Abstract 842: Increased irradiation-induced senescence in Fanconi anemia (FA) mice

Abstract

Abstract Senescence is an irreversible cell cycle arrest induced by various triggers, including aging and irradiation. Fanconi Anemia (FA) is a genetic disorder associated with defects in 22 proteins in the scaffold DNA double strand break repair. FA mice develop anemia and marrow failure and have been considered as a potential model for accelerated aging. Fancd2-/- murine bone marrow (BM) stromal cell lines are radiosensitive in clonogenic survival curves compared to wild type (Berhane et al, Radiat Res, 2014). We tested the Fanca-/- mouse model for irradiation induced senescence. 129Sv Fanca+/+ and Fanca-/- BM stromal cell lines were irradiated to 5Gy, then assayed for senescence at various timepoints. Cells were stained with a chromogenic beta-galactosidase assay. Cells were also stained by immunofluorescence (IF) to determine percent of cells positive for p16 and p21. For total body irradiation (TBI) studies, 129Sv Fanca+/+ and Fanca-/- mice were given 7Gy. Organs were harvested at Day 7, then assayed for senescence. Statistical analysis was carried out using student's t-test. Baseline levels of beta-gal positive Fanca-/- BM stromal cells were not significantly different from Fanca+/+ (respectively, 0.88% ± 0.22% vs 0.74% ± 0.23%). Fanca-/- and Fanca+/+ IF also showed no significant differences in baseline p16 levels (4.98%±2.5 vs 14.37%± 4.06%) and p21 levels (14.57% ± 7.61% vs. 11.7% ± 3.48%). In contrast, Fanca-/- cells showed significantly higher beta-gal staining compared to Fanca+/+ at days 1, 3, and 5 after irradiation (p=0.0042, p<0.0001, p=0.0011 respectively). By day 3 after 5Gy irradiation, Fanca-/- cells showed significant senescence increase compared to control non-irradiated cells for p16 (29.54%± 4.11%) and p21 (84.27±10.40). At day 3 after 5Gy irradiation, In Fanca+/+ cells there was less profound but also significant induction of p16 (17.78%± 3.99%) and p21 (62.08±84.27). At Day 7 after irradiation, beta-gal positive cells were significantly increased compared to control non-irradiated in WT (5.943% ± 0.96%, p<0.001) and Fanca-/- (9.07% ± 1.45%, p<0.0001) cells. At Day 7 after 7Gy TBI, kidney, liver, lung, skin, and brain did not show induction of beta-gal staining. The spleen, a radiosensitive organ, displayed increased beta-gal staining, with a larger increase in Fanca-/- spleen compared to Fanca+/+. Expression of p21 mRNA was significantly induced in Fanca-/- (5.8x increase, p=0.0305) and Fanca+/+ (7.3x increase, p=0.0357) compared to non-irradiated control spleens. Expression of p16 mRNA was increased in both genotypes. Therefore, Fanca-/- mouse organs in vivo and cell lines in vitro display increased irradiation induced senescence compared to Fanca+/+ mice. Sorting live senescent cells will confirm the increase in irradiation-induced senescent cells, and functional outcomes in FA mice. Citation Format: Aranee P. Sivananthan, Xichen Zhang, Wen Hou, Donna Shields, Renee Fisher, Michael Epperly, Joel S. Greenberger. Increased irradiation-induced senescence in Fanconi anemia (FA) mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 842.