Independent Actions on Cyclin-Dependent Kinases and Aryl Hydrocarbon Receptor Mediate the Antiproliferative Effects of Indirubins

Independent actions on cyclin-dependent kinases and aryl hydrocarbon receptor mediate the antiproliferative effects of indirubins

Marie Knockaert1, Marc Blondel1, Ste´ phane Bach1, Maryse Leost1, Cem Elbi2, Gordon L Hager2, Scott RNagy 3, Dalho Han3, Michael Denison3, Martine Ffrench4, Xiaozhou P Ryan5, Prokopios Magiatis6, Panos Polychronopoulos6, Paul Greengard5, Leandros Skaltsounis6 and Laurent Meijer*,1,5

1C.N.R.S., Cell Cycle Group & UPS-2682, Station Biologique, BP 74, 29682 ROSCOFF cedex, Bretagne, France; 2Laboratory of Receptor Biology & Gene Expression, Bldg 41, Room B602, National Cancer Institute, NIH, Bethesda, MD 20892-5055, USA; 3Department of Environmental Toxicology, Meyer Hall, One Shields Avenue, University of California, Davis, CA 95616-8588, USA; 4Universite´ Claude Bernard, Laboratoire de Cytologie Analytique et Cytoge´ne´tique Mole´culaire, 8 Avenue Rockefeller, 69373 Lyon Cedex 08, France; 5Laboratory of Molecular & Cellular Neuroscience, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA; 6Division of Pharmacognosy and Natural Products Chemistry, Department of Pharmacy, University of Athens, Panepistimiopolis Zografou, GR-15771 Athens, Greece

Indirubin, a bis-indole obtained from various natural Introduction sources, is responsible for the reported antileukemia activity of a Chinese Medicinal recipe, Danggui Longhui The bis-indole indirubin has been identified as the main Wan. However, its molecular mechanism of action is still active ingredient of a traditional Chinese medicinal not well understood. In addition to inhibition of cyclin- recipe, Danggui Longhui Wan, used to treat various dependent kinases and glycogen synthase kinase-3, diseases including chronic myelocytic leukemia (reviews indirubins have been reported to activate the aryl in Tang and Eisenbrand, 1992; Xiao et al., 2002). hydrocarbon receptor (AhR), a cotranscriptional factor. Indirubin can be obtained from four different natural Here, we confirm the interaction of AhR and indirubin sources: (1) a variety of indigo-producing plants using a series of indirubin derivatives and showthat their (Balfour-Paul, 1998; Maugard et al., 2001), (2) a number binding modes to AhR and to protein kinases are of mollusks, essentially belonging to the Muricidae unrelated. As reported for other AhR ligands, binding of family (reviewed in Cooksey, 2001), (3) urine of healthy indirubins to AhR leads to its nuclear translocation. and diseased patients (Dealler et al., 1988; Adachi et al., Furthermore, the apparent survival of AhRÀ/À and 2001 and references therein), and (4) various natural or þ / þ cells, as measured by the MTT assay, is equally recombinant bacteria (reviewed in Chotani et al., 2000; sensitive to the kinase-inhibiting indirubins. Thus, the Gillam et al., 2000; MacNeil et al., 2001). Indirubin is a cytotoxic effects of indirubins are AhR-independent and dark red isomer of the blue indigo. Both derive from the more likely to be linked to protein kinase inhibition. In spontaneous, nonenzymatic dimerization of isatin and contrast, a dramatic cytostatic effect, as measured by indoxyl, two colorless precursors found either free or actual cell counts and associated with a sharp G1 phase conjugated to carbohydrates. Interest in this family of arrest, is induced by 1-methyl-indirubins, a subfamily of compounds increased when indirubin and analogues AhR-active but kinase-inactive indirubins. As shown for (collectively referred to as indirubins) were found to TCDD (dioxin), this effect appears to be mediated inhibit the cell cycle regulating cyclin-dependent kinases through the AhR-dependent expression of p27KIP1. Alto- (CDKs) (Hoessel et al., 1999), and glycogen synthase gether these results suggest that AhR activation, rather kinase -3 (GSK-3) (Leclerc et al., 2001). Evidence than kinase inhibition, is responsible for the cytostatic suggests that the antiproliferative effects of indirubins effects of some indirubins. In contrast, kinase inhibition, derive from their ability to inhibit CDKs (Damiens et al., rather than AhR activation, represents the main mechan- 2001; Marko et al., 2001). However, the exact mechanism underlying the cytotoxic properties of this class of isms underlying the antimitotic and antitumor proper- promising antitumor molecules. ties of indirubins remain to be identified. Oncogene (2004) 23, 4400–4412. doi:10.1038/sj.onc.1207535 Indirubin and indigo have recently been discovered as Published online 12 April 2004 potent ligands of the aryl hydrocarbon receptor (AhR), also known as the dioxin receptor (Adachi et al., 2001; Keywords: aryl hydrocarbon receptor; indirubin; cyclin- Kawanishi et al., 2003). AhR, a member of the bHLH/ dependent kinase; glycogen synthase kinase; GSK-3b; PAS family of transcriptional regulators, mediates the kinase inhibitor; cancer effects of many xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and indole-containing compounds (reviewed in Hankinson, 1995; Denison and *Correspondence: L Meijer; E-mail: [email protected] Nagy, 2003). However, despite the large number of Received 5 August 2003; revised 10 November 2003; accepted 16 January compounds found to interact with AhR, its natural 2004; Published online 12 April 2004 ligands remain to be identified (Denison and Nagy, Indirubins, kinases and aryl hydrocarbon receptor M Knockaert et al 4401 2003). The binding of AhRto a ligand leads to its scription. Using the same yeast reporter assay, we tested translocation from the cytoplasm to the nucleus, a series of indirubin analogues for their ability to followed by complex formation with the aryl hydro- activate AhR-dependent transcription (Table 1). Addi- carbon receptor nuclear translocator (ARNT). This tionally, we also tested these compounds in a newly complex then binds to xenobiotic-responsive element described AhRagonist detection bioassay. In this assay, (XRE) and stimulates the transcription of a wide variety ligand and AhR-dependent transcription is measured by of genes, including cytochrome P450 Cyp1A1, p27kip1, the amount of enhanced green fluorescent protein myristoyltransferase, etc. (Rowlands and Gustafsson, (EGFP) expression which is stably transfected into a 1997; Denison et al., 1998; Kolluri et al., 1999, 2001; mouse hepatoma cell line (Nagy et al., 2002a, b) Santini et al., 2001). Several arguments support a link (Table 1, Figure 1). TCDD was used as a positive between AhRactivation and cell cycle control (Ge and control. Our results confirmed that indirubins, including Elferink, 1998; Elferink et al., 2001; reviewed in indirubin-30-oxime (IO), the analogue commonly used in Elferink, 2003). The recent discovery of the interaction cell cycle studies (Damiens et al., 2001; Marko et al., of indirubin with AhRopened the possibility that 2001), are potent AhRagonists, although much less indirubins prevent cell proliferation via an action than TCDD in the mammalian reporter assay (Table 1, through AhR. This could raise some concern about Figure 1). the potential use of these compounds since AhR We also tested the effect of indirubin analogues on activation has been directly linked to carcinogenesis two protein kinases, CDK1/cyclin B and GSK-3 (Hankinson, 1995; Shimizu et al., 2000; Safe, 2001; (Table 1). Interestingly, the indirubin structure/activity Andersson et al., 2002). In contrast, some AhRagonists relationships with respect to AhRbinding and kinase have also been reported to cause growth inhibition of inhibition were only distantly comparable (Table 1) (see various tumor cell lines and as such could be evaluated Discussion). for their use in antitumor therapy (Bradshaw et al., As revealed by cocrystal structures (Hoessel et al., 2002; Koliopanos et al., 2002; Safe and McDougal, 1999; Davies et al., 2001; Meijer et al., 2003) the 2002). indirubin N1 acts as a H-bond donor in the indirubin/ In this article, we report the results of experiments kinase binding. Consequently, methylation at this designed to investigate the contribution of indirubin/ site should clearly eliminate an essential bonding AhRinteraction to the antiproliferative properties of and inactivate indirubins as kinase inhibitors indirubins. We first confirm the interaction between (Meijer et al., 2003; Polychronopoulos et al., AhRand a series of indirubin analogues using two 2004). Based on this observation, we envisaged the distinct AhRreporter systems. We show that the synthesis of 1-methyl-indirubin, 1-methyl-indirubin-30- survival of AhR À/À and þ / þ cells, as measured by oxime (MeIO), 1-methyl-6-bromo-indirubin and 1- the MTT assay, is equally sensitive to the kinase- methyl-6-bromo-indirubin-30-oxime (MeBIO). Indeed, inhibiting indirubins and thus largely AhR-independent, these compounds were found to be the most striking therefore more likely linked to protein kinase inhibition. examples of selectivity, as they were very potent in the In contrast, the kinase-inactive, but AhR-active, 1- AhRassays while being essentially inactive on CDKs methyl-indirubins, show a striking AhR-dependent and GSK-3, compared to their nonmethylated counter- cytostatic effect leading to an arrest of cells in G1. This parts, indirubin (IO) and 6-bromo-indirubin-30-oxime effect appears to be directly linked to the AhR- (BIO), respectively. dependent expression of p27KIP1, as it is not observed These results show that indirubins are potent in AhR À/À cells. Altogether these results suggest that AhRligands in both reporter systems, and that their kinase inhibition, rather than AhRactivation, repre- binding modes to AhRand to protein kinases are sents the main mechanism underlying the cytotoxic unrelated. properties of this class of promising antitumor molecules. In contrast, AhRactivation, rather than kinase Indirubins trigger cytoplasm to nucleus translocation of inhibition, is responsible for the cytostatic effects AhR induced by some indirubins. To confirm the interaction of indirubins with AhRin living cells, we investigated their effect on AhR intracellular distribution (Figure 2). The nuclear trans- Results location of AhRafter treatment with a ligand is a well- Indirubins interact with AhR, yet less potently than known consequence of AhR/ligand interaction (Elbi TCDD et al., 2002). Wild-type Hepa-1 and ARNT-mutant cells were treated for 90 min with various indirubins, Human AhRand ARNThave been coexpressed stably TCDD or DMSO as a control. The cells were then fixed in yeast and the interaction was coupled to a b- and examined by indirect immunofluorescence micro- galactosidase reporter system allowing the detection of scopy following staining with an anti-AhRantibody AhRagonists (Miller, 1997). Using this reporter assay (Figure 2). As expected, in the absence of ligand, AhR to screen for natural AhRagonists in human urine, showed both cytoplasmic and nuclear distribution in Adachi et al. (2001) reported that both indirubin and wild-type Hepa-1 (Figure 2a–c) and in ARNT-mutant indigo were potent activators of AhR-dependent tran- cells (Figure 2g, j).

Oncogene Indirubins, kinases and aryl hydrocarbon receptor M Knockaert et al 4402 Table 1 Effects of indirubins and TCDD on AhRand two protein kinases

No. Compound AhR (yeast) AhR (hepatoma cell line) CDK1/cyclin B GSK-3

1 Indirubin 0.006 0.059 10.000 1.000 2 Indirubin-30-oxime (IO) 0.025 0.720 0.180 0.022 3 1-Methyl-indirubin 0.006 0.830 4100.000 4100.000 4 1-Methyl-indirubin-30-oxime (MeIO) 0.008 0.129 73.000 4100.000 5 4-Chloro-indirubin 40.100* 0.310 10.000 4 100.000 6 5-Methyl-indirubin 0.040 0.025 0.280 0.062 7 5-Bromo-indirubin 0.035 0.007 0.230 0.055 8 5-Chloro-indirubin 0.040 0.052 0.280 0.050 9 5-Iodo-indirubin 0.030 0.030 0.220 0.068 10 5-Iodo-indirubin-30-oxime 0.050 0.005 0.025 0.009 11 6-Bromo-indirubin 0.020 0.008 90.000 0.045 12 1-Methyl-6-bromo-indirubin X0.100 0.050 4100.000 4100.000 13 6-Bromo-indirubin-30-oxime (BIO) 0.009 0.720 0.320 0.005 14 1-Methyl-6-bromo-indirubin-30-oxime (MeBIO) 0.020 0.093 92.000 44.000 15 6-Bromo-indirubin-30-methoxime 40.100* 0.030 3.400 0.030 16 6-Bromo-indirubin-30-acetoxime X0.100 0.160 63.000 0.010 17 6-Chloro-indirubin 0.030 0.007 4100.000 0.140 18 6-Chloro-indirubin-30-oxime 0.004 0.200 0.650 0.020 19 6-Iodo-indirubin 0.040 0.017 1.600 0.055 20 6-Iodo-indirubin-30-oxime 40.100* 0.440 1.300 0.010 21 50-Bromo-indirubin X0.100 0.051 0.510 0.350 22 5,50-Dibromo-indirubin 40.200a 0.047 600.000 0.250 23 60-Bromo-indirubin 0.100 1.200 4100.000 22.000 24 6,60-Dibromo-indirubin X100 410.000 4100.000 4.500 25 TCDD 0.025 0.000015 415.000 415.000

A series of indirubin analogues and TCDD were tested at various concentrations in two AhRinteraction reporter systems and in two kinase assays, as described in the Materials and methods section. EC50 (AhR) and IC50 (kinases) values were calculated from the dose–response curves and are reported in mM. aInactive at the indicated highest concentration tested

Upon addition of IO, AhRrapidly localized to the indirubins, we used IO, MeIO, BIO and MeBIO and nucleus (Figure 2d–f). MeBIO treatment also caused two mouse hepatoma cell lines, 5L (AhR þ / þ ) and the redistribution of AhRto the nucleus, and the nuclear AhR-deficient subclone, BP8 (AhR À/À) (Weiss et al, translocation was independent of the presence of 1996; Kolluri et al.,. 1999). We first confirmed the AhR functional ARNT (Figure 2i, l). This finding was similar status in these two cell lines by Western blotting using to the nuclear translocation kinetics of AhRobserved in anti-AhRantibody (Figure 3). In contrast to BP8 cells, TCDD-treated wild-type Hepa-1 cells and ARNT AhRexpression was detected in 5L cells. mutant cells (Figure 2h, k). All other indirubins tested, We next investigated the effects of IO and MeIO such as BIO, MeBIO and 5-iodo-indirubin-30-oxime, (20 mM) on the proliferation rate of 5L and BP8 cell also triggered ARNT-independent nuclear redistribu- lines, by counting directly the number of live cells after tion of AhR(data not shown). These results demon- 24 and 48 h of exposure to the indirubins (Figure 4). strate that indirubins are potent and functional ligands This approach clearly showed that IO blocks the for AhRin living cells and that ARNTis not necessary proliferation of both cell types, independent of AhR for indirubin-induced nuclear translocation of AhR. expression. In contrast, MeIO, a potent AhRligand, but kinase-inactive indirubin, reduced the rate of prolifera- Cell cycle inhibitory properties of indirubins are AhR- tion only in 5L cells (Figure 4a), and did not effect the proliferation of AhR / cell line BP8 that was, in fact, dependent, but kinase inhibition-independent À À slightly stimulated (Figure 4b). This result indicates that To investigate the relative contributions of AhR MeIO has an AhR-dependent and kinase-independent signaling and kinase inhibition to the cellular effects of antiproliferative effect in mouse hepatoma cells.

Oncogene Indirubins, kinases and aryl hydrocarbon receptor M Knockaert et al 4403

Figure 1 Indirubins are potent AhRagonists. Increasing concentrations of indirubins and TCDD were tested in a hepatoma cell line reporter system, expressing EGFP under the control of a dioxin-responsive element that binds to ligand–AhR–ARNT complex. EGFP activity is reported as a percentage of maximal activity obtained with 1 nM TCDD

We next investigated the effects of indirubins on cell was apparently not affected by MeIO and MeBIO, cycle distribution of AhR þ / þ and À/À cells by FACS the kinase inactive, yet AhR-active, indirubins analysis. Both 5L and BP8 cell lines were treated with (Figure 6b, d). The possibility remains that MeIO 10 mM IO, MeIO, BIO, MeBIO or 100 nM TCDD for and MeBIO block cell cycle without affecting the 24 h. The AhR-active indirubins MeIO (Figure 5a, b) mitosis-associated increase in mitochondria number, and MeBIO (Figure 5b) induce a very striking accumu- on which the MTT assay is essentially based. These lation of AhR þ / þ cells (5L) but not of AhR À/À cells results indicate that IO and BIO have a kinase- (BP8) in the G1 phase (Figure 5b). Comparison of the dependent but AhR-independent cytotoxic effect in effects of different indirubins showed that this G1 mouse hepatoma cells. accumulation is modest (but detectable) with IO and BIO, but highly pronounced with the more potent AhR- Indirubins stimulate p27KIP1 expression in an AhR- active indirubins MeIO and MeBIO (Figure 5a, b). dependent manner TCDD also triggered an AhR-dependent arrest in G1 (Figure 5b), as previously reported (Elferink, 2003). Activation of AhRleads to the enhanced expression of a Altogether these results show that AhR-active indir- variety of genes (review in Elferink, 2003), including the ubins arrest cells in G1 in an AhR-dependent, but cytochrome P450 CYP1A1 (Santini et al., 2001) and the kinase-independent manner. CDK inhibitor p27KIP1 (Kolluri et al., 1999). We first confirmed by Western blotting that TCDD upregulates KIP1 Cytotoxic properties of indirubins are AhR-independent, the expression of both CYP1A1 and p27 in the 5L but kinase inhibition-dependent but not in the BP8 cells (Figure 7; data not shown). Furthermore dose-dependent upregulation of p27KIP1 To investigate the effects of indirubins on cell survival, was also found in 5L cells, but not BP8 cells, exposed 5L and BP8 cells were exposed to a range of concentra- to the AhR-active MeIO (Figure 7a). After testing tions of each indirubin for 24 and 48 h and the global various indirubins, and comparing them to TCDD, we survival rate was estimated using the MTT assay, an found that the level of p27KIP1 induction correlated assay of cell viability based on a mitochondrial enzyme significantly with the potency of each indirubin to assay (Figure 6). Results are expressed as a percentage interact with AhRrather than its ability to inhibit of control, untreated cells. Two main conclusions stem cyclin-dependent kinases (Figure 7b). Both MeIO and from these experiments. First, the survival of both AhR MeBIO were very effective p27KIP1 inducers, while IO À/À and AhR þ / þ cells was equally sensitive to IO and BIO induced a modest, but detectable increase in and BIO, as shown by dose–response curves obtained p27KIP1 level. These results show that indirubins activate after 24 and 48 h (data not shown) exposure to the drugs p27KIP1 expression in an AhR-dependent, but kinase- (Figure 6a, b). Second, the survival of both cell types independent manner.

Oncogene Indirubins, kinases and aryl hydrocarbon receptor M Knockaert et al 4404

Figure 2 Indirubins induce nuclear translocation of AhR. Upper panel: Wild-type Hepa-1 cells were treated for 90 min with either DMSO as a control (a–c)or25mM IO (d–f). The cells were ﬁxed and AhRdistribution was detected by indirect immunoﬂuorescence microscopy using anti-AhRantibody ( a, c and d, f). Total DNA was stained with DAPI (b, c and e, f). Lower panel: Wild-type Hepa-1 (g–i) and ARNT mutant (j–l) cells were treated for 90 min with either DMSO as a control (g, j)or10nM TCDD (h, k)or25mM MeBIO (i, l). Intracellular distribution of AhRwas analysed as described above. Scale bar, 10 mm

Discussion AhR þ / þ , but not in AhR À/À cells. Our results demonstrate that indirubins clearly act as functional Indirubins are functional AhR ligands AhRligands in mammalian cells. Using the yeast reporter assay, we found that some Using a yeast reporter assay for AhRsignaling, Adachi indirubins are more potent AhRactivators than TCDD et al. (2001) reported the identification of indirubin as a (Table 1). However, the yeast reporter assay was clearly potent AhRligand, confirming an earlier observation by less sensitive to TCDD than the mammalian reporter Rannug et al. (1992). In this study, we present several assay. In the mammalian reporter assay, indirubins lines of evidence, which clearly support and extend appear to be much less potent AhRactivators than previous findings: (1) determination of the binding TCDD. In fact, only a limited correlation was observed characteristics of indirubin analogues to AhRby use between the EC50 values obtained using the two reporter of a mammalian reporter system based on AhR- systems with 24 indirubins (Table 1). There are several dependent stable expression of EGFP in mouse hepa- reasons that could explain why the AhRreporter assays toma cell line; (2) visualization of the effect of indirubins are only partially quantitative and poorly comparable: on the intracellular localization of AhR; (3) the effects (1) other indirubin-interacting proteins, such as kinases, of indirubins on p27KIP1 expression and G1 arrest in are competing with AhRfor indirubin binding; (2)

Oncogene Indirubins, kinases and aryl hydrocarbon receptor M Knockaert et al 4405 is complex as the intensity of maximal AhRactivation varies from compound to compound, some indirubins even being inhibitory at higher concentrations (data not shown), and furthermore, as some ligands may induce their own degradation through AhR-dependent induction of cytochrome P450 (Spink et al., 2003). Structure/activity relationship analysis carried out with a large number of synthetic ligands suggests that Figure 3 Characterization of AhR À/À (BP8) and AhR þ / þ the AhRligand-binding pocket can accommodate (5L) cell lines. The presence and absence of AhRexpression in 5L planar ligands with maximal dimensions of and BP8 cells was confirmed by Western blotting using anti-AhR ˚ ˚ ˚ antibody. Equal amounts of protein were loaded in each lane 14 A Â 12 A Â 5A (for a review, see Denison and Nagy, 2003). AhR-interacting indirubins, such as MeBIO (12.6 A˚ Â 7.9 A˚ Â 1.8 A˚ ), clearly meet these requirements. In the absence of an available AhR/ligand cocrystal structure, we can only speculate on the atomic basis of AhR/indirubin interactions. Ligand binding has been mapped to the second PAS domain of the AhR protein (Fukunaga et al., 1995). A model for recognition of TCDD by AhR, based on the crystal structure of the conserved PAS domain of the heme-binding domain of the bacterial O2 sensing FixL protein, has been proposed (Procopio et al., 2002). Another 3D model of AhRhas been published recently (Jacobs et al., 2003). According to these models, AhRshould be able to accommodate the flat hydrophobic indirubins in a way similar to TCDD. According to these models also, indirubins meet the requirements for AhRbinding, that is, a nucleophilic site in the central part of the ligand and electrophilic sites at the sides of the principal molecular axis. Interestingly, there is little correlation between the potency of indirubins as AhRactivators and kinase inhibitors. Although methylation at N1 provides kinase- inactive indirubins but potent AhRagonists, we were unable to find kinase-inhibiting indirubins that were inactive on AhR. Most ATP-competing kinase inhibitors are planar, hydrophobic, small molecular weight compounds, like AhRligands. This raises the intriguing possibility that, besides indirubins, AhRmay actually bind other kinase inhibitors. This is supported by the very recent observation showing that the Jun N-terminal kinase inhibitor SP600125 is a ligand of AhR(Joiakim et al., 2003).

Indirubins as mimetics of natural AhR ligands AhRwas discovered as the receptor for dioxin, Figure 4 Effects of indirubins on the proliferation of AhR À/À (BP8) and AhR þ / þ (5L) cells. 5L (a) and BP8 (b) cells were benzo[a]pyrene and other related, man-made pollutants. treated with 20 mM IO, 20 mM MeIO or a corresponding amount of However, several arguments suggest that AhRis the the carrier DMSO (untreated) for indicated times. Cell prolifera- receptor for endogenous ligands (reviewed in Denison tion was estimated by direct counting and the graph shows a and Nagy, 2003): (1) a phylogenetic survey indicates that representative of three independent experiments with each data point done in triplicates (average and s.e.) the AhRarose over 450 million years ago, that is, in a variety of organisms evolving in different chemical environments, long before environmental pollution by conversely, unidentiﬁed endogenous AhRligands may anthropogenic compounds started (Hahn, 2002); (2) be competing to a variable extent with indirubins in the AhRis expressed in various tissues and at different reporter systems; (3) the solubility of and the perme- developmental time points, which is inconsistent with a ability of cells to these hydrophobic compounds and unique role in defence against environmental chemicals their intracellular distribution is not known, but these (Jain et al., 1998; Kuchenhoff et al., 1999); (3) the loss of factors are very likely to inﬂuence the fraction of function studies that were carried out in mice by indirubins which interacts with AhRand is thus detected different laboratories suggest that AhRplays a role in in the assays; (4) the response of these reporter systems development, in the absence of exogenous ligand

Oncogene Indirubins, kinases and aryl hydrocarbon receptor M Knockaert et al 4406

Figure 5 AhR-active, but kinase-inactive indirubins induce an AhR-dependent arrest in G1 phase. (a) 5L and BP8 cells were cultured in the absence (control) or presence of 10 mM MeIO for 24 h, and the G1, S and G2/M cycle phase distribution was determined by FACS analysis as described in the Materials and methods. (b). The cell cycle phase distribution of 5L and BP8 cells was quantiﬁed following exposure to 0.1 mM TCDD or 10 mM IO, BIO, MeIO or MeBIO for 24 h

(Fernandez-Salguero et al., 1995; Schmidt et al., 1996; porcine lung (Song et al., 2002) and (3) the tryptophan Lahvis et al., 2000); (4) disruption of AhRexpression photoproduct FICZ (6-formylindolo[3,2-b]carbazole) with antisense oligonucleotides or siRNA results in (Wei et al., 2000; Bergander et al., 2003). However, decreased mouse blastocyst development (Peters and none of these indoles is an unequivocal endogenous Wiley, 1995) and abnormalities in cell cycle regulation AhRligand. For example, it cannot be excluded (Ma and Whitlock, 1996; Weiss et al., 1996, Abdelrahim that ITE is a microbial contaminant. Similarly, indir- et al, 2003, for a review, see Elferink, 2003). ubins present in mammals clearly derive from gut Indoles (Miller, 1997; Heath-Pagliuso et al., 1998), microflora. Nevertheless, indirubins may represent tetrapyroles, and arachidonic acid metabolites have mimetics of endogenous indoles acting selectively on been described as natural AhRligands (reviewed in AhRfor specific physiological functions. One hypoth- Denison and Nagy, 2003). Many natural AhRligands esis that deserves further exploration is that, under actually derive from dietary indoles (tryptophan, indole- specific circumstances, cells produce endogenous 3-carbinol) or flavonoids. Among indoles found in AhR-regulating bis-indoles that are chemically related mammals are: (1) indirubin, purified from human urine to indirubins. The demonstration of indirubin and from fetal calf serum (Adachi et al., 2001), (2) the synthesis from indoles by human cytochrome P450 recently described ITE (2-(10H-indole-30-carbonyl)-thia- enzymes is particularly relevant in that respect (Gillam zole-4-carboxylic acid methyl ester), isolated from et al., 2000).

Oncogene Indirubins, kinases and aryl hydrocarbon receptor M Knockaert et al 4407

Figure 6 Effects of indirubins on the survival of AhR À/À (BP8) and AhR þ / þ (5L) cells. 5L and BP8 cells were maintained in the presence of increasing concentrations of IO, BIO, MeIO and MeBIO for 48 h. The cell survival was determined by the MTT assay as described in the Materials and Methods section and is presented as a percent of control, nontreated cells. The graphs show a representative of three independent experiments with each data point conducted in triplicates (average and s.e.)

what is observed depends on: (1) whether cells are presynchronized or not, and (2) the cell type, probably reflecting the specific proportion and distribution of each individual enzymatic target, which comprise protein kinases such as CDK1/cyclin B, CDK2/cyclin A–E, GSK-3, and other kinase targets (Leclerc et al., 2001; Bain et al., 2003). Indirubins appear to bind in a manner similar to CDK2 (Hoessel et al., 1999; Davies et al., 2001), CDK5 and GSK-3 (Meijer et al., 2003; Polychronopoulos et al., 2004) and probably to other kinases as well, given the conserved structure of the ATP-binding pocket. Among the essential indirubin/ kinase bonds, the lactam amide nitrogen of indirubins (N1) donates a hydrogen bond to the backbone oxygen of Glu81 (CDK2), Glu81 (CDK5) or Asp 133 (GSK-3), three amino acids that occupy a homologous position in Figure 7 AhR-active, but kinase-inactive indirubins induce an these kinases. Methylation of indirubins on N1 prohibits KIP1 AhR-dependent upregulation of p27 .(a) 5L and BP8 cells were this interaction and inactivates the inhibitory properties treated with increasing concentrations of MeIO or 0.1 mM TCDD for 24 h. The expression level of p27KIP1 was determined by Western of indirubins towards these and probably other kinases blotting using a specific antibody. (b) Determination of p27KIP1 (Meijer et al., 2003; Polychronopoulos et al., 2004). levels in 5L and BP8 cells by Western blotting following exposure Compared to the nonmethylated indirubins, N1-methy- to 0.1 mM TCDD or 10 mM IO, BIO, MeIO or MeBIO for 24 h lated indirubins have little effect on cell survival as measured by the MTT assay, whether cells express AhR (5L) or not (BP8) (Figure 6). Importantly, these N1- Biochemical and cellular effects of indirubins methylated indirubins are potent AhRagonists. As such they affect cell proliferation by arresting cells in G1 in a Indirubins affect cell proliferation differently according strictly AhR-dependent manner (Figure 5). These results to their concentration, to cell types, and to experimental suggest that indirubins such as IO and BIO reduce cell conditions. Both a G1/S and a G2/M arrest have been survival by a mechanism essentially independent from observed (Hoessel et al., 1999; Damiens et al., 2001; AhR(since similar rates of survival were observed in Marko et al., 2001). Therefore, IO (and BIO) have the both 5L and BP8 cells), a mechanism likely to be a direct capacity to arrest cells both in G1 and in G2/M, and or indirect consequence of kinase inhibition. However,

Oncogene Indirubins, kinases and aryl hydrocarbon receptor M Knockaert et al 4408 both IO and BIO have a modest, but not insignificant N1-methylated indirubins thus provide very interest- effect on AhRas evidenced by the nuclear translocation ing tools to challenge the functions of AhRin cell of AhRthey trigger and the limited (but clearly AhR- cycle regulation. These compounds are potent inducers dependent) p27KIP1 induction and G1 arrest they induce. of p27KIP1, and this provides an explanation for Nevertheless, this limited AhRagonist effect does not the observed G1 arrest. However, we cannot exclude contribute to the antiproliferative effects of these the possibility that the cellular effects derive from the indirubins based on the experimental conditions tested expression of other target genes. Differences in the in this study. mechanism of action of methylated and nonmethylated The cellular response to a particular indirubin thus indirubins could be explained possibly by their differ- depends on its individual affinity for AhRand kinases ential metabolism. AhRagonists are indeed known to and on its partition between the different targets in induce their own degradation, through the induction of the cell. This depends essentially on three factors: (1) cytochrome P450s. Although we have not investigated the intracellular distribution of the indirubin; (2) the this point, it is possible that the N1-methylated intracellular distribution of the targets (the cell response indirubins are only short-lived, and the cellular response will depend on the targets that interact first with the they induce only depends on the half-life of p27KIP1 they indirubin); (3) the expression and activity levels of each have induced. In contrast, the nonmethylated indirubins individual target. In this respect, we have observed large may be much more stable in the cell environment, as variations in the expression of AhRamong different cell they induce much less metabolizing cytochrome P450s. lines (data not shown). Similarly, the position of cells in Being trapped in the ATP-binding pocket of various the cell division cycle certainly plays a role in the activity kinases, indirubins may have long-lasting effects on of each CDK/cyclin complex. cells, resulting in irreversible effects and diminished cell In some case, the effect on AhRwill predominate viability. (cytostatic effect due to a G1 arrest) (Figure 8), while in other case, the effect on kinases will prevail (leading to The pharmacological use of indirubins a cytotoxic effect). Kinase-inactive indirubins will essentially act through AhR. Other indirubins seem to Given the diversity of targets, the use of indirubins as act more through kinase inhibition, although we have pharmacological tools to investigate cell function is not identified so far an AhR-inactive indirubin with limited and requires careful control experiments (such as strong kinase inhibitory properties to demonstrate this cell lines in which the putative target has been knocked possibility. out). However, it is undoubtedly possible to develop indirubin sub-families with greatly enhanced selectivity for specific targets such as GSK-3 (6-bromo-indirubins) (Meijer et al., 2003; Polychronopoulos et al., 2004) or AhR(N1-methylated indirubins) (this article). Indiru- bins can then be evaluated for potential therapeutic applications according to their selectivity (GSK-3: type II diabetes; GSK-3 & CDK5: neurodegenerative dis- orders; CDKs: cancer; AhR: cancer) (Knockaert et al., 2002). In the case of applications in the nervous system, AhRactivation should be given careful attention, as AhR, ARNT and indigo-responsive XRE binding are widely distributed in the brain (Petersen et al., 2000; Kuramoto et al., 2003). There are several arguments that support the inves- tigation of AhRligands as antitumor agents (see reviews in Bradshaw et al., 2002; Safe and McDougal, 2002). In particular, pancreatic tumor cell lines and human pancreatic cancer tissues express high levels of AhR and pancreatic cancer cell growth is inhibited by TCDD and other AhRligands (Koliopanos et al., 2002). Figure 8 Model for the dual mechanism of action of indirubins in Pancreatic tumors thus represent a promising therapeu- dividing cells. Indirubins with a high affinity for AhRand low tic target for AhR-selective indirubins. Furthermore affinity for kinases bind to and activate AhRmore readily. The interference with the AhRsignaling pathway contributes AhR/indirubin complex translocates to the nucleus and forms a to leukemogenesis, as shown by an acute myeloblastic dimer with ARNT. This complex then binds to xenobiotic- responsive elements (XRE) present in the regulatory domains of leukemia in which a fusion protein between translocated numerous target genes and directly or indirectly stimulates the ETS leukemia (TEL) and ARNT is expressed (Salomon- expression of target genes, e.g. p27KIP1. p27KIP1 is a potent inhibitor Nguyen et al., 2000), by the constitutive activation of of CDK2, leading to a marked arrest in G1 and a subsequent AhRin adult T-cell leukemia (ATL) (Hayashibara et al., cytostatic effect. Indirubins with a higher affinity for protein kinases inactivate CDKs, GSK-3, and probably other kinases. This 2003) and by the benzene-induced AhR-dependent induces cell cycle arrest at multiple stages, ultimately leading to hematotoxicity/leukemogenesis (Yoon et al., 2002). reduced cell survival The AhR-binding properties of indirubins may thus

Oncogene Indirubins, kinases and aryl hydrocarbon receptor M Knockaert et al 4409 contribute to their long-reported effects on leukemia was added and the mixture was heated under reflux (1201C) for (see reviews in Tang and Eisenbrand, 1992; Xiao et al., 1.5 h. The solvent was evaporated under reduced pressure and 2002). the residue was washed with water and c-hexane to afford quantitatively MeBIO (14) (15 mg). 1H NMR(DMSO, 400 MHz, d ppm, J in Hz) 13.69 (1H, br s, NOH), 11.78 (1H, s, N0-H), 8.61 (1H, d, J ¼ 8.1Hz, H-4), 8.23 Materials and methods (1H, d, J ¼ 7.3 Hz, H-40), 7.44 (2 H, m, H-60,70), 7.31 (1H, s, H- 7), 7.17 (1H, d, J ¼ 8.1Hz, H-5), 7.07 (1H, br s, H-50), 3.32 (3 H, þ Chemistry s, N-CH3); CI-MS m/z 370, 372 (M þ H) . The synthesis of the nonmethylated indirubins used in this study have been published elsewhere (Polychronopoulos et al., Biochemistry 2004). Compounds 3, 5, 6, 7, 8, 9, 19 and 20 were kindly Biochemical reagents Sodium ortho-vanadate, EGTA, provided by Dr G Eisenbrand (Department of Chemistry, EDTA, Mops, b-glycerophosphate, phenylphosphate, sodium Food, Chemistry and Environmental Toxicology, University fluoride, dithiothreitol (DTT), glutathione-agarose, glu- of Kaiserslautern, Erwin-Schrodinger-Strasse, 67663 Kaiser- ¨ tathione, bovine serum albumin (BSA), nitrophenylphosphate, slautern, Germany). Their synthesis was described previously leupeptin, aprotinin, pepstatin, soybean trypsin inhibitor, (Leclerc et al., 2001). benzamidine, histone H1 (type III-S) were obtained from Sigma Chemicals. [g-32P]ATP (PB168) was obtained from 1-Methyl-indirubin (3) Methanol (25 ml) was vigorously Amersham. The GS-1 peptide (YRRAAVPPSPSLSRHSSPH stirred under nitrogen for 20 min, then 1-methyl-isatin QSpEDEEE) was synthesized by the Peptide Synthesis Unit (128 mg, 0.8 mmol), 3-acetoxyindol (92 mg, 0.53 mmol) was (Institute of Biomolecular Sciences, University of South- added and stirring was continued for 5 min during which time ampton, Southampton, UK). the compounds dissolved. Anhydrous Na2CO3 (213 mg, 2 mmol) was added and the stirring continued for 3 h. The Buffers Homogenization buffer:60mM b-glycerophosphate, dark precipitate was filtered, washed with aqueous methanol 15 mM p-nitrophenylphosphate, 25 mM Mops (pH 7.2), 15 mM (1:1, 10 ml) and CH Cl (10 ml) to give 3 (65 mg, 0.24 mmol, 2 2 EGTA, 15 mM MgCl ,1mM DTT, 1 mM sodium vanadate, 44%). 2 1mM NaF, 1 mM phenylphosphate, 10 mg leupeptin/ml, 10 mg 1H NMR(DMSO, 400 MHz, d ppm, J in Hz) 11.07 (1H, s, 0 1 1 aprotinin/ml, 10 mg soybean trypsin inhibitor/ml and 100 mM N -H), 8.80 ( H, d, J ¼ 7.5 Hz, H-4), 7.66 ( H, d, J ¼ 7.5 Hz, H- benzamidine. 40), 7.59 (1H, t, J ¼ 7.5 Hz, H-60), 7.42 (1H, d, J ¼ 7.5 Hz, H-70), 1 1 Buffer A:10mM MgCl2,1mM EGTA, 1 mM DTT, 25 mM 7.35 ( H, t, J ¼ 7.5 Hz , H-6), 7.10 ( H, t, J ¼ 7.5 Hz, H-5), 7.08 Tris-HCl pH 7.5, 50 mg heparin/ml. (1H, d, J ¼ 7.5 Hz, H-7), 7.03 (1H, t, J ¼ 7.5 Hz , H-50), 3.28 (3H, þ Buffer C: homogenization buffer but 5 mM EGTA, no NaF s, N-CH3); CI-MS m/z 277 (M þ H) . and no protease inhibitors.

6-Bromo-1-methyl-indirubin (12) Methanol (20 ml) was vig- Kinase preparations and assays Kinase activities were assayed orously stirred under nitrogen for 20 min, then 6-bromo-1- in Buffer A or C, at 301C, at a final ATP concentration of methyl-isatin (106 mg, 0.44 mmol), 3-acetoxyindol (61 mg, 15 mM. Blank values were subtracted and activities calculated 0.35 mmol) was added and stirring was continued for 5 min as picomoles of phosphate incorporated for a 10-min incuba- during which time the compounds dissolved. Anhydrous tion. The activities are usually expressed in % of the maximal Na2CO3 (120 mg) was added and the stirring continued for activity, that is, in the absence of inhibitors. Controls were 3 h. The dark precipitate was filtered, washed with aqueous performed with appropriate dilutions of dimethylsulfoxide. methanol (1:1, 10 ml) and CH2Cl2 (10 ml) to give 12 (60 mg, GSK-3 a/b was purified from porcine brain by affinity 0.17 mmol, 49%). chromatography on immobilized axin (Primot et al., 2000). It 1H NMR(DMSO, 400 MHz, d ppm, J in Hz) 11.13 (1H, s, was assayed, following a 1/100 dilution in 1 mg BSA/ml 10 mM N0-H), 8.70 (1H, d, J 8.2 Hz, H-4), 7.66 (1H, d, J 7.8 Hz, H- ¼ ¼ M 0 1 0 1 0 DTT, with 5 ml40m GS-1 peptide as a substrate, in buffer A, 4 ), 7.60 ( H, t, J ¼ 7.8 Hz, H-6 ), 7.43 ( H, d, J ¼ 7.8, Hz, H-7 ), 32 1 1 1 1 in the presence of 15 mM [g- P]ATP (3000 Ci/mmol; 1 mCi/ml) 7.35 ( H, d, J ¼ Hz, H-7), 7.29 ( H, dd, J ¼ 8.2, Hz, H-5), 7.04 1 1 0 3 in a final volume of 30 ml. After 30-min incubation at 30 C, ( H, t, J ¼ 7.8, Hz, H-5 ), 3.28 ( H, s, N-CH3); CI-MS m/z 355, þ 25 ml aliquots of supernatant were spotted onto 2.5 Â 3cm 357 (M þ H) . pieces of Whatman P81 phosphocellulose paper, and, 20 s later, the filters were washed five times (for at least 5 min each 1-Methyl-indirubin-30-oxime (4) (MeIO) 1-Methyl-indirubin time) in a solution of 10 ml phosphoric acid/liter of water. The (35 mg, 0.13 mmol) was dissolved in pyridine (3 ml). Then wet filters were counted in the presence of 1 ml ACS hydroxylamine hydrochloride (20 mg) was added and the (Amersham) scintillation fluid. mixture was heated under reflux (1201C) for 1.5 h. The solvent CDK1/cyclin B was extracted in homogenization buffer was evaporated under reduced pressure and the residue was from M phase starfish (Marthasterias glacialis) oocytes and washed with water and c-hexane to afford quantitatively MeIO purified by affinity chromatography on p9CKShs1-sepharose (4) (37 mg). beads, from which it was eluted by free p9CKShs1 as previously 1H NMR(DMSO, 400 MHz, d ppm, J in Hz) 13.56 (1H, br s, described (Borgne and Meijer, 1996). The kinase activity was NOH), 11.74 (1H, s, N0-H), 8.69 (1H, d, J ¼ 7.8 Hz, H-4), 8.23 assayed in buffer C, with 1 mg histone H1/ml, in the presence 1 0 0 0 1 ( H, d, J ¼ 7.5 Hz, H-4 ), 7.41 (2 H, m, H-6 ,7), 7.23 ( H, t, of 15 mM [g-32P]ATP (3000 Ci/mmol; 1 mCi/ml) in a final 3 0 3 J ¼ 7.8, Hz, H-6), 7.04 ( H, m, H-5 , 5, 7), 3.31 ( H, s, N-CH3); volume of 30 ml. After 10-min incubation at 301C, 25 ml CI-MS m/z 292 (M þ H) þ . aliquots of supernatant were spotted onto P81 phosphocellulose papers and treated as described above. 1-Methyl-6-bromo-indirubin-30-oxime (14) (MeBIO) 1- CDK5/p25 was reconstituted by mixing equal amounts of Methyl-6-bromo-indirubin (15 mg, 0.042 mmol) was dissolved recombinant mammalian CDK5 and p25 expressed in in pyridine (2 ml). Then hydroxylamine hydrochloride (15 mg) Escherichia coli as glutathione-S-transferase (GST) fusion

Oncogene Indirubins, kinases and aryl hydrocarbon receptor M Knockaert et al 4410 proteins and purified by affinity chromatography on glu- (RFUs). Samples were run in triplicate and the fluorescent tathione–agarose (vectors kindly provided by Dr JH Wang) activity present in wells containing media only was subtracted (p25 is a truncated version of p35, the 35-kDa CDK5 from the fluorescent activity in all samples. activator). Its activity was assayed in buffer C as described for CDK1/cyclin B. Cell proliferation analysis The mouse 5L hepatoma cell line (AhR þ / þ ) and BP8 (an Cell biology AhR À/À subclone) were kindly provided by Dr M Gottlicher Chemical reagents 2,3,7,8-Tetrachlorodibenzo-p-dioxin (Forschungszentrum Karlsruhe, Institute of Genetics, Karls- (TCDD) was a kind gift from Dr Steve Safe (Veterinary ruhe, Germany). Cells were cultured in Dulbecco’s modified Physiology and Pharmacology, Texas A&M University, Eagle medium (DMEM) (Biowhittaker) supplemented with College Station, TX, USA). 2mML-glutamine (Eurobio), 10% fetal calf serum (FCS), and gentamycin (Gibco BRL) at 371C in an atmosphere of 7% CO2. Indirubin treatments were performed on 50–60% AhR assays confluent cultures at the indicated time and concentrations Yeast AhR reporter assay The Saccharomyces cerevisiae (Damiens et al., 2001). Control experiments were carried out strain used for the yeast assay for AhRligand activity was using appropriate dilutions of DMSO. YCM3, derived from YPH499 (mat a, ade2-101, his3D200, leu2-1, lys2-801, trp-63, ura3-52) (Miller, 1997; Adachi et al., Cell viability assay 2001). It was kindly provided by Dr CA Miller III (Depart- ment of Environmental Health Sciences and Tulane-Xavier To quantify the toxicity of indirubins on 5L and BP8 cells, we Center for Bioenvironmental Research, Tulane University measured the inhibition of cellular reduction of MTT to MTT School of Public Health and Tropical Medicine, New Orleans, formazan according to Mosmann (1983). After treatment with LA, USA). Standard yeast growth conditions and genetic indirubins, cells were incubated with 0.5 mg MTT/ml fresh 1 manipulations were previously described (Guthrie and Fink, medium at 37 C for 1 h. The formazan products were dissolved 1991). Yeast cells expressing both AhRand ARNTfrom the in DMSO and quantified by measurement of the absorbance at inducible GAL promoter were grown to early log phase at 562 nm. 251C in selective media containing raffinose (2% final concentration), at which time galactose (2%) was added for FACS analysis 3 h. Various concentrations of indirubins (in DMSO) were Flow cytometry analysis (Coulter EPIX XL2, Beckman, CA, then added and the cells were grown for an additional 3 h and USA) of cellular DNA content was performed on ethanol- b-galactosidase activity was determined (adapted from Miller, fixed cell suspensions after ribonuclease A III treatment 1999). Briefly, 1 ml of cell culture was centrifuged and cells (Sigma) and propidium iodide (Sigma) staining, as previously were washed and resuspended in 150 ml of Z-buffer (60 mM described (Ffrench et al., 1985). Percentages of cells in G0/G1, Na2HPO4,40mM NaH2PO4,1mM MgSO4,10mM KCl, S and G2/M phases of cell cycle were then calculated on the 40 mM b-mercaptoethanol). After addition of 50 ml chloroform basis of DNA distribution histograms provided by the and 10 ml of 0.1% SDS, the mix was vortexed for 15 s and the software manufacturer. reaction was started by addition of 700 mlofo-nitrophenol-D- galactopyranoside (1 mg/ml solution in Z-buffer) and further Indirect immunofluorescence microscopy incubated for 10 min at 301C. The reaction was then stopped by addition of 500 mlof1M Na2CO3. b-Galactosidase (referred The wild-type mouse hepatoma cells (Hepa-1) and its variant to as lacZ units) was measured at 420 nm and calculated ARNT mutant cells (lacking functional ARNT) were grown in according to the following formula: absorbance at 420 nm Â minimum essential medium (InVitrogen, Carlsbad, CA, USA) 1000/(absorbance at 600 nm Â ml of cell suspension added Â supplemented with 6% FBS (Hyclone Laboratories, Logan, min of reaction time). UT, USA). Cells were routinely maintained in a 371C incubator with 5% CO2. Cells were treated for 90 min with AhR-dependent EGFP expression assay in a stably transfected control vehicle, DMSO or with ligands, TCDD at 10 nM and mouse hepatoma cell line Mouse hepatoma (H1G1.1c3) cells indirubins at 25 mM. The cells were fixed in 2% paraformalde- containing a stably transfected plasmid expressing XRE-driven hyde and processed for indirect immunofluorescence micro- EGFP fusion gene were maintained in selective media (aMEM scopy as previously described (Elbi et al., 2002). Polyclonal containing 10% fetal bovine serum and 968 mg/l G418). XREs anti-AhRprimary antibody was used at 1 : 400 (Biomol are high-affinity binding sites for AhR/ARNT transcription Research Laboratories, Plymouth, PA, USA). The cells were factor complex and they mediate ligand- and AhR-dependent mounted using Prolong (Molecular Probes, Eugene, OR, transcriptional activation (Nagy et al., 2002a, b). Cells were USA) and were observed on a Nikon E800 microscope plated into black, clear-bottomed 96-well tissue culture dishes equipped with 63 Â 1.35 NA, oil immersion Plano Nikon (Corning, San Mateo, CA, USA) at 75,000 cells per well and objective and a Photometrics MicroMax cooled CCD camera. allowed to attach for 24 h. The selective medium was then Images were collected by using the MetaMorph software replaced with 100 ml of media (lacking G418) containing the (Universal Imaging, Downingtown, PA, USA). DMSO (1% final solvent concentration) or the test chemical at the indicated concentration. EGFP levels were measured in living cells at 24 h using a Tecan Genios microplate fluorom- Abbreviations eter with an excitation wavelength of 485 nm (25 nm band- AhR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon width) and an emission wavelength of 515 nm (10 nm receptor nuclear translocator; bHLH/PAS, basic helix–loop– bandwidth). In order to normalize results between experi- helix/period-aryl hydrocarbon receptor nuclear translocator- ments, the instrument fluorescence gain setting was adjusted so single minded; BIO, 6-bromo-indirubin-30-oxime; BSA, bovine that the level of EGFP induction by 1 nM TCDD produced a serum albumin; CDK, cyclin-dependent kinase; DAPI, diami- relative fluorescence of 9000 relative fluorescence units nophenylindole, dihydrochloride; DTT, dithiothreitol; EGFP,

Oncogene Indirubins, kinases and aryl hydrocarbon receptor M Knockaert et al 4411 enhanced green ﬂuorescent protein; FCS, fetal calf serum; 9, 19, 21, 22), Dr Martin Go¨ ttlicher (5L and BP8 cell lines), FICZ, 6-formylindolo[3,2-b]carbazole; GSK-3, glycogen Dr Charles A Miller III (AhRagonists reporter yeast synthase kinase -3; IO, indirubin-30-oxime; MeBIO, strain), and Dr Steve Safe (TCDD). This research was 1-methyl-6-bromo-indirubin-30-oxime; MeIO, 1-methyl-indir- also supported by grants from the ‘Association pour la ubin-30-oxime; MTT, (4,5-dimethylthiazol-2-yl)-2,5-diphenyl- Recherche sur le Cancer’ ARC5732 (LM), ARC5812 tetrazolium-bromide; TCDD, 2,3,7,8-tetrachlorodibenzo- (MB) and ARC5664 (MF), the National Institutes of p-dioxin; XRE, xenobiotic-responsive element. Environmental Health Sciences NIH5P42ES04699 (MD) and by a grant (‘Mole´ cules & Cibles The´ rapeutiques’) Acknowledgements from the ‘Ministe` re de la Recherche/INSERM/CNRS’ We are most thankful to our colleagues for providing (LM). reagents: Dr Gerhard Eisenbrand (compounds 3, 5, 6, 7, 8,

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