Abstract 1 Distinctive and Differentiation-State Dependent DNA Damage Response of Hematopoietic Stem Cells to Genotoxic Noxae

INTRODUCTION: Hematopoietic stem cells (HSCs) sustain lifelong blood production and can be used in allogenic stem cell transplantation for treatment of hematological malignancies, yet the mechanisms of their DNA damage response (DDR) remain largely unexplored. Genotoxic damage can be induced by chemotherapeutical agents (eg, etoposide, a topoisomerase II inhibitor) or by methylating agents (eg, nitrosamines). Related double strand breaks (DSBs) at the level of immature cells are thus presumed to trigger leukemia. OBJECTIVE: Human quiescent and cycling HSCs were reported to exhibit distinct responses to genotoxic stress upon in vitro exposure to sublethal doses of etoposide or N-nitroso-N-methylurea (MNU). In this context, we aim to identify and compare the mechanisms involved in the determination of HSC stem and progenitor cell fate upon DNA damage to gain more insights into cytotoxic anticancer drug- and contaminant-related hematological toxicity. METHODS: Quiescent human HSCs (CD34(+)) were isolated from umbilical cord blood, sorted and directly exposed to etoposide or MNU. Besides, CD34(+) were expanded in-vitro in medium containing broad acting cytokines for 5 days prior genotoxic exposure. Sublethal doses were assessed by Alamar Blue assay. Propidium iodide staining and colony-forming unit assay (CFU) were used for cell cycle analysis and tracking of cell functionality. RESULTS: In vitro inhibitory concentrations were identified for human HSCs treated with etoposide (IC(50) = 5 µM) and MNU (IC(50) = 1 mM). Treatment of quiescent HSCs with lower genotoxin concentrations induces a cell cycle arrest in the G(0)/G(1)-phase, whereas higher concentrations activate apoptosis-related genes and completely abrogate further differentiation into colony-forming progenitors. In contrast, cycling HSCs exhibit an impaired proliferation, upregulation of autophagy-related genes, reduced proportions of viable CD34(+) cells, and similar effects regarding differentiation capacity into colony-forming progenitors. However, cells exposed to lower genotoxin doses exhibit a higher regeneration capacity 3 days upon treatment. DISCUSSION: These results confirm that the DDR following DSB-induction differs between quiescent and cycling HSCs. Quiescent HSCs exhibit a stronger DDR, probably related to their longer lifespan. To avoid malignant transformation, it is necessary to preclude damage accumulation through cell cycle withdrawal and apoptosis. In contrast, more committed cells rather inline toward damage repair and restoration through activation of, for example, non-homologous end joining or mismatch repair genes.