Accumulation of IR-induced DNA damage impairs the function of Gadd45a^−/−HSCs and promotes leukemogenesis. (A) Schematic diagram for study design. Wild-type mice were fully reconstituted with either Gadd45a^+/+ or Gadd45a^−/− BM and were named as R-1, which were exposed to 4.5-Gy IR 4 months after transplantation. Four thousand donor-derived LSK cells, along with 0.5 × 10⁶ competitors, were subjected to secondary transplantation into R-2 mice 4 months after IR. (B) The cell number of total BM cells in R-1 mice 1 month after 4.5-Gy IR is shown (G^+/+, n = 5; G^−/−, n = 6). (C) Apoptosis in the indicated cell populations of the R-1 mice was detected with Annexin V/DAPI staining 1 month after 4.5-Gy IR (G^+/+, n = 5; G^−/−, n = 6). (D) Isolated LSK cells from R-1 mice 3 months after 4.5-Gy IR were subjected to the alkaline comet assay. The DNA was stained with DAPI, and representative examples are shown. The olive tail moment represents DNA damage level. (E) Isolated LSK cells from R-1 mice 3 months after 4.5-Gy IR were stained by γ-H2AX antibody and DAPI. (F) The percentage of donor-derived B cells in PB of R-1 (post-IR) or R-2 mice is shown (R-1, n = 5; R-2, n = 16). (G) Representative image showing the differences between Gadd45a^+/+ donor-derived control and Gadd45a^−/− donor-derived leukemia: hemogram of PB, 53BP1 foci, BrdU incorporation, and percentage of B cells in BM. (H) Gadd45a expression (relative to GAPDH) was measured in BM cells of AML patients, who were separated by survival time of 25 months (<25 months, n = 10; >25 months, n = 7) (*P < .05, **P < .01).