Radiosensitivity of Fancd2^-/- mouse Bone Marrow Stromal Cells Is Altered By Abrogation of TGF-β Signaling

Both marrow-transplanted and non-transplanted Fanconi Anemia (FA) patients are often radiosensitive. Due to an increased risk of developing secondary malignancies, these patients require dose and volume modification during radiotherapy. To determine whether abrogation of TGF-β signaling alters the radiation sensitivity of Fancd2^-/- mice, cell lines derived from double knockout (DKO) (SMAD3^-/- Fancd2^-/-) mice were compared with those from Fancd2^-/-, SMAD3^-/-, and wild-type mice for ionizing irradiation sensitivity.

Bone marrow stromal cell lines were derived from long-term bone marrow cultures of DKO, Fancd2^-/-, SMAD3^-/-, and wild-type SMAD3^+/+ (129/Sv) X Fancd2^+/+ (B6) F1 mice. Radiation sensitivity was determined using clonogenic irradiation survival curves. There was no significant difference in radiosensitivity comparing DKO cells (Do = 1.95 ± 0.06 Gy, ň = 4.3 ± 0.7) to the wild type SMAD3^+/+ (129/Sv) X Fancd2^+/+ (B6) F1 cell line (Do = 2.00 ± 0.11 Gy, and ň = 5.1 ± 0.7, p = 0.7003 and 0.4820, respectively). The Fancd2-/- cell line was more radiosensitive with a Do of 1.37 ± 0.09 Gy compared to 1.95 ± 0.07 and 2.00 ± 0.11 for DKO and wild type cells (p = 0.0063 and 0.0360, respectively. In contrast, the SMAD3-/- cell line was more radioresistant with an increased shoulder on the irradiation survival curve (ň = 12.1 ± 2.9) compared to the DKO or wild type SMAD3^+/+ (129/Sv) X Fancd2^+/+ (B6) F1 cell lines (ň = 4.335 ± 0.7 or 5.1 ± 0.7, p = 0.00277 or 0.0426, respectively). This confirms and extends results with SMAD3^-/- mouse derived cell lines on another background strain (C57BL/6J) (Epperly, et al., Radiation Research, 165:671-677, 2006). TGF-β signaling was abrogated in both DKO and SMAD3^-/- mouse cell lines (measured by TGF-β inhibition of fresh marrow CFU-GEMM in vitro), confirming the phenotype of altered TGF-β signaling.

Therefore, radiosensitivity associated with the Fancd2-/- genotype was abrogated by interruption of the TGF-β signaling pathway in the same cells.

Supported by research grant NIAID/NIH, U19A168021.