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Assessment of mitomycin C sensitivity in Fanconi anemia complementation group C gene (Fac) knock-out mouse cells
1Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Abstract:
Fanconi anemia (FA) is a genetic disorder defined by cellular hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). MMC causes increased FA cell death, chromosome breakage, and accumulation in the G2 phase of the cell cycle. Recently, Fanconi anemia complementation group C (fac) gene knock-out mice have been developed, and SV40-transformed fibroblasts were established from fac homozygous knock-out (-/-), heterozygous (+/-), and wild-type mice (+/+). MMC sensitivity of these cell lines was assessed by three methods: colony-formation assay in the presence of MMC, chromosome breakage, and cell cycle analysis to detect G2 phase arrest. The fac knock-out fibroblasts (-/-) showed a significantly higher sensitivity to MMC than did fibroblasts from wild-type (+/+) or heterozygous (+/-) mice (three experiments). In addition, we analyzed hematopoietic progenitor colony assays of bone marrow cells from fac knock-out (-/-) and heterozygous (+/-) mice. CFU-E, BFU-E, and CFU-GM colony formation from fac nullizygous mouse progenitors was markedly diminished by MMC when compared to growth of progenitors from heterozygous mice. These results show that fac knock-out mouse cells mimic the behavior of human FA-C patient cells in terms of MMC hypersensitivity. The fac knock-out mouse may be used to model some aspects of human FA and should be useful for understanding the function of the FAC protein.
Insights
Fanconi anemia (FA) mouse models exhibit hypersensitivity to DNA cross-linking agents like mitomycin C (MMC). These findings demonstrate that the fac knock-out mouse accurately models human FA-C patient cells.
Area of Science:
- Genetics
- Molecular Biology
- Hematology
Background:
- Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA cross-linking agents.
- Mitomycin C (MMC) exposure in FA cells leads to increased cell death, chromosomal damage, and G2 cell cycle arrest.
Purpose of the Study:
- To establish and characterize Fanconi anemia complementation group C (fac) gene knock-out mice.
- To assess the MMC sensitivity of fac knock-out mouse fibroblasts and hematopoietic progenitors.
- To validate the fac knock-out mouse as a model for human FA-C.
Main Methods:
- Developed SV40-transformed fibroblasts from fac homozygous knock-out, heterozygous, and wild-type mice.
- Assessed MMC sensitivity using colony-formation assays, chromosome breakage analysis, and cell cycle analysis.
- Performed hematopoietic progenitor colony assays on bone marrow cells from fac knock-out and heterozygous mice.
Main Results:
- Fac knock-out fibroblasts (-/-) exhibited significantly higher MMC sensitivity compared to wild-type (+/+) and heterozygous (+/-) cells.
- MMC markedly diminished colony formation (CFU-E, BFU-E, CFU-GM) in fac nullizygous mouse progenitors compared to heterozygous progenitors.
- The cellular responses of fac knock-out mice mirrored those of human FA-C patient cells.
Conclusions:
- Fac knock-out mouse cells replicate the MMC hypersensitivity observed in human FA-C patient cells.
- The fac knock-out mouse serves as a valuable preclinical model for studying Fanconi anemia.
- This model facilitates a deeper understanding of the functional role of the FAC protein in DNA repair and cellular maintenance.
