Hematopoietic Stem Cells Repair Ionizing Radiation-Induced DNA Double Strand Breaks in a Cell Cycle-Dependent Manner.

Abstract 3241

Poster Board III-178

Mice with mutations in various DNA repair genes exhibit accelerated aging due to hematopoietic stem cell (HSC) premature exhaustion, indicating that DNA repair is crucial for the maintenance of HSC self-renewal and hematopoietic function. In addition, some of these mutated mice are highly susceptible to the development of leukemia and lymphoma due to an increase in genomic instability in HSCs. However, how HSCs respond to genotoxic stress and repair DNA damage have not been well established and thus, were investigated in the present study using a mouse model. Specifically, DNA damage and repair were analyzed by gH2AX immunofluorescent staining and neutral comet assay to quantify IR-induced DNA double strand breaks (DSBs) in HSCs (Lin^- c-kit⁺ Sca1⁺ cells or LKS⁺ cells) and hematopoietic progenitor cells (HPCs; Lin^- c-kit⁺ Sca1^- cells or LKS^- cells) isolated from adult mouse bone marrow (BM) after they were exposed to ionizing radiation (IR). The results showed that exposure to IR induced a similar number of DSBs in HSCs and hematopoietic progenitor cells (HPCs) isolated from adult mouse BM. However, HPCs repaired the damage within 6 h after IR, whereas more than 50% DSBs were unrepaired by HSCs even at 24h after IR, indicating that HSCs are highly deficient in repair of IR-induced DSBs. The deficient DSBs repair in HSCs is attributable to their quiescence, as sorted quiescent Pyronin Y^low HSCs were more deficient in repairing the damage than cycling Pyronin Y^high HSCs. This suggestion is further supported by the observations that proliferating HSCs such as fetal liver HSCs and HSCs isolated from 5-FU-treated adult mouse BM repaired the damage as efficiently as HPCs. In addition, incubation of quiescent Pyronin Y^low HSCs from adult BM with stem cell factor and thrombopoietin for 48 h stimulated the cell cycle entry and DNA damage repair function. These findings indicate that stimulation of cell cycling can promote HSCs to repair DNA damage. The difference in repair of IR-induced DSBs between quiescent and cycling HSCs is not because they express different levels of key proteins (such as Ku70, Ku80, DNA-PKcs, Lig4, XRCC4, Dclre1c, Nhej1, Brac-1, Brac-2, MRE11a, Nbs1, Rad50, Rad51, and ATM) involved in non-homologous end joining (NHEJ) and homologous recombination (HR). Instead, quiescent HSCs exhibited an insignificant activation of DNA-PK and minimal formation of XRCC4 and Rad51 foci after exposure to IR, suggesting that quiescent HSCs are deficient in DSBs repair through the NHEJ and HR pathways. However, quiescent HSCs exhibited similar levels of phosphorylation of ATM and p53 after IR compared to cycling HSCs and HPCs, indicating that quiescent HSCs are proficient in sensing DNA damage to initiate DNA damage responses. These findings provide crucial insights into how HSCs respond to and repair DNA damage, which could significantly advance our understanding on how HSCs maintain their genomic stability.