10 research outputs found
Biomarkers of Disease and Treatment in Murine and Cynomolgus Models of Chronic Asthma
Background
Biomarkers facilitate early detection of disease and measurement of therapeutic efficacy, both at clinical and experimental levels. Recent advances in analytics and disease models allow comprehensive screening for biomarkers in complex diseases, such as asthma, that was previously not feasible.
Objective
Using murine and nonhuman primate (NHP) models of asthma, identify biomarkers associated with early and chronic stages of asthma and responses to steroid treatment.
Methods
The total protein content from thymic stromal lymphopoietin transgenic (TSLP Tg) mouse BAL fluid was ascertained by shotgun proteomics analysis. A subset of these potential markers was further analyzed in BAL fluid, BAL cell mRNA, and lung tissue mRNA during the stages of asthma and following corticosteroid treatment. Validation was conducted in murine and NHP models of allergic asthma.
Results
Over 40 proteins were increased in the BAL fluid of TSLP Tg mice that were also detected by qRT-PCR in lung tissue and BAL cells, as well as in OVA-sensitive mice and house dust mite-sensitive NHP. Previously undescribed as asthma biomarkers, KLK1, Reg3γ, ITLN2, and LTF were modulated in asthmatic mice, and Clca3, Chi3l4 (YM2), and Ear11 were the first lung biomarkers to increase during disease and the last biomarkers to decline in response to therapy. In contrast, GP-39, LCN2, sICAM-1, YM1, Epx, Mmp12, and Klk1 were good indicators of early therapeutic intervention. In NHP, AMCase, sICAM-1, CLCA1, and GP-39 were reduced upon treatment with corticosteroids.
Conclusions and clinical relevance
These results significantly advance our understanding of the biomarkers present in various tissue compartments in animal models of asthma, including those induced early during asthma and modulated with therapeutic intervention, and show that BAL cells (or their surrogate, induced sputum cells) are a viable choice for biomarker examination
A Functional Approach Reveals a Genetic and Physical Interaction between Ribonucleotide Reductase and CHK1 in Mammalian Cells
<div><p>Ribonucleotide reductase (RNR) enzyme is composed of the homodimeric RRM1 and RRM2 subunits, which together form a heterotetramic active enzyme that catalyzes the de novo reduction of ribonucleotides to generate deoxyribonucleotides (dNTPs), which are required for DNA replication and DNA repair processes. In this study, we show that ablation of RRM1 and RRM2 by siRNA induces G1/S phase arrest, phosphorylation of Chk1 on Ser345 and phosphorylation of γ-H2AX on S139. Combinatorial ablation of RRM1 or RRM2 and Chk1 causes a dramatic accumulation of γ-H2AX, a marker of double-strand DNA breaks, suggesting that activation of Chk1 in this context is essential for suppression of DNA damage. Significantly, we demonstrate for the first time that Chk1 and RNR subunits co-immunoprecipitate from native cell extracts. These functional genomic studies suggest that RNR is a critical mediator of replication checkpoint activation.</p></div
RRM1 and RRM2 subunits of Ribonucleotide reductase interact with Chk1 and Polα.
<p>(a) At 30 h after siRNA transfections of the luciferase, RRM1, and RRM2 siRNA duplexes, extracts were prepared for immunoprecipitations. Cells transfected with Luciferase were incubated with 1 mM HU for 30 min. Chk1 was immunoprecipitated from luciferase (positive control), Luciferase + HU, RRM1, and RRM2 depleted cells with Chk1 antibodies (MAb58D7) cross-linked to protein A and were immunoblotted as indicated. Chk1 immunoprecipitations were peptide blocked as a negative control. (b) Polα was immunoprecipitated from luciferase (positive control), luciferase +HU, RRM1, and RRM2 depleted cells depleted cells with Polα antibody (SJK132-20) cross-linked to protein G and Western blots were immunoblotted as indicated. (c) Whole cell extracts were immunoblotted as indicated.</p
% H2AX phosphorylation.
<p>Data represents 2-independent experiments performed in triplicates.</p><p>± denotes standard deviation.</p><p>% H2AX phosphorylation.</p
Quantitation (AUC) of dNTP & NTP pools in the U20S cells.
<p>Analysis was done in triplicate.</p><p>± denotes standard deviation.</p><p>nd  =  not detected.</p><p>Units of measurement are AUC (Area under the curve).</p><p>Quantitation (AUC) of dNTP & NTP pools in the U20S cells.</p
Percentage of cells in G1, S, or G2 phase.
<p>Percentage of cells in G1, S, or G2 phase.</p
RRM1/Chk1 and RRM2/Chk1 co-depletion enhances the accumulation of DNA damage, apoptotic response and effects cell proliferation in U2OS cells.
<p>Cells were transfected with Chk1, RRM1, RRM2, RRM1/Chk1, RRM2/Chk1, and Luciferase control (untreated or treated with 1 mM HU for the last 8 h) before harvesting at 30 h. At 30 h after siRNA transfections, (a) extracts were prepared and immunoblotted with the indicated antibodies. (b) DNA damage was assessed for γ-H2AX phosphorylation using flow cytometry. (c) Cells were collected at indicated time points indicated and analyzed for activated caspases. Data performed in duplicates. Error bars represent SD between experiments. (d) Cell proliferation was assessed with clonogenicity assay.</p
Quantitation of dNTPs and γ-H2AX phosphorylation in U20S cells following depletion of RRM1 and RRM2 subunits of Ribonucleotide reductase.
<p>Cells were transfected with RRM1, RRM2 and Luciferase control (untreated or treated with 1 µM GEM or 1 µM CAFdA for the last 2 h) before harvesting at 30 h. At 30 h after siRNA transfections, (a) NTP/dNTP extractions were prepared and quantified. (b) DNA damage was assessed for γ-H2AX phosphorylation using flow cytometry. Data performed in triplicates. Error bars represent standard deviations (SD) between experiments.</p
Boronotyrosine, a Borylated Amino Acid Mimetic with Enhanced Solubility, Tumor Boron Delivery, and Retention for the Re-emerging Boron Neutron Capture Therapy Field
Boron
neutron capture therapy (BNCT) is a re-emerging binary cellular
level cancer intervention that occurs through the interaction of a
cancer-specific 10boron (10B) drug and neutrons.
We created a new 10B drug, 3-borono-l-tyrosine
(BTS), that improves on the characteristics of the main historical
BNCT drug 4-borono-l-phenylalanine (BPA). BTS has up to 4
times greater uptake in vitro than BPA and increased cellular retention.
Like BPA, BTS uptake is mediated by the l-type amino acid
transporter-1 (LAT1) but is less sensitive to natural amino acid competition.
BTS can be formulated and bolus dosed at much higher levels than BPA,
resulting in 2–3 times greater boron delivery in vivo. Fast
blood clearance and greater tumor boron delivery result in superior
tumor-to-blood ratios. BTS boron delivery appears to correlate with
LAT1 expression. BTS is a promising boron delivery drug that has the
potential to improve modern BNCT interventions