Radiation response in vitro of fibroblasts from a fanconi anemia patient with marked clinical radiosensitivity.
BACKGROUND
. Fanconi anemia (FA) is an autosomal recessive chromosome instability disorder characterized by progressive pancytopenia and cancer susceptibility. The risks of radiation therapy in FA patients who have cancer remain to be investigated. Recently, Marcou et al. (2001) reported a case of severe clinical radiosensitivity in a female FA patient with a tonsillar squamous cell carcinoma treated by radiotherapy. By contrast, her in vitro irradiated skin fibroblasts revealed nearly normal radiosensitivity as determined by the colony survival assay.


METHODS
. In view of this discrepancy, the radiation response of this particular FA fibroblast strain (designated 425BR) was further analyzed in the present study by means of the alkaline single-cell gel electrophoresis (Comet) assay, and also by the cytochalasin-blocked micronuclei (MN) test. In addition, the expression levels of DNA repair proteins, hMre11, Rad50, and Rad51, were investigated using Western blot and foci immunofluorescence staining.


RESULTS
. The Comet assay revealed that the initial DNA fragmentation in irradiated FA cells was two times higher and the DNA rejoining process was three times slower than that in control (1BR3) fibroblasts. Moreover, although the baseline level of MNs was lower in FA cells than in controls, the FA fibroblasts were more prone (about two times) to MN production than control cells when irradiated with 2-4 Gy. Western blot analysis of the DNA repair proteins (hMre11, Rad50, and Rad51) did not reveal any abnormalities in protein expression levels or their migration patterns in the fibroblasts derived from an FA patient either before or after irradiation. At the same time, in vitro irradiated cells from the FA patient exhibited a significantly reduced number of nuclei with focally concentrated DNA repair Rad51 protein than in control cells.


CONCLUSIONS
. The increased DNA damage and MN induction in irradiated FA fibroblasts, and the reduction of the formation of DNA repair foci containing Rad51 suggest a possible link to the profound clinical radiosensitivity reported earlier for this FA patient. The findings on this particular FA cell strain presented in the study point toward the difficulties involved in the prediction of the radiation response of cell lines and tumors based solely on the colony survival test.
Djuzenova
Cholpon
C
Department of Radiotherapy, University of Würzburg, Würzburg, Germany. djuzenova_t@klinik.uni-wuerzburg.de
Flentje
Michael
M
Plowman
Pierce Nicholas
PN
D000328:Adult; D002470:Cell Survival / Q000528:radiation effects; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000528:radiation effects*; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D005199:Fanconi Anemia / Q000235:genetics* / Q000473:pathology*; D005260:Female; D005347:Fibroblasts / Q000473:pathology / Q000528:radiation effects*; D006801:Humans; D048629:Micronuclei, Chromosome-Defective / Q000528:radiation effects*; D011829:Radiation Dosage; D011836:Radiation Tolerance
D004247:DNA