Diminished Oxidative Stress Responses in Bone Marrow Stromal Cell Lines Derived From Fanconi Anemia (Fanc-D2−/−) Mice

Abstract 4398

Fanconi Anemia (FA) is an inherited cancer susceptibility syndrome associated with a defect in one or more of the 15 FA pathway gene products leading to bone marrow failure, increased cellular sensitivity to DNA cross-linking agents and increased risk of myeloid leukemia. Underlying DNA repair vulnerabilities render FancD2−/−, a homozygous deletion recombinant negative mice, sensitive to whole body irradiation. We quantitated hematopoiesis in long term bone marrow cultures, and the radiosensitivity and oxidative responses of derived marrow stromal and IL-3 dependent hematopoietic progenitor cell lines derived from FancD2−/− mice on a C57BL/6 background (generously provided by Drs. A. D'Andrea and K. Parmar, Dana-Farber cancer Inst., Boston, MA). Long-term bone marrow culture stromal cell lines derived from FancD2−/− were compared to FancD2+/− or FancD2+/+. FancD2−/− long term marrow cultures showed decreased cobblestone islands, representative of hematopoietic stem cell generating foci, and production of non-adherent cells compared to wild type FancD2+/+ or heterozygous FancD2 +/− cultures (p = 0.0148) and produced fewer colony forming cells on day 7 and 14 compared to FancD2 +/− or FancD2 +/+ cell lines (p < 0.0001). FancD2−/− stromal cell lines showed decreased DNA repair by comet assay after irradiation to 5Gy (P<0.0001). Following irradiation to 5 or 10 Gy, FancD2−/− cells had an increased cell doubling time (44.1 + 0.7 and 54.2 + 7.8) compared to FancD2 +/+ cells (35.5 + 1.5hrs and 33.6 + 1.3,hrs p = 0.006 and 0.051, respectively). FancD2−/− cells had decreased baseline antioxidant levels compared to the FancD2 +/+ cells (0.55 + 0.11 and 0.91 + 0.17 trolox units, respectively). FancD2−/− cells at 24 hr following 10 Gy irradiation had 0.39 + 0.03 trolox units compared to 0.76 + 0.07 trolox units in FancD2 +/+ cells (p = 0.006). In clonogenic irradiation survival curve assays, FancD2−/− marrow stromal cells were radiosensitive (Do = 1.43 + 0.06 Gy, n = 4.98 + 0.65) compared to FancD2 +/+ (Do 1.70 + 0.09 Gy and n = 8.33 + 0.72, p = 0.0395 and 0.0040, respectively) or FancD2 +/− cells (Do = 1.67 + 0.08 and n = 3.63 + 0.44, p = 0.0348 and 0.1365, respectively). Furthermore, FancD2 +/− cells were more radiosensitive compared to FancD2 +/+ cells ((n = 3.63 + 0.44 or 8.33 + 0.72), p = 0.0003). Thus, the FancD2 gene controls several parameters of sensitivity to oxidative stress and ionizing radiation as demonstrated in mouse long-term marrow cultures and derived cell lines.