Induction of toll-like receptors and NALP/PAN/PYPAF family members by modified oligonucleotides in lung epithelial carcinoma cells

ISIS 199044 is a chimeric 2'-O-methyl-containing oligonucleotide that produces toxicity in several cultured cell lines. Upon investigation into the mechanism of cytotoxicity, we discovered that treatment of lung epithelial carcinoma cells, A549, with ISIS 199044 and several other cytotoxic oligonucleotides induces a group of genes that are not normally expressed in these cells. These genes are involved in host response to foreign materials. Among them were toll-like receptor 7 (TLR7) and TLR9, members of the toll-like receptor family, responsible for immune response to nucleic acids and cryopyrin, a member of NALP/PAN/PYPAF family, which is known to assemble with ASC and regulate NF-kappaB activation and to modulate apoptosis. Maximal induction occurred 12-24 hours posttreatment with 500 nM oligonucleotide in the presence of Lipofectin reagent. Furthermore, we have shown that this induction is chemistry dependent; it can be negated by certain modifications, such as replacement of 2'-O-methyl with 2'-O-methoxyethyl groups or substitution of phosphorothioates with phosphodiester linkages. DNA microarray analysis identified additional genes modulated by ISIS 199044, particularly genes involved in DNA damage/repair.     

An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis

High levels of plasma apolipoprotein B-100 (apoB-100), the principal apolipoprotein of LDL, are associated with cardiovascular disease. We hypothesized that suppression of apoB-100 mRNA by an antisense oligonucleotide (ASO) would reduce LDL cholesterol (LDL-C). Because most of the plasma apoB is made in the liver, and antisense drugs distribute to that organ, we tested the effects of a mouse-specific apoB-100 ASO in several mouse models of hyperlipidemia, including C57BL/6 mice fed a high-fat diet, Apoe-deficient mice, and Ldlr-deficient mice. The lead apoB-100 antisense compound, ISIS 147764, reduced apoB-100 mRNA levels in the liver and serum apoB-100 levels in a dose- and time-dependent manner. Consistent with those findings, total cholesterol and LDL-C decreased by 25-55% and 40-88%, respectively. Unlike small-molecule inhibitors of microsomal triglyceride transfer protein, ISIS 147764 did not produce hepatic or intestinal steatosis and did not affect dietary fat absorption or elevate plasma transaminase levels. These findings, as well as those derived from interim phase I data with a human apoB-100 antisense drug, suggest that antisense inhibition of this target may be a safe and effective approach for the treatment of humans with hyperlipidemia.     

Inhibition of proteasomal function by curcumin induces apoptosis through mitochondrial pathway

Curcumin is a natural polyphenolic compound having an antiproliferative property, which recent evidence suggests is due to its ability to induce apoptosis. However, the molecular mechanisms through which curcumin induces apoptosis are not fully understood. Here, we report that the curcumin-induced apoptosis is mediated through the impairment of the ubiquitin-proteasome system. Exposure of curcumin to the mouse neuro 2a cells causes a dose-dependent decrease in proteasome activity and an increase in ubiquitinated proteins. Curcumin exposure also decreases the turnover of the destabilized enhanced green fluorescence protein, a model substrate for proteasome and cellular p53 protein. Like other proteasome inhibitors, curcumin targets proliferative cells more efficiently than differentiated cells and induces apoptosis via mitochondrial pathways. Addition of curcumin to neuro 2a cells induces a rapid decrease in mitochondrial membrane potential and the release of cytochrome c into cytosol, followed by activation of caspase-9 and caspase-3.     

A curvature-mediated mechanism for localization of lipids to bacterial poles

Subcellular protein localization is a universal feature of eukaryotic cells, and the ubiquity of protein localization in prokaryotic species is now acquiring greater appreciation. Though some targeting anchors are known, the origin of polar and division-site localization remains mysterious for a large fraction of bacterial proteins. Ultimately, the molecular components responsible for such symmetry breaking must employ a high degree of self-organization. Here we propose a novel physical mechanism, based on the two-dimensional curvature of the membrane, for spontaneous lipid targeting to the poles and division site of rod-shaped bacterial cells. If one of the membrane components has a large intrinsic curvature, the geometrical constraint of the plasma membrane by the more rigid bacterial cell wall naturally leads to lipid microphase separation. We find that the resulting clusters of high-curvature lipids are large enough to spontaneously and stably localize to the two cell poles. Recent evidence of localization of the phospholipid cardiolipin to the poles of bacterial cells suggests that polar targeting of some proteins may rely on the membrane's differential lipid content. More generally, aggregates of lipids, proteins, or lipid-protein complexes may localize in response to features of cell geometry incapable of localizing individual molecules.     

Investigation of bacteriophage MS2 viral dynamics using model discrimination analysis and the implications for phage therapy

Lytic phages infect their bacterial hosts, use the host machinery to replicate, and finally lyse and kill their hosts, releasing progeny phages. Various mathematical models have been developed that describe these phage-host viral dynamics. The aim of this study was to determine which of these models best describes the viral dynamics of lytic RNA phage MS2 and its host Escherichia coli C-3000. Experimental data consisted of uninfected and infected bacterial cell densities, free phage density, and substrate concentration. Parameters of various models were either determined directly through other experimental techniques or estimated using regression analysis of the experimental data. The models were evaluated using a Bayesian-based model discrimination technique. Through model discrimination it was shown that phage-resistant cells inhibited the growth of phage population. It was also shown that the uninfected bacterial population was a quasispecies consisting of phage-sensitive and phage-resistant bacterial cells. When there was a phage attack the phage-sensitive cells died out and the phage-resistant cells were selected for and became the dominant strain of the bacterial population.     

A Naturally Occurring Variant in Human TLR9, P99L, Is Associated with Loss of CpG Oligonucleotide Responsiveness

The innate immune system employs Toll-like receptors (TLRs) for the detection of invading microorganisms based on distinct molecular patterns. For example, TLR9 is activated by microbial DNA and also by short therapeutic CpG-containing oligonucleotides (CpG-ODN). TLR9 activation leads to the production of interferons and the priming of humoral adaptive immune responses. Unfortunately, the principles of ligand recognition by TLR9 are poorly understood, and genetic variants of TLR9, which may affect its function, have not been characterized systematically on the molecular level. We therefore sought to functionally characterize reported single nucleotide polymorphisms of TLR9 in the HEK293 model system. We discovered that two variants, P99L and M400I, are associated with altered receptor function regarding NF-κB activation and cytokine induction. Our investigations show that for the most functionally impaired variant, P99L, the ability to respond to physiological and therapeutic TLR9 ligands is severely compromised. However, CpG-ODN binding is normal. CpG-ODN recognition by TLR9 thus appears to involve two separate events, CpG-ODN binding and sensing. Our studies highlight Pro-99 as a residue important for the latter process. In genotyping studies, we confirmed that both M400I (rs41308230) and P99L (rs5743844) are relatively rare variants of TLR9. Our data add rs41308230 and rs5743844 to the list of functionally important TLR variants and warrant further research into their relevance for infectious disease susceptibility or responsiveness to CpG-ODN-based therapies.     

Development and characterization of a human articular cartilage‐derived chondrocyte cell line that retains chondrocyte phenotype

Chondrocytes, the only cell type present in articular cartilage, regulate tissue homeostasis by a fine balance of metabolism that includes both anabolic and catabolic activities. Therefore, the biology of chondrocytes is critical for understanding cartilage metabolism. One major limitation when studying primary chondrocytes in culture is their loss of phenotype. To overcome this hurdle, limited attempts have been made to develop human chondrocyte cell lines that retain the phenotype for use as a good surrogate model. In this study, we report a novel approach to the establishment and characterization of human articular cartilage‐derived chondrocyte cell lines. Adenoviral infection followed by culture of chondrocytes in 3‐dimensional matrix within 48 h post‐infection maintained the phenotype prior to clonal selection. Cells were then placed in culture either as monolayer, or in 3‐dimensional matrix of alginate or agarose. The clones were characterized by their basal gene expression profile of chondrocyte markers. Based on type II collagen expression, 21 clones were analyzed for gene expression following treatment with IL‐1 or BMP‐7 and compared to similarly stimulated primary chondrocytes. This resulted in selection of two clones that retained the chondrocyte phenotype as evidenced by expression of type II collagen and other extra‐cellular matrix molecules. In addition, one clone (AL‐4‐17) showed similar responses as primary chondrocytes when treated with IL‐1 or BMP‐7. In summary, this report provides a novel procedure to develop human articular cartilage‐derived chondrocyte cell lines, which preserve important characteristics of articular chondrocytes and represent a useful model to study chondrocyte biology. J. Cell. Physiol. 222: 695–702, 2010. © 2009 Wiley‐Liss, Inc.     

Eicosapentaenoic and docosahexaenoic acids enriched polyunsaturated fatty acids from the coastal marine fish of Bay of Bengal and their therapeutic value

Eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) enriched polyunsaturated fatty acids (PUFA) significantly present in marine fish oil emerge as preventive agents for combating many health problems specially in chronic or metabolic disorders. The fish in the coastal area of Bay of Bengal has remained unexplored with respect to EPA/DHA enriched PUFA content in its oils, although it may be a potential source in harnessing the health benefit. In this study, seven varieties of the coastal fish were analysed for the content of EPA/DHA. The one locally known as lotte, (Harpadon nehereus) though has low content of total lipids, was found to have high EPA/DHA in its oil. The phospholipids rich fraction was extracted from the total fish oil. The EPA/DHA enriched PUFA was isolated to investigate the potential use for health benefits. EPA/DHA is found to act as protective agent against mercury poisoning studied in cell culture as well as in animal mode. It is found to be highly preventive in diabetes. The lotte is available in the coastal area of Bay of Bengal adjoining West Bengal, India in large scale and it is the first report showing EPA/DHA enriched PUFA in these fish oil that can be availed to harness in important health benefits.     

S-phase arrest by reactive nitrogen species is bypassed by okadaic acid, an inhibitor of protein phosphatases PP1/PP2A

In mammalian cells DNA damage activates a checkpoint that halts progression through S phase. To determine the ability of nitrating agents to induce S-phase arrest, mouse C10 cells synchronized in S phase were treated with nitrogen dioxide (NO(2)) or SIN-1, a generator of reactive nitrogen species (RNS). SIN-1 or NO(2) induced S-phase arrest in a dose- and time-dependent manner. As for the positive controls adozelesin and cisplatin, arrest was accompanied by phosphorylation of ATM kinase; dephosphorylation of pRB; decreases in RF-C, cyclin D1, Cdc25A, and Cdc6; and increases in p21. Comet assays indicated that RNS induce minimal DNA damage. Moreover, in a cell-free replication system, nuclei from cells treated with RNS were able to support control levels of DNA synthesis when incubated in cytosolic extracts from untreated cells, whereas nuclei from cells treated with cisplatin were not. Induction of phosphatase activity may represent one mechanism of RNS-induced arrest, for the PP1/PP2A phosphatase inhibitor okadaic acid inhibited dephosphorylation of pRB; prevented decreases in the levels of RF-C, cyclin D1, Cdc6, and Cdc25A; and bypassed arrest by SIN-1 or NO(2), but not cisplatin or adozelesin. Our studies suggest that RNS may induce S-phase arrest through mechanisms that differ from those elicited by classical DNA-damaging agents.     

C/EBPβ regulates TNF induced MnSOD expression and protection against apoptosis

The CCAAT enhancer binding protein-β (C/EBPβ) is a critical regulator of many cellular processes. Exposure of C/EBPβ-deficient fibroblasts to tumor necrosis factor-α (TNF) resulted in their death due to apoptosis. While, the expression of Bad, Bcl-2, Bcl-x, CAS, and hILP/XIAP, as well as the nuclear translocation of NF-κB was normal in C/EBPβ-deficient cells, induction of manganous superoxide dismutase (MnSOD) gene did not occur. Ectopic expression of C/EBPβ in C/EBPβ–deficient fibroblasts prevented TNF-induced apoptosis. C/EBPβ complemented cells were able to induce MnSOD in response to TNF, ruling out the possibilities that C/EBPβ could render protection by regulating early apoptotic gene expression and/or NF-κB p65 expression. Moreover, C/EBPβ-deficient cells stably transfected with an MnSOD expression vector bypassed the requirement of C/EBPβ in protection against TNF-induced cell death, suggesting that C/EBPβ protects TNF-induced apoptotic cell death through its role in activating MnSOD expression. Mechanistically, C/EBPβ was required for induced NF-κB p65 binding to MnSOD’s intronic TNF response element and indispensable for histone acetylation of the element in response to TNF. These results suggest a role for C/EBPβ in MnSOD regulation through remodeling of local chromatin structure. This work was supported by a grant from the National Institutes of Health, CA96810.