Abstract
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 dose-dependent 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.
Highlights
Therapy-related myelodysplasia/acute myeloid leukemia is a major complication of autologous-hematopoietic stem cell transplant
A lower expression of genes implicated in DNA repair in CD34+ cells in peripheral blood stem cell products from patients pre-autologous-hematopoietic stem cell transplant (aHCT) was associated with the later development of t-MDS/AML, an association that persisted in bone marrow cells at the time of diagnosis
The original transfection control SV40-GFP in the pCMS constructs was poorly expressed in primary cells and was replaced by tdTomato expressed under the spleen focus-forming virus (SF) promoter [10,11] to allow a balanced expression of both transgenes in hematopoietic cells and to improve discrimination between transfection control and repair signals (Figure S3)
Summary
Therapy-related myelodysplasia/acute myeloid leukemia (tMDS/AML) is a major complication of autologous-hematopoietic stem cell transplant (aHCT). 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. 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
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