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DNA Repair Capacity for Personalizing Risk and Treatment Response - Assay Development and Optimization in Human Peripheral Blood Mononuclear Cells (PBMCs).

ABSTRACT: DNA repair capacity (DRC) is the ability of a cell to repair DNA damage. Differential DRC plays an important role in human disease, including lung and other cancers. Measuring DRC could aid in translational disease research and in personalizing treatment. We developed and optimized a flow cytometry-based assay to measure individual DRC using GFP-expressing plasmids modified by ultraviolet (UV) light for nucleotide excision repair (NER) and restriction enzyme digestion to induce a blunt double-strand cut between promoter and GFP expression regions for nonhomologous end joining (NHEJ). Cryopreserved peripheral blood mononuclear cells (PBMCs) from healthy volunteers were used to measure DRC and optimize the assay. Pathway specificity of the NHEJ DRC assay was confirmed using Ku80-/- MEF cells, which showed a 6-fold reduction in NHEJ compared to Ku80+/+. Using a cell mixing assay, we show a linear correlation between NHEJ DRC and the expected concentration of Ku80. NHEJ DRC measurements in cryopreserved PBMCs are quantifiable with low interindividual and inter-assay variability, and a titratable decrease in NHEJ activity was observed in PBMCs treated with the DNA-PK inhibitor NU7441. Pathway specificity of the NER DRC assay was confirmed by a decrease in measured NER activity in human XPC deficient compared to XPC proficient fibroblasts, with a linear correlation measured between NER DRC and expected XPC concentration by cell mixing assay. NER DRC is quantifiable, reproducible, and titratable in PBMCs from healthy volunteers. We measured both NER and NHEJ DRC in PBMCs obtained from newly diagnosed, untreated lung cancer patients; measured DRC differed in these PBMCs compared to healthy volunteers. With further investigation, measurement of NER and NHEJ DNA repair capacity may be useful in personalizing disease risk and response to DNA damaging therapies and small molecular inhibitors of DNA repair pathways using readily available human PBMCs.

SUBMITTER: Nasrallah NA

PROVIDER: S-EPMC9248805 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Autism is one of the most common neurological developmental disorder associated with social isolation and restricted interests in children. The etiology of this disorder is still unknown. There is neither any confirmed laboratory test nor any effective therapeutic strategy to diagnose or cure it. To search for biomarkers for early detection and exploration of the disease mechanisms, here, we investigated the protein expression signatures of peripheral blood mononuclear cells (PBMCs) in autistic children compared with healthy controls by using isobaric tags for relative and absolute quantitation (iTRAQ) proteomics approach. The results showed a total of 41 proteins as differentially expressed in autistic group as compared to control. These proteins are found associated with metabolic pathways, endoplasmic reticulum (ER) stress and protein folding, endocytosis, immune and inflammatory response, plasma lipoprotein particle organization, and cell adhesion. Among these, 17 proteins (13 up-regulated and four down-regulated) are found to be linked with mitochondria. Eight proteins including three already reported proteins in our previous studies were selected to be verified. Five already reported autism associated pro-inflammatory cytokines [interferon-γ (IFN-γ), interleukin-1β (IL-1β), IL-6, IL-12, and tumor necrosis factor-α (TNF-α)] were detected in plasma by enzyme-linked immunosorbent assay (ELISA) analysis. The results were consistent with proteomic results and reports from previous literature. These results proposed that PBMCs from autistic children might be activated, and ER stress, unfolded protein response (UPR), acute-phase response (APR), inflammatory response, and endocytosis may be involved in autism occurrence. These reported proteins may serve as potential biomarkers for early diagnosis of autism. More specifically, simultaneous detection of three proteins [complement C3 (C3), calreticulin (CALR), and SERPINA1] in the plasma and PBMCs could increase the authenticity of detection.

Project description:BackgroundAlzheimer's disease (AD) is the most common form of dementia, and it affects more women than men. Mitochondrial dysfunction (MD) plays a key role in AD, and it is detectable at an early stage of the degenerative process in peripheral tissues, such as peripheral mononuclear blood cells (PBMCs). However, whether these changes are also reflected in cerebral energy metabolism and whether sex-specific differences in mitochondrial function occur are not clear. Therefore, we estimated the correlation between mitochondrial function in PBMCs and brain energy metabolites and examined sex-specific differences in healthy participants to elucidate these issues.MethodsThe current pilot study included 9 male and 15 female healthy adults (mean age 30.8?±?7.1 years). Respiration and activity of mitochondrial respiratory complexes were measured using a Clarke-electrode (Oxygraph-2k system), and adenosine triphosphate (ATP) levels were determined using a bioluminescence-based assay in isolated PBMCs. Citrate synthase activity as a mitochondrial marker was measured using a photometric assay. Concentrations of brain energy metabolites were quantified in the same individuals using 1H-magnetic resonance spectroscopy (MRS).ResultsWe detected sex-associated differences in mitochondrial function. Mitochondrial complexes I, I+II, and IV and uncoupled respiration and electron transport system (ETS) capacity in PBMCs isolated from blood samples of females were significantly (p?<?0.05; p?<?0.01) higher compared to males. ATP levels in the PBMCs of female participants were approximately 10% higher compared to males. Citrate synthase (CS) activity, a marker of mitochondrial content, was significantly (p?<?0.05) higher in females compared to males. Sex-associated differences were also found for brain metabolites. The N-acetylaspartate (NAA) concentration was significantly higher in female participants compared to males in targeted regions. This difference was observed in white matter (WM) and an area with a high percentage (>?50%) of gray matter (GM) (p?<?0.05; p?<?0.01). The effect sizes indicated a strong influence of sex on these parameters. Sex-associated differences were found in PBMCs and brain, but the determined parameters were not significantly correlated.ConclusionsOur study revealed sex-associated differences in mitochondrial function in healthy participants. The underlying mechanisms must be elucidated in more detail, but our study suggests that mitochondrial function in PBMCs is a feasible surrogate marker to detect differences in mitochondrial function and energy metabolism in humans and it underscores the necessity of sex-specific approaches in therapies that target mitochondrial dysfunction.

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DNA Repair Capacity for Personalizing Risk and Treatment Response - Assay Development and Optimization in Human Peripheral Blood Mononuclear Cells (PBMCs).

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