Mouse Fanconi Anemia (FA) Fancd2-/- Bone Marrow Stromal Cells Demonstrate Ionizing Irradiation Induced Senescence

Abstract

Senescent cells accumulate in tissues affected by aging, ionizing irradiation, and loss of telomere length and other sources of DNA damage signaling. FA mice deficient in the Fancd2 protein display reduced homologous recombination and increased nonhomologous end joining for repair of DNA double strand breaks. Studies also show a role for Fancd2 in the ALT pathway of telomere maintenance. Fancd2-/- bone marrow stromal cell lines are radiosensitive compared to Fancd2+/+ marrow stromal cells (Do = 1.4 ± 0.1 Gy vs 1.6 ± 0.1 Gy, p = 0.0124), and by comet assay where Fancd2-/- cells display slower DNA repair after irradiation (p < 0.0001) (Berhane, et. al., Radiation Research 181:76-89, 2014). We investigated whether Fancd2-/- cells were more susceptible to induction of senescence. Fancd2+/+ control and Fancd2-/- murine bone marrow stromal cell lines were analyzed for irradiation induced senescence. Bone marrow stromal cell lines derived from long term bone marrow cultures of Fancd2+/+, and Fancd2-/- marrow (129/Sv) were plated in 6 well tissue culture plates and irradiated to 5 or 10 Gy. At 24, or 48 hr after irradiation, cells were stained for senescence biomarkers B-Gal and p21. Fancd2+/+ and Fancd2-/- bone marrow stromal cells stained for percent B-Gal positive cells following 0, 5 or 10 Gy at 24 and 48 hr after irradiation showed that Fancd2-/- cells had increased B-Gal staining compared to Fancd2+/+ cells at 0 Gy (10 ± 1% vs 1 ± 1%, p<0.0001). At 24 and 48 hr after 5 and 10 Gy Fancd2-/- cells had increased B-Gal staining (14 ± 1% at 24 hr after 5 Gy, 18 ± 1% at 24 hr after 10 Gy, 18 ± 1% at 48 hr after 5 Gy and 23 ± 1% at 48 hr after 10 Gy, p < 0.0123 compared to 0 Gy). In contrast increased B-Gal staining was delayed in FancA+/+ cells to 48 hr after both 5 and 10 Gy (7 ± 4% at 24 hr after 5 Gy, 3 ± 1% at 48 hr after 10 Gy, 3 ± 1% at 48 hr after 5 Gy, 4 ± 1% at 48 hr after 10 Gy, p = 0.0027 and < 0.0001 comparing 5 and 10 Gy at 48 hr to 0 Gy, respectively). The percent of nonirradiated Fancd2-/- cells positive for p21 was statistically increased compared to Fancd2+/+ cells (16 ± 2% vs 4 ± 1%, p = 0.0007). Both control Fancd2+/+ and Fancd2-/- cells had increased p21 positive cells at 24 and 48 hr following 5 or 10 Gy (p < 0.05) was always greater in Fancd2-/- cells . At 24 and 48 hrs after 5 Gy, Fancd2-/- cells had 45 ± 5% and 39 ± 1% positive p21 cells compared to 9 ± 1% and 5 ± 1% for Fancd2+/+ cells (p < 0.0001). At 24 and 48 hours after 10 Gy the percent of Fancd2-/- cells positive for p21 was 58 ± 3% and 48 ± 5%, respectively, compared to 11 ± 1% and 7 ± 1%, respectively, for Fancd2+/+ cells (p < 0.0001). The radiosensitivity of Fancd2-/- bone marrow stromal cells increases senescence in vitro as measured by both B-Gal and p21. Study of FA tissues in vivo after TBI should reveal organ specific acceleration of senescence and provide insight into late irradiation effects in marrow transplanted FA patients.