TNF-α induces leukemic clonal evolution ex vivo in Fanconi anemia group C murine stem cells
J. Clin. Invest. June Li, et al. 117:3283 doi:10.1172/JCI31772 [
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Figure 5The role of TNF-α–induced ROS in genetic instability. (
A) 1 × 10
6 TNF-α–treated WT cells or
Fancc–/– preleukemic cells were injected i.v. into lethally irradiated recipients, which after 10 days were injected i.p. with 1 dose of TNF-α (100 μg/kg) followed by NAC (100 mg/kg/d) administration for 10 days. Control mice were injected with PBS only. The mice were sacrificed 24 hours after the last NAC injection, and BM cells were analyzed for DNA strand breaks by the comet assay. The mean tail moment of WT control sample is expressed as 100%. Larger tail moments represent higher levels of DNA damage. Three recipient mice from each group were analyzed, and 50 cells per mouse were scored from random sampling. (
B) Tissue sections of liver and lung from recipient mice described in
A were stained with anti–8-oxodG antibody and counterstained with H&E. Original magnification, ×40. (
C) WT and
Fancc–/– preleukemic cells were cultured in the presence of TNF-α (10 ng/ml), and protein extracts were prepared 0, 1, 2, and 4 hours after TNF-α treatment and analyzed by immunoblotting with anti–phospho-p53
Ser20, anti-γH2AX, and anti-actin antibodies. Extracts were also prepared from cells 2 hours (2–) after TNF-α withdrawal after the cells had been treated with TNF-α for 4 hours. (
D) BM cells from recipient mice described in
A were analyzed for p53
Ser20 and γH2AX expression. (
E) Examples of metaphase chromosomes prepared from donor-derived WT and
Fancc–/– BM cells. Chromosomal aberrations (arrows) were noted in
Fancc–/– cells but rarely in WT cells.
