Interlaboratory comparison of high-throughput protein biomarker assay quantifications for radiation exposure classification.

In the event of a widespread radiological incident, thousands of individuals will require rapid assessment of exposure using validated biodosimetry assays to inform clinical triage. In this scenario, multiple biodosimetry laboratories may be necessary for large-volume sample processing. To meet this need, we have developed a high-throughput assay for the rapid measurement of intracellular protein biomarkers in human peripheral blood samples using an Imaging Flow Cytometry (IFC) platform. The objective of this work was to harmonize and validate the reproducibility of our blood biomarker assay for radiation exposure across three IFC instruments, two located at Columbia University (CU) and the third at Health Canada. The Center for Radiological Research (CRR) at CU served as the central laboratory and reference instrument, where samples were prepared in triplicate, labeled with two radiation responsive leukocyte biomarkers (BAX and phosphor-p53 (Ser37)), and distributed for simultaneous interrogation by each IFC. Initial tests showed that significantly different baseline biomarker measurements were generated on each instrument when using the same acquisition settings, suggesting that harmonization of signal intensities is necessary. Subsequent tests harmonized biomarker measurements after irradiation by modulating laser intensity using two reference materials: unstained samples and standardized rainbow beads. Both methods generated measurements on each instrument without significant differences between the new and references instruments, allowing for the use of one master template to quantify biomarker expression across multiple instruments. Deming regression analyses of 0-5 Gy dose-response curves showed overall good correlation of BAX and p53 values across new and reference instruments. While Bland-Altman analyses indicated low to moderate instrument biases, ROC Curve analyses ultimately show successful discrimination between exposed and unexposed samples on each instrument (AUC values > 0.85).

Development of a human peripheral blood ex vivo model for rapid protein biomarker detection and applications to radiation biodosimetry.

In the event of a widespread radiological incident, thousands of people may be exposed to a wide range of ionizing radiation. In this unfortunate scenario, there will be a need to quickly screen a large number of people to assess the amount of radiation exposure and triage for medical treatment. In our earlier work, we previously identified and validated a panel of radiosensitive protein biomarkers in blood leukocytes, using the humanized-mouse and non-human primate (NHP) models. The objective of this work was to develop a high-throughput imaging flow-cytometry (IFC) based assay for the rapid measurement of protein biomarker expression in human peripheral blood samples irradiated ex vivo. In this assay design, peripheral human blood samples from healthy adult donors were exposed to 0-5 Gy X-irradiation ex vivo and cultured for up to 2 days. Samples were stained with a cocktail of surface antigens (CD66b, CD20, and CD3), fixed and permeabilized, and intracellularly stained for BAX (Bcl-2-associated X) protein, used here as a representative biomarker. Samples were interrogated by IFC, and a uniform analysis template was created to measure biomarker expression in heterogeneous and specific leukocyte subtype populations at each time point. In this human blood ex vivo model, we show that within gated populations of leukocyte subtypes, B-cells are highly radiosensitive with the smallest surviving fraction, followed by T-cells and granulocytes. Dose-dependent biomarker responses were measured in the lymphocytes, B-, and T-cell populations, but not in the granulocytes, with dose-response curves showing increasing fold changes in BAX protein expression up to Day 2 in lymphocyte populations. We present here the successful use of this ex vivo model for the development of radiation dose-response curves of a candidate protein biomarker towards future applications of dose reconstruction and biodosimetry.

Machine learning approach for quantitative biodosimetry of partial-body or total-body radiation exposures by combining radiation-responsive biomarkers.

During a large-scale radiological event such as an improvised nuclear device detonation, many survivors will be shielded from radiation by environmental objects, and experience only partial-body irradiation (PBI), which has different consequences, compared with total-body irradiation (TBI). In this study, we tested the hypothesis that applying machine learning to a combination of radiation-responsive biomarkers (ACTN1, DDB2, FDXR) and B and T cell counts will quantify and distinguish between PBI and TBI exposures. Adult C57BL/6 mice of both sexes were exposed to 0, 2.0-2.5 or 5.0 Gy of half-body PBI or TBI. The random forest (RF) algorithm trained on ½ of the data reconstructed the radiation dose on the remaining testing portion of the data with mean absolute error of 0.749 Gy and reconstructed the product of dose and exposure status (defined as 1.0 × Dose for TBI and 0.5 × Dose for PBI) with MAE of 0.472 Gy. Among irradiated samples, PBI could be distinguished from TBI: ROC curve AUC = 0.944 (95% CI: 0.844-1.0). Mouse sex did not significantly affect dose reconstruction. These results support the hypothesis that combinations of protein biomarkers and blood cell counts can complement existing methods for biodosimetry of PBI and TBI exposures.

Role of deubiquitinases in DNA damage response.

DNA damage response (DDR) serves as an integrated cellular network to detect cellular stress and react by activating pathways responsible for halting cell cycle progression, stimulating DNA damage repair, and initiating apoptosis. Efficient DDR protects cells from genomic instability while defective DDR can allow DNA lesions to go unrepaired, causing permanent mutations that will affect future generations of cells and possibly cause disease conditions such as cancer. Therefore, DDR mechanisms must be tightly regulated in order to ensure organismal health and viability. One major way of DDR regulation is ubiquitination, which has been long known to control DDR protein localization, activity, and stability. The reversal of this process, deubiquitination, has more recently come to the forefront of DDR research as an important new angle in ubiquitin-mediated regulation of DDR. As such, deubiquitinases have emerged as key factors in DDR. Importantly, deubiquitinases are attractive small-molecule drug targets due to their well-defined catalytic residues that provide a promising avenue for developing new cancer therapeutics. This review focuses on the emerging roles of deubiquitinases in various DNA repair pathways.

A Rapid and Precise Mutation-Activated Fluorescence Reporter for Analyzing Acute Mutagenesis Frequency.

Mutagenesis reporters are critical for quantifying genome stability. However, current methods rely on cell survival/death to report mutation, which takes weeks and prevents evaluation of acute or time-dependent changes. Existing methods also have other limitations, such as cell type restrictions. Using our discovery that mCherryFP fluorescence depends on residue Trp98, we replaced this codon with a stop codon to generate a mutation biosensor (termed CherryOFF), with a green fluorescence protein (GFP) as an internal control. We found that the red fluorescence of this biosensor is activated by a specific A/T-G/C nucleotide transition. Compared with the established hypoxanthine phosphoribosyl transferase assay, our reporter has similar or better ability to detect changes of mutation frequency induced by physical/chemical mutagens or manipulation of mutation-related genes. Furthermore, CherryOFF-GFP can report mutagenesis independently of cell-death events, can be adapted to many cell types, and can generate readouts within 1 day for the measurement of acute or time-dependent events.

XPC: Going where no DNA damage sensor has gone before.

XPC has long been considered instrumental in DNA damage recognition during global genome nucleotide excision repair (GG-NER). While this recognition is crucial for organismal health and survival, as XPC's recognition of lesions stimulates global genomic repair, more recent lines of research have uncovered many new non-canonical pathways in which XPC plays a role, such as base excision repair (BER), chromatin remodeling, cell signaling, proteolytic degradation, and cellular viability. Since the first discovery of its yeast homolog, Rad4, the involvement of XPC in cellular regulation has expanded considerably. Indeed, our understanding appears to barely scratch the surface of the incredible potential influence of XPC on maintaining proper cellular function. Here, we first review the canonical role of XPC in lesion recognition and then explore the new world of XPC function.

The deubiquitinating enzyme USP24 is a regulator of the UV damage response.

Regulation of p53 by ubiquitination and deubiquitination is important for its function. In this study, we demonstrate that USP24 deubiquitinates p53 in human cells. Functional USP24 is required for p53 stabilization, and p53 destabilization in USP24-depleted cells can be corrected by the forced expression of USP24. We show that USP24 depletion renders cells resistant to apoptosis after UV irradiation, consistent with the requirement of USP24 for p53 stabilization and PUMA activation in vivo. Additionally, purified USP24 protein is able to cleave ubiquitinated p53 in vitro. Importantly, cells with USP24 depletion exhibited significantly elevated mutation rates at the endogenous HPRT locus, implying an important role for USP24 in maintaining genome stability. Our data reveal that the USP24 deubiquitinase regulates the DNA damage response by directly targeting the p53 tumor suppressor.

Whole genome expression profiling shows that BRG1 transcriptionally regulates UV inducible genes and other novel targets in human cells.

UV irradiation is known to cause cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), and plays a large role in the development of cancer. Tumor suppression, through DNA repair and proper cell cycle regulation, is an integral factor in maintaining healthy cells and preventing development of cancer. Transcriptional regulation of the genes involved in the various tumor suppression pathways is essential for them to be expressed when needed and to function properly. BRG1, an ATPase catalytic subunit of the SWI/SNF chromatin remodeling complex, has been identified as a tumor suppressor protein, as it has been shown to play a role in Nucleotide Excision Repair (NER) of CPDs, suppress apoptosis, and restore checkpoint deficiency, in response to UV exposure. Although BRG1 has been shown to regulate transcription of some genes that are instrumental in proper DNA damage repair and cell cycle maintenance in response to UV, its role in transcriptional regulation of the whole genome in response to UV has not yet been elucidated. With whole genome expression profiling in SW13 cells, we show that upon UV induction, BRG1 regulates transcriptional expression of many genes involved in cell stress response. Additionally, our results also highlight BRG1's general role as a master regulator of the genome, as it transcriptionally regulates approximately 4.8% of the human genome, including expression of genes involved in many pathways. RT-PCR and ChIP were used to validate our genome expression analysis. Importantly, our study identifies several novel transcriptional targets of BRG1, such as ATF3. Thus, BRG1 has a larger impact on human genome expression than previously thought, and our studies will provide inroads for future analysis of BRG1's role in gene regulation.