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Background:Elevated APOBEC3B expression in tumours correlates with a kataegic pattern of localised hypermutation. We assessed the cellular phenotypes associated with high-level APOBEC3B expression and the influence of p53 status on these phenotypes using an isogenic system.Methods:We used RNA interference of p53 in cells with inducible APOBEC3B and assessed DNA damage response (DDR) biomarkers. The mutational effects of APOBEC3B were assessed using whole-genome sequencing. In vitro small-molecule inhibitor sensitivity profiling was used to identify candidate therapeutic vulnerabilities.Results:Although APOBEC3B expression increased the incorporation of genomic uracil, invoked DDR biomarkers and caused cell cycle arrest, inactivation of p53 circumvented APOBEC3B-induced cell cycle arrest without reversing the increase in genomic uracil or DDR biomarkers. The continued expression of APOBEC3B in p53-defective cells not only caused a kataegic mutational signature but also caused hypersensitivity to small-molecule DDR inhibitors (ATR, CHEK1, CHEK2, PARP, WEE1 inhibitors) as well as cisplatin/ATR inhibitor and ATR/PARP inhibitor combinations.Conclusions:Although loss of p53 might allow tumour cells to tolerate elevated APOBEC3B expression, continued expression of this enzyme might impart a number of therapeutic vulnerabilities upon tumour cells.

The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell.

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Fibrocartilage degeneration is the earliest pathologic finding in navicular disease but remains difficult to detect, even with magnetic resonance (MR) imaging. We hypothesized that injection of the navicular bursa with saline would improve accuracy of MR imaging evaluation of palmar fibrocartilage. Thoracic limbs were collected from 11 horses within 6 h of death. Imaging was performed with a 1.5 T magnet using sagittal 2D proton density and transverse 3D FLASH sequences with fat saturation. For the purpose of determining sensitivity and specificity of the MR images, fibrocartilage was classified as normal or abnormal, based on combination of the findings of gross and microscopic pathology. Thickness of fibrocartilage was measured on histologic sections and corresponding transverse FLASH MR images before and after injection of saline. A paired Student's t-test was used for comparison of measurements. Partial thickness fibrocartilage loss was present in 6 of 22 limbs. Sensitivity of precontrast MR images for detection of lesions was 100% while specificity was 6%. Saline MR arthrography resulted in both sensitivity and specificity of 100% based on consensus review. Mean histologic fibrocartilage thickness was 0.75 AE 0.12 mm. Mean fibrocartilage thickness on precontrast transverse FLASH images was 0.93 AE 0.065 and 0.73 AE 0.09 mm on postsaline images. The histologic cartilage thickness was signficantly different from that in precontrast images (Po0.001) but not in images acquired after saline injection (P ¼ 0.716). Based on our results, and using pulse sequences as described herein, navicular fibrocartilage can only be evaluated reliably for the presence of partial thickness lesions after intrabursal injection of saline.

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