Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism

Phenotype-dependent production of des-gamma-carboxy prothrombin in hepatocellular carcinoma

Background Des-gamma-carboxy prothrombin (DCP) is an established tumor marker for hepatocellular carcinoma (HCC), but the precise mechanism of its production remains unknown. We have recently demonstrated that cytoskeletal rearrangement during the phenotypic changes involved in epithelial mesenchymal transition (EMT) plays a crucial role in DCP production through the impairment of vitamin K uptake. However, DCP production in long-lasting severe hypoxic conditions with nutrient deprivation, such as transarterial embolization, remains unknown.Methods We examined the effects of long-lasting hypoxia with nutrient deprivation, as well as the constitutive expression of hypoxia-inducible factor (HIF)-1-alpha, on EMT status, DCP production, and protein synthesis in human hepatoma cell lines by enzyme-linked immunosorbent assay, immunofluorescent studies, and western blotting. Immunohistochemistry findings for DCP, anti-hepatocyte paraffin 1 (Hep Par 1), and vimentin were examined using human resected HCC samples.Results Both severe hypoxia with nutrient deprivation and HIF-1-alpha transfection led to the cessation of DCP production, by attenuating protein synthesis through the hypophosphorylation of mammalian target of rapamycin and its effector proteins, indicative of a further phenotypic shift involving impaired liver-specific protein synthesis. In immunohistochemistry, the distribution of DCP- and Hep Par 1-positive HCC cells was mostly similar and vimentin-positive HCC cells were frequently observed in the areas that were negative for Hep Par 1 and/or DCP.Conclusions HCC cells produce DCP when they undergo mild phenotypic changes. However, when HCC cells adopt mesenchymal properties they lose their capacity for protein synthesis, and the production of DCP is attenuated. Building upon our previous works, it appears that DCP could be a unique tumor marker that reflects the stepwise phenotypic changes of HCC

A modified protocol for accurate detection of cell fusion-mediated premature chromosome condensation in human peripheral blood lymphocytes

When interphase cells are fused with mitotic cells, loosely distributing chromatins in nuclei are induced to form prematurely condensed chromosomes. In this paper we report a modified protocol to unequivocally detect prematurely condensed chromosomes in human peripheral blood lymphocytes that were fused with mitotic Chinese hamster ovary (CHO) cells. To examine cell fusion-mediated premature chromosome condensation (PCC), we conducted morphological analysis by differential interference contrast microscopy and molecular cytogenetic analysis by fluorescence in situ hybridization using pan-centromeric and telomeric peptide nucleic acid (PNA) probes. These modified procedures may serve to improve the usefulness of the technique of PCC in cytogenetic investigations

Outline of the TEPCO Fukushima Dai-ichi Nuclear Power Plant accident.

The 2011 Off the Pacific Coast of Tohoku Earthquake struck the northeast part of Japan at 14:46 JST on 11 March 2011, triggering a tsunami with over 10m-height. The earthquake caused serious damages to the TEPCO Fukushima nuclear power plants (NPPs). Although the reactors at the NPPs automatically had shut down after the earthquake, the cooling systems and its power supply were damaged. The troubles in the cooling systems led to hydrogen explosions and core melting, resulting in a large amount of radioactive materials being released into the environment. The deposition of major radioactive materials, I-131, Cs-134 and Cs-137, on the ground induced the high ambient dose of radiation around the NPP, especially within 20 km radius, and even further distance in the northwest direction. Therefore, many people had to evacuate or stay indoors. Various agricultural and marine products were contaminated with radioactive materials; radioactive concentrations in some of them exceed over the provisional regulation values. Thus, the situation caused an anxiety among public

Clinical Impact of Radiation-Resistant Mesenchymal Stem Cells in Bone Marrow Deduced from Preclinical Studies

Mesenchymal stem cells in the bone marrow have attracted great interest over the past decades, not only as a basic scientific subject but also as a novel and advanced clinical tool. More than 300 mesenchymal stem cell-related clinical trials are currently registered in the world. Hematopoietic stem cells in bone marrow are extremely radiation-sensitive, whereas mesenchymal stem cells show considerable radiation-resistance. Intrinsic cellular mechanisms, including highly efficient reactive oxygen species-scavenging ability and active DNA damage response pathways, have been reported to explain the radiation-resistance of mesenchymal stem cells in the bone marrow. The precise interactions between residual host mesenchymal stem cells and donor mesenchymal stem cells at the time of bone marrow transplantation following whole-body irradiation, however, remain unknown. This short review summarizes our current understanding of the clinical impact of the radiation-resistance of endogenous mesenchymal stem cells in the bone marrow

Characteristics of three-dimensional prospectively isolated mouse bone marrow mesenchymal stem/stromal cell aggregates on nanoculture plates

Three-dimensional (3-D) aggregate culturing is useful for investigating the functional properties of mesenchymal stem/stromal cells (MSCs). For 3-D MSC analysis, however, pre-expansion of MSCs with two-dimensional (2-D) monolayer culturing must first be performed, which might abolish their endogenous properties. To avoid the need for 2-D expansion, we used prospectively isolated mouse bone marrow (BM)-MSCs and examined the differences in the biological properties of 2-D and 3-D MSC cultures. The BM-MSCs self-assembled into aggregates on nanoculture plates (NCP) that have nanoimprinted patterns with a low-cellular binding texture. The 3-D MSCs proliferated at the same rate as 2-D-cultured cells by only diffusion culture and secreted higher levels of pro-angiogenic factors such as vascular endothelial growth factor and hepatocyte growth factor (HGF). Conditioned medium from 3-D MSC cultures promoted more capillary formation than that of 2-D MSCs in an in vitro tube formation assay. Matrigel-implanted 3-D MSC aggregates tended to induce angiogenesis in host mice. The 3-D culturing on NCP induced alpha-fetoprotein (AFP) expression in MSCs without the application of AFP- or endodermal-inducible factors, possibly via an HGF-autocrine mechanism, and maintained their differentiation ability for adipocytes, osteocytes, and chondrocytes. Prospectively isolated mouse BM-MSCs expressed low/negative stemness-related genes including Oct3/4, Nanog, and Sox2, which were not enhanced by NCP-based 3-D culturing, suggesting that some of these cells differentiate into meso-endodermal layer cells. Culturing of prospectively isolated MSCs on NCP in 3-D allows the analysis of the biological properties of more closely endogenous BM-MSCs and might contribute to tissue engineering and repair

Developmentally regulated role for Ras-GRFs in coupling NMDA glutamate receptors to Ras, Erk and CREB

p140 Ras-GRF1 and p130 Ras-GRF2 constitute a family of calcium/calmodulin-regulated guanine–nucleotide exchange factors that activate the Ras GTPases. Studies on mice lacking these exchange factors revealed that both p140 Ras-GRF1 and p130 Ras-GRF2 couple NMDA glutamate receptors (NMDARs) to the activation of the Ras/Erk signaling cascade and to the maintenance of CREB transcription factor activity in cortical neurons of adult mice. Consistent with this function for Ras-GRFs and the known neuroprotective effect of CREB activity, ischemia-induced CREB activation is reduced in the brains of adult Ras-GRF knockout mice and neuronal damage is enhanced. Interestingly, in cortical neurons of neonatal animals NMDARs signal through Sos rather than Ras-GRF exchange factors, implying that Ras-GRFs endow NMDARs with functions unique to mature neurons