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HEMATOPOIESIS
From the Oregon Cancer Center, Department of Medicine
(Division of Hematology and Medical Oncology) and Department of
Molecular and Medical Genetics, Oregon Health Sciences University,
Portland, OR, and Molecular Hematopoiesis Laboratory, VA Medical
Center, Portland, OR.
The Fanconi anemia (FA) group C gene product (FANCC) functions to
protect cells from cytotoxic and genotoxic effects of cross-linking agents. FANCC is also required for optimal activation of STAT1 in
response to cytokine and growth factors and for suppressing cytokine-induced apoptosis by modulating the activity of
double-stranded RNA-dependent protein kinase. Because not all FANCC
mutations affect STAT1 activation, the hypothesis was considered that
cross-linker resistance function of FANCC depends on structural
elements that differ from those required for the cytokine signaling
functions of FANCC. Structure-function studies were designed to test
this notion. Six separate alanine-substituted mutations were generated in 3 highly conserved motifs of FANCC. All mutants complemented mitomycin C (MMC) hypersensitive phenotype of FA-C cells and corrected aberrant posttranslational activation of FANCD2 in FA-C mutant cells.
However, 2 of the mutants, S249A and E251A, failed to correct defective
STAT1 activation. FA-C lymphoblasts carrying these 2 mutants
demonstrated a defect in recruitment of STAT1 to the interferon Fanconi anemia (FA) is an autosomal recessive
disease characterized by bone marrow failure, variable congenital
anomalies, and a predisposition to leukemia.1-3 Cells from
patients with FA exhibit hypersensitivity to alkylating agents such as
mitomycin C (MMC) and diepoxybutane (DEB). Indeed, the hypersensitivity to cytotoxic effects of DNA cross-linking agents is currently used as
the basis for the diagnostic tests for FA.4,5 It is known
that FA is genetically heterogeneous, with at least 7 complementation
groups (A-G) identified thus far.6,7 The genes encoding
the groups A (FANCA),8 C
(FANCC),9 D2
(FANCD2),10 G
(FANCG),11 and F
(FANCF)12 have been cloned. The group C locus,
FANCC, encodes a 63-kd protein of unknown
function.9 Expression of the normal FANCC
complementary DNA (cDNA) in FA-C cells prevents MMC-induced chromosomal
aberrations, prevents G2 arrest in cell cycle, and prevents
growth inhibition and apoptosis.13,14 FANCC forms
complexes with at least 3 other FA proteins, namely FANCA, FANCF, and
FANCG.15-17 Such complexes are believed to function in DNA
damage repair or toleration in part by facilitating posttranslational activation of FANCD2, which can then translocate to nuclear damage foci.18
Although the FANCC protein can be found in the
nucleus,19 it is principally
cytoplasmic.19-22 Moreover, evidence indicates that FANCC
binds to a number of cytosolic proteins in vitro, including the mitotic
cyclin-dependent kinase cdc2,23 the molecular chaperone GRP94,24 NADPH cytochrome P-450 reductase,25
and the signal transducer and activator of transcription
STAT1.26 Although the functional consequences of these
molecular interactions are not clear, they suggest that FANCC may play
roles in cell cycle control, protein transport, regulation of
detoxification, and survival signal transduction.
In our view, the role of FANCC in suppressing apoptotic responses in
hematopoietic cells may account for the nearly universal development of
bone marrow failure in patients with inactivating mutations. This
notion derives from reports that suppression of FANCC gene
expression represses clonal growth of normal erythroid and
granulocyte-macrophage progenitor cells and that disruption of the
FANCC gene, in mice, rendered hematopoietic progenitor cells
hypersensitive to the apoptotic effects of interferon One function of FANCC that may account for its importance in
hematopoietic control is its role in governing proper activation of
STAT molecules. Recently, we reported that lymphoblasts from FA-C
patients were defective in STAT1 activation in response to stimulation
with IFN- Cell cultures and IFN- Construction of site-directed FANCC mutants
MMC sensitivity assay and cytogenetic analysis The pLXSN36 plasmids containing the normal FANCC or its mutant versions (10 µg) were transduced into lymphoblasts by a previously described protocol.26 Sets of isogenic lines were selected for G418 (300 µg/mL) resistance, and cellular sensitivity to MMC was assayed by plating cells at a density of 2 × 105/mL in 24-well plates. After incubation with various concentrations of MMC for 5 days, viable cell counts by trypan blue exclusion were performed. Each sample was tested in duplicate or triplicate. Cytogenetic analysis for chromosomal aberrations was performed on the HSC536N cell lines with and without the addition of MMC and DEB. MMC and DEB were added at a final concentration of 40 ng/mL and 100 ng/mL, respectively, and the cultures, protected from light, were incubated at 37°C for 48 hours. After exposure to 0.25 µg/mL colcemid (Gibco-BRL, Grand Island, NY) for 1 hour, cells were treated with 0.075 M KCl for 10 minutes and then fixed with a 3:1 mixture of methanol/acetic acid. Slides of fixed cells were stained with Wright stain and about 50 metaphases from each sample were scored for chromosomal breaks and radial formation.Subcellular fractionation, immunoprecipitation, and immunoblotting Cells were lysed with lysis buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.6, 1% sodium deoxycholate, 1% Triton X-100, and 0.1% sodium dodecyl sulfate [SDS]) supplemented with the following protease inhibitors: 1% aprotinin, 1 µg/mL leupeptin, 1 mM phenylmethylsulfonylfluoride (PMSF), and 2 mM sodium orthovanadate. Cellular fractionation was performed as described elsewhere.19 For cell lysates used specifically to assess IFN receptor complex, cells were lysed in digitonin lysis buffer (1% digitonin, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 50 mM NaF, and 5 mM sodium pyrophosphate) containing the protease inhibitors mentioned above. Whole cell lysates ( WCLs; 1 mg total protein) were precleared with 50 µL 50% protein A-Sepharose suspension (Pharmacia Biotech, Piscataway, NJ) for 1 hour at 4°C. After separation of the protein A-Sepharose the lysate was incubated with anti-IFN- R (2 µg/mL) (Antigenix America Antibodies, New York, NY) for 3 to 5 hours
at 4°C. Immunocomplexes were then bound to protein A-Sepharose beads
(50 µL 50% slurry) during a 1- to 2-hour incubation at 4°C,
recovered by centrifugation at 1000 rpm for 1 minute, and washed with
0.1% digitonin wash buffer. For immunoblotting, samples were heated at
94°C for 5 minutes in 2 × Laemmli SDS sample buffer, separated by
SDS-polyacrylamide gel electrophoresis (PAGE), and transferred onto
nitrocellulose membranes. Immunoblots were subsequently incubated with
anti-tyrosine phosphorylated STAT1 (P-STAT1) (New England Biolabs,
Beverly, MA), anti-STAT1 / polyclonal antibody (Santa Cruz
Biotechnology, Santa Cruz, CA), or polyclonal antisera against
FANCD218 or FANCC.19,37 After washing, the
blots were incubated with appropriate secondary antibody for 30 minutes
at room temperature, and developed by using an enhanced
chemiluminescence (ECL) kit (Amersham, Buckinghamshire, United Kingdom).
GST-FANCC fusion protein purification and in vitro binding assays The GST-FANCC fusion constructs were created by insertion of the entire open reading frame (ORF) of FANCC and its mutant versions into the BamHI and SmaI sites of a yeast overexpression vector pGST.38 Expression and purification of the GST-fusion proteins were performed as described elsewhere.26 For binding, about 10 µg GST-fusion proteins bound to glutathione-Sepharose beads was incubated with WCLs from untreated or IFN--treated JY cells (1 mg total proteins)
prepared in 1% digitonin buffer. The final concentration of digitonin
in the binding mixture was adjusted to 0.5%. After shaking at 4°C
for 40 minutes, the beads were recovered and washed 3 times with 0.1%
digitonin buffer before boiling in Laemmli SDS sample buffer, and
immunoblotted as described above.
Analysis of apoptosis To quantify apoptotic cells, we used a polyclonal antibody to the active form of caspase 3 in a flow cytometric assay to detect cells in the early stages of apoptosis. The flow cytometric assays were performed by the procedure described elsewhere.32Electromobility shift assay Nuclear extracts were prepared by the method of Dignam and coworkers.39 Oligonucleotides encoding the-IFN
activation site (GAS) from the IRF1 gene40 and
the p21 gene41 labeled with [-32P]-ATP to 2.5 × 104 cpm/ng using T4
polynucleotide kinase (Boehringer Mannheim, Indianapolis, IN).
Binding reactions (20 µL) contained 5 µg nuclear extract and 0.2 ng
labeled oligo were incubated at room temperature for 30 minutes, then
resolved on a 4% nondenaturing polyacrylamide gel. In competition
reactions, 25-fold molar excess of unlabeled oligonucleotides carrying
the GAS were incubated with the binding mixtures. For supershift
assays, binding reactions were incubated for a further 45 minutes in
the presence of 5 µg STAT1 antibody before gel loading.
Strategy for FANCC mutagenesis The location of mutations in the FANCC gene found in FA-C patients reveal that both termini as well as the central region of the FANCC protein are critical for normal biologic function.1,2 We identified 3 motifs, located in the amino- or carboxyl-terminal and central regions of the FANCC protein (Figure 1), which are highly conserved among FANCC homologs of human, mouse, rat, and bovine origin.9,42,43 Sequence analysis of these conserved motifs by the nnpredict program44 predicted helical (motifs I and II) or-strand (motif II) structures that have a
propensity to interact with nucleic acids or proteins. Although such
analyses are of a subjective nature, they served as a guide in our
studies because analysis of FANCC primary sequence shows no homologies to known proteins or known functional motifs. We made alanine substitutions at amino acid positions F64 and T66 (motif I), S249 and
E251 (motif II), and F529 and Y531 (motif III) in the human FANCC
protein (Figure 1). The alanine mutagenesis strategy presumes that,
although a functional group of the natural amino acid may be removed,
alanine substitutions are less apt to perturb the overall structure or
stability of the protein.45 This method thus allows for
identification of amino acid residues within functional motifs of
potential importance in the FANCC protein.
Complementation activity of the FANCC mutants To examine the abilities of the FANCC mutants to complement the characteristic MMC hypersensitivity of FA cells, alanine mutant FANCC cDNAs, as well as the wild-type FANCC gene, were subcloned into the retroviral vector pLXSN36 and transduced into lymphoblasts derived from FA-C patients. The PD4 lymphoblast cell line is heterozygous for a frameshift mutation in one allele, predicting a truncated peptide of the amino-terminal 44 amino acids, and a nonsense mutation in the other allele, predicting a terminated peptide of 185 residues.20 The HSC536N cell line contains a T to C substitution in one allele, resulting in an L554P amino acid change; the other allele is not expressed because it is deleted.9,46 As shown in Figure 2, PD4 cells and HSC536N cells transduced with the normal FANCC gene corrected the defect of the mutant FA-C cells in resistance to MMC exposure. Parental cells alone or transduced with the retroviral vector exhibited hypersensitivity to MMC with an effective drug concentration yielding 50% reduction in cell viability (EC50) of 5 nM MMC for PD4 cells (Figure 2A) and approximately 2 nM MMC for HSC536N cells (Figure 2B). All 6 alanine-substituted mutants conferred MMC resistance in that the survival curves in both PD4 and HSC536N cells were similar to those of the parental cell lines transduced with the normal FANCC gene. Although these alanine-substituted mutants were able to complement the MMC hypersensitivity in mutant PD4 and HSC536N cells, their ability to exhibit a dominant negative phenotype in vivo could not be excluded. For instance, mutations in the ATP- binding domain of DNA mismatch repair protein MSH2 do not affect mismatch recognition but cause a dominant negative effect when overexpressed in wild-type cells.47 Therefore, we introduced the 6 mutants along with the normal FANCC as well as the retroviral vector alone into the normal lymphoblast cell line JY. As shown in Figure 2C, no alanine-substituted mutation influenced survival of JY cells exposed to MMC. Cytogenetic analysis has shown that FA-C lymphoblasts had an increased frequency of chromosomal aberrations induced by DNA cross-linking agents. We analyzed radial chromosomes formed in retroviral-transduced HSC536N cells treated with MMC. As shown in Table 1, whereas HSC536N and HSC536N/neo cells had a characteristic high frequency of radial formation (> 20%), all 6 alanine-substituted mutant-transduced cell lines as well as HSC536N/FANCC had significantly reduced numbers of chromosomal radials (< 5%). Similar results were obtained with analysis of chromosomal breaks induced by MMC and DEB in these retrovirally transduced cell lines (data not shown). These cytogenetic data are consistent with the MMC survival curves demonstrated in Figure 2. Taken together, these results indicate that the alanine substitutions in the 3 conserved motifs (Figure 1) of the FANCC protein are not defective in their capacity to protect FA-C cells from cross-linking agent-induced toxicity.
Because an intact FA protein complex is essential for cellular
resistance to MMC, we further tested whether these alanine mutations
affected the FA complex formation. Because the formation of a
functional FA complex is required for the posttranslational activation
of FANCD2,18 we chose to examine the activation state of
FANCD2 in nuclear extracts of lymphoblasts bearing these alanine mutants as the most direct way to demonstrate the function of the FA
complex. As shown in Figure 3, the
corrected FA-C cells (HSC/FANCC) and all 6 alanine mutants contained 2 nuclear FANCD2 isoforms: the short form (FANCD2-S) which is the primary
translation product,10 and posttranslationally modified
(by monoubiquitination18) FANCD2-L form (lanes 8-12). FA-C
lymphoblasts (HSC536N) expressed only nuclear FANCD2-S (lane 7). As
expected, no activated FANCD2-L was observed in any cytoplasmic sample
(lanes 1-6). This result suggests that these alanine-substituted FANCC
proteins are normally assembled into the multisubunit nuclear complex
that consequently promotes the activation of FANCD2 and functions to
protect cells from MMC genotoxicity.
Effects of FANCC mutations on STAT1 phosphorylation in HSC536N cells The ability of the 6 alanine-substituted mutants to complement the MMC hypersensitivity of FA-C cells is an indication that the mutants function normally in facilitating DNA damage responses or DNA repair. However, a different situation may exist for the signaling functions of FANCC. We have previously shown that STAT1 phosphorylation induced by IFN- is markedly reduced in HSC536N cells when compared to wild-type
cells and the complemented HSC536N/FANCC cells, and FANCC acts to
facilitate the docking of STAT1 to the activated IFN-
receptor.3,26 We sought to elucidate the effects of the
FANCC mutant proteins on STAT1 phosphorylation defects in FA-C cells.
Retrovirally transduced HSC536N cells were serum-starved for 2 hours
then treated with 1 ng/mL IFN- for 5 minutes, and STAT1
phosphorylation was determined by immunoblot analysis with an antibody
specific for tyrosine-phosphorylated STAT1 (Figure 4). Despite correction of the MMC
hypersensitivity phenotype by all 6 alanine mutations, immunoblot
analysis of cell lysates revealed that defective STAT1 phosphorylation
was not corrected in HSC536N cells transduced with FANCC
cDNAs containing the 2 mutations (S249A and E251A) located in the
motif II of FANCC (Figure 4, lanes 10 and 12). In contrast, mutations
located in the amino-terminal motif (F64A and T66A) and in the
carboxyl-terminal motif (F525A and Y531A) complemented the STAT1
phosphorylation defect (lanes 6, 8 and 14, 16, respectively). Results
were identical in 4 separate experiments. This suggests that the
central region of the FANCC protein is required for STAT1 activation in
response to IFN- stimulation. The 6 HSC536N cell lines transduced
with alanine mutants of FANCC mutations were also tested for expression
of FANCC protein. All lines expressed more FANCC protein than that detected in the parent line (data not shown).
FANCC mutant protein expression in PD4 cells To rule out the possibility that the reduced amount of STAT1 phosphorylation in the 2 mutants located in the middle domain was simply due to reduced stability of these mutant proteins, we expressed the 6 mutant FANCC proteins in PD4 cells. PD4 cells do not express endogenous full-length FANCC protein because they contain 2 naturally occurring truncated FANCC mutations (delG322, and C to T at bp 808).20 Cell lysates from PD4 cells carrying the FANCC mutants were probed with antiserum generated against an N-terminal polypeptide to the FANCC protein revealed varying amounts of FANCC protein in some of the cell lines (Figure 5). All effectively corrected the MMC hypersensitivity phenotype in PD4 cells (Figure 2A) despite the fact that some expressed low levels of mutant FANCC protein (Figure 5, T66A [lane 4] and Y531A [lane 8]). Variable amounts of protein expression did not correlate with reduced STAT1 phosphorylation seen in the HSC536N cells (Figure 4), because the quantity of FANCC protein in the S249A and E251A mutants was not reduced (Figure 5, lanes 5 and 6).
Effects of FANCC mutations on STAT1 phosphorylation in PD4 FA-C cells The FANCC mutant-expressing PD4 cells above were further tested for STAT1 phosphorylation defects. Unlike HSC536N cells, PD4 cells do not exhibit suppressed STAT1 phosphorylation in response to IFN (Figure 6, lanes 1 and 2). The expression of FANCC mutant proteins (lanes 5-16) and wild-type FANCC (lanes 3 and 4) neither suppresses nor augments induced STAT1 phosphorylation. The S249A and E251A constructs, which failed to complement STAT1 phosphorylation in the HSC536N cells, are not dominant negative mutants for STAT1 activation in PD4 cells (lanes 10 and 12). Based on results of studies described below, the gene product derived from the 322delG mutation by reinitiation at residue 55 (M55), explains the normal STAT1 phosphorylation phenotype detected in the PD4 cells. This reinitiation product loses only a small portion of the amino-terminus of the FANCC protein leaving the motif II domain and its surrounding peptide sequences intact (see below).
Effects of FANCC mutations on STAT1 phosphorylation in normal JY cells To test whether these FANCC mutations disrupt normal cell functions, we introduced the mutant cDNAs into normal JY lymphoblasts. Immunoblot analysis of cell lysates indicated that STAT1 phosphorylation in JY cells is unaffected by the expression of the FANCC mutant proteins (Figure 7). The S249A and E251A mutations have no detectable effect on STAT1 phosphorylation in these normal cells and are therefore not negatively dominant for STAT1 activation. Reduced amounts of phosphorylated STAT1 in lanes 8 and 10 result from reduced total STAT1 protein in this particular experiment.
FANCC mutations are defective in STAT1
recruitment to the IFN- -stimulated cells latent
STAT1 is recruited to a docking site on IFN-R, where STAT1 is then activated by tyrosine phosphorylation.48,49
Consistent with this, STAT1 has been found associated with
tyrosine-phosphorylated IFN-R both in vitro and in
vivo.50-52 We have shown that FANCC can facilitate STAT1
activation by recruiting STAT1 to the IFN- receptor.26
The apparent failure of S249A and E251A mutants to correct the STAT1
defect is an indication that in these 2 mutants STAT1 may not be
recruited to the receptor. To confirm this notion we stimulated the
retrovirally transduced cells with IFN-, prepared WCLs in 1%
digitonin lysis buffer, and performed coimmunoprecipitation using
antibody to IFN R. The presence of STAT1 in anti-IFN-R immunoprecipitates was detected by probing with anti-STAT1 antibody. Whereas HSC536N cells transduced with the vector alone (HSC536N/neo) did not contain STAT1 in the anti-IFN-R precipitate (Figure 8, top, lanes 1 and 2), IFN- induced
coimmunoprecipitation of STAT1 with IFN-R in the normal
FANCC-transduced cell line HSC536N/FANCC (lane 4). Whereas
the 4 alanine-substituted mutations in both termini of FANCC showed
normal STAT1 recruitment, cells transduced with the 2 alanine-substituted mutations located in the central region of FANCC,
S249A and E251A, contained markedly less STAT1 in the receptor complex
(lanes 6, 8, 14, and 16 versus 10 and 12). This result correlated with
the observation in which S249A and E251A mutants only partially
corrected the deficiency of STAT1 activation in HSC536N cells (Figure
4). All lanes contained approximately equal amounts of
immunoprecipitated IFN-R protein loaded onto the gels (Figure
8, bottom).
FANCC mutations suppress FANCC-STAT1 interaction The FANCC protein has been shown to associate with STAT1 in WCLs from IFN--stimulated cell lines.26 We therefore
examined the effect of the alanine substitutions on FANCC-STAT1
interaction. We stimulated the normal lymphoblasts (JY) with IFN-
and incubated WCLs with the normal or mutated FANCC proteins bound to
glutathione-Sepharose beads through GST. FANCC-interacting cellular
proteins were then isolated from WCLs by glutathione-Sepharose affinity
precipitation. Western blotting with antibody to P-STAT1 showed that
IFN- induced tyrosine phosphorylation of STAT1 in
IFN--stimulated WCLs (Figure 9, top
panel, lane 2). GST-fusion protein affinity binding assays showed that
IFN- induced the association of STAT1 with the normal FANCC and the
alanine-substituted mutants F64A and Y531A, but significantly less
STAT1 association occurred with the S249A and E251A mutants (lanes 6, 8 and 14, 16 versus 10 and 12). STAT1 showed an even weaker association
with GST-fused L554P, a naturally occurring FANCC mutation
found in FA-C patients46 (lane 16). STAT1 did not bind to
GST alone (lanes 3 and 4). Reprobing the blot with antibody to total
STAT1 indicated that IFN- did not have measurable effect on the
expression of the STAT1 protein (Figure 9, middle panel, lanes 1 and
2), and confirmed that FANCC-STAT1 interaction was suppressed by S249A,
E251A, and L554P mutations (lanes 10, 12, and 16). The bottom panel of
Figure 8 demonstrates that input amounts of GST-fusion proteins were
comparable in the binding reactions.
The results described above implicate a potential defect in
survival signaling in S249A and E251A mutants. We thus sought to
identify the functional significance of these alanine mutations. We
expressed mutant FANCC proteins in HSC536N lymphoblasts, then tested
the transductants for sensitivity to IFN-
M55 mutation restores STAT1 activation but fails to correct MMC hypersensitivity of FA-C cells We next sought to determine whether MMC and STAT functions could be disconnected by naturally occurring FANCC mutants. We investigated a naturally occurring FANCC truncated mutant, M55, which lacks the amino-terminal 54 residues owing to an abnormal initiation at methionine 55.35 Note that the deletion point of M55 is close to motif I but leaves intact the central motif II (Figure 11A), which is required for optimal STAT1 activation (Figures 4, 8, and 9) and antiapoptotic function. We determined the effect of the M55 truncated mutant on STAT1 activation and MMC sensitivity in FA-C lymphocytes. Electromobility shift assay (EMSA) using a GAS (a STAT1 binding site from the IRF1 gene40) oligonucleotide probe, showed that STAT1 DNA-binding activity was restored when M55 was expressed in IFN--stimulated HSC536N cells. Control HSC536N cells were
completely defective in STAT1 DNA-binding (Figure 11B, compare lane 1 with lane 2). Nuclear extracts from PD4 cells also showed GAS-binding
activity (lane 4), consistent with the result described in Figure 6.
The complexes formed with the GAS in extracts from FANCC-corrected
HSC536N and PD4 cells were disrupted by excess unlabeled GAS (Figure
11B, lanes 5 and 6). The identity of STAT1-DNA complexes was confirmed
by a STAT1-specific antibody that altered the migration of the
STAT1-DNA band in the EMSA (lane 7). Furthermore, EMSA using the GAS
sequences derived from the p21 gene promoter41 produced
essentially the same results (Figure 11C) as demonstrated in Figure
11B. FA-C mutant HSC536N cells transduced with the M55 cDNA did not
correct MMC hypersensitivity of FA-C cells as compared to the parental
mutant cells (Figure 11D). The slight increase in survival of
M55-transduced HSC536N cells probably reflects a dose effect because
transduction of HSC536N cells even with the noncomplementing L554P
mutant cDNA also resulted in slight resistance to MMC exposure (Figure
11D). Neither the M55 nor the L554P mutant corrected MMC-induced
cytogenetic abnormalities in HSC536N cells. In addition, expression of
M55 did not reduce survival of the normal JY lymphocytes exposed to MMC
(data not shown). Although Youssoufian and colleagues53 reported that overexpression of the L554P mutant rendered human 293 cells hypersensitive to MMC, we did not observed this phenomenon in
L554P-expressing lymphoblasts (Figure 11D) and HeLa cells (data not
shown). One possible explanation for the differences is the expression
levels of the mutant protein. Youssoufian and coworkers used expression
vector pED6, which resulted in 4- to 15-fold higher levels of
expression than the endogenous FANCC gene. We used
retroviral vector pLXSN that expressed only 1- to 2-fold higher FANCC
mutant proteins relative to the endogenous FANCC proteins, which
apparently were not sufficient to cause a dominant negative effect.
Another possibility is that we used different cell lines.
An increasingly persuasive body of evidence indicates that FANCC
functions in distinct pathways controlling protective responses to
alkylating agents, and survival responses to cytokines and human growth
factors.1,3,5,26,32 The functional heterogeneity of the
Fanconi proteins, known to exist as multimeric complexes with each
other,54,55 resembles the heterogeneity of the ATM protein. The latter plays roles in signal transduction, cell cycle checkpoints, and DNA damage response.56,57 Through the use of alanine-scanning mutagenesis, we have identified herein a central motif of the FANCC protein that appears to be required to mediate (1)
the recruitment of STAT1 to the IFN- We also determined that certain mutations in FA-C patients might result in the disconnection of one function of FANCC from another. Specifically, a naturally occurring mutant of FANCC, M55, was capable of restoring STAT1 activation in FA-C cells, but failed to correct MMC hypersensitivity of FA-C cells (Figure 11). Moreover, lymphoid cells from a child bearing this mutation exhibit normal STAT1 activation responses to IFN. Based on these findings, we argue that FANCC structural determinants for damage/repair responses may differ from those for STAT1 signaling. Although alanine substitutions usually preserve the native
conformation of the wild-type protein,45 such
substitutions may remove the contacts that mediate interactions between
the 2 interacting proteins or between protein and DNA. The loss of
critical amino acid-amino acid contacts due to the alanine
substitutions may explain our observation that the S249A and E251A
substitutions in the central motif suppress the FANCC-STAT1 interaction
(Figures 8 and 9). Although the substitutions themselves are not bulky, the mutations might, nonetheless, influence tertiary structure of
FANCC. Specifically, the highly conserved S249 and E251 residues, which
are polar and charged, respectively, exist within a predicted High degrees of homology between FANCCs from various organisms is scattered in small blocks of several amino acid residues and spans the entire peptide sequence. When we tested the alanine-substituted mutants for responses to the cytotoxic effects of MMC and DEB, we were surprised to find that this function of FANCC is remarkably resistant to alanine substitutions (Figure 2 and Table 1). The ability of the FANCC protein to accommodate these substitutions without an impact on the MMC resistance function of the mutants may explain the recent finding that bovine FANCC is able to complement a FA-C patient-derived lymphoblast cell line HSC536N, even though the sequence between the 2 FANCCs are only 72% identical.42 Similarly, introduction of the normal human FANCC cDNA into a mouse hematopoietic progenitor cell line suppressed apoptosis induced by growth factor deprivation29 and into a mouse FA-C mutant fibroblast cell line rescued normal resistance to MMC (M. C. Heinrich and G.C.B., unpublished data, 1999), despite the fact that the human and mouse FANCCs are even more distant (62% identical). Our results indicate that hypersensitivity to MMC or DEB in FA cells is not coupled to the cytokine signaling functions of FANCC, including the defect of STAT1 activation because the 2 alanine mutants in the central region of FANCC retain the ability to confer normal resistance to the cytotoxic effects of DNA cross-linking agents (Figure 2 and Table 1) but render cells defective in the activation of STAT1 (Figures 4, 8, and 9). Furthermore, we confirmed that FANCC mutations with "disconnected" functions actually occur in nature. The data obtained with a naturally occurring FANCC mutant, presented in Figure 11, demonstrate that the truncated M55 mutant preserves the ability to facilitate STAT1 activation but fails to correct MMC hypersensitivity of FA-C cells. Taken together, our results suggest that separate structural elements of FANCC permit the protein to specifically respond to different extracellular signals. It is likely that FANCC acts coordinately with additional, as yet unrecognized factors, and with other FA proteins, by using separate structural elements, to function in different cellular processes including the response or repair (or both) of specific DNA damage and the control of cell growth by facilitating the cellular response to growth and survival factors. Growth factor signal transduction is defective in FA cells, a
finding that may contribute to the bone marrow failure characteristic of this disease. Several lines of evidence indicate that bone marrow
failure in FA results from increased apoptosis owing to hypersensitivity to inhibitory factors and diminished responses to
survival cues.3,26,29,59 Our results, together with those of others, support a model in which one of the functions of FANCC is to
ensure optimal cellular responses to growth factors, at least in part
by facilitating STAT1 activation. It is known that depletion of growth
factors or cytokines leads to progressive apoptosis in many cell types
including fibroblast60 and hematopoietic cells,29,61 which can be rescued by the addition of
defined factors. These factors include STAT1 activators such as
IFN- Regardless of the precise mechanism, domain II is unambiguously essential for the antiapoptotic function of FANCC. In fact, our results also provide a potential molecular basis for the clinical observation that bone marrow failure seems milder in children with the 322delG mutation in which all 3 conserved motifs, including motif II, remains intact. Finally, the current diagnostic tests for FA would necessarily fail to identify lesions (eg, point mutations in domain II) that disrupt signaling functions of Fanconi proteins without interdicting their capacity to protect cells from cross-linking agents. Although we have not yet identified such mutations in nature, evidence of such lesions should be now be sought in children whose phenotype is suggestive of FA but whose MMC or DEB tests are normal.
We thank Dr Manuel Buchwald for providing the lymphoblast cell line HSC536N from a type C Fanconi anemia patient, Dr A. D. Miller for the retroviral vector pLXSN, Dr David C. Hinkle for the pGST vector, Dr Brian Druker for anti-phosphotyrosine antibody 4G10 and for helpful discussions, and Tara Koretsky for valuable technical assistance. We thank Dr Alan D'Andrea for providing the M55 FANCC cDNA and anti-FANCD2 antibody as well as for helpful discussions.
Submitted December 12, 2000; accepted May 1, 2001.
Supported by National Institutes of Health grant HL48546 and a Department of Veterans Affairs Merit Review Grant to G.C.B.
Q.P. and T.A.C. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Grover C. Bagby, MD, Oregon Cancer Center, Department of Medicine (Division of Hematology and Medical Oncology) and Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, OR 97201; e-mail: grover{at}ohsu.edu.
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© 2001 by The American Society of Hematology.
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  M. D. Milsom, B. Schiedlmeier, J. Bailey, M.-O. Kim, D. Li, M. Jansen, A. M. Ali, M. Kirby, C. Baum, L. J. Fairbairn, et al. Ectopic HOXB4 overcomes the inhibitory effect of tumor necrosis factor-{alpha} on Fanconi anemia hematopoietic stem and progenitor cells Blood, May 21, 2009; 113(21): 5111 - 5120. [Abstract] [Full Text] [PDF]
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K. Bijangi-Vishehsaraei, M. R. Saadatzadeh, A. Werne, K. A. W. McKenzie, R. Kapur, H. Ichijo, and L. S. Haneline Enhanced TNF-{alpha}-induced apoptosis in Fanconi anemia type C-deficient cells is dependent on apoptosis signal-regulating kinase 1 Blood, December 15, 2005; 106(13): 4124 - 4130. [Abstract] [Full Text] [PDF]
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M. R. Saadatzadeh, K. Bijangi-Vishehsaraei, P. Hong, H. Bergmann, and L. S. Haneline Oxidant Hypersensitivity of Fanconi Anemia Type C-deficient Cells Is Dependent on a Redox-regulated Apoptotic Pathway J. Biol. Chem., April 16, 2004; 279(16): 16805 - 16812. [Abstract] [Full Text] [PDF]
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I. Brodeur, I. Goulet, C. S. Tremblay, C. Charbonneau, M.-C. Delisle, C. Godin, C. Huard, E. W. Khandjian, M. Buchwald, G. Levesque, et al. Regulation of the Fanconi Anemia Group C Protein through Proteolytic Modification J. Biol. Chem., February 6, 2004; 279(6): 4713 - 4720. [Abstract] [Full Text] [PDF]
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M. W. Lensch, M. Tischkowitz, T. A. Christianson, C. A. Reifsteck, S. A. Speckhart, P. M. Jakobs, M. E. O'Dwyer, S. B. Olson, M. M. Le Beau, S. V. Hodgson, et al. Acquired FANCA dysfunction and cytogenetic instability in adult acute myelogenous leukemia Blood, July 1, 2003; 102(1): 7 - 16. [Abstract] [Full Text] [PDF]
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T. Taniguchi and A. D. D'Andrea The Fanconi anemia protein, FANCE, promotes the nuclear accumulation of FANCC Blood, September 18, 2002; 100(7): 2457 - 2462. [Abstract] [Full Text] [PDF]
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T. R. Rutherford, N. E. Myatt, F. M. Gibson, A. A. Clarke ;, and G. C. Bagby Jr The Fanconi anemia cell line HSC536N is not sensitive to interferon-gamma and does not cleave PARP in response to Fas-mediated cell killing Blood, April 1, 2002; 99(7): 2627 - 2630. [Full Text] [PDF]
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Top
Abstract
Introduction
, treated; 
, untreated). Treatments of
HSC/VEC and HSC/FANCC with Camptothecin were used as positive controls,
50 mM for 9 hours) and induced positivity of 65.1% and 22.5%,
respectively. Total events of 10 000 for each sample were
analyzed.
