Simvastatin inhibits the proliferation of human prostate cancer PC-3 cells via down-regulation of the insulin-like growth factor 1 receptor

Recently, statins have been being studied for their proapoptic and antimetastatic effects. However, the exact mechanisms of their anticancer action are still unclear. Dolichyl phosphate is a nonsterol isoprenoid derivative in the mevalonate pathway that affects the expression of the Insulin-like growth factor 1 receptor (IGF-1R). IGF-1R activation is required for prostate cell proliferation; therefore, IGF-1R inhibitory agents may be of preventive and/or therapeutic value. In this study, the effects of simvastatin on IGF-1R signaling in prostate cancer PC-3 cells were examined. Simvastatin suppressed proliferation and induced apoptosis of PC-3, and the expression of IGF-1R was suppressed by simvastatin. Knockdown of IGF-1R by siRNA led to inhibition of proliferation of PC-3. Simvastatin also inhibited IGF-1-induced activation of both ERK and Akt signaling and IGF-1-induced PC-3 cell proliferation. Our results suggest statins are potent inhibitors of the IGF-1/IGF-1R system in prostate cancer cells and may be beneficial in prostate cancer treatment.     

Acquired Thermotolerance and Temperature-Induced Protein Accumulation in the Extremely Thermophilic Bacterium Rhodothermus obamensis

Temperature-induced changes in thermotolerance and protein composition were examined in heat-shocked cells and high-temperature-grown cells of the extremely thermophilic bacterium Rhodothermus obamensis. The survival at temperatures superoptimal for growth (90 and 95°C) was enhanced in both heat-shocked cells and high-temperature-grown cells relative to that of cells grown at optimal temperatures. In a comparison of protein composition using two-dimensional gel electrophoresis, putative heat shock proteins (HSPs) and high-temperature growth-specific proteins (HGPs) were detected. N-terminal amino acid sequence analysis revealed that the putative HSPs were quite similar to the ATP-binding subunits of ABC transporters and the HGPs were proteins corresponding to domains II and III of elongation factor Tu. These results suggested that this extreme thermophile has developed temperature-induced responses that include increased survival under hyperthermal conditions, changes in protein composition, and also the production of novel HSPs.     

Increase in the ability of human cancer cells to induce cytotoxic T lymphocytes by ultraviolet irradiation

The roles of ultraviolet-B (UV) radiation in the immunogenicity of human cancer cells have not been fully studied. We have investigated the effects of UV radiation on metastatic melanoma and renal cell carcinoma cells with regard to MHC antigen expression and the ability to induce cytotoxic T lymphocyte (CTL) activity in peripheral blood mononuclear cells (PBMC) or tumor-infiltrating lymphocytes (TIL) against untreated autologous tumor cells. UV radiation respectively decreased or increased MHC class I expression of freshly isolated tumor cells or cultured tumor cells, and also decreased MHC class I expression of starved cultured tumor cells. It increased the ability of both freshly isolated and cultured tumor cells to induce CTL activity from PBMC against untreated autologous tumor cells. UV-irradiated subclones that were more susceptible to CTL lysis were more potent for CTL induction from TIL than either an untreated parental clone or a UV-irradiated subclone that was resistant to CTL lysis. In summary, UV radiation increased the ability of tumor cells to induce CTL activity without a corresponding effect on MHC antigen expression.This work was supported in part by a grant CA47891 from the National Cancer Institute, USA, a grant-in-aid of the comprehensive 10-years strategy for cancer control from ministry of a Health and Welfare, Japan, and the Ishibashi Research Fund, Japan     

Induction of Manganese Superoxide Dismutase by Cytokines and Lipopolysaccharide in Cultured Mouse Astrocytes

Abstract: To determine whether cytokines or lipopolysaccharide (LPS) are involved in the induction of superoxide dismutase (SOD) in the nervous system, we examined the effects of these substances on the levels of SOD in cultured mouse astrocytes. Treatment of astrocytes with 10² to 10⁴ U/ml tumor necrosis factor-α for 3 days increased the levels of Mn SOD in a dose- and time-dependent manner to as much as six times the level under nontreated conditions. Treatment with 1.0 µg/ml LPS for 3 days elicited a fourfold increase in levels of Mn SOD, and the effect of LPS was also dose dependent. Furthermore, Mn SOD in astrocytes was induced by a 3-day exposure to interleukin-1α at concentrations of 10² or 10³ U/ml. However, these stimuli had no effect on levels of copper-zinc SOD (Cu/Zn SOD) in astrocytes. By contrast, interferon-γ did not change the levels of either Mn or Cu/Zn SOD in the cells. The results indicate that the selective induction of Mn SOD by cytokines and LPS, which has been observed in nonnervous tissues, may also occur in nervous tissues. The induction of Mn SOD may represent a mechanism for protection of cells from oxidative stress.     

Linear Ubiquitination of NEMO Negatively Regulates the Interferon Antiviral Response through Disruption of the MAVS-TRAF3 Complex

The RIG-I/Mda5 sensors recognize viral intracellular RNA and trigger host antiviral responses. RIG-I signals through the adaptor protein MAVS, which engages various TRAF family members and results in type I interferon (IFNs) and proinflammatory cytokine production via activation of IRFs and NF-κB, respectively. Both the IRF and NF-κB pathways also require the adaptor protein NEMO. We determined that the RIG-I pathway is differentially regulated by the linear ubiquitin assembly complex (LUBAC), which consists of the E3 ligases HOIL-1L, HOIP, and the accessory protein SHARPIN. LUBAC downregulated virus-mediated IFN induction by targeting NEMO for linear ubiquitination. Linear ubiquitinated NEMO associated with TRAF3 and disrupted the MAVS-TRAF3 complex, which inhibited IFN activation while stimulating NF-κB-dependent signaling. In SHARPIN-deficient MEFs, vesicular stomatitis virus replication was decreased due to increased IFN production. Linear ubiquitination thus switches NEMO from a positive to a negative regulator of RIG-I signaling, resulting in an attenuated IFN response.     

Linear polyubiquitin chains: A new modifier involved in NFκB activation and chronic inflammation including dermatitis

The ubiquitin conjugation system regulates a wide variety of biological phenomena, including protein degradation and signal transduction, by regulating protein function via polyubiquitin conjugation in most cases. Several types of polyubiquitin chains exist in cells, and the type of polyubiquitin chain conjugated to a protein seems to determine how that protein is regulated. We identified a novel linear polyubiquitin chain and the ubiquitin-protein ligase complex that assembles it, designated LUBAC. Both were shown to have crucial roles in the canonical NFκB activation pathway. This year, three groups, including our laboratory, identified SHARPIN as a new subunit of LUBAC. Of great interest, Sharpin was identified as a causative gene of chronic proliferative dermatitis in mice (cpdm), which is characterized by numerous inflammatory symptoms including chronic dermatitis, arthritis and immune disorders. Deletion of SHARPIN drastically reduces the amount of LUBAC and attenuates signal-induced NFκB activation. The pleomorphic symptoms of cpdm mice suggest that LUBAC-mediated NFκB activation may play critical roles in mammals and be involved in various disorders. A forward look into the linear polyubiquitin research is also discussed.Key words: ubiquitin, linear ubiquitination, NFκB, LUBAC, SHARPIN, cpdm, chronic dermatitis, TNFα     

A novel induction mechanism of the rat CYP1A2 gene mediated by Ah receptor-Arnt heterodimer

We have identified an enhancer responsible for induction by 3-methylcholanthrene in the upstream region of the CYP1A2 gene. The enhancer does not contain the invariant core sequence of XREs that are binding sites for the Ah receptor (AhR) and Arnt heterodimer. The enhancer did not show any inducible expression in Hepa-1-derived cell lines, C4 and C12, deficient of Arnt and AhR, respectively. On the other hand, bacterially expressed AhR-Arnt heterodimer could not bind to the enhancer. Mutational analysis of the enhancer revealed that a repeated sequence separated by six nucleotides is important for expression. A factor binding specifically to the enhancer was found by using gel shift assays. Bacterially expressed AhR-Arnt heterodimer interacted with the factor. A dominant negative mutant of the AhR to XRE activated the enhancer. Collectively, these results demonstrate that a novel induction mechanism is present in which the AhR-Arnt heterodimer functions as a coactivator.     

Porcine brain natriuretic peptide-like immunoreactivity in rat tissues

The presence of immunoreactive porcine brain natriuretic peptide in rat tissues was studied with a specific radioimmunoassay for porcine brain natriuretic peptide-26. The cross-reactivity of the antiserum used was less than 0.001% with rat atrial natriuretic peptide, rat brain natriuretic peptide-32 and rat brain natriuretic peptide-45. Immunoreactive porcine brain natriuretic peptide was detectable in various tissues of the rat, and high concentrations of immunoreactive porcine brain natriuretic peptide were found in the brain and cardiac atrium, with the highest level in the hypothalamus (159±30 fmol/gram wet tissue, mean±SEM, n=4). Reverse phase high performance liquid chromatography showed that the immunoreactive porcine brain natriuretic peptide of the whole brain and heart extracts eluted mainly at an identical position to synthetic porcine brain natriuretic peptide-26. These findings indicate that porcine brain natriuretic peptide-like substance, distinct from rat brain natriuretic peptide, is present in high concentrations in the rat brain and cardiac atrium.     

An alternative synthetic method for 4'-C-ethynylstavudine by means of nucleophilic substitution of 4'-benzoyloxythymine nucleoside

For the synthesis of 2',3' -didehydro-3' -deoxy-4' -C-ethynylthymidine (8: 4' -Ed4T), a recently reported promising anti-HIV agent, a new approach was developed. Since treatment of 1-(2,5-dideoxy-beta-l-glycero-pent-4-enofuranosyl)thymine with Pb(OBz)4 allowed the introduction of a 4'-benzoyloxy leaving group, nucleophilic substitution at the 4' -position became feasible for the first time. Thus, reaction between the 4'-benzoyloxy derivative (11) and Me3SiC identical with CAl(Et)Cl as a nucleophile led to the isolation of the desired 4'-"down"-ethynyl derivative (15) stereoselectively in 62% yield.     

Microbial Community in Black Rust Exposed to Hot Ridge Flank Crustal Fluids

During Integrated Ocean Drilling Program Expedition 301, we obtained a sample of black rust from a circulation obviation retrofit kit (CORK) observatory at a borehole on the eastern flank of Juan de Fuca Ridge. Due to overpressure, the CORK had failed to seal the borehole. Hot fluids from oceanic crust had discharged to the overlying bottom seawater and resulted in the formation of black rust analogous to a hydrothermal chimney deposit. Both culture-dependent and culture-independent analyses indicated that the black-rust-associated community differed from communities reported from other microbial habitats, including hydrothermal vents at seafloor spreading centers, while it shared phylotypes with communities previously detected in crustal fluids from the same borehole. The most frequently retrieved sequences of bacterial and archaeal 16S rRNA genes were related to the genera Ammonifex and Methanothermococcus, respectively. Most phylotypes, including phylotypes previously detected in crustal fluids, were isolated in pure culture, and their metabolic traits were determined. Quantification of the dissimilatory sulfite reductase (dsrAB) genes, together with stable sulfur isotopic and electron microscopic analyses, strongly suggested the prevalence of sulfate reduction, potentially by the Ammonifex group of bacteria. Stable carbon isotopic analyses suggested that the bulk of the microbial community was trophically reliant upon photosynthesis-derived organic matter. This report provides important insights into the phylogenetic, physiological, and trophic characteristics of subseafloor microbial ecosystems in warm ridge flank crusts.