Davy Jones

A mutation found in the promoter region of the human survivin gene is correlated to overexpression of survivin in cancer cells.

Survivin, a unique antiapoptotic factor, plays an important role in cell cycle regulation. Numerous clinical studies have shown that survivin is markedly overexpressed in most common types of cancer, suggesting that transcriptional deregulation is a major mechanism involved in aberrant expression of survivin in cancers. In this study, we have identified several polymorphisms in the survivin gene promoter. One of these polymorphisms is located at CDE/CHR repressor elements, and appears to be a common mutation with high frequency among cancer cell lines compared to normal cell line controls. The presence of the mutation was correlated in these cell lines with increased survivin expression at the both mRNA and protein levels. Furthermore, gel mobility shift analysis and transcriptional analysis showed the mutation changed cell cycle-dependent transcription by modifying the binding motif of the CDE/CHR repressor. These results indicate that the high level of survivin in some cancers is, at least in part, due to a genetic defect in the promoter region of the human survivin gene, which causes derepression of survivin transcription apparently due to the mutated CDE/CHR repressor binding motifs.

Juvenile hormone III-dependent conformational changes of the nuclear receptor ultraspiracle

The identification of potential endogenous or synthetic ligands for orphan receptors in the steroid receptor superfamily is important both for discerning endogenous regulatory pathways and for designing receptor inhibitors. The insect nuclear receptor Ultraspiracle (USP), an ortholog of vertebrate RXR, has long been treated as an orphan receptor. We have tested here the fit of terpenoid ligands to the JH III-binding site of monomeric and homo-oligomeric USP from Drosophila melanogaster (dUSP). dUSP specifically bound juvenile hormone III (JH III), but not control farnesol or JH III acid, and also specifically changed in conformation upon binding of JH III in a fluorescence binding assay. Juvenile hormone III binding caused intramolecular changes in receptor conformation, and stabilized the receptors dimeric/oligomeric quaternary structure. In both a radiometric competition assay and the fluorescence binding assay the synthetic JH III agonist methoprene specifically competed with JH III for binding to dUSP, the first demonstration of specific binding of a biologically active JH III analog to an insect nuclear receptor. The recombinant dUSP bound with specificity to a DR12 hormone response element in a gel shift assay. The same DR12 element conferred enhanced transcriptional responsiveness of a transfected juvenile hormone esterase core promoter to treatment of transfected cells with JH III, but not to treatment with retinoic acid or T3. The activity of JH III or JH III-like structures, but not structures without JH III biological activity, to bind specifically to dUSP and activate its conformational change, provide evidence of a terpenoid endogenous ligand for Ultraspiracle, and offer the prospect that synthetic, terpenoid structures may be discovered that can agonize or antagonize USP function in vivo.

The retinoid-X receptor ortholog, ultraspiracle, binds with nanomolar affinity to an endogenous morphogenetic ligand

The in vivo ligand‐binding function and ligand‐binding activity of the Drosophila melanogaster retinoid‐X receptor (RXR) ortholog, ultraspiracle, toward natural farnesoid products of the ring gland were assessed. Using an equilibrium fluorescence‐binding assay, farnesoid products in the juvenile hormone (JH) biosynthesis pathway, and their epoxy derivatives, were measured for their affinity constant for ultraspiracle (USP). Farnesol, farnesal, farnesoic acid and juvenile hormone III exhibited high nanomolar to low micromolar affinity, which in each case decreased upon addition of an epoxide across a double bond of the basic farnesyl structure. Similar analysis of the substitution on C1 of methyl ether, alcohol, aldehyde, and carboxylic acid showed that each conferred weaker affinity than that provided by the methyl ester. Attention was thus focused for a ring‐gland farnesoid product that possesses the features of methyl ester and lack of an epoxide. A secreted product of the ring gland, methyl farnesoate, was identified possessing these features and exhibited an affinity for ultraspiracle (Kd = 40 nm) of similar strength to that of RXR for 9‐cis retinoic acid. Mutational analysis of amino acid residues with side chains extending into the ligand‐binding pocket cavity (and not interacting with secondary receptor structures or extending to the receptor surface to interact with coactivators, corepressors or receptor dimer partners) showed that the mutation C472A/H475L strongly reduced USP binding to this ring gland product and to JH III, with less effect on other ring‐gland farnesoids and little effect on binding by (the unnatural to Drosophila) JH I. Along with the ecdysone receptor, USP is now the second arthropod nuclear hormone receptor for which a secreted product of an endocrine gland that binds the receptor with nanomolar affinity has been identified.

Comparative studies on the protein composition of hymenopteran venom reservoirs

The proteins of venom reservoirs from 25 hymenopteran species from 21 genera were investigated with regard to their protein composition and immunological similarities. It was found that low mol. wt proteins and polypeptides are typical for the venoms of ants, social wasps and bees. Six species of ichneumonoid parasitic wasps lack these low mol. wt proteins. Except for bee venoms none of the low mol. wt proteins contain mannose. The venoms of ichneumonoid parasites and ants contained primarily acidic proteins, whereas those of social wasps and bees contained abundant neutral and basic proteins. Immunological analysis showed that a number of venom proteins from parasitic braconid (Chelonus) wasps shared conserved immunoreactive determinants with the venom proteins of higher Hymenoptera, including some not of parasitic habit. Implications of phylogenetic relationships among hymenopteran species are discussed.

Pseudoparasitism of host Trichoplusia ni by Chelonus spp. as a new model system for parasite regulation of host physiology

Abstract Most host insect species parasitized as eggs by wasps of the subfamily Cheloninae initiate precocious metamorphosis many days later as preultimate—instar larvae. However, development is then suppressed early in the precocious prepupal stage. These events occur even in “pseudoparasitized” hosts containing no live or functional parasite at the time the endocrine lesions become manifest. The endocrine bases for these effects have been determined using a Trichoplusia ni-Chelonus spp. model system. Using hosts pseudoparasitized by C. near curvimaculatus or C. insularis it was determined that the entire feeding stage developmental pattern of the final instar is precociously expressed in penultimate-instar hosts. The precocious expression of the final-instar developmental pattern may be due to alteration or re-programming of tissues involved in establishment of critical growth thresholds. The second endocrine lesion is due to a lower than normal titre of juvenile hormone in precocious prepupae, leading to a lower than normal ecdysteroid titre. The validity of the concept of parasite regulation of host physiology, and its necessary distinction from indirect stress effects, are discussed. In few systems suggestive of parasite regulation of the host has the hypothesis been tested at a biochemical level. It is proposed that the T. ni-Chelonus spp. interaction is an excellent model system for such an analysis. The current results using this system already have implications for basic knowledge on host-parasite interactions, development of chemicals to disrupt insect growth, biological control strategies and new experimental probes for investigations of normal insect physiology.

Participation of Survivin in mitotic and apoptotic activities of normal and tumor‐derived cells*

Survivin is a member of the inhibitor of apoptosis (IAP) gene family, containing a single baculovirus IAP repeat (BIR) and no RING finger, that is expressed in many human cancers. Although it has been proposed to be involved in mitotic and cytokinetic processes, its functional subcellular distribution in the cytoplasm and nucleus, and its binding to centrosomes, spindle fibers, and centromeres in relation to these processes, is not fully resolved. We have analyzed the localization of Survivin in normal (Detroit 551, IMR‐90) and tumor‐derived (HeLa, Saos‐2) cell lines, and found that it does colocalize with centrosomes in the cytoplasm during interphase, then moves to centromeres during mitosis, and finally localizes to the midbody spindle fibers during telophase. However, Taxol, a popular microtubule stabilizing agent that is frequently used in the study of these processes, severely disrupted the localization of Survivin. Taxol treatment of cells promoted extensive relocalization of Survivin with α‐tubulin on microtubules during either interphase or mitosis. Survivin antisense oligonucleotide markedly sensitized HeLa cells to cell death induced by agents acting at the level of cell surface receptor (Fas pathway) or at the level of mitochondria (etoposide). HeLa cell death induced by Survivin antisense oligonucleotide could be partially complemented by Deterin, the Drosophila homolog of Survivin (Jones et al. [ 2000 ] J. Biol. Chem. 275:22157–22166). Reciprocally, a chimera of the Deterin BIR domain and Survivin C‐terminus could rescue Drosophila Kc cells from death induced by transfection of a human caspase‐7‐expressing plasmid. These results indicate common components of Survivin and Deterin antiapoptotic action in the vertebrate and invertebrate phyla. J. Cell. Biochem. 83: 342–354, 2001.

Interactions of ultraspiracle with ecdysone receptor in the transduction of ecdysone‐ and juvenile hormone‐signaling

Analyses of integration of two‐hormone signaling through the vertebrate nuclear hormone receptors, for which the retinoid X receptor is one partner, have generated a number of mechanistic models, including those described as ‘subordination’ models wherein ligand‐activation of one partner is subordinate to the liganded state of the other partner. However, mechanisms by which two‐hormone signaling is integrated through invertebrate nuclear hormone‐binding receptors has not been heretofore experimentally elucidated. This report investigates the integration of signaling of invertebrate juvenile hormone (JH) and 20‐OH ecdysone (20OHE) at the level of identified nuclear receptors (ultraspiracle and ecdysone receptor), which transcriptionally activate a defined model core promoter (JH esterase gene), through specified hormone response elements (DR1 and IR1). Application of JH III, or 20OHE, to cultured Sf9 cells transfected with a DR1JHECoreLuciferase (or IR1JHECoreLuciferase) reporter promoter each induced expression of the reporter. Cotreatment of transfected cells with both hormones yielded a greater than additive effect on transcription, for especially the IR1JHECoreLuciferase reporter. Overexpression in Sf9 cells of recombinant Drosophila melanogaster ultraspiracle (dUSP) fostered formation of dUSP oligomer (potentially homodimer), as measured by coimmunoprecipitation assay and electrophoretic mobility assay (EMSA) on a DR1 probe, and also increased the level of transcription in response to JH III, but did not increase the transcriptional response to either 20OHE treatment alone or to the two hormones together. Inapposite, overexpression of recombinant D. melanogaster ecdysone receptor (dEcR) in the transfected cells generated dUSP/dEcR heterodimer [as measured by EMSA (supershift) on a DR1 probe] and increased the transcriptional response to 20OHE‐alone treatment, but did not increase the transcriptional response to the JH III‐alone treatment. Our studies provide evidence that in this model system, JH III‐activation of the reporter promoter is through USP oligomer (homodimer) that does not contain EcR, while the 20OHE‐activation is through the USP/EcR heterodimer. These results also show that the integration of JH III and 20OHE signaling is through the USP/EcR heterodimer, but that when the EcR partner is unliganded, the USP partner in this system is unable to transduce the JH III‐activation.

Considerations on the structural evidence of a ligand-binding function of ultraspiracle, an insect homolog of vertebrate RXR

This analysis considers the structural evidence of a ligand-binding function of the nuclear receptor ultraspiracle (USP). The positions and nature of residues in the ligand-binding domain of USP from six higher insects is evaluated in comparison to the function of conserved residues vertebrate receptors that have been co-crystallized with ligand. USP appears to conserve residues that in vertebrate receptors (1) form the hydrophobic ligand-binding pocket, (2) contact oxygen-containing moieties on ligands, such as hydroxyl, keto and carboxyl groups, and (3) in response to ligand-binding conformationally change to form a multi-helix hydrophobic groove for recruitment of transcriptional co-activators. These structural features are consistent with the recent report that USP can bind the epoxymethylfarnesoates (juvenile hormones) and thereupon is induced to change conformation.

Chelonus sp.: Suppression of host ecdysteroids and developmentally stationary pseudoparasitized prepupae

When eggs of the caterpillar Trichoplusia ni are stung by Chelonus sp. (near C. curvimaculatus) (Braconidae), the developing host larvae precociously spin a cocoon but then remain developmentally stationary in the prepupal stage. The latter event happens even in hosts which were stung and precociously spin cocoons but which, upon dissection, contain no obvious parasite. Injection of radiolabeled ecdysone into either pseudoparasitized or allatectomized larvae demonstrates suppressed rates of conversion of ecdysone to 20-hydroxyecdysone when compared with controls. The data indicate that the occurrence of developmentally stationary pseudoparasitized prepupae is due to less production of ecdysteroid and less conversion of ecdysone to 20-hydroxyecdysone, both probably as a result of suppressed juvenile hormone titer.

The endocrine basis for developmentally stationary prepupae in larvae ofTrichoplusia ni pseudoparasitized byChelonus insularis

Larvae of the cabbage looper,Trichoplusia ni, precociously initiate metamorphosis in the penultimate instar when parasitized byChelonus insularis. Some larvae developing from stung eggs precociously spin cocoons, but upon dissection contain no live or obvious parasites. Such ‘pseudoparasitized’ larvae greatly slow down in development as prepupae, due to a suppressed ecdysteroid titer which in turn may be caused by a suppressed juvenile hormone titer.