The GCN2/eIF2αK stress kinase regulates PP1 to ensure mitotic fidelity.

GCN2/eIF2αK4 is exclusively seen as an eIF2α kinase, which regulates reprogramming of protein translation in response to stress. Here, we show that GCN2 has an unexpected role in unstressed cells as a regulator of mitosis. This function is not through its canonical role in translation reprogramming, but through the regulation of two previously unidentified substrates, PP1α and γ. In the absence of GCN2 function, timing and levels of phosphorylation of key mitotic players are altered, leading to aberrant chromosome alignment, missegregating chromosomes, elevated number of tripolar spindles, and a delay in progression through mitosis. Pharmacological inhibition of GCN2 results in similar effects and is synergistic with Aurora A inhibition in causing more severe mitotic errors and cell death. We suggest that GCN2-dependent phosphorylation of PP1α and γ restrains their activity and this is important to ensure the timely regulation of phosphorylation of several PP1 substrates during early mitosis. These findings highlight a druggable PP1 inhibitor and open new avenues of research on the therapeutic potential of GCN2 inhibitors.

Predictive biomarkers for 5-ALA-PDT can lead to personalized treatments and overcome tumor-specific resistances.

BACKGROUND: Photodynamic therapy (PDT) is a minimally invasive, clinically approved therapy with numerous advantages over other mainstream cancer therapies. 5-aminolevulinic acid (5-ALA)-PDT is of particular interest, as it uses the photosensitiser PpIX, naturally produced in the heme pathway, following 5-ALA administration. Even though 5-ALA-PDT shows high specificity to cancers, differences in treatment outcomes call for predictive biomarkers to better stratify patients and to also diversify 5-ALA-PDT based on each cancer's phenotypic and genotypic individualities. AIMS: The present study seeks to highlight key biomarkers that may predict treatment outcome and simultaneously be exploited to overcome cancer-specific resistances to 5-ALA-PDT. METHODS AND RESULTS: We submitted two glioblastoma (T98G and U87) and three breast cancer (MCF7, MDA-MB-231, and T47D) cell lines to 5-ALA-PDT. Glioblastoma cells were the most resilient to 5-ALA-PDT, while intracellular production of 5-ALA-derived protoporphyrin IX (PpIX) could not account for the recorded PDT responses. We identified the levels of expression of ABCG2 transporters, ferrochelatase (FECH), and heme oxygenase (HO-1) as predictive biomarkers for 5-ALA-PDT. GPX4 and GSTP1 expression vs intracellular glutathione (GSH) levels also showed potential as PDT biomarkers. For T98G cells, inhibition of ABCG2, FECH, HO-1, and/or intracellular GSH depletion led to profound PDT enhancement. Inhibition of ABCG2 in U87 cells was the only synergistic adjuvant to 5-ALA-PDT, rendering the otherwise resistant cell line fully responsive to 5-ALA-PDT. ABCG2 or FECH inhibition significantly enhanced 5-ALA-PDT-induced MCF7 cytotoxicity, while for MDA-MB-231, ABCG2 inhibition and intracellular GSH depletion conferred profound synergies. FECH inhibition was the only synergism to ALA-PDT for the most susceptible among the cell lines, T47D cells. CONCLUSION: This study demonstrates the heterogeneity in the cellular response to 5-ALA-PDT and identifies biomarkers that may be used to predict treatment outcome. The study also provides preliminary findings on the potential of inhibiting specific molecular targets to overcome inherent resistances to 5-ALA-PDT.

Photodynamic Efficacy of Cercosporin in 3D Tumor Cell Cultures.

In the present work, we study the photodynamic action of cercosporin (cerco), a naturally occurring photosensitizer, on human cancer multicellular spheroids. U87 spheroids exhibit double the uptake of cerco than T47D and T98G spheroids as shown by flow cytometry on the single cell level. Moreover, cerco is efficiently internalized by cells throughout the spheroid as shown by confocal microscopy, for all three cell lines. Despite their higher cerco uptake, U87 spheroids show the least vulnerability to cerco-PDT, in contrast to the other two cell lines (T47D and T98G). While 300 µm diameter spheroids consistently shrink and become necrotic after cerco PDT, bigger spheroids (>500 µm) start to regrow following blue-light PDT and exhibit high viability. Cerco-PDT was found to be effective on bigger spheroids reaching 1mm in diameter especially under longer exposure to yellow light (~590 nm). In terms of metabolism, T47D and T98G undergo a complete bioenergetic collapse (respiration and glycolysis) as a result of cerco-PDT. U87 spheroids also experienced a respiratory collapse following cerco-PDT, but retained half their glycolytic activity.

Cytotoxic and Photocytotoxic Effects of Cercosporin on Human Tumor Cell Lines.

Cercosporin is a naturally occurring perylenequinone. Although other perylenequinones have been extensively studied as photosensitizers in photodynamic therapy of cancer (PDT), cercosporin has been studied in this light only within the remits of phytopathology. Herein, we investigated the photocytotoxicity of cercosporin against two glioblastoma multiforme (T98G and U87) and one breast adenocarcinoma (MCF7) human cell lines. Cercosporin was found to be a potent singlet oxygen producer upon 532 nm excitation, while its cell loading was similar for MCF7 and U87, but approximately threefold higher for T98G cells. The subcellular localization of cercosporin was in all cases in both mitochondria and the endoplasmic reticulum. Light irradiation of cercosporin-incubated cells around 450 nm showed that T98G cells were more susceptible to cercosporin PDT, mainly due to their higher cercosporin uptake. Metabolic studies before and 1 h following cercosporin PDT showed that cercosporin PDT instigated a bioenergetic collapse in both the respiratory and glycolytic activities of all cell lines. In the dark, cercosporin exhibited a synergistic cytotoxicity with copper only in the most respiratory cell lines (MCF7 and T98G). Cercosporin is a potent photosensitizer, but with a short activation wavelength, mostly suitable for superficial PDT treatments, especially when it is necessary to avoid perforations.

Cellular reactions and compensatory tissue re-organization during spontaneous recovery after spinal cord injury in neonatal mice.

Following incomplete spinal cord injuries, neonatal mammals display a remarkable degree of behavioral recovery. Previously, we have demonstrated in neonatal mice a wholesale re-establishment and reorganization of synaptic connections from some descending axon tracts (Boulland et al.: PLoS One 8 (2013)). To assess the potential cellular mechanisms contributing to this recovery, we have here characterized a variety of cellular sequelae following thoracic compression injuries, focusing particularly on cell loss and proliferation, inflammation and reactive gliosis, and the dynamics of specific types of synaptic terminals. Early during the period of recovery, regressive events dominated. Tissue loss near the injury was severe, with about 80% loss of neurons and a similar loss of axons that later make up the white matter. There was no sign of neurogenesis, no substantial astroglial or microglial proliferation, no change in the ratio of M1 and M2 microglia and no appreciable generation of the terminal complement peptide C5a. One day after injury the number of synaptic terminals on lumbar motoneurons had dropped by a factor of 2, but normalized by 6 days. The ratio of VGLUT1/2+ to VGAT+ terminals remained similar in injured and uninjured spinal cords during this period. By 24 days after injury, when functional recovery is nearly complete, the density of 5-HT+ fibers below the injury site had increased by a factor of 2.5. Altogether this study shows that cellular reactions are diverse and dynamic. Pronounced recovery of both excitatory and inhibitory terminals and an increase in serotonergic innervation below the injury, coupled with a general lack of inflammation and reactive gliosis, are likely to contribute to the recovery. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 928-946, 2017.

Epigenetic regulation of nestin expression during neurogenic differentiation of adipose tissue stem cells.

Adipose-tissue-derived stem cells (ASCs) have received considerable attention due to their easy access, expansion potential, and differentiation capacity. ASCs are believed to have the potential to differentiate into neurons. However, the mechanisms by which this may occur remain largely unknown. Here, we show that culturing ASCs under active proliferation conditions greatly improves their propensity to differentiate toward osteogenic, adipogenic, and neurogenic lineages. Neurogenic-induced ASCs express early neurogenic genes as well as markers of mature neurons, including voltage-gated ion channels. Nestin, highly expressed in neural progenitors, is upregulated by mitogenic stimulation of ASCs, and as in neural progenitors, then repressed during neurogenic differentiation. Nestin gene (NES) expression under these conditions appears to be regulated by epigenetic mechanisms. The neural-specific, but not muscle-specific, enhancer regions of NES are DNA demethylated by mitogenic stimulation, and remethylated upon neurogenic differentiation. We observe dynamic changes in histone H3K4, H3K9, and H3K27 methylation on the NES locus before and during neurogenic differentiation that are consistent with epigenetic processes involved in the regulation of NES expression. We suggest that ASCs are epigenetically prepatterned to differentiate toward a neural lineage and that this prepatterning is enhanced by demethylation of critical NES enhancer elements upon mitogenic stimulation preceding neurogenic differentiation. Our findings provide molecular evidence that the differentiation repertoire of ASCs may extend beyond mesodermal lineages.