Rebecca A Randle

Selina Raguz

Christopher F Higgins

Ernesto Yagüe
email: ernesto.yague@csc.mrc.ac.uk

Role of the highly structured 5'-end region of MDR1 mRNA on Pglycoprotein expression

Keywords: cytotoxic stress / drug resistance / eIF4E / mRNA translation / mRNA hairpin EBV, Epstein Barr virus, eIF, eukaryotic initiation factor, MAPK, mitogen-activated protein kinase, Mnk, MAP kinase-interacting kinase, mTOR, mammalian target of rapamycin kinase, PI3-K, phosphatidylinositol 3

Overexpression of P-glycoprotein, encoded by the MDR1 gene, is often responsible for multidrug resistance in acute myeloid leukemia. We have shown previously that MDR1 (P-glycoprotein) mRNA levels in K562 leukemic cells exposed to cytotoxic drugs are up-regulated but P-glycoprotein expression is translationally blocked. Here we show that cytotoxic drugs down-regulate the Akt signaling pathway leading to hypophosphorylation of the translational repressor 4E-BP and decreased eIF4E availability. The 5'-end of MDR1 mRNA adopts a highly-structured fold. Fusion of this structured 5'-region upstream of a reporter gene impeded its efficient translation, specifically under cytotoxic stress, by reducing its competitive ability for the translational machinery. The effect of cytotoxic stress could be mimicked in vivo blocking the phosphorylation of 4E-BP by mTOR using rapamycin or eIF4E siRNA, and relieved by over-expression of either eIF4E or constitutively-active Akt. Upon drug exposure MDR1 mRNA was up-regulated, apparently stochastically, in a small proportion of cells. Only in these cells could MDR1 mRNA compete successfully for the reduced amounts of eIF4E and translate P-glycoprotein. Consequent drug efflux and restoration of eIF4E availability results in a feed-forward relief from stressinduced translational repression and to the acquisition of drug resistance.

INTRODUCTION

Multidrug resistance, the phenomenon by which cancer cells treated with a single chemotherapeutic agent become cross-resistant to multiple functionally and structurally unrelated compounds, is most frequently associated with up-regulation of P-glycoprotein. This membrane protein, coded by the MDR1 (ABCB1) gene, is an ATP-dependent efflux pump that maintains intracellular drug concentrations below cytotoxic levels [START_REF] Gottesman | Mechanisms of cancer drug resistance[END_REF] We have used a K562 myelogenous leukaemia cell model to determine the mechanisms by which MDR1/P-glycoprotein-mediated multidrug resistance is acquired [START_REF] Yagüe | P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation[END_REF], with the aim of developing therapeutic strategies that will target upregulation of P-glycoprotein expression. The K562 cell line is a useful model system as drug-resistant sub-lines can readily be derived by selection with clinically-relevant doses of chemotherapeutic agents [START_REF] Yagüe | P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation[END_REF]. Short-term (1-3 days) exposure of naïve K562 cells to a range of cytotoxic agents results in up-regulation of MDR1 mRNA steadystate levels by stabilization of the otherwise short-lived MDR1 mRNA. However, this stabilized mRNA is not associated with translating polyribosomes and P-glycoprotein is not synthesised. Selection for drug resistance, by long-term exposure to drug, leads to resistant cells in which this translational block is overcome. Translational repression of MDR1 mRNA has also been described in EBV-transformed human B lymphocytes and in leukaemic CEM cells [START_REF] Yagüe | P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation[END_REF][START_REF] Baker | Epigenetic changes to the MDR1 locus in response to chemotherapeutic drugs[END_REF], and the well-documented lack of correlation between MDR1 mRNA and P-glycoprotein levels in leukaemia [START_REF] Chaudhary | Expression and activity of Pglycoprotein, a multidrug efflux pump, in human hematopoietic stem cells[END_REF] implies that translational regulation represents a general regulatory step in P-glycoprotein expression.

The rate-limiting step in translation is initiation and is the most common target for translational control. For the majority of eukaryotic mRNAs, initiation of protein synthesis occurs via a cap-dependent mechanism [START_REF] Gebauer | Molecular mechanisms of translational control[END_REF]. The initial binding of the translation apparatus at the 5'-end of an mRNA begins with the formation of a ternary complex (eIF2, GTP and a tRNA initiator) which is recruited by the small ribosomal subunit (40S) together with its associated initiation factors to form the 43S pre-initiation complex which recognizes the 5'-cap of an mRNA. This requires the eIF4F protein complex which comprises eIF4E (which recognises and binds to the 5'cap structure), eIF4A, and eIF4G. Since the levels of eIF4E are normally low, assembly of the eIF4F complex and its interaction with the 5'-mRNA cap provides the rate-limiting step in translation initiation. Once bound to the mRNA cap, the 43S pre-initiation complex migrates along the 5'-untranslated region (UTR) in an ATPdependent process known as scanning, until it reaches the translation initiation codon (AUG) [START_REF] Gebauer | Molecular mechanisms of translational control[END_REF][START_REF] Kozak | Pushing the limits of the scanning mechanism for initiation of translation[END_REF]. For a proportion of cellular mRNAs, cap-independent translation involves internal ribosomal entry sites [START_REF] Stoneley | Cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression[END_REF].

Several features of an mRNA molecule can influence its translation [START_REF] Gebauer | Molecular mechanisms of translational control[END_REF]. Loop structures involving the 5'-UTR of mRNAs with free energy values of less than between -30 to -50 Kcal/mol, generally inhibit translation [START_REF] Pickering | The implications of structured 5' untranslated regions on translation and disease[END_REF]. In vitro RNase mapping has shown that the 5'-terminal region of MDR1 mRNA (nucleotides 1-289) is highly structured [START_REF] Kostenko | Secondary structure of the 5'-region of PGY1/MDR1 mRNA[END_REF]. This 289-nucleotide region includes the 140-nucleotide 5'-UTR which folds with the following 149 nucleotides of the MDR1 coding sequence to form a stable structure with the initiation codon located in a hexanucleotide loop at its centre (Figure 1A). Here we show that the highly structured region at the 5'-end of MDR1 mRNA affects translation of reporter transcripts and that this down-regulation is more pronounced under cytotoxic stress, when the Akt pathway is down-regulated and available levels of eIF4E low. Drug resistance is acquired by up-regulation, by an apparently stochastic mechanism, of MDR1 mRNA in a small subpopulation of cells. This allows effective competition for eIF4E, translation of P-glycoprotein, and consequent removal of cytotoxic drugs from the cell restoring eIF4E availability. This positive feed-forward cycle leads to the development and maintenance of multidrug resistance.

EXPERIMENTAL

Cells

Human myelogenous leukaemic K562 cells and its colchicine-resistant derivative KC40, have been described before [START_REF] Yagüe | P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation[END_REF]. KC40 cells were routinely maintained in the presence of 20 ng/ml colchicine. Cells were transfected using an Amaxa Nucleofector, following the manufacturers' recommendations. For transient transfections, 4.5 µg of the test plasmid were cotransfected with 0.5 µg phRGTK (expressing Renilla luciferase to normalize for transfection efficiency) (Promega).

For stable transfections, plasmids (2 µg) were linearised with PvuI, cotransfected as above with 0.1 µg pGK-puro, and transfectants selected with 2 µg/ml puromycin.

Generation of drug resistant K562 cells

Cells were seeded at 5 x10 5 cells/ml in 50 ml culture medium containing 20 ng/ml colchicine. At days 3, 10 and 14, 50 ml of fresh culture medium without drug were added. At day 18 cells were spun down and resuspended in 160 ml culture medium with 20 ng/ml colchicine. From day 25-40 the colchicine-containing medium was changed routinely twice per week.

Plasmids

The constitutively-active Akt expression plasmid (pCAAKT) was from Upstate. The constitutively-active Mnk-1 expression plasmid (pCAMNK) [START_REF] Waskiewicz | Phosphorylation of the cap-binding protein eukaryotic translation initiation factor 4E by protein kinase Mnk1 in vivo[END_REF], and the MDR1 downstream promoter-luciferase reporter plasmid pMDR1(-1202) [START_REF] Scotto | Transcriptional regulation of the MDR1 gene by histone acetyltransferase and deacetylase is mediated by NF-Y[END_REF] have been described. The 289-nucleotide MDR1 5'-structure (consisting of the 140-nucleotide 5'-UTR and 149 nucleotides of proximal coding region) was amplified from reversetranscribed KC40 mRNA using primers OLEY24bis and OLEY26 (all oligonucleotide sequences are listed in Supplementary Table 1). The 140-nucleotide 5'-UTR was amplified with primers OLEY24bis and OLEY25. PCR products were inserted into pGL3Control (Promega) upstream of the luciferase gene, using the NcoI and EcoRI sites, generating pGL-Loop and pGL-UTR, respectively. The luciferase translation initiation codon within pGL-UTR was then mutated to that found in the native MDR1 using primers OLEY176 and OLEY177, and the Quikchange Site Directed mutagenesis kit (Stratagene), according to manufacturer's instructions. The translation start codon and two other out of frame-AUGs in the MDR1 coding region, but upstream from the luciferase gene in pGL-Loop, were sequentially mutated by site-directed mutagenesis as above with oligonucleotides OLEY27 and OLEY28, OLEY41 and OLEY42, and OLEY43 and OLEY44, respectively, to generate pGL-LoopM. Thus, pGL-UTR, pGL-Loop and pGL-LoopM had the same 5'-UTR length and sequence surrounding the translation initiation codon.T7 promoter sequences, produced by annealing the complementary oligonucleotides OLEY203 and OLEY204, were inserted into EcoRI -digested pGL-UTR and pGL-Loop to generate T7pGL-UTR and T7pGL-Loop, respectively. eIF4E cDNA was obtained from reverse transcribed K562 mRNA by PCR using high fidelity Expand (Roche Diagnostics) and primers OLEY184 and OLEY185. The resulting 1391-base pairs DNA fragment was inserted into the pEF6/V5-His-TOPO plasmid vector (Invitrogen) to generate peIF4E.

Genomic DNA and Southern blotting

Genomic DNA was digested with MfeI (for luciferase) or EcoRI (for β-globin), blotted and hybridized to radioactive probes, derived from pGL3control (luciferase coding sequence) or the β-globin locus as described [START_REF] Yagüe | P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation[END_REF].

Luciferase expression

A Dual-Luciferase Reporter Assay was used to measure both firefly and Renilla luciferase in lysates of transiently transfected cells 16 h after transfection (Promega).

Protein content was measured using a DC protein assay (Bio-Rad).

De novo protein synthesis

Incorporation of [ 35 S]Met into nascent proteins was determined by trichloroacetic acid precipitable counts as previously described [START_REF] Yagüe | P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation[END_REF].

RNA analysis

Luciferase mRNA analysis was performed by semi quantitative RT-PCR. Briefly, after reverse transcription [START_REF] Yagüe | P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation[END_REF], cDNA was amplified by PCR with OLEY218 and OLEY219 for luciferase, and OLEY8 and OLEY70 for GAPDH normalization [START_REF] Raguz | Activation of the MDR1 upstream promoter in breast carcinoma as a surrogate for metastatic invasion[END_REF] for 20 to 29 cycles. Detection of amplified cDNA was by Southern blotting and hybridization to radioactively end-labelled internal oligonucleotides, essentially as described [START_REF] Yagüe | P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation[END_REF]. Quantification was with a PhosphorImager (Molecular Dynamics). MDR1 mRNA analysis was by reverse transcription and real-time quantitative PCR (RT-Q-PCR), as described [START_REF] Yagüe | Complete reversal of multidrug resistance by stable expression of small interfering RNAs targeting MDR1[END_REF]. mRNA half-life was determined as described [START_REF] Yagüe | P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation[END_REF] except that Id2 mRNA turnover was determined by RT-Q-PCR with primers Id2F and Id2R (PrimerBank ID: 31982933a1) [START_REF] Wang | A PCR primer bank for quantitative gene expression analysis[END_REF] and normalized to 18S ribosomal RNA (18Sf and 18Sr primers). m 7 GTP pull-down eIF4E-containing protein complexes were isolated by binding to m 7 GTP Sepharose as described [15].

In vitro transcription, in vitro translation and RNA competition

T7pGL-UTR and T7pGL-Loop plasmids were linearized downstream from the luciferase stop codon and in vitro transcribed using a mMessage mMachine T7 Ultra kit (Ambion). A cap analogue and a poly(A) tail were added to the 5'-and 3'-ends, respectively, following the manufacturer's recommendations. In vitro translation was carried out with 0.4 µg of in vitro-transcribed mRNA and 0-20 µg competitor RNA (isolated either from K562 cells or from yeast) in a rabbit reticulocyte cell-free system (Promega), and luciferase activity measured after 90 min at 30°C following manufacturer's recommendations. Where indicated, in vitro transcription and translation was also carried out using a TNT Quick Coupled Transcription and Translation Rabbit Reticulocyte System (Promega). All experiments were performed in triplicate on at least three independent occasions.

RNA interference

siRNA (2.7 µg) targeted to eIF4E (Cell Signalling Technology), or non-specific AllStars negative control (Qiagen), were transfected into cells using Amaxa Nucleofector Kit V. Decrease in cellular eIF4E protein levels were assayed by western blotting 24 h after transfection.

Antibodies and flow cytometry

All antibodies against translation factors were from Cell Signalling. Detection of plasma membrane P-glycoprotein with antibody UIC2, BODIPY-taxol efflux assays, and western blotting were performed as previously described [START_REF] Yagüe | P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation[END_REF][START_REF] Elliott | Multidrug transporter activity in lymphocytes[END_REF]. Cell viability was determined by TOTO-3 exclusion.

Biochemical Journal Immediate Publication. Published on 15 Jun 2007 as manuscript BJ20070235

RESULTS

The 5'-MDR1 mRNA structure down-regulates translation of a reporter gene

The structure of the 5'-end of the MDR1 mRNA, nucleotides 1-289, [START_REF] Kostenko | Secondary structure of the 5'-region of PGY1/MDR1 mRNA[END_REF] (Fig 1A ), has a calculated free energy value of less than -100 Kcal/mol [START_REF] Hofacker | Vienna RNA secondary structure server[END_REF]. We hypothesized that if formed in vivo, such a structure would down-regulate translation from a linked reporter. The 289-base pairs region and the 140-base pairs region corresponding to the 5'-UTR were cloned upstream from the luciferase reporter gene in pGL3control, generating pGL-Loop and pGL-UTR, respectively (Fig 1B). The luciferase activity of pGL-UTR was approximately 2.5 fold above pGL3control in transiently transfected K562 cells (Figure 1C). This may be due to the longer 5'-UTR sequence in pGL-UTR transcripts (138 nucleotides compared to 35 nucleotides in pGL3control mRNAs), resulting in a greater translation efficiency of the luciferase mRNA [START_REF] Kozak | Effects of long 5' leader sequences on initiation by eukaryotic ribosomes in vitro[END_REF]. The presence of the 289-bp structured region (pGL-Loop) resulted in a significant reduction in luciferase activity (5-to 7-fold) relative to pGL-UTR (Figure 1C) indicating that this region of MDR1 mRNA is inhibitory to reporter gene expression. Changes in luciferase activity were due to changes at the translational level and not at the levels of transcription or mRNA stability since the steady state levels of luciferase mRNA, as detected by semi-quantitative RT-PCR, were comparable (Figure 1D). Equally, the small fusion protein formed between the first amino acids of P-glycoprotein and firefly luciferase in pGL-Loop did not affect luciferase enzymatic activity (data not shown). Following site-directed mutagenesis to eliminate AUGs in the loop structure (pGL-LoopM), the predicted fold and free energy was unchanged (data not shown).

Therefore, we reasoned that if the loop at the 5'-end of MDR1 mRNA were formed in vivo, luciferase translation from pGL-LoopM, when compared to pGL-Loop, would be greatly reduced. Transfection of pGL-LoopM into K562 cells showed that luciferase activity was completely absent in pGL-LoopM-expressing cells (Figure 1C). This suggests that the 43S pre-initiation complex cannot scan or shunt through the loop region present in pGL-LoopM, preventing translation of the downstream luciferase reporter. In contrast, in pGL-Loop-expressing cells, the initiation codon in the hexanucleotide loop at the centre of the structure improves the translatability of these transcripts, presumably because the 43S pre-initiation complex has to scan or shunt through a shorter region before reaching the starting AUG. Thus, the 5'-end of MDR1 mRNA is highly structured in vivo and down-regulates translation of a downstream reporter gene.

The loop at the 5'-end of MDR1 mRNA specifically down-regulates translation of a luciferase reporter gene upon treatment with cytotoxic drugs

To test the effects of cytotoxic stress on the translation of transcripts with the 5'-MDR1 structure, we generated stable K562 cell lines with similar numbers of copies of pGL3control, pGL-UTR or pGL-Loop, named K-pGL3, K-pGL-UTR and K-pGL-Loop cells, respectively (Figure 2A). Treatment of each of these cell lines with 1 µM colcemid for 3 days resulted in a similar decrease in the incorporation of 35 S[Met] into nascent proteins (Figure 2B). Luciferase activity of K-pGL3 and K-pGL-UTR cells decreased by 25%. However, in K-pGL-Loop cells, which include the entire 5'-MDR1 structure upstream of the luciferase reporter, luciferase activity decreased by 75% (Figure 2B). This effect was due to a decrease in translation, and not transcription of the transgenes, since the steady-state levels of the corresponding mRNAs did not change significantly upon drug treatment (Figure 2C). Cytotoxic stress results in a global reduction in translation [START_REF] Holcik | Translational control in stress and apoptosis[END_REF]. While the observed decrease in luciferase activity in K-pGL3 and K-pGL-UTR cells following colcemid treatment probably reflects this generalized effect, the greater decrease observed in K-pGL-Loop cells indicates a specific effect of the MDR1 5'-mRNA structure on translation of the luciferase reporter under cytotoxic stress.

Down-regulation of signaling pathways due to cytotoxic stress

Since control of protein synthesis is generally achieved by changes in the phosphorylation state of translation initiation factors, or the regulators that interact with them, we examined several signaling pathways known to affect translation following cytotoxic stress. A number of kinases, activated by various types of stresses, can phosphorylate eIF2α at Ser51 and down-regulate global translation [START_REF] Proud | eIF2 and the control of cell physiology[END_REF].

Following exposure of K562 cells to 1µM colcemid for 3 days, an increase in the phosphorylation of eIF2α was observed (Figure 3A To confirm that the drug-induced hypophosphorylation of 4E-BP affected the formation of translation initiation complexes we pulled-down eIF4E and associated proteins from colcemid-treated K562 cell extracts using a 5'-end mRNA cap analogue (m 7 GTP-Sepharose). Bound proteins were analyzed by western blotting. Treatment with colcemid resulted in a time-dependent increase in the binding of 4E-BP to eIF4E, and a reduction in the binding of eIF4G (Figure 3C). Thus, cytotoxic stress in K562 cells leads to decrease in the ability of eIF4E to associate with eIF4G resulting in down-regulation of translation.

Down-regulation of available eIF4E following cytotoxic stress decreases the translation efficiency of transcripts with the MDR1 mRNA 5'-structure

To ask how the changes in 4E-BP phosphorylation and eIF4E availability affect the translation of luciferase reporter genes in K-pGL-UTR and K-pGL-Loop cells following cytotoxic drug exposure, we transfected these lines transiently with plasmids expressing constitutively-active forms of Mnk1 (pCAMNK) and Akt (pCAAKT), singly or together, and measured luciferase activity following treatment of the cells with 1 µM colcemid for 3 days. Expression of constitutively-active Mnk1 increased the phosphorylation of eIF4E after drug exposure in both cell lines, as expected (Figure 4A). Expression of constitutively-active Akt also increased 4E-BP phosphorylation after drug exposure, but to a lesser extent. This is probably because, unlike Mnk1 which phosphorylates eIF4E directly, Akt phosphorylates and activates mTOR which then in turn phosphorylates 4E-BP (Figure 4B) [START_REF] Raught | eIF4E activity is regulated at multiple levels[END_REF]. There was no significant difference in the steady state-levels of the reporter mRNAs in each transfected cell line upon drug exposure (data not shown). Since expression of constitutively-active Mnk1 did not increase expression of luciferase from pGL-Loop under cytotoxic stress (Figure 4A,C,D) we can infer that, at least under these conditions, the phosphorylation status of eIF4E does not contribute to the downregulation of translation of pGL-Loop transcripts. In contrast, translation of pGL-Loop transcripts following cytotoxic stress was enhanced significantly in cells expressing constitutively-active Akt (Figure 4D).

We confirmed the importance of Akt by down-regulating mTOR with rapamycin in K-pGL-UTR and K-pGL-Loop cells. Concentrations of rapamycin used in these assays were not toxic to K562 cells, as determined by proliferation analyses (data not shown). Western blot analysis demonstrated that treatment with rapamycin decreased the level of phosphorylated 4E-BP in cells, as expected (Figure 4E).

Similarly, steady-state levels of luciferase reporter mRNAs (data not shown) and the translation of pGL-UTR transcripts (Figure 4F) did not change significantly.

Nonetheless, rapamycin strongly repressed translation of pGL-Loop transcripts (Figure 4F). Thus, translational repression of transcripts containing the 5'-structure of MDR1 mRNA is very likely due to a decrease in Akt activity and 4E-BP phosphorylation. In conclusion, inhibition of the PI3K/Akt/mTOR/4E-BP phosphorylation cascade, and subsequent decrease in the availability of eIF4E in drug exposed cells, specifically reduces the translation of transcripts including the structured 5'-end of MDR1 mRNA.

The 5'-end MDR1 mRNA loop renders chimaeric luciferase reporter mRNAs poorly competitive for eIF4E

The limiting factor for translation initiation is eIF4E, and transcripts with long and structured 5'-UTRs are generally considered to be poorly competitive [START_REF] Pickering | The implications of structured 5' untranslated regions on translation and disease[END_REF][START_REF] De Benedetti | eIF4E expression in tumors: its possible role in progression of malignancies[END_REF]. We tested whether the 5'-MDR1 mRNA structure affected mRNA competitiveness in vitro. Luciferase transcripts were synthesized in vitro with either the MDR1 5'-UTR or the intact 5'-MDR1 loop structure from T7pGL-UTR and T7pGL-Loop, respectively. Translation of these transcripts in the presence of increasing amounts of total K562 RNA was performed in a rabbit reticulocyte cell-free system. Luciferase mRNA with the 5'-UTR of MDR1 mRNA (T7pGL-UTR) competed efficiently with increasing amounts of RNA up to 7 µg. Conversely, just a small increase (0.1 µg) in the amount of competitor RNA negatively affected translation from luciferase mRNA with the 5'-end MDR1 mRNA structure (T7pGL-Loop). A progressive increase in the amount of competitor RNA, decreased luciferase translation from T7pGL-Loop (Figure 5A). Similar results were obtained when in vitro transcription and translation was performed in a single reaction using a TnT system (Promega) (data not shown).

Equally, yeast RNA could be substituted for K562 RNA as competitor, thus ruling out the effect of any putative miRNA from K562 cells (data not shown).

We hypothesized that over-expression of eIF4E would compensate for loss of available endogenous eIF4E through repression by 4EBP, and so would increase the competitive ability of pGL-Loop transcripts under cytotoxic stress. We therefore transfected K-pGL-UTR and K-pGL-Loop cells with an eIF4E expression plasmid (Figure 5B) and treated them with 1 µM colcemid for 3 days. m 7 GTP pull-down of translation initiation complexes confirmed that, upon cytotoxic treatment, more eIF4G was associated with eIF4E in cells transfected with the eIF4E expression plasmid (Figure 5C). As before, luciferase activity in transfected K-pGL-UTR and K-pGL-Loop cells was measured to estimate the relative translation efficiencies of pGL-UTR and pGL-Loop transcripts. Although translation from pGL-UTR was not affected by over-expression of eIF4E, translation from pGL-Loop was increased by approximately 40%. There was no significant difference in the steady-state levels of luciferase mRNA in each transfected cell line upon drug exposure (data not shown).

Further confirmation of the poor competitiveness of pGL-Loop transcripts was obtained by RNA interference. Transfection of siRNA targeting eIF4E in K-pGL-UTR and K-pGL-Loop cells decreased eIF4E levels equally in both cells (Figure 5E).

Although the eIF4E decrease in unstressed K562 cells did not affect global protein synthesis or translation from pGL-UTR transcripts, it decreased specifically the translation from pGL-Loop transcripts (by approximately 25%) (Figure 5F). Thus, reporter transcripts with the 5'-structure from MDR1 mRNA compete inefficiently for the limiting amounts of eIF4E in K562 cells such that, under cytotoxic stress which decreases the availability of eIF4E in the cells, translation from MDR1 transcripts is diminished.

MDR1 mRNA is translated when global translation is not down-regulated

How do drug resistant K562 cells arise and overcome the translational block in MDR1 mRNA to translate P-glycoprotein in conditions of cytotoxic stress when eIF4E is downregulated? A mechanism that would allow efficient translation of MDR1 mRNA in drug resistant K652 cells would be by increasing the levels of phosphorylated 4E-BP, with the consequent increased availability of eIF4E and enhanced translation initiation. However, western blot analysis showed that the levels of phosphorylated 4E-BP are similar in both naïve and drug resistant K562 cells (data not shown).

Therefore, to translate MDR1 mRNA drug-resistant K562 cells must increase the steady-state levels of this transcript, enabling it to compete effectively for the translation machinery.

If the above competition model is correct, MDR1 mRNA should be translated into P-glycoprotein in cells in which eIF4E availability has not been down-regulated (i.e. in cells not exposed to cytotoxic stress). Phorbol ester (TPA) treatment of K562 cells up-regulates MDR1 mRNA steady-state levels to those found in P-glycoproteinexpressing, drug-resistant KC40 cells (Supplementary Figure 1A). This up-regulation is due both to an increase in transcription from the MDR1 promoter (Supplementary Figure 2A) and to specific stabilization of MDR1 mRNA, increasing its half-life to 20 h (Supplementary Figure 2B). TPA treatment did not affect the turnover of other short-lived mRNA like Id2 [START_REF] Yagüe | P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation[END_REF] (Supplementary Figure 2C) or alter the MDR1 transcriptional start point (data not shown), ensuring the same structured mRNA is formed as from P-glycoprotein-expressing drug-resistant K562 cells. Treatment with TPA did not increase the phosphorylation status of eIF2α or alter the phosphorylation status of eIF4E and 4E-BP1, which remained similar to that of drug naïve cells, as opposed to drug-induced K562 cells (Supplementary Figure 1B).

After 16 h treatment with 32 nM TPA, approximately 30% K562 cells expressed P-glycoprotein on the plasma membrane, at 20% of the levels of KC40 cells (Supplementary Figure 1C). TPA-treated K562 cells were sorted by flow cytometry into P-glycoprotein positive and negative populations. These populations showed differential up-regulation of MDR1 mRNA. Steady-state MDR1 mRNA levels were up-regulated approximately 20-fold in P-glycoprotein positive cells when compared with P-glycoprotein negative cells (Supplementary Figure 1D). In addition, Biochemical Journal Immediate Publication. Published on 15 Jun 2007 as manuscript BJ20070235 although MDR1 mRNA levels in P-glycoprotein negative TPA-treated K562 cells were 10-fold greater than in naïve cells, this degree of up-regulation was insufficient to increase the competitive ability of MDR1 transcripts (Supplementary Figure 1D). Thus, only in the subpopulation of TPA-treated K562 cells that up-regulate levels of MDR1 mRNA sufficiently to increase its competitive ability for eIF4E, is Pglycoprotein translated.

Drug resistant K562 cells originate from a small subpopulation which differentially up-regulates MDR1 mRNA

We next asked whether differential up-regulation of MDR1 mRNA in a subpopulation of drug-treated K562 cells might occur during selection for drug resistance. We followed cultures of cells for 37 days after treatment with colchicine, in the presence and absence of 10 µM verapamil (a competitive inhibitor of P-glycoprotein). Four parameters were monitored: changes in MDR1 mRNA levels were detected by RT-Q-PCR; the percentage of viable cells (TOTO-3 negative), P-glycoprotein expression (antibody binding) and P-glycoprotein activity (BODIPY-taxol efflux) were detected by flow cytometry. Treatment with 20 ng/ml colchicine resulted in progressive cell death (Figure 6A), reaching a maximum at 26 days when only approximately 5% of the cells were alive. After longer incubation times, colchicine-resistant clones began to emerge, and after an additional 10 days most of the culture was composed of viable, colchicine-resistant cells. The first clones of colchicine-resistant cells were discernible at day 12, consisting of at least 8 cells. Assuming that the growth rate of these cells was equivalent to that of KC40 (approx 2 days doubling time), we can estimate that the first colchicine-resistant cells appear as early as day 6, equivalent to approximately 5 cells per million in the original population. While addition of verapamil alone (10-25 µM) had no effect on the growth of K562 cells, its inclusion during the selection process abolished the appearance of colchicine resistant cells, implying that P-glycoprotein function is essential for the development of drug resistance in these cells (Figure 6 and data not shown).

MDR1 mRNA levels increased slightly during the first 20 days of culture (from 0.1% to 3% of the level found in KC40), and dramatically thereafter reaching similar levels to those of KC40 cells at day 37 (Figure 6B). Similarly, the percentage of cells expressing surface P-glycoprotein (UIC2 positive), and the percentage of cells showing P-glycoprotein-mediated drug efflux (BODIPY-taxol), remained at basal levels for the first 14 days and then increased progressively until day 37 where a population phenotypically similar to KC40 was obtained (Figure 6C,D). Verapamil abolished the appearance of P-glycoprotein and BODIPY-taxol efflux positive cells.

We sorted by flow cytometry the small percentage of UIC2 positive cells (3-4%) after 7 days of treatment with 20 ng/ml colchicine and determined their MDR1 mRNA levels by RT-Q-PCR. As observed for TPA-treated K562 cells (Supplementary Figure 1D), there was a differential up-regulation of MDR1 mRNA (approximately 5-fold) between P-glycoprotein positive and negative cells. Levels of MDR1 mRNA in P-glycoprotein negative and positive cells were approximately 25and 125-fold those of naïve K562 cells, respectively (Figure 6E).

We next asked whether the naïve K562 cell line was a heterogeneous cell population (i.e. not clonal) and the cells up-regulating MDR1 mRNA were predetermined. For that we isolated, by serial dilution, 20 clones each derived from a single cell, exposed them to 1 µM colcemid for 3 days and determined the percentage of P-glycoprotein-positive cells as above. The K562 cell line and its 20 single cell derived clones all expressed equivalent percentages of P-glycoprotein-positive cells following drug exposure (data not shown). This indicates that the small percentage of cells which differentially up-regulate MDR1 mRNA and synthesize very small amounts of P-glycoprotein is not predetermined and is a stochastic process within a clonal population. Thus, differential up-regulation of MDR1 mRNA occurs stochastically in a small subpopulation of K562 cells upon exposure to cytotoxic drugs.

DISCUSSION

Acquisition of the multidrug resistance phenotype appears to be a multistep process involving many different regulatory steps and may differ between cell types [26 -28]. We have previously shown that, upon low level drug selection, multidrugresistant K562 leukemic cells arise due to overexpression of P-glycoprotein.

However, this only occurs in a small proportion of cells within the drug-exposed population which can overcome a translational block of P-glycoprotein synthesis.

This block prevents MDR1 mRNA associating with polysomes [START_REF] Yagüe | P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation[END_REF]. Here we determine how this translational block is overcome and define the mechanism, at least for this cell type, by which drug resistance develops.

Several features of an mRNA can influence its translation. mRNA transcribed from the MDR1 downstream promoter, which is the only promoter active in K562 cells [START_REF] Raguz | Activation of the MDR1 upstream promoter in breast carcinoma as a surrogate for metastatic invasion[END_REF], does not have uORFs in the 5'-UTR, and other features influencing translation such as the poly(A) tail length and polyadenylation site are unchanged irrespective of the translational status of MDR1 mRNA (data not shown). Secondary structures in the 5'-UTR, such as hairpins, are known to block translation [START_REF] Gebauer | Molecular mechanisms of translational control[END_REF].

Although the short 5'-UTR of MDR1 mRNA appears to have no negative effects on translation, it has previously been shown, by RNase mapping, that the 5'UTR can fold with the first 149 nucleotides of coding region into three highly structured hairpins with several internal loops [START_REF] Kostenko | Secondary structure of the 5'-region of PGY1/MDR1 mRNA[END_REF]. We have shown that, in vivo, when this entire 5'-MDR1 structure is cloned upstream of a reporter gene it reduces translation, specifically under conditions of cytotoxic stress.

Following cytotoxic stress, we show that eIF2α is phosphorylated and that there is a down-regulation in global cellular translation. In addition, the Akt signalling pathway is down-regulated, leading to hypophosphorylation of 4E-BP.

Hypophosphorylation of 4E-BP increases its affinity for, and sequestration of, the cap binding protein eIF4E [START_REF] Gebauer | Molecular mechanisms of translational control[END_REF]. eIF4E is generally considered rate-limiting for translation.

Crucially, not all mRNAs are affected equally by down-regulation in available eIF4E levels. It has been proposed that transcripts with long and structured 5'-UTRs are poor competitors for limiting eIF4E [START_REF] Clemens | Translational regulation in cell stress and apoptosis. Roles of the eIF4E binding proteins[END_REF][START_REF] Graff | Translational control and metastatic progression: enhanced activity of the mRNA cap-binding protein eIF-4E selectively enhances translation of metastasis-related mRNAs[END_REF], and it has long been known that mRNAs containing extensive secondary structure in their 5'-UTR translate efficiently when eIF4E is over-expressed [START_REF] Koromilas | mRNAs containing extensive secondary structure in their 5' non-coding region translate efficiently in cells overexpressing initiation factor eIF-4E. Embo J. 11[END_REF]. We demonstrate that, under cytotoxic stress, there is a down-regulation of the available eIF4E levels, and that the highly structured 5'-end MDR1 mRNA competes poorly for eIF4E and its translation is repressed, specifically.

This poor competitiveness explains the translational block of P-glycoprotein synthesis under conditions of cytotoxic stress. This suggests that compounds that mimic the 5'end cap of mRNA, such as ribavirin, or which inhibit mTOR or Akt, could decrease the competitive ability of MDR1 mRNA, preventing translation of P-glycoprotein and thereby avoiding of drug resistance.

Activation of Akt had a greater effect on the translation of MDR1 5'-end reporter mRNAs than did over-expression of eIF4E. In the same way, inhibition of mTOR activity by rapamycin had a greater effect in the translation of the same transcripts than specifically targeting eIF4E with siRNA. This suggests that other targets of mTOR (such as eIF4G and p70 S6 kinase), whose role in the activation of translational initiation is well established [31,[START_REF] Keiper | Protein synthesis initiation factor 4G[END_REF], may also play a role in MDR1 mRNA translational control.

The PI3K-Akt signalling pathway is over-activated in a wide range of tumour types promoting a variety of responses from cell growth and proliferation to survival and motility, leading to tumour progression [33]. Akt activation has also been associated with tumour metastasis and resistance to chemotherapy [START_REF] West | Activation of the PI3K/Akt pathway and chemotherapeutic resistance[END_REF]. It has been suggested that activation of the PI3K -Akt pathway in tumours leads to an increase in the availability of eIF4E and this in turns activates the translation of poorly competitive mRNAs, many of which are important for cell growth [START_REF] De Benedetti | eIF4E expression in tumors: its possible role in progression of malignancies[END_REF][START_REF] Graff | Translational control and metastatic progression: enhanced activity of the mRNA cap-binding protein eIF-4E selectively enhances translation of metastasis-related mRNAs[END_REF][START_REF] Clemens | Translational control: the cancer connection[END_REF]. Our data suggests a similar phenomenon for MDR1 mRNA, implying a mechanistic link between uncontrolled growth and drug resistance.

The poor competitiveness of MDR1 mRNA implies that, in order to develop drug resistance in the presence of drugs which reduce the availability of eIF4E, MDR1

mRNA must be up-regulated above a certain threshold in order to compete effectively for eIF4E and translate P-glycoprotein. An alternative hypothesis would be that an inhibitory factor, such as a miRNA, is out-competed when the transcripts reach a critical level. We have found different putative miRNAs targeting MDR1 3'-UTR when using the miRBase (http://microrna.sanger.ac.uk) or TargetScanS (http://genes.mit.edu/tscan/) databases, but none of them are shared between the five vertebrate target sequences available (data not shown). Thus, although we cannot rule out the possibility that some as yet uncharacterized miRNA negatively regulates MDR1 mRNA translation in K562 cells, the in vitro competition data using rabbit retyculocyte lysates (Figure 5A, and data not shown) rule out that the effect of the 5'end MDR1 loop involves a miRNA.

Under conditions in which the levels of eIF4E are not affected, such as following TPA treatment, we have shown that for P-glycoprotein to be translated, MDR1 mRNA levels must be increased at least 10-fold above those in naïve K562 cells. The threshold for MDR1 mRNA translation would be expected to be even higher under conditions, such as cytotoxic drug exposure, in which eIF4E is sequestered. On a population basis, the increase in MDR1 mRNA levels, due to increased stability, is 60 to 70-fold. Importantly, we show here that the up-regulation of MDR1 mRNA in response to cytotoxic stress is not homogeneous but that, in a small percentage of cells (less than 5%) there is a large increase in mRNA levels of up to 125-fold. This is stochastic, in that fluctuation tests showed that the small subpopulation of cells in which MDR1 mRNA is stabilised following drug treatment is not predetermined. The mechanism of this stochastic stabilisation is unknown.

However, the consequence is that this subpopulation of cells produces sufficient MDR1 mRNA to effectively compete for the reduced eIF4E, enabling a small amount of P-glycoprotein to be synthesised. The active P-glycoprotein reduces intracellular concentrations of the cytotoxic drug, leading to increased availability of eIF4E. This, in turn, increases the ability of MDR1 mRNA to compete and translate more Pglycoprotein. This positive feed-back loop, which is abolished by inhibiting Pglycoprotein function, leads to the appearance and maintenance of drug resistance.

  left panel), which is probably responsible for the down-regulation of35 S[Met] incorporation into nascent proteins reported by us previously[START_REF] Yagüe | P-glycoprotein (MDR1) expression in leukemic cells is regulated at two distinct steps, mRNA stabilization and translational initiation[END_REF] and shown in Figure2B. Extracellular stimuli also modulate the activities of both ERK1 and p38 MAP kinase (MAPK), which activate MAP kinase-interacting kinase 1 (Mnk-1) which phosphorylates eIF-4E at Ser209 [21, 22]. Colcemid treatment of K562 cells resulted in down-regulation of phosphorylated Mnk and p38 MAPK, but not of Erk, leading to hypophosphorylation of eIF4E (Figure 3A middle panel). Activation of the phosphatidylinositol 3-kinase (PI3-K)-Akt signalling pathway and phosphorylation by mTOR (mammalian target of rapamycin kinase) reduces the affinity of eIF4E-binding proteins (4E-BPs, a family of translational repressors that bind and sequester eIF4E) for eIF4E, allowing binding to eIF4G and translation inititation. Many cellular stresses also inhibit 4E-BP phosphorylation leading to increased binding of eIF4E, reduced availability of eIF4E and ultimately a decrease in the levels of translation [21, 23, 24]. Exposure of K562 cells to colcemid led to down-regulation of the PI3-K / Akt / mTOR signalling pathway and hypophosphorylation of 4E-BP (Figure 3A right panel). The effect on the phosphorylation status of eIF4E and 4E-BP was independent of the drug used, since doxorubicin and cytarabine produced similar down-regulation in phosphorylation (Figure 3B).

Figure 1

 1 Figure 1 The 5'-end of MDR1 mRNA is highly structured (A) Two-dimensional fold of the 5'-end of MDR1 mRNA, based on in vitro RNA mapping data as described [9]. The four lines joining the two first loops represent a pseudo-knot. Nucleotide numbering is in italics with the translation initiation codon at the centre of the structure, indicated as +1 Met. AUG codons are shown in black. The location of the loop within MDR1 mRNA is indicated in the diagram: white regions, UTRs; black region: MDR1coding region forming the loop with the 5'-UTR; grey region: remaining of the MDR1coding region. (B) The principal plasmids used in this study. In all cases, the position of the translation start codon is indicated (ATG). pGL-UTR has only the 5'-UTR of MDR1 mRNA (140 nucleotides) in front of the luciferase gene, whereas in pGL-Loop the entire 5'-MDR1 mRNA structure (comprising the 140-nucleotide 5'-UTR and the contiguous 149 nucleotides of coding sequence) is cloned in-frame with the luciferase gene. In pGL-LoopM, the ATGs in the loop structure have been mutated (marked as X) so that translation starts at the luciferase gene ATG. (C) Luciferase expression of K562 cells after transient transfection and phRGTK (expressing Renilla luciferase to normalize for transfection efficiency). Luciferase activity is expressed relative to that obtained with pGL3control. Data are the mean of three experiments; error bars represent the standard deviation. (D) RT-PCR analysis of luciferase transcripts from cells transfected with the luciferase reporter plasmids. Three different cycle numbers were used. As a control, GAPDH transcripts were also amplified by RT-PCR.

Figure 2

 2 Figure 2 The structure at the 5'-end of MDR1 mRNA down-regulates translation specifically upon treatment with cytotoxic drugs Stably transfected K562 cell lines were generated using pGL3, pGL-UTR, and pGL-Loop plasmids to generate K-pGL3, K-pGL-UTR, and K-pGL-Loop cells, respectively. (A) Southern blot analysis of the luciferase and β-globin loci in the three stable cell lines. All lines had similar copy number of integrated luciferase plasmid (Luciferase: β-globin ratio). (B and C) K-pGL3, K-pGL-UTR, and K-pGL-Loop cells were treated with 1 µM colcemid for 3 days. (B) Luciferase activity (white boxes) was used as a surrogate for the translation efficiency of the luciferase reporters whereas [ 35 S]Met incorporation (grey boxes) was used to determine global translation efficiency. Both are expressed as the percentage of those found in untreated cells. (C) Steady-state luciferase mRNA levels relative to those of untreated cells. The data are the mean of three experiments. Error bars represent the standard deviation. * indicates p<0.05.

Figure 3

 3 Figure 3 Down-regulation of signaling pathways controlling the phosphorylation of eIF4E and 4E-BP (A) K562 cells were treated with 1 µM colcemid for 3 days and the status of several pathways controlling the phosphorylation of the indicated translation factors analyzed by western blotting. (B) Western blot showing the phosphorylation status of eIF4E and 4E-BP after treatment of K562 cells with 0.35 µM doxorubicin and 100 µM cytarabine for 3 days. (C) m 7 GTP-sepharose pull-down and western blot analysis of initiation complexes from K562 cells treated with 1 µM colcemid for the indicated time.

Figure 4

 4 Figure 4 The phosphorylation status of 4E-BP controls the translation efficiency of transcripts with the structured MDR1 mRNA 5'-end K-pGL-UTR and K-pGL-Loop cells were transiently transfected with vector only or plasmids expressing constitutively-active forms of Mnk (pCAMNK), Akt (pCAAKT), or both, and treated with 1 µM colcemid for 3 days. Western blot analyses (A-C) were used to verify the effect of the constitutive kinase activity on the phosphorylation status of eIF4E (Mnk) and 4E-BP (Akt). Luciferase activity (D) was used as a surrogate for translation efficiency and is expressed as the percentage of that found in colcemid-treated untransfected cells. (E, F) The mTOR inhibitor rapamycin downregulates translation from pGL-Loop transcripts. K-pGL-UTR and K-pGL-Loop cells were treated with rapamycin for 8 hours. (E) Western blot analysis indicating the inhibition of 4E-BP phosphorylation by mTOR in both cell lines. (F) Relative luciferase activity (expressed as the percentage of that found in cells not treated with rapamycin) as a surrogate of pGL-UTR and pGL-Loop translation. All numerical data are the mean of three experiments; error bars represent the standard deviation. * indicates p<0.01.

Biochemical Journal Immediate Publication. Published on 15 Jun 2007 as manuscript BJ20070235

Biochemical Journal Immediate Publication. Published on 15 Jun 2007 as manuscript BJ20070235 © 2007 The Authors Journal compilation © 2007 Biochemical Society

© 2007 The Authors Journal compilation © 2007 Biochemical Society

ACKNOWLEDGEMENTS

We thank J.A. Cooper, A Willis and K. Scotto for reagents and E. O'Connor and E.

Ng for advice with FACS.


Sara Fröjdö

Delphine Cozzone

Hubert Vidal

Luciano Pirola
email: luciano.pirola@sante.univ-lyon1.fr

Resveratrol is a

Keywords: resveratrol, insulin signalling, phosphoinositide 3-kinase, sirtuins, human primary myotubes PI3K, phosphoinositide 3-kinase, PKB, protein kinase B, SIR, silent information regulator, MAPK, mitogen activated protein kinase, LPA, lysophospatidic acid, IRS, insulin receptor substrate, IC 50 , inhibitory concentration 50

Introduction

Resveratrol is a phytoalexin found in vegetal dietary sources that has been shown to possess chemopreventive and chemotherapeutic properties as well as capability to increase stress resistance and extend lifespan in eukaryotes by acting as a caloric restriction mimetic [START_REF] Baur | Therapeutic potential of resveratrol: the in vivo evidence[END_REF].

The chemopreventive/chemotherapeutic properties of resveratrol are correlated to 1) its inhibitory action on cyclooxygenase COX-1 [START_REF] Jang | Cancer chemopreventive activity of resveratrol, a natural product derived from grapes[END_REF] and other enzymes involved in prostaglandin synthesis acting downstream of COX proteins [START_REF] Sexton | Resveratrol interferes with AKT activity and triggers apoptosis in human uterine cancer cells[END_REF] and 2) its capability to decrease PI3K-controlled glucose metabolism and the activatory phosphorylation of the anti-apoptotic kinase PKB/Akt [START_REF] Sexton | Resveratrol interferes with AKT activity and triggers apoptosis in human uterine cancer cells[END_REF]4].

The function of resveratrol as a caloric restriction mimetic is though to occur via activation of Sir2-family proteins (silent information regulators, collectively termed sirtuins). Sirtuins are NAD + -dependent deacetylases involved in transcriptional silencing, genomic stability and DNA repair, p53-mediated apoptosis, lipid mobilisation and gluconeogenesis [START_REF] Yang | SIRT1 and endocrine signaling[END_REF]. These functions depend on a deacetylase activity that is not limited to histone proteins but also directed to other targets including p53 and FOXO-family forkhead transcription factors [START_REF] Vaziri | hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase[END_REF][START_REF] Brunet | Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase[END_REF].

Yeast Sir2 -as well as the metazoan SIR-2.1, Sir2 and SIRT1 orthologs from Caenorhabditis elegans, Drosophila melanogaster and mammalians respectively -are involved in lifespan control and define a major genetic pathway promoting aging [START_REF] Guarente | Genetic pathways that regulate ageing in model organisms[END_REF]. High-throughput screening aimed at discovering sirtuin activators, using a fluorescently-modified p53 peptide substrate, identified resveratrol as an in vitro activator of SIRT1 and yeast Sir2 [START_REF] Howitz | Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan[END_REF]. Resveratrol administration induced lifespan extension in yeast, C. elegans, D. melanogaster and mice fed on a high-calorie diet [START_REF] Howitz | Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan[END_REF][10][11]. Yet a caveat on the causal relationship between resveratrol-activated SIRT1/Sir2 and lifespan prolongation exists. Indeed, while in vivo sirtuin activation by resveratrol has been related to decreased PGC-1 acetylation status [12], the in vitro activatory effects of resveratrol are reportedly dependent on the fluorescently-modified substrate used in the screening [START_REF] Kaeberlein | Substratespecific activation of sirtuins by resveratrol[END_REF]14].

A further pathway central to the control of lifespan implicates insulin and insulin-like signalling -through the PI3K/PKB cascade and the downstream target forkhead transcription factors FOXO/DAF16 [START_REF] Guarente | Genetic pathways that regulate ageing in model organisms[END_REF]. Mild mutations lowering insulin-like signalling -either at the receptor level or at intracellular signalling components -lead to sirtuin-independent lifespan prolongation in eukaryotes [START_REF] Guarente | Genetic pathways that regulate ageing in model organisms[END_REF]15,[START_REF] Fabrizio | Regulation of longevity and stress resistance by Sch9 in yeast[END_REF], including mammals [17,18]. FOXO/DAF-16 may represent a convergence point of lifespan control by the insulinlike and sirtuins pathways. In fact, C. elegans SIR-2.1 -through interaction with 14-3-3 proteinsregulates DAF-16 and extends lifespan [19].

Besides controlling lifespan, SIRT1 acts as a regulator of insulin action. In hepatocytes, SIRT1 promotes gluconeogenesis via the induction of PGC-1 dependent gluconeogenic genes [START_REF] Rodgers | Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1[END_REF]; in adipose tissue, it Biochemical Journal Immediate Publication. Published on 6 Jun 2007 as manuscript BJ20070236 © 2007 The Authors Journal compilation © 2007 Biochemical Society inhibits adipogenesis and favours free fatty acid mobilisation by repressing PPAR [START_REF] Picard | Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma[END_REF] and, in pancreatic beta cells, it improves insulin secretion via repression of the UCP-2 gene [START_REF] Bordone | Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells[END_REF]23].

In an effort to uncover possible functions of SIRT1 in muscle insulin signalling we treated human primary myotubes and muscle-derived cell lines with resveratrol -in the micromolar concentration rangeand studied insulin signalling events. Here, we show that resveratrol inhibits the PI3K-PKB-FOXO signalling cascade induced by insulin without affecting the insulin induced MAPK cascade. This inhibition -rather than depending on sirtuin activation -is due to a direct inibitory action of resveratrol on class IA PI3K catalytic subunits p110 and p110 .

Class IA heterodimeric PI3Ks of the p85/p110 type are activated by tyrosine kinase receptor signalling and are involved in mitogenic signalling and metabolic control while class IB PI3K p110 is activated by G-protein coupled receptor and is mainly involved in immune responses [24,[START_REF] Vanhaesebroeck | The PI3K-PDK1 connection: more than just a road to PKB[END_REF].

Our data define class IA PI3Ks as an additional target on which resveratrol could act upon to induce lifespan extension, affect insulin action and exert its chemopreventive function.

Experimental procedures

Materials -Resveratrol, recombinant human insulin, L--phosphatidylinositol (PtdIns), L-phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), bovine brain crude phosphoinositides (PIs), 3-snphosphatidyl-L-serine (PS), sodium cholate, 2-deoxy-D-glucose and lysophosphatidic acid (LPA) were purchased from Sigma. Inositol lipids and 3-sn-phosphatidyl-L-serine were dissolved in chloroform and stored at -80 °C until use. 32 P -ATP ( 220 TBq/mmol) and [1,2 3 H] 2-deoxy-D-glucose (259 GBq/mmol) were from Amersham and NEN respectively. The following antibodies were used: anti-phospho-Ser-473 PKB, anti-phospho-Thr-308 PKB and anti-phospho-FOXO 1/4 (Cell Signaling Technology, Danvers, MA), anti PKB, anti IRS1 and anti IRS2, anti SIRT1 (Santa Cruz Biotechnology, Santa Cruz, CA), antiphospho-MAP Kinase1/2 and anti-p85 (Upstate Biotechnology, Lake Placid, NY). The adenovirus expressing constitutively activated p110 CAAX was described previously [START_REF] Pirola | Phosphoinositide 3-kinase-mediated reduction of insulin receptor substrate-1/2 protein expression via different mechanisms contributes to the insulin-induced desensitization of its signaling pathways in L6 muscle cells[END_REF]. Expression plasmids for p85 , p110 and GST-p110 , p110 , GST-p110 were previously described [START_REF] Wymann | Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction[END_REF][28][START_REF] Bondeva | Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK[END_REF].

Cell culture -L6 and CCL 136, RD cells (hereby referred as CCL), were both from ATCC, American Type Culture Collection) were cultured by standard cell culture procedures in Dulbecco's modified Eagle's Medium (DMEM), containing 4,5 g/l glucose, and supplemented with 10% Fetal Calf Serum (FCS); cells were starved in the same medium without FCS. Human primary myotube cultures were prepared as described and kept in DMEM, containing 1 g/l glucose, and supplemented with 10% FCS [START_REF] Roques | A phosphatidylinositol 3-Kinase/p70 ribosomal S6 protein kinase pathway is required for the regulation by insulin of the p85alpha regulatory subunit of phosphatidylinositol 3-kinase gene expression in human muscle cells[END_REF].

Western blot, Immunoprecipitations and PI3K assays -Cells were lysed in a lysis buffer containing 20 -5-mM EDTA, 1 mM Na 3 VO 4 , 20 mM NaF, 1 mM dithiothreitol, protease inhibitor cocktail (Sigma P2714), pH 8.0. Lysates were kept on ice for 15 minutes and cleared by centrifugation at 13 000 x g for 15 minutes. Protein concentrations were determined by the Bio-rad protein assay reagent (Bio-rad).

When subjected to immunoprecipitation, cell lysates were incubated with protein A sepharose and antibodies to p85 , IRS-1 or IRS2 as indicated.

Cell lysates (10 µg to 30 µg of protein) were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Perkin Elmer), and blocked in 3% (w/v) bovine serum albumin or 4% skimmed dry milk in Tris buffered saline / 0,5% tween-20. Membranes were incubated overnight at 4 °C with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies and Enhanced Chemiluminescence (Pierce) detection. PI3K protein kinase assays on overexpressed PI3Ks were performed as described [START_REF] Wymann | Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction[END_REF].

PI3K lipid kinase assays on IRS1 immunoprecipitates and overexpressed PI3Ks were performed as follows: protein A-bound p85 /p110 , p85 /p110 and IRS1 immune-complexes or gluthatione sepharose-bound p85 /GST-p110 and GST-p110 were washed twice with ice-cold PI3K lipid kinase buffer (10 mM HEPES, 5 mM MgCl 2 .) After washing, Protein A / gluthatione sepharose beads containing residual lipid kinase buffer ( 5 µl) were incubated on ice in 25 µl lipid kinase buffer containing 2x concentrated resveratrol for 30 min. 10 µl sonicated lipids (see below for their preparation) were added for 10 min (on ice) and phosphorylation reactions were started by adding 10 µ l of 50 µ M ATP supplemented with 0.5 µCi/µl 32 P -ATP (final ATP concentration = 10 µM, supplemented with 5 µCi 32 P -ATP/reaction). Reactions were let to proceed for 20 min at room temperature on a shaking platform.

Lipid mixes were prepared as follows: chloroform was removed under vacuum from stock solution.

PtdIns (10 µg/reaction) was resuspended in lipid kinase buffer prior to sonication. Bovine brain crude phosphoinositides (PIs, 10 µg/reaction) and PtdIns(4,5)P 2 / PS mix (5 µg and 10 µg/reaction respectively) were resuspended in lipid kinase buffer containing 2,5% (w/v) sodium cholate prior to sonication.

Reactions were stopped by the addition of 50 µl 1M HCl. Phosphorylated lipids were separated by the unreacted ATP by addition of 200 µl methanol/chloroform 1:1 (v/v). After vortexing and centrifugation, the lipid-containing lower phase was collected and dried under vacuum.

Extracted lipids from reactions using PtdIns(4 ,5)P 2 or PIs as substrate were separated on oxalate-treated silica 60 TLC plates (Merck) with chloroform/acetone/methanol/acetic acid/water (80:30:26:24:14, v/v/v/v/v). Extracted lipids from reactions using PtdIns as substrate were separated on silica 60 TLC plates (Merck) with chloroform/methanol/water/ammonia (45/35/8.5/1.5, v/v/v/v). Phosphorylated lipids were detected by autoradiography on Kodak X-Omat films and quantified by phosphorimaging.

Biochemical Journal Immediate Publication. Published on 6 Jun 2007 as manuscript BJ20070236

© 2007 The Authors Journal compilation © 2007 Biochemical Society 2-deoxyglucose uptake in 3T3-L1 adipocytes -3T3-L1 preadipocytes seeded in 12-wells plates were differentiated as previously described [START_REF] Orlicky | Construction of stable coxsackievirus and adenovirus receptor-expressing 3T3-L1 cells[END_REF]. Once differentiated, cells were starved overnight in DMEM without FCS. Insulin-induced 2-deoxyglucose uptake was determined as previously described [START_REF] Pirola | Phosphoinositide 3-kinase-mediated reduction of insulin receptor substrate-1/2 protein expression via different mechanisms contributes to the insulin-induced desensitization of its signaling pathways in L6 muscle cells[END_REF].

RNA interference -The short interfering RNA duplex targeting SIRT1 was purchased from Qiagen (Courtaboeuf, France). Transfection with siRNAs was performed by the calcium phosphate method as described [START_REF] Pirola | Phosphoinositide 3-kinase-mediated reduction of insulin receptor substrate-1/2 protein expression via different mechanisms contributes to the insulin-induced desensitization of its signaling pathways in L6 muscle cells[END_REF]. The sense sequence of the siRNA for SIRT1 was as follows: 5'

CCCUGUAAAGCUUUCAGAAdTdT.

Statistical analysis -Unless otherwise indicated, all experiments were performed at least three times.

Values are expressed as means ± standard deviation (SD) or standard error of the mean (SEM). Data were analysed by unpaired Student's t test (unless otherwise indicated) and differences were considered as statistically significant when p<0.05.

Results:

Resveratrol inhibits PI3K activity and PI3K downstream targets in human primary myotubes and muscle derived cell lines. The deacetylase SIRT1 has been implicated in the control of insulin secretion by pancreatic beta cells [START_REF] Bordone | Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells[END_REF]23] and has been shown to affect insulin signalling in hepatocytes and adipocytes [START_REF] Rodgers | Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1[END_REF][START_REF] Picard | Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma[END_REF]. To investigate the possible function of SIRT1 in muscle insulin signalling we used the small polyphenolic compound resveratrol, reported to activate SIRT1 [START_REF] Howitz | Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan[END_REF].

In human primary myotubes, insulin-induced PKB Serine-473 and Threonine-308 phosphorylations were fully prevented by a 30 minute pre-treatment with 100 µM resveratrol as was Serine-193 phosphorylation of the PKB downstream target FOXO4 (Figure 1A). Such a complete inhibitory effect -obtained with resveratrol concentrations employed to activate SIRT1 -is reminiscent of that observed upon treatment of cells with the PI3K inhibitors wortmannin or LY294002.

Upon insulin stimulation, PI3K associates to the insulin receptor substrate molecules IRS1 and IRS2 [START_REF] White | The insulin signalling system and the IRS proteins[END_REF].

We thus evaluated the in vitro inhibitory effect of resveratrol on the IRS1-PI3K complex immunoprecipitated following insulin stimulation of primary myotubes. Increasing concentrations of resveratrol were added to IRS1 immunoprecipitates, that were subsequently subjected to a PI3K assay.

Resveratrol inhibited IRS1-associated PI3K activity with an IC 50 of 25 µM (Figure 1B). To corroborate the above findings, we tested the effect of resveratrol on insulin-signalling in rat L6 and human rhabdomyosarcoma CCL muscle-derived cell lines. As in primary myotubes, 100 µM resveratrol pretreatment fully inhibited insulin-induced PKB Serine-473 and Threonine 308 phosphorylations and, in both cell lines, the IC 50 for such inhibition was < 10 µM (Figure 2A and2C). In L6 cells, insulin-induced activation of the MAPK pathway was only mildly affected by the highest concentration of resveratrol employed (Figure 2A), and the insulin-induced interaction between the PI3K p85 adapter subunit and 2A) suggesting that resveratrol exerts its inhibitory action downstream of IRS1/2. Since an inhibitory effect of resveratrol on PI3K was also observed in IRS1-PI3K immune complexes from L6 cells (Figure 2B), PI3K is the likely target of resveratrol.

A major biological response induced by insulin via PI3K is the translocation of the glucose transporter GLUT4 at the plasma membrane and a consequential increase in glucose uptake [START_REF] Bryant | Regulated transport of the glucose transporter GLUT4[END_REF]. We next tested the effect of resveratrol on insulin-induced 2-deoxyglucose uptake in 3T3-L1 adipocytes, a cell line displaying an increase in 2-deoxyglucose uptake after insulin stimulation. Insulin treatment induced a 40.0 % increase of 2-deoxyglucose uptake (p<0.05, paired t-Test, n=6). 100 µM resveratrol pre-treatment yielded a 82.7% decrease of insulin-induced 2-deoxyglucose transport in comparison to insulin treated cells (p<0.01, paired t-Test, n=6) thus lowering the overall uptake below the basal level. This indicates that resveratrol not only affects insulin-stimulated glucose uptake but also -as previously reported in U937 and HL-60 cells [START_REF] Park | Inhibition of glucose and dehydroascorbic acid uptakes by resveratrol in human transformed myelocytic cells[END_REF] -the basal GLUT-1 dependent glucose transport.

In C. Elegans, the SIRT1 ortholog SIR-2.1 interacts with 14-3-3 proteins to mediate lifespan extension [19]. As 14-3-3 proteins reportedly interact with IRS1 [35, 36], IRS1 could in principle coimmunoprecipitate SIRT1 that would be targeted by resveratrol and cause -perhaps by an indirect mechanism -PI3K inhibition. However, we have been unable to detect SIRT1 in IRS1 immune complexes neither before nor after insulin stimulation (Figure 7A).

We next asked whether the inhibitory action of resveratrol on PI3K and PKB occurs when cells are subjected to a stimulus other than insulin. Adenovirus-mediated expression of a membrane-targeted consitutively active PI3K (p110 CAAX) induced a PKB serine-473 phosphorylation -independently from an extracellular stimulation -that was inhibited by resveratrol treatment (Figure 3A), suggesting a direct inhibitory effect of resveratrol upon p110 PI3K. As observed with insulin treatment, stimulation of resveratrol-treated L6 cells with 10% FCS resulted in a complete blockade of PKB phosphorylation but not MAPK phosphorylation (Figure 3A). Likewise, resveratrol inhibited LPA-mediated PKB phosphorylation (Figure 3B).

Resveratrol targets the class IA PI3K ATP-binding site in a competitive and reversible fashion. To gain insight into isoform-specificity, class IA p85 /p110 , p85 /p110 and class IB GST-p110 were overexpressed in 293 cells and their sensitivity to resveratrol was tested in in vitro lipid kinase assays by using PtdIns as substrate. p85 /p110 was inhibited by resveratrol with an IC 50 of 25µM, the IC 50 towards p85 /p110 was 50 µM, while class IB GST-p110 was less sensitive to resveratrol, retaining 75% of the activity when treated with 100 µM resveratrol (IC 50 > 100 µM, Figure 4A). While PtdIns is routinely used to test in vitro PI3K activity, the PI3K physiological substrate is PtdIns(4,5)P 2 [24]. We thus tested the inhibitory action of resveratrol on p85 /GST-p110 by using PtdIns(4,5)P 2 as substrate (Figure 4B), and likewise observed an IC 50 of 25µM and 90% inhibition at 100 µM resveratrol. Furthermore, comparison of the lipid kinase activities of p85 /GST-p110 versus GST-p110 by using PtdIns(4,5)P 2 as substrate confirmed the modest inhibitory action of resveratrol towards GST-p110 (Supplementary figures 1A and 1B). As a final approach to determine the inhibitory action of resveratrol towards PI3K, we performed lipid kinase assays using bovine brain crude phosphoinositides (PIs), which contain a mixture of PtdIns, PtdIns4P and PtdIns(4,5)P 2 . Inhibition of p85 /GST-p110 activityby resveratrol was observed using either substrate (PtdIns(4,5)P 2 , or PIs, Figure 4C).

Finally, inhibition of class IA p85 /p110 , p85 /p110 , p85 /GST-p110 and class IB GST-p110 by100 µM resveratrol was evaluated by using PIs as substrate. As observed with PtdIns and PtdIns(4,5)P 2 , inhibition was maximal for class IA p110 isoforms and moderate for the class IB p110 isoform (Supplementary figure 1C,D).

The same differential sensitivity towards inhibition by resveratrol was observed in relation to the PI3K protein kinase activities of p110 /p110 versus p110 . While p85 /GST-p110 autophosphorylation was inhibited by resveratrol in a concentration-dependent manner, GST-p110 autophosphorylation was not affected by resveratrol (Figure 5 and Supplementary figure 2A) and both p85 /p110 [START_REF] Dhand | PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity[END_REF], p85 /p110 [28] autophosphorylation activities were inhibited by 100µM resveratrol, albeit to a smaller extent than the inhibition observed by LY treatment (Supplementary figure 2B).

The two most widely used PI3K inhibitors, wortmannin and LY, differ in their mode of action. Both inhibitors target competitively the ATP/substrate binding site. However, wortmannin binds covalently to a lysine involved in the phosphotransfer reaction (lysine 802 in p110 ) causing an irreversible inhibition, while LY binding to the catalytic region is reversible [START_REF] Vlahos | A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002)[END_REF]. Pre-treatment of L6 cells with 100 nM wortmannin or 50 µM LY resulted in the complete inhibition of insulin-stimulated PKB phosphorylations on Serine 473 and Threonine 308. Medium replacement between inhibitor treatment and insulin stimulation led to the loss of the inhibitory action of LY but not wortmannin (Figure 6B). Under these experimental conditions, the mode of action of resveratrol resembles that of LY, and thus does not occur through a covalent binding to the p110 catalytic subunit.

To assess whether resveratrol inhibits PI3K in a competitive fashion, p85 /p110 lipid kinase activity was tested in the presence of 50 µM resveratrol and increasing concentrations of ATP (Figure 6A). The inhibitory action of resveratrol was diminished by increasing ATP concentrations, indicating that resveratrol acts as a PI3K inhibitor by competing with ATP for the catalytic site.

Biochemical Journal Immediate Publication. Published on 6 Jun 2007 as manuscript BJ20070236 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Inhibition of PI3K by resveratrol is sirtuin-independent. As discussed above, IRS1-associated PI3K activity is inhibited by resveratrol, albeit SIRT1 does not co-purify in IRS1 immune complexes, suggesting a SIRT1-independent inhibition directly targeting PI3K. To conclusively demonstrate that resveratrol-mediated PI3K inhibition is sirtuin-independent, we downregulated endogenous SIRT1 by RNA interference obtaining a >80% decrease in expression. Under these conditions, resveratrol inhibition of insulin-stimulated PKB serine-473 phosphorylation was unaltered (Figure 7B), demonstrating the lack of involvement of SIRT1 in the observed inhibition.

Discussion

Resveratrol, a polyphenolic compound found in appreciable amounts in grapes and red wine, is a currently widely investigated molecule for its potentially beneficial effects on health and its capability to promote longevity [11].

In yeast, contrasting reports exist as to whether resveratrol administration increases replicative lifespan (Reviewed in [START_REF] Longo | Sirtuins in aging and age-related disease[END_REF]). In animal models, from lower metazoans to vertebrates, including small mammals, resveratrol administration has the remarkable property to prolong lifespan [10, 11, 40].

Several genes have been identified that play a part in the control of lifespan, including genes implicated in insulin-like signalling and genes coding for the Sir2/SIRT1 sirtuin family of deacetylases [START_REF] Guarente | Genetic pathways that regulate ageing in model organisms[END_REF]. Highthroughput screening for sirtuin activators identified resveratrol as an activator of SIRT1 (13.4 fold at 100 µM) and Sir2 (2 fold at 2-5 µM) [START_REF] Howitz | Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan[END_REF]. This finding, combined with genetic evidence of the involvement of Sir2/SIRT1 in lifespan prolongation led to the suggestion that resveratrol acts through enzymatic activation of sirtuins. [START_REF] Howitz | Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan[END_REF]10]. Yet, recent studies suggest that resveratrol-induced increase of sirtuin enzymatic activity is dependent on the modified substrate used in the in vitro screening [START_REF] Kaeberlein | Substratespecific activation of sirtuins by resveratrol[END_REF]14].

Beside sirtuins being bona fide enzymes activated by resveratrol, other targets have been proposed that may explain the aging-promoting, antioxidant and chemopreventive/chemotherapeutic properties of resveratrol, including cyclooxygenases [START_REF] Jang | Cancer chemopreventive activity of resveratrol, a natural product derived from grapes[END_REF], intracellular signalling cascades activating nuclear factor-kB (NF-kB) and activator protein-1 (AP-1) [START_REF] Kundu | Molecular basis of chemoprevention by resveratrol: NF-kappaB and AP-1 as potential targets[END_REF], AMPK [11] as well as the insulin-IGF-1 signalling pathways [START_REF] Zhang | Resveratrol inhibits insulin responses in a SirT1-independent pathway[END_REF]. Intriguingly, in the report by Zhang [START_REF] Zhang | Resveratrol inhibits insulin responses in a SirT1-independent pathway[END_REF] describing the inhibition of both the PKB and MAPK signalling pathways by resveratrol in primary hepatocytes and hepatocyte-derived cell lines [START_REF] Zhang | Resveratrol inhibits insulin responses in a SirT1-independent pathway[END_REF], resveratrol appears to work through inhibition of the insulin-stimulated interaction between IRS1 and PI3K and between IRS-1 and Grb-2 [START_REF] Zhang | Resveratrol inhibits insulin responses in a SirT1-independent pathway[END_REF]. On the contrary, we failed to observe inhibition of the IRS1/PI3K p85 interaction (Figure 2A In most of the above-mentioned resveratrol-modulated pathways, the genuine molecular target has not been identified or it is controversial. In our investigation we obtained data indicating that i) the catalytic subunits p110 and p110 of class IA PI3Ks are inhibited in vitro by resveratrol in the micromolar concentration range with an IC 50 25 mM; ii) PI 3-kinase -dependent phosphorylation of downstream targets (including PKB and FOXO) is inhibited by resveratrol with an IC 50 < 10 µM for insulin induced PKB serine-473 phosphorylation. This inhibition occurred in several muscle-derived cell lines, including rat L6, rabdomyosarcoma CCL and human primary myotubes derived from several donors. The different IC 50 s observed in PI3K in vitro assays versus PKB serine-473 measurements may be a reflection of a different affinity of resveratrol towards PI3K in a cell culture setting versus the test-tube. Finally, iii) an insulin-dependent end-point response such as insulin-induced glucose uptake in 3T3-L1 adipocytes was likewise inhibited by resveratrol.

Both the lipid-and protein kinase activities of p110 and p110 catalytic subunits are affected by resveratrol and this inhibition explains the in vivo blockade of the PI3K-PKB signalling pathway induced by insulin, LPA or stimulation via FCS in human primary myotubes and muscle-derived cell lines.

Overexpression of both class IA and class IB PI3Ks followed by in vitro lipid-kinase and protein-kinase assays allowed us to show that resveratrol is a class IA specific PI3K inhibitor. Nonetheless, further investigation will be necessary to define whether the class IB PI3K p110 relative refractivity to resveratrol inhibition is maintained in intact cells. Based on competition studies with ATP, resveratrol appeared to act as a competitive inhibitor targeting the ATP binding pocket. By comparing the inhibition of insulin-induced PKB phosphorylation by LY294002, wortmannin and resveratrol, we show that inhibition by resveratrol is reversible.

The original screening leading to the identification of resveratrol identified other molecules structurally related to resveratrol capable to activate Sir2/SIRT1, including quercetin. Quercetin is a naturally occurring flavonoid reported to be a broad-spectrum protein kinase inhibitor [START_REF] Srivastava | Inhibition of phosphorylase kinase, and tyrosine protein kinase activities by quercetin[END_REF] IC 50 s as class IA PI3K. Additionally to a possible inhibitory activity on other kinases, the pleiotropic effects of resveratrol are best underscored by the fact that it also activates AMPK in C2C12 myotubes [11], a phenomenon that we also observed in human primary myotubes and L6 cells (data not shown).

In conclusion, our observation that class IA PI3K is inhibited by resveratrol in the micromolar range usually employed in lifespan extension, stress resistance, and chemoprevention studies define a further target on which resveratrol could act upon and has several implications: i) In cancer, the PI3K/PKB pathway is virtually always activated [START_REF] Hennessy | Exploiting the PI3K/AKT pathway for cancer drug discovery[END_REF]. The chemopreventive and/or chemotherapeutic properties of resveratrol -in addition to its inhibitory action on COX [START_REF] Jang | Cancer chemopreventive activity of resveratrol, a natural product derived from grapes[END_REF] -could be consequential to the negative regulation of the PI3K pathway. ii) Active research is currently engaged in the discovery of PI3K inhibitors with therapeutic potential. While several synthetic class-and isoform-specific PI3K inhibitors have been described [47-49], resveratrol provides an example of a natural and nontoxic molecule endowed with isoform-selectivity towards class IA PI3K. iii) Finally, given the interplay of the insulinlike and sirtuin pathways in ageing regulation, resveratrol may exert its action on both pathways. Further research is thus warranted to determine to which extent each of the two pathways is modulated by resveratrol and whether resveratrol inhibits PI3K in lower metazoans and unicellular eukaryotes.

Biochemical

  Journal Immediate Publication. Published on 6 Jun 2007 as manuscript BJ20070236 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  ) at concentrations up to 100 µM. Such discrepancy is probably dependent on the different cell types investigated and a direct comparison of the action of resveratrol in hepatocytes versus muscle cells is warranted. Biochemical Journal Immediate Publication. Published on 6 Jun 2007 as manuscript BJ20070236 © 2007 The Authors Journal compilation © 2007 Biochemical Society

  and a PI 3-kinase inhibitor (IC 50 = 3,8 µM) [44]. The crystal structures of the tyrosine kinase Hck [45] and PI3K p110 [44] complexed with quercetin showed that the 4' hydroxy group make a hydrogen bond to the catalytic phosphoacceptor Hck lysine-295 and p110 lysine-833. By comparing the structures of resveratrol and quercetin we hypothesise that the 4' hydroxyl group of resveratrol makes a hydrogen bond to the p110 and p110 catalytic lysines in the ATP-binding site. Taken together, our data demonstrate that resveratrol targets class IA PI3Ks and consequently inhibit their downstream signalling molecules. However, given the broad spectrum of kinases inhibited by the related molecule quercetin, we cannot discount that other kinases might be inhibited by resveratrol with similar Biochemical Journal Immediate Publication. Published on 6 Jun 2007 as manuscript BJ20070236 © 2007 The Authors Journal compilation © 2007 Biochemical Society Frojdo et. al; resveratrol inhibits PI 3-kinase -11-

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Inhibition of the PI3K-PKB pathway, but not of the MAPK pathway, by resveratrol in L6 and CCL cells. (A) L6 myoblasts were serum-starved overnight, pre-treated for 30 min with the indicated concentrations of resveratrol and subsequently stimulated for 10 min with 1000 nM insulin. Separated total proteins were immunoblotted with antibodies to PKB pS473, PKB and pMAPK. Furthermore, equal amounts of lysates were immunoprecipitated with antibodies to IRS1 and IRS2 and the associated PI3K visualised by immunoblotting with anti-p85 antibodies (I.P., immunoprecipitation; IgG, immunoglobulin G). (B) IRS1 associated PI3K activity was measured in IRS1 immunoprecipitates obtained from starving (-) or insulin-stimulated L6 cells (+, 1000 nM insulin, 10 minutes). Immunecomplexes were pre-treated with the indicated concentrations of resveratrol. 32 P-labelled PtdIns3P was separated by TLC as in Figure 1. (C) Resveratrol dose-response inhibition of insulin-induced PKB S473 and T308 phosphorylations in CCL cells. Equal loading was verified by immunoblotting of total PKB.

Figure 3 .

 3 Figure 3. Resveratrol inhibits PKB S473 phosphorylation induced by various agonists. (A) L6 cells were infected for 48h with an adenovirus expressing a constitutively active p110 (Adp110 CAAX) as indicated. After overnight serum-starvation, cells were pretreated for 30 min with 100 µM resveratrol (Res.) as indicated and subsequently stimulated with 10% fetal calf serum (FCS). Cell lysates were immunoblotted with antibodies to PKB pS473 and pMAPK. (B) CCL cells were starved overnight, pretreated for 30 min with the indicated concentrations of resveratrol and subsequently stimulated with 10

Figure 4 .

 4 Figure 4. Resveratrol inhibits recombinant class IA, but not class IB, PI3K lipid kinase activity. (A) Relative dose-response inhibition of class IA p85 /p110 , p85 /p110 and class IB GST-p110 in lipid kinase assays using PtdIns as substrate. Representative TLCs for each PI3K isoform are shown on the bottom. Assays for each experimental condition were performed at least four times. Activity in the absence of resveratrol was normalised at 100%. (B) Relative dose-response inhibition of class IA p85 /GST-p110 in a lipid kinase assay using PtdIns(4,5)P 2 as substrate and representative TLC. Assays for each experimental condition were performed in triplicate. (C) Inhibition of class IA p85 /GSTp 1 1 0 by 100µM resveratrol by using PtdIns(4,5)P 2 or PIs as substrates. Data are from at least 3 independent assays, activity in the absence of resveratrol was normalised at 100%. Bars indicate the standard deviation. The significance of inhibition was compared to the assays without resveratrol using Student's t test. *P<0.05, **P<0.01, ***P<0.001.

Figure 5 .

 5 Figure 5. Resveratrol inhibits recombinant class IA, but not class IB, PI3K protein kinase activity. Relative dose-response inhibition of class IA p85 / G S T -p 1 1 0 and class IB GST-p110 autophosphorylation activities. % activity refers to the incorporated radioactivity into p85 and GST-p110 bands relative to the autophosphorylation reaction without resveratrol. Bottom: quantification of incorporated radioactivity from three independent experiments. Bars indicate the standard deviation. The significance of inhibition was compared to the assays without resveratrol using Student's t test. *P<0.1, **P<0.05. p85 /GST-p110 and GST-p110 expression levels were visualised by coomassie-blue staining (see supplementary figure 2A).

Figure 6 .

 6 Figure 6. Resveratrol targets the PI3K ATP-binding site in a non-covalent fashion. (A) The lipid kinase activity of class IA p85 /p110 was assayed in the absence (-) or in the presence (+) of 50 µM resveratrol (Res.) and with increasing ATP concentrations as indicated. The experiment shown is representative of two independent ones in which each condition was tested in duplicate. % activity is the mean from 4 independent reactions (B) Serum-starved L6 cells were pretreated with 50 µM LY294002 (LY), 100 µM resveratrol (R) or 100 nM wortmannin (W) as indicated for 30 min. Prior to a 10 min stimulation with 1000 nM insulin, the medium was replaced in the lanes indicated (washout), thus allowing the elimination of non-covalently bound inhibitor. Separated proteins were immunoblotted with antibodies to PKB pS473, PKB pT308 and total PKB to monitor equal loading.

Figure 7 .Figure 1 B

 71 Figure 7. SIRT1 is not found in IRS1 immune complexes and resveratrol inhibits the insulin-activated PKB independently of sirtuin expression. (A) L6 myoblasts were serum-starved overnight and subsequently stimulated for 10 min with 1000 nM insulin. Separated total proteins were immunoblotted with antibodies to p85 (that also recognises the short-spliced forms p50 and p55 ) and SIRT1. In parallel, equal amounts of lysates were immunoprecipitated with antibodies to IRS1 and immunoblotted with anti-p85 and -SIRT1 antibodies (I.P., immunoprecipitation; IgG, immunoglobulin G). (B) L6 myoblasts were transfected with siRNAs directed to SIRT1. After an overnight starving and 30 minutes 100 µM resveratrol pretreatment as indicated cells were stimulated with 1000 nM insulin for 10 minutes.Total lysates were immunoblotted with antibodies to PKB pS473 and SIRT1.

Biochemical Journal Immediate Publication. Published on 6 Jun 2007 as manuscript BJ20070236 © 2007 The Authors Journal compilation © 2007 Biochemical Society

	Frojdo et. al; resveratrol inhibits PI 3-kinase
	mM Tris-HCl, 138 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 5% (v/v) glycerol, 1% (v/v) Nonidet P-40, 5

Biochemical Journal Immediate Publication. Published on 6 Jun 2007 as manuscript BJ20070236 © 2007 The Authors Journal compilation © 2007 Biochemical Society

	IRS1 or IRS2 was unaffected (Figure

Biochemical Journal Immediate Publication. Published on 6 Jun 2007 as manuscript BJ20070236 © 2007 The Authors Journal compilation © 2007 Biochemical Society Frojdo et. al; resveratrol inhibits PI 3-kinase

Biochemical Journal Immediate Publication. Published on 6 Jun 2007 as manuscript BJ20070236 © 2007 The Authors Journal compilation © 2007 Biochemical Society

This work has been supported by an ARD/PNRD (Association pour la Recherche sur le Diabète and Programme national de recherche sur le diabète) grant (to L.P.). We thank Bernd Nürnberg (University of Duesseldorf, Germany) for providing the pcDNA3-p110 plasmid. We thank Christine Durand for technical assistance.