Granulocytes Affect Double-Strand Break Repair Assays in Primary Human Lymphocytes

Citation: Lacoste S, Bhatia R, Bhatia S, O'Connor TR ( Granulocytes Affect Double-Strand Break Repair Assays in Primary Human Lymphocytes Sandrine Lacoste 0 Ravi Bhatia 0 Smita Bhatia 0 Timothy R. O'Connor 0 Jian Jian Li, University of California Davis, United States of America 0 1 Department of Cancer Biology, Beckman Research Institute, Duarte, California, United States of America, 2 Division of Hematology and Hematopoietic Cell Transplantation, City of Hope Medical Center, Duarte, California, United States of America, 3 Department of Population Sciences, City of Hope Medical Center , Duarte, California , United States of America Patients who develop therapy-related myelodysplasia/acute myeloid leukemia after autologous-hematopoietic stem cell (aHCT) transplant show lower expression levels of DNA repair genes in their pre-aHCT CD34+ cells. To investigate whether this leads to functional differences in DNA repair abilities measurable in patients, we adapted two plasmid-based host-cell reactivation assays for use in primary lymphocytes. Prior to applying these assays to patients who underwent aHCT, we wanted first to verify whether sample preparation affected repair measurements, as patient samples were simply depleted of erythrocytes (with hetastarch) prior to freezing, which is not the classical way to prepare lymphocytes prior to DNA repair experiments (with a density gradient). We show here that lymphocytes from healthy donors freshly prepared with hetastarch show systematically a higher level of double-strand break repair as compared to when prepared with a density gradient, but that most of this difference disappears after samples were frozen. Several observations points to granulocytes as the source for this effect of sample preparation on repair: 1) removal of granulocytes makes the effect disappear, 2) DSB repair measurements for the same individual correlate to the percentage of granulocytes in the sample and 3) nucleofection in presence of granulocytes increases the level of reactive oxygen species (ROS) in neighboring lymphocytes in a dosedependent manner (R2 of 0.95). These results indicate that co-purified granulocytes, possibly through the release of ROS at time of transfection, can lead to an enhanced repair in lymphocytes that obfuscates any evaluation of inter individual differences in repair as measured by host-cell reactivation. As a result, hetastarch-prepared samples are likely unsuitable for the assessment of DSB repair in primary cells with that type of assay. Granulocyte contamination that exists after a density gradient preparation, although much more limited, could have similar effects, but might be circumvented by freezing cells prior to analysis. - Funding: Funding was provided by the NIH SPORE grant (5P50CA107399, S. Forman, http://www.nih.gov/index.html) and by the City of Hope Comprehensive Cancer Center Grant (NCI 5P30CA033572-27, S. Rosen, http://www.cancer.gov/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Therapy-related myelodysplasia/acute myeloid leukemia (tMDS/AML) is a major complication of autologous-hematopoietic stem cell transplant (aHCT). Samples from patients who received aHCT for a relapsed or refractory Hodgkins or non-Hodgkins lymphoma have been collected for a prospective longitudinal study with the objective to identify new markers that help predict patients at risk of t-MDS/AML [1,2]. Expression microarrays show differences between patients from the cohort that did or did not later develop t-MDS/AML [3]. Notably, a lower expression of genes implicated in DNA repair in CD34+ cells in peripheral blood stem cell products from patients pre-aHCT was associated with the later development of t-MDS/AML, an association that persisted in bone marrow cells at the time of diagnosis. Our ultimate goal is to verify if these differences result in functional changes in DNA repair capacities that could be more easily evaluated in a clinical setting. Many assays exist that can be used to evaluate inter-individual differences in repair abilities. Among those, host-cell reactivation assays have the advantage to directly measure repair and can be adapted to study specific repair pathways. Moreover, the damage is generated in vitro prior to the introduction in the cells where the repair will be measured by the reactivation of a transgene, avoiding as much as possible concerns about cytotoxicity associated with the damage. Host-cell reactivation assays can be performed on any cell type that can be transfected, including cryopreserved primary lymphocytes [4]. Multiple population studies have used host-cell reactivation assays to evaluate DNA repair as a risk factor for several types of cancer (reviewed in [5]). We show here two host-cell reactivation assays to study independently the two pathways of double-strand bread (DSB) repair that are prevalent in non-cycling primary lymphocytes: non-homologous end-joining (NHEJ) and single-strand annealing (SSA). These assays, that we adapted for use in primary lymphocytes, can provide reproducible results in triplicates for both type of repair in 48 h starting from the cells obtained from 2.5 ml of blood, indicating that they could be applied to patient samples. However, the patients samples we want to analyze were not prepared with this specific application in mind, but to preserve all white blood cells (WBCs) lineages for subsequent study of the progression of the disease after aHCT. To that effect, patients blood samples were only treated with hetastarch in order to remove most of the red blood cells (RBCs) and simply frozen afterwards. But the method of choice to investigate DNA repair in peripheral blood lymphocytes is usually a density gradient that recovers mostly mononuclear cells (lymphocytes and monocytes), whereas RBCs and granulocytes sediment at the bottom of the gradient. Therefore, the main difference between the two types of preparation is related to the presence of granulocytes in addition to the lymphocytes to be studied. Granulocytes constitute 3580% of leukocytes in the blood and are therefore the predominant cell type recovered when all WBCs are preserved, like after hetastarch aggregation. Granulocytes co-purified with peripheral blood mononuclear cells are thought to be responsible for some T-cell dysfunctions observed in samples that were not processed within 68 h from collection [68]. So prior to applying the new assays to patient cells, we used the blood of healthy volunteers to determine whether the type of sample preparation (density gradient or hetastarch with or without freezing), and subsequent differences in cell composition, affected DNA repair measurements. Materials and Methods Ethics statement Use of human blood samples was approved by the City of Hope Internal Review Board: IRB protocol #98 (...truncated)