Dataset Information

Compromised DNA Repair and Signalling in Human Granulocytes.

ABSTRACT: In previous studies, we showed impaired DNA repair in human monocytes. Here, we addressed the question of whether human neutrophilic granulocytes that arise from the same precursor as monocytes exhibit a similar phenotype and are impaired in repairing their DNA. We show that neutrophilic granulocytes isolated from peripheral blood display a lack of the same repair proteins that are missing in monocytes and do not show repair of their DNA when damaged by ionising radiation (IR) or chemical ROS. Contrary to T cells, we observed no decline in the number of single-strand breaks following ?-radiation. Also, granulocytes did not show ?H2AX foci formation while T cells and peripheral blood lymphocytes (PBL) responded. In comparison to PBL, XRCC1, PARP-1 and ligase III were not expressed and there was also no discernible signal for key damage response proteins ATM, ATR and DNA-PKCS as well as ?H2AX in neutrophils. Time course and dose-response experiments confirmed the absence of H2AX phosphorylation after radiation treatment although an accumulation of double-strand breaks was detected in the neutral Comet assay. Overall, the data indicate that terminally differentiated neutrophilic granulocytes in the peripheral blood display strong downregulation of DNA repair and DNA damage response factors, which should be taken into account if studies with whole peripheral blood containing granulocytes are performed, causing a significant intra-experimental variation in the cellular repair capacity.

SUBMITTER: Ponath V

PROVIDER: S-EPMC6738182 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Compromised DNA Repair and Signalling in Human Granulocytes.

Ponath Viviane V Heylmann Daniel D Haak Tobias T Woods Kevin K Becker Huong H Kaina Bernd B

Journal of innate immunity 20181008 1

In previous studies, we showed impaired DNA repair in human monocytes. Here, we addressed the question of whether human neutrophilic granulocytes that arise from the same precursor as monocytes exhibit a similar phenotype and are impaired in repairing their DNA. We show that neutrophilic granulocytes isolated from peripheral blood display a lack of the same repair proteins that are missing in monocytes and do not show repair of their DNA when damaged by ionising radiation (IR) or chemical ROS. C ...[more]

PMID: 30296787

Similar Datasets

Project description: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.

Dataset Information

Compromised DNA Repair and Signalling in Human Granulocytes.

Publications

Compromised DNA Repair and Signalling in Human Granulocytes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets