Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair.
Bottom Line: Individuals with Fanconi anemia (FA) are susceptible to bone marrow failure, congenital abnormalities, cancer predisposition and exhibit defective DNA crosslink repair.This cooperativity of FancC and Mus81 in developmental outcome was also mirrored in response to crosslink damage and chromosomal integrity.Thus, our findings reveal that both pathways safeguard against DNA damage from exceeding a critical threshold that triggers proliferation arrest and apoptosis, leading to compromised in utero development.
Affiliation: Department of Pharmacology and Toxicology, University of Toronto, Toronto, M5S 1A8, Canada.Show MeSH
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Mentions: Primary cultures of fibroblasts from FhetMhet, FhetMko, FkoMhet and FkoMko sibling embryos were propagated in order to establish if the observed defects in FkoMko mice reflected a cell-intrinsic mechanism. Compared to fibroblasts from sibling controls, a pronounced proliferation defect was apparent in FkoMko primary fibroblasts at each time of assessment (P between 0.05 and < 0.001 dependent upon passage; Figure 5A). Reversal of the growth defect was observed in FkoMko fibroblasts following immortalization with SV40 (Figure 5B), suggesting that the observed loss of proliferation is at least partly due to p53-mediated checkpoint activation. Compared to FhetMhet, FhetMko and FkoMhet cells, FkoMko primary fibroblasts also showed a tendency to accumulate in the G2/M phases of the cell cycle, with increased arrest occurring in a passage-dependent manner (P < 0.001 for P4 G2/M, P < 0.05 for P4 S-phase; Figure 5C). Furthermore, FkoMko primary fibroblasts showed a greater tendency to undergo passage-dependent apoptosis in culture (P < 0.01; Figure 5D). As FkoMko embryos exhibited an increased incidence of γH2AX-positive cells, we examined whether elevated levels of spontaneous DNA damage were mirrored at the cellular level through quantification of micronuclei. We determined that primary FkoMko fibroblasts exhibited an increased incidence of micronuclei compared to all controls (P < 0.05; Figure 5E, left panel). Strikingly, this susceptibility to accumulated DNA damage was also observed in the erythrocytes of FkoMko adult mice (aged 6–8 months) enumerated for micronuclei (Howell-Jolly bodies), suggesting that the observed susceptibility to DNA damage was not specific to embryonic cells (P < 0.001 versus FhetMhet,P = 0.005 versus FhetMko,P = 0.030 versus FkoMhet; Figure 5E, right panel).
Affiliation: Department of Pharmacology and Toxicology, University of Toronto, Toronto, M5S 1A8, Canada.