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. 2009 Dec 15;69(24):9431-8.
doi: 10.1158/0008-5472.CAN-09-2452.

Embryonic lethality after combined inactivation of Fancd2 and Mlh1 in mice

Henri J van de Vrugt  1 , Laura Eaton, Amy Hanlon Newell, Mushen Al-Dhalimy, R Michael Liskay, Susan B Olson, Markus Grompe
Affiliations

Embryonic lethality after combined inactivation of Fancd2 and Mlh1 in mice

Henri J van de Vrugt et al. Cancer Res. .

Abstract

DNA repair defects are frequently encountered in human cancers. These defects are utilized by traditional therapeutics but also offer novel cancer treatment strategies based on synthetic lethality. To determine the consequences of combined Fanconi anemia (FA) and mismatch repair pathway inactivation, defects in Fancd2 and Mlh1 were combined in one mouse model. Fancd2/Mlh1 double-mutant embryos displayed growth retardation resulting in embryonic lethality and significant underrepresentation among progeny. Additional inactivation of Trp53 failed to improve the survival of Fancd2/Mlh1-deficient embryos. Mouse fibroblasts were obtained and challenged with cross-linking agents. Fancd2-deficient cells displayed the FA-characteristic growth inhibition after mitomycin C (MMC) exposure. In primary fibroblasts, the absence of Mlh1 did not greatly affect the MMC sensitivity of Fancd2-deficient and Fancd2-proficient cells. However, in Trp53 mutant immortalized fibroblasts, Mlh1 deficiency reduced the growth-inhibiting effect of MMC in Fancd2 mutant and complemented cells. Similar data were obtained using psoralen/UVA, signifying that MLH1 influences the cellular sensitivity to DNA interstrand cross-links. Next, the effect of MLH1 deficiency on the formation of chromosomal aberrations in response to cross-linking agents was determined. Surprisingly, Mlh1 mutant fibroblasts displayed a modest but noticeable decrease in induced chromosomal breakage and interchange frequencies, suggesting that MLH1 promotes interstrand cross-link repair catastrophe. In conclusion, the combined inactivation of Fancd2 and Mlh1 did not result in synthetic lethality at the cellular level. Although the absence of Fancd2 sensitized Mlh1/Trp53 mutant fibroblasts to MMC, the differential survival of primary and immortalized fibroblasts advocates against systemic inactivation of FANCD2 to enhance treatment of MLH1-deficient tumors.

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Figures

Figure 1
Figure 1. Embryonic lethality in Fancd2/Mlh1 double mutant mice
Image of embryos around 12 dpc depicting a representative litter from Fancd2/Mlh1 double heterozygous breeding pairs. The severely underdeveloped Fancd2/Mlh1 double mutant is undergoing absorption. In this litter the Fancd2 mutant Mlh1 heterozygous embryo also shows developmental defects. Respective genotypes are indicated; HET: heterozygous, MUT: mutant.
Figure 2
Figure 2. Clonal survival of primary fribroblasts in addition of MMC
Fancd2 mutant fibroblasts display a characteristic FA-like hypersensitivity to MMC. Additional inactivation of Mlh1 does not alter the MMC sensitivity of Fancd2 mutant primary fibroblasts. Fancd2/Mlh1 double heterozygous (-×-), double mutant (-□-), Fancd2 heterozygous Mlh1 mutant (-•-), and Fancd2 mutant Mlh1 heterozygous (-Δ-), error bars indicate standard error.
Figure 3
Figure 3. Clonal survival of immortalized fibroblasts with Fancd2 and Mlh1 defects in addition of 6-TG, MMC, psoralen or angelicin plus UVA
A) MLH1 complementation restores 6-thioguanine sensitivity in immortalized double mutant Fancd2/Mlh1 cells. Concurrent expression of FANCD2 made cells even more sensitive to 6-TG at concentrations of 0.5 and 2.5 μM (p≤ 0.005). B) Fancd2/Mlh1 double mutant fibroblasts show a remarkable resistance to MMC displaying clonal survival frequencies close to FANCD2 and MLH1complemented cells. While FANCD2 complementation mediates MMC resistance, expression of MLH1 greatly enhances MMC sensitivity of Fancd2-deficient immortalized fibroblasts, p<0.05 at 5 and 10 nM MMC, p=0.01 at 20 and 50 nM MMC, when compared to clonal survival of double mutant fibroblasts. C) Clonal survival after exposure to psoralen plus UVA irradiation. FANCD2 complemented and Fancd2/Mlh1 double mutant cells are resistant to psoralen/UVA ICL damage. In contrast, Fancd2 mutant fibroblasts expressing functional MLH1 displayed significantly reduced clonal growth after psoralen UVA exposure at concentration of 0.2 and 0.5 ng/ml in comparison to double mutant cells (p<0.05). D) In parallel with psoralen/UVA, cells were exposed to angelicin/UVA and subsequent clonal survival was determined. No clear differences were observed among the clonal survival of the isogenic cell lines. FANCD2 / MLH1 complemented (-×-), double mutant mock complemented (-□-), FANCD2 complemented, Mlh1-deficient (-•-), and Fancd2-deficient MLH1 complemented (-Δ-), error bars indicate standard error.
Figure 4
Figure 4. MlH1 increases chromosomal aberrations in immortal fibroblasts after exposure to MMC or DEB
A, B) Frequencies of chromosomal interchanges after 48 hr of continuous exposure to 0, 5 or 15 ng/ml MMC or 0, 50 or 100 ng/ml DEB. 1 At 100 ng/ml DEB excessive chromosomal damage was observed in one of the averaged experiments and the outcome was set to 100% chromosomal aberrations which is an underrepresentation of the actual damage level. +: complemented with FANCD2 or MLH1, -: mock complemented, error bars indicate standard error.
Figure 5
Figure 5. Mlh1 increases chromosomal damage in primary fibroblasts after exposure to MMC or DEB
A, B) Frequencies of chromosomal interchanges after 48 hr of continuous exposure to 0, 5 or 15 ng/ml MMC or 0, 50 or 100 ng/ml DEB. 1 At 100 ng/ml DEB excessive chromosomal damage was observed in one of the averaged experiments and the outcome was set to 100% aberrations for breaks and interchanges which is an underrepresentation of the actual damage level. H: heterozygous for Fancd2 or Mlh1, M: mutant for Fancd2 or Mlh1, error bars indicate standard error.
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