Reduced FANCD2 influences spontaneous SCE and RAD51 foci formation in uveal melanoma and Fanconi anaemia

Abstract

Uveal melanoma (UM) is unique among cancers in displaying reduced endogenous levels of sister chromatid exchange (SCE). Here we demonstrate that FANCD2 expression is reduced in UM and that ectopic expression of FANCD2 increased SCE. Similarly, FANCD2-deficient fibroblasts (PD20) derived from Fanconi anaemia patients displayed reduced spontaneous SCE formation relative to their FANCD2-complemented counterparts, suggesting that this observation is not specific to UM. In addition, spontaneous RAD51 foci were reduced in UM and PD20 cells compared with FANCD2-proficient cells. This is consistent with a model where spontaneous SCEs are the end product of endogenous recombination events and implicates FANCD2 in the promotion of recombination-mediated repair of endogenous DNA damage and in SCE formation during normal DNA replication. In both UM and PD20 cells, low SCE was reversed by inhibiting DNA-PKcs (DNA-dependent protein kinase, catalytic subunit). Finally, we demonstrate that both PD20 and UM are sensitive to acetaldehyde, supporting a role for FANCD2 in repair of lesions induced by such endogenous metabolites. Together, these data suggest FANCD2 may promote spontaneous SCE by influencing which double-strand break repair pathway predominates during normal S-phase progression.

Figure 1

FANCD2 expression is reduced in UM. (a) Western blot for FANCD2 in the cutaneous melanoma cell line WM793, two UM-derived cell lines SOM157d and SOM196b, the colorectal cancer cell line HCT116 and the transformed fibroblast cell line MRC5VA. (b) Western blot for FANCD2 in primary cells extracted from different UM patients (labelled Mel and then the sample number) and control cells WM793 and MRC5VA. (c) Quantification of endogenous FANCD2 foci formation in WM793, SOM157d, SOM196b and MRC5VA cells. Average and s.d. of at least three repeats is shown. Significance was determined using Student's t-test; **P<0.01. Representative images of immunofluorescent staining for FANCD2 (green) and 4,6-diamidino-2-phenylindole (DAPI; blue) are shown.

Figure 2

Promoter methylation and E2F-1 binding is altered in UM. (a) Relative expression of FANCD2 RNA compared with the level in WM793 cells and measured by real-time PCR. Average and s.d. of three repeats is shown. Significance was calculated using Student's t-test; ***P<0.001. (b) FANCD2 promoter region showing C residues −698 and −688 bp upstream of the FANCD2 start site (red) and site of putative E2F binding (underlined). These residues were unmethylated in UM and methylated in control cells. (c) Amount of real-time PCR product spanning the putative E2F binding site immunoprecipitated with E2F-1 antibody relative to input. Average and s.d. of two repeats is shown. Significance was determined using Student's t-test; **P<0.01. (d) Proposed model of dysregulation of expression of FANCD2 in UM; (i) in wild-type cells, the E2F-1 binding site in the promoter is methylated, preventing E2F-1 binding and allowing expression of FANCD2. (ii) In UM, methylation is absent, E2F-1 binds and expression is repressed.

Figure 3

Complementing UM with FANCD2 overcomes low SCE. (a) Western blot for FANCD2 in the cutaneous melanoma cell line WM793, parental UM-derived cell line SOM196b, SOM196b retrovirally transfected with FANCD2 (196b-D2), SOM196b retrovirally transfected with empty vector (196b-pMMP), the colorectal cancer cell line HCT116 and the transformed fibroblast cell line MRC5VA. (b) Representative images of immunofluorescent staining for FANCD2 (green) and 4,6-diamidino-2-phenylindole (DAPI; blue). (c) Quantification of endogenous FANCD2 foci formation in WM793, SOM196b, 196b-D2 and 196b-pMMP. (d) SCE formation in the parental UM-derived cell line SOM196b, SOM196b retrovirally transfected with FANCD2 (196b-D2), SOM196b retrovirally transfected with empty vector (196b-pMMP). Average and s.d. of three repeats is shown. Significance was calculated using Student's t-test; NS, not significant; *P<0.05, **P<0.01 and ***P<0.001.

Figure 4

Spontaneous RAD51 foci formation is reduced in UM. (a) Quantification of RAD51 foci in the parental UM-derived cell line SOM196b, SOM196b retrovirally transfected with FANCD2 (196b-D2) and SOM196b retrovirally transfected with empty vector (196b-pMMP). (b) Representative images from (a). (c) Quantification of RAD51 foci in the cutaneous melanoma cell line WM793, two UM-derived cell lines SOM157d and SOM196b, the transformed fibroblast cell line MRC5VA, the osteocarcinoma cell line U2OS, primary cells from a conjunctive melanoma (conj Mel 568) and primary cells from two UMs (SOM569 and SOM571). Average and s.d. of three repeats is shown. Significance was calculated using Student's t-test; NS, not significant; *P<0.05 and **P<0.01. SCE is expressed as the number of SCE observed/diploid genome. Where polyploidy existed, numbers were adjusted accordingly.

Figure 5

Endogenous RAD51 foci and SCE levels are decreased in FANCD2-deficient PD20 and FANCD2-depleted MRC5VA cells. (a) Quantification of RAD51 and (b) SCE formation in the FA-derived cell line PD20 and wild-type FANCD2 (PD20-D2) and FANCD2 monoubiquitination mutant (PD20-K561R) complemented PD20 cells. (c) Western blot for FANCD2 in MRC5VA cells treated for 48 h with scrambled or FANCD2 siRNA. (d) Quantification of RAD51 foci and (e) SCE formation in FANCD2-depleted MRC5VA cells. Average and s.d. of three repeats is shown. Significance was calculated using Student's t-test; **P<0.01 and ***P<0.001. SCE is expressed as the number of SCE observed/diploid genome. Where polyploidy existed, numbers were adjusted accordingly.

Figure 6

UM- and FANCD2-deficient FA patient cells are sensitive to acetaldehyde. (a) Fraction of cells surviving compared with equivalent untreated control 4 days after exposure to 10 mM acetaldehyde and (b) quantification of RAD51 foci formation, in the parental UM-derived cell line SOM196b, SOM196b retrovirally transfected with FANCD2 (196b-D2), the FA-derived cell line PD20 and wild-type FANCD2 (PD20-D2) and FANCD2 monoubiquitination mutant (PD20-K561R) complemented PD20 cells. *P<0.05, **P<0.01 and ***P<0.001.