) describes the cloning of a fulllength cDNA corresponding to a candidate cell surface HP/HS interacting protein, HIP, expressed by a variety of human epithelia. A synthetic peptide was synthesized corresponding to an amino acid sequence predicted from the cDNA sequence and used to prepare a rabbit polyclonal antibody. This antibody reacted with a protein with an apparent M r of 24,000 by SDS-polyacrylamide gel electrophoresis that was highly enriched in the 100,000 ؋ g particulate fraction of RL95 cells. This molecular weight is similar to that of the protein expressed by 3T3 cells transfected with HIP cDNA. HIP was solubilized from this particulate fraction with NaCl concentrations 0.8 M demonstrating a peripheral association consistent with the lack of a membrane spanning domain in the predicted cDNA sequence. HIP was not released by heparinase digestion suggesting that the association is not via membrane-bound HS proteoglycans. NaCl-solubilized HIP bound to heparin-agarose in physiological saline and eluted with NaCl concentrations of 0.75 M and above. Furthermore, incubation of Heparan sulfate proteoglycans (HSPGs) 1 located either on cell surfaces or in extracellular matrices are found in nearly all mammalian tissues (1-5). Functionally, HSPGs and a variety of HP/HS-binding proteins have been shown to participate in a diverse range of biological processes such as cell attachment, growth factor binding, cell proliferation, migration, morphogenesis, and viral pathogenicity (6 -8). Several lines of evidence indicate that HSPGs play an important role during the initial attachment of the apical plasma membrane of trophectodermal cells of the blastocyst to the apical plasma membrane of the uterine epithelium. In mice, HSPGs are expressed on the cell surfaces of two-cell stage and post-implantation stage embryos (9). Furthermore, blastocyst attachment to laminin, fibronectin, and isolated mouse uterine epithelial cells is inhibited by HP. Embryo attachment also is inhibited by the treatment of embryos with HP/HS lyases or inhibitors of proteoglycan biosynthesis (10, 11). Immunological studies of murine embryo implantation sites indicated that the core protein of the basement membrane HSPG, perlecan, and HP/HS chains are located between the apical cell surfaces of trophectodermal cells and uterine epithelial cells during the peri-implantation stage (12). Expression of perlecan on the external trophectodermal surface correlates with acquisition of attachment competence in vitro as well. Externally disposed H/HS-binding sites have been described on the cell surface of mouse uterine epithelial cells (13). Furthermore, using a heterologous cell adhesion assay, we demonstrated that HP/HS-like glycosaminoglycans participate in the initial attachment between two human cell lines, JAR and RL95, used to mimic the initial attachment of the human embryonic trophectoderm to human uterine epithelial cells, respectively (14). As is the case for mouse uterine epithelia, the human uterine epithelial cell line, RL95, has specific,...

p53 is an important mediator of the cellular stress response with roles in cell cycle control, DNA repair, and apoptosis. 53BP2, a p53-interacting protein, enhances p53 transactivation, impedes cell cycle progression, and promotes apoptosis through unknown mechanisms. We now demonstrate that endogenous 53BP2 levels increase following UV irradiation induced DNA damage in a p53-independent manner. In contrast, we found that the presence of a wild-type (but not mutant) p53 gene suppressed 53BP2 steady-state levels in cell lines with defined p53 genotypes. Likewise, expression of a tetracycline-regulated wild-type p53 cDNA in p53-null fibroblasts caused a reduction in 53BP2 protein levels. However, 53BP2 levels were not reduced if the tetracycline-regulated p53 cDNA was expressed after UV damage in these cells. This suggests that UV damage activates cellular factors that can relieve the p53-mediated suppression of 53BP2 protein. To address the physiologic significance of 53BP2 induction, we utilized stable cell lines with a ponasterone A-regulated 53BP2 cDNA. Conditional expression of 53BP2 cDNA lowered the apoptotic threshold and decreased clonogenic survival following UV irradiation. Conversely, attenuation of endogenous 53BP2 induction with an antisense oligonucleotide resulted in enhanced clonogenic survival following UV irradiation. These results demonstrate that 53BP2 is a DNA damage-inducible protein that promotes DNA damage-induced apoptosis. Furthermore, 53BP2 expression is highly regulated and involves both p53-dependent and p53-independent mechanisms. Our data provide new insight into 53BP2 function and open new avenues for investigation into the cellular response to genotoxic stress. p53 is mutated in more than 50% of human cancers and plays a pivotal role in mediating cellular responses to stress signals such as DNA damage and hypoxia (24,25

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This study explores the changes in expression of microRNA (miRNA) and related genes under simulated microgravity conditions. In comparison with static 1 ؋ g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. miRNA has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. However, very little is known about the effect of altered gravity on miRNA expression. To test the hypothesis that the miRNA expression profile would be altered in zero gravity resulting in altered regulation of gene expression leading to metabolic or functional changes in cells, we cultured TK6 human lymphoblastoid cells in a high aspect ratio vessel (bioreactor) for 72 h either in the rotating condition to model microgravity in space or in the static condition as a control. Expression of several miRNAs was changed significantly in the simulated microgravity condition including miR-150, miR-34a, miR-423-5p, miR-22, miR-141, miR-618, and miR-222. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA microarray and validated the related genes using quantitative RT-PCR. Expression of several transcription factors including EGR2, ETS1, and c-REL was altered in simulated microgravity conditions. Taken together, the results reported here indicate that simulated microgravity alters the expression of miRNAs and genes in TK6 cells. To our knowledge, this study is the first to report the effects of simulated microgravity on the expression of miRNA and related genes.

Living organisms are constantly threatened by environmental DNA-damaging agents, including UV and ionizing radiation (IR). Repair of various forms of DNA damage caused by IR is normally thought to follow lesion-specific repair pathways with distinct enzymatic machinery. DNA double strand break is one of the most serious kinds of damage induced by IR, which is repaired through double strand break (DSB) repair mechanisms, including homologous recombination (HR) and non-homologous end joining (NHEJ). However, recent studies have presented increasing evidence that various DNA repair pathways are not separated, but well interlinked. It has been suggested that non-DSB repair mechanisms, such as Nucleotide Excision Repair (NER), Mismatch Repair (MMR) and cell cycle regulation, are highly involved in DSB repairs. These findings revealed previously unrecognized roles of various non-DSB repair genes and indicated that a successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems. One of our recent studies found that suppressed expression of non-DSB repair genes, such as XPA, RPA and MLH1, influenced the yield of IR induced micronuclei formation and/or chromosome aberrations, suggesting that these genes are highly involved in DSB repair and DSB-related cell cycle arrest, which reveals new roles for these gene products in the DNA repair network. In this review, we summarize current progress on the function of non-DSB repair-related proteins, especially those that participate in NER and MMR pathways, and their influence on DSB repair. In addition, we present our developing view that the DSB repair mechanisms are more complex and are regulated by not only the well known HR/NHEJ pathways, but also a systematically coordinated cellular network.