An anti-parallel triple helix motif with oligodeoxynucleotides containing 2'-deoxyguanosine and 7-deaza-2'-deoxyxanthosine.

Triple helix formation of oligodeoxynucleotides (ODNs) with a 15 base pair poly-purine DNA target in the HER2 promoter was examined by footprinting analysis. 7-deaza-2'-deoxyxanthosine (dzaX) was identified as a purine analogue of thymidine (T) which forms dzaX:A-T triplets. ODNs containing 2'-deoxyguanosine (G) and dzaX were found to form triple helices in an anti-parallel orientation, with respect to the poly-purine strand of the target DNA. In comparative studies under physiological K+ and Mg++ concentrations and at pH 7.2, the ODNs containing G and dzaX showed high affinity to the target sequence while the ODNs containing G and T were not able to bind. In the absence of added monovalent salts both ODNs showed high affinity to the target sequence. The substitution of 7-deaza-2'-deoxyguanosine for G substantially decreased the capacity of the ODNs to form triple helices under physiological conditions, indicating that dzaX may be unique in its ability to enhance triple helix formation in the anti-parallel motif.     

Allelic variation of the type 2 tumor necrosis factor receptor gene

The nucleotide sequence data reported in this paper have been submitted to the EMBL Data library and have been assigned the accession number X76401.     

Biophysical and antisense properties of oligodeoxynucleotides containing 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines.

The synthesis of 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines is described. The nucleosides were synthesized, functionalized into the phosphoramidites and incorporated into oligodeoxynucleotides. Spectroscopic melting experiments against complementary RNA showed increases of 3-4 degreesC per modification for single substitutions and smaller increases per incorporation for multiple substitutions relative to unmodified control sequences. The 7-propyne and 7-iodo nucleosides were incorporated into antisense sequences targeting the 3'-UTR of murine C- raf mRNA. Both nucleosides demonstrated substitution-dependent potency. The sequences with three and four substitutions of the 7-propyne-7-deaza-2-amino-2'-deoxyadenosine exhibited a 2-3-fold increase in potency over unmodifed controls.     

Growth inhibition of cervical tumor cells by antisense oligodeoxynucleotides directed to the human papillomavirus type 16 E6 gene.

Human papillomavirus type 16 (HPV-16) is the HPV type most frequently associated with cervical carcinomas. Based on our previous research with anti-HPV ribozymes, we developed a 16-nucleotide antisense oligodeoxynucleotide (AntiE6) able to direct RNase H activity on full-length HPV-16 E6/E7 mRNA. Although the precise mechanism is not completely understood, addition of 50 microM AntiE6 oligodeoxynucleotide in sterile water caused a significant decrease in the growth rate of CaSki and QGU cervical tumor cell lines. In contrast, addition of a mismatched mutant oligodeoxynucleotide (M7) did not affect cell growth after 72 hours. Treatment with AntiE6 resulted in down-regulation of E6/E7 mRNA and an increase in p53 levels in QGU cells. AntiE6 was also able to (>70%) inhibit significantly growth of transplanted cervical tumors in nude mice after 2 weeks treatment using constant delivery by osmotic pumps. These results indicate that the AntiE6 antisense oligodeoxynucleotides can act as a therapeutic agent against cervical carcinomas.     

Induction of interleukin 2 receptor (TAC) by tumor necrosis factor in YT cells

The effect of human recombinant tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) on interleukin 2 receptor (IL-2R) expression on YT cells was examined. IL-2R expression was assessed by flow cytometric analysis with a monoclonal antibody to IL-2R (anti-TAC). TNF-alpha, like IL-1 beta, induced increased levels of IL-2R expression on YT cells with similar kinetics of induction. Maximum induction occurred at 20 to 30 hr. On a molar basis. TNF was less active than IL-1 beta. RNA isolated from TNF-alpha- or IL-1 beta-treated YT cells contained increased levels of IL-2R-specific mRNA as indicated by slot blot analysis by using an IL-2R-specific mRNA probe. Kinetic and IL-1 beta mRNA expression studies indicated that the TNF effect was a direct one. Because IL-2R expression is known to be associated with lymphocyte activation, the present results suggest that TNF-alpha may play a role in the regulation of immune responses.     

Characterization of a recombinant extracellular domain of the type 1 tumor necrosis factor receptor: evidence for tumor necrosis factor-alpha induced receptor aggregation.

An expression plasmid encoding the extracellular portion of the human tumor necrosis factor (TNF) type 1 receptor (TNF-R1) was constructed and used to generate a stable cell line secreting soluble TNF-R1 (sTNF-R1). The sTNF-R1 was purified, and its biochemical properties and its interactions with human TNF-alpha were examined. SDS-PAGE resolved the purified sTNF-R1 into three bands of approximate Mr 24,200, 28,200, and 32,800. Sedimentation equilibrium analysis gave a molecular weight of 25,000 for sTNF-R1 whereas the molecular weight obtained by gel filtration chromatography was approximately 55,000-60,000. Scatchard analysis of [125I]TNF-alpha binding to sTNF-R1 revealed high-affinity binding (Kd = 93 pM), comparable to that observed for the intact receptor on whole cells. Competitive binding experiments showed that sTNF-R1 has a 50-60-fold higher affinity for TNF-alpha than for TNF-beta, in contrast to the equal affinities of TNF-alpha and TNF-beta for the full-length TNF-R1 transiently expressed in mammalian cells. The sTNF-R1 was found to block the cytotoxicity of TNF-alpha and TNF-beta on a murine L-M cell assay. The sizes of the sTNF-R1.TNF-alpha complex determined by gel filtration chromatography and sedimentation equilibrium were approximately 141 and 115 kDa, respectively. The stoichiometry of the complex was examined by Scatchard analysis, size-exclusion chromatography, HPLC separation, amino acid composition, sequence analysis, and sedimentation equilibrium. The data from these studies suggest that at least two molecules of sTNF-R1 can bind to a single TNF-alpha trimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triple-helix formation in the antiparallel binding motif of oligodeoxynucleotides containing N(9)- and N(7)-2-aminopurine deoxynucleosides

Triplex-forming oligodeoxynucleotide 15mers, designed to bind in the antiparallel triple-helical binding motif, containing single substitutions (Z) of the four isomeric αN⁷-, βN⁷-, αN⁹- and βN⁹-2-aminopurine (ap)-deoxyribonucleosides were prepared. Their association with double-stranded DNA targets containing all four natural base pairs (X-Y) opposite the aminopurine residues was determined by quantitative DNase I footprint titration in the absence of monovalent metal cations. The corresponding association constants were found to be in a rather narrow range between 1.0 × 10⁶ and 1.3 × 10⁸ M^–1. The following relative order in Z × X-Y base-triple stabilities was found: Z = αN⁷ap: T-A > A-T> C-G ~ G-C; Z = βN⁷ap: A-T > C-G > G-C > T-A; Z = αN⁹ap: A-T = G-C > T-A > C-G; and Z = βN⁹ap: G-C > A-T > C-G > T-A.     

Ectodomain shedding of preadipocyte factor 1 (Pref-1) by tumor necrosis factor alpha converting enzyme (TACE) and inhibition of adipocyte differentiation

Preadipocyte factor 1 (Pref-1), an epidermal growth factor repeat containing transmembrane protein found in the preadipocytes, inhibits adipocyte differentiation in vitro and in vivo. Here, we examined the processing of membrane form of Pref-1A to release the 50-kDa soluble form that inhibits adipocyte differentiation. The ectodomain cleavage of Pref-1 is markedly enhanced by phorbol 12-myristate 13-acetate in a dose- and time-dependent manner. The basal and stimulated cleavage is inhibited by the broad metalloproteinase inhibitor GM6001, a fact that suggests that cleavage of membrane Pref-1A is dependent on a metalloproteinase. Next, we showed that release of soluble Pref-1A is inhibited by TAPI-0 and by a tissue inhibitor of metalloproteinase-3, TIMP-3, that can inhibit tumor necrosis factor alpha converting enzyme (TACE), but not by TIMP-1 or TIMP-2. On the other hand, overexpression of TACE increases Pref-1 cleavage to produce the 50-kDa soluble form. Furthermore, this cleavage was not detected in cells with TACE mutation or with TACE small interfering RNA. TACE-mediated shedding of Pref-1 ectodomain inhibits adipocyte differentiation of 3T3-L1 cells and in Pref-1-null mouse embryo fibroblasts transduced with Pref-1A. Identification of TACE as the major protease responsible for conversion of membrane-bound Pref-1 to the biologically active diffusible form provides a new insight into Pref-1 function in adipocyte differentiation.     

Epstein-Barr virus immediate-early protein BZLF1 inhibits tumor necrosis factor alpha-induced signaling and apoptosis by downregulating tumor necrosis factor receptor 1

Tumor necrosis factor alpha (TNF-alpha) is a key mediator of host immune and inflammatory responses and inhibits herpesvirus replication by cytolytic and noncytolytic mechanisms. TNF-alpha effects are primarily mediated through the major TNF-alpha receptor, TNF-R1, which is constitutively expressed in most cell types. Here we show that the Epstein-Barr virus (EBV) immediate-early protein BZLF1 prevents TNF-alpha activation of target genes and TNF-alpha-induced cell death. These effects are mediated by down-regulation of the promoter for TNF-R1. Additionally, we demonstrate that expression of TNF-R1 is downregulated during the EBV lytic replication cycle. Thus, EBV has developed a novel mechanism for evading TNF-alpha antiviral effects during lytic reactivation or primary infection.     

The gene for the type 1 tumor necrosis factor receptor (TNF-R1) is localized on band 12p13

Summary The gene coding for the type I (p55) tumor necrosis factor receptor (TNF-R1) has been localized on human chromosome 12, band 12p13.2, by in situ hybridisation using a biotinylated genomic probe.     

Induction of prostaglandin I(2) receptor by tumor necrosis factor alpha in osteoblastic MC3T3-E1 cells.

Mouse osteoblastic cells MC3T3-E1 produced prostaglandin E(2) via the reaction of cyclooxygenase-2 enzyme induced by tumor necrosis factor alpha (TNFalpha). Originally, the mRNA level for prostaglandin I(2) receptor (IP) was low in the cells. However, the addition of TNFalpha brought about a marked increase in the IP mRNA with a lag of about 3 h up to an about 8-fold higher level for 24 h. In addition, the induction of IP was supported by a binding experiment of [(3)H]iloprost (a stable analogue of prostaglandin I(2)). The amount of iloprost bound to the TNFalpha-stimulated cell membranes increased to a saturation level around 30 nM. Dexamethasone, cycloheximide and cyclooxygenase inhibitor suppressed the IP mRNA induction. The finding with the latter two compounds suggested a TNFalpha-dependent de novo synthesis of a protein, which is involved in the IP mRNA induction and may be attributed partially to the induced cyclooxygenase-2.     

Ablation of tumor necrosis factor receptor type I (p55) alters oxygen-induced lung injury

Hyperoxic lung injury, believed to be mediated by reactive oxygen species, inflammatory cell activation, and release of cytotoxic cytokines, complicates the care of many critically ill patients. The cytokine tumor necrosis factor (TNF)-alpha is induced in lungs exposed to high concentrations of oxygen; however, its contribution to hyperoxia-induced lung injury remains unclear. Both TNF-alpha treatment and blockade with anti-TNF antibodies increased survival in mice exposed to hyperoxia. In the current study, to determine if pulmonary oxygen toxicity is dependent on either of the TNF receptors, type I (TNFR-I) or type II (TNFR-II), TNFR-I or TNFR-II gene-ablated [(-/-)] mice and wild-type control mice (WT; C57BL/6) were studied in >95% oxygen. There was no difference in average length of survival, although early survival was better for TNFR-I(-/-) mice than for either TNFR-II(-/-) or WT mice. At 48 h of hyperoxia, slightly more alveolar septal thickening and peribronchiolar and periarteriolar edema were detected in WT than in TNFR-I(-/-) lungs. By 84 h of oxygen exposure, TNFR-I(-/-) mice demonstrated greater alveolar debris, inflammation, and edema than WT mice. TNFR-I was necessary for induction of cytokine interleukin (IL)-1beta, IL-1 receptor antagonist, chemokine macrophage inflammatory protein (MIP)-1beta, MIP-2, interferon-gamma-induced protein-10 (IP-10), and monocyte chemoattractant protein (MCP)-1 mRNA in response to intratracheal administration of recombinant murine TNF-alpha. However, IL-1beta, IL-6, macrophage migration inhibitory factor, MIP-1alpha, MIP-2, and MCP-1 mRNAs were comparably induced by hyperoxia in TNFR-I(-/-) and WT lungs. In contrast, mRNA for manganese superoxide dismutase and intercellular adhesion molecule-1 were induced by hyperoxia only in WT mice. Differences in early survival and toxicity suggest that pulmonary oxygen toxicity is in part mediated by TNFR-I. However, induction of specific cytokine and chemokine mRNA and lethality in response to severe hyperoxia was independent of TNFR-I expression. The current study supports the prediction that therapeutic efforts to block TNF-alpha receptor function will not protect against pulmonary oxygen toxicity.     

Linkage mapping of the tumor necrosis factor receptor 2 (TNFR2) gene to 1p36.2 using the single-strand conformation polymorphism technique

We identified two informative polymorphisms in the transcribed 3 untranslated region of the tumor necrosis factor receptor 2 (TNFR2) gene using the poly-merase chain reaction (PCR) with the single-strand conformation polymorphism (SSCP) technique. These poly-morphisms demonstrated Mendelian inheritance and were useful for linkage analysis. TNFR2 was very closely linked to the pronatriodilatin gene (PND), and the TNFR2 SSCP polymorphisms were much more informative than the restriction fragment length polymorphisms currently available for the PND locus. In addition, we have demonstrated that genotyping could be performed with DNA obtained from paraffin-embedded tissue.     

Lack of tumor necrosis factor receptor type 1 inhibits liver fibrosis induced by carbon tetrachloride in mice

Chronic liver injury causes liver regeneration, resulting in fibrosis. The proinflammatory cytokine tumor necrosis factor (TNF) is involved in the pathogenesis of many acute and chronic liver diseases. TNF has pleiotropic functions, but its role in liver fibrosis has not been clarified. Chronic repeated injection of CCl4 induces liver fibrosis in mice. We examined whether signaling through TNF receptors was critical for this process, using mice lacking either TNF receptor (TNFR) type 1 or TNFR type 2 to define the pathophysiologic role of TNFR signals in liver fibrosis. Liver fibrosis caused by chronic CCl4 exposure was TNF-dependent; histological fibrosis was seen in wild-type (WT) and TNFR-2 knockout (KO) mice, but not in TNFR-1 KO mice. Furthermore, a marked reduction in procollagen and TGF-beta synthesis was observed in TNFR-1 KO mice, which also had little detectable NF-kappa B, STAT3, and AP1 binding, and reduced levels of liver interleukin-6 (IL-6) mRNA compared to WT and TNFR-2 KO mice. In conclusion, our results indicate the possibility that NF-kappa B, STAT3, and AP1 binding by signals transduced through TNFR-1 plays an important role in liver fibrosis formation.     

Novel DNA polymorphism in the mouse tumor necrosis factor receptors type 1 and type 2

The introduction of the polymerase chain reaction (PCR) provides an entirely new means of analyzing DNA polymorphism and makes practical the analysis of length variation in simple-sequence tandem repeats of dinucleotides. In the process of cloning and sequencing the mouse genomic DNA for tumor necrosis factor (TNF) receptors type 1 and type 2, we identified two simple dinucleotide repeats within the noncoding regions of TNF receptor type 1 and three such sequences within TNF receptor type 2. PCR analysis of these sequences, using genomic DNA from 21 different inbred and wild mouse strains, as demonstrated by running the amplified products on sequencing gels, showed that the repeats are highly polymorphic. We identified seven alleles of TNF receptor type 2 and five alleles of TNF receptor type 1. Using these polymorphic markers in two sets of recombinant inbred strains of mice, the chromosomal localization of Tnfr-1 was mapped to mouse chromosome 6 and Tnfr-2 was located to the distal portion of mouse chromosome 4.     

Rb-independent induction of apoptosis by bovine papillomavirus type 1 E7 in response to tumor necrosis factor alpha

Bovine papillomavirus type 1 (BPV-1) is a small DNA virus that causes fibropapillomas of the host. BPV-1 has served as the prototype for studies of the molecular biology of the papillomaviruses. BPV-1 efficiently induces anchorage-independent growth and focus formation in murine C127 cells. The transforming properties of BPV-1 primarily reside in two genes, E5 and E6. Each of these genes is sufficient to transform cells. Although no independent transformation activity has been detected for E7, it was shown to be required for full transformation of C127 by BPV-1. We investigated the biological activities of BPV-1 E7 in several assays. Our results indicate that expression of BPV-1 E7 sensitizes cells to tumor necrosis factor alpha (TNF)-induced apoptosis. The TNF-induced apoptosis in E7-expressing cells was accompanied by increased release of arachidonic acid, indicating that phospholipase A(2) was activated. Unlike the E7 proteins from the cancer-related human papillomaviruses, the BPV-1 E7 protein does not associate efficiently with the retinoblastoma protein (pRB) in vitro, nor does it significantly affect the pRB levels in cultured cells. Furthermore, BPV-1 E7 sensitizes Rb-null cells to TNF-induced apoptosis. These studies indicate that BPV-1 E7 can sensitize cells to apoptosis through mechanisms that are independent of pRB.     

Phosphorylation of tumor necrosis factor receptor 1 (p55) protects macrophages from silica-induced apoptosis

Macrophages play a fundamental role in silicosis in part by removing silica particles and producing inflammatory mediators in response to silica. Tumor necrosis factor alpha (TNFalpha) is a prominent mediator in silicosis. Silica induction of apoptosis in macrophages might be mediated by TNFalpha. However, TNFalpha also activates signal transduction pathways (NF-kappaB and AP-1) that rescue cells from apoptosis. Therefore, we studied the TNFalpha-mediated mechanisms that confer macrophage protection against the pro-apoptotic effects of silica. We will show that exposure to silica induced TNFalpha production by RAW 264.7 cells, but not by IC-21. Silica-induced activation of NF-kappaB and AP-1 was only observed in RAW 264.7 macrophages. ERK activation in response to silica exposure was only observed in RAW 264.7 macrophages, whereas activation of p38 phosphorylation was predominantly observed in IC-21 macrophages. No changes in JNK activity were observed in either cell line in response to silica exposure. Silica induced apoptosis in both macrophage cell lines, but the induction of apoptosis was significantly larger in IC-21 cells. Protection against apoptosis in RAW 264.7 cells in response to silica was mediated by enhanced NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNFalpha receptor. Inhibition of these two protective mechanisms by specific pharmacological inhibitors or transfection of dominant negative mutants that inhibit IkappaBalpha or ERK phosphorylation significantly increased silica-induced apoptosis in RAW 264.7 macrophages. These data suggest that NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNF receptor are important cell survival mechanisms in the macrophage response to silica exposure.