FANCD2-deficient human fibroblasts are hypersensitive to ionising radiation at oxygen concentrations of 0% and 3% but not under normoxic conditions.
OBJECTIVE
Individuals suffering from Fanconi Anemia (FA) exhibit a pronounced hypersensitivity to agents that cause DNA inter-strand crosslinks and frequently also to ionising radiation. However, fibroblast lines derived from FA patients generally show little or no radiosensitivity in vitro. Here, we sought to elucidate the role of the central FA protein D2 (FANCD2) in determining cellular radioresistance.


METHODS
Clonogenic radiation survival was assessed in an isogenic pair of human fibroblasts with or without wild-type FANCD2 under varying oxygen concentrations. Additional endpoints included single-cell gel electrophoresis, RAD51 foci formation, and apoptosis.


RESULTS
At 20% oxygen, there was no reduction in the survival of FANCD2-deficient fibroblasts compared to wild-type complemented cells. However, at 0% oxygen FANCD2-deficient cells were more radiosensitive than wild-type cells. Interestingly, at 3% oxygen, which more closely resembles the physiological environment in human tissues, the difference in radiosensitivity was maintained. Our data also suggest that the increased radiosensitivity of FANCD2-deficient cells seen under conditions of reduced oxygen is associated with apoptotic cell death, but not secondary to a defect in the homologous recombination repair pathway that is required for crosslink repair.


CONCLUSIONS
Our data may help explain the previously described discrepancy between the clinical and cellular radiosensitivity of FA patients.
Kuhnert
Verena M
VM
Department of Radiation Oncology, University of Lübeck, Lübeck, Germany.
Kachnic
Lisa A
LA
Li
Li
L
Purschke
Martin
M
Gheorghiu
Liliana
L
Lee
Richard
R
Held
Kathryn D
KD
Willers
Henning
H
D000818:Animals; D017209:Apoptosis / Q000528:radiation effects; D002460:Cell Line; D052236:Fanconi Anemia Complementation Group D2 Protein / Q000172:deficiency* / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism / Q000528:radiation effects*; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology*; D051135:Rad51 Recombinase / Q000235:genetics; D011836:Radiation Tolerance / Q000235:genetics; D011839:Radiation, Ionizing; D011995:Recombination, Genetic / Q000528:radiation effects; D015203:Reproducibility of Results
D052236:Fanconi Anemia Complementation Group D2 Protein; D051135:Rad51 Recombinase; D010100:Oxygen