Spontaneous malignant transformation of human ovarian surface epithelial cells in vitro.

PURPOSE: Epithelial ovarian cancer has no reliable marker for early detection and no known specific premalignant changes. Human ovarian surface epithelial (HOSE) cells expressing human papillomavirus type 16 (HPV-16) E6/E7 genes undergo crisis, and surviving cells exhibit an immortalized phenotype. Cells show an increasingly invasive phenotype on collagen rafts over time. To ascertain the nature of this aberrant growth, we characterized this spontaneous progression of HOSE cells from a benign to an invasive phenotype using histopathology, immunophenotyping, and tumorigenesis assays. EXPERIMENTAL DESIGN: At various passages, cells were monitored for growth on collagen, response to tumor necrosis factor alpha and daunorubicin, immunohistochemistry and Western blot analysis of E-cadherin and beta-catenin, growth in soft agar, and tumor formation in immunodeficient mice. RESULTS: As passage number increased, cells became increasingly aggressive on collagen, with more pronounced focal stratification and invasion. Furthermore, late-passage cells were more resistant to the apoptotic effects of TNF-alpha and daunorubicin than earlier-passage cells. E-cadherin expression was limited to early-passage cells, whereas beta-catenin was expressed regardless of passage. Cells invading collagen formed colonies in soft agar at low efficiency but were not tumorigenic in immunodeficient mice. Some cultures recovered from colonies grew in soft agar at high efficiencies, and one was tumorigenic. CONCLUSIONS: HOSE cells expressing E6/E7, over time, develop characteristics of malignant cells and produce tumors consistent with an ovarian surface epithelium lineage. Progression of HOSE cells from a benign to an invasive phenotype in vitro may provide a model to dissect the progression of ovarian cancer.

Modulation of intracellular beta-catenin localization and intestinal tumorigenesis in vivo and in vitro by sphingolipids.

Sphingolipid consumption suppresses colon carcinogenesis, but the specific genetic defect(s) that can be bypassed by these dietary components are not known. Colon tumors often have defect(s) in the adenomatous polyposis coli (APC)/beta-catenin regulatory system. Therefore, C57Bl/6J(Min/+) mice with a truncated APC gene product were fed diets supplemented with ceramide, sphingomyelin, glucosylceramide, lactosylceramide, and ganglioside G(D3) (a composition similar in amount and type to that of dairy products) to determine whether tumorigenesis caused by this category of genetic defect is suppressed. Sphingolipid feeding reduced the number of tumors in all regions of the intestine, and caused a marked redistribution of beta-catenin from a diffuse (cytosolic plus membrane) pattern to a more "normal" localization at mainly intercellular junctions between intestinal epithelial cells. The major digestion product of complex sphingolipids is sphingosine, and treatment of two human colon cancer cell lines in culture (SW480 and T84) with sphingosine reduced cytosolic and nuclear beta-catenin, inhibited growth, and induced cell death. Ceramides, particularly long-chain ceramides, also had effects. Thus, dietary sphingolipids, presumably via their digestion products, bypass or correct defect(s) in the APC/beta-catenin regulatory pathway. This may be at least one mechanism whereby dietary sphingolipids inhibit colon carcinogenesis, and might have implications for dietary intervention in human familial adenomatous polyposis and colon cancer.

Evaluation of the 5-French saline paediatric gastric tonometer.

OBJECTIVE: To evaluate the paediatric 5-French (Fr) saline-filled gastric tonometer. DESIGN: (a) In vitro comparison of saline bath reference pCO2 with tonometric pCO2 measured by normal saline-filled and phosphate-buffered saline-filled 5-Fr tonometers, and by a recirculating gas tonometer. ( b) In vivo comparison of gastric intramucosal pCO2i, measured by normal saline-filled 5-Fr tonometer (NST) and simultaneously by recirculating gas tonometer (RGT) in ten paediatric intensive care patients. (c) In vivo comparison of pCO2i measured simultaneously by 2 NST 5-Fr tonometers, before and after enteral feeding, in ten paediatric intensive care patients. MEASUREMENTS AND MAIN RESULTS: (a) Twenty consecutive measurements of pCO2 were made at constant reference pCO2 of 19, 38, 56, and 75 mmHg (2.5, 5.0, 7.5, and 10.0 kPa), respectively. The NST tonometer underestimated reference pCO2 by mean bias (limits of agreement) of 58% (20%), and the phosphate-buffered saline-filled tonometer by 6% (26%). The RGT showed mean bias 5.7% with narrow limits of agreement (1.5%). (b) In 50 paired (NST vs. RGT) in vivo measurements over pCO2i range 23-73 mmHg (3.0-9.7 kPa), the NST underestimated RGT pCO2i by a mean bias of 10 mmHg (1.3 kPa), with limits of agreement +/-10 mmHg (1.5 kPa). This resulted in NST consistently overestimating pHi and underestimating pCO2 gap (both P < 0.001). (c) One hundred simultaneous paired NST measurements were assessed (50 without, and 50 with enteral feeding). The mean biases (limits of agreement) were identical in the fasted and fed states 0.4+/-6 mmHg, with no difference between the fed and fasting states (P = 0.7). CONCLUSIONS: There are inherent problems in the methodology of saline tonometry, which adversely affect the accuracy and reliability of the 5-Fr paediatric gastric tonometer in comparison to recirculating gas tonometry.

Essential role for the dsRNA-dependent protein kinase PKR in innate immunity to viral infection.

The double-stranded (ds) RNA-dependent protein kinase PKR is considered to play an important role in interferon's (IFN's) response to viral infection. Here, we demonstrate that mice lacking PKR are predisposed to lethal intranasal infection by the usually innocuous vesicular stomatitis virus, and also display increased susceptibility to influenza virus infection. Our data indicate that in normal cells, PKR primarily prevents virus replication by inhibiting the translation of viral mRNAs through phosphorylation of eIF2alpha, while concomitantly assisting in the production of autocrine IFN and the establishment of an antiviral state. These results show that PKR is an essential component of innate immunity that acts early in host defense prior to the onset of IFN counteraction and the acquired immune response.

Alpha/beta interferons potentiate virus-induced apoptosis through activation of the FADD/Caspase-8 death signaling pathway.

Interferon (IFN) mediates its antiviral effects by inducing a number of responsive genes, including the double-stranded RNA (dsRNA)-dependent protein kinase, PKR. Here we report that inducible overexpression of functional PKR in murine fibroblasts sensitized cells to apoptosis induced by influenza virus, while in contrast, cells expressing a dominant-negative variant of PKR were completely resistant. We determined that the mechanism of influenza virus-induced apoptosis involved death signaling through FADD/caspase-8 activation, while other viruses such as vesicular stomatitis virus (VSV) and Sindbis virus (SNV) did not significantly provoke PKR-mediated apoptosis but did induce cytolysis of fibroblasts via activation of caspase-9. Significantly, treatment with IFN-alpha/beta greatly sensitized the fibroblasts to FADD-dependent apoptosis in response to dsRNA treatment or influenza virus infection but completely protected the cells against VSV and SNV replication in the absence of any cellular destruction. The mechanism by which IFN increases the cells' susceptibility to lysis by dsRNA or certain virus infection is by priming cells to FADD-dependent apoptosis, possibly by regulating the activity of the death-induced signaling complex (DISC). Conversely, IFN is also able to prevent the replication of viruses such as VSV that avoid triggering FADD-mediated DISC activity, by noncytopathic mechanisms, thus preventing destruction of the cell.

Oligonucleotide-based inhibition of embryonic gene expression.

We describe a technique to define gene function using antisense oligonucleotide (AS-ODN) inhibition of gene expression in mice. A single intravenous injection of an AS-ODN targeting vascular endothelial growth factor (VEGF) into pregnant mice between E7.5-8.5 resulted in a lack of primary angiogenesis. This enabled us to define the critical window required to inhibit VEGF expression and recapitulate the primary loss of function phenotype observed in VEGF (-/-) embryos. This phenotype was sequence-specific and time- and dose-dependent. Injection of an AS-ODN targeting a second gene, E-cadherin, into pregnant mice at E10 confirmed a hypothesized secondary phenotype. This is the first report of AS-ODN inhibition of gene expression in utero and provides a new strategy for target validation in functional genomics.

Vesicular stomatitis virus G protein acquires pH-independent fusion activity during transport in a polarized endometrial cell line.

Entry of vesicular stomatitis virus (VSV), the prototype member of the rhabdovirus family, occurs by receptor-mediated endocytosis. Subsequently, during traversal through the endosomal compartments, the VSV G protein acquires a low-pH-induced fusion-competent form, allowing for fusion of the viral membrane with endosomal and lysosomal membranes. This fusion event releases genomic RNA into the cytoplasm of the cell. Here we provide evidence that the VSV G protein acquires a fusion-competent form during exocytosis in a polarized endometrial cell line, HEC-1A. VSV infection of HEC-1A cells results in high viral yields and giant cell formation. Syncytium formation is blocked in a concentration-dependent manner by treatment with the lysosomotropic weak base ammonium chloride, which raises intravesicular pH. Virus release is somewhat delayed by treatment with ammonium chloride, but virus yields gradually reach those of control cells. In addition, inhibition of vacuolar H(+)-ATPases by treatment with bafilomycin A1 also inhibited cell to cell fusion without altering virus yields. Virions released from infected HEC cells were themselves not fusion competent, since viral entry required an active H(+)-ATPase and a low-pH-induced conformational change in the viral G protein. Thus, the conformation change leading to fusion competence during exocytotic transport is reversible and reverts during or after release of the virion from the infected cell.

Host cell dependence of viral morphology.

The morphology of influenza virions was found to depend on cellular determinants. Influenza viral filaments up to 30 microm in length were observed to form at high levels on surfaces of various polarized epithelial cell types infected with the A/Udorn/72 virus. In contrast, virions produced by nonpolarized cell types infected with this virus were almost exclusively of spherical morphology. Disruption of the actin microfilament array by cytochalasin D treatment of polarized MDCK cells had a profound effect on viral morphology. Although virus titers and release of spherical particles were not reduced in the presence of cytochalasin D, we observed a 15-fold reduction in the release of filamentous particles. In contrast, the ratio of filaments to spheres produced by infected MDCK cells was not altered by the microtubule-disrupting agent nocodazole. These observations indicate that the polarized cell phenotype and the integrity of the actin microfilament network are important cellular determinants of the morphology of a filamentous influenza virus.

Induction of apoptosis by fumonisin B1 in HT29 cells is mediated by the accumulation of endogenous free sphingoid bases.

Fumonisin B1 (FB1) and aminopentol (AP1) (which is formed by hydrolysis of FB1) are found in corn contaminated with some strains of Fusarium moniliforme. Incubation of HT29 cells (a human colonic cell line) with FB1 or AP1 caused a significant reduction in cell number; AP1 was less potent, with 50 microM AP1 causing the same reduction (ca. 30% after 24 h) as 10 microM FB1. The reduction in cell number reflected increases in DNA fragmentation and the percentage of apoptotic cells. Both FB1 and AP1 caused the accumulation of sphinganine (25- and 35-fold by 10 microM FB1 and 50 microM AP1, respectively); thus, concentrations of FB1 and AP1 that caused comparable reductions in cell number were also similar with respect to elevation of sphinganine, a compound that is growth inhibitory and cytotoxic. Inhibition of the first step of sphingolipid biosynthesis with ISP-1 prevented the elevation in sphinganine, DNA fragmentation, and apoptosis induced by FB1. Therefore, these effects of FB1 on HT29 cells can be attributed to the accumulation of sphinganine. Since consumption of food contaminated with Fusarium moniliforme (Sheldon) exposes colonic cells to these mycotoxins, the possibility that FB1 and AP1 are toxic for intestinal cells in vivo should be evaluated, especially in the light of the recent report (Bhat et al., Clin. Toxicol. 35, 249, 1997) describing intestinal disturbances in humans after consumption of moldy corn and sorghum containing fumonisins.

The M1 and M2 proteins of influenza A virus are important determinants in filamentous particle formation.

Influenza A virus is highly pleomorphic with particles exhibiting either spherical or filamentous morphology. The mechanisms behind this pleomorphism and its importance in viral pathogenesis are not clearly understood. We have observed that budding of filamentous influenza A/Udorn virus particles can be readily visualized by immunofluorescence microscopy. Filamentous particle formation was inhibited by treatment of cells with the anti-M2 14C2 antibody, but was not inhibited with the isotype identical 5C4 anti-M2 antibody or by anti-neuraminidase antibody. To further explore the viral determinants of filamentous particle formation, we investigated the morphology and growth characteristics of three variants of A/Udorn/72 virus, which had previously been selected for their resistance to growth inhibition by the 14C2 anti-M2 monoclonal antibody. Two of the variant viruses, 5A and 10A, contain single amino acid substitutions in the cytoplasmic domain of the M2 protein, whereas the 1A variant contains a single amino acid substitution in the viral matrix protein, M1. Variants 5A and 10A both were found to retain the filamentous particle phenotype found in the parental strain A/Udorn/72, and the production of filamentous virions by both variants was resistant to inhibition by the 14C2 antibody. However, immunofluorescence and electron microscopy revealed that the variant 1A was composed almost exclusively of spherical particles. The 1A variant displayed higher viral yields and a larger plaque size than the filamentous viruses. In addition, we separated distinct populations highly enriched in spherical or filamentous particles by velocity gradient centrifugation. Analysis of the protein compositions of these particles revealed that the NP:M1 or NP:HA ratios in filamentous particles were significantly lower than in spherical particles, but the filaments have higher levels of NP per particle. The spherical and filamentous particles were found to have similar specific infectivity. These results indicate that the filamentous morphology of the A/Udorn virus depends upon the matrix (M1) and/or M2 proteins.

Effects of antibody to the influenza A virus M2 protein on M2 surface expression and virus assembly.

We have investigated the effect of a monoclonal antibody on influenza virus release and the cell surface expression of M2, comparing virus strains which were observed previously to be sensitive (A/Udorn) or resistant (A/WSN and A/Udorn variants) to growth inhibition by M2 antibody 14C2. Incubation of A/Udorn virus-infected cells in the presence of the inhibitory M2 antibody resulted in a significant reduction in the yield of virus, as measured by infectivity assays as well as by the release of purified virions. The release of A/Udorn virus was not inhibited by the presence of monovalent 14C2 Fab, in contrast to IgG, indicating that a bivalent structure is essential for 14C2 antibody-mediated viral growth restriction. The level of M2 surface expression in A/Udorn virus-infected MDCK cells was found to be reduced to approximately 60% of control levels in cells incubated with the 14C2 antibody. In contrast, M2 surface expression levels in A/WSN virus-infected cells were decreased by only approximately 5-15%, and A/WSN virus assembly appeared to be unaffected by the M2 antibody treatment. M2 antigen associated with cell membranes and virus particles was redistributed into clusters after M2 antibody treatment in infected cells. Incubation in the presence of the 14C2 antibody also reduced M2 surface expression by approximately 40-50% in cells infected with a recombinant vaccinia virus that expresses the M2 A/Udorn protein. These results demonstrate that M2 antibody reduces the level of influenza virus particle formation in a single cycle of infection and suggest that inhibition of A/Udorn virus replication by the 14C2 antibody is related to the reduced cell surface expression and redistribution of the M2 protein induced by the antibody treatment.

Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin.

The role of three N-linked glycans which are conserved among various hemagglutinin (HA) subtypes of influenza A viruses was investigated by eliminating the conserved glycosylation (cg) sites at asparagine residues 12 (cg1), 28 (cg2), and 478 (cg3) by site-directed mutagenesis. An additional mutant was constructed by eliminating the cg3 site and introducing a novel site 4 amino acids away, at position 482. Expression of the altered HA proteins in eukaryotic cells by a panel of recombinant vaccinia viruses revealed that rates and efficiency of intracellular transport of HA are dependent upon both the number of conserved N-linked oligosaccharides and their respective positions on the polypeptide backbone. Glycosylation at two of the three sites was sufficient for maintenance of transport of the HA protein. Conserved glycosylation at either the cg1 or cg2 site alone also promoted efficient transport of HA. However, the rates of transport of these mutants were significantly reduced compared with the wild-type protein or single-site mutants of HA. The transport of HA proteins lacking all three conserved sites or both amino-terminally located sites was temperature sensitive, implying that a polypeptide folding step had been affected. Analysis of trimer assembly by these mutants indicated that the presence of a single oligosaccharide in the stem domain of the HA molecule plays an important role in preventing aggregation of molecules in the endoplasmic reticulum, possibly by maintaining the hydrophilic properties of this domain. The conformational change observed after loss of all three conserved oligosaccharides also resulted in exposure of a normally mannose-rich oligosaccharide at the tip of the large stem helix that allowed its conversion to a complex type of structure. Evidence was also obtained suggesting that carbohydrate-carbohydrate interactions between neighboring oligosaccharides at positions 12 and 28 influence the accessibility of the cg2 oligosaccharide for processing enzymes. We also showed that terminal glycosylation of the cg3 oligosaccharide is site specific, since shifting of this site 4 amino acids away, to position 482, yielded an oligosaccharide that was arrested in the mannose-rich form. In conclusion, carbohydrates at conserved positions not only act synergistically by promoting and stabilizing a conformation compatible with transport, they also enhance trimerization and/or folding rates of the HA protein.

Evaluation of the elderly driver with arthritis.

Examination focusing upon the two clusters of function necessary for turning and braking is important as is nonthreatening questioning about driving. The effects of drugs used for arthritis on the CNS and a heightened emphasis upon the psychosocial implications of immobility complicate the management of older drivers with arthritis. Assuming optimal treatment of the arthritis, the two most important management tools are actually power-steering and automatic transmission. Less expensive adaptive available thorough rehabilitation services (e.g., auxiliary mirrors) are also of value.

Three adjacent genes of African swine fever virus with similarity to essential poxvirus genes.

Nucleotide sequencing of the right end of the SalIj fragment of the highly virulent Malawi Lil20/1 strain of African swine fever virus (ASFV) has revealed three adjacent genes with similarity to: serine-threonine protein kinases; members of the putative helicase superfamily SF2; and the vaccinia virus 56 kDa abortive late protein. All three genes are transcribed to the left with respect to the orientation of the ASFV genome. Gene L19IL predicts a protein similar to serine-threonine protein kinases including vaccinia virus gene B1R. Gene L19KL predicts a protein that is likely to be a nucleic acid-dependent ATPase, as it has similarity to both the poxvirus 70 kDa early transcription factor subunit and the poxvirus nucleoside triphosphatase I gene. Gene L19LL has extensive similarity to the vaccinia virus 56 kDa abortive late protein.

A rapid, quantitative assay for titration of bovine virus diarrhoea-mucosal disease virus.

An end point dilution microtitration assay is described that can be used for the titration of both cytopathic and non-cytopathic isolates of bovine virus diarrhoea-mucosal disease virus. Indirect immunofluorescence is used to detect infected MDBK cells in the wells of Terasaki plates. The virus titre is derived from the number of uninfected wells, using the Poisson distribution. The assay is simple, fast and economical. Titres of cytopathic virus determined by the microtitration assay and standard plaque assay are equivalent.

Expression of Kirsten murine sarcoma virus in transformed nonproducer and revertant NIH/3T3 cells: evidence for cell-mediated resistance to a viral oncogene in phenotypic reversion.

Expression of the provirus in a clonally related series of Kirsten murine sarcoma virus-transformed NIH/3T3 nonproducer cell lines was examined at both the transcriptional and translational levels. All cells expressed high levels of genome-sized viral RNA with little variation between cell lines despite differences in provirus integration site and copy number. Expression of K-ras RNA was estimated to be at least 10- to 20-fold higher than that of the mouse cellular homolog of the viral transforming gene. Levels of the virus-coded transforming protein, p21, were similarly elevated, with little variation between nonproducer cells. In two revertant cell lines containing a normal provirus and a rescuable transforming gene, no impairment in expression at either the transcriptional or translational level was found. After superinfection with Kirsten murine sarcoma virus, one revertant became more tumorigenic, whereas the other remained nontumorigenic. These results show that cell transformation by Kirsten murine sarcoma virus is invariably associated with elevated expression of the virus-coded oncogene and that one of the revertants is resistant to the action of the viral transforming gene.