Abstract
Accumulation of damaged DNA in hematopoietic stem cells (HSC) is associated with chromosomal abnormalities, genomic instability, and HSC aging and might promote hematological malignancies with age. Despite this, the regulatory pathways implicated in the HSC DNA damage response have not been fully elucidated. One of the sources of DNA damage is reactive oxygen species (ROS) generated by both exogenous and endogenous insults. Balancing ROS levels in HSC requires FOXO3, which is an essential transcription factor for HSC maintenance implicated in HSC aging. Elevated ROS levels result in defective Foxo3-/- HSC cycling, among many other deficiencies. Here, we show that loss of FOXO3 leads to the accumulation of DNA damage in primitive hematopoietic stem and progenitor cells (HSPC), associated specifically with reduced expression of genes implicated in the repair of oxidative DNA damage. We provide further evidence that Foxo3-/- HSPC are defective in DNA damage repair. Specifically, we show that the base excision repair pathway, the main pathway utilized for the repair of oxidative DNA damage, is compromised in Foxo3-/- primitive hematopoietic cells. Treating mice in vivo with N-acetylcysteine reduces ROS levels, rescues HSC cycling defects, and partially mitigates HSPC DNA damage. These results indicate that DNA damage accrued as a result of elevated ROS in Foxo3-/- mutant HSPC is at least partially reversible. Collectively, our findings suggest that FOXO3 serves as a protector of HSC genomic stability and health.
Highlights
Accumulation of damaged DNA in hematopoietic stem cells (HSC) is associated with chromosomal abnormalities, genomic instability, and HSC aging and might promote hematological malignancies with age
Damaged DNA accumulated in Foxo3Ϫ/Ϫ HSPC subpopulations, including long term repopulating HSC (LT-HSC; LSK Flk2ϪCD34Ϫ) and c-Kitϩ (LinϪSca1Ϫc-Kitϩ) multipotent progenitor cells but not in Foxo3Ϫ/Ϫ total bone marrow (BM) control cells (Fig. 1,C for gating strategy, and D; n ϭ 4 per genotype); ␥H2AX was relatively increased in lineage negative cells depleted of mature blood cells and enriched for hematopoietic stem and progenitor cells, it did not reach significance in the samples evaluated
We showed that the damaged DNA in homeostatic Foxo3Ϫ/Ϫ HSPCs is mediated by both elevated endogenous reactive oxygen species (ROS) and a defective base excision
Summary
Foxo3Ϫ/Ϫ Hematopoietic Stem and Progenitor Cells Accumulate Oxidative DNA Damage at the Steady State—Foxo3Ϫ/Ϫ LSK cells (LinϪSca1ϩc-Kitϩ) enriched for HSC accumulate. Using a specific probe that detects the main DNA oxidation lesion, 8-hydroxyguanosine (8-OHdG), by flow cytometry, we noted 8-OHdG levels were increased in Foxo3Ϫ/Ϫ LSK cells as compared with WT cells (Fig. 2C), the difference did not reach significance in the replicates analyzed (n ϭ 3) These results indicate that Foxo3Ϫ/Ϫ HSPC DNA accumulates high levels of oxidative insults. To address a possible DNA repair defect, we used a recently developed [39] BER molecular beacon assay to quantitatively evaluate APE1 endonuclease activity and OGG1-mediated glycosylase activity for removal of the 8-oxo-dG lesion in an 8-oxodG/A base pair (Fig. 5F, schematic) Using this fluorescent real time quantitative assay, we found no difference in the APE1 endonuclease activity of WT and Foxo3Ϫ/Ϫ primitive hematopoietic cells (data not shown).
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