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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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The FANCC Gene and Its Products

* and .

* Corresponding Author: Program in Genetics and Genomic Biology, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. Email: sgordon@sickkids.ca

Fanconi anaemia (FA) is an autosomal recessive disorder characterized by progressive pancytopaenia and predisposition to malignancy, often accompanied by congenital malformations. The cellular phenotype of FA includes increased chromosomal instability and accumulation in the G2 phase of the cell cycle, both of which are exacerbated by the hallmark sensitivity of FA cells to DNA crosslinking agents such as mitomycin C (MMC) and diepoxybutane (DEB). FA is genetically heterogeneous, consisting of at least eleven complementation groups, the genes for eight of which have been cloned. FANCC was the first gene causal for FA to be identified, and consequently has been the most intensively studied. Loss of function studies have demonstrated an important role for FANCC in the proliferation of germ cells and haematopoietic stem cells (HPCs). Together with the protein products of at least five other FA genes, FANCC participates in the formation of a nuclear protein complex, the formation of which is required for monoubiquitination of the FANCD2 protein. This cooperative action of the FA proteins fits well with the indistinguishable clinical presentation and universal cellular crosslinker sensitivity of the complementation groups. However, despite its ability to participate in a nuclear protein complex and possible involvement in nuclear activities such as DNA repair and transcriptional regulation, FANCC is unique among the FA proteins in having a predominantly cytoplasmic cellular localization. Investigation of possible cytoplasmic roles for FANCC have revealed it to be a multifunctional protein involved in the suppression of cell death in response to a wide range of stimuli including DNA-crosslinking agents, factor withdrawal, dsRNA, stimulatory cytokines and Fas ligation, as well as a having a possibly interrelated role in maintaining of the redox state of the cell.

Cloning and Characteristics of the FANCC Gene

The FANCC gene was cloned by using a transfected episomal cDNA expression library to functionally complement the crosslinker sensitivity of the FA-C lymphoblastoid cell line (LCL) HSC536.1 Three complementing plasmids with inserts sized 4.6, 3.2 and 2.3 kb were obtained, all containing the same predicted open reading frame (ORF) of 1678bp with alternatively processed untranslated regions (UTRs). Assuming use of the first in frame ATG, this ORF encodes a 558 amino acid polypeptide with a Mr of 63kD. The predicted protein sequence has no homology to other proteins, although it is evolutionarily conserved in vertebrates, with homology searches finding significant matches in animals as distant as ray-finned fish.2 Two different 5' UTRs that converge 77bp upstream of the initiation codon represent alternatively spliced exons designated exon -1 and exon -1a. Three different 3' UTRs corresponding to identical sequences truncated at different points generated the 2.3, 3.2 and 4.6 kb transcripts all of which were present on a Northern blot of lymphocytes with the largest being the most abundant.1

The human FANCC gene was localized to chromosome 9q22.3 by in situ hybridization3 and Southern blot analysis suggests that the FANCC gene is at least 40kb.4 The FANCC coding sequence contains 14 exons ranging in size from 53bp to 204bp.5 Skipping of exon 13 appears to be a relatively common alternative splicing event, seen in a proportion of transcripts in both patients and normal controls, although it has not been established whether this in-frame deletion results in a functional protein.6

Further characterization of the 5' end of the human FANCC gene revealed that exon -1 is located 5' to exon -1a, separated by a small intron. There are consensus splice donor sites downstream of both exons, whereas only exon -1 and not -1a has an upstream acceptor splice site, suggesting exon -1 is not spliced onto -1a.7 The large majority of transcripts in lymphocytes contain exon -11 and only transcript containing exon -1 and not exon -1a was detected in CD34+ bone marrow cells, even after exposure to growth factors.8 The sequences upstream of exons -1 and -1a have no TATA or CAAT boxes, but, as often seen in widely expressed genes, CpG islands are present around the putative transcription start sites. Putative cis-acting binding sites are found between exons -1 and -1a and also upstream of exon -1. The genomic region extending from 1500bp 5' of the putative transcriptional start site of exon -1 through the 3' end of exon -1a is able to drive expression of a luciferase reporter in CaCo2 cells. Further deletion analysis revealed strong positive regulatory ability upstream of exon -1 but also suggested the presence of a negative regulatory element in the intron between exons -1 and -1a or in exon -1a itself.7

The human FANCC gene contains two p53 binding sites, one in the promoter region (-1295 to -1266) and a second in exon 14.9 Although p53 has been shown to bind the consensus in the promoter in vitro, luciferase reporter assays showed deletion of this site made no difference in FANCC promoter activity in cell lines overexpressing wild type p53. Wild-type p53 overexpression in human fibroblast and lymphoblast cell lines is nevertheless able to induce transcription of FANCC, although p53 mutants incapable of DNA binding retain part of this activity, suggesting activation of transcription is not entirely due to p53 binding elements in the FANCC gene.9

Mammalian Homologs of FANCC

Three overlapping clones representing murine FANCC were isolated from a mouse liver cDNA library.10 While two clones contained a putative open reading frame of 558 amino acids, the third contained an additional 99bp inserted at nucleotide 1849, resulting in an in frame insertion of 33 residues that extends the predicted ORF length to 591 amino acids. The position of the insertion corresponds to a splice junction in the human gene, suggesting it is an alternatively spliced exon.10 The predicted amino acid sequence is 66% identical to human, with 79% similarity allowing for conservative amino acid substitutions. Although this is low in comparison to other DNA repair genes, exogenous expression of either the 558 or 591 residue coding regions in a human FA-C LCL corrects the sensitivity to DEB and MMC.10 The murine FANCC gene has been assigned to chromosome 13 by interspecific backcross analysis.11

Cloning of rat and bovine FANCC homologs12 has provided further insight into the evolutionary conservation of FANCC. The predicted open reading frames of the rat and bovine cDNA are 1674 and 1701bp respectively, with the extra length of the bovine ORF located primarily at the 3' end. An alignment of ORFs from all four mammalian species revealed a 65% nucleotide identity overall. The mouse and bovine 5' UTRs obtained most resemble human exon -1 as opposed to exon -1a. The 3' UTR is not conserved between species, although all contain multiple polyadenylation signals generating transcripts of different lengths.12

Alignment of the predicted polypeptides from all four species revealed only 53% amino acid identity or 67% similarity allowing for conservative substitutions (fig. 1), although all have a similar predicted secondary structure and preponderance of hydrophobic amino acids. Apart from short stretches of 6 to 9 conserved amino acids the degree of similarity is uniform across the sequence.10 Four putative CK-2 and two putative PKC phosphorylation sites are conserved across species,12 although an attempt to detect FANCC phosphorylation in multiple cells lines did not generate any evidence that it is a phosphoprotein.13 The p53 binding consensus in exon 14 of the human sequence is not conserved in the bovine sequence. The bovine FANCC cDNA is, however, able to correct the MMC sensitivity of the HSC536 FA-C cell line.12

Figure 1. Alignment of the predicted protein products of the human, mouse, rat and bovine FANCC genes.

Figure 1

Alignment of the predicted protein products of the human, mouse, rat and bovine FANCC genes. Residues identical in all species are indicated by dark shading, with lighter shading indicating similarity if conservative amino acid substitutions are permitted. (more...)

FANCC Gene Mutations

Presence of mutations in the FANCC coding region has confirmed its loss of function as the genetic defect in FA-C patients. The HSC536 cell line used in the gene cloning experiments has a maternally inherited L554P missense mutation predicted to disrupt alpha helical secondary structure.1 FANCC transcripts derived from the paternal allele contain a 327bp deletion resulting in removal of exons 1 and 2.14 Screening of unclassified FA cell lines and patient samples has identified further mutations in FANCC for a total of at least ten, with some clustering in exons 1 and 14 (fig. 2).

Figure 2. The spectrum of FANCC gene mutations observed in patients.

Figure 2

The spectrum of FANCC gene mutations observed in patients. Clustering of mutations in exons 1 and 14 is observed. The IVS4 + 4 A>T mutation results in the in-frame skipping of exon 4 or a smaller 40bp deletion likely resulting from the use of (more...)

Exon 1 mutations include nonsense mutations Q13X15 and W22X,16 as well as the deletion of a single G at base 322 (322delG) predicted to generate a truncated peptide of 44 amino acids.1 The IVS4 + 4 A>T mutation in intron 417 generates transcripts with either an in-frame deletion of the entire exon 4 or a smaller 40bp deletion likely resulting from use of a cryptic slice site and causing a frame shift. A nucleotide substitution in exon 5 near the intron 5 boundary is expected to generate a stop codon (R174X), but appears to instead result in exon 6 skipping that maintains the reading frame, with arginine changed to tryptophan.18 A base pair change resulting in a nonsense mutation in exon 6 (R185X) also results in exon 6 being spliced out of a proportion of transcripts, suggesting this mutation may alter splice site selection.6 Mutations in the C-terminal part of FANCC include L496R in exon 1316 and the exon 14 mutations R548X,15 L554P 1 and insertion of adenine at position 1806 (1806insA) expected to generate a frameshift and truncated protein of 526 residues.19

Failure to complement MMC sensitivity of the HSC536 FA-C cell line by exogenous expression confirmed the pathogenicity of 1806insA, R548X,20 L496R21 and L554P.22 Remarkably, two siblings homozygous for the L496R (1749T>G) mutation became somatic mosaics when an additional 1748C>T change occurred in vivo at one allele creating a substitution of cysteine instead of an arginine. Complementation analysis demonstrated restoration of FANCC functional activity by this secondary substitution.21 Overexpression of the L554P mutant several fold in 293 cells has also been shown to induce a level of MMC hypersensitivity similar to that seen in FA cells.23

Linkage analysis with three microsatellite markers flanking the FANCC gene showed linkage to this locus for 8% of FA families analysed.24 Large scale screening of FA patients to identify FANCC mutations assigned 5.3%16 to 14.4% 25 of patients to group C, depending on the population used. Seven of the first 100 patients assigned to complementation groups by the European Fanconi Anaemia Research Programme were FA-C.2

The two most frequent mutations in FANCC are IVS4 + 4 A>T in intron 4 and 322delG in exon1. The 322delG mutation is associated with a mild phenotype, including both fewer somatic abnormalities and later age of aplastic anemia onset compared to all FA patients.26 Cell lines from patients with 322delG do not express full length FANCC, but do express a 50-kD amino-truncated FANCC polypeptide resulting from reinitiation of translation at methionine 55 (M55). Overexpression of the M55 FANCC cDNA in an FA-C cell line lacking M55 partially corrected the MMC sensitivity phenotype, suggesting the presence of M55 could potentially mediate the FA phenotype in patients with exon 1 mutations upstream of the reinitiation site.27

The IVS4 + 4 A T mutation is found in 80% of patients of Ashkenazi Jewish descent,17 with a carrier frequency greater than 1% in this ethnic group indicating a possible founder effect.25 In these patients the mutation is associated with a severe phenotype including an earlier age of diagnosis25 and higher prevalence of several abnormalities including CNS defects. 26 More recently, Japanese patients homozygous for IVS4 + 4 A>T have also been identified.28 In contrast to Ashkenazi patients with this mutation, no significant difference in severity of clinical phenotype, including age of onset of haematological disease, number of major congenital malformations or development of myelodysplastic syndrome and AML, was seen between IVS4 + 4 A>T homozygotes and other patients. Analysis of surrounding markers did not reveal a founder haplotype in the Japanese patients.28

As may be expected given the ability of FANCC mammalian homologs with relatively low sequence identity to complement the MMC sensitivity of FA-C cells,10 the human FANCC gene is quite polymorphic. Non-silent alterations causing changes to the coding sequence include G139E,17 S26F, D195V,25 L190F, I312V, Q465R, V449M,16 V60I, E417L29 and P211R30 (fig. 1).

FANCC variants have also been identified in sporadic pancreatic cancers.31 One clearly inactivating homozygous variant, a somatic 5bp deletion of nt 1903-7 in exon 14 causing a frameshift, has been identified. Another tumour was homozygous for D195V, a variant previously shown to complement the MMC sensitivity of an FA-C LCL. This suggests that this mutation is not causal for FA, but the possibility that it interferes with other aspects FANCC function cannot be ruled out. The functional significance of a heterozygous E521K variant and homozygous M350V variant is unclear, as neither has been previously reported as either a mutation or polymorphism.31 In another study, a pair of siblings with T-ALL and MDS, one of whom later developed AML, were found to be heterozygous for a 377-378delGA frameshift mutation in exon 4.32

Expression of the FANCC Gene Products

mRNA Expression

Human FANCC1 and murine FANCC10 transcripts are detected by RT-PCR in a wide variety of tissues, indicative of ubiquitous expression. RNA in situ hybridization in adult mouse tissues reveals a uniform pattern of expression in all cells,10 whereas during mouse embryogenesis there is an overall pattern of strong expression in proliferating and differentiating cells that diminishes as cells terminally differentiate.33 Expression is first detected in the mesenchyme that gives rise to skeletal tissues at embryonic day 8 (E8). FANCC is also expressed in non-skeletal mesenchyme including the gut, lung, kidney and site of whisker follicles. Mesenchymal expression generally becomes less abundant as embryogenesis progresses. During the development of bone by endochondral ossification (E13 to E19.5) FANCC expression is detected in the actively dividing and differentiating cells of the inner perichondrium, the osteoprogenitors of the inner periosteal layer and in the zone of endochondral ossification, which contains cells derived from both osteogenic and hematopoietic lineages. No FANCC expression was detected in relatively mature and differentiated cells such as chondrocytes or osteocytes. FANCC expression in the perichondrium of developing digits of the forelimb and in cranial and facial bones formed by intramembranous ossification, is consistent with skeletal abnormalities seen in FA.33

In analysis of single-cell murine haematopoietic precursors, FANCC is expressed strongly, although at low frequency, in pluripotential precursors and weakly in samples from growing, terminally differentiating populations of monocytes and lymphocytes.34 RT-PCR to assess mRNA levels in sorted murine haematopoietic stem cell fractions also revealed high FANCC expression in lineage-depleted CD34+ cells as opposed to low expression in CD34- cells, suggesting a role at this stage of differentiation.35 Human FANCC mRNA expression in haematopoietic cells is also highest in CD34+ cells from bone marrow, although levels do not change as committed progenitors differentiate in vitro upon exposure to growth factors.8

FANCC Protein Expression and Stability

As seen with mRNA expression, exposure to varying levels of TGF-β, IFN-γ, MMC, heat, gamma-irradiation or hydrogen peroxide does not alter FANCC protein levels in LCLs.8 This is perhaps not surprising given that sensitivity to ICL-inducers is corrected completely by low levels of FANCC protein expression, with greater levels of expression not increasing resistance.36FANCC protein levels also do not change in response to hypoxia mimicking agents in a hypoxia responsive cell line.37 The half-life of wild type FANCC protein in transiently transfected COS cells is approximately 45 minutes, a relatively rapid turnover during which no change in cellular localization and protein interactions are observed with or without MMC treatment.23

In synchronized cells, whereas levels of exogenous and endogenous FANCC mRNA expression38,39 are constant throughout the cell cycle, expression of exogenous FANCC protein is lowest at G1/S, increases during S-phase, is maximal at G2/M, and declines during mitosis.38,39 Regulated expression of exogenously expressed FANCC requires only the coding sequence and not the 5' or 3' UTR. Inhibitors of the 26S proteasome prevent the reduction in expression at G1/S, indicating this regulation of FANCC expression is dependent on proteasome function.38

The discovery of ubiquitinated FANCC also suggests a role for the proteasome in regulation of FANCC. Treatment of normal LCLs with proteasome inhibitors results in cytoplasmic accumulation of an ubiquitinated C-terminal proteolytic fragment of FANCC (p47) that correlates temporally with apoptotic induction.40 Caspase inhibitors prevent accumulation of p47 as does mutagenesis of putative caspase-8 cleavage sites. Mutation of cleavage sites does not affect the ability of FANCC to restore FANCD2 monoubiquitination and correct MMC sensitivity, and actually enhances the ability of FANCC to delay the onset of apoptosis in response to etoposide. In contrast, exogenous expression of p47 increases sensitivity of LCLs to apoptosis, suggesting that cleavage eliminates the ability of FANCC to suppress apoptosis or that p47 itself is proapoptotic.40

Intracellular level of FANCC may also be regulated via its interaction with GRP94, a stress-inducible molecular chaperone.41 Reduction of GRP94 expression in the rat NRL cell line significantly reduces FANCC protein levels, and induces development of MMC sensitivity in this relatively resistant cell line. Part of the central domain of FANCC that binds GRP94 is spliced out of the IVS-4 + 4 A>T mutant, eliminating the interaction.41

FANCC Subcellular Localization

FANCC contains no recognizable nuclear localization sequences and early studies revealed predominantly cytoplasmic localization of the protein in several cell lines,42-44 with some reports of a smaller amount associated with internal membranes.42,44 Additional refinement in more recent studies has demonstrated that approximately 10% of endogenous FANCC in LCLs is reproducibly found in the nuclear fractions.45 Immunofluorescent analysis of exogenously expressed FANCC is highly variable, ranging from predominantly cytoplasmic to predominantly nuclear.38,45,46 However, FANCC is consistently excluded from nucleoli.47 Exclusively cytoplasmic localization of the FANCC-L554P mutant45,46 suggests nuclear localization is not an artifact of overexpression.

In an asynchronous population, FANCC nuclear foci were observed in approximately 25% of cells.38,47 The exogenously expressed FANCC also colocalizes with endogenous FANCE in nuclear foci.48 Exogenous expression of FANCE, which contains a putative nuclear localization signal49 and binds directly to FANCC,50,51 results in dramatic nuclear accumulation of wild type FANCC but not FANCC containing disease-associated mutations that prevent interaction with FANCE. It is not clear whether this promotion of FANCC nuclear accumulation involves nuclear import or retention.48 Exclusion of endogenous FANCC from the cytoplasm13 and overall lower level of FANCC expression in FA-E cell lines is also corrected by reintroduction of FANCE.52

FANCC is involved with other FA proteins in a protein complex, the formation of which is required for the monoubiquitination of FANCD2 and correction of MMC sensitivity.53 Most studies have focused primarily on a nuclear form of the complex containing FANCA, FANCC, FANCE, FANCF, FANCG and FANCL.13,48,54-57 Recent work has demonstrated that this FA protein complex has at least four forms, all of which contain at least FANCA, FANCC, FANCF and FANCG. There are two cytoplasmic forms sized 500-600kD and 750kD, the larger of which is only present during mitosis, as well as 1MD and 2MD nuclear forms, the smaller of which is chromatin-associated.58 FANCE is notably absent from the cytoplasmic complexes.58 The presence of FANCC in cytoplasmic FA protein complexes is not surprising, as FANCC force-targeted to the nucleus is not able to correct crosslinker-induced cytotoxicity, demonstrating that the role of FANCC in mediating this response requires cytoplasmic localization at some point.36

Other investigations of FANCC subnuclear localization have revealed its presence in the chromatin and nuclear matrix together with at least FANCA and FANCG. The amounts present in this compartment increase in response to MMC with a time course that suggests cells must first encounter crosslinks during S-phase.59 Synchronized cells show little change in FA protein content in chromatin and nuclear matrix fractions during G1, S or G2 phase but FANCC and the other FA proteins are not detected at mitosis, implying they are absent from condensed chromosomes.59 This finding, together with discovery of a distinct mitotic FA complex in the cytoplasm,58 is consistent with an earlier study of exogenously expressed FANCC in HeLa cells that suggested cells with strong cytoplasmic localization were undergoing or had just completed mitosis,46 although another study found cell cycle position could not account for FANCC subcellular localization.38 FANCC from HeLa cell chromatin-associated protein extracts, together with nonerythroid alpha spectrin (αSPIIΣ*), FANCA and FANCG, complexes with a DNA substrate containing interstrand crosslinks generated by psoralen + UVA. It is not clear whether FANCC and the other FA proteins are able to bind directly to the DNA substrate or whether alpha spectrin acts as a scaffold to mediate the interaction.60

FANCC has also been shown to directly bind to and partially colocalize in nuclear foci with at least one protein of demonstrable nuclear function, the transcriptional repressor FAZF.47 FAZF is homologous to and can heterodimerize with the promyelocytic leukemia zinc finger (PLZF), known to play a role in limb patterning via chromatin remodelling.61 The fact that FAZF protein in CD34+ haematopoietic progenitor cells is highly expressed during early differentiation, declining as myeloid and erythroid cells terminally differentiate, is also intriguing given the phenotype of haematopoietic failure caused by the absence of its interacting partner FANCC.62

FANCC in the Cellular Response to ICL Inducers

Cell Cycle Abnormalities

Treatment of FA-C LCLs with low dose MMC causes protracted G2/M arrest mediated by persistent inactivation of the cyclin B1/cdc2 kinase complex preceding or accompanying the induction of apoptosis. In corrected cells, G2/M arrest and inactivation of the cyclin B/cdc2 complex is transient, after which cells resume cycling.63 Despite a report that FANCC associates with cdc2 in HeLa cell lysates,39 in uncorrected FA-C cells caffeine-dependent activation of cdc2 releases the G2/M block but does not prevent apoptosis. Thus while the FANCC protein may influence the cdc2 kinase complex to allow G2 to M progression after low-dose MMC, the apoptotic response to low-dose MMC is not directly caused by inhibition of cdc2 kinase activation. At high doses of MMC both FA-C and wild type cells undergo S-phase arrest and high levels of apoptosis.63

When exposed to equitoxic amounts of MMC, a similar degree and rate of G2/M arrest is observed in FA-C LCLs compared with corrected isogenic or normal LCLs. This implies that that G2/M arrest in FA-C cells is not an abnormal response at this point in the cell cycle, but a normal response to excessive DNA damage following low-dose crosslinkers.64 However, while normal and corrected FA-C cells had increased percent of cells in S phase with a discrete peak in late S, uncorrected FA-C cells had a decrease in the proportion of cells in S phase.64

Further evidence of an S-phase defect is observed using bivariate DNA distribution methods to analyze the response of LCLs to psoralen plus UVA.65 Two populations of S-phase cells can be distinguished, replicating and arrested. Normal and corrected FA-C cells are arrested 24 hours after treatment but in FA-C cells replication is continued in S-phase at 24 and even 48 hours. This suggests G2 accumulation is a normal response to excessive damage that reaches that compartment after failure to arrest replication in S-phase.65 Furthermore, in FA-C as in normal cells, passage through S-phase is required for cell cycle arrest and chromosomal abnormalities in response to crosslinking agents.66

Apoptosis

Comparison of the cellular proliferation and viability of FA-C and isogenic LCLs expressing FANCC cDNA upon exposure to a wide spectrum of DNA-damaging agents demonstrated that increased cytotoxicity of FA-C cells is limited to cross linking agents such as MMC, cisplatin and nitrogen mustard, whereas other agents including potent free radical producers and non-crosslinking analogs of nitrogen mustard only capable of forming monoadducts did not elicit a differential response.67,68

In response to low dose MMC treatment, FA-C LCLs show increased induction of p53 expression accompanied by higher levels of apoptosis compared with isogenic corrected cell lines,67,69 whereas at higher MMC concentrations similar levels of p53 induction and apoptosis are observed. Despite the observed p53 induction, overexpression of a dominant negative p53 mutant did not increase cell survival, suggesting that p53 is not crucial for MMC-dependent apoptosis in immortalized LCLs,69 although this appears to differ from the situation in vivo, as discussed later.70

It is important to note that not all studies of the response of FA-C LCLs to MMC have agreed that apoptosis is the primary cause of MMC-induced cell death. One study found that at equimolar high doses of MMC, FA-C and normal cells undergo apoptosis at a similar rate, but at equitoxic doses of MMC the level of apoptosis in FA-C LCLs was similar to or slightly less than in normal controls.71 Further studies using isogenic cell lines failed to detect some of the classical features of apoptosis such as apoptotic body formation, DNA fragmentation and PARP cleavage72,73 in LCLs following MMC treatment, suggesting toxicity may be mediated by a necrosis-like pathway of programmed cell death.

FANCC Loss of Function

Use of an antisense oligodeoxynucleotide to repress FANCC gene expression increased MMC-induced chromosomal breakage in normal lymphocytes and inhibited clonal growth of haematopoietic progenitor cells (HPCs) in a dose dependent manner. No growth-inhibitory effect was observed in fibroblasts or endothelial cells. As the FANCC repression did not affect growth factor expression in bone marrow cells, the function of FANCC in regulating proliferation of HPCs is likely direct.74

Further loss of function studies have been facilitated by the generation of two Fancc-deficient mouse models by use of gene targeting to replace either exon 875 or exon 976 of Fancc with a neo cassette. Homozygous null (Fancc-/-) mice are viable, with no malformations and no haematological failure or development of malignancy in the first year. Primary cultures of splenic cells and skin fibroblasts from -/- mice show an increase in spontaneous and DEB- and MMC- induced chromosomal aberrations.75,76 Immortalized fibroblastoid cell lines developed from Fancc-/- mice also show significant hypersensitivity to MMC relative to Fancc+/- and Fancc+/+ as shown by reduced cell viability, induction of chromosomal abnormalities, and G2 accumulation.77,78

Fancc Loss of Function and Germ Cell Development

Fancc-/- mice, particularly females, have reduced fertility.75,76 The small ovaries of females have deficient germinal stroma with very few developing follicles. Male mice have testicular atrophy and a mosaic pattern of seminiferous tubules with normal spermatogenesis and abnormal tubules with only Sertoli cells. Both male and female abnormalities are seen in newborns, suggesting germ cell loss is a developmental defect.76

Fancc expression is detected by RT-PCR in murine genital ridges throughout gonadal development in both sexes.79 Comparison of both male and female germ cell numbers during embryonic development revealed a dramatic reduction at all stages examined in the Fancc-/- mice relative to Fancc+/-. No defect in the migration of germ cells was observed, though a 35% reduction in mitotic index of Fancc-/- PGCs at E12.5 compared to FANCC+/- reveals a proliferative defect. An additional germ cell survival defect is suggested by the continued decrease in female Fancc-/- germ cell numbers after proliferation has ceased.79

Fancc Loss of Function and Haematopoiesis

Cultured bone marrow cells from Fancc-/- mice have similar erythroid and myeloid colony growth at 2 and 4 months, but a significant reduction at 6 and 11 months compared with heterozygotes.76 Hypersensitive reduction in progenitor growth in vitro in response to IFN-γ76 TNFα or MIP-1α (macrophage inflammatory protein-1α) 80 was not age dependent, suggesting cytokine sensitivity could be causal in the progressive depletion of growth potential. Fancc is required for prevention of apoptosis in response to cytokines in immature myeloid haematopoeitic cells. Despite elevated apoptotic response, no difference in absolute number of splenic or bone marrow progenitors per organ was observed. Increased suicide in multipotential and lineage specific progenitors implies that a higher proportion of progenitors are cycling in vivo to compensate for the increased apoptosis.80

Fancc loss of function is also associated with loss of haematopoietic stem cell repopulating ability in vivo, 81 with long term reconstitution and secondary repopulating ability particularly affected.82 This is suggestive of a defect in stem cell development potential, an idea also supported by lower differentiation potential of Fancc-/- HPCs measured in a single cell culture system.83 A possible link to the enhanced Fas receptor (CD95) expression in CD34+ Fancc-/- HPCs in response to IFN-γ84 has been suggested.82 Despite lower overall engraftment, contribution of the Fancc-/- cells to lymphoid and myeloid lineages is similar, also indicative of a defect in the pluripotential stem cells.81 Retroviral-mediated gene transfer of Fancc to Fancc-/- HPCs enhances repopulating ability.85

Haematopoietic progenitor colony assay of bone marrow cells treated with MMC in vitro revealed a marked reduction of erythroid and myeloid colony growth.78 The effect of MMC in vivo was studied by administration of MMC to Fancc-/- mice.86 Chronic exposure to nonlethal doses of MMC results in a progressive pancytopaenia resulting from a reduction in the number of early and committed progenitor cells not seen in Fancc+/+.86 MMC treatment of bone marrow cells results in a dramatic reduction in number of CD34+ cells but only a slight decrease in CD34- cells, although it is not clear whether the CD34+ population is more susceptible to cell death or whether there is a defect in differentiation from CD34- to CD34+ after MMC treatment.82 MMC sensitivity of bone marrow cells from Fancc-/- mice transduced with retrovirus carrying human FANCC cDNA and transplanted to marrow ablated recipients is phenotypically corrected in vivo as assessed by peripheral blood counts following MMC dosing.87

As p53 behaviour may be altered by the EBV-immortalization of LCLs, p53 requirement for apoptosis in FA-C cells has been tested in vivo by generation of mice deficient for both genes.70 The TNFα sensitivity and apoptosis seen in Fancc-/- haematopoietic progenitor cells and MEFs is completely eliminated in double Trp53-/- Fancc-/- knockouts, suggesting it is p53 dependent. Low-dose MMC-induced reduction of progenitors is also p53 dependent, although rescue was not maintained at high doses, suggesting a role for a p53 independent pathway. Absence of the Fancc gene also significantly reduced the latency of tumour formation in both p53 null and heterozygote mice. In addition, some tumour types not observed in Trp53+/- or Trp53-/- mice were seen in the double mutants, including malignancies observed in FA patients.70

FANCC and Cytokine Signalling

Retroviral mediated overexpression of FANCC in factor-dependent human and murine haematopoeitic progenitor cell (HPC) lines suppresses the onset of apoptosis following IL-3 withdrawal, leading to the suggestion that FANCC may suppress apoptosis in response to normal fluctuations of cytokine levels in the bone marrow microenvironment.88

Similarly, HPCs from transgenic mice overexpressing human FANCC were protected in vitro against Fas-mediated cell death induced by either a combination of IFN-γ and TNFα, known to upregulate Fas receptor (CD95) expression, or by a Fas-agonist antibody.89 Conversely, treatment of Fancc-/- murine HPCs with TNFα and Fas-agonist antibody inhibits colony formation in vitro compared to Fancc+/-, with BFU-E reduced by TNFα alone.84 Fas receptor (CD95) expression in the CD34+ fraction of untreated Fancc-/- bone marrow is similar to wild type, but its induction in response to TNFα is elevated, with a corresponding increase in apoptotic induction. The effect of TNFα in vivo is similar, with Fancc-/- mice transgenic for overexpression of TNFα having increased CD95 in CD34+ cells and spontaneous reduction in BFU-E.84

HPCs from an FA-C patient and Fancc-/- mice are hypersensitive to the mitotic inhibitory effects of IFN-γ, and IFN-γ-induced priming of the Fas pathway occurs at lower doses of IFN-γ. Colony growth of CD34+ bone marrow cells from an FA-C patient and Fancc-/- mice is suppressed by a Fas-agonist antibody whereas Fas blocking antibody abrogates the inhibitory effect of low dose IFN-γ on Fancc-/- CD34+ cells.90 No increased expression of IFN receptor-α or members of the TNF receptor superfamily is observed in patient-derived lymphoblasts or bone marrow cells of Fancc-/- mice, either constitutively or in response to IFN-γ treatment, suggesting FANCC suppresses apoptosis via an intracellular mechanism.91

The in vitro growth suppressive effects IFN-γ and Fas-agonist antibodies in both murine and human FA-C bone marrow cells are blocked by caspase-3 inhibitors. In FA-C lymphoblasts, inhibitors of caspase-8 are able to suppress caspase-3 activation, suggesting that FANCC functions upstream of caspase-3 in suppression of IFN-γ-induced apoptotic pathway.92 However there is disagreement as to whether the level of Fas-mediated cell death in FA-C LCLs is actually elevated 71,73 and the high constitutive level of Fas expression in these cells has been proposed to interfere with the response to Fas-priming.93

STAT1 activation in response to IFN-γ is suppressed in the FA-C cell line HSC536, and STAT1 induction is corrected by reintroduction of FANCC.90 In response to IFN-γ FANCC is required for trafficking of STAT1 to the IFN-γ receptor complex at which point it becomes activated. As STAT1 activation requires FANCC, STAT1 signaling is suppressed in FA-C cells and therefore the excessive apoptosis is STAT1-independent.94 Despite lack of proper STAT1 activation, protein levels of IFN-γ-inducible genes known to influence haematopoietic cell survival including IFN regulatory factor-1 (IRF-1), IFN-γ stimulated gene factor 3 gamma subunit (ISGF3γ), and the cyclin-dependent kinase inhibitor p21waf1 are all constitutively expressed at high levels in FA-C LCLs, bone marrow cells and MEFs. Forced expression of the negative transacting regulator ICSCP (IFN-γ-inducible factor IFN consensus sequence binding protein) in FA-C LCLs does not downregulate the expression of IFN-γ-inducible genes, so FANCC modulates these genes independently of both the STAT1 pathway and ICSBP. 95

Not all FANCC mutations affect STAT1 activation. The PD4 FA-C cell line, which is heterozygous for the 322delG mutation and therefore contains a mutant form of FANCC protein initiated at methionine 55 (M55), has normal STAT1 activation. Exogenous expression of M55 restores the STAT-1 signaling pathway (STAT-1 DNA binding) in the completely defective HSC536 cell line. This suggests that structural elements of the FANCC protein required for cytokine signalling are, at least in part, different than those required for mediating resistance to DNA cross-linking agents, and may also contribute to the milder phenotype seen in patients with the 322delG mutation.96 Conversely, alanine mutagenesis within motifs highly conserved in known mammalian FANCC sequences identified mutants able to correct crosslinker sensitivity and restore FANCD2 monoubiquitination but unable to return STAT1 phosphorylation to normal levels in the deficient HSC536 cell line. Lack of STAT1 phosphorylation by these S249A and E251A mutants correlated with reduced FANCC STAT1 interaction, markedly less STAT1 trafficked to the IFN-γ receptor, and increased apoptosis induced by IFN-γ and TNF-γ.96

The RNA-dependent protein kinase (PKR), an IFN-γ. and TNFα-inducible protein that influences Fas activity and activates caspases-3 and -8, is constitutively phosphorylated and displays increased dsRNA binding activity in FA-C LCLs and embryonic fibroblasts from Fancc-/- mice (MEFs) upon exposure to IFN-γ or dsRNA. Overexpression of wild-type PKR sensitized Fancc-/- MEFs to caspase-3 activation whereas inhibition of PKR activity by a dominant-negative PKR mutant reduced IFN-γ and dsRNA hypersensitivity to a greater extent in Fancc-/- than Fancc+/+ MEFs.97 Modulation of PKR activation in FANCC-/- cells is mediated by interaction of Fancc with Hsp70, a protein previously shown to suppress PKR activity and caspase-3 activation.98 Hsp70, FANCC and PKR have been shown to form a ternary complex in LCLs.99 Mutants of Fancc blocking interaction with Hsp70 fail to inhibit PKR kinase activity and do not give protection from IFN-γ /TNFα induced cytotoxicity. Repression of HSP70 expression in normal lymphoblasts causes greater susceptibility to IFN-γ and TNFα, but does not further sensitize FA-C mutant cells suggesting they are already fully compromised. 98 Interestingly, the ability of Fancc to inhibit the kinase activity of PKR is independent of the ability of Fancc to interact with other FA proteins.99

Surprisingly, given this involvement of FANCC in cytokine signaling pathways, in vivo administration of IFN-γ to Fancc-/- mice with and without subsequent Fas-ligation, did not induce bone marrow failure as shown by lack of effect on blood counts, progenitor colony formation and marrow cellularity.100

FANCC in DNA Damage and Repair

The much lower doses of MMC required to generate an equivalent amount of DNA interstrand crosslinks (ICLs) in FA-C cells compared to corrected isogenic counterparts and non-FA cells suggests a pre-repair defect. The efficiency of ICL repair in corrected FA-C, normal and mock transfected FA-C LCLs differentially treated to generate similar levels of ICL damage is similar, suggesting repair is not affected by the presence of FANCC.36

Other studies have implicated FANCC in the repair of specific types of DNA damage. A plasmid-based DNA end-joining assay in FA-C LCLs revealed that error-free processing of blunt-ended DSB is greatly reduced, resulting in both increased frequency and size of deletions. Complementation with FANCC cDNA restores fidelity of this blunt-ended DSB processing.101 An FA-C LCL has also been shown to repair the oxidative DNA damage in plasmid DNA extracellularly treated with potassium permanganate less efficiently than did non-FA cells, and this defect is also complemented by exogenous expression of FANCC.102

FA-C LCLs, as well as cells from other FA subtypes, are unable to activate the assembly of RMN complex proteins (RAD50/MRE11/NBS1) into subnuclear foci in response to ICL inducers. DSB formation and unhooking of ICLs in response to MMC is normal, indicating that absence of RMN assembly is not simply due to absence of DNA ends produced as intermediates of crosslinking. MMC treatment induces phosphorylation of NBS1 in corrected cells, but not in FA-C cells. Notably, phosphorylated NBS1 participates in activation of DNA-damage dependent S-phase arrest and FA cells are deficient in ICL-dependent S-phase checkpoint activation.65 In contrast, formation of RMN foci and phosphorylation of NBS-1 in response to ionizing radiation was normal in FA cells.103

On exposure to MMC, Rad51 foci formation in normal and corrected FA-C LCLs increases; however, in uncorrected FA-C cells this response is reduced and delayed, despite normal levels of Rad51 protein. BRCA1 foci formation is also delayed in response to MMC treatment in FA-C cells. Rad51 foci formation in response to gamma-irradiation was normal in FA-C LCLs,103 although another study using primary fibroblasts found significantly reduced Rad51 foci formation in response to ionizing radiation.104

FANCC and Oxidative Stress

At ambient 20% oxygen, when MMC is expected to generate free radicals through redox cycling, FA-C LCLs increase induction of apoptosis in response to low-dose MMC. In contrast, at hypoxic 5% oxygen, when the metabolic activation of MMC for crosslinking is facilitated, levels of apoptosis observed are not different from normal cells.105

FANCC binds to NADPH: cytochrome c (P450) reductase (RED), a microsomal membrane protein involved in electron transfer, including the bioreductive activation of MMC. The interaction, which involves the cytosolic membrane proximal domain of the reductase that contains a flavin mononucleotide (FMN) binding site, is disrupted by addition of FMN to lysates. The catalytic activity of RED is attenuated by overexpression of FANCC, but cannot be suppressed completely. Thus a role for FANCC in fine-tuning of the redox state of cell has been proposed.106

Association of FANCC with glutathione S-transferase P1-1 (GSTP1) increases GSTP1 activity in murine haematopoietic cells particularly after induction of apoptosis by factor withdrawal. FANCC functions by preventing the formation of inactivating disulphide bonds within GST during apoptosis.107 FANCC overexpression attenuates glutathione (GSH) depletion after apoptotic induction, and if GSH is artificially depleted the antiapoptotic effect of GSTP1 appears dependent on total amount FANCC available to keep it in reduced state. The eight cysteine residues conserved in FANCC across species 12 have been proposed as possible participants in thiol-disulphide exchange.107

In vivo alteration of redox state in Fancc-/- mice was suggested by the result a cross to mice with targeted mutagenesis of the Cu/Zn superoxide dismutase gene (Sod1). Although not displaying developmental defects or increased chromosomal aberrations typical of FA, Fancc-/- Sod1-/- mice had a phenotype of bone marrow hypocellularity with bicytopaenia. Absolute numbers of primitive haematopoeitic progenitors was similar to normal but a decrease in committed progenitor populations was demonstrated by severely reduced in vitro clonogenic potential of committed myeloid and lymphoid progenitors compared to Fancc-/- and Sod-/- null mutants.108

A link between reactive oxygen species (ROS) induction and Fas-priming by IFN has been suggested by increased ROS accumulation and decreased intracellular glutathione levels in FA-C LCLs primed with IFN-γ and treated with Fas-agonist antibody compared to isogenic corrected cells. The antioxidant dehydroascorbic acid (DHA) reduced the Fas-priming effect of IFN-γ confirming ROS as a mediator of the exaggerated IFN-γ response.109 IFN-γ induced ROS accumulation in FA-C LCLs is accompanied by activation of the stress-activated protein kinases JNK and p38 and prevention the stress kinase activation by DHA implies it is due to ROS. Partial inhibition of caspase-3 activation by inhibitors of p38 but not JNK suggests IFN-γ and Fas ligation can mediate signals for apoptosis in FA-C cells via p38 but that other pathways must also be involved. Hydrogen peroxide exposure also results in ROS accumulation with p38 and JNK activation in FA-C cell lines, but not in corrected cells or in uncorrected FA-C cells pretreated with DHA.110

Nitric oxide (NO) is also a factor in the sensitivity of Fancc-/- bone marrow progenitor cells to cytokines. In vitro cytokine-mediated growth inhibition of Fancc-/- bone marrow progenitor cells is prevented by inhibition of NO synthase activity, whereas growth inhibition by two different NO-generating agents is greater in Fancc-/- haematopoeitic cells than in wild type cells. Furthermore, stimulation of Fancc-/- bone marrow-derived macrophages with IFN-γ and bacterial lipopolysaccharide results in increased inducible nitric oxide synthase (iNOS) levels and increased nitrite release.111

Fancc-/- HPCs and MEFs are highly sensitive to oxidant stimuli as shown by clonogenic progenitor assays of cells exposed to hydrogen peroxide (H2O2). Oxidant-mediated apoptosis is increased in Fancc-/- MEFs and treatment of these MEFs with anti-oxidants before culture with H2O2 reduces apoptosis and returns survival to wild type levels.112 The serine-threonine kinase apoptosis signal-regulating kinase 1 (ASK1), a MAPKKK able to activate the JNK and p38 signalling cascades113 active in FA-C LCLs treated with H2O2 or IFN-γ and Fas ligation, 110 is hyperactivated in H2O2-treated Fancc-/- MEFs. Inhibition of ASK1 activity by a dominant negative ASK1 mutant, or reduction of ASK1 expression by siRNA alleviated H2O2 induced apoptosis, suggesting it is ASK1 dependent. Transduction of Fancc-E251A has the same protective effect on viability of Fancc-/- MEFs as wild-type Fancc, whereas Fancc-322delG gave no protection against H2O2. This implies the role of Fancc in HSP70/PKR signaling is not required for the protective effect, another example of the multiplicity of Fancc function.112

Conclusions

The understanding of FANCC protein function has improved considerably since the initial discovery that loss of function of a previously unknown protein with no obvious functional motifs was causal in the FA-C defect. The generation of isogenic LCLs with and without FANCC expression has allowed controlled examination of many aspects of the FA cellular phenotype. Discoveries such as the defect in arrest of S-phase replication 65 and the altered behaviour of the RMN complex proteins in response to ICL-inducers,103 have provided important clues to FANCC function in mediating cellular response to DNA-damaging agents. The generation of Fancc-/- mice has also been invaluable, allowing the use of primary non-immortalized cellular materials that are rarely, if ever, available from patients, and making it possible to perform in vivo studies of haematopoiesis.

Although the number of possible cellular roles for FANCC seems ever-increasing, new discoveries often link back to previous findings and recurrent themes have emerged. The recent demonstration of the cooperativity of p53 and FANCC in suppression of tumorigenesis70 is interesting given that p53 can be activated by PKR,114 which is in turn inhibited by FANCC interaction with HSP70.99 The ASK1 apoptotic program, observed in Fancc-/- MEFs treated with H2O2,112 is known to be initiated when redox sensitive proteins that bind to and inactivate ASK1 become oxidated and dissociate. Among these redox sensitive proteins are the GSTs,115,116 at least one class of which, GSTP1, is maintained in a reduced state by FANCC.107

Even as discoveries seem to connect together, some diversity in FANCC function is suggested by the multiple subcellular localizations of the protein, as well as overlap in the residues of FANCC critical for mediating different protein-protein interactions (fig. 3). Multiplicity of function is also demonstrated by the differential ability of certain FANCC mutants to correct FA-C cellular phenotypes such as MMC sensitivity, failure of FA protein complex formation, lack of STAT1 activation, hyperactivation of PKR, and elevated ASK1-mediated apoptosis. This finding is of particular interest in the assessment of genotype-phenotype correlations. For example, the M55 mutant found in FA-C cells with a 322delG mutation allows for normal STAT1 activation, which may help to account for the relatively mild phenotype seen in these patients.96 Of equal interest is that the converse may also be true. Although FANCC mutants that prevent HSP70 interaction but correct MMC sensitivity have so far been artificially generated in alanine mutagenesis studies, the possibility that such mutations exist in nature, but have not yet been discovered due to failure to produce a classical FA phenotype, is very real. Testing of FANCC variants identified in sporadic cancers31,117 may be of particular interest in this regard.

Figure 3. Amino acid residues in FANCC identified as critical for direct and indirect protein interactions.

Figure 3

Amino acid residues in FANCC identified as critical for direct and indirect protein interactions. FANCC residues implicated in binding are indicated in brackets beside the name of the interacting protein. In the case of other interacting proteins, either (more...)

Studies of FANCC expression, subcellular localization, protein interactions, and gain and loss of function have demonstrated roles for FANCC in a wide range of cellular activities, some of which may be interconnected. One major role for FANCC is cooperation with other FA proteins in a protein complex in both the nuclear and cytoplasmic compartments. Other largely cytoplasmic roles may be unique to FANCC, including protection against the cytotoxicity of IFN-γ and TNFα via association with HSP70 and PKR, and maintenance of the redox state of the cell via interactions with RED and GSTP1. Defects in the differentiation and proliferation of HPCs and germ cells seen with Fancc loss of function may be mediated by both nuclear and cytoplasmic activities of the FANCC protein.

Acknowledgments

Work in our laboratory has been supported by the Canadian Institutes of Health Research (CIHR) and the Hospital for Sick Children Foundation. MB holds the Lombard Insurance Chair in Pediatric Research, Hospital for Sick Children and University of Toronto.

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