C, the percentage of cell death as measured by
the sulforhodamine B binding assay. Note the absence of dead cells in
the Cys-mut-expressing cells. Data from a 7-day growth assay in the
presence of IFN/RA combination are shown. D, IFN/RA-induced
apoptosis. MCF-7 cells were exposed to various treatments for 5 days
and then stained with propidium iodide and FITC-labeled
annexin V. Annexin-positive cells were scored by FACS analysis and
expressed as a percentage of the total number of cells. E,
enzymatic activity of thioredoxin reductase in cells expressing various
mutants of TR. Data indicates mean ± S.E. of triplicates. In
every case the background enzymatic activity (caused by endogenous Trx)
was subtracted from the experimental values.
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Fig. 4.
Effect of caspase inhibitors on
IFN/RA-induced death. The sulforhodamine B binding assay was
performed with cells exposed to IFN- (500 units/ml), RA (1 µM), or their combination for 5 days. MCF-7 cells were
pretreated with the indicated doses of Z-VAD-fmk and then with the
IFN/RA combination. The 0 and 100% growth values in this experiment
are 0.147 and 0.893, respectively. B, MCF-7 cells stably
transfected with the expression vector or caspase 8 analog
c-FLIP or v-FLIP were exposed to the IFN/RA
combination and/or individual drugs for 5 days. The 0 and 100% growth
values in this experiment are: 0.167 and 0.912 (for vector); 0.174 and
1.078 (for c-FLIP); and 0.182 and 0.995 (for
v-FLIP), respectively. Open bars, IFN-
;
filled bars, RA; striped bars, IFN/RA
combination. C, upper panel, the Western blot
analysis of cells expressing FLIP proteins with the indicated
antibodies. The FLAG-specific antibody detects the FLIP proteins.
C, lower panel, the same blot probed with
actin-specific antibodies. D, annexin V binding to the MCF-7
cells expressing various TR constructs. The cells were treated for 5 days with IFN/RA combination as described above prior to FACS analysis
with FITC-labeled annexin V. Empty and filled
bars, untreated and Z-VAD-fmk-treated (50 µM) cells,
respectively. WT, wild-type TR. E, MCF-7 cells
overexpressing the FLIP proteins were treated with the IFN-
/RA
combination as described above. FACS analysis was performed as
described under "Materials and Methods." The percentage of annexin
V-positive cells is presented.
or RA did
not cause cell death (Fig. 4B). However, the IFN/RA
combination induced death in vector-transfected cells but not in
c-FLIP- or v-FLIP-expressing cells.
Interestingly, c-FLIP strongly inhibited the death-inductive
effect of the IFN/RA combination compared with v-FLIP. The
expression of FLIPs in cells was confirmed by Western blot analysis
with FLAG epitope-specific antibodies (Fig. 4C). The
c-FLIP and v-FLIP code for 55- and 26-kDa
proteins, respectively.
-galactosidase and the expression vectors for
wild-type TR or the Cys-mut. Three days after transfection, the cells
were fixed and stained with X-gal to determine the percentage of dead
cells. Blue-colored cells with a normal epithelial morphology were
considered live, and those with a rounded appearance were considered
dead. The percentage of cell death was determined from the ratio of
dead blue cells to the total nuber of blue cells. As shown in Fig. 5A, wild type but not Cys-mut
caused a dose-dependent increase in cell death. Vector
alone did not cause significant cell death. Similar data were obtained
in T47D, COS-7, and HeLa cells (data not shown). Thus, TR, when
overexpressed, activates cell death in different tumor cell lines.
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Fig. 5.
Induction of cell death by TR in MCF-7 cells
as measured by a transfection assay. A, cells were
transfected with a -galactosidase reporter and expression vectors
for TR or Cys-mut. Forty hours later, the cells were stained with
x-gal, and the number of dead cells was determined. Numbers on the
x axis indicate the micrograms of the expression plasmid
that were transfected. The total amount of DNA transfected into cells
was kept constant (4 µg) by adding pCXN2-Myc vector where required.
WT, wild-type TR. B, inhibition of TR-induced
cell death by a mutant caspase 8 (Casp8-mut) or
c-FLIP. MCF-7 cells were transfected with a
-galactosidase reporter (1 µg) of TR (2 µg). Along with these, 1 µg of either a mutant caspase 8 or c-FLIP was
cotransfected. The percentage of death was calculated as described
above. pcDNA 3.1 was used to normalize the total amount of DNA
transfected.
-galactosidase expression vector and wild-type TR cDNA. Along with these plasmids the expression vector carrying a catalytically inactive caspase 8 mutant (C
A) or c-FLIP was also transfected. The
percentage of death was determined after staining with X-gal (Fig.
5B). Transfection of TR caused 34% death. However,
co-expression of the caspase 8 mutant strongly suppressed such cell
death. Expression of the caspase 8 mutant did not cause any significant
death compared with the vector-transfected control. In a similar
manner, c-FLIP also inhibited TR-activated cell death. These
data suggest that TR-induced cell death depends on caspases, in
particular caspase 8. In the subsequent experiments, only wild-type TR
and Cys-mut were employed, because there was no significant difference
between the death-promoting activities of TK-pm and
wild-type TR.
-galactosidase and
caspase 8 expression vectors. Along with these, either a wild-type TR
or Cys-mut expression vector was cotransfected. Thirty-six hours later,
cells were stained with X-gal, and the percentage of cell death was
determined as described under "Materials and Methods." As shown in
Fig. 6A, transfection of
wild-type TR caused a significant cell death. In this experiment, we
employed a suboptimal concentration of wild-type TR to distinguish its
stimulatory effect on caspase 8-induced death. Caspase 8 overexpression
itself caused a strong cell death. Cotransfection of the pCXN2-Myc
vector alone did not augment caspase 8-induced cell death. However,
cotransfection of the pCXN2-Myc vector containing wild-type TR
augmented cell death significantly higher than did either plasmid
alone, indicating a synergistic effect. In contrast, the Cys-mut
strongly inhibited the death-inductive effect of caspase 8.
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Fig. 6.
Effect of TR on caspase-induced cell
death. Expression vectors for caspase 8 (Casp8,
A and B) or caspase 9 (Casp9,
C) were co-expressed along with the -galactosidase
reporter gene into MCF-7 cells. WT, wild-type TR. One
microgram each of caspase and
-galactosidase reporters was used in
this experiment. Specific effector plasmids, indicated on the
x axis, were co-expressed to determine their effects on
caspase-induced cell death. The amount (0.5 µg) of TR or Cys-mut
transfected was kept at a suboptimal level to distinguish the
differences between various controls. The total amount of DNA
transfected into cells was kept constant (3 µg) by adding pCXN2-Myc
vector where required. B, an increasing molar fold of
Cys-mut was cotransfected with a caspase 8 expression vector, and the
percentage of dead cells was calculated as described above.
C, the effect of TR on caspase 9-induced death.
-galactosidase and caspase 8 expression vectors as
described above. Along with these plasmids, various molar amounts of
the Cys-mut expression vector were transfected. The percentage of cell
death was determined 36 h post-transfection. The Cys-mut inhibited
caspase 8-induced cell death in a concentration-dependent manner. In the presence of a 3-fold molar excess of Cys-mut, caspase 8-induced death was completely ablated.
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Fig. 7.
MCF-7 cells stably transfected with various
TR expression vectors were treated with the IFN/RA combination for 5 days. Cell lysates were prepared and Western blot analyses, with
the indicated antibodies, were performed using (100 µg) of protein
from each sample. Cleavage products are indicated with
arrows. C, untreated control;
IR, IFN/RA combination; WT, wild-type TR.
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Fig. 8.
Effect of TR on mitochondrial damage.
A, the release of cytochrome c from mitochondria
in response to IFN/RA treatment in MCF-7 cells. Cells expressing
various TR mutants were treated with the IFN/RA combination for the
days indicated, and the cytosolic and membrane fractions were prepared
as described under "Materials and Methods" and in Ref. 42.
A comparable amount of total protein (120 µg) from each preparation
was subjected to Western blot analysis with antibodies specific to
cytochrome c (Cyt C). WT, wild-type
TR. A and B, the Western blot analysis of
cytosolic and total cytochrome c, respectively.
C, a quantification of the amount of cytochrome c
detected in the cytosol. Each error bar is the mean ± S.E. of triplicate measurements.
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Fig. 9.
Caspase activation in cells expressing
various TR mutants. Cell lysates were prepared after various days
of IFN/RA treatment. Equal quantities of total protein (25 µg) from
each sample were incubated for 3 h with substrates specific for
each caspase coupled with a pNA group. Absorbance of the
chromophore, pNA, released after enzymatic digestion of
these substrates was monitored at 405 nm. Each error bar
represents the mean ± S.E. of triplicate
measurements.WT, wild-type TR.
, and
the IFN/RA combination, total RNA was extracted and employed in a
ribonuclease protection assay (RPA) that allows the detection of
death-specific mRNAs (Fig.
10A). In this assay multiple
32P-labeled probes corresponding to caspase 8, decoy
receptor 1, DR3, DR4, DR5, Fas, Fas ligand, TRAIL, TNF receptor p55,
TNF receptor-associated protein with a death domain, and
receptor-interacting protein genes were first hybridized to the
cellular RNA and subjected to RNase digestion. The protected RNA
species were visualized after resolving the bands on a polyacrylamide
gel. These analyses revealed that IFN/RA treatment causes an
up-regulation of mRNAs corresponding to death receptors DR3, DR4,
DR5, and Fas and death-activating ligands Fas ligand and TRAIL. IFN-
alone induced the expression of DR3 and DR5 but not the other
mRNAs. RA did not significantly induce these genes. Caspase 8 mRNA levels remained unchanged with any of the treatments. The TNF
receptor p55, TRADD, and receptor-interacting protein mRNAs
remained unaltered under these conditions. All the lanes had a
comparable amount of RNA as shown by internal controls, ribosomal L32,
and GAPDH. These data indicate that the IFN/RA combination alone
induces the certain death receptors and their ligands.
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Fig. 10.
Induction of death-associated gene
expression by the IFN/RA combination. A, MCF-7 cells
were treated with IFN- , RA, or their combination for 72 h prior
to harvesting total RNA. A comparable amount of RNA (100 µg) was
subjected to RPA after hybridizing to a set of commercially available
death-associated gene probes (PharMingen) labeled with
[32P]. After the assay, the protected RNAs were resolved
on a polyacrylamide gel, dried, and autoradiographed to detect bands.
The positions of death-associated genes and the internal controls,
ribosomal protein L32 and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), are indicated in the middle. A negative
control reaction with yeast tRNA was also run, and none of these bands
were detectable in that sample (data not shown). Casp 8,
caspase 8; FasL, Fas ligand; TNRF p55, TNF
receptor p55; TRADD, TNF receptor-associated protein with a
death domain; RIP, receptor-interacting protein.
B, the effect of the IFN/RA combination on death-associated
gene expression in MCF-7 cells expressing various TR constructs
(indicated above the panel). Cells were treated with the IFN/RA
combination as described above. An equal amount of total RNA (100 µg)
was employed in RPA. The minus and plus signs
indicate treatment with IFN/RA. C, Western blot analysis of
death-associated protein expression. A comparable amount of total
protein (75 µg) from the indicated cell lines was used, and the blots
were probed with the indicated monoclonal antibodies. D,
quantification of TRAIL and Fas protein expression using laser
densitometry. Various treatments and the cell lines are indicated on
the x axis. The band intensity ± S.E. in arbitrary
units is shown on the y axis.
-gal expression
vector was also included to detect the transfected cells. Dead and live
cells were enumerated after in situ X-gal staining as
described under "Materials and methods." The transfection of
wild-type TR or Trx caused a 3-4-fold increase in cell death. Neither
TR Cys-mut nor the Trx mutant caused cell death above the levels
observed with the expression vector alone. However, transfection of TR
and Trx together synergistically induced a 12-fold increase in cell
death. Under the same conditions, the presence of a mutant Trx blocked
wild-type TR-induced death. Similarly, the Cys-mut failed to augment
Trx-dependent death. Thus, TR requires its substrate Trx1
to transduce electrons to components of death machinery. Lastly, we
have determined whether IFN/RA caused an increase in Trx expression
using a Western blot analysis (Fig. 10B). Trx protein levels
were increased ~5-fold by 24 h and remained constant until
72 h, the time at which significant death could be detected in
the culture.
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Fig. 11.
Thioredoxin plays an important role in
promoting the death stimulatory effects of TR through caspase 8. A, MCF-7 cells were transfected with various combinations of
TR (0.5 µg) or Trx (Trx-mut, 0.5 µg) mutants along with
the CMV- -galactosidase expression vector (1 µg). The total amount
of DNA transfected was kept constant by adding the expression vector
pCXN2-Myc where required. Dead cells were enumerated afterward by
in situ
-galactosidase staining as described under
"Materials and Methods." Important experimental samples are
boxed. B, Western blot analysis of Trx 1 expression. Cells were treated with the IFN/RA combination for the
indicated times. Total protein (50 µg) from each sample was separated
on a 15% SDS-polyacrylamide gel and electroblotted onto nylon
membranes. The blots were probed by a Trx1-specific monoclonal
antibody. The same blot was stripped and probed with a monoclonal
antibody against actin. C, catalytically active TR and Trx
are necessary for caspase 8 function. MCF-7 cells were transfected with
the indicated plasmids. Concentrations of these plasmids were kept
suboptimal for distinguishing the differences between various controls.
TR (0.5 µg), Trx (0.5 µg), and caspase 8 (1 µg) were used in this
experiment. All transfection mixtures also contained the CMV-
-gal (1 µg) plasmid. The total amount of DNA (3 µg) transfected into the
cells was kept constant by adding pCXN2-Myc where required. The
percentage of dead cells was determined as described under "Materials
and Methods."
View larger version (16K):
[in a new window]
Fig. 12.
A schematic model indicating the steps
through which TR influences the "core apoptotic machinery."
The IFN/RA combination activates caspase 8, which in turn
induces the mitochondrial damage leading to the release of cytochrome
c. Cytochrome c in association with Apaf-1
activates caspase 9. Caspase 9 in turn activates the cleavage of
proteins required for the cellular viability.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-induced cell death (39), and certain tumor cells that
hyper-produce Trx grow poorly (63). Although overexpression of Trx1 in
clonal isolates of certain transformed cells promotes growth, the
majority of the cells overexpressing Trx1 did not survive (64). Lastly,
enforced expression of Trx1 causes cell death in lung epithelial
cells.2 Collectively, these
data indicate that TR1:Trx1 can regulate cell death. In contrast, some
studies have shown that certain human T cell leukemia
virus-transformed lymphocytes secrete Trx in high
amounts and grow rapidly, indicating a cytokine-like function for this
protein (65). It should be noted, however, the protein once secreted
may be subjected to an extracellular oxidizing environment. Thus, the
growth-promoting effect of such protein may be different from that of
intracellular protein.
A mutants of caspase 8 blocked
TR1-induced death (Fig. 5). Because all caspases require the cysteine
for their function activity, it is important to know whether all or
selected caspases are targets of TR1 during cell death. The data
obtained with the limited number of caspases used in this study show
that caspase 8, but not caspase 9, is inhibited in the presence of a
mutant TR1 (Figs. 6 and 7). It would seem that the redox state of
caspase 9 is controlled by an other undefined factor(s). TR1-modulated
death seems to depend on the redox status of caspase 8 and is mediated
by Trx1. Consistent with this, mutational inactivation of the critical cysteine residues in all three proteins results in the suppression of
cell death (Fig. 11). We speculate that in the absence of an active TR1
or Trx1, caspase 8 exists in an oxidized form, thus precluding cell
death. We were unable to detect a physical interaction between Trx and
caspase 8 by coimmunoprecipitation (data not shown). This may be caused
by the transient nature of their interaction and the instability of the
caspase 8-Trx complex upon lysis. However, consistent with our
observations, a recent study has shown that caspase 3 activity may be
restored after incubation with Trx1 (70). Indeed, the blockade of Trx1
also blocks the IFN/RA-induced death
pathway.3 Conversely, nitric
oxide-mediated oxidation and S-nitrosylation of the active
site cysteine have been implicated in down-regulation of caspase 3 activity (71, 72). Depletion of cellular glutathione levels enhances
the apoptotic effect of TNF-
in certain hepatoma cells (73).
Treatment of cells with reducing agents augmented Fas-induced cell
death and enhanced caspase 3 activity (74). These observations suggest
that the redox factors can exert positive and negative effects on the
death machinery.
(76), and AP1 (77) critically
depend on their redox status, and Trx seems to play an important role
in these processes. Although IFN to some extent induces DR3 and DR5
gene expression, it does not induce the death stimulatory ligands. RA
did not induce any of the DRs. However, it may affect the death program
at a separate step. The precise role of RA in promoting cell death in
the presence of IFN-
requires a detailed investigation.
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ACKNOWLEDGEMENTS |
---|
We thank E. Alnemri, J. Tschopp, and J. Yodoi for providing several important plasmids used in the study and Martin Flajnik and Bret Hassel for a critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by National Cancer Institute Grants CA 78282 and CA 71401 (to D. V. K.). S. P. M. R. is supported by National Institutes of Health Grant HL-58122.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.
§ Present address: Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, OH 44195.
¶ Present address: The Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724.
** To whom correspondence should be addressed. Tel.: 410-328-1396; Fax: 410-328-1397; E-mail: dkalvako@umaryland.edu.
Published, JBC Papers in Press, April 30, 2001, DOI 10.1074/jbc.M100380200
2 S.P.M. Reddy and R. Wu, unpublished observation.
3 Ma, X., Karra, S., Lindner, D. J., Hu, J., Reddy, S. P. M., Kimchi, A., Yodoi, J., and Kalvakolanu, D. V. (2001) Oncogene, in press.
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ABBREVIATIONS |
---|
The abbreviations used are: STAT, signal transducing activator of transcription; RA, all-trans-retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; TR, thioredoxin reductase; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; PARP, poly(ADP-ribose) polymerase; FLIP, Fas-associated death domain protein-like interleukin-1 converting enzyme inhibitory protein; c-FLIP and v-FLIP, cellular and viral FLIP, respectively; PCR, polymerase chain reaction; Cys-mut, TR mutant lacking active site cysteines; TK, tyrosine kinase; TK-pm, point mutant lacking potential tyrosine phosphorylation sites; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate; CMV, cytomegalovirus; Trx, thioredoxin; pNA, p-nitroanilide; Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; RPA, ribonuclease protection assay; DR, death receptor.
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