Insect Biochemistry and Molecular Biology 31 (2001) 1057–1064 www.elsevier.com/locate/ibmb
Periodic expression of an ecdysteroid-induced nuclear receptor in a lepidopteran cell line (IAL-PID2) Ste´phane Debernard a
a,*
, Franc¸oise Bozzolan a, Line Duportets b, Patrick Porcheron
a
Laboratoire de Physiologie Cellulaire des Inverte´bre´s, Universite´ Pierre et Marie Curie, 12 rue Cuvier, 75005 Paris, France b Laboratoire de Biologie Cellulaire, INRA Centre de Versailles, Route de Saint Cyr, 78026 Versailles, France Received 29 May 2000; received in revised form 24 November 2000; accepted 13 January 2001
Abstract A set of DNA primers was designed within the DNA-binding domain of the Manduca hormone receptor 3 (MHR3) cDNA. These primers were used in RT-PCR to isolate a 204 bp cDNA fragment from IAL-PID2 cells exposed to 10⫺6 M 20-hydroxyecdysone (20E) for 12 h. The amino acid sequence deduced from the cDNA fragment presented 100% identity with the zinc finger domain of Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Choristoneura hormone receptor 3 (CHR3). This cDNA fragment was used as a probe on total RNA from IAL-PID2 cells exposed to 20E and hybridized to mRNA, the size of which was close to 4.5 kb and named Plodia hormone receptor 3 (PHR3). Kinetics of induction of PHR3 mRNA were similar to that of HR3 genes but varied according to the position of cells in their cell cycle. The non-steroidal ecdysone agonist, RH-5992 induced the expression of PHR3 at lower concentrations than 20E. From sequence similarity, mRNA size, 20E and RH5992 inducibilities, we conclude that PHR3 transcript could encode a Plodia hormone receptor 3 involved in the genetic signalling cascade of 20E. Thanks to its periodic expression, this putative orphan nuclear receptor could serve as a suitable cellular marker for studying changes of epidermal cell sensitivity to 20E during the cell cycle. 2001 Elsevier Science Ltd. All rights reserved. Keywords: 20-Hydroxyecdysone; Plodia interpunctella; IAL-PID2 cells; Steroid hormone receptor superfamily; Plodia hormone receptor 3; Cell cycle
1. Introduction Through molting, insects periodically shed their rigid exoskeleton to accommodate growth during the larval life. They also exhibit sequential polymorphism changes from larvae to pupae and then to the adult stage. The steroid hormone 20-hydroxyecdysone (20E) and juvenile hormone (JH) are the main hormones that regulate molting and metamorphosis (Riddiford and Truman, 1993). This radical reorganization in body form during metamorphosis involves coordination of diverse 20E-regulated biological responses within a single animal. 20E activates a genetic signalling cascade leading to both the histolysis of larval structures and the differentiation and morphogenesis of adult structures. These responses occur at various times during metamorphosis in a cell* Corresponding author. Tel.: +33 1 44 27 65 93; fax: +33 1 44 27 65 09. E-mail address:
[email protected] (S. Debernard).
and tissue-specific manner, thus imposing multiple levels of regulation to integrate the temporal and spatial patterns of specificity required to orchestrate this developmental transition. In Chironomus fumiferma (Clever and Karlson, 1960) and Drosophila melanogaster (Ashburner et al., 1974; Richards, 1976a,b), studies of the salivary gland polytene chromosome puffing hierarchy have provided significant insights into the mechanisms of the 20E-mediated signalling cascade. A hierarchical model was proposed in which 20E binds to its nuclear receptor to form a 20E-receptor complex that activates directly and hence rapidly a few early puffs while repressing the late puffs. When the proteins encoded by the early puffs become sufficiently abundant, they both repress their own promoters and activate the late genes. Many of these puff genes encode members of the nuclear receptor superfamily, EcR (Koelle et al., 1991), USP (Yao et al., 1992), E75 (Segraves and Hogness, 1990), E78B (Stone and Thummel, 1993), DHR3 (Koelle et al., 1992) and FTZ-F1 (Lavorgna et al., 1993). During the onset of Drosophila metamorphosis, a precise
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sequential activation of these nuclear receptors contribute to the orchestration of the 20E-induced signalling cascade (Huet et al., 1995). Most of these Drosophila genes have been characterized from tissues of other species (Henrich and Brown, 1995), indicating that the overall ecdysteroid regulatory pathway may be shared by all insects. A group of genes induced by 20E in the same way as the DHR3 early late gene belong to the subfamily hormone receptor 3 (HR3) including Manduca HR3 (Palli et al., 1992), Galleria HR3 (Jindra et al., 1994), Choristoneura HR3 (Palli et al., 1996). Developmental studies have shown that the HR3 genes seem to play a crucial role in the coordination of the 20Einducible hierarchy. DHR3 has been implicated as a regulator of the larval-to-prepupal transition during the onset of metamorphosis. DHR3 exerts this function by both repressing early genes in newly formed prepupae and inducing the FTZ-F1 competence factor in mid prepupae, thus arresting the late larval genetic response to ecdysone and preparing for the prepupal response (Kevin et al., 1997; Lam et al., 1999; White et al., 1997). Other studies have shown that the combinatorial expression cascade of 20E-induced nuclear receptors may be different according to the tissue and developmental stage (Talbot et al., 1993), thus contributing to the spatial and temporal diversity of the response to 20E. Using a 20E responsive epidermal cell line, we were interested in understanding the dynamic of the 20Einduced genetic signalling cascade at the cellular level by the characterization of putative nuclear receptors involved in this cascade at different periods of the cell cycle. An Indian meal-moth cell line, IAL-PID2, isolated from imaginal wing discs of Plodia interpunctella (Lynn and Oberlander, 1983) responds to 20E by an arrest of cell proliferation and biochemical and morphological differentiation (Cassier et al., 1991; Porcheron et al., 1991). The characteristic phenotypic responses of our cell line were similar to those reported in other cell lines (Judy, 1969; Courgeon, 1972; Dinan et al., 1990; Palli et al., 1995a,b; Fretz and Spindler, 1999). In this study, using a set of DNA primers designed within the DNAbinding domain of MHR3, we cloned and characterized the zinc finger domain of a putative ecdysone-induced Plodia hormone receptor 3 (PHR3) from the IAL-PID2 cell line by RT-PCR. Using this cloned fragment as a probe, we report periodic expression of PHR3 during the cell cycle under 20E action. Therefore, PHR3 is identified as a suitable molecular marker for both defining the period of sensitivity of the cells to 20E and for studying the changes of 20E-induced signalling cascades with regards to the cell cycle.
2. Materials and methods 2.1. Tools The MHR3 cDNA was isolated from abdominal epidermal RNA of the tobacco hornworn, Manduca sexta (Palli et al., 1991). This cDNA encodes a member of the steroid hormone receptor superfamily which is indeed similar to the Drosophila hormone receptor 3 (DHR3). This cDNA was a generous gift of Dr L. Riddiford at the University of Washington, Seattle. 2.2. Cell culture The IAL-PID2 cell line was established from imaginal wing discs of last instar larval of Plodia interpunctella Hu¨ bner, the Indian meal-moth (Lynn and Oberlander, 1983). The cell line kept its sensitivity to 20E. Cells grow as a loosely attached monolayer. We maintained them at 26°C in 75 cm2 tissue culture flasks with 12 ml of antibiotic-free Grace’s medium (Life Technologies, Cergy Pontoise, France) supplemented with 10% heatinactivated foetal bovine serum (FBS) (Roche, Molecular Diagnostics, Meylan, France) and 1% bovine serum albumin (BSA) (Fraction V, Sigma, Saint Quentin Fallavier, France). Cells were subcultured weekly to a near confluent monolayer. Cells were rinsed off the bottom of the flask in a gentle stream of culture medium and resuspended. Cell density was estimated by counting the cells in an aliquot of the suspension in a Mallassez hemocytometer under the microscope. All the cultures were initiated by seeding flasks with 1.5×106 cells. For use in culture, 20-hydroxyecdysone, anisomycin (ASN) and tebufenozide (RH-5992) were dissolved in ethanol and diluted in appropriate volumes of sterile Grace’s medium before addition to the culture medium. Final ethanol concentration in all treatments and control cultures was maintained under 0.1% in order to prevent any toxic effect of the solvent. 2.3. Synchronization of cells by serum deprivation The cells were first cultured in flasks under standard conditions for 72 h. They were then deprived of serum through the following successive steps. The cells were first rinsed for a few minutes and placed in 12 ml Grace’s medium containing 1% BSA but no FBS. Serum deprivation for 24–48 h brought the cells to quiescence phase at the border of the G1 compartment of their cycle (Hatt et al., 1994, 1997). After 48 h of serum deprivation, the cells were replaced in Grace’s medium supplemented with both 1% BSA and 10% FBS. The addition of FBS allowed cell cycle resumption. More than 80% of the cells entered in phase S within less than 2 h and they after went through G2 phase and mitosis in good synchrony (Hatt et al., 1997).
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2.4. Isolation of RNA
2.8. Northern blotting
Total RNA from cells was isolated by guanidinium isothiocyanate–phenol–chloroform extraction method (Chomczynski and Sacchi, 1987). RNA concentration was determined spectrophotometrically and the quality of RNA was checked by electrophoresis on a formaldehyde–agarose gel (1%).
Northern blot hybridization analysis was performed according to the manufacturer’s instructions. RNA samples (15 g) were denatured with formamide (50%) and formaldehyde (2.2 M), separated on 1% denaturating agarose gel and transferred to a Boerhinger Mannheim positively charged nylon membrane. Blotted RNA was hybridized overnight at 42°C with the DIG labeled PHR3 probe. An immunological signal detection by chemiluminescence was performed as described in Roche’s DIG system User’s Guide for filter hybridization. A molecular RNA marker ladder DIG-labeled (Roche, Meylan, France) was run in parallel on Northern blot to determine the molecular weight of hybridizing RNAs.
2.5. Reverse transcriptase-polymerase chain reaction (RT-PCR) Using the first strand cDNA synthesis kit (Roche, Meylan, France), 1 g total RNA was reverse transcribed into single-stranded cDNA with reverse transcriptase AMV and Oligo-p(dT)15 as primer. The resulting cDNAs were amplified by PCR with a set of DNA primers (A11, A12) on a Techne programmable thermal cycler using recombinant Pfu DNA polymerase which creates blunt-ended fragments for cloning. Reactions were carried out in 100 l final volume including 10 mM KCl, 6 mM ammonium sulfate, 20 mM Tris– HCl (pH 8), 2.5 mM MgCl2 with 2.5 units of Pfu DNA polymerase and 25% of the cDNA. The primers A11 5⬘Forward primer (5⬘-CCG TGC AAA GTT TGC GGC G-3⬘) and A12 5⬘-Reverse primer (5⬘-GCT CAT GCC GAG TTT GAG GC-3⬘) were added thereafter at 1 M and each dNTP at 0.8 mM. Following an initial 5 min denaturation at 94°C, the thermal amplification procedure included the five first cycles of denaturation for 1 min at 94°C, annealing at 50°C for 30 s and an elongation at 68°C for 90 s. The reaction was prolonged for 30 cycles with an annealing temperature of 45°C. 2.6. PCR product cloning and sequencing The blunt-ended PCR product was purified by agarose gel electrophoresis and cloned with Stratagene’s pCRScript TM SK (+) cloning kit following the manufacturer’s instructions. After colony isolation, DNA minipreps were prepared and correct insertion was determined by restriction enzyme analysis. The DNA clone containing the proper insert was sequenced by the dideoxy chain termination method (Sanger et al., 1977) (Genome Express, Grenoble, France). 2.7. Generation of DIG-labeled probe Using the PCR DIG probe synthesis kit (Roche, Meylan France), a probe was generated by PCR amplification of the cDNA fragment obtained by RT-PCR. The probe was labeled by incorporation of digoxigenin-11-dUTP during polymerase chain reactions (PCR). The DIG-labeled probe was used at a concentration of 25 ng/ml in hybridization solution.
2.9. Dot blotting With the PR600 Dot Blot Filtration Manifolds, 20 g total denatured RNA were loaded into each well to be immobilized on a positively charged nylon membrane by vacuum blotting. After hybridization, the membrane was treated, washed and immunological detection was made according to procedures as described under the Northern blotting.
3. Results 3.1. Cloning of PHRX cDNA fragment To begin our investigation of the molecular signalling cascade induced by 20E in the IAL-PID2 cells, we investigated the expression of a putative ecdysteroid-induced nuclear receptor belonging to the steroid hormone receptor superfamily. Total RNA isolated from IAL-PID2 cells treated with 10⫺6 M 20E for 12 h was converted to cDNA. The cDNA was then used as a template in a PCR amplification procedure with a set of DNA primers A11 and A12, derived from sequences within the DNAbinding domain of cDNA MHR3. Only one amplification product of expected size (204 bp) was obtained (Fig. 1). This PCR product named PHRX was purified, cloned into pCR-Script vector and the clone was sequenced in both directions. Fig. 2(A) shows both the nucleotide and deduced amino acid sequence of 204 bp PHRX located between the DNA primers A11 and A12. 3.2. Sequence comparison Comparison of the deduced amino acid sequence of PHRX with sequences obtained through Genbank showed that this sequence had significant amino acid identity with the DNA-binding domain of several members of the steroid hormone receptor superfamily (Evans,
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1988). The alignment of the deduced amino acid sequence of PHRX with the amino acid sequence of Manduca hormone receptor 3 (MHR3) (Palli et al., 1992), Drosophila hormone receptor 3 (DHR3) (Koelle et al., 1992), Choristoneura hormone receptor 3 (CHR3a) (Palli et al., 1996), Caenorhabditis hormone receptor 3 (CHR3b) (Kostrouch et al., 1995) and Galleria hormone receptor 3 (GHR3) (Jindra et al., 1994) is presented in Fig. 2(B). The 55 amino acid region of PHRX showed 100% amino acid identity with MHR3, GHR3, CHR3a zinc finger domains whereas it presented 96% amino acid identity with DHR3 and 91% with CHR3b. The amino acid identities thus suggested the expression of an ecdysteroid-inducible nuclear receptor belonging to the subfamily of hormone receptor 3 (HR3). 3.3. Isolation and characterization of PHR3 mRNA
Fig. 1. Cloning of PHRX cDNA. Ten microliters of PHRX amplified with DNA primers (A11, A12) were resolved on 1.5% agarose gel. Total RNA isolated from IAL-PID2 cells treated with 20E at 10⫺6 M for 12 h (lane 1) or without treatment to 20E (lane 2) were converted to cDNAs that were subsequently used as templates. Fragments size (nucleotides) given on the right are determined according to the mobility of DNA fragments in the 1 Kb ladder coelectrophoresed on the same gel (lane 3).
To verify the expression of this putative receptor, total RNAs were isolated from IAL-PID2 cells cultured in the presence of 20E at 10⫺6 M for 12 h and examined by northern blot hybridization using the PHRX as a probe at a hybridization temperature at 42°C. The northern blot analysis showed that no hybridization was observed in absence of 20E and only one transcript was detected with 20E (Fig. 3). Its size was close to 4.5 kb. This 20Einduced transcript could thus encode a putative receptor that we named Plodia hormone receptor 3 (PHR3) in IAL-PID2 cells.
Fig. 2. (A) Nucleotide and deduced amino acid sequences of PHRX. Nucleotide numbers are given on the left and the amino acid numbers on the right. The reverse primer (A12) and forward primer (A11) derived from sequences within the DNA-binding domain of cDNA MHR3 and used for the RT-PCR are designed in bold type and underlined. (B) Alignment of the deduced amino acid sequence of PHRX with Manduca hormone receptor 3 (MHR3; Palli et al., 1992), Galleria hormone receptor 3 (GHR3; Jindra et al., 1994), Choristoneura hormone receptor 3 (CHR3a; Palli et al., 1996), Drosophila hormone receptor 3 (DHR3; Koelle et al., 1992) and Caenorhabditis hormone receptor 3 (CHR3b; Kostrouch et al., 1995). Asteriks indicate identical residues and the dots indicate conserved substitutions. Multiple sequence alignment was performed using the CLUSTAL program (Higgins and Sharp, 1988).
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Fig. 3. Isolation of PHR3 mRNA. Fifteen micrograms of total RNA from IAL-PID2 cells cultured with 10⫺6 M 20E for 12 h were separated on a agarose (1%) formaldehyde gel, transferred to Nylon membrane and hybridized with 204 bp PHR3 probe at a hybridization temperature at 42°C. Lane 1: Cells cultured without 20E. Lane 2: Cells cultured with 10⫺6 M 20E for 12 h. RNA sizes (kb) of RNA ladder (Boehringer Mannheim) are given on the left.
3.4. Effect of 20E and ANS on induction of PHR3 To follow the pattern of PHR3 mRNA induction by 20E, IAL-PID2 were cultured in Grace’s medium containing 20E at 10⫺6 M for different continuous time exposures. The PHR3 transcript was already detectable at 2 h, reached peak level by 6 h and declined by 12 h [Fig. 4(a)]. To determine the minimal concentration of 20E required for the induction of PHR3 mRNA, IALPID2 cells were exposed to various concentrations of 20E for 6 h. The northern blot analysis showed that a significant induction of PHR3 mRNA was first observed at 10⫺6 M with an increase up to 10⫺5 M [Fig. 4(b)]. The range of activity for 20E is both close to the physiological concentration of ecdysteroids in lepidopteran insects such as the spruce budworm, Choristoneura fumiferama (Palli et al., 1995a,b) and to the peak hemolymph ecdysteroid concentration during the fourth larval molt determined in Manduca sexta (Curtis et al., 1984). Therefore, we used a 10⫺6 M concentration of 20E in all following experiments. To determine whether 20E directly initiated PHR3 mRNA transcription, we checked the effect of protein synthesis inhibitor, anisomycin (ANS). The IAL-PID2 cells were cultured in Grace’s medium containing (10⫺6 M) 20E with ANS (5 g/ml). Under these culture conditions, the presence of ANS for 6 h caused 94% inhibition of protein synthesis (N=4)
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Fig. 4. Induction of PHR3 mRNA by 20E. Fifteen micrograms of total RNA from IAL-PID2 cultured in Grace’s medium with 20E at 10⫺6 M for various times of exposure (a) or with 20E for 6 h at different concentrations (b) were separated on a agarose (1%) formaldehyde gel, transferred to Nylon membrane and hybridized with 204 bp PHR3 probe at a hybridization temperature at 42°C.
and the cells remained viable even 24 h after ANS removal. Fig. 5 shows that both ANS caused a slight reduction in the level of PHR3 mRNA for 3 and 6 h but neither completely prevented the induction by 20E. Furthermore, the observed decline in PHR3 mRNA levels after continuous exposure to 20E alone for more than 6 h did not occur in the presence of ANS. These observations indicated that the majority of the induction of PHR3 mRNA by 20E was independent from protein synthesis and thus probably due to direct action of 20E on the PHR3 gene. Whether the reduction in the induction of mRNA PHR3 in the presence of ANS is due to the
Fig. 5. Effect of ANS on PHR3 mRNA induction. Twenty micrograms of total RNA from IAL-PID2 cells cultured in Grace’s medium with 20E at 10⫺6 M or with 10⫺6 M 20E and 5 g/ml ANS for various times were analysed by dot blots and hybridized with 204 bp PHR3 probe.
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toxicity of ANS or that the full response requires protein synthesis, is not clear from these data. On the other hand, the decrease in PHR3 mRNA in the continuous presence of 20E probably requires synthesis of new protein(s). 3.5. Induction of PHR3 and the cell cycle We wondered whether the induction of the PHR3 transcript by 20E could depend on the state of the cells regarding their position in the cell cycle. The cells were thus synchronized at the end of their G1 phase by serum deprivation. Their cyclic activity resumed after addition of FBS. The vast majority of cells entered S phase in synchrony after such treatment. Cells were pretreated with 10⫺6 M 20E for 4 h at 2, 6, 13, 16, 28, and 31 h after FBS supplementation. Northern blot analysis showed that PHR3 mRNA was induced at 16, 28 and 31 h after cell cycle resumption (Fig. 6). 3.6. Induction of PHR3 by RH-5992 Ring-substituted dibenzoylhydrazines are well known non-steroidal ecdysone agonists. We therefore tried to determine if RH-5992 could mimic 20E action on PHR3 mRNA induction. The cells were synchronized in G1 phase and 16 h after the addition of FBS, 20E or RH5992 was added to the culture medium for 4 h, at concentrations ranging from 10⫺5 M to 10⫺9 M. The dose– response curve (Fig. 7) demonstrated that RH-5992 was able to induce PHR3 mRNA at very low concentrations (10⫺8 M, 10⫺7 M) whereas a response could hardly be seen for 20E. As higher concentrations (10⫺6 M, 10⫺5 M), 20E yielded a greater induction response for PHR3 mRNA than RH-5992. A Northern blot hybridization allowed us to verify the induction by RH-5992 of a single transcript whose size was similar to that of PHR3
Fig. 6. Induction of PHR3 mRNA and the cell cycle. At different times after the serum supplementation, the synchronized IAL-PID2 cells were exposed to 20E at 10⫺6 M for 4 h. Fifteen micrograms of total RNA from IAL-PID2 cells were separated on a agarose (1%) formaldehyde gel, transferred to Nylon membrane and hybridized with 204 bp PHR3 probe at a hybridization temperature at 42°C.
Fig. 7. Induction of PHR3 mRNA by RH-5992. Sixteen hours after the serum supplementation, the synchronized IAL-PID2 cells were exposed to 20E or RH-5992 at different concentrations for 4 h. For each concentration, 20 g of total RNA from IAL-PID2 were analysed by dot blots and hybridized with 204 pb probe.
mRNA (data not shown). When cells were treated with 10⫺6 M RH-5992, an inhibition of cell proliferation was observed and cytoplasmic extensions and cells attachment started appearing within 2 days post treatment (data not shown).
4. Discussion The first aim of this paper was to begin to apprehend the genetic signalling cascade of 20E in our cell line by the characterization of a putative hormone receptor 3 (HR3) involved in this cascade. The second goal was to check the dynamics of the expression of this receptor according to the cell cycle. Our approach parallels investigations by other groups who study the expression of these receptors, either in vivo or in vitro on whole tissue, but with a slightly different perspective. The use of an ecdysone responsive cell line that retains the ability to respond to the insect molting hormone (Cassier et al., 1991) and that can be synchronized, provides us large quantities of cells in controlled conditions resulting in homogeneous and reproducible material for experiments. Moreover, the ability to experimentally manipulate the dynamic of the cells allows us to follow in parallel the cell changes in response to 20E and the 20E-induced genetic signalling cascade according to the phases of cell cycle. The first aim of this work was to clone and to characterize a selected region of HR3 from RNA of IAL-PID2 cells pretreated with 20E. We used a strategy consisting of the amplification of the DNA binding domain, a region which is the most conserved for all members of the steroid hormone receptor superfamily (Evans, 1988). The amplification was realized by using a pair of specific oligonucleotide primers to the cDNA of MHR3. The position of these primers at the ends of the DNA binding domain was designed on the basis of conserved amino acid and nucleotide sequences from the members of the
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subfamily HR3 that have been cloned from Drosophila melanogaster, Galleria mellonella, Caenorhabditis elegans and Choristoneura fumiferana. Thanks to this approach, we have successfully cloned a IAL-PID2 cell cDNA fragment named PHRX. The deduced amino acid sequence of PHRX revealed the presence of two zinc fingers with two short sequences of six amino acids corresponding to the “P-box” and “D-box” [Fig. 2(A)]. PHRX has a deduced amino acid sequence 100% identical to the lepidopteran HR3s. One of the two amino acids changes from threonine in PHR3 to a serine in DHR3 is maintained in lepidopteran and dipteran HR3. As might be expected, analysis of the amino acid sequence of PHRX suggested that PHRX represented the DNA binding domain of a putative ecdysteroid-inducible nuclear receptor belonging to the subfamily of HR3. We then reported the detection by northern hybridization of a 4.5 kb PHR3 mRNA in 20E treated IAL-PID2 cells. This result agrees with previous expression studies performed in vitro on GHR3, CHR3 and Malacosoma disstria HR3 (MdHR3) in which 20E induced a 4.5 kb mRNA in isolated abdomens from G. mellonella (Jindra et al., 1994), in the CF-203 cell line from C. fumiferana (Palli et al., 1996), in the MD-66 cell line from Malacosoma disstria (Palli et al., 1995a,b) and in the GV1 cell line from Manduca sexta (Lan et al., 1997). The expression of PHR3 was directly induced by 20E and protein synthesis was necessary for its subsequent decline. Thus, PHR3 expression was similar to that of the early genes (Ashburner et al., 1974; Ashburner, 1974) and its appearance after 2 h was slower than that of the early genes in Drosophila (E74, Thummel et al., 1990; E75, Segraves and Hogness, 1990), more in consonance with MHR3, CHR3, MdHR3 and GHR3 early late genes. RH-5999 exhibited an in vitro activity comparable with that of 20E on the IAL-PID2 cell line, inducing PHR3 mRNA, inhibiting cellular proliferation and long term forming pseudoepithelial aggregates structures (Cassier et al., 1991). This PHR3 mRNA induction by RH-5992 suggested an involvement of PHR3 in this genetic cascade. The pattern of PHR3 mRNA induction proceeded with a different stepwise sensitivity for RH-5992 as compared to that for 20E. This result is in agreement with those obtained in dose response experiments for the induction of Choristoneura HR3 mRNA by RH-5992 in CF-203 cells (Sundaram et al., 1998) and on the selective action of RH-5992 on lepidopterans insects. Independently, Wing (1988) found that the IAL-PID2 cells displayed ecdysteroid receptor-like activity with the appropriate saturability and specificity for both the ringsubstituted dibenzolhydrazines and the ecdysteroids. The differential induction by 20E of PHR3 mRNA over time suggests that the sensitivity of IAL-PID2 cells to 20E could depend on the position of the cells in their cell cycle. The maximum induction of PHR3 mRNA
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observed at 16 h after cell cycle resumption corresponded to the passage of the cells from S to G2 phase, whereas that observed at 28 h and 31 h coincided with the end of mitosis and entry in G1 (Hatt et al., 1997). Hence, 20E could selectively induce a genetic cascade and the expression of a nuclear receptor belonging to the HR3 subfamily when the IAL-PID2 cells pass through both growth phases of their cycle, i.e. G1 and G2 phases. The genetic cascade induced in G2 could easily be related to cell arrest and subsequent biochemical and morphological changes already described for insect cell lines and tissues exposed to ecdysteroids (Judy, 1969; Courgeon, 1972; Cherbas et al., 1980; Barbara et al., 1982; Dinan et al., 1990; Cassier et al., 1991; Porcheron et al., 1991; Palli et al., 1995a,b; Fretz and Spindler, 1999), but the role of the G1 response remains to be understood. However, the expression of PHR3 could be the first molecular marker for studying 20E action at level of the cell cycle. Experiments are in progress to specify the variations of PHR3 mRNA induction to the phases of cell cycle using cytological markers. We plan to isolate early genes and other early late genes of the 20E-induced genetic cascades and to define the role of each cascade in the 20E regulated biological response at the level of the cell cycle. Acknowledgements We thank Dr Rene´ Lafont and his colleagues (Department of Biology, CNRS-ENS) for their support and technical advices in the early steps of this work. References Ashburner, M., 1974. Sequential gene activation by ecdysone in polytene chromosomes of Drosophila melanogaster. II. The effects of inhibitors of protein synthesis. Dev. Biol. 39, 141–157. Ashburner, M., Chihara, C., Meltze, P., Richards, G., 1974. on the temporal control of puffing activity in polytene chromosomes of Drosophila melanogaster. Cold Spring Harbor Symp. Quant. Biol. 38, 665. Barbara, J., Graves, B., Schubiger, G., 1982. Cell cycle changes during growth and differentiation of imaginal leg discs in Drosophila melanogaster. Dev. Biol. 94, 104–110. Cassier, P., Serrant, P., Garcia, R., Coudouel, N., Andre´ , M., Guillaumin, D., Porcheron, P., Oberlander, H., 1991. Morphological and cytochemical studies of the effects of ecdysteroids in a lepidopteran cell line (IAL-PID2). Cell Tissue Res. 265, 361–369. Cherbas, L., Yonge, C.D., Cherbas, P., Williams, C.M., 1980. The morphological response of Kc-H cells to ecdysteroids: hormonal specificity. Wilhelm Roux’s Archives 189, 1–15. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem. 162, 156–159. Clever, U., Karlson, P., 1960. Induktion von Puff-Veranderungen in den Speicheldrusen-chromosomen von Chironomus tentans durch Ecdyson. Exp. Cell Res. 20, 623–626.
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