Arsenic is widely distributed in nature and all organisms possess regulatory mechanisms to evade toxicity and acquire tolerance. Yet, little is known about arsenic sensing and signaling mechanisms or about their impact on tolerance and detoxification systems. Here, we describe a novel role of the S. cerevisiae mitogen-activated protein kinase Hog1p in protecting cells during exposure to arsenite and the related metalloid antimonite. Cells impaired in Hog1p function are metalloid hypersensitive, whereas cells with elevated Hog1p activity display improved tolerance. Hog1p is phosphorylated in response to arsenite and this phosphorylation requires Ssk1p and Pbs2p. Arsenite-activated Hog1p remains primarily cytoplasmic and does not mediate a major transcriptional response. Instead, hog1⌬ sensitivity is accompanied by elevated cellular arsenic levels and we demonstrate that increased arsenite influx is dependent on the aquaglyceroporin Fps1p. Fps1p is phosphorylated on threonine 231 in vivo and this phosphorylation critically affects Fps1p activity. Moreover, Hog1p is shown to affect Fps1p phosphorylation. Our data are the first to demonstrate Hog1p activation by metalloids and provides a mechanism by which this kinase contributes to tolerance acquisition. Understanding how arsenite/antimonite uptake and toxicity is modulated may prove of value for their use in medical therapy.

Neutrophil apoptosis is important for the termination of inflammatory reactions, in that it ensures placid clearance of these potently cytotoxic cells. Various proinflammatory cytokines delay neutrophil apoptosis, which may result in accumulation of these cells, sometimes accompanied by tissue destruction, potentially leading to various inflammatory disease states. Rheumatoid arthritis (RA) is characterized frequently by elevated levels of the acute-phase reactant serum amyloid A (SAA) in circulation and in tissues. SAA is emerging as a cytokine-like molecule with the ability to activate various proinflammatory processes, many of which involve signaling via the formyl peptide receptor-like 1 (FPRL1). In this study, we show that SAA, purified from plasma from RA patients or in recombinant form, suppressed apoptosis of human neutrophils. Blocking FPRL1 did not lessen the antiapoptotic effects of SAA, implying the action of a receptor distinct from FPRL1. In contrast, antagonists of the nucleotide receptor P2X7 abrogated the antiapoptotic effect of SAA completely but did not block intracellular calcium transients evoked by SAA stimulation. Based on these results and also the finding that blocking P2X7 inhibited antiapoptotic actions of unrelated stimuli (LPS and GM-CSF), we propose that P2X7 is a general mediator of antiapoptotic signaling in neutrophils rather than a bona fide SAA receptor.

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The mut-T homolog-1 (MTH1) inhibitor TH588 has shown promise in preclinical cancer studies but its targeting specificity has been questioned. Alternative mechanisms for the anti-cancer effects of TH588 have been suggested but the question remains unresolved. Here, we performed an unbiased CRISPR screen on human lung cancer cells to identify potential mechanisms behind the cytotoxic effect of TH588. The screen identified pathways and complexes involved in mitotic spindle regulation. Using immunofluorescence and live cell imaging, we showed that TH588 rapidly reduced microtubule plus-end mobility, disrupted mitotic spindles, and prolonged mitosis in a concentration-dependent but MTH1-independent manner. These effects activated a USP28-p53 pathway – the mitotic surveillance pathway – that blocked cell cycle reentry after prolonged mitosis; USP28 acted upstream of p53 to arrest TH588-treated cells in the G1-phase of the cell cycle. We conclude that TH588 is a microtubule-modulating agent that activates the mitotic surveillance pathway and thus prevents cancer cells from re-entering the cell cycle.

IntroductionPotential stem cell niches (SNs) were recently reported in intervertebral discs (IVDs) and knee joints (KJs) in different mammals (located adjacent to the epiphyseal plate; EP). The aim here was to examine further possible cellular migration and migration directions of cells originating from niches possibly involved in regeneration of cartilaginous tissues in the IVD and in the KJ regions in adult mammals.MethodsIn total, 33 rabbits were used in studies A through C.A. IVD cells were sorted; fluorescence-activated cell sorting (FACS) by size (forward scatter; ≤10 μm or >10 μm or GDF5+ cells (anti-GDF5 antibody). Sorted cells, labeled with cell tracer (carboxyfluorescein-diacetate-succinimidyl ester; CDFA-SE) were applied on IVD explants in vitro. Migrating cells/distance was evaluated by fluorescence- and confocal-microscopy (FC).B. DNA labeling was performed with BrdU (oral administration). Animals were killed (14 to 56 days), KJs collected, and BrdU+ cells visualized with immunohistochemistry (IHC)/anti-BrdU antibody in SN and articular cartilage (AC).C. Cell tracer: (Fe-nanoparticles: Endorem) were injected into SNs of IVDs (LI-LV) and KJs (tibia). Animals were killed after 2 to 6 weeks. Fe-labeled cells were traced by ferric-iron staining (Prussian blue reaction; Mallory method).ResultsA. GDF5+ cells and ≤10-μm cells displayed the best migration capability in IVD explants. GDF5+ cells were detected at a tissue depth of 1,300 μm (16 days). B. BrdU+ cells were observed in early time points in niches of KJs, and at later time points in AC, indicating a gradual migration of cells. C. Fe+ cells were detected in IVDs; in annulus fibrosus (AF) in 11 of 12 animals and in nucleus pulposus (NP) in two of 12 animals. In AC (tibia), Fe+ cells were detected in six of 12 animals. In the potential migration route (PMR), from niches toward the IVD, Fe+ cells (three of 12 animals) and in PMR toward AC (KJs) (six of 12 animals) were detected.ConclusionsResults indicate similar cellular migration patterns in cartilage regions (IVD and KJs) with migration from stem cell niche areas into the mature cartilaginous tissues of both the KJs and the IVD. These findings of a cellular migration pattern in mature cartilage are of interest from tissue-repair and engineering perspectives.