Effect of ecdysone receptor gene switch ligands on endogenous gene expression in 293 cells Siva K Panguluri1, Bing Li2*, Robert E Hormann2 and Subba R Palli1 Department of Entomology, College of Agriculture, University of Kentucky, Lexington, KY, USA Intrexon Corporation, Norristown, PA, USA Keywords diacylhydrazine; ecdysone; gene therapy; microarray; RSL-1 Correspondence S R Palli, Department of Entomology, College of Agriculture, University of Kentucky, Lexington, KY 40546 USA Fax: +1 859 323 1120 Tel: +1 859 257 4962 E-mail: rpalli@uky.edu *Present address MicroBiotiX, Inc., Worcester, MA, USA (Received 24 June 2007, revised 13 August 2007, accepted September 2007) doi:10.1111/j.1742-4658.2007.06089.x Regulated gene expression may substantially enhance gene therapy Correlated with structural differences between insect ecdysteroids and mammalian steroids, the ecdysteroids appear to have a benign pharmacology without adversely interfering with mammalian signaling systems Consequently, the ecdysone receptor-based gene switches are attractive for application in medicine In the present study, the effect of inducers of ecdysone receptor switches on the expression of endogenous genes in HEK 293 cells was determined Four ligand chemotypes, represented by a tetrahydroquinoline (RG-120499), one amidoketone (RG-121150), two ecdysteroids [20-hydroxyecdysone (20E) and ponasterone A (Pon A)], and four diacylhydrazines (RG-102240, RG-102277, RG-102398 and RG-100864), were tested in HEK 293 cells The cells were exposed to ligands at concentrations of lm (RG-120499) or 10 lm (all others) for 72 h and the total RNA was isolated and analyzed using microarrays Microarray data showed that the tetrahydroquinoline ligand, RG-120499 caused cell death at concentrations ‡ 10 lm At lm, this ligand caused changes in the expression of genes such as TNF, MAF, Rab and Reprimo At 10 lm, the amidoketone, RG-121150, induced changes in the expression of genes such as v-jun, FBJ and EGR, but was otherwise noninterfering Of the two steroids tested, 20E did not affect gene expression, but Pon A caused some changes in the expression of endogenous genes At lower concentrations pharmacologically relevant for gene therapy, intrinsic gene expression effects of ecdysteroids and amidoketones may actually be insignificant A fortiori, even at 10 lm, the four diacylhydrazine ligands did not cause significant changes in expression of endogenous genes in 293 cells and therefore should have minimum pleiotropic effects when used as ligands for the ecdysone receptor gene switch Gene therapy is used to correct a defect in the expression of a gene by transferring a gene expression cassette containing a promoter, a terminator, and the coding region of a gene whose absence or defect causes a disease Current technology uses constitutive promoters, such as the cytomegalovirus promoter, for expression of transgenes Such an ‘always on’ arrangement is not desirable because it can exacerbate pleiotropic effects and also leaves no option for remediation in the event of medical complications due to transgene expression To address this issue, regulated expression of the additive or corrective gene becomes attractive for various gene therapy applications Despite extra complexity, the regulated expression system can Abbreviations AMK, amidoketone; qRT-PCR, quantitative real-time reverse transcription PCR; STAT 6, signal transducer and activator of transcription 6; THQ, tetrahydroquinoline FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5669 Ecdysone receptor gene switch ligands S K Panguluri et al substantially raise the level of safety and might even be essential to control the expression levels of proteins that have narrow therapeutic indices, such as cytokinins and hormones The main purpose of a regulated gene expression system is to control the timing and levels of transgene expression in vivo Whether the expressed protein remains within the cell or, more commonly, is secreted and ⁄ or distributed in extracellular compartments, it will undergo elimination according to pharmacokinetc principles [1] Thus, too little protein will be subtherapeutic, too much will be potentially toxic Therefore, a successful therapy would be characterized by regulated expression of the transgene finely tuned to the changing clinical state of a patient Several gene switches have been developed for regulating the expression of transgenes in humans [2] More specifically, ecdysone receptor (EcR) gene switches for medicinal purposes have been reported by several laboratories [3–9] Among these switches, EcRbased gene switches display particularly low basal activity in the absence of an inducer and strong inducible activity in the presence of an inducer [2,8,10] The relative structural dissimilarity of ecdysteroids and mammalian steroids might suggest that binding of the former to vertebrate steroid receptors would be too weak for pharmacological effects, particularly adverse ones In support of this proposition, humans consume significant amounts of phytoecdysteroids contained in dietary vegetables seemingly without any apparent detrimental effects [11] However, Oehme et al [12] reported recently that ecdysteroids and ecdysone mimics can induce and ⁄ or suppress endogenous genes in RKO and other mammalian cells and promote apoptosis A more comprehensive understanding of possible pleiotropic effects of ligand inducers and ⁄ or the switch components is essential for successful use of the EcR gene switch for in vivo applications such as gene therapy The diacylhydrazine [13,14] nonsteroidal ecdysone agonists, such as Rheoswitch ligand (RSL-1; Fig 1), are reported to be an excellent inducer for EcR gene switches, supporting up to 9000-fold induction of reporter activity [15] Other steroidal ligands, such as ponasterone A (Pon A; Fig 1), have also been reported as potential inducers of EcR-based gene OH OH O H R N O N H HO H HO R1 OH R2 H O Diacylhydrazines (DAH) Ecdysteroids (ECD) 20-hydroxyecdysone R = OH ponasterone R=H RG-100864 RG-102398 RG-102240 RG-102277 R1=Cl R2=H R1=2-CH3, 3-OCH3 R2=3,5-di-CH3 R1=2-CH2CH3, 3-OCH3 R2=3,5-di-CH3 R1=2-CH2CH3, 3-OCH3 R2=3-CH3, 5-C(O)NH2 F O N H HN O F O N O O F Amidoketone (AMK) RG-121150 Tetrahydroquinoline (THQ) RG-120499 5670 Fig EcR ligands analyzed by microarray and qRT-PCR analysis FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS S K Panguluri et al switches [8,14] In addition, other chemotypes, such as amidoketones (AMK) and tetrahydroquinolines (THQ), are also being developed as inducers of EcR gene switches [16–20] (Fig 1) The main goal of the present study was to determine the intrinsic gene expression effects of EcR switch inducers in mammalian cells We studied the effect of eight EcR ligands: four diacylhydrazines, two ecdysteroids, one THQ and one AMK ligand (Fig 1) on the expression of endogenous genes in HEK 293 cells using microarray and quantitative real-time reverse transcription PCR (qRT-PCR) THQ ligand caused changes in the expression of genes such as TNF, MAF, Rab and Reprimo The AMK ligand induced changes in the expression of genes such as v-jun, FBJ and EGR 20-Hydroxyecdysone (20E) did not affect gene expression, but Pon A caused some changes in the expression of endogenous genes At lower ligand concentrations applicable for therapeutic use, potential pleiotropic effects may or may not be observed The four diacylhydrazine ligands did not cause significant changes in the expression of endogenous genes Results and Discussion THQ ligand, RG-120499, affects 293 cells via many pathways Incubation of 293 cells with the THQ compound RG120499 at 10 lm for days resulted in the death of 70% of the cells as indicated by observation of cell morphology To determine the possible genes and pathways that are affected by this ligand, we performed a microarray experiment using RNA isolated from 293 cells treated with lm RG-120499 A total of 1171 genes were up-regulated and 443 genes were downregulated in 293 cells treated with this compound compared to the cells treated with dimethylsulfoxide (Fig 2A) Among these, 115 genes showed P £ 0.01 with a two-fold or more greater in expression levels in ligand-treated cells compared to the levels in dimethylsulfoxide-treated cells (Table 1) Among these 115 genes, 55 genes showed signal detection values of more than 100 We selected v-maf, TNF, PNAS13, Rab, Reprimo, DNAH and KIF9 genes for qRT-PCR The qRTPCR data (Fig 2B,C) showed that v-maf, TNF, PNAS13 and DNAH mRNA levels increased, and Rab and Reprimo mRNA levels decreased in RG-120499 treated cells, compared to the levels in dimethylsulfoxide-treated cells The microarray and qRT-PCR data showed perfect correlation for these six genes The twofold down-regulation of the KIF9 gene observed in microarray analyses was not confirmed by qRT-PCR Ecdysone receptor gene switch ligands because this method showed a ten-fold up-regulation of this gene in the presence of RG-102499 MAF [v-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian)] is a basic-leucine zipper transcription factor that plays crucial roles in gene regulation, differentiation, oncogenesis and development in many organisms [21] v-maf is a viral oncogene encoding a leucine zipper motif that forms heterodimers with the protein products of maf-related genes or other proteins such as fos, jun and myc oncogenes that have leucine zipper motifs [22] Our microarray and qRT-PCR data showed that, in 293 cells, RG-120499 up-regulates v-maf gene expression by 2.4- and 2.3-fold, respectively Nishizawa et al [22] have demonstrated that the human cellular counterpart of the v-maf (c-maf) gene is conserved across species Additionally, Massrieh et al [21] reported that the MAF transcription factor transcript levels are induced by proinflammatory cytokines in PHM1-31 myometrial cells and that MAF transcription factor mRNA is rapidly induced by IL-1B and TNF in primary myometrial and PHM1-13 cells Our data also indicate up-regulation of the TNF gene in microarray and qRT-PCR data (by 2.2- and 5.5-fold, respectively), an observation consistent with TNF-induced up-regulation of MAF transcript levels Zheng et al [23] reported that the tumor necrosis factor (TNF) can mediate mature T-cell receptor-induced apoptosis through the p75 TNF receptor This may be the possible reason for the death of 293 cells when treated with 10 lm RG-120499 ligand In addition to the aforementioned up-regulated genes, two genes, namely, Rab and Reprimo, were down-regulated by this THQ ligand in 293 cells, as observed by both microarray and qRT-PCR techniques The Rab proteins constitute a subfamily of Ras-related GTP-binding proteins that are localized in distinct intracellular compartments [24] Mutations in the Rab gene can alter the morphology of entire organelles by blocking protein transport along the exocytic and endocytic pathways because Rab proteins plays a key role in membrane trafficking [25] Barbosa et al [26] reported that mutations in the Rab gene(s) can cause irregularities in the protein transport machinery leading to the formation of giant lysosomes in mouse beige (bg) mutant and other mutant mice It has been suggested that Chediak–Higashi syndrome, a rare autosomal recessive disorder in humans, is the consequence of a mutation to a homolog of bg Our microarray and qRT-PCR data indicate that the rab gene was down-regulated in 293 cells by two- and 23-fold, respectively, by the THQ, RG-120499 Mutation studies of the Rab gene suggest that the Rab gene down-regulation by FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5671 S K Panguluri et al 1E-05 Ecdysone receptor gene switch ligands 1E-00 1E-01 1E-02 1E-03 1E-04 Significance (t test p-value) A x4 /2 /1.5 x1.5 Fold Suppression/Induction v-Maf TNF KIF 0.015 0.01 0.005 5.5 -23 4.3 -2 10 23 v-Maf TNF THQ THQ RAB Reprimo DNAH DMSO THQ DMSO THQ DMSO THQ PNAS DMSO THQ THQ THQ Reprimo DNAH DMSO THQ DMSO THQ RAB DMSO DMSO THQ PNAS -2 5.1 -2 DMSO THQ 2.2 DMSO THQ 2.4 2.3 0.02 DMSO -2 x16 0.025 THQ C 2.7 x8 DMSO /4 DMSO 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 DMSO Signal Values B /8 Relative Expression /16 KIF Fig The THQ ligand RG-120499 affects 293 cells via many pathways (A) The V-plot of differentially expressed genes from microarray data The P-values of t-test are plotted against fold suppression or induction The horizontal bar in the plot represents the nominal significant level 0.001 for the t-test under the assumption that each gene has a unique variance The vertical bars represent the genes that are minimum of two-fold up- or down-regulated compared to the control dimethylsulfoxide (DMSO) (B) The signal values of v-maf, TNF, PNAS, Rab, Reprimo, DNAH and KIF9 genes from the microarray The signal values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide (C) The relative expressions of v-maf, TNF, PNAS, Rab, Reprimo, DNAH and KIF9 gene transcripts in qRT-PCR The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide this ligand may be a reason for cell death in addition to modulation of the v-maf and TNF genes Tumor suppressor genes that encode transcriptional factors can affect a variety of cellular mechanisms underlying growth, differentiation, and apoptosis [27,28] Also, when cells were exposed to DNA damage-inducing agents or other noxious stress, the p53 protein, which is the most commonly mutated gene in human cancer, is induced and ⁄ or activated, resulting in cell cycle arrest or apoptosis [29–31] Reprimo is a highly glycosylated protein, which will localize in the cytoplasm and induce G2 arrest of the cell cycle when expressed ectopically [32] In the present study, it was observed that RG-120499 down-regulates the Reprimo gene by two-fold as measured by both microarray and qRT-PCR techniques From these observations and 5672 previous reports, we suggest that the down-regulation of the Reprimo gene may cause loss of DNA repair, which, in turn, is passed on to the next generations, thereby accumulating DNA defects RG-120499 modulates the expression of other genes as well The PNAS123 gene (transformation related protein 11) is up-regulated by 2.7- and 4.3-fold as measured by microarray and qRT-PCR, respectively The pathways in which these genes are involved are not known RG120499 also triggers five- and 23-fold up-regulation of the DNAH (human axonemal dynein heavy chain) gene as measured by microarray and qRT-PCR techniques, respectively DNAH is a microtubule associated motor protein that moves cilia and flagella [33,34] Afzelius [35,36] showed that patients suffering from Kartagener syndrome have cilia lacking dynein FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS S K Panguluri et al Ecdysone receptor gene switch ligands Table Differentially expressed genes in 293 cells treated with different ligands Total genes Genes with signal values 100 or more Ligand P-value Up-regulated Down-regulated Total Up-regulated Down-regulated Total RG-120499 (THQ) < < < < < < < < < < < < < < < < < < < < < < < < 12 71 26 15 20 19 20 27 21 26 31 30 15 15 25 14 18 17 97 13 57 45 35 34 45 41 39 39 0 0 0 0 0 0 0 11 0 0 0 2 46 17 0 0 0 RG-121150 (AMK) 20E (steroid) Pon A (steroid) RG-100864 (halofenozide, diacylhydrazine) RG-102398 (methoxyfenozide, diacylhydrazine) RG-102240 (RSL-1, diacylhydrazine) RG-102277 (diacylhydrazine) 0.0001 0.001 0.01 0.0001 0.001 0.01 0.0001 0.001 0.01 0.0001 0.001 0.01 0.0001 0.001 0.01 0.0001 0.001 0.01 0.0001 0.001 0.01 0.0001 0.001 0.01 arms This disease is characterized by chronic respiratory tract infections, altered position of internal organs, and infertility arising from immotile sperm Milisav and Affara [37] reported that the human dynein-related gene DNEL2 may play an important role specifically in sperm motility and is not involved in the movement of cilia In summary, the use of the THQ gene switch ligand RG-120499 may cause significant changes in the expression of host genes AMK, RG-121150, affects gene expression in 293 cells The affect of the AMK RG-121150 on gene expression in 293 cells was analyzed using microarray and qRTPCR In microarray analysis, a total of 636 genes were up-regulated and 604 genes were down-regulated by this ligand (Fig 3A) Among these genes, 71 genes showed P £ 0.01 and a two-fold or greater change in expression (Table 1) Among 71 genes, 24 genes showed signal detection values greater than 100 Among these are hypothetical proteins, nuclear proteins, transcriptional factors, glycogen phosphorylase, hormone degrading enzymes, kinases and some solute carrier proteins To validate microarray data with qRT-PCR, the primers for early growth response gene (EGR), FBJ, v-jun, and a hypothetical protein gene were designed For three of the four genes, the microarray data was confirmed by qRT-PCR The data showed that the hypothetical protein gene was up-regulated by two- and 1.5-fold in microarray and qRT-PCR experiments, respectively EGR and v-jun showed suppression of their expression by the ligand in analyses by both methods (Fig 3B,C) The gene FBJ mRNA levels were down-regulated in microarray experiments and upregulated in qRT-PCR experiments The EGR gene product is a transcription factor that plays a role in differentiation and growth EGR genes are transiently and coordinately induced upon activation of peripheral blood T lymphocytes [38,39] These EGR genes are also expressed in a wide range of cell types, including lymphoid cells, myeloid cells such as thymocytes, B cells, monocytes, and nonlymphoid cells such as fibroblasts, kidney cells and neurons [40,41] Huang et al [42,43] showed that the expression of the EGR gene exogenously in various tumor cells unexpectedly and markedly reduces growth and tumorigenicity, whereas the suppression of endogenous EGR by antisense RNA enhances growth and promotes phenotypic transformation From our microarray and qRT-PCR data, the AMK RG-121150 caused downregulation of this EGR gene by five- and six-fold, FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5673 1E-04 1E-03 1E-02 Significance (t test p-value) 1E-00 1E-01 A S K Panguluri et al 1E-05 Ecdysone receptor gene switch ligands EGR v-jun 0.2000 0.1500 0.1000 -6.14 0.0500 -9 1.5 3.3 Hypo EGR v-jun Amido DMSO Amido DMSO Amido 0.0000 DMSO Amido ketone FBJ x16 0.2500 DMSO -58 DMSO DMSO Amido ketone DMSO Amido ketone Hypo Amido ketone -21 Relative Expression -5.4 2.05 x8 /4 /2 /1.5 x1.5 x4 Fold Suppression/Induction C 18000 16000 14000 12000 10000 8000 6000 4000 2000 DMSO Signal Values B /8 Amido /16 FBJ Fig AMK RG-121150 affects endogene expression in 293 cells (A) The V-plot of differentially expressed genes from microarray analysis The P-values of t-test are plotted against fold suppression or induction The horizontal bar in the plot represents the nominal significant level 0.001 for the t-test under the assumption that each gene has a unique variance The vertical bars represent the genes that are minimum of twofold up- or down-regulated compared to the control dimethylsulfoxide (DMSO) (B) The signal values of hypothetical protein, EGR, v-jun and FBJ genes from microarray The signal values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide (C) The relative expressions of hypothetical protein, EGR, v-jun and FBJ gene transcript levels in qRT-PCR The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide respectively, indicating a possible effect RG-121150 on this pathway Overall, these studies showed that AMK ligand RG-121150 could cause significant changes in the expression of genes in host cells However, the corresponding toxicological potential could be substantially mitigated or even erased by the likelihood that actual gene therapy dosage would be significantly lower than the 10 lm studied here Likewise, judicious choice of the specific member of the AMK chemotype may confer intrinsic benignity Ecdysteroid ligand, 20E, shows little effect on 293 cells but Pon A affects cell division Two steroid ligands Pon A and 20E were tested in 293 cells to determine their effect on gene expression 5674 using microarray and qRT-PCR In 20E-treated 293 cells, a total of 542 genes were up-regulated and 627 genes were down-regulated compared to dimethylsulfoxide-treated cells (Fig 4A) Out of these, 51 genes showed P £ 0.01 and a change in gene expression of two-fold or greater (Table 1) Among these 51 genes, only five genes had signal detection values greater than 100 We selected signal transducer and activator of transcription (STAT6) genes to perform qRT-PCR analyses Although the STAT6 gene was down-regulated by 20E in microarray analysis, the qRT-PCR analyses showed that the mRNA levels did not change in treated cells compared to untreated cells (Fig 4B,C) Experiments in our laboratory (S R Palli, unpublished results) showed that, in mammalian cells, 20E does not induce reporter genes placed under the FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS Ecdysone receptor gene switch ligands 1E-04 1E-03 1E-02 1E-01 1E-00 Significance (t test p-value) A 1E-05 S K Panguluri et al /16 /8 /4 /2 /1.5 x1.5 Fold Suppression/Induction C 200 150 -2.86 100 50 20E DMSO x8 x16 0.006 Relative Expression Signal Values B 250 x4 -0.7 0.005 0.004 0.003 0.002 0.001 20E DMSO STAT6 STAT6 Fig Steroidal ligand 20E does not have much effect on 293 cells (A) The V-plot of differentially expressed genes from microarray analysis The P-values of t-test are plotted against fold suppression or induction The horizontal bar in the plot represents the nominal significant level 0.001 for the t-test under the assumption that each gene has a unique variance The vertical bars represent the genes that are minimum of two-fold up- or down-regulated compared to the control dimethylsulfoxide (DMSO) (B) The signal values of STAT6 gene from microarray The signal values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide (C) The relative expressions of STAT6 RNA transcripts as determined by qRT-PCR The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide control of the EcR gene switch The microarray data corroborate evidence for an overall benign influence of 20E on gene expression and metabolism in mammalian cells [44] It is conceivable that 20E is excluded by mammalian cells, or perhaps some other factors in the mammalian cells inhibit the activity of 20E A total of 639 genes were up-regulated and 638 were down-regulated in 293 cells treated with Pon A compared to the cells treated with dimethylsulfoxide (Fig 5A) Among these, 41 genes showed P £ 0.01 and two-fold or greater induction (up ⁄ down-regulated; Table 1) Only three genes showed signal detection values greater than 100 Out of these three genes, we selected tousled-like kinase (Tlk) gene for qRT-PCR analysis The data from both methods showed that the Tlk gene was induced by Pon A by two-fold (Fig 5B,C) In proliferating human cells, Tlks are maximally active during the S phase but rapidly inactivated in response to inhibitors of DNA replication [45] Sillje and Nigg [46] showed that the Asf1 (antisilencing function 1) proteins are phosphorylated by Tlks both in vivo and in vitro during the S phase and dephosphorylated with inactivation of Tlks Constantino et al [47] used the ecdysone inducible gene expression system in hematopoietic cells and found that the two steroids, muristerone A and Pon A, altered the signaling pathways induced by IL-3 in the pro-B cellline, Ba ⁄ F3 Indeed, they also showed that these ecdysteroids potentiate the IL-3-dependent activation of the PI 3-kinase ⁄ Akt pathway, an effect that could ultimately interfere with the growth, and ⁄ or survival of these cells Our data not reveal any affect of Pon A on the PI 3-kinase ⁄ Akt pathway in 293 cells A possible reason is that these genes might be expressed only FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5675 /16 /8 Signal Values B 90 /4 /2 /1.5 x1.5 x4 Fold Suppression/Induction PonA DMSO x8 x16 C 80 70 60 50 40 30 20 10 Relative Expression Significance (t test p-value) A S K Panguluri et al 1E-00 1E-01 1E-02 1E-03 1E-04 1E-05 Ecdysone receptor gene switch ligands 0.0025 0.002 0.0015 0.001 0.0005 1.99 PonA DMSO TLK TLK Fig Steroidal ligand Pon A may affect cell division in 293 cells (A) The V-plot of differentially expressed genes from microarray analysis The P-values of t-test are plotted against fold suppression or induction The horizontal bar in the plot represents the nominal significant level 0.001 for the t-test under the assumption that each gene has a unique variance The vertical bars represent the genes that are minimum of two-fold up- or down-regulated compared to the control dimethylsulfoxide (DMSO) (B) The signal values of Tlk gene from microarray The signal values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide (C) The relative expression of Tlk transcripts as determined by qRT-PCR The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide in a particular type of cells, such as hematopoietic cells, but not 293 cells These observations, in conjunction with our microarray and qRT-PCR data, indicate that Pon A may affect cell division in mammalian cells Diacylhydrazine ligands not induce significant changes in gene expression Diacylhydrazine nonsteroidal ligands have been subjected to extensive studies concerning their toxicological effects on vertebrates for the purposes of Environmental Protection Agency registration as commercial insecticides [48,49] We selected four representatives from the diacylhydrazine gene switch chemotype for evaluation of their effects on expression of endogenous genes in 293 cells: the lepidopteran insecticide, methoxyfenozide (RG-102398); the coleopteran insecticide, halofenozide (RG-100864); the RheoSwitchÒ ligand RSL-1 (RG-102240, also known as 5676 GSTM-E); and, finally, a more polar and water-soluble variant of RSL-1, namely RG-102277 First, we determined the effect of dimethylsulfoxide itself on the expression of genes in 293 cells The expressions of a total of 43 genes were modulated with P £ 0.01 with a two-fold change in expression Among the affected genes, 38 were up-regulated and five of them were down-regulated Interestingly, only 13 up-regulated genes and one down-regulated gene showed signal detection values of more than 100 in the dimethylsulfoxide-treated 293 cells In our experience, the genes that show signal detection values less than 100 are not reliable indicators of gene expression; therefore, we did not consider these in our analyses Cells treated with the diacylhydrazine RG-102240 resulted in up-regulation of a total of 865 genes; on the other hand, 411 genes were down-regulated in 293 cells treated with RG-102240 (Fig 6A) From these, we considered for further analysis only the genes with P £ 0.01 FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 1E-04 1E-03 1E-02 1E-01 1E-00 Significance (t test p-value) A Ecdysone receptor gene switch ligands 1E-05 S K Panguluri et al /8 /16 x4 x8 x16 C -3.91 -2.11 DMSO GSE DAB DMSO GSE Relative Expression 1200 1000 800 600 400 200 Signal Values B /2 /1.5 x1.5 Fold Suppression/Induction /4 0.008 0.007 0.006 0.005 0.004 0.003 0.002 0.001 Kinesine 3.9 DMSO GSE DAB -2.8 DMSO GSE Kinesine Fig RSL-1 ligand does not cause significant changes in gene expression in 293 cells (A) The V-plot of differentially expressed genes from microarray analysis The P-values of t-test are plotted against fold change in gene expression The horizontal bar in the plot represents the significant level of 0.001 for the t-test under the assumption that each gene has a unique variance The vertical bars represent the genes that are minimum of two-fold up- or down-regulated compared to the control dimethylsulfoxide (DMSO) (B) The signal values of Dab and KIF9 genes from the microarray analysis The signal values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide (C) The relative expressions of Dab and KIF9 genes as determined by qRT-PCR analysis The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide with a two-fold change in expression compared to the expression in dimethylsulfoxide-treated cells A total of 46 genes met these criteria However, only six of these genes showed signal detection values greater than 100 (Table 1) The annotations for these genes were developed by using NIH david ease software (http://david niaid.nih.gov/david/ease.htm) Only two out of six were annotated; these were identified as DAB2 interacting protein gene and kinesin family member-9 (KIF9) The reliability of microarray results depends on several factors such as array production, RNA extraction, probe labeling, hybridization conditions and image analysis [50–53] Therefore, the genes identified as differentially expressed by this method must be validated with another method such as qRT-PCR, which is quantitative, rapid, and requires 1000-fold less RNA than conventional assays [54] For this reason, we designed primers for DAB2 interacting protein gene and KIF9 from their cDNA sequences for qRT-PCR analysis Their expression levels were measured in dimethylsulfoxide- and RG-102240-treated 293 cells and compared to microarray data The DAB2 interacting protein gene showed two-fold down-regulation by microarray and a four-fold increase by qRT-PCR analysis in RG-102240-treated cells compared to the expression in dimethylsulfoxide-treated cells (Fig 6B,C) The KIF9 gene showed 3.9-fold down-regulation by microarray and 2.8-fold down-regulation by qRT-PCR analysis in RG-102240-treated cells compared to the expression in dimethylsulfoxide-treated cells (Fig 6B,C) The Drosophila melanogaster Dab (Disabled) interacting protein is the mammalian ortho- FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5677 Ecdysone receptor gene switch ligands S K Panguluri et al logue of D melanogaster DabIP The DabIP participates in a signaling complex containing various proteins involved in brain development as well as other aspects of adult brain function [55] Although the microarray showed two-fold down-regulation of the Dab gene, the qRT-PCR data showed four-fold up-regulation The reasons for difference in the fold regulation between the two techniques are not readily apparent KIF9 protein is found to interact with Raslike GTPase Gem and is involved in cell shape remodeling [56] Both microarray data and qRT-PCR data, showed similar results (3.9-fold and 2.8-fold down-regulation, respectively) in KIF9 gene expression levels Over-expression of the KIF9 mutant did not cause any significant difference in Gem-induced cell elongation and it does not appear to be essential for the Gem1E-05 1E-04 1E-03 1E-02 1E-01 1E-00 Significance (t test p-value) A induced phenotypic changes [56] From these observations, it can be concluded that, although the RSL-1 ligand down-regulates the KIF9 gene, there may not be significant changes in cell shape remodeling Treatment of HEK 293 cells with RG-102398 resulted in a total of 598 down-regulated and 388 up-regulated genes (Fig 7A) Among these, 52 genes indicated P £ 0.01 and a change in expression of twofold or greater compared to dimethylsulfoxide-treated cells (Table 1) Only three genes [ribosomal protein L13 (RPL), hypothetical protein gene FLJ38705 and leptin receptor] out of these 52 showed signal detection values greater than 100 The qRT-PCR and microarray analysis of RPL gene expression are weakly correlated The RPL gene was down-regulated by two-fold by microarray, but was up-regulated 1.25-fold by /16 /8 /4 /2 /1.5 x1.5 x4 x8 x16 Fold Suppression/Induction C 250 Relative expression Signal values B 200 150 -2.05 -2.27 100 50 DMSO RG-102398 RPL DMSO RG-102398 Zink Finger 0.10000 0.09000 0.08000 0.07000 0.06000 0.05000 0.04000 0.03000 0.02000 0.01000 0.00000 -1.49 1.25 DMSO RG-120398 RPL DMSO RG-120398 Zink Finger Fig Effect of RG-102398 on 293 cells (A) The V-plot of differentially expressed genes from microarray analysis The P-values of t-test are plotted against fold suppression or induction The horizontal bar in the plot represents the nominal significant level 0.001 for the t-test under the assumption that each gene has a unique variance The vertical bars represent the genes that are minimum of two-fold up or down regulated compared to the control dimethylsulfoxide (DMSO) (B) The signal values of RPL and Zink finger protein genes The signal values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide (C) The relative expressions of RPL and Zink finger protein RNA levels as determined by qRT-PCR The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide 5678 FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS S K Panguluri et al Ecdysone receptor gene switch ligands qRT-PCR analysis in RG-102398-treated cells compared to the expression in dimetylsulfide-treated cells (Fig 7B,C) By contrast, a good correlation was observed between the microarray and qRT-PCR data with the hypothetical protein gene (down-regulated by two-fold and 1.4-fold by microarray and qRT-PCR analyses, respectively) (Fig 7B,C) The hypothetical protein FLJ38705 is zinc finger protein 41 homolog (mouse) The pathways and interactions of this hypothetical protein are not known RLP is a ribosomal protein which usually decorates the rRNA cores of the subunits Approximately two-thirds of the mass of the ribosome consists of RNA and one-thirds comprises protein The proteins are named in accordance with the subunit of the ribosome to which they belong: the small (S1 to S31) and the large (L1 to L44) Many of ribosomal proteins, particularly those of the large subunit, feature a globular, surface-exposed domain with 1E-05 1E-04 1E-03 1E-02 Significance (t test p-value) 1E-00 1E-01 A long finger-like projections that extend into the rRNA core to stabilize its structure Most of the proteins interact with multiple RNA elements, often from different domains The crucial activities of decoding and peptide transfer are RNA-based; proteins play an active role in functions that may have evolved to streamline the process of protein synthesis In addition to their function in the ribosome, many ribosomal proteins have some function ‘outside’ the ribosome Although the RLP13 gene was affected by the RSL ligand RG-102398 in the present study, further studies are needed to confirm its effects, as these two techniques indicate opposite regulation Cells treated with RG-100864 showed a total of 566 up-regulated genes and 398 down-regulated genes (Fig 8A) Among these, only 38 genes showed P £ 0.01 and two-fold or greater induction (Table 1) Only three of these genes showed signal detection /8 Signal Values B /4 /2 /1.5 x1.5 x4 Fold Suppression/Induction -2.08 140 120 100 80 60 40 20 DMSO RG-100864 x8 x16 C Relative Expression /16 0.0160 0.0140 0.0120 0.0100 0.0080 0.0060 0.0040 0.0020 0.0000 Hypo -2.6 DMSO RG-100864 Hypo Fig Effect of RG-100864 on 293 cells (A) The V-plot of differentially expressed gene from microarray analysis The P-values of t-test are plotted against fold suppression or induction The horizontal bar in the plot represents the nominal significant level 0.001 for the t-test under the assumption that each gene has a unique variance The vertical bars represent the genes that are minimum of two-fold up- or downregulated compared to the control dimethylsulfoxide (DMSO) (B) The signal values of hypothetical protein gene from the microarray The signal values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide (C) The relative expressions of hypothetical protein transcript levels as determined by qRT-PCR The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5679 Ecdysone receptor gene switch ligands S K Panguluri et al 1E-04 1E-03 1E-02 Significance (t test p-value) 1E-00 1E-01 A KIAA0515] had assigned cDNA sequences MRC expression, showed a positive correlation between qRT-PCR and microarray data (2.8- and 3.3-fold down-regulated, respectively; Fig 9B,C) MRC, also known as Endo 180, is a 180 kDa transmembrane glycoprotein that is a part of trimolecular cell surface complex with urokinase-type plasmogen activator and its receptor [57] This trimolecular complex plays an important role in cell guidance and chemotaxis during normal and pathological events [58] The expression of MRC is restricted to stromal cells, macrophages, endothelial cells, tumor endothelium and breast cancer tissue in normal conditions [59–61] The up-regulation of this MRC is observed in tumor endothelial cells [62], which have a potential role in the regulation of tumor neoangiogenesis The ligand RG-102277 showed downregulation of this MRC gene in 293 cells as measured by both analytical techniques Because only up-regula- 1E-05 values greater than 100 A cDNA sequence is available for only one gene out of these three: hypothetical protein gene FLJ22344, to which we have designed primers and performed qRT-PCR The microarray data and qRT-PCR data for this hypothetical protein gene are negatively correlated In the microarray experiment, the hypothetical protein gene showed two-fold up-regulation compared to two-fold down-regulation in qRTPCR analysis (Fig 8B,C) The pathways and interactions of this hypothetical protein are not known In 293 cells treated with RG-102277, 512 genes were up-regulated and 456 genes were down-regulated compared to dimethylsulfoxide-treated cells (Fig 9A) Among these genes, 45 genes showed P £ 0.01 and two-fold or greater induction (up ⁄ down regulated) (Table 1) Only five out of these 45 genes showed signal detection values greater than 100, of which two genes [mannose receptor C type (MRC) and /16 /8 /2 /1.5 x1.5 x4 /4 Fold Suppression/Induction Signal Values 160 140 120 100 80 60 40 20 2.01 -3.35 DMSO RG102277 MRC DMSO RG102277 KIAA Relative expression C B x8 x16 0.160000 0.140000 0.120000 0.100000 0.080000 0.060000 0.040000 0.020000 0.000000 -1.3 -2.8 DMSO RGDMSO RG102277 102277 KIAA MRC Fig Effect of RG-102277 on 293 cells (A) The V-plot of differentially expressed genes from microarray analysis The P-values of t-test are plotted against fold suppression or induction The horizontal bar in the plot represents the nominal significant level 0.001 for the t-test under the assumption that each gene has a unique variance The vertical bars represent the genes that are minimum of two-fold up- or down-regulated compared to the control dimethylsulfoxide (DMSO) (B) The signal values of MRC and KIAA genes from the microarray The signal values from the microarray analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide (C) The relative expressions of MRC and KIAA transcripts as determined by qRT-PCR The relative expression values from the qRT-PCR analysis were plotted for each gene are indicated as mean ± SD (n ¼ 3) The numbers above the bar represents the fold changes with this ligand against dimethylsulfoxide 5680 FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS S K Panguluri et al tion will cause tumor neoangiogenesis, the downregulation of this gene by RG-102277 probably does not contraindicate its use Overall, the effect of diacylhydrazines on the expression of genes in 293 cells was minimal as shown by microarray as well as by qRT-PCR data There appear to be differences among the individual diacylhydrazines, but it may be premature to interpret these data in terms of structure–activity relationships No significant pathway is affected by these ligands Only one or two genes have a cDNA sequence available for qRTPCR analysis and only half of the genes studied showed positive correlation between microarray and qRT-PCR analysis The poor correlation between the microarray and qRT-PCR data for these genes is due to the fact that the small variations in the mRNA levels can be accurately detected by qRT-PCR, but might not be accurately reflected in microarray expression scores, especially for genes expressed at low levels (approximately three or four copies per cell) [63] Etienne et al [64] have hypothesized that, in addition to genes with low expression levels, those with very high expression levels or a greater percent of absent calls may show lower levels of correlation between microarray and semiquantitative qRT-PCR data The level of expression differences between microarray and qRT-PCR may also be due to the lack of specificity in the primers designed to discriminate gene family members at the level of primary screening by DNA arrays [54] Therefore, to the degree of resolution afforded by combined qRT-PCR and microarray analysis, we concluded that these diacylhydrazine ligands are safe for use as inducers of gene switches, especially because the concentrations anticipated for gene therapy would likely be lower than the lm examined in the present study Conclusion The present investigation analyzed the effects of EcR gene switch ligands on the expression of endogenous genes Two widely used analytical techniques, oligo nucleotide probe microarray and qRT-PCR, were employed to perform the analyses Keeping in mind that the ligand concentrations in this investigation are high relative to the anticipated pharmacologically relevant blood levels for gene therapy, the THQ RG120499 caused significant changes in the expression of genes such as v-maf, TNF, PNAS13, Rab, Reprimo and DNAH at lm An AMK, RG-121150, affected the expression of genes such as EGR, FBJ and v-jun at 10 lm Also at 10 lm, the steroidal ligand, Pon A induced only the Tlk gene, known to interfere with the growth and ⁄ or survival of cells Otherwise, like RG- Ecdysone receptor gene switch ligands 121150, Pon A also appears to be a benign substance The homologue 20E did not cause significant changes in gene expression at all Likewise, at 10 lm, four diacylhydrazine nonsteroidal ligands did not cause significant changes in gene expression, except for minor effects on a few genes of lesser significance, KIF9, RPL and MRC The relatively minor perturbation of gene expression in 293 cells for the diacylhydrazines corroborates on a gene expression level the known favorable mammalian toxicology Experimental procedures Sources, preparation, and characterization of ligands General procedures Most reagents were purchased from Aldrich (Milwaukee, WI, USA), VWR (West Chester, PA, USA), or Fisher Scientific (Pittsburg, PA, USA) Pon A and 20E were purchased from Alexis Biochemicals (Lausen, Switzerland) Both substances were assayed without further purification Solvents were reagent grade unless otherwise stated Anhydrous solvents were used as purchased Analytical TLC was performed on Macherey–Nagel PolygramÒ Sil G ⁄ UV254 0.2 mm plates Most plates were visualized by UV light; some plates were developed using iodine or phosphomolybdic acid Silica gel chromatography was performed using Aldrich silica gel ˚ (230–400 mesh, 60 A) in glass columns under a N2 or argon head pressure of approximately 30 psi Melting points were measured in glass capillary tubes and are uncorrected Most H NMR spectra were recorded at 400.13 MHz with a Bruker DPX-400; 13C NMR spectra were recorded at 100.6 MHz with a Bruker DPX-400 at NMR Services in Rochester, NY Some 1H NMR was performed at 200 MHz on a Varian instrument (Palo Alto, CA, USA) or 300 MHz on a Bruker instrument (Billerica, MA, USA) Unless otherwise stated, internal reference is solvent LC-MS analysis was performed using an Agilent 1100 LC stack coupled with an Agilent (Foster City, CA, USA) single quad mass spectrometer Solvents were (A) H2O ⁄ 0.1% formic acid and (B) ACN ⁄ 0.1% formic acid in a gradient of T ¼ 15% B to T ¼ 10 98% B and a stop time of 20 on a 75 mm · 2.1 mm C18 column with a ow rate ẳ 0.2 mLặ min)1 Exact mass analysis were performed by direct infusion into an Agilent ESI ⁄ TOF mass spectrometer Mass spectrometry was performed by the Scripps Research Institute mass spectroscopy service (La Jolla, CA, USA) Preparation and purification of ligands The diacylhydrazine RG-102277 (Fig 10) was prepared in a manner analogous to literature precedent [13] with adjustments in the final phase of the synthesis to incorporate the FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5681 Ecdysone receptor gene switch ligands S K Panguluri et al O COOH N H a, b O NH O N H g O COCl COOH c, d, e, f N O O Br Br h, i, j, k O O N H N O l NH2 O O N H O O R RG-102277 N R = CH2OH CHO C(O)OH C(O)OPh-F5 Fig 10 Synthesis of the diacylhydrazine RG-102277 (a) SOCl2, dimethylformamide; (b) t-BuNHNH3Cl, aqueous K2CO3, CH2Cl2; (c) CH3OH, Et3N, CH2Cl2; (d) N-bromosuccinimide, AIBN, CCl4; (e) aqueous HBr, 100 °C h; (f) SOCl2, dimethylformamide, CHCl3; (g) aqueous K2CO3, CH2Cl2; (h) CaCO3, H2O, dioxane; (i) PCC, CH2Cl2; (j) KMnO4, t-BuOH; (k) PfOH, N¢,N¢-dicyclohexylcarbodiimide; (l) NH4OH, dioxane, 60 °C, h amide group 2-Ethyl-3-methoxybenzoic acid was converted to the corresponding acid chloride and coupled with t-butylhydrazine under controlled conditions to form a mono-acylated hydrazine The alternate ring was constructed from 3,5-dimethyl benzoic acid Rather than direct bromination of the acid [14], we found that esterification to the methyl ester followed by controlled bromination with N-bromosuccinimide and distillation provides pure and plentiful quantities of 3-bromomethyl-5-methyl benzoate Subsequent de-esterification using HBr smoothly yields the corresponding carboxylic acid The bromomethyl benzoic acid, in turn, was converted to the acid chloride using thionyl chloride and then coupled with the aforementioned mono-hydrazide The benzylic bromide was converted to the benzyl alcohol using calcium carbonate, and the alcohol was then oxidized first to the aldehyde and then to the carboxylic acid using pyridinium chlorochromate and potassium permanganate, respectively [13] In our hands, this two-step oxidation provided purer product in higher yields than did direct oxidation The diacylhydrazine carboxylic acid was then esterified to an activated ester with pentafluorophenol using N¢,N¢-dicyclohexylcarbodiimide as a coupling agent Finally, the pentafluorophenol was displaced with ammonia in dioxane ⁄ water to provide RG-102277 The diacylhydrazines, as well as the THQ RG-120499 and AMK RG-121150 were purified by flash chromatography and ⁄ or crystallization to 99% purity or greater as judged by 400 MHz 1H NMR The ecdysteroids were used in the form obtained from the commercial vendor 5682 [6-Fluoro-4-(4-fluoro-phenylamino)-2-methyl-3,4dihydro-2H-quinolin-1-yl]-(3-fluoro-4-methyl-phenyl)methanone (RG-120499) RG-120499 was prepared according to the previously described procedure [17,18] 1H NMR (400 MHz, dimethylsulfoxide-d6) d: 7.22 (t, J ¼ 8.0 Hz, 1H), 7.11 (d, J ¼ 10.4 Hz, 1H), 7.04–6.96 (m, 4H), 6.88–6.79 (m, 3H), 6.64 (dd, J ¼ 4.8, 8.4 Hz, 1H), 6.02 (d, J ¼ 8.4 Hz, 1H), 4.78– 4.69 (m, 1H), 4.66–4.59 (m, 1H), 2.77 (ddd, J ¼ 3.6, 8.8, 12.8 Hz, 1H), 2.23 (s, 3H), 1.26 (ddd, J ¼ 3.2, 8.8, 12.0 Hz, 1H), 1.20 (d, J ¼ 5.6 Hz, 3H); 13C NMR (100 MHz, dimethylsulfoxide-d6) d: 167.60, 161.53, 161.11, 158.99, 158.82, 156.27, 153.86, 144.86, 140.28, 136.37, 133.40, 131.60, 128.61, 126.88, 126.62, 124.67, 116.05, 115.83, 115.68, 115.44, 114.16, 114.09, 113.70, 113.48, 111.31, 111.07, 49.25, 48.81, 21.48, 14.38; HRES (ESI) m ⁄ z calculated for C24H22F3N2O [M+ + H]+ 411.1684, found 411.1653, calculated for C24H21F3N2NaO [M+ + Na]+ 433.1504, found 433.1508 5-Ethyl-2,3-dihydro-benzo[1,4]dioxine-6-carboxylic acid[1-(3,5-dimethyl-benzoyl)-cyclopentyl]-amide (RG-121510) RG-121510 was prepared according to the previously described procedure [15,16] 1H NMR (400 MHz, dimethylsulfoxide-d6) d: 7.52 (s, 2H), 7.15 (s, 1H), 6.68 (d, J ¼ 8.4 Hz, 1H), 6.57 (d, J ¼ 8.4 Hz, 1H), 6.30 (s, 1H), 4.28 (s, FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS S K Panguluri et al 4H), 2.68 (m, 2H), 2.42–2.35 (m, 8H), 2.15–2.10 (m, 2H), 1.94–1.87 (m, 4H), 1.00 (t, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz, dimethylsulfoxide-d6) d: 201.68, 168.63, 144.67, 141.65, 137.30, 136.85, 133.10, 132.28, 129.20, 125.99, 119.12, 114.38, 70.91, 64.22, 37.78, 24.90, 21.35, 19.62, 14.70; HRES (ESI) m ⁄ z calculated for C25H30NO4 [M++ H]+ 408.2175, found 408.2177, calculated for C25H29NNaO4 [M++ Na]+ 430.1994, found 430.1991 Benzoic acid N-tert-butyl-N¢-(4-chloro-benzoyl)hydrazide (RG-100864) RG-100864 was prepared according to the previously described procedure [65,66] 1H NMR (400 MHz, dimethylsulfoxide-d6) d: 10.75 (s, 1H), 7.55–7.49 (m, 5H), 7.45 (d, J ¼ 6.8 Hz, 2H), 7.40 (dd, J ¼ 2.4, 9.6 Hz, 2H), 1.52 (s, 9H); 13C NMR (400 MHz, dimethylsulfoxide-d6) d: 171.32, 166.35, 136.98, 134.16, 132.52, 132.32, 128.92, 128.81, 127.97, 127.40, 60.91, 27.78; HRMS (ESI) m ⁄ z calculated for C18H19ClN2NaO2 [M++Na] 353.1022, found 353.1033 3,5-Dimethyl-benzoic acid N-tert-butyl-N¢(3-ethoxy-2-methyl-benzoyl)-hydrazide (RG-102398) RG-102398 was prepared according to the previously described procedure [67] 1H NMR (400 MHz, dimethylsulfoxide-d6) d: 10.48 (s, 1H), 7.14 (t, J ¼ 8.0 Hz, 1H), 7.09 (s, 2H), 7.07 (s, 1H), 7.00 (d, J ¼ 8.0 Hz, 1H), 6.28 (d, J ¼ 7.6 Hz, 1H), 3.78 (s, 3H), 3.29 (s, 6H), 1.75 (s, 3H), 1.50 (s, 9H); 13C NMR (100 MHz, dimethylsulfoxide-d6) d: 172.57, 167.72, 157.60, 138.34, 137.02, 136.11, 130.86, 126.85, 124.97, 124.07, 118.63, 112.21, 60.11, 55.75, 28.19, 21.21, 11.52; HRMS (ESI) m ⁄ z calculated for C22H29N2O3 [M+ + H] 369.2178, found 369.2169, calculated for C22H28N2NaO3 [M+ + Na] 391.1998, found 391.1985 3,5-Dimethyl-benzoic acid N-tert-butyl-N¢(3-methoxy-2-ethyl-benzoyl)-hydrazide (RG-102240) RG-102240 was prepared according to the previously described procedure [67] 1H NMR (400 MHz, dimethylsulfoxide-d6) d: 10.50 (s, 1H), 7.12 (t, J ¼ 8.0 Hz, 1H), 7.09 (s, 3H), 7.00 (d, J ¼ 8.0 Hz, 1H), 6.20 (d, J ¼ 7.6 Hz, 1H), 3.77 (s, 3H), 2.34–2.25 (m, 1H), 2.29 (s, 6H), 2.13– 2.01 (m, 1H), 1.52 (s, 9H), 0.88 (t, J ¼ 7.2 Hz, 3H); 13 C NMR (100 MHz, dimethylsulfoxide-d6) d: 172.61, 167.98, 157.30, 138.32, 137.09, 135.73, 130.78, 130.40, 126.81, 124.87, 118.64, 112.44, 60.52, 56.07, 28.17, 21.08, 19.98, 15.31; HRMS (ESI) m ⁄ z calculated for C23H31N2O3 [M++ H] 383.2335, found 383.2320, calculated for C23H30N2O3 [M+ + Na] 405.2154, found 405.2140 Ecdysone receptor gene switch ligands 2-Ethyl-3-methoxybenzoyl chloride 2-ethyl-3-methoxybenzoic acid [68] (60 g, 330 mmol) was suspended in 80 mL CHCl3 in a 500 mL round-bottom flask equipped with condensor, addition funnel, thermometer, and KOH trap Dimethylformamide (0.5 mL) was added, and the mixture was heated at 30 °C, thereby dissolving much of the acid Thionyl chloride (30 mL) was added through the addition funnel over a period of approximately 0.5 h, maintaining temperature at approximately 30 °C The reaction was heated at reflux for approximately h A Dean-Stark trap was attached, and excess CHCl3 and SOCl2 was removed by distillation The Dean-Stark trap was removed, and a short distillation head was attached Product acid chloride was distilled under vacuum (approximately mm) Two fractions of 2-ethyl-3-methoxybenzoyl chloride were collected: a forerun (6.46 g, 9.8%, b.p 93–95 °C at approximately torr) possibly containing a small amount of SOCl2, and a main fraction (56.36 g, 86%, b.p 93–95 °C at approximately torr) The product crystallized as it cooled to room temperature H NMR (400 MHz, CDCl3) d: 7.67 (d,1H), 7.29 (t, 1H), 7.08 (d, 1H), 3.87 (s, 3H), 2.88 (q, 2H), 1.15 (t, 3H) 2-Ethyl-3-methoxy-benzoic acid N¢-tert-butyl-hydrazide (2) A 500 mL, three-neck flask, equipped with magnetic stir bar and thermometer was charged with 100 mL CH2Cl2 and aqueous K2CO3 (37.4 g, 270.6 mmol in 60 mL in water) The biphasic mixture was cooled in an ice bath and solid t-butylhydrazine hydrochloride (26.87 g, 215.6 mmol) was added in portions The mixture was cooled to )3 °C, and vigorously stirred as 2-ethyl-3-methoxybenzoyl chloride (prepared from 2-ethyl-3-methoxybenzoic acid, 24.4 g, 135.4 mmol, after removal of thionyl chloride and without further purification) was added dropwise as a solution in 50 mL CH2Cl2, while maintaining the temperature below )2 °C After the addition was completed, stirring was continued at )2 °C to )5 °C for another h, and then at room temperature overnight Water was added to dissolve salts, the mixture was shaken in a separatory funnel, and the aqueous phase was removed The remaining CH2Cl2 phase was washed twice with water and then shaken vigorously with 100 mL 10% NaOH This aqueous NaOH phase was chilled on ice, acidified with 10% HCl, and extracted three times with ethyl acetate The ethyl acetate solution was then dried over MgSO4, and solvent was evaporated to yield 2-ethyl3-methoxy-benzoic acid N¢-tert-butyl-hydrazide as a slightly orange solid (10.9 g, 43.5 mmol, 32.1% yield), quite pure as analyzed by TLC (Rf ¼ 0.28, : CH2Cl2: EtOAc) The original CH2Cl2 phase, now dark in color, was dried over MgSO4, filtered and solvent was removed FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5683 Ecdysone receptor gene switch ligands S K Panguluri et al in vacuum to yield 26 g of crude product This material was chromatographed on silica gel using : CH2Cl2: EtOAc as eluant, to provide an additional 18.5 g (73.9 mmol, 54.7% yield) purified product Mp ¼ 90– 91 °C 1H NMR (200 MHz, CDCl3) d: 7.20 (t, 1H), 7.0 (br s, 1H), 6.92 (d, 2H), 4.95 (br, 1H), 3.85 (s, 3H), 2.75 (q, 2H), 1.12 (t, 3H), 1.2 (s, 9H); 13C NMR (100 MHz, CDCl3) d: 169.4, 157.8, 135.1, 131.6, 126.8, 119.1, 112.3, 56.2, 55.6, 26.9, 20.7, 14.9 A secondary fraction, 4.5 g of less pure product was obtained Methyl 3,5-dimethylbenzoate 3,5-dimethylbenzoyl chloride (268 g, 1.59 mol) was dissolved in 400 mL methylene chloride in a L round bottom flask The mixture was cooled in an ice ⁄ water bath to °C Methanol (1.91 mol, 61 g, 1.2 equivalents) was added with stirring A mixture of 241 g (2.38 mol) triethylamine in 100 mL methylene chloride was then carefully added via dropping funnel over a period of approximately 45 The mixture was stirred at °C for approximately 30 min, then permitted to warm to room temperature and stirred for h The mixture was then poured into a separatory funnel and shaken once with 10% HCl, once with water, once with aqueous NaHCO3, and finally with brine The organic phase was dried over Na2SO4, and solvent was removed in vacuo., to provide a light yellow solid (225 g, 97% yield) Methyl 3-bromomethyl-5-methylbenzoate Methyl 3,5-dimethylbenzoate (255 g, 1.55 mol) was dissolved in 300 mL CCl4 in a L round bottom flask with mechanical stirring and heated to reflux N-bromosuccinimide (212 g, 1.19 mol) and 2,2¢-azobis(2-methylpropionitrile) (AIBN, g) were added portionwise over a period of approximately h The reaction was monitored by GC and stopped when the quantity of dibromide approximately equaled unreacted starting material The heterogeneous mixture was filtered through a bed of silica gel and solvent was removed in vacuo The crude product was distilled under vacuum (b.p 130 °C, approximately mmHg) and recrystallized from pentane to yield 65 g of white solid (23% yield, 97–99% pure) 1H NMR (200 MHz, CDCl3) d: 7.90 (s, 1H), 7.70 (s, 1H), 7.4 (s, 1H), 4.5 (s, 2H), 3.92 (s, 3H), 2.4 (s, 3H) Significantly larger quantities of slightly less pure material were also obtained Higher boiling fractions contained methyl 3,5-bromomethylbenzoate which was also recrystallized from pentane: (25 g, 6.5% yield, 99% pure) 3-Bromomethyl-5-methylbenzoic acid Methyl 3-bromomethyl-5-methylbenzoate (38 g, 160 mmol), 48% HBr (300 g), and 100 mL water were mixed in a 5684 500 mL round bottom flask and stirred at 105 °C for h, then at room temperature overnight The mixture was poured onto ice, and the precipitate was collected on a sintered funnel, washed with water and pentane, and air-dried to yield 31 g (85% yield) 3-bromomethyl-5-methylbenzoic acid 1H NMR (200 MHz, CDCl3) d: 7.97 (s, 1H), 7.90 (s, 1H), 7.47 (s, 1H), 4.5 (s, 2H), 2.45 (s, 3H) 3-Bromomethyl-5-methylbenzoyl chloride (4) 3-bromomethyl-5-methylbenzoic acid (31 g 140 mmol), oxalyl chloride (86 g, 680 mmol), and CHCl3 (10 mL) were mixed in a 300 mL round bottom flasked and stirred at 50 °C for h, then at room temperature overnight As the reaction was incomplete, two drops of dimethylformamide were added and the mixture heated at 55 °C for h Excess oxalyl chloride and CHCl3 were removed in vacuo, CCl4 was added, and volatiles were removed again The crude product was recrystallized from hexane, then washed with pentane to yield 10.5 g of pale yellow solid, m.p 45–46 °C H NMR (200 MHz, CDCl3) d: 7.95 (s, 1H); 7.90 (s, 1H); 7.55 (s, 1H); 4.50 (s, 2H); 2.45 (s, 3H) An additional approximately 20 g of product was recovered by repeated recrystallizations N-(3-bromomethyl-5-methyl-benzoyl)-N-tert-butyl-N¢(2-ethyl-3-methoxy-benzoyl)-hydrazide (5) The literature procedure was followed [19] Into a 100 mL round bottom flask containing 2.50 g (10 mmol) of 2-ethyl3-methoxy-benzoyl-N¢-tert-butyl-hydrazide was added 15 mL of methylene chloride, 2.60 g (10.5 mmol) of 3-bromomethyl-5-methylbenzoyl chloride in mL of methylene chloride and a solution of 2.76 g (20 mmol) of potassium carbonate in 15 mL of water The reaction mixture was stirred overnight at room temperature, then diluted with 20 mL of methylene chloride and transferred to a separatory funnel The methylene chloride layer was separated and dried, and the solvent was removed in vacuo The crude product was purified by column chromatography to yield 4.01 g of N-(3-bromomethyl-5-methyl-benzoyl)-N-tertbutyl-N¢-(2-ethyl-3-methoxy-benzoyl)-hydrazide (87% yield) 1H NMR (300 MHz, CDCL3) d: 7.41 (s, 1H), 7.1 (m, 3H), 7.02 (t, 1H), 6.082 (d, 1H), 6.08 (d, 1H), 4.41 (s, 2H), 3.78 (s, 3H), 2.4 (m, 1H), 2.31 (s, 3H), 2.25 (m, 1H), 1.60 (s, 9H), 1.01 (t, 3H) N-(3-hydroxymethyl-5-methyl-benzoyl)-N-tert-butylN¢-(2-ethyl-3-methoxy-benzyl)-hydrazide (6) The literature procedure was followed [19] To 4.00 g (8.68 mmol) of N-(3-bromomethyl-5-methyl-benzoyl)-Ntert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)-hydrazide, contained in a 250 mL round bottom flask, were added 40 mL FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS S K Panguluri et al of dioxane, 40 mL of water, and 4.34 g of calcium carbonate The reaction flask was placed into an 85 °C oil bath and the reaction was stirred and heated for 18 h The reaction mixture was cooled, transferred to a larger flask with ethyl acetate and most of the dioxane was evaporated The reaction mixture was shaken with about 100 mL of ethyl acetate and filtered The ethyl acetate layer was separated and the aqueous layer extracted twice with ethyl acetate Ethyl acetate extract was dried and evaporated to yield 2.07 g of N-(3-hydroxymethyl-5-methyl-benzoyl)-N-tertbutyl-N¢-(2-ethyl-3-methoxy-benzyl)-hydrazide (60% yield) H NMR (300 MHz, CDCl3) d: 7.78 (s, 1H), 7.1–7.4 (3 s, 3H), 6.96 (t, 1H), 6.8 (d, 1H), 6.08 (d, 1H), 4.53 (s, 2H), 3.77 (s, 3H), 2.35 (m, 1H), 2.32 (s, 3H), 2.2 (m, 1H), 1.60 (s, 9H), 0.96 (t, 3H) 3-Formyl-5-methyl-benzoic acid N-tert-butyl-N¢(2-ethyl-3-methoxy-benzoyl)-hydrazide (7) The literature procedure was followed [19] To 2.00 g (5.02 mmol) of N-(3-hydroxymethyl-5-methyl-benzoyl)-Ntert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)-hydrazide placed in a 250 mL round bottom flask, were added 100 mL of methylene chloride and 1.16 g of pyridinium chlorochromate (PCC) The reaction mixture was refluxed for approximately h, at which time TLC (1 : ethyl acetate: hexane) indicated the formation of product (Rf ¼ 0.5) The reaction mixture was concentrated to about 20 mL and then chromatographed on silica gel Elution with 30–35% ethyl acetate in hexane yielded 1.75 g (88%) 3-formyl-5-methylbenzoic acid N-tert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)hydrazide as a white solid 1H NMR (300 MHz, CDCl3) d: 9.93 (s, 1H), 7.6–7.8 (3 s, 3H), 7.0 (t, 1H), 6.82 (d, 1H), 6.19 (d, 1H), 3.77 (s, 3H), 2.42 (s, 3H), 2.3 (m, 1H), 2.0 (m, 1H), 1.62 (s, 9H), 0.90 (t, 3H) 3-[N-tert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)hydrazinocarbonyl]-5-methyl-benzoic acid (8) In a 200 mL round-bottom flask, 3-formyl-5-methylbenzoic acid N-tert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)hydrazide 3.74 g (9.43 mmol) was dissolved in 25 mL warm t-BuOH Phosphate buffer (25 mL, pH 7.2, Aldrich 31925.2) was added, and the mixture was stirred while 1.65 g (10.4 mmol) KMnO4 was added The reaction was then stirred at 45–50 °C for h The reaction mixture was poured into 300 mL cold water Up to approximately 50 mL of 0.5% NaOH was used to wash glassware The aqueous phases were combined and filtered through Whatman #3 filter paper to remove most of the MnO2 sludge The brown aqueous filtrate was extracted thrice with 150 mL CH2Cl2 The organic extracts were dried over MgSO4, treated with charcoal, and evaporated to provide 2.81 g (72.2% yield) 3-[N-tert-butyl-N¢-(2-ethyl-3-methoxy- Ecdysone receptor gene switch ligands benzoyl)-hydrazinocarbonyl]-5-methyl-benzoic acid as a cream-colored solid Since the product did not seem to be very soluble in CH2Cl2, the original aqueous solution was again extracted with 250 mL ethyl acetate This was evaporated to yield an additional 0.14 g (3.6% yield) of product H NMR (300 MHz, dimethylsulfoxide) d: 7.8 (br s, 2H), 7.5 (s, 1H), 7.15 (t, 1H), 7.0 (d, 1H), 6.22 (d, 1H), 4.2 (br, 1H), 3.74 (s, 3H), 2.42 (s, 3H), 2.2 (m, 1H), 1.87 (m, 1H), 1.51 (s, 9H), 0.8 (t, 3H) 3-[N-tert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)hydrazinocarbonyl]-5-methyl-benzoic acid pentafluorophenyl ester (9) Pentafluorophenol (0.74 g, mmol as a 50% solution in ethyl acetate) was added to a solution of 3-[N-tert-butylN¢-(2-ethyl-3-methoxy-benzoyl)-hydrazinocarbonyl]-5-methylbenzoic acid (1.5 g, 36.4 mmol) in 30 mL ethyl acetate N¢,N¢-Dicyclohexylcarbodiimide (3.7 mL of a m solution in CH2Cl2, 3.7 mmol) was added and the mixture was stirred at room temperature for days The reaction mixture was partitioned between water and ethyl acetate The organic phase was separated, dried over MgSO4, and filtered Solvent was removed in vacuo to give 3.0 g of a thick oily solid This material was purified by silica gel chromatography using a gradient of 0–40% ethyl acetate in hexanes to provide 1.76 g (84% yield) 3-[N-tert-butyl-N¢-(2ethyl-3-methoxy-benzoyl)-hydrazinocarbonyl]-5-methyl-benzoic acid pentafluorophenyl ester 1H NMR (400 MHz, CDCl3) d: 8.2 (s, 1H), 8.1 (s, 1H), 7.75 (s, 1H), 7.6 (br s, 1H), 7.1 (t, 1H), 6.9 (d, 1H), 6.3 (d, 1H), 3.8 (s, 3H), 2.5 (s, 3H), 2.4 (m, 1H), 2.15 (m, 1H), 1.62 (s, 9H), 0.95 (t, 3H) 3-[N-tert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)hydrazinocarbonyl]-5-methyl-benzamide (RG-102277) 3-[N-tert-butyl-N¢-(2-ethyl-3-methoxy-benzoyl)-hydrazinocarbonyl]-5-methyl-benzoic acid pentafluorophenyl ester (150 mg, 0.259 mmol) was stirred in a solution of mL concentrated NH4OH and mL dioxane at 60 °C for h The mixture was partitioned with water, and the organic layer was concentrated The residue was purified by silica gel column chromatography using 100% ethyl acetate with 0.1–2% of triethylamine to provide 3-[N-tert-butyl-N¢-(2ethyl-3-methoxy-benzoyl)-hydrazinocarbonyl]-5-methyl-benzamide (46 mg, 43% yield) TLC Rf ¼ 0.06, : ethyl acetate:hexane + triethylamine) 1H NMR (400 MHz, CDCl3) d: 7.9 (s,1H), 7.75 (s 1H), 7.58 (s, 1H), 7.55 (s, 1H), 7.0 (t, 1H), 6.85 (d, 1H), 6.3 (br, 1H), 6.2 (d, 1H), 5.6 (br, 1H), 3.77 (s, 3H), 2.4 (s, 3H), 2.38 (m, 1H), 2.1 (m, 1H), 1.60 (s, 9H), 0.95 (t, 3H) 1H NMR (400 MHz, dimethylsulfoxided6) d: 10.58 (s, 1H), 7.96 (s, 1H), 7.78 (s, 2H), 7.44 (s, 1H), 7.37 (s, 1H), 7.12 (t, J ¼ 8.0 Hz, 1H), 7.01 (d, J ¼ 8.0 Hz, 1H), 6.23 (d, J ¼ 7.2 Hz, 1H), 3.77 (s, 3H), 2.39 (s, 3H), FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS 5685 Ecdysone receptor gene switch ligands S K Panguluri et al 2.24 (dt, J ¼ 7.2, 14.4 Hz, 1H), 1.89 (dt, J ¼ 7.2, 12.8 Hz, 1H), 1.55 (s, 9H), 0.84 (t, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz, dimethylsulfoxide-d6) d: 171.57, 167.20, 157.69, 138.34, 137.46, 135.71, 134.04, 130.49, 129.35, 127.07, 123.87, 118.71, 112.40, 61.01, 56.08, 27.81, 21.16, 20.12, 14.99; HRMS (ESI) m ⁄ z calculated for C23H30N3O4 [M+ + H] 412.2236, found 412.2229, calculated for C23H29N3NaO4 [M+ + Na] 434.2056, found 434.2026 Cell lines, reagents, and ligand treatment The human embryonic kidney cell cultures (HEK 293) were propagated in DMEM supplemented with 10% fetal bovine serum All media and serum were purchased from Life Technologies (Rockville, MD, USA) The cells were cultured to 20% confluency in 25 cm2 flasks and ligands (1 or 10 lm) or dimethylsulfoxide control were added to each flask The cells were grown for three days in a CO2 incubator at 37 °C and cell morphology was observed daily under a light microscope Isolation of total RNA The cells were harvested at days after the addition of ligand (10 lm for all ligands except RG-120499 which was added at lm) The cells were collected by scraping with a rubber policeman and centrifuged at 2300 g for The medium was discarded and 500 lL of Triazol reagent (Molecular Research Center Inc., Cincinnati, OH, USA) was added to the cell pellet The cells were homogenized, 100 lL of chloroform was added, and the mixture was gently agitated while keeping at room temperature for min, and then centrifuged at 15 800 g for 15 at °C The supernatant was transferred into a fresh tube, 500 lL isopropanol was added, and the mixture incubated at room temperature for The mixture was then centrifuged at 15 800 g for 15 at °C The supernatant was discarded slowly and the total RNA pellet was washed with 70% ethanol, dried and dissolved in nuclease-free water The RNA was quantified both visually (agarose gel electrophoresis) and UV-spectrophotometer cDNA synthesis by reverse transcription was perfoemd using lg of DNAse-1 (Ambion Inc., Austin, TX, USA)-treated RNA and iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) template in the subsequent in vitro transcription reactions The in vitro transcription reaction was carried out in the presence of T7 RNA polymerase and a biotinylated nucleotide analog ⁄ ribonucleotide mix for complementary RNA (cRNA) amplification and biotin labeling The biotinylated cRNA targets were then purified, fragmented, and hybridized to GeneChip expression arrays Human U133 plus 2.0 (Affymetrix Inc., Santa Clara, CA, USA) Array images were processed to determine signals and detection calls (P, present; A, absent; M, marginal) for each probe set using a GeneChip Operating Software (GCOS) Computer Workstation (Affymetrix Inc.) Microarray analyses used three chips per each treatment Data analysis Data analysis was performed using SAS (SAS Institute, Cary, NC, USA), r for Windows GUI front-end (http:// www.r-project.org), and q-value software [69] The probe sets with absent calls across all samples were removed to reduce the multiple-testing problem The expression levels were normalized to the chip median and log transformed Two-way analysis of variance tests are carried out to identify differentially expressed genes For each probe set, the model yijk ¼ l + + bj + cij + eijk was fit, where yijk is the log-transformed expression level of the kth chip in the ith treatment and the jth replicate The variable l represents the grand mean expression, is the effect due to the treatment, bj is the effect due to the replicate, cij is the interaction effect between treatment and replicate, and eijk is an error term, which is assumed to be normally distributed with mean and variance r2 Q-values computed using Q-value software indicates the false detection rate for each probe set Ratio comparison was performed by dividing expression levels of expression in ligand treated cells with the expression levels in dimethylsulfoxide-treated cells The gene sets that showed two-fold induction or two-fold suppression were transferred to separate up and down lists, respectively The genes sets with a P £ 0.01 were identified in these lists and gene annotation was performed for these probe sets at Affymetrix NetAffx analysis center (http://www.affymetrix.com/analysis/index.affx) Functional classification of select probe sets was performed at the NIH DAVID server (http://apps1.niaid.nih.gov/david/ upload.asp) Volcano plots were prepared using the r program Microarray analysis The microarray analysis was performed at the UK microarray core facility (University of Kentucky, Lexington, KY, USA) The total RNA was converted into double-stranded cDNA using a T7-oligo (dT) promoter primer Following RNase H-mediated second-strand cDNA synthesis, the double-stranded cDNA was purified and served as a 5686 qRT-PCR analysis The genes that showed signal detection values more than 100, P £ 0.01 and a change in gene expression of more than two-fold compared to the control (dimethylsulfoxide) were selected to study their relative expressions by qRT-PCR using a MyiQ single color real-time PCR detection system FEBS Journal 274 (2007) 5669–5689 ª 2007 The Authors Journal compilation ª 2007 FEBS S K Panguluri et al (Bio-Rad Laboratories) qRT-PCR was performed in a total of 20 lL reaction volume containing lL of cDNA, lL of each of forward and reverse sequence specific primers (from 10 lm primer stock), 10 lL of supermix (Bio-Rad Laboratories) and lL of nuclease free water All the qRT-PCR reactions were performed using the following reaction conditions: Initial incubation of 95 °C for followed by 45 cycles of 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s A fluorescence reading determined the extent of 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RG-121150, affects gene expression in 293 cells The affect of the AMK RG-121150 on gene expression in 293 cells was analyzed using microarray and qRTPCR In microarray analysis, a total of 636 genes were... water-soluble variant of RSL-1, namely RG-102277 First, we determined the effect of dimethylsulfoxide itself on the expression of genes in 293 cells The expressions of a total of 43 genes were modulated