Granulocyte Colony-Stimulating Factor Induced Proliferation Leads to An Increase in Radical Oxygen Species Production and DNA Damage in Hematopoietic Stem Cells
Authors: Souza Liliana , Silva Erica , Calloway Elissa , Rossi Michael , McLemore Morgan L. 
Published: October 2019

Abstract
Abstract 1352
Patients with severe congenital neutropenia (SCN) require continuous G-CSF treatment throughout their lifetime. Such prolonged treatment increases the incidence of myelodysplastic syndromes/acute myeloid leukemia (MDS/AML) in this population. We hypothesize that excessive hematopoietic stem cell (HSC) proliferation can lead to DNA damage, cause an increase in radical oxygen species (ROS) production and DNA instability. This can lead to the impairment of stem cell function or leukemogenesis. We utilized wild-type (WT) and the p53 heterozygous (+/−) mouse model to test our hypothesis. Previous studies have shown that the p53 tumor suppressor gene regulates several functions of hematopoietic cell proliferation, differentiation, apoptosis, and aging. In response to DNA damage, p53 can either elicit cell-cycle arrest or apoptosis. Additionally, p53 deletions and mutations have been found at high frequency in blast crisis chronic myelogenous leukemia and with some frequency in acute leukemia and MDS. We treated wild- type (WT) and p53+/− mice daily with G-CSF for different time periods. Animals were euthanized after 1 and 3 months of daily G-CSF treatment and the lin-ckit+sca+ (KLS) bone marrow population was isolated. The results show a 3 fold increase in the KLS population of WT mice and a 4 fold increase in the mutant mice. These KLS cell populations were then analyzed for the amount of DNA double strand breaks (DSB) via the presence of nuclear pH2AX. Our results show that there is a 2 fold increase in the DSB of the WT cells and a 3 fold increase in the p53+/− when compared to the levels of DSB of untreated mice. Our results also indicate that the fold increase of the DSB in KLS cells was the same in animals treated with G-CSF for 3 months as the animals treated for 1 month. However, the levels of pH2AX and ROS increased. Even though the basal levels of ROS in p53+/− were higher than in the WT, G-CSF treatment increased OH− levels in both groups significantly. Therefore, to analyze the increasing levels of ROS and DNA damage caused chromosome alterations, we extracted DNA from hematopoietic progenitor cells from animals treated with G-CSF for 3 months. We then performed a CGH array analyses. Our results point out variations in gains and losses of several chromosome regions implicated in hematological malignant diseases. The results of a competitive transplant assay with WT and p53+/− bone marrow, demonstrate an advantage of p53+/− HSC over WT HSC that became exacerbated after G-CSF treatment. An increase in DNA damage and ROS production in KLS cells was also detected post transplant. These increases were intensified after the transplanted animals were treated with G-CSF. Our results suggest that excessive proliferation induced by long term G-CSF treatment can contribute to an increase in ROS production, DSBs in HSC's, and genomic instability that may impair HSC function. Understanding the causes and trends of chromosomal instability could improve our knowledge of leukemogenesis and potentially reveal novel treatment strategies for severe congenital neutropenia patients.

Disclosures:
No relevant conflicts of interest to declare.