Poriferan survivin exhibits a conserved regulatory role in the interconnected pathways of cell cycle and apoptosis.

Survivin orchestrates intracellular pathways during cell division and apoptosis. Its central function as mitotic regulator and inhibitor of cell death has major implications for tumor cell proliferation. Analyses in early-branching Metazoa so far propose an exclusive role of survivin as a chromosomal passenger protein, whereas only later during evolution a complementary antiapoptotic function might have arisen, concurrent with increased organismal complexity. To lift the veil on the ancestral function(s) of this key regulator, a survivin-like protein (SURVL) of one of the earliest-branching metazoan taxa was identified and functionally characterized. SURVL of the sponge Suberites domuncula shares considerable similarities with its metazoan homologs, ranging from conserved exon/intron structure to presence of protein-interaction domains. Whereas sponge tissue shows a low steady-state level, SURVL expression was significantly upregulated in rapidly proliferating primmorph cells. In addition, challenge of tissue and primmorphs with heavy metal or lipopeptide stimulated SURVL expression, concurrent with the expression of a newly discovered caspase. Complementary functional analyses in transfected HEK-293 cells revealed that heterologous expression of a SURVL-EFGP fusion not only promotes proliferation but also enhances resistance to cadmium-induced cell death. Taken together, these results suggest both a deep evolutionary conserved dual role of survivin and an equally conserved central position in the interconnected pathways of cell cycle and apoptosis.

Pharmacological characterisation of pyrimidinoceptor responses in NG108-15 cells.

In the present study, the P2Y receptor(s) mediating the effects of the pyrimidines UTP and UDP on phospholipase C activation in the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 was investigated. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis detected transcripts for the P2Y(6) and P2Y(2) receptors, but not for P2Y(1) and P2Y(4.) UTP and UDP were equipotent agonists and their effects were partially additive. Suramin, reactive blue 2 and pyridoxal phosphate-6-azophenyl-2',4'disulfonic acid (PPADS) antagonised the phospholipase C response to both UTP and UDP. High micromolar concentrations of adenosine, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), 2',3'-O-isopropylideneadenosine (iPAdo) and adenosine 3':5'-cyclic monophosphate (3',5'-cAMP) were able to antagonise the effect of UTP on phospholipase C but not that of UDP. The additivity of the UTP and UDP responses, novel P2 receptor antagonist profile and the distinguishing action of adenosine may indicate the expression of a pyrimidine selective P2Y receptor in addition to the P2Y(6) type in these cells.

Adenosine-derived non-phosphate antagonists for P2Y(1) purinoceptors.

Novel type antagonists for P2Y(1) adenine nucleotide receptors were synthesized by coupling of adenosine 5'-OH group with oligo-aspartate chain via a carbonyl linker. All these conjugates (AdoOC(O)Asp(n), n = 1-4) inhibited the 2MeSADP-stimulated synthesis of inositol phosphates in 1321N1 human astrocytoma cells stably expressing human P2Y(1) receptors. This inhibitory effect followed the rank order AdoOC(O)Asp(2)> AdoOC(O)Asp(3)> AdoOC(O)Asp(1)> AdoOC(O)Asp(4) with antagonistic constant pA(2) = 5.4 for AdoOC(O)Asp(2). Potency of this non-phosphate inhibitor was comparable with the previously known adenosine 3',5'- and 2', 5'-bisphosphates. Chemical and biological stabilities of these novel adenosine derived antagonists of the nucleotide receptor provide perspectives of their pharmacological implication.

Expression and activity of 2-5A synthetase in the course of differentiation and apoptosis of PC12 cells.

The role of IFN-induced 2-5A system in cell differentiation has not been elucidated. While studying differentiation of PC12 cells we found that the simultaneous treatment of cells with NGF and IFN-gamma in serum-containing medium resulted first in the extension of neurites and then apoptosis. On the contrary, in serum-free medium the cells underwent a more rapid neuronal differentiation. Only the doses of NGF which induced the outgrowth of neurites from the cells were able to induce rapid cell death in combined treatment. When the cells were treated subsequently with NGF and IFN-gamma, the induction of death was observed with NGF post-treatment, but not with NGF pretreatment. Relying on these alternative biological responses, we studied the changes in 2-5A synthetase activity and its 43 kDa isoform expression in the course of differentiation and death of PC12 cells. The results of the present work showed that NGF-induced differentiation of the cells did not evoke any increase in 2-5A synthetase activity or any increase in the expression of its 43 kDa isoform. Moreover, the obtained results demonstrated that NGF could not significantly affect the IFN-induced signalling pathway leading to the activation of 2-5A synthetase gene, at least regarding the studied enzyme activity.

Origin of the interferon-inducible (2'-5')oligoadenylate synthetases: cloning of the (2'-5')oligoadenylate synthetase from the marine sponge Geodia cydonium.

In vertebrates cytokines mediate innate (natural) immunity and protect them against viral infections. The cytokine interferon causes the induction of the (2'-5')oligoadenylate synthetase [(2-5)A synthetase], whose product, (2'-5')oligoadenylate, activates the endoribonuclease L which in turn degrades (viral) RNA. Three isoforms of (2-5)A synthetases exist, form I (40-46 kDa), form II (69 kDa), and form III (100 kDa). Until now (2-5)A synthetases have only been cloned from birds and mammals. Here we describe the cloning of the first putative invertebrate (2-5)A synthetase from the marine sponge Geodia cydonium. The deduced amino acid sequence shows signatures characteristic for (2-5)A synthetases of form I. Phylogenetic analysis of the putative sponge (2-5)A synthetase indicates that it diverged first from a common ancestor of the hitherto known members of (vertebrate) (2-5)A synthetases I, (2-5)A synthetases II and III. Moreover, it is suggested that the (2-5)A synthetases II and III evolved from this common ancestor (very likely) by gene duplication. Together with earlier results on the existence of the (2'-5')oligoadenylates in G. cydonium, the data presented here demonstrate that also invertebrates, here sponges, are provided with the (2-5)A system. At present, it is assumed that this system might be involved in growth control, including control of apoptosis, and acquired its additional function in innate immune response in evolutionarily younger animals, in vertebrates.

Only pyrimidinoceptors are functionally expressed in mouse neuroblastoma cell lines.

The ability of UTP, UDP, ATP, and ADP to influence inositol phospholipid hydrolysis in neuroblastoma origin cell lines was assessed. The mouse neuroblastoma lines N1E 115, Neuro 2a, and NB4 1A3 and the rat glioma/mouse neuroblastoma hybrid line NG108-15 gave robust responses to both UTP and UDP, which were essentially equipotent. Thus a range of cell lines of mouse neuroblastoma origin express a pyrimidine-selective P2Y receptor. The NG108-15 cells were the only cell type tested at which ATP and ADP displayed activity with EC50 values of greater than 100 microM, compared with values of 0.58 and 1.25 microM for UTP and UDP, respectively. In contrast to the cell lines derived from mouse neuroblastoma, the human neuroblastoma lines SH-SY5Y and SK-N-SH did not respond to any nucleotides, although both responded well to carbachol.

[3'-Deoxy-3'-fluoro analogs of core trimers of 2-5A: effect on lytic activity of human NK-lymphocytes]. / 3'-Dezoksi-3'-ftoranalogi korovykh trimerov 2-5A: vliianie na liticheskuiu aktivnost' NK-limfotsitov cheloveka.

The effect of core trimers, (2'-5')-analogues of oligoadenylic acid containing 9-(3-deoxy-3-fluoro-beta-D-xylofuranosyl)adenine (AF) and 3'-deoxy-3'-fluoroadenosine (AF) in various positions of the oligomer chain, on the lytic activity of human natural killer cells (NK cells) was studied in three different ways. The cellular cytotoxicity was determined using a highly sensitive nonradioactive approach employing a chelate europium-diethylenetriamino-pentaacetic acid complex (Eu-DTPA). It was shown that all fluorodeoxyanalogues enhance the lytic activity of intact NK lymphocytes, which follows from the lysis rate constant k2. At the same time, the substitution of either the central adenosine fragment or (to a greater extent) the 5'-terminal residue of (2'-5')A3 with AF causes a decrease in the number of active NK cells, which, unlike the case of the natural core trimer, leads to a loss of the capacity to increase the activity of NK. By contrast, isomeric ribo-analogues. (2'-5')(AF)A2 and (2'-5')A(AF)A, and trimers with the 2'(3')-terminal nucleotide substituted by AF or AF increased the activity of NK cells with an effectiveness close to or higher than the natural trimer (2'-5')A3. Inasmuch as isomeric xylo- and ribo-3'-deoxy-3'-fluoroanalogues of (2'-5')A3 are stereochemically modified oligomers, the data unambiguously suggest that the spatial structure of these trimers affects the increase in the lytic activity of NK cells.

[Interaction of 3'-fluoro-3'-deoxyanalogs of a trimer of (2'-5')oligoadenylic acid with (2'-5')A3-antibodies: stereochemical aspect]. / Vzaimodeistvie 3'-ftor-3'-dezoksianalogov trimera (2'-5')oligoadenilovoi kisloty s (2'-5')A3-antitelami: stereokhimicheskii aspekt.

Mouse antibodies to (2'-5')oligoadenylates were obtained by the immunization of animals with the (2'-5')oligoadenylic acid trimer conjugated with bovine serum albumin through a 2',3'-levulinic acid residue. Using radioimmunoassay, the reactivity of mouse polyclonal antibodies to the (2'-5')oligoadenylic acid trimer was studied for the trimer analogues containing 9-(3-deoxy-3-fluro-beta-D- xylofuranosyl)adenine and 3'-deoxy-3'-fluoro-adenosine in various positions of the chain. It was found that (a) the three-dimensional structure of short oligonucleotides is an important factor in the antibody recognition; (b) antibodies are more sensitive to modifications of the 5'-terminal and central ribose fragments of the (2'-5')oligoadenylic acid trimer; (c) the 3'-hydroxyl group plays a secondary role in the formation of the antigen determinant.

Pyrimidinoceptor potentiation by ATP in NG108-15 cells.

Regulation of inositol phospholipid hydrolysis by UTP and UDP in neuroblastoma x glioma hybrid cell line NG108-15 was potentiated in the presence of ATP. The effect of ATP was dose dependent and shifted the EC50 value for these uracil nucleotides up to three powers of magnitude, having no influence on the maximal value of the response. Adenine nucleotides (ADP, AMP, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), beta,gamma-methyleneadenosine 5'-triphosphate (betagammaMeATP), 3'-O-(4-benzoyl)benzoyl ATP (BzATP) and 3'-deoxyadenosine 5'-O-(1-thio)triphosphate (dATPalphaS)) as well as adenosine, had no influence on the pyrimidinoceptor response. The potentiation effect was abolished by excess of EDTA. The results were in agreement with the hypothesis of pyrimidinoceptor affinity regulation via extracellular phosphorylation of the receptor protein, initiated by ATP. This mechanism may have physiological implication for functioning of uracil nucleotides as endogenous signaling molecules.

Identification of the reaction products of (2'-5')oligoadenylate synthetase in the marine sponge.

Previously we reported on the presence of a high (2'-5')oligoadenylate synthetase activity in the marine sponge Geodia cydonium [Kuusksalu, A., Pihlak, A., Müller, W. E. G. & Kelve, M. (1995) Eur. J. Biochem. 232, 351-357]. The presence of (2'-5')oligoadenylates [(2'-5')A] in crude sponge extract was shown by radioimmunoassay and by their HPLC comigration with authentic (2'-5')A oligomers. In addition, the sponge (2'-5')oligoadenylates displayed biological activity, as determined by inhibition studies of protein biosynthesis in rabbit reticulocyte lysate. In the present study individual (2'-5')oligoadenylates synthesized by sponge enzyme were separated by HPLC. The exact composition of every oligonucleotide peak eluted was determined by matrix-assisted laser-desorption-ionization mass spectrometry (MALDI-MS) analysis. The 2'-5' phosphodiester bond in oligoadenylates was verified by NMR analysis. Based on the high concentration of (2'-5')A oligomers in G. cydonium and their similarity with those found in mammals we propose that the (2'-5')A system is involved in a cytokine-mediated pathway and/or in a protection system against viruses, present in the marine environment.

Monoclonal antibodies specific for Semliki Forest virus replicase protein nsP2.

A panel of monoclonal antibodies (MAbs) was raised against Semliki Forest virus (SFV) nonstructural protein nsP2, which is a protease, an NTPase, a putative RNA helicase, and a regulator of the synthesis of the subgenomic 26S mRNA encoding the structural proteins. nsP2, used for immunization, was expressed as a histidine fusion protein in Escherichia coli and purified by metal affinity chromatography. Dot-blot assay, using a membrane fraction from SFV-infected cells as antigen, gave 33 positive clones. Of these, 30 MAbs recognized nsP2 in Western immunoblotting, and 25 showed positive indirect immunofluorescence (IFAT) in SFV-infected cells; 15 MAbs stained the cytoplasmic vacuoles (CPVI), which are the sites of viral RNA synthesis in alphavirus-infected cells. MAb 3B5 recognized only CPVIs, as shown by double immunofluorescence staining with polyclonal anti-nsP3 antiserum. Most of the MAbs (20/33) recognized the nuclear form of nsP2, which may be associated with SFV neurovirulence. Immunoprecipitation with MAbs revealed that the SFV nonstructural proteins are associated with each other. None of the MAbs recognized Sindbis virus nsP2 in immunoblotting, indicating that they were directed to non-conserved sequences specific for SFV. Interestingly, these epitopes were located mostly within the N-terminal half of nsP2. Unexpectedly, the anti-nsP2 MAb 1E9 cross-reacted strongly with a host protein of 78 kDa from uninfected human, murine, avian and insect cells. This protein was identified as the immunoglobulin binding protein, BiP, by 2-D gel mapping and reaction with anti-BiP antiserum.

Neurotoxicity in rat cortical cells caused by N-methyl-D-aspartate (NMDA) and gp120 of HIV-1: induction and pharmacological intervention.

Incubation of highly enriched neurons from rat cerebral cortex with the human immunodeficiency virus type 1 (HIV-1) coat protein gp120 for 18 h results in fragmentation of DNA at internucleosomal linkers, a feature of apoptosis. We report that neurons respond to exposure to gp120 with an increased release of arachidonic acid via activation of phospholipase A2. This process is not inhibited by antagonists of the N-methyl-D-aspartate (NMDA) receptor channels. To investigate the influence of arachidonic acid on the sensitivity of NMDA receptor towards its against, low concentrations of NMDA were coadministered with arachidonic acid. Under these conditions the NMDA-mediated cytotoxicity was enhanced. We conclude that gp120 causes an activation of phospholipase A2, resulting in an increased release of arachidonic acid which in turn sensitizes the NMDA receptor. Two compounds were found to act cytoprotectively against the deleterious effect caused by gp120 on neurons: Memantine [1-amino-3,5-dimethyladamantane] and Flupirtine [2-amino-3-ethoxycarbonylamino-6-(4-fluoro-benzyl-amino)-pyridine maleate]. Both compounds have been found to display a potent cytoprotective effect on neurons treated with the excitatory amino acid NMDA or with the human immunodeficiency virus type 1 (HIV-1) coat protein gp120. The NMDA antagonist Memantine, a drug currently used in the therapy of spasticity and Parkinson's disease, prevented the effects of gp120 at micromolar concentrations. Flupirtine was previously found to be a centrally acting, nonopiate analgesic agent which additionally possesses anticonvulsant and muscle-relaxant activity at doses similar to those producing analgesia. The cytoprotective effect of Flupirtine in vitro was significant (above 10 microM). Considering the fact that both Memantine and Flupirtine display almost no clinical side effects, these drugs may prove useful both in preventing primary infection of brain cells with the HIV virus, as well as in treating the neurological disorders often associated with the immunodeficiency syndrome such as AIDS-related dementia.

Flupirtine partially prevents neuronal injury induced by prion protein fragment and lead acetate.

Flupirtine belongs to the class of triaminopyridines and is successfully applied clinically as a non-opiate analgesic drug with additional muscle relaxant properties. Recently it was reported that flupirtine acts like an antagonist of the N-methyl-D-aspartate (NMDA) receptor complex in neuronal cells both in vitro and in vivo. Here we have used primary cortical cells from rat embryos to demonstrate that this compound is also neuroprotective against the toxic effects caused by the prion agent PrPSc and lead acetate (Pb). These two agents display pleiotropic effects on neurons, which include activation of the NMDA receptor complex. At concentrations above 30 microM the toxic-peptide fragment of PrPSc causes apoptotic fragmentation of DNA and is consequently neurotoxic. Pb is neurotoxic at concentrations above 10 microM. Co-administration of flupirtine (10 microM) with either of these agents resulted in reduced neurotoxicity. These data indicate that the cytoprotective effect of flupirtine is measurable in vitro against these noxious agents which show their effects, including modulation of the NMDA receptor complex, pleiotropically.

The (2'-5')oligoadenylate synthetase is present in the lowest multicellular organisms, the marine sponges. Demonstration of the existence and identification of its reaction products.

We have proved the presence of (2'-5')oligoadenylates [(2'-5')An] and oligoadenylate synthetase [(2'-5')An synthetase] in the marine sponge Geodia cydonium. (2'-5')An isolated from sponge crude extract competed with authentic (2'-5')An for binding to polyclonal antiserum against (2'-5')An. HPLC analysis revealed the presence of nucleotides eluting with molecular markers for (2'-5')A oligomers. The biological activity of sponge (2'-5')An was demonstrated by inhibiting the protein biosynthesis in rabbit reticulocyte lysate. The activity of the (2'-5')An synthetase, present in crude sponge extract, was found to be high compared to that in mammalian interferon-treated cell extract. The (2'-5')An synthetase from sponge extract binds to poly(I).poly(C) as does the mammalian enzyme. Western blot analysis with antibodies to recombinant rat 43-kDa (2'-5')An synthetase revealed in sponge immunologically related proteins with molecular masses of approximately 110, 65, 61 and 34 kDa. We conclude, that the (2'-5')An system has evolved from receptors and enzymes involved in cell adhesion and/or growth control.

Impairment of intracellular antiviral defense with age: age-dependent changes in expression of interferon-induced and double-stranded RNA-activated 2-5A synthetase in rat.

The 2',5'-oligoadenylate (2-5A) system is involved in the defense of mammalian cells against virus infection. In a previous study [25], we demonstrated that the activities of the enzymes which synthesize and degrade 2-5A [2-5A synthetase (2-5OAS) and 2',3'-exoribonuclease] and of the enzyme that is activated by 2-5A (ribonuclease L) change during aging and development in different tissues of rat. The age-dependent decrease in 2-5OAS activity and increase in 2-5A nuclease activity results in a decrease in the cellular 2-5A content, suggesting that the efficiency of the antiviral 2-5A system is impaired in aged rats. Here we determined the age-dependent changes in the level of mRNA coding for the class I isoenzyme of 2-5OAS (M(r) 40-46 kDa) in rat liver and brain using a cDNA which was recently cloned from rat hippocampus. We found that the decrease in 2-5OAS activity is accompanied by a decrease in the level of 2-5OAS mRNA; in old animals (32-33 months old), the amount of 2-5OAS mRNA was reduced to 20-30% compared to young adult (2-3 months old) (100%) and middle-aged adult animals (12 months old) (110-120%). In addition, Western-blotting experiments revealed that the amount of class I 2-5OAS capable of binding to its activator, poly(I).poly(C), is also diminished in the livers and brains of old rats compared to those of young adult and middle-aged adult animals.

Rapid reduction of mRNA coding for 2'-5'-oligoadenylate synthetase in rat pheochromocytoma PC12 cells during apoptosis.

Apoptosis is a form of physiological cell death, characterized by DNA fragmentation, which often depends on RNA and protein synthesis. Because cellular RNA is also degraded during apoptosis we studied the role of the 2'-5'-oligoadenylate (2-5A) synthetase in this process. The product of the synthetase, 2-5A, stimulates endoribonuclease-L-mediated controlled RNA degradation. Here we show that apoptosis is induced in rat phenochromocytoma PC12 cells by tributyltin (TBT) at low concentrations (1 nM); already 5-10 min. after addition of this compound DNA fragmentation resulting in a stepladder-like gel pattern was observed. The level of mRNA coding for 2-5A synthetase was determined using a cloned cDNA from rats. Sequence analyses of the rat 2-5A synthetase (M(r) 40-46,000) revealed high homology to other members of class I synthetase cloned from mouse and human. Applying the rat cDNA as a probe we found that parallel with degradation of DNA the level of mRNA coding for 2-5A synthetase decreased already 7.5 min. after induction of apoptosis by TBT the amount of 2-5A synthetase mRNA was reduced by 60%. This finding indicates that this enzyme is among those mRNAs which are degraded during apoptosis and it suggests that 2-5A synthetase, which is involved in the antiviral response of cells and most likely in the control of cell growth and differentiation, does not play an active role during this process.

Inhibition of DNA topoisomerase I activity by 2',5'-oligoadenylates and mismatched double-stranded RNA in uninfected and HIV-1-infected H9 cells.

2',5'-Oligoadenylates (2-5As) inhibit the type I DNA topoisomerase activity both in uninfected and HIV-1-infected human T cell line H9 as well as the purified enzyme (calf thymus). Topoisomerase I activity was determined by measuring the relaxation of negatively supercoiled pBR322 DNA. Inhibition of topoisomerase I by 2-5A depends on the chain length of the oligomer and the presence of 5'-phosphate. The 5'-triphosphate of the 2-5A hexamer was most active (almost total inhibition of DNA relaxation at 10 microM concentration); the 2-5A core trimer (at 100 microM) displayed no significant effect. In crosslinking and immunoprecipitation experiments we present evidence that 2-5A (32P-labelled 2-5A derivative, ppp(A2'p)3 A[32P]pCp) is able to bind to nuclear topoisomerase I. The mismatched dsRNA, poly(I).poly(C12U) (Ampligen), exhibited a strong anti-HIV-1 activity. However, our data show that this antiviral effect is not related to topoisomerase I inhibition. On the other hand, we did observe the production of longer oligomers of 2-5A in cells treated with poly(I).poly(C12U). It remains speculative, whether the in vivo effect could be catalyzed by this activity of poly(I).poly(C12U). In addition we could show that 2-5A also inhibits topoisomerase I activity associated with isolated HIV-1 particles.

The 2-5A system and HIV infection.

2',5'-Oligoadenylates (2-5A) have an essential role in the establishment of the antiviral state of a cell exposed to virus infection. The key enzymes of the 2-5A system are the 2-5A forming 2',5'-oligoadenylate synthetase (2-5OAS), the activity of which depends on the presence of viral or cellular double-stranded RNA (dsRNA), and the 2-5A-activated ribonuclease (RNase L). Basic research in recent years has shown that the 2-5A system is a promising target for anti-HIV chemotherapy, particularly due to its interaction with double-stranded segments within HIV RNA. Two new strategies have been developed which yield a selective antiviral effect of 2-5A against HIV-1 infection: (1) development of 2-5A analogues displaying a dual mode of action (activation of RNase L and inhibition of HIV-1 RT) and (2) intracellular immunization of cells against HIV-1 infection by application of the HIV-1-LTR--2-5OAS hybrid gene. A further strategy is the inhibition of DNA topoisomerase I by longer 2-5A oligomers.