Regulation of Interferon and Retinoic Acid-induced Cell Death Activation through Thioredoxin Reductase*

Xinrong MaDagger , Sreenivasu KarraDagger , Wei GuoDagger , Daniel J. LindnerDagger §, Jiadi HuDagger , Jon E. AngellDagger , Edward R. HofmannDagger , Sekhar P. M. ReddyDagger ||, and Dhananjaya V. KalvakolanuDagger **

From the Dagger  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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Interferons (IFNs) and retinoids are potent biological response modifiers. The IFN-beta 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma 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-gamma -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.

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-RARalpha , 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-alpha (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).

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.

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Reagents-- Restriction and DNA-modifying enzymes (New England Biolabs); G418 sulfate, isopropyl-1-thio-beta -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-beta 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.

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 10-11 M estradiol during treatment with IFN-beta and RA. The cells were grown in phenol red-free medium for 24 h before treatments were initiated.

Plasmids-- The mammalian expression vector pCXN2-Myc contains a Myc epitope tag in its multiple cloning site. The chicken actin promoter and the rabbit beta -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).

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 beta -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 beta -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.

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).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Generation of Mutant TR1 Proteins-- We have shown earlier that down-regulation of cellular TR gene expression by its antisense mRNA inhibits IFN-beta /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-beta 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.

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.


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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.

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-beta /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-beta (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-beta 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.

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.


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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-beta (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-beta ; 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-beta /RA combination as described above. FACS analysis was performed as described under "Materials and Methods." The percentage of annexin V-positive cells is presented.

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-beta 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.

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-beta -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 beta -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 beta -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.

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-beta -galactosidase expression vector and wild-type TR cDNA. Along with these plasmids the expression vector carrying a catalytically inactive caspase 8 mutant (Cright-arrowA) 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.

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-beta -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 beta -galactosidase reporter gene into MCF-7 cells. WT, wild-type TR. One microgram each of caspase and beta -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.

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-beta -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.

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.


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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.

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).


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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.

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).


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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.

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-beta , 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-beta 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-beta , 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.

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-beta -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.

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.


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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-beta -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 beta -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-beta -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."


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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

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-gamma -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.

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, Cright-arrowA 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-alpha 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.

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-1alpha (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-beta requires a detailed investigation.

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).

    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.

    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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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