Figure 2. Rapamycin protects in vivo hematopoietic cells against radiation exposure.

(A,B) Mice in the treatment group were given 4 mg/kg of rapamycin by i.p. injection five times every other day before total body irradiation, whereas mice in the control group were injected with carrier DMSO. After irradiation, peripheral blood counts were performed at the indicated time points. In the rapamycin-treated group, white blood cell, lymphocyte, red blood cell, and platelet numbers were markedly higher than those of the control group soon after irradiation (A), but only white blood cell and lymphocyte counts were higher than those of the control for the long period post-irradiation (B). (C) Expression of γH2A.X was measured by flow cytometry in total bone marrow cells, lineage-positive cells, lineage-negative cells, and LSK cells of the two mouse groups following 24 h ex vivo incubation after whole body irradiation of the mice. DNA damage was significantly reduced in the rapamycin-treated group (right panel). Left panels are representative flow histograms of the treatment groups. (D) Rapamycin improved bone marrow progenitors’ colony formation under radiation exposure. Mice were pretreated with carrier or rapamycin for five times before γ-irradiation. Hematopoietic mononuclear cells were then collected from the bone marrow of the sacrifice mice at day 1 after 0 Gy, 1 Gy and 3 Gy by Ficoll gradient centrifugation. Clonogenic progenitors were determined in methylcellulose medium using 6 × 10³ bone marrow mononuclear cells per 35 mm dishes and incubated for 7 days. The number of colonies containing more than 50 cells was determined. Colony numbers in the rapamycin-treated group were significantly increased after the radiation exposure. (E) LSK cells (Lin⁻Sca-1⁺c-Kit⁺) were sorted with fluorescent-activated cell sorting followed by magnetic-activated cell sorting for lineage-negative cells. Representative LSK sorting gates are shown (left and middle panels). The percentage number of LSK cells, expressed as a percentage against the number of total bone marrow mononuclear cells, was higher in the rapamycin-treated group than in the control group at day 3 after 5 Gy whole body irradiation (right panel). Data are mean ± SD from at least three independent experiments. n ≥ 5, *p < 0.05 and **p < 0.01.