Excessive Hematopoietic Stem Cell Proliferation Leads to Chromosomal Instability

Truncation mutations in the granulocyte colony stimulating factor receptor (G-CSFR), common in severe congenital neutropenia (SCN), lead to excessive stem cell proliferation in response to G-CSF. These G-CSFR mutants are (at least indirectly) implicated in the progression of these patients to acute leukemia. Since SCN patients require continuous G-CSF treatment throughout their lifespan, we hypothesize that excessive stem cell proliferation can lead to DNA damage. Stem cells are relatively quiescent and rarely enter the cell cycle under normal conditions. During the cell cycle cells generate approximately 5000 single strand DNA lesions per nucleus (Vilenchik and Knudson, 2003). Approximately 1% of these lesions are ultimately converted to double strand DNA breaks (DSBs). Hematopoietic stem cells are found within the Sca+ ckit+ Lin- (KLS) population. Wild type and mice bearing a mutant G-CSFR similar to that found in patients with SCN were treated with G-CSF. After 21 days of treatment with G-CSF (10 ug/kg/day), the KLS population in the bone marrow increased four-fold in wild type mice and eight-fold in mutant mice. We isolated Lin-Sca+ bone marrow cells from these G-CSF treated mice and evaluated for the presence of double stranded DNA breaks by staining with anti-phospho-H2AX by immunofluorescence. H2AX is a histone whose phosphorylated form localizes to the site of double stranded DNA breaks. The results showed that there is an 8-fold increase in the DSB in wild type Lin-Sca+ and 10-fold in mutant Lin-Sca+ when compared to cells from untreated mice. This data suggests that excessive proliferation can contribute to an increase in DSBs in hematopoietic stem cells. Investigation of potential mechanisms contributing to DSB formation are ongoing. Understanding the causes and trends of chromosomal instability would improve our understanding of leukemogenesis and potentially reveal novel treatment strategies.