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A higher throughput assay for quantification of melphalan-induced DNA damage in peripheral blood mononuclear cells.

ABSTRACT: Inter-individual differences in DNA adduct formation and repair influence the response to melphalan treatment, however, further clinical investigation of this variability requires a logistically feasible and reproducible bioassay. Our improved fluorescence-based QPCR-block assay is robust, has good precision, and improved throughput. It also incorporates direct PCR amplification from melphalan exposed PBMC using commercially available blood tubes and extraction kits to maximise the utility of this assay for future clinical studies. Using this assay we have demonstrated reproducible inter-individual differences in melphalan-induced QPCR-block across individual PBMC donors. As proof-of-principle we assessed nine healthy donors and found a 7.8 fold range in sensitivity following exposure of PBMC ex vivo. This likely reflects differences in melphalan transport into cells as well as differences in DNA adduct repair proficiency. This improved bioassay may be useful for assessment of these processes in patients about to receive melphalan treatment.

SUBMITTER: van Kan M

PROVIDER: S-EPMC6906414 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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A higher throughput assay for quantification of melphalan-induced DNA damage in peripheral blood mononuclear cells.

van Kan Maia M Burns Kathryn E KE Browett Peter P Helsby Nuala A NA

Scientific reports 20191211 1

Inter-individual differences in DNA adduct formation and repair influence the response to melphalan treatment, however, further clinical investigation of this variability requires a logistically feasible and reproducible bioassay. Our improved fluorescence-based QPCR-block assay is robust, has good precision, and improved throughput. It also incorporates direct PCR amplification from melphalan exposed PBMC using commercially available blood tubes and extraction kits to maximise the utility of th ...[more]

PMID: 31827154

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Project description:OBJECTIVES:Metabolic derangements in sepsis stem from mitochondrial injury and contribute significantly to organ failure and mortality; however, little is known about mitochondrial recovery in human sepsis. We sought to test markers of mitochondrial injury and recovery (mitochondrial biogenesis) noninvasively in peripheral blood mononuclear cells from patients with sepsis and correlate serial measurements with clinical outcomes. DESIGN:Prospective case-control study. SETTING:Academic Medical Center and Veterans Affairs Hospital. PATIENTS:Uninfected control patients (n = 20) and septic ICU patients (n = 37). INTERVENTIONS:Blood samples were collected once from control patients and serially with clinical data on days 1, 3, and 5 from septic patients. Gene products for HMOX1, NRF1, PPARGC1A, and TFAM, and mitochondrial DNA ND1 and D-loop were measured by quantitative reverse transcriptase-polymerase chain reaction. Proinflammatory cytokines were measured in plasma and neutrophil lysates. MEASUREMENTS AND MAIN RESULTS:Median (interquartile range) Acute Physiology and Chronic Health Evaluation II and Sequential Organ Failure Assessment scores were 21 (8) and 10 (4), respectively, and 90-day mortality was 19%. Transcript levels of all four genes in peripheral blood mononuclear cells were significantly reduced in septic patients on day 1 (p < 0.05), whereas mitochondrial DNA copy number fell and plasma D-loop increased (both p < 0.05), indicative of mitochondrial damage. D-loop content was directly proportional to tumor necrosis factor-α and high-mobility group protein B1 cytokine expression. By day 5, we observed transcriptional activation of mitochondrial biogenesis and restoration of mitochondrial DNA copy number (p < 0.05). Patients with early activation of mitochondrial biogenesis were ICU-free by 1 week. CONCLUSIONS:Our findings support data that sepsis-induced mitochondrial damage is reversed by activation of mitochondrial biogenesis and that gene transcripts measured noninvasively in peripheral blood mononuclear cells can serve as novel biomarkers of sepsis recovery.

Project description:Endogenous damage associated molecular pattern molecules (DAMPs) released from necrotic, damaged or stressed cells are associated with an inflammatory response. Whether the microRNA (miR) expression signature of this response is different from that of a pathogen associated molecular pattern (PAMP)-stimulated inflammatory response is unknown. We report here that miR-34c and miR-214 are significantly expressed in fresh human peripheral blood mononuclear cells (PBMCs) exposed to DAMP-containing freeze-thaw lysates, or to conditioned media from serum-starved and glucose-deprived cells (p<6×10(-4) and p<3.7×10(-3)), respectively. Interestingly, only miR-34c expression was differentially expressed in PBMCs exposed to freeze-thaw lysates or conditioned media from wildtype High Mobility Group B1 (HMGB1(+/+)) mouse embryonic fibroblast (MEF) cells, when compared to cultures exposed to lysates or conditioned media from HMGB1(-/-) MEFs. miR-155 expression in these cultures was negligible, but was significantly expressed in PBMCs stimulated with Lipopolysaccahride (LPS) or most other Toll-like receptor (TLR) ligands, making it the prototypic "PAMPmiR". Exposure to a damaged human colorectal carcinoma cell line lysate (HCT116) similarly resulted in increased miR-34c and miR-214 levels. When PBMCs were pre-transfected with anti-miR-34c and then exposed to lysate, expression levels of IKKγ mRNA, a putative target of miR-34c, increased, while protein levels of IKKγ in cultures transfected with a pre-miR-34c were abrogated. Levels of miR-34c expression (as well as pro-inflammatory cytokines, IL-1β and TNFα) decreased when PBMC cultures were briefly pre-incubated with the K(+) channel (inflammasome) inhibitor, glybenclamide, suggesting that inflammasome activation is upstream of miR-34c expression in response to DAMPs. Our findings demonstrate that a specific microRNA expression signature is associated with the inflammatory response to damaged/injured cells and carries implications for many acute and chronic inflammatory disorders.

Dataset Information

A higher throughput assay for quantification of melphalan-induced DNA damage in peripheral blood mononuclear cells.

Publications

A higher throughput assay for quantification of melphalan-induced DNA damage in peripheral blood mononuclear cells.

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