Search

Investigation of characteristics of cell- and nuclear-penetrating anti-double stranded (ds)DNA autoantibodies (autoAbs) is important to understand pathogenesis of lupus nephritis, but has not been clearly explored. The present study reports that three anti-dsDNA monoclonal autoAbs, which contain more than two arginine residues in their CDR3s of variable heavy domain (VH), penetrated into living murine mesangial cells and the cell nuclei. However, an anti-dsDNA monoclonal Ab (mAb) having only one arginine in the CDR3-VH did not penetrate cells. To assess the contribution of antigen-binding sites, especially the VH, in cell- and nuclear-penetration, we evaluated the characteristics of recombinant single chain Fv(scFv), VH, and variable light domain (VL) of a penetrating mAb. The scFv and VH domain, containing arginine in CDR3-VH maintained the ability to penetrate cells and the cell nuclei, whereas the VL domain, having no arginine in CDR3, did not penetrate cells. The penetratingm Abs, scFv, and VH activated ERK and increased cellular protein levels of Bcl-2, whereas the non-penetrating Ab and VL did not. The cell survival was decreased by the penetrating mAbs, scFv and VH, not by the non-penetrating mAb and VL. The data indicate that an antigen-binding site is required for cell-penetration and that positively-charged arginine residues in CDR3-VH contribute to the cell- and nuclear-penetrating ability of a subset of anti-dsDNA autoAbs. Furthermore,the nuclear-penetrating anti-dsDNA autoAbs could possibly function as a pathogenic factor for lupus nephritis by up-regulating ERK activation and Bcl-2 production in mesangial cells. The cell- and nuclear-penetrating VH domain may be exploited as a vehicle for the intra cellular delivery of various useful molecules. [ABSTRACT FROM AUTHOR]

HT22 immortalized hippocampal neurons serve as a cellular model system to study oxidative stress, an imbalance of cellular redox homeostasis. Glutamate induces HT22 cell death by inhibiting the uptake of cystine into the cells via the cystine/glutamate transport system xc-, thus leading to reduced levels of glutathione. Here, we show that glutamate-induced cell death is attenuated in HT22 cells overexpressing heat shock protein 70 or heme oxygenase-1. Moreover, supplementing the culture medium with sodium selenite completely protected HT22 against oxidative glutamate toxicity. In contrast, neither heat shock protein 70 nor heme oxygenase-1 expression or increased concentrations of sodium selenite protected HT22 cells against serum withdrawal-induced cell death. These data indicate that glutamate-induced cell death differs substantially from that induced by growth factor deprivation. [Copyright &y& Elsevier]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to exert potent cytotoxic activity against many tumor cells but not normal cells. However, some tumor cells are resistant to TRAIL, and it has not been determined how this occurs. In the present study, we obtained three subgroups of Jurkat clones with TRAIL-sensitive, -partial resistant and -resistant phenotypes. We found that most TRAIL-resistant and -partial resistant clones expressed low levels of DR5, whereas most TRAIL-sensitive clones expressed high levels of Death Receptor (DR5). However, there were clones with a range of different TRAIL-sensitivities that had similar levels of DR5 expression. The expression levels of DR4 and the decoy receptors, DcR1 and DcR2, did not correlate with TRAIL sensitivities. We also compared the subgroups in terms of the expression of Fas-associated death domain protein (FADD), the levels of activation of Receptor Interacting Protein (RIP) and caspases, and cleavage of Poly (ADP-Ribose)Polymerase (PARP). Basal expression levels of FADD were not significantly different among the subgroups. After treatment with TRAIL, both TRAIL-sensitive and partial resistant clones showed high levels of activation of caspase-3, caspase-8, RIP and PARP. Relative basal level and induced level of Phosphoprotein over Expressed in Diabetes/Phosphoprotein Enriched in Astrocytes (PED/PEA-15) after TRAIL treatment were compared in the clones. Basal levels of PED/PEA-15 expression were similar among sensitive, partial resistant and resistant clones. TRAIL did not change the PED/PEA-15 level in the clones. In addition, transduction and expression of the dominant negative form of the I-kBα gene did not change TRAIL-sensitivities. Our results showed that the expression levels of DR5, the activation levels of caspase-8, -3 and RIP were critical factors in determining TRAIL-sensitivities in Jurkat cells. The results of our study also suggest that cells with different TRAIL-sensitivities arise through multiple mechanisms even within a single cell line. [Copyright &y& Elsevier]

Purpose: Whole-body radiation therapy can cause severe injury to the hematopoietic system, and therefore it is necessary to identify a novel strategy for overcoming this injury. Methods and Materials: Mice were irradiated with 4.5 Gy after heat shock protein 25 (HSP25) gene transfer using an adenoviral vector. Then, peripheral blood cell counts, histopathological analysis, and Western blotting on bone marrow (BM) cells were performed. The interaction of HSP25 with Tie2 was investigated with mouse OP9 and human BM-derived mesenchymal stem cells to determine the mechanism of HSP25 in the hematopoietic system. Results: HSP25 transfer increased BM regeneration and reduced apoptosis following whole-body exposure to ionizing radiation (IR). The decrease in Tie2 protein expression that followed irradiation of the BM was blocked by HSP25 transfer, and Tie2-positive cells were more abundant among the BM cells of HSP25-transferred mice, even after IR exposure. Following systemic RNA interference of Tie2 before IR, HSP25-mediated radioprotective effects were partially blocked in both mice and cell line systems. Stability of Tie2 was increased by HSP25, a response mediated by the interaction of HSP25 with Tie2. IR-induced tyrosine phosphorylation of Tie2 was augmented by HSP25 overexpression; downstream events in the Tie2 signaling pathway, including phosphorylation of AKT and EKR1/2, were also activated. Conclusions: HSP25 protects against radiation-induced BM damage by interacting with and stabilizing Tie2. This may be a novel strategy for HSP25-mediated radioprotection in BM. [ABSTRACT FROM AUTHOR]

Abstract: Metallothioneins (MTs) are intracellular low-molecular-weight, cysteine-rich proteins with potent metal-binding and redox functions, but with limited membrane permeativity. The aim of this study was to investigate whether we could enhance delivery of MT-1 to pancreatic islets or β cells in vitro and in vivo. The second goal was to determine whether increased MT-1 could prevent cellular toxicity induced by high glucose and free fatty acids in vitro (glucolipotoxicity) and ameliorate the development of diabetes induced by streptozotocin in mice or delay the development of diabetes by improving insulin secretion and resistance in the OLETF rat model of type 2 diabetes. Expression of HIV-1 Tat–MT-1 enabled efficient delivery of MT into both INS-1 cells and rat islets. Intracellular MT activity increased in parallel with the amount of protein delivered to cells. The formation of reactive oxygen species, glucolipotoxicity, and DNA fragmentation due to streptozotocin decreased after treating pancreatic β cells with Tat–MT in vitro. Importantly, in vivo, intraperitoneal injection resulted in delivery of the Tat–MT protein to the pancreas as well as liver, muscle, and white adipose tissues. Multiple injections increased radical-scavenging activity, decreased apoptosis, and reduced endoplasmic reticulum stress in the pancreas. Treatment with Tat–MT fusion protein delayed the development of diabetes in streptozotocin-induced mice and improved insulin secretion and resistance in OLETF rats. These results suggest that in vivo transduction of Tat–MT may offer a new strategy to protect pancreatic β cells from glucolipotoxicity, may improve insulin resistance in type 2 diabetes, and may have a protective effect in preventing islet destruction in type 1 diabetes. [Copyright &y& Elsevier]

Abstract: In the present study, we investigated the substrate specificity of catalytic activity of a cytotoxic anti-DNA monoclonal autoantibody, G1-5, which was obtained from an MRL-lpr/lpr mouse by hybridoma technology. The antibody catalyzed hydrolysis of single- and double-stranded DNA with a higher substrate specificity for thymine than adenine by either β-glycosidic or phosphodiester bond cleavage. The hydrolysis rate (k cat) showed maximum at acidic pH conditions, suggesting that the catalytic site of the antibody contains essential carboxylic group(s). Treatment of cells with the antibody promoted cell death and induced the activation of caspases. The cell death induced by the antibody was inhibited by the pan-caspase inhibitor. Furthermore, the antibody binds to cell membrane and penetrates into the cells. Our results suggest that the cell death is initiated by antibodies penetrating to cells and nucleus, hydrolyzing considerable amount of DNA, and mediating the caspase-dependent apoptotic pathway. [Copyright &y& Elsevier]

HSP25 has been shown to induce resistance to radiation and oxidative stress; however, its exact mechanisms remain unclear. In the present study, a high concentration of H2O2 was found to induce DNA fragmentation in L929 mouse fibroblast cells, and HSP25 overexpression attenuated this phenomenon. To elucidate the mechanisms of H2O2-mediated cell death, ERK1/2, p38 MAPK, and JNK1/2 phosphorylation in the cells after treatment with H2O2 were examined. ERK1/2 and JNK1/2 were activated by H2O2; ERK1/2 activation was inhibited in HSP25-overexpressed cells, while JNK1/2 was indifferent. Inhibition of ERK1/2 activation by treatment of the cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced cell death; similarly treated HSP25-overexpressed cells were not at all affected. Moreover, inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 transfection did not affect H2O2-mediated cell death in control cells. Dominant-negative Ras or Raf transfection inhibited H2O2-mediated ERK1/2 activation and cell death in control cells. On the contrary, HSP25-overexpressed cells did not show any differences. Upstream pathways of H2O2-mediated ERK1/2 activation and cell death involved both tyrosine kinase (PDGFβ receptor and Src) and PKCδ, while in HSP25-overexpressed cells these kinases did not respond to H2O2 treatment. Since HSP25 overexpression reduced reactive oxygen species (ROS), increased manganese superoxide dismutase (MnSOD) gene expression, and increased enzyme activity, involvement of MnSOD in HSP25-mediated attenuation of H2O2-mediated ERK1/2 activation and cell death was examined. Blockage of MnSOD with antisense oligonucleotides prevented DNA fragmentation and returned the ERK1/2 activation to the control level. Indeed, when MnSOD was overexpressed in L929 cells, similar to in HSP25-overexpressed cells, DNA fragmentation and ERK1/2 activation were reduced. From the above results, we suggest for the first time that reduced oxidative damage by HSP25 was due to MnSOD-mediated downregulation of ERK1/2. [Copyright &y& Elsevier]