Effects of a novel tyrosine kinase inhibitor in rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: Biologicals have revolutionised the treatment of rheumatoid arthritis (RA). However, progressive joint destruction can still be observed in many patients and the search for novel molecular therapies targeting specific signalling pathways is ongoing. In the present study, we investigated the effects of GW282974, a novel compound directed against tyrosine kinase activity with respect to the potential suppression of inflammation and destruction. METHODS: Synovial tissue specimens were obtained from RA patients undergoing surgical joint replacement. Rheumatoid arthritis synovial fibroblasts (RASFs) were stimulated with cytokines and GW282974 was added in different concentrations. Gene expression was checked by TaqMan PCR, using 18S as housekeeping gene. Protein analysis was quantified by ELISA. Cell growth and proliferation was measured using the "ViaLight" proliferation assay. RESULTS: EGF had no effect on the gene expression profile of RASFs when used as single stimulatory agent. In combination with pro-inflammatory mediators however, EGF showed a synergistic effect. The expression of matrix metalloproteinases, inflammatory cytokines and cyclooxygenase-2 on mRNA levels was strongly increased, whereas the addition of GW282974 abrogated these effects in a dose-dependent manner. These data could be confirmed on protein/lipid levels analysing the supernatants of RASFs by ELISA. Similarly, cell growth and proliferation of RASFs were inhibited by GW282974 in a dose- and time-dependent manner. By contrast, no cytotoxic effects were seen within the concentrations used. DISCUSSION: GW282974 appears to interfere with the inflammatory and the destructive pathways in RASFs and might therefore be used as novel therapeutic strategy for the treatment of RA.

Recreation of neuronal Kv1 channel oligomers by expression in mammalian cells using Semliki Forest virus.

The multiple roles of voltage-sensitive K(+) channels (Kv1 subfamily) in brain are served by subtypes containing pore-forming alpha (1.1-1.6) and auxiliary beta subunits, usually in an (alpha)(4)(beta)(4) stoichiometry. To facilitate structure/activity analysis, combinations that are prevalent in neurones and susceptible to alpha-dendrotoxin (alphaDTX) were reproduced in mammalian cells, using Semliki Forest virus. Infected Chinese hamster ovary cells expressed N-glycosylated Kv1.1 and 1.2 alpha subunits (M(r) approximately 60 and 62 K) that assembled and bound [(125)I]-alphaDTX with high affinity; an appreciable proportion appeared on the cell surface, with Kv1.2 showing a 5-fold enrichment in a plasma membrane fraction. To obtain 'native-like' alpha/beta complexes, beta1.1 or 2.1 (M(r) approximately 42 and 39 K, respectively) was co-expressed with Kv1.1 or 1.2. This slightly enhanced N-glycosylation and toxin binding, most notable with beta2. 1 and Kv1.2. Solubilization of membranes from cells infected with Kv. 1.2 and beta2.1, followed by Ni(2+) chromatography, gave a purified alpha1.2/beta2.1 complex with a size of approximately 405 K and S(20, W) = 15.8 S. Importantly, these values indicate that four alpha and beta subunits co-assembled as in neurones, a conclusion supported by the size ( approximately 260 K) of the homo-tetramer formed by Kv1.2 alone. Thus, an authentic K(+) channel octomer has been reconstructed; oligomeric species were also found in plasma membranes. To create 'authentic-like' hetero-oligomeric channels, Kv1.1 and 1.2 were co-expressed and shown to have assembled by the precipitation of both with IgGs specific for either. Consistently, confocal microscopy of cells labeled with these antibodies showed that the relatively low surface content of Kv1.1 was increased by Kv1.2. [(125)I]-alphaDTX binding to these complexes was antagonized by DTX(k), a probe selective for Kv1.1, in a manner that mimicks the pattern observed for the Kv1.1/1.2-containing channels in neuronal membranes.

Human Bak induces cell death in Schizosaccharomyces pombe with morphological changes similar to those with apoptosis in mammalian cells.

Apoptosis as a form of programmed cell death (PCD) in multicellular organisms is a well-established genetically controlled process that leads to elimination of unnecessary or damaged cells. Recently, PCD has also been described for unicellular organisms as a process for the socially advantageous regulation of cell survival. The human Bcl-2 family member Bak induces apoptosis in mammalian cells which is counteracted by the Bcl-x(L) protein. We show that Bak also kills the unicellular fission yeast Schizosaccharomyces pombe and that this is inhibited by coexpression of human Bcl-x(L). Moreover, the same critical BH3 domain of Bak that is required for induction of apoptosis in mammalian cells is also required for inducing death in yeast. This suggests that Bak kills mammalian and yeast cells by similar mechanisms. The phenotype of the Bak-induced death in yeast involves condensation and fragmentation of the chromatin as well as dissolution of the nuclear envelope, all of which are features of mammalian apoptosis. These data suggest that the evolutionarily conserved metazoan PCD pathway is also present in unicellular yeast.

Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family.

Site-specific RNA cleavage generates the 3' end of a poxvirus late mRNA.

The cowpox virus late mRNAs encoding the major protein of the A-type inclusions have 3' ends corresponding to a single site in the DNA template. The DNA sequence of the Alu I-Xba I fragment at this position encodes an RNA cis-acting signal, designated the AX element, which directs this RNA 3' end formation. In cells infected with vaccinia virus the AX element functions independently of either the nature of the promoter element or the RNA polymerase responsible for generating the primary RNA. At late times during virus replication, vaccinia virus induces or activates a site-specific endoribonuclease that cleaves primary RNAs within the AX element. The 3' end produced by RNA cleavage is then polyadenylylated to form the 3' end of the mature mRNA. Therefore, the poxviruses employ at least two mechanisms of RNA 3' end formation during the viral replication cycle. One mechanism, which is operative at early times in viral replication, involves the termination of transcription [Rohrmann, G., Yuen, L. & Moss, B. (1986) Cell 46, 1029-1035]. A second mechanism, which is operative at late times during viral replication, involves the site-specific cleavage of primary RNAs.

Vaccinia virus directs the synthesis of early mRNAs containing 5' poly(A) sequences.

mRNAs transcribed from late promoters of several poxvirus genes contain 5' poly(A) sequences that are not complementary to the viral DNA. In contrast, early mRNAs containing 5' poly(A) sequences have not previously been identified. Modifications to the sequence of the promoter of an early gene of cowpox virus enable this promoter to direct the synthesis of RNAs containing 5' poly(A) sequences. When the sequence 3'-ATTTA-5', which is present at the RNA start-sites of several late promoters, is positioned such that the RNA start-site of the early promoter is at the first thymidylate in this sequence, this early promoter directs the synthesis of early RNAs containing 4-11 adenylates at their 5' ends. When two of the thymidylates in the sequence 3'-ATTTA-5' are removed, the promoter directs the synthesis of early RNAs lacking 5' poly(A) sequences. These results are consistent with the proposal that 5' polyadenylylation of poxvirus RNAs occurs by repetitive transcription of thymidylates in the sequence 3'-ATTTA-5' often present at the sites of transcriptional initiation. In addition, these results demonstrate that 5' polyadenylylation of viral RNAs is not exclusively a late function. The promoter regions of a few early genes of vaccinia virus contain the sequence 3'-ATTTA-5'. Analyses of the transcripts of one of these genes, the D5 gene, indicated that these mRNAs contain 5' poly(A) sequences, suggesting that early mRNAs of a subset of viral genes contain 5' poly(A) sequences.

Transcription of a poxvirus early gene is regulated both by a short promoter element and by a transcriptional termination signal controlling transcriptional interference.

The promoter region of an early gene (38K gene) of cowpox virus has been characterized by deletion and linker scanning mutational analyses. Modified versions of this promoter region were placed into the genome of vaccinia virus, and their transcriptional efficiencies were assessed by quantifying RNAs transcribed from these sequences. These analyses showed that the sequences in the region between 33 and 4 base pairs upstream of the transcriptional start site affect the efficiency of transcription from this promoter. Linker scanning mutations in the -27 to -10 region inhibited transcription. This region contains the sequence 5'-GAAAATATATT-3', which is present in at least two other early genes in the same positions (-21 to -11) relative to the transcriptional start sites of these genes. Elements of this sequence are similarly positioned in the promoter regions of several other poxvirus genes, suggesting that this sequence represents a transcriptional control element of at least a subset of poxvirus genes. The -8 to -2 sequence (5'-TTTTTAT-3') contains a transcriptional termination signal. Mutation of this sequence had two separate effects: (i) it reduced the efficiency of transcription from the promoter by approximately 30%, and (ii) it prevented this sequence from terminating the transcription from upstream genes. When overlapping transcription from upstream genes was not prevented by a termination signal present either within the 38K promoter or upstream of the promoter, transcription from this promoter was reduced by about 30%. This indicates that transcriptional termination has a role in the regulation of viral gene expression by controlling transcriptional interference.

Hemorrhage in lesions caused by cowpox virus is induced by a viral protein that is related to plasma protein inhibitors of serine proteases.

Several recombinant cowpox viruses were constructed and used to identify a viral gene that controls the production of hemorrhage in lesions caused by the Brighton Red strain of cowpox virus (CPV-BR). This gene is located in the KpnD fragment of CPV-BR DNA, between 31 and 32 kilobases from the end of the genome. This position corresponds well with that predicted from analyses of the DNA structures of spontaneously generated deletion mutants. The gene responsible for hemorrhage encodes a 38-kDa protein that is one of the most abundant early gene products. The 11-base-pair sequence GAAAATATATT present 84 base pairs upstream of its coding region is also present upstream of three other early genes of vaccinia virus; therefore, this sequence may be involved in the regulation of transcription. There is extensive similarity between the predicted amino acid sequence of the 38-kDa protein and the amino acid sequences of several plasma proteins that are inhibitors of various serine proteases involved in blood coagulation pathways. This suggests that the viral protein may possess a similar biological activity, which may enable it to effect hemorrhage by inhibiting one or more of the serine proteases involved in the host's normal processes of blood coagulation and wound containment.

Spontaneous deletions and duplications of sequences in the genome of cowpox virus.

Examination of the genomes of 10 white-pock variants of cowpox virus strain Brighton red (CPV-BR) revealed that 9 of them had lost 32 to 38 kilobase pairs (kbp) from their right-hand ends and that the deleted sequences had been replaced by inverted copies of regions from 21 to 50 kbp long from the left-hand end of the genome. These variants thus possess inverted terminal repeats (ITRs) from 21 to 50 kbp long; all are longer than the ITRs of CPV-BR (10 kbp). The 10th variant is a simple deletion mutant that has lost the sequences between 32 and 12 kbp from the right-hand end of the genome. The limits of the inner ends of the observed deletions (between 32 and 38 kbp from the right-hand end of the CPV-BR genome) appear to be defined by the location of the nearest essential gene on the one hand and the location of the gene that encodes "pock redness" on the other. The genomes of the deletion/duplication white-pock variants appear to have been generated either by single crossover recombinational events between two CPV-BR genomes aligned in opposite directions or by the nonreciprocal transfer of genetic information. The sites where such recombination/transfer occurred were sequenced in four variants. In all of them, the sequences adjacent to such sites show no sequence homology or any other unusual structural feature. The analogous sites at the internal ends of the two ITRs of CPV-BR also were sequenced and also show no unusual features. It is likely that the ITRs of CPV-BR and of its white-pock variants, and probably those of other orthopox-virus genomes, arise as a result of nonhomologous recombination or by random nonreciprocal transfer of genetic information.

Benzyl derivative facilitation of transcription in Escherichia coli at the ara and lac operon promoters: metabolite gene regulation (MGR).

A number of benzyl derivatives have been tested for their ability to induce the expression of the araBAD operon in an Escherichia coli K-12 strain. Those derivatives shown to be stimulatory include: benzoic acid (BA), para-amino benzoic acid (PABA), para-hydroxy benzoic acid (PHBA), ortho-amino benzoic acid (OABA), 3-hydroxy-4-methoxy phenylethylamine (MTA), and 4-hydroxy-3-methoxyphenol acetic acid (HVA). The araC gene product was necessary to facilitate the induction. To further characterize if the inductive effect was mediated at the level of transcription, an araBAD-tetracycline resistant (Tcr) operon fusion plasmid (pAP-B) was employed. Benzyl derivatives which induce expression of the araBAD operon in situ also induced a Tcr phenotype with pAP-B. Both indole acetic acid (IAA) and imidazole (IM), which were previously shown to circumvent the necessity for cAMP in the induction of the araBAD operon, also induced a Tcr phenotype with pAP-B. Induction of lac or other cAMP responding operons with the inducing molecules at the chromosomal level was not detectable when assessed by carbon utilization. However, a lacZYA-Tcr operon fusion plasmid (pLPI) did respond to IAA and several of the inducing benzyl derivatives. Catabolite repression of chromosomal araBAD expression was reversed when the exogenous concentration of OABA was elevated. Similar effects on the Tcr phenotypes conferred by pAP-B and pLP1 were observed when OABA or several other inducing benzyl derivatives were present exogenously.