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HiIDDD: a high-throughput imaging pipeline for the quantitative detection of DNA damage in primary human immune cells.

ABSTRACT: DNA damage is a prominent biomarker for numerous diseases, including cancer, as well as for the aging process. Detection of DNA damage routinely relies on traditional microscopy or cytometric methods. However, these techniques are typically of limited throughput and are not ideally suited for large-scale longitudinal and population studies that require analysis of large sample sets. We have developed HiIDDD (High-throughput Immune cell DNA Damage Detection), a robust, quantitative and single-cell assay that measures DNA damage by high-throughput imaging using the two major DNA damage markers 53BP1 and [Formula: see text]-H2AX. We demonstrate sensitive detection with low inter-assay variability of DNA damage in various types of freshly isolated and cryopreserved primary human immune cells, including CD4 + and CD8 + T cells, B cells and monocytes. As proof of principle, we demonstrate parallel batch processing of several immune cell types from multiple donors. We find common patterns of DNA damage in multiple immune cell types of donors of varying ages, suggesting that immune cell properties are specific to individuals. These results establish a novel high-throughput assay for the evaluation of DNA damage in large-scale studies.

SUBMITTER: Gallant K

PROVIDER: S-EPMC9022135 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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HiIDDD: a high-throughput imaging pipeline for the quantitative detection of DNA damage in primary human immune cells.

Gallant Kelsey K Bektas Arsun A Kaileh Mary M Lustig Ana A Ferrucci Luigi L Pegoraro Gianluca G Misteli Tom T

Scientific reports 20220415 1

DNA damage is a prominent biomarker for numerous diseases, including cancer, as well as for the aging process. Detection of DNA damage routinely relies on traditional microscopy or cytometric methods. However, these techniques are typically of limited throughput and are not ideally suited for large-scale longitudinal and population studies that require analysis of large sample sets. We have developed HiIDDD (High-throughput Immune cell DNA Damage Detection), a robust, quantitative and single-cel ...[more]

PMID: 35428779

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Project description:BackgroundSporadic breast cancer like many other cancers is proposed to be a manifestation of abnormal genetic and epigenetic changes. For the past decade our laboratory has identified genes involved in DNA damage response (DDR), apoptosis and immunosurveillance pathways to influence sporadic breast cancer risk in north Indian population. Further to enhance our knowledge at the epigenetic level, we performed DNA methylation study involving 17 gene promoter regions belonging to DNA damage response (DDR) and death receptor apoptotic pathway in 162 paired normal and cancerous breast tissues from 81 sporadic breast cancer patients, using a high throughput quantitative DNA methylation analysis technology.ResultsThe study identified five genes with statistically significant difference between normal and tumor tissues. Hypermethylation of DR5 (P=0.001), DCR1 (P=0.00001), DCR2 (P=0.0000000005) and BRCA2 (P=0.007) and hypomethylation of DR4 (P=0.011) in sporadic breast tumor tissues suggested a weak/aberrant activation of the DDR/apoptotic pathway in breast tumorigenesis. Negative correlation was observed between methylation status and transcript expression levels for TRAIL, DR4, CASP8, ATM, CHEK2, BRCA1 and BRCA2 CpG sites. Categorization of the gene methylation with respect to the clinicopathological parameters showed an increase in aberrant methylation pattern in advanced tumors. These uncharacteristic methylation patterns corresponded with decreased death receptor apoptosis (P=0.047) and DNA damage repair potential (P=0.004) in advanced tumors. The observation of BRCA2 -26 G/A 5'UTR polymorphism concomitant with the presence of methylation in the promoter region was novel and emerged as a strong candidate for susceptibility to sporadic breast tumors.ConclusionOur study indicates that methylation of DDR-apoptotic gene promoters in sporadic breast cancer is not a random phenomenon. Progressive epigenetic alterations in advancing tumors result in aberrant DDR-apoptotic pathway thereby promoting tumor development. We propose, since pathological epigenetic changes of the DDR-apoptotic genes are reversible modifications, these could further be targeted for therapeutic interventions.

Dataset Information

HiIDDD: a high-throughput imaging pipeline for the quantitative detection of DNA damage in primary human immune cells.

Publications

HiIDDD: a high-throughput imaging pipeline for the quantitative detection of DNA damage in primary human immune cells.

Similar Datasets

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