Human FANCC is hypomorphic in murine Fancc-deficient cells

Fanconi anemia (FA) is characterized by bone marrow failure (BMF), leukemia, and epithelial malignancies and is caused by inactivation of one of 14 different genes, including FANCC.^1,2  Although FA proteins clearly prevent cross-linking-agent-induced genotoxicity, there is substantial evidence some of these proteins are multifunctional.^3,4  For example, 2 human FANCC (hFANCC) point mutants have been described that fully complement cross-linking agent hypersensitivity (CAH) in hFANCC-deficient cells without altering other functions of hFANCC.³  What is less certain is the relevance of each disparate hFANCC function to hematopoietic stem cell (HSC) homeostasis.

Fancc^−/− mice^5,6  display CAH⁷  and endotoxin (lipopolysaccharide [LPS]) hypersensitivity (EH),⁸  and both cross-linking agents and LPS induce BMF at doses that are tolerated in wild-type mice. HSCs from Fancc^−/− mice display reduced multilineage-repopulating ability in vivo,^9,10  a defect that can be restored by ectopic hFANCC expression.¹¹  Likewise, hFANCC protects against endotoxin-induced BMF.⁸  Although it has been assumed by some in this field that the sole function of FA proteins is genome stabilization and that the FA hematopoietic defect is simply a downstream consequence of genomic instability, no group has tested this notion directly in vivo. We therefore planned to do so by transducing Fancc-deficient HSCs with hFANCC separation-of-function mutants.³  Preparing for this study, we had to assure that hFANCC and murine Fancc (mFancc) were equivalent in a murine context, so we compared the relative efficacies of hFANCC and mFancc to complement CAH in Fancc-deficient cells. Surprisingly, we found that hFANCC, unlike mFancc, does not fully correct CAH, but that both hFANCC and mFancc correct EH.

Primary (GFAC2 and 5) and SV40-transformed (MEF 11.1 and 61) embryonic fibroblasts (MEFs) were derived from wild-type and Fancc-deficient mice, respectively.¹²  SV40-transformed-tongue-epithelial cells (6640SV) were isolated from Fancc-deficient mice by trypsinization. Progenitors and primary (NFF-6, 11Lu) and transformed (NFF6-U195, PD331T) fibroblasts were isolated and grown as previously described.^11,13  Mitomycin C (MMC) and breakage assays were performed as previously described.¹⁴  Institutional Animal Care and Use Committee approval was given for isolating primary tissue after death and deriving cell lines.

hFANCC cDNA was isolated previously.¹⁵  Cloning of mFancc, virion production,^16,17  and transduction methods are described in supplemental data (available on the Blood Web site; see the Supplemental Materials link at the top of the online article).

Whole-cell lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis¹⁴  and incubated with rabbit-polyclonal antibodies: anti-hFANCC (1:1000; Oregon Health & Science University FA Antibody Project) or anti-mFancd2 (1:200; kindly provided by Dr Alan D'Andrea, Harvard). Densitometry was measured using Gel Documentation System (Bio-Rad) and analyzed with Quantity One software.

Cells were plated at 25 000 cells/well in 24-well dishes and treated plus or minus LPS (Sigma-Aldrich). After 24 hours, interleukin-6 (IL-6) was measured from supernatants with Mouse IL-6 Quantikine ELISA Kit (R&D Systems).

We ectopically expressed hFANCC and mFancc cDNA in Fancc-deficient MEFs (MEF61) and compared their capacity to complement CAH. We found that mFancc fully complemented the CAH to wild-type levels, but hFANCC had a marginal effect on MMC hypersensitivity (Figure 1A). Specifically MEF61 and hFANCC-transduced MEF61 (MEF61/hFANCC) cells showed similar EC₅₀ (half-maximal effective concentration) values (≅ 20 and 30nM, respectively), but mFancc-transduced MEF61 cells (MEF61/mFancc) were fully complemented (EC₅₀ ≅ 100nM) to wild-type levels (MEF11.1; EC₅₀ ≅ 70nM). Chromosomal breakage (Figure 1B) mirrored the results discussed earlier in this paragraph. Specifically, MEF61/hFANCC cells displayed 2- to 3-fold more breaks than MEF61/mFancc or MEF11.1 and higher levels of quadriradial forms than MEF61/mFancc cells (Figure 1C and Figure 1D, respectively). Interestingly, the reverse was not true. We confirm, as reported previously,¹⁸  that mFancc fully complements CAH in hFANCC-deficient cells (supplemental Figure 1).

Although our study does show marginal complementation by hFANCC, our results do not support the conclusions of Gush et al¹⁹  who reported that hFANCC is fully proficient in protecting Fancc-deficient cells from MMC-induced damage. We attribute the differences to the facts that in their study only one MMC dose was measured in vitro, low doses of MMC were used for the in vivo study, no chromosomal breakage was reported, and a direct mFancc comparison was not done.

Failure of hFANCC to correct CAH was not cell type–specific. We measured CAH of Fancc-deficient tongue epithelial cells (6640SV; Figure 2A) and hematopoietic progenitors (HPC-M; Figure 2B) transduced with hFANCC or mFancc. 6640SV/hFANCC cells (EC₅₀ ≅ 30nM) were significantly more sensitive to MMC than 6640SV/mFancc (EC₅₀ ≅ 80nM). Likewise, HPC-M/hFANCC cells (EC₅₀ ≅ 10nM) were more sensitive than HPC-M/mFancc (EC₅₀ ≅ 30nM) and wild-type (HPC-W; EC₅₀ ≅ 60nM).

Our results did not stem from low hFANCC expression in Fancc-deficient cells. We quantified both hFANCC mRNA and hFANCC protein levels. Although transduced hFANCC mRNA was approximately 3-fold lower than mFancc levels in MEF61 and 6640SV cells, hFANCC transcripts were approximately 67-fold higher than levels of endogenous mFancc in wild-type cells (supplemental Figure 2 and data not shown). Full-length hFANCC protein was also expressed at high levels in MEF61/hFANCC cells (Figure 2C top, lane 3) and by densitometry was more than 8-fold higher than endogenous hFANCC levels in normal human fibroblasts (lanes 5-7). Thus, hFANCC protein is expressed in murine cells at levels that would be more than sufficient to confer a normal MMC response.

Mono-ubiquitination of Fancd2 (mediated by Fancc) is required for cross-linking agent resistance.⁴  As expected, mFancc-transduced cells showed high levels of both nonubiquitinated and mono-ubiquitinated Fancd2 (Figure 2C bottom; Fancd2-S and -L, respectively; lanes 3 and 4). However, Fancd2-L was undetectable in hFANCC-transduced epithelial cells (lanes 5 and 6). Similar results were found using MEFs (supplemental Figure 3). Therefore, the lack of CAH complementation by hFANCC in mFancc-deficient cells is associated with its inability to facilitate mFancd2 ubiquitination. Although hFANCC was incapable of correcting the MMC phenotype, we did find that EH was completely corrected. Because endotoxin-exposed Fancc-deficient mice are known to produce extraordinarily high IL-6 levels,⁸  we confirmed that Fancc-deficient MEFs secreted more IL-6 after LPS stimulation than did wild-type cells (Figure 2D) and next showed that MEFs transduced with either hFANCC or mFancc were fully complemented.

hFANCC and mFancc share 79% amino acid similarity and 66% identity (supplemental Figure 4), which is similar to other FA core-complex proteins but substantially less than other DNA-repair proteins.^18,20  Because hFANCC retains the ability to completely suppress EH in murine cells, our results suggest that requisite domains for this function remain intact in hFANCC, but domains required for optimal DNA repair in the context of a murine cell do not.

In light of the fact that that levels of hFANCC/mFancc RNA and hFANCC/mFancc protein in transduced MEFs and epithelial cells are so much higher than endogenous levels and that transduction efficiencies in hematopoietic cells are similar (74% and 79%, respectively), we conclude that hFANCC is hypomorphic in murine cells because the DNA damage defect persists and therefore argue that EH does not devolve simply from genomic instability. That hFANCC ameliorates the repopulating defect of Fancc-deficient HSCs to levels approximating wild-type cells,^11,17  despite significantly reduced DNA-repair capacity (Figure 2B), indicates that noncanonical functions of FANCC/Fancc may be critical for supporting optimal hematopoiesis. To formally distinguish relative contributions of CAH and EH to BMF in mice, identification of separation-of-function mutants will be required. In effect, hFANCC cDNA meets this standard in murine cells and provides added opportunities to identify unique functional domains. Although such experiments will be informative, we also define 2 caveats for future research with murine FA models. First, it will be essential to confirm that any human FA cDNA is equivalent to murine cDNA in comprehensive functional assays. Second, to dissect Fancc's structure-function relationships, separation-of-function mutations must be developed using mFancc because hFANCC is hypomorphic. It will also be of interest to create murine homologs of known human FA mutations because, in light of our results, some of them might not inactivate mFancc function.

The authors thank Sunchin Kim, who provided technical assistance.

This work was supported by Fanconi Anemia Research Fund (L.E.H.), the Knight Cancer Institute at Oregon Health & Science University, Veterans Association Merit Review (G.C.B.), National Institutes of Health/National Heart, Lung, and Blood Institute (5P01-HL48546; G.C.B., S.B.O.), and National Institutes of Health/National Cancer Institute (1-R01-CA138237-01; D.W.C., G.C.B.).

National Institutes of Health

Contribution: L.E.H., G.C.B., and D.W.C. designed the study; L.E.H. and G.C.B. wrote the manuscript; L.E.H., W.W.K., J.E.Y., R.K.R., T.K., and Z.S. performed key experiments; S.B.O. conducted breakage and helped analyze data; and D.W.C. conducted hematopoietic progenitor assays and helped analyze data.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Laura E. Hays, 3710 SW US Veterans Hospital Rd, Portland Veterans Administration Medical Center, R&D-2, Portland, OR 97239; e-mail: haysl@ohsu.edu.