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Originally published In Press as doi:10.1074/jbc.M313721200 on February 5, 2004

J. Biol. Chem., Vol. 279, Issue 16, 16805-16812, April 16, 2004
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Oxidant Hypersensitivity of Fanconi Anemia Type C-deficient Cells Is Dependent on a Redox-regulated Apoptotic Pathway*

M. Reza Saadatzadeh{ddagger}, Khadijeh Bijangi-Vishehsaraei{ddagger}, Ping Hong, Heidi Bergmann, and Laura S. Haneline§

From the Departments of Pediatrics and Microbiology/Immunology, Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana 46202-5254

Received for publication, December 15, 2003 , and in revised form, January 30, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fanconi anemia is a genetic disorder characterized by bone marrow failure. Significant evidence supports enhanced apoptosis of hematopoietic stem/progenitor cells as a critical factor in the pathogenesis of bone marrow failure in Fanconi anemia. However, the molecular mechanism(s) responsible for the apoptotic phenotype are incompletely understood. Here, we tested whether alterations in the activation of a redox-dependent pathway may participate in the pro-apoptotic phenotype of primary Fancc -/- cells in response to oxidative stress. Our data indicate that Fancc -/- cells are highly sensitive to oxidant stimuli and undergo enhanced oxidant-mediated apoptosis compared with wild type controls. In addition, antioxidants preferentially enhanced the survival of Fancc -/- cells. Because oxidative stress activates the redox-dependent ASK1 pathway, we assessed whether Fancc -/- cells exhibited increased oxidant-induced ASK1 activation. Our results revealed ASK1 hyperactivation in H2O2-treated Fancc -/- cells. Furthermore, using small interfering RNAs to decrease ASK1 expression and a dominant negative ASK1 mutant to inhibit ASK1 kinase activity, we determined that H2O2-induced apoptosis was ASK1-dependent. Collectively, these data argue that the predisposition of Fancc -/- hematopoietic stem/progenitor cells to apoptosis is mediated in part through altered redox regulation and ASK1 hyperactivation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fanconi anemia (FA)1 is a heterogeneous bone marrow (BM) failure syndrome with cellular abnormalities that include chromosomal instability, increased apoptosis, and cell cycle control defects (1-5). The diversity of clinical presentation in children with FA is related, in part, to the existence of multiple complementation types, with eight FA complementation group cDNAs being identified thus far (FANCA, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, and FANCL) (6-14). Despite some clinical variability between individuals with specific FA gene mutations (15, 16), the major cause of mortality in all FA complementation types is BM failure (1, 2, 5). These studies suggest that apoptotic loss of hematopoietic stem/progenitor cells has a key pathogenetic role in this disorder. Thus, understanding molecular mechanisms involved in the predisposition of FA cells to apoptosis is of critical importance to improve current treatment approaches for children with FA.

Numerous studies show that FANCA, FANCC, FANCE, FANCF, and FANCG interact in a multimeric nuclear protein complex (17-23), the formation of which is required for FANCL to monoubiquitinate FANCD2 (14), a post-translational modification that signals relocalization of FANCD2 into nuclear foci containing BRCA1 (24). However, the majority of FANCC resides in the cytoplasm (25, 26), and cytoplasmic localization is required for protection against genotoxin-induced apoptosis (27). Interestingly, cytoplasmic FANCC interacts with heat shock protein 70 (HSP70) to protect cells from interferon-{gamma}/TNF-{alpha}-induced apoptosis, yet FANCC-HSP70 interaction is dispensable for protection against genotoxic stress (28-30). Together these data support an additional cytoplasmic role for FANCC in suppressing apoptosis.

Participation of FANCC in redox metabolism has been proposed previously and is supported by functional interactions with cytochrome P-450 reductase (CPR) (31) and glutathione S-transferase P1 (GSTP1) (32). Evidence of oxygen sensitivity was first provided by Joenje et al. (34), who demonstrated that the chromosomal instability of primary FA cells could be reduced if grown at lowered oxygen tension (33). Several conflicting studies have been reported since this original description, although most analyses were conducted in immortalized cell lines or in cells with unidentified FA complementation type and/or mutation (34-36). Now with the availability of murine models, the issue of whether FA cells have altered redox regulation is being readdressed in primary cells. Using mice deficient in the murine FANCC homologue (Fancc), Hadjur et al. (37) showed that mice mutant at both the Fancc and superoxide dismutase 1 (SOD1) loci exhibit severe defects in hematopoiesis, including histological evidence of BM hypoplasia, an observation not detected in singly mutant mice. Although these data provide strong genetic evidence that Fancc -/- cells are hypersensitive to endogenously generated oxidants, it is unknown whether the molecular mechanism responsible for this hyper-sensitive response is due to altered redox signaling.

Redox signaling has a critical role in controlling multiple complex cellular processes including apoptosis, proliferation, senescence, and differentiation (38-44). This highly conserved regulatory process involves maintenance of the intracellular environment in an overall reduced state. Cellular oxidative stress results in the oxidation of key cysteine residues on redox-sensitive proteins, a post-translational modification that affects intracellular signaling pathways in a fashion similar to phosphorylation. A notable example of redox apoptotic signaling involves the serine-threonine kinase apoptosis signal-regulating kinase 1 (ASK1). In the normal reducing environment of a cell, ASK1 activity is inhibited via binding to thioredoxin, glutaredoxin, and glutathione S-transferases (45-51). After direct or indirect oxidant stress (i.e. H2O2, TNF-{alpha}, glucose/serum deprivation, and heat shock), these proteins are oxidized forming intramolecular disulfide bonds, which result in a conformational change and dissociation from ASK1. Unbound ASK1 is then available to autophosphorylate and subsequently phosphorylate downstream kinases, initiating an apoptotic program.

To extend our understanding of oxidant hypersensitivity in FA, we investigated whether primary Fancc -/- cells exhibit alterations in the redox-dependent ASK1 apoptotic pathway. Our data demonstrate that Fancc -/- cells exhibit ASK1 hyperactivation after H2O2 treatment. In addition, we show that enhanced H2O2-induced apoptosis in Fancc -/- cells is ASK1-dependent and that pretreatment with antioxidants preferentially protects Fancc -/- cells from apoptosis induced by H2O2 as compared with controls. Collectively, these data argue that the predisposition of primary Fancc -/- cells to oxidant-induced apoptosis is mediated through hyperactivation of a redox-dependent apoptotic signaling pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Mice—Fancc +/- mice in a C57Bl/6J genetic background were bred to generate Fancc -/- and wild type (WT) mice for hematopoietic progenitor assays and timed embryos for murine embryo fibroblast (MEF) cell lines as previously described (52, 53). All of the studies were approved by the Indiana University Laboratory Animal Research Center.

Hematopoietic Progenitor Assays—WT and Fancc -/- BM low density mononuclear and ckit+lin- cells were prepared as previously described (54, 55). Cells from WT and Fancc -/- mice were resus-pended in Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 20% fetal calf serum (Biowhittaker, Walkersville, MD) and then exposed to H2O2 (Sigma) for 1 h. After oxidant treatment, the cells were washed and plated in clonogenic assays as described previously (55). For hyperoxia exposure, low density mononuclear cells were placed in an airtight chamber before infusing with a gas mixture containing 50% O2, 5% CO2, and 45% N2 (Praxair, Indianapolis, IN). The chamber was then incubated for 4 or 16 h at 37 °C before the cells were harvested for clonogenic assays. An O2 analyzer was used to measure the O2 concentration before and after each incubation period (50 + 3%) to ensure an airtight culture system. Control cultures were incubated at 21% O2 for 4 or 16 h.

MEF Survival Assays—MEFs were maintained as previously described (53). All of the studies were conducted in at least two or three different MEF cell lines/genotype, and only MEFs that were less than passage 5 were utilized. To assess H2O2 sensitivity, WT and Fancc -/- MEFs were cultured with H2O2 for 24 h before assessing viability by trypan blue exclusion. In some experiments, MEFs were pretreated with 20 µM selenomethionine (SeMet; Sigma) overnight or 4 mM N-acetylcysteine (NAC; Sigma) for 1 h prior to culturing with H2O2. To evaluate apoptosis, MEFs were treated with 100 µM H2O2 for 4-6 h and analyzed by the terminal deoxynucleotidyltransferase-mediated nick-end labeling (TUNEL) assay as previously described (55, 56).

Retroviral Constructs and Transduction—PG13 retroviral packaging cells containing the FANCC mutants (FANCC-E251A and FANCC-del322G) in the pLXSN backbone were generously provided by Dr. Grover C. Bagby, Jr. (Oregon Health Sciences, Portland, OR) (29). Retroviral supernatants were harvested and utilized to transduce GP+E86 retroviral packaging cells as previously described (52) to pseudotype viral particles with an ecotropic envelope. The MFG-FAC retrovirus encoding the FANCC cDNA was used as a control, which previously was shown to correct mitomycin C sensitivity of Fancc -/- cells to WT levels (52). The dominant negative ASK1 cDNA (ASK1-K709M) (57) was generously provided by Dr. Hidenori Ichijo (University of Tokyo, Tokyo, Japan) in a pcDNA plasmid. The ASK1-K709M cDNA was subcloned into the NotI site of the bicistronic retroviral plasmid MIEG3 (58), which is 5' to an internal ribosomal entry site-enhanced green fluorescent protein (EGFP) cassette. A GP+E86 packaging cell line was developed for MIEG3 and MIEG3-ASK1-K709M as previously described (52). Retroviral supernatants were collected, filtered, and stored at -80 °C until utilized for transduction of MEFs. Early passage MEFs (P0-P1) were transduced with retroviral supernatants four times over 2 consecutive days in the presence of polybrene as previously described (52, 53).

ASK1 in Vitro Kinase Assay—ASK1 kinase activity was determined by depriving MEFs of serum for 4 h followed by treatment with 100 µM H2O2 for 5 min. MEFs were then washed twice with cold phosphate-buffered saline containing 1 mM sodium orthovanadate and lysed in nonionic lysis buffer. The protein concentrations were determined using the BCA assay (Pierce). The ASK1 immunoprecipitations were conducted using protein A Sepharose beads (Amersham Biosciences) and anti-ASK1 antibody (Cell Signaling, Beverly, MA). Immunobeads were subjected to an in vitro kinase reaction using either myelin basic protein (Sigma) or MKK4 (Upstate Biotechnologies, Inc.) as substrates for ASK1. The kinase mixtures contained 20 mM MgCl2, 0.1 M sodium orthovanadate, 1 M dithiothreitol, 30 mM {beta} glycerol phosphate, 5 mM EGTA, 20 mM MOPS, 1 µM ATP, and 10 µg of substrate/sample before adding 2.5 µCi of [{gamma}-32P]ATP/sample. The kinase reaction buffer was added to each sample and incubated at 30 °C for 30 min. The reactions were terminated by the addition of sample buffer. The protein samples were separated on a 12% SDS-PAGE gel (Invitrogen), transferred to a nitrocellulose membrane, and subjected to autoradiography.

Western Blotting—Equivalent amounts of protein (200-500 µg) were separated on a 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane. For immunodetection of FANCC mutants, a primary rabbit anti-FANCC antibody, previously generated by our laboratory (52), and a secondary anti-rabbit horseradish peroxidase antibody (Amersham Biosciences) were used as described (52) before visualizing by chemiluminescence (Amersham Biosciences). To document equal protein loading, the membrane was stripped and reprobed with {beta}-actin (Sigma). For immunodetection of ASK1, rabbit anti-ASK1 antibody (Cell Signaling) was used at a 1:200 dilution before incubating with the secondary antibody anti-rabbit horseradish peroxidase (1:2000 dilution).

Small Interfering RNA Transfection Protocol—Table I lists the RNA sequences used for these studies. The ASK1 siRNA sequence targeted nucleotides 570-590 of the ASK1 mRNA and was designed according to the manufacturer's recommendations (Dharmicon, Lafayette, CO). Either sense or scrambled oligonucleotides were used as a control for every transfection experiment. WT and Fancc -/- MEFs were cultured in a 6-well tissue culture dish to 30-50% confluency. The RNA oligonucleotides were diluted in Opti-MEM (Invitrogen) to obtain a 250 nM solution per Dharmicon's recommendations. Oligofectamine transfections were conducted exactly per the manufacturer's recommendations (Invitrogen). Following the transfection, 500 µl of Dulbecco's modified Eagle's medium (Invitrogen) containing 30% fetal calf serum was added without removing the transfection mixture. The cells were incubated for 72 h at 37 °C before harvesting for H2O2 cytotoxicity assays and ASK1 Western blotting. Four independent transfections were conducted with similar results.


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  		TABLE I
siRNA sequences

 
Statistical Analyses—For all data shown, a Student's t test was conducted to evaluate for differences between treatment groups, and a p value <= 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Fancc -/- Hematopoietic Progenitors and MEFs Are Hyper-sensitive to Oxidants—Because FA patients have severe defects in hematopoietic stem/progenitor cell function, we initially tested whether Fancc -/- progenitors were hypersensitive to oxidant stress. To accomplish this aim, WT and Fancc -/- BM low density mononuclear cells were cultured either with H2O2 or in hyperoxic conditions (50% O2) before plating in clonogenic progenitor assays. For studies utilizing H2O2, Fancc -/- progenitors were significantly more sensitive to multiple H2O2 doses as compared with controls (Fig. 1A). In addition, Fancc -/- progenitors exposed to 50% O2 for 4 or 16 h exhibited a marked reduction in colony formation as compared with WT control cultures (Fig. 1B). BM low density mononuclear cells are a heterogeneous cell population that includes a significant proportion of differentiated cells compared with the relatively low frequency of clonogenic progenitor cells (0.01-0.5%). Given our previous observations that Fancc -/- progenitors are exquisitely sensitive to multiple inhibitory cytokines such as interferon-{gamma} and TNF-{alpha} (55), together with the knowledge that inflammatory cells such as lymphocytes and granulocytes are major sources of secreted inhibitory cytokines, it was crucial to eliminate these differentiated cells from our culture system. To test whether the observed oxidant hypersensitivity was due to an intrinsic abnormality in Fancc -/- progenitor cells and not secondary to accessory cells present in BM low density mononuclear cell populations, WT and Fancc -/- ckit+lin- cells were purified by fluorescence cytometry, treated with 100 µM H2O2, and plated in colony assays. This phenotypically defined cell population enriches for immature hematopoietic stem and progenitor cells and excludes differentiated progeny cells. Similar to prior studies with low density mononuclear cells, Fancc -/- ckit+lin- cells were hypersensitive to H2O2 (Fig. 1C), supporting an intrinsic hematopoietic progenitor cell defect.



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  		FIG. 1.
Fancc -/- hematopoietic progenitors are hypersensitive to oxidants. A, H2O2 sensitivity of low density mononuclear cells. WT and Fancc -/- low density cells were treated for 1 h with increasing concentrations of H2O2 before assaying for progenitors in triplicate cultures. The percentage of untreated control was determined by dividing the number of colonies scored at each H2O2 concentration by untreated controls. The data shown represent one of four independent experiments with similar results. B, 50% O2 sensitivity of low density mononuclear cells. WT and Fancc -/- low density BM cells were cultured for 4 or 16 h at 50% O2 before plating in triplicate progenitor assays. The percentage base-line calculation was determined by dividing the number of colonies scored in hyperoxic conditions by the number of colonies counted from room air cultures. The data shown represent one of four independent experiments with similar results. C, H2O2 sensitivity of ckit+lin- cells. After fluorescence-activated cell sorting, ckit+lin- cells from Fancc -/- and WT mice were treated with 100 µM H2O2 for 1 h. The cells were washed and plated in progenitor assays as described above. The data represent one of three independent experiments. All of the error bars represent S.E. *, p <= 0.02.

 
Because of the difficulty in obtaining sufficient numbers of primary hematopoietic progenitor cells, we established WT and Fancc -/- MEFs to utilize as a cellular model system for evaluation of alterations in oxidant responsiveness in Fancc -/- cells. Initial studies determined that Fancc -/- MEFs exhibited an enhanced sensitivity to H2O2 (Fig. 2A), comparable with that observed in Fancc -/- progenitors. TUNEL assays confirmed that the H2O2 hypersensitivity in Fancc -/- MEFs was due to enhanced apoptosis (Fig. 2B). In addition, we observed that Fancc -/- MEFs exhibit a slight increase in apoptosis when grown in basal conditions, similar to previous data in cultured Fancc -/- hematopoietic cells (52). To test whether antioxidants protect Fancc -/- MEFs from oxidant exposure, MEFs were pretreated with either SeMet or NAC before culturing with H2O2. Consistent with an altered redox state, Fancc -/- MEFs pretreated with SeMet or NAC were protected from H2O2 treatment compared with control Fancc -/- MEFs (Fig. 2C). Importantly, survival of Fancc -/- MEFs was restored to WT levels after SeMet and NAC pretreatment. Furthermore, SeMet pretreatment of Fancc -/- MEFs reduced apoptosis in basal and H2O2-treated conditions (Fig. 2D).



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  		FIG. 2.
Antioxidants decrease H2O2-induced apoptosis in Fancc -/- MEFs to WT levels. A,H2O2 sensitivity of MEFs. WT and Fancc -/- MEFs were treated with H2O2 before assaying viability by trypan blue exclusion. The percentage of untreated control was determined by dividing the number of MEFs scored at each H2O2 concentration by untreated controls. The data shown represent the means of four experiments. *, p <= 0.02. B, H2O2-induced apoptosis. WT and Fancc -/- MEFs were grown on coverslips before treating with 100 µM H2O2. Apoptosis was analyzed 4-6 h after H2O2 treatment using a TUNEL assay. For each experiment, at least 100 cells were evaluated (DAPI+) to determine the percentage of apoptotic cells (TUNEL+) in each condition. The mean of three independent experiments is shown. *, p <= 0.01. C, antioxidants and MEF viability assays. MEFs were either grown in normal conditions, pretreated with 20 µM SeMet, or pretreated with 4 mM NAC prior to 100 µM H2O2 treatment. Viability was assessed by trypan blue exclusion. The data shown represent the means of four experiments. *, p <= 0.04; **, p <= 0.006. D, SeMet pretreatment and apoptosis. MEFs were either grown in normal conditions or pretreated with 20 µM SeMet prior to 100 µM H2O2 treatment. Apoptosis was analyzed 4-6 h after H2O2 treatment using a TUNEL assay. For each experiment, at least 100 cells were evaluated (DAPI+) to determine the percentage of apoptotic cells (TUNEL+) in each condition. The mean of three independent experiments is shown. *, p <= 0.01; **, p <= 0.002.

 
Correction of H2O2 Hypersensitivity in Fancc -/- MEFs Requires the N-terminal Domain of FANCC—Previously reported structure-function studies showed that FANCC has at least two independent functions, separable by FANCC mutant cDNAs (29). One recognized function is to maintain normal alkylating agent sensitivity, which is associated with restoration of nuclear FA protein complex formation (corrected by FANCC-E251A mutant). The second identified function is to sustain normal PKR-mediated apoptotic signaling via an interaction with HSP70 (corrected by FANCC-del322G mutant, an FA patient-derived mutation resulting in deletion of amino acids 1-54). To determine whether oxidant hypersensitivity in Fancc -/- MEFs segregates with a known FANCC function, we transduced Fancc -/- MEFs with a retrovirus encoding either FANCC, FANCC-E251A, or FANCC-del322G. Mock infected Fancc -/- MEFs were utilized as a control. FANCC expression in transduced MEFs was confirmed by Western blotting (Fig. 3A) before evaluating H2O2 sensitivity (Fig. 3B). The H2O2 dose-response curves for MEFs transduced with a retrovirus containing either FANCC or FANCC-E251A were indistinguishable, suggesting that the role of FANCC in regulating HSP70/PKR signaling is not required for protection against H2O2. In marked contrast, MEFs transduced with the FANCC-del322G mutant exhibited no protection against H2O2, as compared with the mock-infected control. These data show that the N-terminal domain of FANCC is required for protection against H2O2 in Fancc -/- cells.



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  		FIG. 3.
The N-terminal domain of FANCC is required to correct H2O2 hypersensitivity in Fancc -/- MEFs. Fancc -/- MEFs were transduced with a retrovirus encoding either the FANCC cDNA, FANCC-E251A, FANCC-del322G, or mock-infected. A, FANCC Western blot of transduced MEFs. The autoradiograph shown is representative of three separate transductions. B, H2O2 dose response of transduced MEFs. Transduced MEFs were treated with increasing H2O2 concentrations, and cell viability was determined by trypan blue exclusion. The data shown represent the means of four experiments. *, p < 0.05.

 
Enhanced H2O2-induced Apoptosis in Fancc -/- MEFs Is ASK1-dependent—Because H2O2-induced apoptosis in MEFs is ASK1-dependent (59) and Fancc -/- MEFs exhibit enhanced H2O2-induced apoptosis (Fig. 2A), we reasoned that Fancc -/- MEFs may demonstrate increased ASK1 activity after H2O2 treatment. To test this hypothesis, we evaluated H2O2-induced ASK1 activation in WT and Fancc -/- MEFs. In vitro ASK1 kinase assays revealed that Fancc -/- MEFs treated with H2O2 exhibited a significant increase in ASK1 activity compared with WT controls (Fig. 4, a experiment representative of five experiments with mean fold induction 2.8 ± 0.5 versus 1.5 ± 0.3 p < 0.05). To determine whether the predisposition of Fancc -/- MEFs to H2O2-induced apoptosis was dependent on ASK1 function, we utilized siRNA oligonucleotides to reduce ASK1 expression. Western blotting confirmed that WT and Fancc -/- MEFs transfected with the ASK1 siRNA oligomer exhibited a significant decrease in ASK1 expression (Fig. 5A, a representative experiment). Interestingly, Fancc -/- MEFs transfected with the ASK1 siRNA oligomer were completely protected against H2O2 treatment compared with untreated controls (Fig. 5B), whereas WT MEFs transfected with the ASK1 siRNA oligomer did not exhibit a significant increase in survival.



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  		FIG. 4.
H2O2 induces hyperactivation of ASK1 in Fancc -/- MEFs. ASK1 kinase activity was evaluated by depriving MEFs of serum for 4 h followed by treatment with 100 µM H2O2 for 5 min. MEFs were then washed and lysed in nonionic lysis buffer. ASK1 immunoprecipitations were subjected to an in vitro kinase reaction as described under "Experimental Procedures." Autoradiography of ASK1 kinase assays, densitometry analyses, and Western blots for total ASK1 are shown. The data shown are representative of five independent experiments.

 



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  		FIG. 5.
H2O2-induced apoptosis is ASK1 dependent in Fancc -/- MEFs. ASK1 siRNA studies. WT and Fancc -/- MEFs were transfected with either ASK1 siRNA oligonucleotides or control oligonucleotides. MEFs were incubated for 72 h at 37 °C before harvesting for ASK1 Western blot (A) and H2O2 cytotoxicity assays (B). The data shown are representative of four independent transfection experiments with similar results. *, p < 0.05; **, p < 0.01. C, dominant negative ASK1 studies. WT and Fancc -/- MEFs were transduced with a retrovirus encoding a catalytically inactive, dominant negative ASK1 (ASK1-K709M) or vector control (control). Transduced MEFs were treated with 100 µM H2O2 for 4 h before evaluating apoptosis by the TUNEL assay (phycoerythrin+). The percentage of apoptotic cells was calculated by dividing EGFP+PE+ cells by total EGFP+ cells for each condition. At least 100 cells were scored per condition per experiment. The data shown represent the means of three experiments. *, p < 0.05.

 
To further test whether H2O2-induced apoptosis in Fancc -/- MEFs was dependent on ASK1 kinase activity, we constructed a bicistronic retroviral vector that encodes a catalytically inactive, dominant negative ASK1 cDNA and EGFP (MIEG3-ASK1-K709M), allowing for selection of transduced cells (EGFP+). WT and Fancc -/- MEFs were transduced with either MIEG3-ASK1-K709M or vector control and then evaluated for H2O2-induced apoptosis. Fancc -/- MEFs transduced with MIEG3-ASK1-K709M exhibited significantly less apoptosis after H2O2 treatment compared with Fancc -/- MEFs transduced with vector alone (Fig. 5C). Consistent with previously published data for ASK1 -/- MEFs, inhibiting ASK1 kinase activity in WT MEFs also resulted in reduced apoptosis after H2O2 treatment, although the inhibition was more dramatic in Fancc -/- MEFs. Collectively, these data show that the predisposition of Fancc -/- MEFs to H2O2-mediated apoptosis is ASK1-dependent and identify ASK1 as a critical mediator of oxidant-induced apoptosis in Fancc -/- cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The first demonstration of oxygen hypersensitivity in FA cells was made over 20 years ago (33); however, since that initial report little progress has been made to understand the molecular mechanisms involved. In fact, contradictory data have led to significant debate (60, 61). Our data in primary Fancc -/- hematopoietic progenitors and MEFs clearly demonstrate hypersensitivity to oxidative agents. Furthermore, these studies are the first to establish an intrinsic defect in the oxidant responsiveness of Fancc -/- progenitors, independent of differentiated hematopoietic cell populations and the BM microenvironment. Our data compliment previous studies showing that Fancc -/- mice devoid of superoxide dismutase 1 expression develop a hypoplastic BM (37) linking increased endogenous oxidant stress with marrow failure in Fancc -/- mice. Together these observations suggest that when Fancc -/- hematopoietic cells encounter an increase in either endogenous or exogenous oxidant stress, enhanced apoptotic cell loss may occur, contributing to the pathogenesis of BM failure in FA.

Numerous reports in immortalized cell lines suggest that the loss of FA protein function may result in a pro-oxidant cellular environment (31-33, 60, 62-71). Furthermore, protein-protein interaction studies provide support for the concept that FANCC may directly participate in redox metabolism by interacting with two cytoplasmic binding proteins, GSTP1 (32) and CPR (31). These studies demonstrated that FANCC regulates the activity of both GSTP1 and CPR, with the loss of FANCC predicting increased GSTP1 oxidation (decreased activity) and increased CPR activity. In a reduced conformation GSTP1 inhibits stress-activated apoptotic signaling (50, 72, 73), suggesting that the loss of FANCC may result in altered redox-dependent stress signaling. In addition, CPR transfers electrons to cytochromes and molecular oxygen (74-77); hence increased CPR activity in FANCC deficient cells may subsequently result in a pro-oxidant cellular environment by generating increased reactive oxygen species. We reasoned that if an altered redox environment was responsible for the oxidant hypersensitivity in Fancc -/- MEFs, pretreatment with antioxidants that provide additional cellular reducing equivalents would preferentially protect Fancc -/- cells compared with WT controls. Indeed, our data showed that SeMet or NAC pretreatment not only preferentially protected Fancc -/- MEFs from H2O2-induced apoptosis but improved viability to WT levels, supporting a potential role for FANCC in redox metabolism.

Interestingly, we and others have previously shown that Fancc -/- cells are exquisitely hypersensitive to TNF-{alpha} (55, 78), a potent activator of ASK1 (46, 59, 79). Pang et al. (28, 29) demonstrated that binding of FANCC to HSP70 acts as a negative regulator for PKR-mediated apoptotic signaling induced by costimulation with interferon-{gamma} and TNF-{alpha}. To evaluate whether H2O2-induced apoptosis was dependent on HSP70 binding to FANCC, we transduced Fancc -/- MEFs with a retrovirus containing a FANCC construct mutated at the HSP70-binding site (FANCC-E251A). These studies demonstrated that the FANCC-E251A mutant completely corrected the sensitivity of Fancc -/- MEFs to H2O2, suggesting a HSP70/PKR independent mechanism. In contrast, the FANCC-del322G mutant did not protect against H2O2-induced apoptosis in Fancc -/- MEFs. This is a particularly intriguing observation because CPR binding to FANCC is predicted to be disrupted by this mutation (31).

Given our data showing that Fancc -/- cells undergo increased H2O2-mediated apoptosis, we investigated whether increased activation of a redox-dependent apoptotic signaling pathway was involved. Our data demonstrate that Fancc -/- MEFs treated with H2O2 exhibit ASK1 hyperactivation. Furthermore, studies utilizing siRNA and dominant negative methodologies to decrease ASK1 activity reveal that H2O2-induced apoptosis in Fancc -/- MEFs is ASK1-dependent. Ichijo et al. (57) identified ASK1 as an important kinase involved in oxidant- and TNF-{alpha}-induced apoptosis. Subsequent studies revealed that thioredoxin, in a reduced conformation, inhibits ASK1-dependent apoptosis by binding to ASK1 (46), which is the first demonstration that an apoptotic cascade is directly controlled by the cellular redox environment. Since this original description, it is now recognized that the redox regulation of ASK1 is complex, involving the direct interaction of ASK1 with multiple redox-dependent binding partners including thioredoxin, glutathione S-transferases, and glutaredoxin (45-51). The precise physiologic role that individual negative regulators have in inhibiting ASK1 activity remains unclear. However, there is evidence that these redox-dependent proteins may act as sensors for specific cellular redox stresses (46, 49, 51). Collectively, these studies suggest that ASK1 functions as a major modulator of apoptotic signaling induced by multiple types of oxidant stress including H2O2, TNF-{alpha}, glucose/serum deprivation, and heat shock (46, 48, 51, 80). In addition, because ASK1 activity is tightly regulated by redox-dependent mechanisms (45-48), these observations support the idea that Fancc -/- cells may exhibit an altered redox environment, which predisposes to ASK1-mediated apoptosis.

An intriguing possible explanation for the enhanced propensity of Fancc -/- MEFs to ASK1-mediated apoptosis may be due to disruption of GSTP1 redox control. Previous studies showed that FANCC expression maintains GSTP1 in a reduced conformation during growth factor withdrawal and subsequently protects from apoptotic cell death (32). Importantly, reduced glutathione S-transferases are critical for inhibition of ASK1 activation and consequently oxidant-induced apoptosis (45, 50, 51). Interestingly, Gilot et al. (50) reported that GSTP1 overexpression in primary hepatocytes protected from ASK1-dependent apoptosis, demonstrating that GSTP1 regulates ASK1 activity. Future investigation of the underlying mechanism that initiates ASK1 hyperactivation in Fancc -/- cells will be important to understand the role that FANCC has in preserving survival after oxidant stress. Regardless, our data in primary Fancc -/- cells identify ASK1 as a potentially important molecular target to defend against an apoptotic fate induced by oxidant stress.

In summary, our data indicate that Fancc -/- progenitors exhibit an intrinsic defect in oxidant responsiveness. In addition, we show that the hypersensitivity of Fancc -/- cells to oxidative stress is ASK1-dependent. Furthermore, our data showing that antioxidants protect Fancc -/- cells from enhanced oxidant-induced apoptosis suggest a potential translational role for antioxidants in the prevention and/or delay of BM failure in FA. Future preclinical studies to test the potential of such an approach will be important.


   FOOTNOTES
 
* This work were supported by United States Public Health Services Grants P01 HL53586, P30 DK49218, and K08 HLDK04071-01, American Cancer Society Grant IRG-84-002-16 and grants from the Showalter Trust, the Clarian Values Fund, and the Riley Children's Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} These authors contributed equally to the work. Back

§ To whom correspondence should be addressed: Cancer Research Institute, 1044 W. Walnut St., Rm. 476, Indianapolis, IN 46202-5254.

1 The abbreviations used are: FA, Fanconi anemia; BM, bone marrow; TNF, tumor necrosis factor; CPR, cytochrome P-450 reductase; GST, glutathione S-transferase; ASK1, apoptosis signal-regulating kinase 1; WT, wild type; MEF, murine embryo fibroblast; SeMet, selenomethionine; NAC, N-acetylcysteine; EGFP, enhanced green fluorescent protein; MOPS, 4-morpholinepropanesulfonic acid; siRNA, small interfering RNA; DAPI, 4',6-diamidino-2'-phenylindole-dihydrochrolide; TUNEL, terminal deoxynucleotidyltransferase-mediated nick-end labeling. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Manuel Buchwald (Hospital for Sick Children, University of Toronto) for providing us with the Fancc +/- mice. We also thank Drs. Yoder, Kelley, Ingram, Clapp, Dinauer, Kapur, and M. Smith (Indiana University) for many valuable discussions and thoughtful critique of the manuscript. We thank Marsha Hippensteel and Arliene Britt for administrative support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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