Synchrotron Microbeam Radiation Therapy (MRT) relies on the spatial fractionation of the synchrotron photon beam into parallel micro-beams applying several hundred of grays in their paths. Several works have reported the therapeutic interest of the radiotherapy modality at preclinical level, but biological mechanisms responsible for the described efficacy are not fully understood to date. The aim of this study was to identify the early transcriptomic responses of normal brain and glioma tissue in rats after MRT irradiation (400Gy). The transcriptomic analysis of similarly irradiated normal brain and tumor tissues was performed 6 hours after irradiation of 9 L orthotopically tumor-bearing rats. Pangenomic analysis revealed 1012 overexpressed and 497 repressed genes in the irradiated contralateral normal tissue and 344 induced and 210 repressed genes in tumor tissue. These genes were grouped in a total of 135 canonical pathways. More than half were common to both tissues with a predominance for immunity or inflammation (64 and 67% of genes for normal and tumor tissues, respectively). Several pathways involving HMGB1, toll-like receptors, C-type lectins and CD36 may serve as a link between biochemical changes triggered by irradiation and inflammation and immunological challenge. Most immune cell populations were involved: macrophages, dendritic cells, natural killer, T and B lymphocytes. Among them, our results highlighted the involvement of Th17 cell population, recently described in tumor. The immune response was regulated by a large network of mediators comprising growth factors, cytokines, lymphokines. In conclusion, early response to MRT is mainly based on inflammation and immunity which appear therefore as major contributors to MRT efficacy.

Most patients with pseudohypoparathyroidism type 1b (PHP-1b) display a loss of imprinting (LOI) encompassing the GNAS locus resulting in PTH resistance. In other imprinting disorders, such as Russell-Silver or Beckwith-Wiedemann syndrome, we and others have shown that the LOI is not restricted to one imprinted locus but may affect other imprinted loci for some patients. Therefore, we hypothesized that patients with PHP-1b might present multilocus imprinting defects. We investigated, in 63 patients with PHP-1b, the methylation pattern of eight imprinted loci: GNAS, ZAC1, PEG1/MEST, ICR1, and ICR2 on chromosome 11p15, SNRPN, DLK1/GTL2 IG-DMR, and L3MBTL1. We found multilocus imprinting defects in four PHP-1b patients carrying broad LOI at the GNAS locus (1) simultaneous hypermethylation at L3MBTL1 differentially methylated region 3 (DMR3), and hypomethylation at PEG1/MEST DMR (n = 1), (2) hypermethylation at the L3MBTL1 (DMR3) (n = 1) and at the DLK1/GTL2 IG-DMR (n = 1), and (3) hypomethylation at the L3MBTL1 DMR3 (n = 1). We suggest that mechanisms underlying multilocus imprinting defects in PHP-1b differ from those of other imprinting disorders having only multilocus loss of methylation. Furthermore, our results favor the hypothesis of "epidominance", that is, the phenotype is controlled by the most severely affected imprinted locus.

Glioblastomas are the most common primary brain tumors, highly vascularized, infiltrating, and resistant to current therapies. This cancer leads to a fatal outcome in less than 18 months. The aggressive behavior of glioblastomas, including resistance to current treatments and tumor recurrence, has been attributed to glioma stemlike/progenitor cells. The transcription factor EGR1 (early growth response 1), a member of a zinc finger transcription factor family, has been described as tumor suppressor in gliomas when ectopically overexpressed. Although EGR1 expression in human glioblastomas has been associated with patient survival, its precise location in tumor territories as well as its contribution to glioblastoma progression remain elusive. In the present study, we show that EGR1-expressing cells are more frequent in high grade gliomas where the nuclear expression of EGR1 is restricted to proliferating/progenitor cells. We show in primary cultures of glioma stemlike cells that EGR1 contributes to stemness marker expression and proliferation by orchestrating a PDGFA-dependent growth-stimulatory loop. In addition, we demonstrate that EGR1 acts as a positive regulator of several important genes, including SHH, GLI1, GLI2, and PDGFA, previously linked to the maintenance and proliferation of glioma stemlike cells.

Synchrotron microbeam radiation therapy (MRT) relies on the spatial fractionation of a synchrotron beam into parallel micronwide beams allowing deposition of hectogray doses. MRT controls the intracranial tumor growth in rodent models while sparing normal brain tissues. Our aim was to identify the early biological processes underlying the differential effect of MRT on tumor and normal brain tissues. The expression of 28,000 transcripts was tested by microarray 6 hr after unidirectional MRT (400 Gy, 50 mm-wide microbeams, 200 mm spacing). The specific response of tumor tissues to MRT consisted in the significant transcriptomic modulation of 431 probesets (316 genes). Among them, 30 were not detected in normal brain tissues, neither before nor after MRT. Areg, Trib3 and Nppb were down-regulated, whereas all others were up-regulated. Twenty-two had similar expression profiles during the 2 weeks observed after MRT, including Ccnb1, Cdc20, Pttg1 and Plk1 related to the mitotic role of the Pololike kinase (Plk) pathway. The up-regulation of Areg expression may indicate the emergence of survival processes in tumor cells triggered by the irradiation; while the modulation of the "mitotic role of Plk1" pathway, which relates to cytokinetic features of the tumor observed histologically after MRT, may partially explain the control of tumor growth by MRT. The identification of these tumor-specific responses permit to consider new strategies that might potentiate the antitumoral effect of MRT.Microbeam radiation therapy (MRT) is a novel form of preclinical radiotherapy based on the spatial fractionation of an incident synchrotron beam into arrays of parallel microbeams, which are typically few tens microns wide and separated on centre by few hundred microns (for review 1 ). This unique irradiation geometry allows the deposition of hectogray doses in tumors. Remarkably, brain tumors can be palliated by MRT in small animals with limited damage to normal cerebral tissues.2,3 This differential effect relates to the radioresistance of normal brain vessels to MRT for doses up to 1,000 Gy 4,5 , whereas MRT induces a denudation of tumor vessel endothelium, a decrease in tumor blood volume, 6,7 and tumor hypoxia.7 However, other mechanisms may contribute to the differential effects of MRT on tumor vs. normal tissues, e.g., direct impact of ionising radiation on tumor cells occurring before the vascular effect 6 and communication between lethally irradiated cells in the microbeam's path and less damaged cells sited in-between.8 All biological mechanisms underlying these differential effects remain to identify.Increasing the knowledge about the differential response between normal and tumor tissue to MRT is crucial because the identification of processes highly specific for the tumor tissue could be exploited to increase tumor control without disturbing the repair and maintenance of surrounding normal brain tissues. Specific cellular and molecular responses could be used to design efficient and safe adjuvant therapies.We have recently d...

There is great interest in understanding how the cancer stem cell population may be maintained in solid tumors. Here, we show that tumor cells exhibiting stem-like properties and expression of pluripotency markers NANOG and OCT4 can arise from original differentiated tumor cells freshly isolated from human glioblastomas (GBM) and that have never known any serum culture conditions. Induction of EGR1 by EGFR/ERK signaling promoted cell conversion from a less aggressive, more differentiated cellular state to a self-renewing and strongly tumorigenic state, expressing NANOG and OCT4. Expression of these pluripotency markers occurred before the cells re-entered the cell cycle, demonstrating their capacity to change and dedifferentiate without any cell divisions. In differentiated GBM cells, ERK-mediated repression of miR-199a-3p induced EGR1 protein expression and triggered dedifferentiation. Overall, this signaling pathway constitutes an ERK-mediated "toggle switch" that promotes pluripotency marker expression and stem-like features in GBM cells.Significance: This study defines an ERK-mediated molecular mechanism of dedifferentiation of GBM cells into a stem-like state, expressing markers of pluripotency.See related commentary by Koncar and Agnihotri, p. 3195

GATA1s-induced stage-specific differentiation delay increases immature megakaryocytes in vivo and in vitro, during development. Differentiation delay is associated with increased numbers of cells in S-phase and reduced apoptosis.

Scientific Category: Haematopoiesis and Stem Cells Key PointsGATA1s-induced stage-specific differentiation delay increases immature megakaryocytes in vivo and in vitro, during development.Differentiation delay is associated with increased numbers of cells in S-phase and reduced apoptosis. AbstractThe megakaryocyte/erythroid Transient Myeloproliferative Disorder (TMD) in newborns with Down Syndrome (DS) occurs when N-terminal truncating mutations of the hemopoietic transcription factor GATA1, that produce GATA1short protein (GATA1s), are acquired early in development. Prior work has shown that murine GATA1s, by itself, causes a transient yolk sac myeloproliferative disorder. However, it is unclear where in the hemopoietic cellular hierarchy GATA1s exerts its effects to produce this myeloproliferative state. Here, through a detailed examination of hemopoiesis from murine GATA1s ES cells and GATA1s embryos we define defects in erythroid and megakaryocytic differentiation that occur relatively in hemopoiesis.GATA1s causes an arrest late in erythroid differentiation in vivo, and even more profoundly in ES-cell derived cultures, with a marked reduction of Ter-119 cells and reduced erythroid gene expression. In megakaryopoiesis, GATA1s causes a differentiation delay at a specific stage, with accumulation of immature, kitexpressing CD41 hi megakaryocytic cells. In this specific megakaryocytic compartment, there are increased numbers of GATA1s cells in S-phase of cell cycle and reduced number of apoptotic cells compared to GATA1 cells in the same cell compartment. There is also a delay in maturation of these immature GATA1s megakaryocytic lineage cells compared to GATA1 cells at the same stage of differentiation. Finally, even when GATA1s megakaryocytic cells mature, they mature aberrantly with altered megakaryocyte-specific gene expression and activity of the mature megakaryocyte enzyme, acetylcholinesterase. These studies pinpoint the hemopoietic compartment where GATA1s megakaryocyte myeloproliferation occurs, defining where molecular studies should now be focussed to understand the oncogenic action of GATA1s.

<p>this text corresponds to the legends of supplemental tables 1 to 3 and supplemental figures 1 to 14</p>