Subba Reddy Palli

Molecular analysis of the mode of action of RH-5992, a lepidopteran-specific, non-steroidal ecdysteroid agonist

Abstract The dibenzoyl hydrazine, RH-5992, induces precocious molting in all lepidopteran larvae tested including the tobacco hornworm, Manduca sexta and the spruce budworm, Choristoneura fumiferana. Synthesis of a new cuticle including formation of a new head capsule that is incompletely sclerotized and lack of ecdysis are some of the major phenotypic effects. Similar to 20E, RH-5992 induces Manduca hormone receptor 3 (MHR3) mRNA and suppresses 14 kDa larval cuticular protein (LCP-14) transcript levels in 4th instar epidermis of Manduca , cultured in vitro . The ED 50 for induction of MHR3 was 1.4 × 10 −7 M, 10 times less than that of 20E. When the epidermis was exposed to 20E for 17 h and then cultured in hormone-free medium for a further 48 h, the LCP-14 mRNA level went up to nearly 60% of the maximal level reached in the untreated control. However, when RH-5992 was used, the level remained low even after 48 h. Dopa decarboxylase (DDC) transcription normally occurs at the end of the molt in preparation for sclerotization and in the epidermis cultured in vitro requires initial exposure to 20E for 17 h followed by its removal. Both in vivo and transient in vitro treatment with RH-5992 prevented DDC expression for up to 48 h after the removal of the compound. Thus, RH-5992 mimics the action of 20E on the expression of these 3 genes, but its effects persist in the tissue much longer than 20E.

Cloning and developmental expression of Choristoneura hormone receptor 3, an ecdysone-inducible gene and a member of the steroid hormone receptor superfamily

Degenerate oligonucleotides and cDNA converted from Choristoneura fumiferana embryonic RNA were used in a polymerase chain reaction (PCR) procedure to isolate a 683 bp cDNA fragment. Comparison of the deduced amino acid sequence of this cDNA fragment showed that it was a region of an MHR3-like gene from C. fumeferana; we therefore named it Choristoneura hormone receptor 3 (CHR3). This CHR3 cDNA fragment was used as a probe to screen a C. fumiferana embryonic cDNA library. Twenty clones were isolated and two overlapping clones were sequenced. The longest open reading frame of CHR3 cDNA codes for 546 amino acids. The deduced amino acid sequence of this open reading frame contained all five regions typical of a steroid hormone nuclear receptor. The C domain showed the highest identity to Manduca hormone receptor 3 (MHR3), Drosophila hormone receptor 3 (DHR3) and Galleria hormone receptor 3 (GHR3). The A/B, D and E domains also showed significant amino acid similarity with MHR3, DHR3 and GHR3. The 683 bp CHR3 cDNA probe detected two mRNAs of 3.8 and 4.5 kb present during the ecdysteroid peaks for embryonic, larval, pupal and adult molts but were not detected during the intermolt periods. In sixth instar larvae, the 3.8 and 4.5 kb mRNA were detected in the epidermis, fat body and midgut tissues and the maximum expression was observed during the prepupal peak of ecdysteroids in the hemolymph. CHR3 mRNA was induced in 20-hydroxyecdysone treated CF-203 cells as well as in the midgut, fat body and epidermis of larvae that were fed the non-steroidal molting hormone agonist, RH-5992. In vitro transcription and translation of the CHR3 cDNA yielded a 61 kDa protein that bound to the retinoid related orphan receptor response element.

The ultraspiracle gene of the Spruce Budworm, Choristoneura fumiferana: Cloning of cDNA and developmental expression of mRNA†

Cloning and characterization of a Choristoneura fumiferana ultraspiracle (Cfusp) cDNA are described. First, a PCR fragment and then a cDNA clone (4.4 kb) were isolated from spruce budworm cDNA libraries. Comparison of the deduced amino acid sequence of this cDNA with the sequences in Genbank showed that this sequence had high homology with the ultraspiracle cDNAs cloned from Drosophila melanogaster (Dmusp), Bombyx mori (Bmusp), Manduca sexta (Msusp), and Aedes aegypti (Aausp). The Cfusp cDNA contained all the regions that are typical for a steroid/thyroid hormone receptor superfamily member. The DNA binding domain or C region was the most conserved sequence among all the usps. The A/B, D, and E regions also showed high amino acid identity with the amino acid sequences of Dmusp, Msusp, Bmusp, and Aausp. The Cfusp 4.5-kb mRNA was present in the embryos, in all larval stages, and in the pupae. The Cfusp mRNA levels in the midgut increased during the sixth-instar larval development and reached peak levels during the ecdysteroid raises for the pupal molt. However, Cfusp mRNA levels remained unchanged in the midgut of fifth-instar larvae, and in the epidermis and fat body of sixth-instar larvae indicating both a tissue- and stage-specific regulation of Cfusp mRNA expression.

Basis for selective action of a synthetic molting hormone agonist, RH-5992 on lepidopteran insects

The non-steroidal ecdysone agonist, RH-5992, induces a precocious incomplete molt in lepidopteran insects but is refractory to insects of other orders. We used two lepidopteran cell lines, FPMI-CF-203 (CF-203) and IPRI-MD-66 (MD-66) and two dipteran cell lines, DM-2 and Kc, to investigate the lepidopteran specificity of RH-5992. The mRNAs for hormone receptor 3 homologues cloned from Drosophila (DHR3) and Choristoneura (CHR3) are directly induced by 20-hydroxyecdysone (20E) and serve as suitable markers for studying ecdysone action. Dose response experiments showed that 10(-7) M 20E induced CHR3 mRNA in CF-203 cell and DHR3 mRNA in DM-2 cells. Concentrations of RH-5992 as low as 10(-10) M induced CHR3 mRNA in CF-203 cells, whereas concentrations as high as 10(-6) M induced only very low levels of DHR3 mRNA in DM-2 cells. Studies using 14C-RH-5992 revealed that lepidopteran cell lines (CF-203 and MD-66) retained more of this compound within the cells than the dipteran cell lines (DM-2 and Kc). The clearance of RH-5992 from DM-2 cells was temperature dependent and was blocked by 10(-5) M ouabain, an inhibitor of Na+, K(+)-ATPase suggesting that the efflux was due to active transport.

Forest insect cell lines responsive to 20-hydroxyecdysone and two nonsteroidal ecdysone agonists, RH-5849 and RH-5992

Abstract The effects of 20-hydroxyecdysone (20E) and two substituted dibenzoylbutylhydrazines, RH-5849 and RH-5992, were investigated in vitro using three forest insect cell lines. Two of these cell lines, IPRI-MD-66 (MD-66) from the forest tent caterpillar, Malacosoma disstria and IPRI-CF-1 (CF-1) from the spruce budworm, Choristoneura fumiferana , grow freely suspended, whereas the cells of the third line, FPMI-CF-70 (CF-70) from C. fumiferana , stay attached to the culture flask. MD-66 cells responded to all three compounds by forming clumps and by producing filamentous extensions. In addition, these compounds produced increased cell attachment and reduced cell proliferation in this cell line. CF-70 cells also responded to all three compounds although to a lesser extent. On the other hand, CF-1 cells showed little or no morphological response. The above effects of RH-5992 on MD-66 cells were both dose and time dependent. This compound also induced the expression of the Malacosoma disstria hormone receptor (MdHR3) in these cells in a dose dependent manner, as was the case with 20E. This result indicated that the effect of RH-5992 on MD-66 cells is specific and related to their response to 20E. These phenotypic and molecular observations indicate that MD-66 cells and RH-5992 will be an excellent in vitro model system for studying the mode of action of ecdysteroid agonists.

Ultrastructural Effects of a Non-Steroidal Ecdysone Agonist, RH-5992, on the Sixth Instar Larva of the Spruce Budworm, Choristoneura fumiferana

Force feeding of RH-5992 (Tebufenozide), a non-steroidal ecdysone agonist to newly moulted sixth instar larvae of the spruce budworm, Choristoneura fumiferana, (Lepidoptera: Tortricidae) initiates a precocious, incomplete moult. Within 6h post treatment (pt) the larva stops feeding and remains quiescent. Around 12hpt, the head capsule slips partially revealing an untanned new head capsule that appears wrinkled and poorly formed. By 24hrpt, the head capsule slippage is pronounced and there is a mid-dorsal split of the old cuticle in the thoracic region but there is no ecdysis. The larva remains moribund in this state and ultimately dies of starvation and desiccation. The temporal sequence of the external and internal changes of the integument were studied using both scanning and transmission electron microscopy. Within 3hpt, there is hypertrophy of the Golgi complex indicating synthetic activity and soon after, large, putative ecdysial droplets are seen. Within 24h, a new cuticle that lacks the endocuticular lamellae is formed. The formation of the various cuticular components, the degradation of the old cuticle and changes in the organelles of the epidermal cells of the mesothoracic tergite are described. The difference between the natural moult and the one induced by RH-5992 are explained on the basis of molecular events that take place during the moulting cycle. The persistence of this ecdysone agonist in the tissues permits the expression of all the genes that are up-regulated by the presence of the natural hormone but those that are turned on in the absence of the hormone are not expressed.

Transcriptional activation of the cloned Heliothis virescens (Lepidoptera) ecdysone receptor (HvEcR) by MuristeroneA

Ecdysteroids play an important role during insect development. We report here the isolation and characterisation of an Ecdysone receptor (EcR) homologue from Heliothis virescens (HvEcR) and present evidence supporting the HvEcR active role as an active component of the native insect receptor. Alignment of the deduced amino acid sequence of HvEcR with those of EcRs from other species confirmed its membership of this family and showed that it is closely related to the B1 isoform of Drosophila melanogaster. Northern blot analysis showed that two transcripts (6.0 and 6.5 kb) were recognised by a probe spanning the DNA and ligand binding domains of the HvEcR. Genomic Southern blots showed that the HvEcR is encoded by a single copy gene. Two lines of evidence towards the functional activity of the HvEcR are presented. In vitro transcribed and translated HvEcR showed specific binding to hsp27 and pall response elements in the presence of CfUSP. Stable expression of HvEcR in 293 cells induced reporter gene activity in the presence of muristeroneA in a dose dependant manner while dexamethasone failed to activate.

Analysis of ecdysteroid action in Malacosoma disstria cells: Cloning selected regions of E75- and MHR3-like genes

IPRI-MD-66 (MD-66) cells respond to 20-hydroxyecdysone (20E, 4 x 10(-6) M) in the medium by producing cytoplasmic extensions, clumping and attaching themselves to the substrate. These morphological changes are at a maximum by 6 days post treatment. Degenerate oligonucleotides, designed on the basis of conserved amino acid sequences in the DNA and ligand binding regions of the members of the steroid hormone receptor superfamily, were used in RNA-PCR to isolate two cDNA fragments, Malacosoma disstria hormone receptor 2 (MdHR2) and Malacosoma disstria hormone receptor 3 (MdHR3) from the MD-66 cells. Comparison of deduced amino acid sequences of these cDNA fragments with the members of the steroid hormone receptor superfamily showed that MdHR2 is most closely related to E75 proteins of Manduca sexta, Galleria mellonella and Drosophila melanogaster. The MdHR3 is most closely related to Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Drosophila hormone receptor 3 (DHR3) proteins. At a concentration of 4 x 10(-6) M, 20E induces the expression of MdHR2 and MdHR3 beginning at 3 h, reaching maximum levels in 12 h and declining in 24 h. MdHR2 binds to a 2.5 kb mRNA, whereas MdHR3 binds to a 4.5 kb mRNA. Based on sequence similarity, RNA size and ecdysone inducibility, we conclude that these cDNA fragments, cloned from MD-66 cells, are regions of E75- (MdHR2) and MHR3- (MdHR3) like genes.

RNA- AND PROTEIN-SYNTHESIS INHIBITORS INDUCE APOPTOSIS IN A MIDGUT CELL LINE FROM THE SPRUCE BUDWORM, CHORISTONEURA FUMIFERANA

Abstract RNA-synthesis inhibitors actinomycin D and dichlorobenzimidazole riboside, and protein-synthesis inhibitors anisomycin and cycloheximide induced apoptosis in FPMI-CF-203 (CF-203), a continuous midgut cell line of Choristoneura fumiferana . Actinomycin D induced apoptosis in more than 90% of the CF-203 cells at a concentration as low as 0.01 μg/ml of medium. Dichlorobenzimidazole riboside, on the other hand, induced apoptosis in 99% of these cells at a concentration of 100 μg/ml or higher. A concentration of 50 μg/ml or higher of anisomycin induced apoptosis in more than 90% of these cells by 24 h after treatment. Cycloheximide was the least effective, requiring 100 and 500 μg/ml to induce apoptosis in 17 and 30% of these cells, respectively, by 24 h after treatment. The cells treated with actinomycin D showed an oligomeric ladder beginning at 6 h after treatment, whereas the cells treated with dichlorobenzimidazole riboside, anisomycin or cycloheximide showed the oligomeric ladder beginning at 12 h after treatment. Some of the CF-203 cells treated with actinomycin D or anisomycin showed the classical apoptotic morphological characteristics such as plasma membrane blebbing, nuclear fragmentation and packaging of cytoplasmic organelles and chromatin into apoptotic bodies by 8 h after treatment, and by 24 h after treatment the effects were evident in most of the cells. Thus, two different classes of chemicals that inhibit RNA synthesis and protein synthesis by different modes of action, induced apoptosis in CF-203 cells.

Selective mechanism of action of tebufenozide on lepidopteran cell lines

The non-steroidal ecdysone agonist, tebufenozide (RH-5992), induces a precocious incomplete molt primarily on lepidopteran insects, but has little or no effect on insects of other orders. 20 Hydroxyecdysone at 10 -7 M induced the transcription factor CHR3 mRNA in CF-203 cells and DHR3 mRNA in DM-2 cells. Tebufenozide even at 10 -10 M induced CHR3 mRNA in lepidopteran CF-203 cells, but even at 10 -5 M it induced only trace levels of DHR3 mRNA in dipteran DM-2 cells. Studies using radiolabelled RH-5992 revealed that lepidopteran cell lines (CF-203 and MD-66) retained more of this compound within the cells than dipteran cell lines (DM-2 and K c ). The efflux of radiolabelled RH-5992 from DM-2 cells was temperature-dependent and was blocked by 10 -5 M ouabain, an inhibitor of Na + , K + -ATPase, suggesting that the efflux was due to active transport.