Stefano Grosso

Viviana Volta

Leonardo A Sala

Pier Marina Vietri

Carlo Marchisio

Dorit Ron

Stefano Biffo
email: biffo.stefano@hsr.it

PKCβII modulates translation independently from mTOR and through RACK1

Keywords: mTOR independent translational control PKC, RACK1, initiation, phorbol esters, hispidin, eIF6 Abbreviations used: PKC,Protein Kinase C

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PKCβII Modulates Translation Independently from mTOR and through RACK1

Stefano GROSSO *, † ¶ , Viviana VOLTA * † ¶ , Leonardo A. SALA * , Marina VIETRI * † , Pier Carlo MARCHISIO ‡ , Dorit RON § , Stefano BIFFO * †1 INTRODUCTION Translation requires at least 11 eIFs (eukaryotic Initiation Factors) controlling rate and accuracy [START_REF] Pestova | Functions of eukaryotic factors in initiation of translation[END_REF]. The global initiation rate is controlled by signaling on the eIF4F complex, regulating cap-dependent translation, and on the eIF2 complex, bringing the ternary complex (tRNA i Met , eIF2 and GTP) on the 40S subunit [START_REF] Holcik | Translational control in stress and apoptosis[END_REF]. The most studied stimulatory pathway on translation is through mTOR (mammalian Target Of Rapamycin) kinase that acts on the formation of the cap complex, by the phosphorylation of the negative regulator 4E-BP1 (eIF4E-Binding Protein 1) [START_REF] Lin | PHAS-I as a link between mitogen-activated protein kinase and translation initiation[END_REF][START_REF] Pause | Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function[END_REF]. The mTOR pathway is activated by growth factors, and optimal nutrient conditions. However, in most cells mTOR inhibition by rapamycin blocks only 15% of the translation rate [START_REF] Jefferies | Rapamycin selectively represses translation of the "polypyrimidine tract" mRNA family[END_REF], suggesting that other pathways parallel to mTOR may be important. For instance, stimulation of translation by adhesion to extracellular matrix is totally independent from mTOR activation [START_REF] Gorrini | Fibronectin controls cap-dependent translation through beta1 integrin and eukaryotic initiation factors 4 and 2 coordinated pathways[END_REF], and translational recovery after hypertonic stress requires Erk1/2 signaling [START_REF] Naegele | Molecular cross-talk between MEK1/2 and mTOR signaling during recovery of 293 cells from hypertonic stress[END_REF]. Thus, dissecting mTOR independent pathways is of major importance, in order to fully grasp the complexity of translational regulation. The protein kinase C superfamily includes different isoforms that are involved in most cellular processes, including cell cycle control, differentiation, and apoptosis. In mammals, eleven PKC isoforms exist, divided into three subgroups on the basis of their responsiveness to general PKC activators [START_REF] Mellor | The extended protein kinase C superfamily[END_REF]. Conventional PKCs (PKCα, PKCβI/II, PKCγ) require both diacylglycerol (DAG) and Ca 2+ for activation. They can also be activated by phorbol esthers, such as phorbol 12-myristate 13-acetate (PMA), in a Ca 2+ -dependent manner. Novel PKCs (PKCδ, PKCε, PKCη, PKCθ) are DAG/PMA sensitive but do not respond to Ca 2+ . Finally, atypical PKCs (PKCζ and PKCι/λ) are unresponsive to DAG and Ca 2+ and are activated by interaction with other signaling proteins. PKCs have long been thought to be involved in translational control, on the basis of the observation that, in general, PKC stimulation by pharmacological agents leads to increased translation [START_REF] Herbert | The extracellular signal-regulated kinase pathway regulates the phosphorylation of 4E-BP1 at multiple sites[END_REF]. In principle, PKC-mediated control of translation can either be due to activation of pathways upstream of initiation factors, namely through the PI3K/Akt/mTOR and MAPK/S6K pathways [START_REF] Hay | Upstream and downstream of mTOR[END_REF], or to the direct phosphorylation of initiation factors and ribosome-associated proteins. As an example of indirect activation of the translational machinery, PKCη acts both on the activation of the PI3K, mTOR cascade [START_REF] Aeder | PKC-eta mediates glioblastoma cell proliferation through the akt and mTOR signaling pathways[END_REF] and the S6K phosphorylation [START_REF] Martin | Phorbol 12-myristate 13-acetate and serum synergize to promote rapamycin-insensitive cell proliferation via protein kinase C-eta[END_REF], therefore triggering eIF4F complex formation. Increased levels of eIF4F then stimulate cap-dependent translation. The direct phosphorylation of components of the translational apparatus by PKC is less clear-cut. Several reports have identified among initiation factors and ribosome-associated factors, potential substrates of PKC, in vitro, but their unequivocal demonstration, in vivo, is lacking. A case is the one of eIF4E that is efficiently phosphorylated by PKC, in vitro, [START_REF] Whalen | Phosphorylation of eIF-4E on serine 209 by protein kinase C is inhibited by the translational repressors, 4E-binding proteins[END_REF] and is found phosphorylated upon PKC activation, in vivo [START_REF] Morley | Differential stimulation of phosphorylation of initiation factors eIF-4F, eIF-4B, eIF-3, and ribosomal protein S6 by insulin and phorbol esters[END_REF]. However, knockout studies showed that Mnk1 and Mnk2 (MAP kinase interacting kinases 1 and 2) are the responsible kinases, in vivo [START_REF] Ueda | Mnk2 and Mnk1 are essential for constitutive and inducible phosphorylation of eukaryotic initiation factor 4E but not for cell growth or development[END_REF]. Similarly, in vitro phosphorylation by PKC has been reported for the elongation factor eEF1 [START_REF] Kielbassa | Protein kinase C delta-specific phosphorylation of the elongation factor eEF-alpha and an eEF-1 alpha peptide at threonine 431[END_REF], and recently for other proteins associated with the translational machinery such as eIF6 [START_REF] Ceci | Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly[END_REF]. This said, the issue of direct substrates of PKC on the ribosomal machinery, in vivo, is complicated by the existence of multiple PKC isoforms differentially expressed and by the fact that it is unclear whether PKCs and ribosome-associated substrates can be in close proximity. In this context, the requirement of membrane-derived DAG for PKC activation brings a positional constraint for the phosphorylation of ribosome-associated factors, and suggests that PKCs may act directly on the ribosomal machinery exclusively in the presence of adaptors or intermediate molecules, or in proximity of membranes. It is therefore important to verify whether specific PKC isoforms can copurify with ribosomes or membrane-ribosome complexes. One putative adaptor between PKC and ribosomes is RACK1.

RACK1 (Receptor for Activated C Kinase) is a conserved protein homologous to the β-subunit of heterotrimeric G proteins. Its sequence contains seven internal Trp-Asp 40 (WD40) domains, devoted to protein-protein interaction. WD40 domains are predicted to fold into a β-propeller structure, each domain forming a blade of the propeller [START_REF] Steele | Identification of a surface on the beta-propeller protein RACK1 that interacts with the cAMP-specific phosphodiesterase PDE4D5[END_REF][START_REF] Sondek | Ggamma-like (GGL) domains: New frontiers in Gprotein signaling and beta-propeller scaffolding[END_REF]. RACK1 has been identified as a receptor for PKC, after its activation by DAG and Ca 2+ [START_REF] Ron | Cloning of an intracellular receptor for protein kinase C: A homolog of the beta subunit of G proteins[END_REF]. Subsequent work has shown that the preferred PKC isoform(s) binding RACK1 are PKCβs [START_REF] Ron | C2 region-derived peptides inhibit translocation and function of beta protein kinase C in vivo[END_REF]. The PKCβ locus generates two isoforms by alternative splicing, PKCβI and PKCβΙΙ. Both bind RACK1, although PKCβΙΙ at higher affinity. Intriguingly, PKCβΙΙ is highly expressed in hypertrophic heart [START_REF] Takeishi | In vivo phosphorylation of cardiac troponin I by protein kinase Cbeta2 decreases cardiomyocyte calcium responsiveness and contractility in transgenic mouse hearts[END_REF][START_REF] Wakasaki | Targeted overexpression of protein kinase C beta2 isoform in myocardium causes cardiomyopathy[END_REF], where translational rates are pathologically increased. RACK1 is very abundant and can function as a general adaptor molecule, binding other membrane-associated proteins such as Src kinase, integrin β1, the NMDA receptor [START_REF] Mccahill | The RACK1 scaffold protein: A dynamic cog in cell response mechanisms[END_REF]. Work in yeast and mammalian cells have demonstrated that RACK1 (Asc1p in yeast) binds the small ribosomal subunit in the cytosol [START_REF] Link | Direct analysis of protein complexes using mass spectrometry[END_REF] and mRNPs [START_REF] Angenstein | A receptor for activated C kinase is part of messenger ribonucleoprotein complexes associated with polyA-mRNAs in neurons[END_REF]. A similar situation has been observed in fission yeast, where Cpc2/RACK1 copurifies with the small subunit [START_REF] Shor | Cpc2/RACK1 is a ribosomeassociated protein that promotes efficient translation in schizosaccharomyces pombe[END_REF]. RACK1 also binds eukaryotic initiation factor 6 (eIF6), and in vitro, in the presence of PKC, can mediate the release of eIF6 from the ribosome [START_REF] Ceci | Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly[END_REF]. These data suggested that RACK1 might function as a PKC adaptor on the ribosomal subunit. More recently, a cryo-EM map of the eukaryotic ribosome has shown that RACK1 is located in proximity of the mRNA exit channel, in close contact with the binding surface of eIF3 complex, and close to mRNA binding proteins [START_REF] Sengupta | Identification of the versatile scaffold protein RACK1 on the eukaryotic ribosome by cryo-EM[END_REF]. Thus, RACK1 may provide a platform for allowing PKC translocation to ribosomes, and a subsequent direct action of PKC on the translational machinery.

We tested the hypothesis that RACK1 acts as a PKCβ kinase platform on ribosomes by a) defining the effect of the pharmacological inhibition of PKCβII on translation, b) characterizing the RACK1/ PKCβII interaction on the ribosomal apparatus, c) studying the effect of inhibition of RACK1/ PKCβII binding, d) studying the effect of exogenous PKCβII overexpression. We report that PKCβII modulates global translational rate and provide evidence supporting a model in which PKCβII/RACK1 interaction has a role in PKC-based stimulation, independent from mTOR.

MATERIALS AND METHODS

Reproducibility

All immunoprecipitations, metabolic assays and polysomal profiles were performed at least three times, with similar outcomes. For immunoprecipitations and polysomal profiles, representative experiments are shown.

Cell lines and treatments

HeLa and HEK293 cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco), glutamine and antibiotics. Transfections were carried out with either calcium phosphate (HEK) or Gene Porter Transfection Reagent (HeLa) according to manufacturer instruction (GTS). All stimulations were performed after 3-6 hours growth factor starving. Cells were either stimulated with 50 nM PMA, 100 nM insulin for 30 min, or pre-treated with 50 µM LY294002, 100 nM BMI, 5 µM hispidin, 50 nM rapamycin 15 min before stimulation.

Drugs and reagents

The following antibodies were used: rabbit polyclonal anti-phospho-Akt (Ser 473), anti-phospho-S6 (Ser 235/236) and anti-rpS6 (Cell Signaling); rabbit polyclonal isoform-specific anti-PKCβII (C-18) (Santa Cruz); mouse monoclonals anti-PKCε, anti-RACK1, anti-PKCβ (BD Transduction Laboratories), anti-KDEL (Stressgen), anti-β actin (Sigma). Rabbit polyconal anti-4E-BP1 was from N. Sonenberg, McGill University, Montreal, Canada [START_REF] Gingras | Activation of the translational suppressor 4E-BP1 following infection with encephalomyocarditis virus and poliovirus[END_REF]. Rabbit polyclonal anti-human β1 integrin was from I. De Curtis, San Raffaele Scientific Institute, Milano, Italy [START_REF] Cattelino | A cellfree system to study regulation of focal adhesions and of the connected actin cytoskeleton[END_REF]. Rabbit polyclonal anti-rpS14 was from T-A. Sato, Columbia University, New York, USA [START_REF] Nadano | Preparation and characterization of antibodies against human ribosomal proteins: Heterogeneous expression of S11 and S30 in a panel of human cancer cell lines[END_REF]. Plasmids: PKCβII from A. Newton [START_REF] Edwards | Carboxyl-terminal phosphorylation regulates the function and subcellular localization of protein kinase C betaII[END_REF]. PMA (phorbol 12-myristate 13-acetate), insulin (human, recombinant), and cycloheximide were from Sigma. The PKC inhibitor BMI (Bisindolyl Maleimide I), PKCβ inhibitor hispidin, mTOR inhibitor rapamycin and BSA (bovine serum albumin) were from Calbiochem, PI3K inhibitor LY294002 was from Alexis Pharmaceuticals. RNasin ® was from Promega.

In vitro binding assay of PKCβII

The in vitro binding of PKCβII to ribosomes was performed as follows. Five pmol purified ribosomal subunit 40S and 60S [START_REF] Pestova | The joining of ribosomal subunits in eukaryotes requires eIF5B[END_REF] and 10 ng recombinant PKCβII and PKCε (CalBiochem) were applied on nitrocellulose membrane (Biorad). The membrane was blocked for 1 h with 5% (w/v) defatted milk in phosphate-buffered saline containing 0.05% (v/v) Tween 20. Membranes were probed with 100 µl of lysate from HEK293 cells, which had been diluted 1:1 with lysis buffer containing 5% (w/v) defatted milk. This step was performed 1 hr RT. Further development of the blots was done exactly as for standard Western blots. The overlay assay of PKCβII with RACK1 was performed as previously described [START_REF] Ron | Cloning of an intracellular receptor for protein kinase C: A homolog of the beta subunit of G proteins[END_REF] with some modifications. Recombinant RACK1 (125 ng) was blotted onto nitrocellulose membrane (Schleicher & Schuell) using a slot blot apparatus (Schleicher & Schuell). Unbound material was removed, and the membrane was incubated in overlay block (0.2 M NaC1, 50 mM Tris-HC1 pH 7.5, 3% bovine serum albumin, and 0.1% polyethylene glycol) for 1 h at room temperature. In a separate tube, PKCβII (1 µg of purified recombinant SF9-expressed protein) was incubated in the presence of 50 µg/ml phosphatidylserine, 0.8 µg/ml DAG, and 1mM calcium in overlay buffer (50 mM Tris-HC1 pH 7.4, 0.1% bovine serum albumin, 5 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor 0.1% polyethylene glycol, 0.2 M NaC1, 0.1 mM CaC1 2 , and 12 mM β-mercaptoethanol) and in the presence or absence of the peptide. The mixture was incubated for 15 min while being rotated at room temperature and then added to immobilized RACK1 for additional rotation of 15 min. Unbound material was removed, and the membrane was washed three times in overlay wash buffer (50 mM Tris-HC1 pH 7.4, 0.1% polyethylene glycol, 0.2 M NaC1, 0.1 mM CaC1 2 , and 12 mM β-mercaptoethanol). Binding of PKCβII was detected using anti-PKCβII polyclonal antibody followed by enhanced chemiluminescent reaction (Amersham Pharmacia Biotech) and processed using the STORM system (Molecular Dynamics).

Polysomal profile and fraction analysis

Polysomal profile was performed as described [START_REF] Ceci | Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly[END_REF] with some modifications. Cells were pre-treated 10 minutes with 100 µg/ml cycloheximide, scraped in 50 mM Tris-HCl pH 7.4, 100 mM NaCl, 30 mM MgCl 2 , 0.1% Igepal, 100 µg/ml cycloheximide, 40 U/ml RNasin ® , protease inhibitor cocktail (Sigma). Whole cell extracts were clarified at 4 °C 10 minutes 15,000 g. Ten OD 254 were layered on a 15-50% sucrose gradient in 50 mM Tris-acetate pH 7.5, 50 mM NH 4 Cl, 12 mM MgCl 2 , and 1 mM DTT and centrifuged at 4 °C in a SW41Ti Beckman rotor for 3.30 h at 39,000 rpm. Absorbance at 254 nm was recorded by BioLogic LP software (BioRad) and fractions collected. Cells were incubated at 37 °C with serum-free medium for 2 hrs and with methionine-free medium for additional 2 hrs. Cells were pulsed with 28 µCi per plate of Promix 35 S-labelled methionine (Amersham) and stimulated with 100 nM insulin and 50 nM PMA for 30 min. Cells were lysed by scraping in 50 µl RIPA buffer without SDS (10 mM Tris-HCl pH 7.5, 1% Na-deoxycholate, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, proteases inhibitor cocktail). Ten µl extracts were TCA-precipitated on glass microfiber filters (Whatman) and counted. Obtained values were normalized on sample protein content, quantified by BiCinchonic Acid (BCA) protein assay (Pierce). Each sample was performed in triplicate and expressed as mean ± SEM. Statistical analysis was performed by ANOVA test.

Immunoprecipitation and Western Blot

Adherent subconfluent HeLa and HEK293 cells were lysed with 10 mM NaCl, 10 mM MgCl 2 , 10 mM TrisHCl pH 7.3, 1% Triton X-100, 1% Na-deoxycholate, 1 mM DTT, 5 mM NaF, 2 mM Na 3 VO 4 , 40 U/ml RNasin ® , protease inhibitor cocktail. The whole cell extract was clarified at 4 °C at 15,000 g for 10 minutes. The amount of protein recovered was quantified by the BCA protein assay. For immunoprecipitation, 3 mg of total proteins were pre-cleared with protein A beads (Amersham), and then incubated for 2 h at 4 °C with anti-PKCβII/anti-PKCβ or with an irrelevant antibody. Protein A beads were added and incubated for 1 h at 4 °C. The beads were washed four times with lysis buffer and resuspended in sample buffer. Immunoprecipitates were resolved on SDS-PAGE, transferred to Immobilon-P membranes (Millipore), and probed with appropriate antibodies. Spleen cells were derived from adult C57 mice as follows. Mice were sacrificed according to the procedures approved by the Institutional Animal Care and Use Commettees (IACUC), and spleens were immediately dissected out. Finely minced spleens were forced through a 40 µm-sieved cell strainer in serum-free RPMI medium (Gibco). Cells were spun down, washed in serum-free RPMI, lysed and immunoprecipitation performed as described as above. Phospho-protein detection was carried out in total lysates prepared in 20 mM HEPES pH 7.4, 100 mM NaCl, 1% Na-deoxycholate, 1% Triton X-100, 2 mM Na 3 VO 4 , 50 mM NaF, 10 mM  βglycerophosphate and protease inhibitor cocktail. The lysate was cleared by centrifugation. Protein content was quantified with BCA as above. Equal amount proteins were analyzed by Western blot. Peptides design R7 peptide (RRFVGHTKDV) was designed on the RACK1 sequence binding PKCβII. R9 peptide (GYTDNLIRVWQVT) was derived from the 7th WD40 repeat of RACK1. Peptides were synthesized as previously described [START_REF] Zhang | C2 region-derived peptides of beta-protein kinase C regulate cardiac Ca2+ channels[END_REF]. Both peptides used in this study were over 90% pure.

RNA interference

The siRNAs targeted to human RACK1 was purchased from Ambion. The sequences are as follows: siRNA identification ID #135615 (siRNA1), 5'-CCAUCAAGCUAUGGAAUACTT-3' (sense) and 5'-GUAUUCCAUAGCUUGAUGGTT-3' (antisense); siRNA ID #135616 (siRNA2), 5'-GCUAUGGAAUACCCUGGGUTT-3' (sense) and 5'-ACCCAGGGUAUUCCAUAGCTT-3' (antisense); and siRNA ID #135617 (siRNA3), 5'-CCUUUACACGCUAGAUGGUTT-3' (sense) and 5'-ACCAUCUAGCGUGUAAAGGTG-3' (antisense). The siRNA negative control was purchased from Ambion (negative control siRNA #1). 1.5x10 5 cells were seeded per plate (35 mm diameter) the day before transfection, and then transfected with 50 nM oligonucleotide using 2 µl Lipofectamine2000 Reagent (Invitrogen). The siRNA-lipofectamine mixture was prepared in Opti-MEM medium (Gibco) and added to cells. After 5 hours the cells were added with normal growing medium. Protein expression was assessed by Western blotting at 48 hours post-transfection and densitometric analysis was performed. Metabolic assay was carried out on siRNA transfected cells as described.

RESULTS

PKCβ isoform can control translational rate in an mTOR-independent fashion

In HEK293 cells, PKC stimulation by PMA causes increase of protein synthesis and phosphorylation of 4E-BPs [START_REF] Herbert | The extracellular signal-regulated kinase pathway regulates the phosphorylation of 4E-BP1 at multiple sites[END_REF]. Indeed, polysomal profile analysis of PMA stimulated HEK293 cells, shows always augmented polysomal peaks and reduced 80S, indicating an increase at the initiation step of translation (Fig. 1A), accompanied by increased methionine incorporation (Fig. 1B). To dissect the underlying PKC pathway, we applied BMI, a broad spectrum PKC inhibitor [START_REF] Toullec | The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C[END_REF] or hispidin, an isoform-specific PKCβ inhibitor [START_REF] Gonindard | Synthetic hispidin, a PKC inhibitor, is more cytotoxic toward cancer cells than normal cells in vitro[END_REF]. In conditions of stimulation, both PKC inhibitors cause decreased methionine incorporation (Fig. 1B). Interestingly, PKCβ inhibition by hispidin represses methionine incorporation as efficiently as global PKC inhibition, suggesting its isoform-specific involvement. The inhibition of either PKC or of mTOR has no effect on nonstimulated cells indicating that the effect we see is specific for the conditions of stimulation (Supplementary Fig. 1). Phorbol ester stimulation of all conventional PKCs causes activation of the ERK-S6K pathway and phosphorylation of 4E-BP1 [START_REF] Herbert | Distinct signalling pathways mediate insulin and phorbol ester-stimulated eukaryotic initiation factor 4F assembly and protein synthesis in HEK 293 cells[END_REF]. Then we analyzed the effect of global PKC inhibition and specific PKCβ inhibition on the phosphorylation status of 4E-BP1 and rpS6, which are downstream targets of mTOR and RSK/S6K [START_REF] Hay | Upstream and downstream of mTOR[END_REF], respectively. Broad PKC inhibitor BMI abolishes PMA-stimulated rpS6 and 4E-BP1 phosphorylation (Fig. 1C, lane 5), but not insulin-induced phosphorylation while PKCβ inhibitor hispidin does not (Fig. 1C, lane 6). Thus PKCβ isoform inhibition impairs translation without affecting the phosphorylation of mTOR targets. In agreement, the mTOR inhibitor rapamycin marginally represses PKC-stimulated translation by PMA, while it represses insulin-stimulated translation more effectively (Fig. 1D). Taken together these data indicate that PKCβ affects translational efficiency, acting either downstream of the PI3K-Akt-mTOR axis or through a parallel pathway. Since PKCβ binds RACK1, one possibility is that PKCβ acts in proximity to ribosomes; thus, we studied the relationships between PKCβ and ribosomes.

PKCβII associates with ribosomes

We first tested whether active PKCβII binds ribosomes directly. We spotted native 40S ribosomal subunits containing RACK1 or 60S subunits on filters, and performed binding with two PKC isoforms, βII and ε. PKCε is a PMA sensitive, Ca 2+ unresponsive PKC isoform that binds monomeric RACK1 [START_REF] Besson | The anchoring protein RACK1 links protein kinase cepsilon to integrin beta chains. requirements for adhesion and motility[END_REF]). We found that PKCβΙΙ specifically bound 40S subunits, but not 60S subunits, whereas PKCε bound neither of them (Fig. 2A). We measured whether PKCβII may transiently interact with ribosomes in vivo. By polysomal profiles, in unstimulated conditions, little or no PKCβII was found to co-sediment with the part of the gradient containing rpS6 (Fig. 2B, left panel). Upon PKC stimulation, we observed a reproducible shift of part of PKCβII towards fractions containing ribosomes (Fig. 2B, right panel, compare with RACK1 and rpS6 containing lanes). The increase of ribosome-associated PKCβII was two-fold (Fig. 2C), and specific for PKCβII since PKCε never co-sedimented with ribosomes (Fig. 2B). Note that polysomal profiles were obtained in native conditions, without crosslinking, suggesting that the shift of PKCβII is comparable to the shift observed for some initiation factors [START_REF] Valasek | In vivo stabilization of preinitiation complexes by formaldehyde cross-linking[END_REF]. This said, the absence of PKCβII from polysomes indicates that PKCβII is not retained during the elongation phase, whereas RACK1 is. To further study the association of PKCβII with ribosomes, we immunoprecipitated endogenous PKCβII from unstimulated and PMA-stimulated cells, and tested for the presence of ribosomal proteins. PKCβII coimmunoprecipitates ribosomal protein rpS6 after stimulation (Fig. 2D). The amount of PKCβII-coprecipitated ribosomal proteins was low, but detegent-resistent. The association of ribosomes with PKCβII was also observed in mouse tissues, e.g. in the mouse spleen, an organ containing high levels of the kinase (Fig. 2E). The coprecipitation data show that in vivo, PKCβΙΙ has the potential to translocate to either ribosomes or ribosome-containing membranes. Since ribosomes can normally associate to ER membranes, reportedly even in the absence of ongoing translation [START_REF] Potter | Endoplasmic reticulum-bound ribosomes reside in stable association with the translocon following termination of protein synthesis[END_REF][START_REF] Stephens | Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation[END_REF], we studied whether PKCβII activation resulted in translocation to membranes, including the endoplasmic reticulum. Indeed, PKCβII activation results in its translocation to endomembranes and E.R. (Suppl. Fig. 2 and [START_REF] Becker | Isoenzyme-specific translocation of protein kinase C (PKC)betaII and not PKCbetaI to a juxtanuclear subset of recycling endosomes: Involvement of phospholipase D[END_REF]). Overall data in Fig. 1 and 2 lead to the hypothesis that PKCβII affects translation without acting on mTOR pathway, but preferentially interacting with the ribosomal machinery, either directly or through RACK1 or upon translocation to endomembranes.

RACK1-PKCβII complex modulates translation

An important issue is to define whether the binding of PKCβII to RACK1 is instrumental to PKCβII action on ribosomes and whether it may explain the localization of PKCβII on ribosomes. To investigate the significance of PKCβII and RACK1 association on translation, we used a RACK1-derived peptide (R7), which mimics the RACK1 binding surface to PKCβII and a control peptide (R9), which is designed on a RACK1 domain not involved in PKCβII binding. In vitro binding asssay of PKCβII on immobilized RACK1 showed that R7 inhibits the association between active PKCβII and RACK1 in a concentration-dependent manner, while R9 is ineffective (Fig. 3, second and third couple bars). Polysomal profiles of cells treated with R7 peptide, which blocks PKCβII binding to RACK1, showed a reduction in polysomal peaks and less ribosome-associated PKCβII, compared to R9 (Fig. 4A andB), without obviously affecting the localization of RACK1. We decided to approach the question whether the change in PKCβII distribution was associated with changes in methionine incorporation, too. Introduction of R7 blocking peptide in HEK293 cells caused a statistically significant reduction of PKC stimulated translation, whereas R9 did not (Fig. 4C; Fig. 6C). To further extend these data, we performed a series of experiments in which after treatment with R7/R9 peptides, polysomal profiles were run, mRNA was extracted from polysomal fractions and blotted for the presence of different mRNAs. We found that treatment with the R7 peptide reduced the polysomal association of several mRNAs (Suppl. Fig. 3), consistently with the effects on methionine incorporation. Hispidin-based PKCβII inhibition caused a reduction in methionine incorporation without inhibition of mTOR activity. We tested whether R7-mediated impairment of translation caused an inhibition of mTOR activity. We found that R7 represses translation (Fig. 4C) without affecting rpS6 and 4E-BP1 phosphorylation (Fig. 4D, lane 5), exactly as hispidin (see Fig. 1B-C). If RACK1 is important in mediating PKC effects, reduction of RACK1 by siRNA may also affect PKC stimulated translation. We tested the hypothesis by RACK1 siRNA. A 70% reduction in RACK1 expression by siRNA (Fig. 4E) impaired responsiveness to PKC-mediated increase of translation (Fig. 4F).

Exogenously expressed PKCβII affects translation inhibited by RACK1-PKCβII disruption

Exogenous PKCβII recapitulated the results observed in HEK293 cells. PKCβII was overexpressed in HeLa cells, which express negligible endogenous PKCβII (Fig. 5A). The integrity and activation property of transfected PKCβII in overexpressing cells was tested by co-immunoprecipitation with RACK1 after activation (Fig. 5B), and by phosphorylation of a specific PKC substrate [START_REF] Soh | Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element[END_REF] (not shown). In HeLa overexpressing PKCβII, stimulation of PKCs resulted in increased cosedimentation of PKCβII with RACK1 containing fractions (Fig. 5C-D). We immunoprecipitated overexpressed PKCβII and blotted for the endogenous ribosomal protein of the 40S subunit rpS14 (Fig. 5E). A small, reproducible pool of rpS14 was associated with PKCβII, similarly to what observed for rpS6 in HEK293 cells. Exogenous overexpression of PKCβII led to increased methionine incorporation in newly translated proteins (Fig. 6A), while mock and PKCι (Fig. 6B) had no effect. Last, we measured translation in cells overexpressing PKCβII with administration of either R7 or R9 peptides. As we observed in HEK293, the stimulation of PKC resulted in increased methionine incorporation in cells without peptides, or cells incubated with the R9 control peptide, but not in cells treated with R7 peptide (Fig. 6C).

Biochemical Journal Immediate Publication. Published on 16 Jun 2008 as manuscript BJ20080463 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080463 S t a g e 2 ( a ) P O S T -P R I N T DISCUSSION One of the most studied pathway regulating translation is the mTOR dependent pathway, which acts mainly through 4E-BP1 phosphorylation. However, as rapamycin-mediated block of mTOR and translation in most cells blocks only 15% of translation, other pathways must exist. Here we have shown that PKCβII, at least in HEK293 and HeLa cells, affect translation through an mTOR independent pathway. Our data suggest that this pathway acts close to the ribosomal machinery through RACK1. The most consistent model explaining our data is that upon activation, PKCβII fulfills its membrane requirement by translocating to endomembranes, and then through its interaction with RACK1 can get in physical contact with the translational apparatus. Alternatively, coprecipitation of PKCβII with ribosomes is due to the fact that they may both reside on membranes (see below). Structural and biochemical work has indicated that PKCβII binds RACK1 with nanomolar affinity, only after PKC activation [START_REF] Stebbins | Binding specificity for RACK1 resides in the V5 region of beta II protein kinase C[END_REF]. The high affinity of binding, the high levels of RACK1 in cells, are consistent with the hypothesis that the RACK1-PKC complex can form in cells, in spite of the transient activation of PKC. The question is whether a RACK1/PKC complex can associate on the ribosome, in vivo and how stable it is. Several aspects must be taken in account: although in vitro PKCβII binds at high affinity RACK1 which in turns binds 40S, the detection of a stoichiometric interaction of PKCβΙΙ with ribosomes in vivo may be complicated by the kinetics of the interaction, possibly too rapid. Furthermore, PKCβII and ribosomes must be in equilibrium with endomembranes to allow PKC activation. Our model is that signaling to the translational machinery occurs at the interface between membranes and ribosomes, and RACK1 acts as a transient scaffold for PKC on the ribosome. In this context, we like to suggest that the presence of endomembranes may be a general feature of translational control: for instance the translational regulator mTORraptor complex, mTORC1, is detergent-sensitive. In addition, work from Nicchitta's lab [START_REF] Potter | Endoplasmic reticulum-bound ribosomes reside in stable association with the translocon following termination of protein synthesis[END_REF][START_REF] Stephens | Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation[END_REF] suggests that most ribosomes are membrane-associated even when translation is blocked. Another alternative is that a specific pool of RACK1, not ribosome-bound, interacts with the activated PKCβII, and then mediates a signal to the translational machinery. In this context the mixture RACK1/PKC is effective in mediating phoshporylation-dependent eIF6 release from 60S in vitro [START_REF] Ceci | Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly[END_REF]. A question to be addressed is which ribosome-associated substrates of RACK1-PKCβII may account for PKCβII-induced translational activation. One substrate that is phosphorylated by RACK1-PKC complex is eIF6 [START_REF] Ceci | Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly[END_REF]. However, the precise contribution of the activity of eIF6 in the control of translational rate, in vivo, is still unclear, due to its pleiotropic action on ribosome biogenesis [START_REF] Sanvito | The beta4 integrin interactor p27(BBP/eIF6) is an essential nuclear matrix protein involved in 60S ribosomal subunit assembly[END_REF][START_REF] Si | The saccharomyces cerevisiae homologue of mammalian translation initiation factor 6 does not function as a translation initiation factor[END_REF], although eIF6 knockout mice have a translation deficit downstream of PMA stimulation (Gandin et al., in preparation). Furthermore, PKC activation results in a strong increase of translation in conditions of overexpression of eIF6 [START_REF] Ceci | Release of eIF6 (p27BBP) from the 60S subunit allows 80S ribosome assembly[END_REF], and an increase in phosphorylated eIF6 [START_REF] Carotenuto | Phosphorylation of p27(BBP)/eIF6 and its association with the cytoskeleton are developmentally regulated in Xenopus oogenesis[END_REF]. As most other kinases, it is likely that RACK1-PKCβII complex may act on other substrates too, which remain to be characterized. Indeed, purified ribosomes show a sustained PKC-like activity (Grosso, unpublished results). Among the putative substrates, eIF4B has a PKC consensus sequence that is phosphorylated in vivo. In conclusion, we provide evidence that a PKC isoform-specific pathway, alternative to the mTOR-4EBP1-S6K cascade, signals to the complexity of initiation factors and mRNA binding proteins acting on translational control.

  Fractions were precipitated with 10% trichloroacetic acid (TCA) according to standard protocol, separated on SDS-PAGE and analysed by Western blot. Densitometric analysis was performed by ImageQuant 5.0 software, Molecular Dynamics. Permeabilization and metabolic assay Subconfluent HEK293 cells grown in 35 mm diameter plates were washed with 1 ml ice-cold HBSS-H (137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl 2 , 1.03 mM MgSO 4 , 0.34 mM Na 2 HPO 4 , 0.44 mM KH 2 PO 4 , 5.6 mM glucose, 20 mM HEPES-NaOH pH 7.4) and then treated 1 min with 50 µg/ml saponin in 1 ml ice-cold ICM buffer without magnesium (see below) in the presence of 10 µM peptide. Cells were recovered for 5 min in 1 ml ice-cold ICM buffer (125 mM KCl, 19 mM NaCl, 10 mM HEPES-KOH pH 7.3, 0.33 mM CaCl 2 , 1.4 mM MgCl 2 ) containing 10 µM peptide.

Biochemical

  Journal Immediate Publication. Published on 16 Jun 2008 as manuscript BJ20080463 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080463 S t a g e 2 ( a ) P O S T -P R I N T Moreover, R7 peptide was made twice (the second one from Calso Laboratory, Lyngby, Denmark), and each batch gave the same biological activities.

Biochemical

  Journal Immediate Publication. Published on 16 Jun 2008 as manuscript BJ20080463 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080463 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 16 Jun 2008 as manuscript BJ20080463 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080463 S t a g e 2 ( a ) P O S T -P R I N T

BiochemicalFig. 1 .

 1 Fig. 1. PKCβ modulates translation without affecting the phosphorylation of mTOR targets. (A) Polysomal profiles of HEK293 cells, unstimulated (left panel) or stimulated with PMA (right panel). Initiation of translation (polysomes) increases upon PKC stimulation. (B) HEK293 cells were treated as indicated and methionine incorporation was counted. The experiment was carried out in triplicate. PMA-insulin mediated translation is inhibited both by the broad PKC inhibitor BMI and by PKCβ specific inhibitor hispidin. (C) HEK293 cells were treated as described and analysed for anti-P-Akt (upstream factor in insulin cascade stimulating translation), anti-4E-BP1 and anti-P-rpS6 (markers for mTOR and S6K1 activation). Anti-rpS6 is used as a loading normalizer. LY294002 inhibits PI3K/mTOR-dependent translation. Note that hispidin impairs translation without affecting the phosphorylation pattern of mTOR targets. (D) HEK293 cells were treated as indicated and methionine incorporation was counted. Note that rapamycin inhibition does not impair PMA dependent translation.

Fig. 2 .

 2 Fig. 2. Endogenous PKCβII is associated to ribosomes. (A) Purified 40S and 60S ribosomal subunits were spotted on nitrocellulose membrane. Overlay assay of PKCβII over immobilized ribosomes shows preferential association with 40S subunit. Recombinant PKCβII and PKCε (novel PKC isoform) were spotted as a control for isoform specificity of PKC antibodies. Note that increased staining of recombinant PKCs is due to their denaturation, whereas ribosomes and PKC on ribosomes is in native form (B) Fractions from polysomal profiles of HEK293 cells, unstimulated (left panel) or stimulated with PMA (right panel) were probed for PKCβII, PKCε, RACK1 and rpS6. PMA treatment shifts PKCβII to the heavier part of the gradient. (C) Densitometric analysis of the percentage of PKCβII cosedimenting with ribosomes by pooling data from independent experiments. Data are expressed as the percentage of the total amount of PKCβII. rpS6-containing fractions define ribosome-bound PKCβII. (D) PKCβII was immunoprecipitated from unstimulated or stimulated HEK293. Input is 10% of IP. Immunocomplexes were probed for PKCβII and rpS6 as a ribosomal marker. (E) The same experiment as in (D) was carried out on mouse spleen. (D) and (E) show co-immunoprecipitation of ribosomes with PKCβII.

Fig. 3 .

 3 Fig.3. R7 inhibits RACK1-PKCβII binding in vitro. Overlay assay of PKCβII on immobilized RACK1 was performed in presence or absence of R7 and R9 peptides as indicated. Phosphatidylserine (PS), diacylglycerol (DAG) and Ca 2+ physiologically activate PKCβII. R7 prevents the binding of PKCβII to RACK1 in concentration dependent manner.

Fig. 4 .

 4 Fig.[START_REF] Pause | Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function[END_REF]. Inhibition of RACK1-PKCβII binding affects translation. (A) HEK293 cells were permeabilized in presence of R7 or R9 peptide and then fully stimulated. The ratio between 40S+60S+80S monosomes (in white) and translating polysomes (in grey) is indicated as an inset. R7 peptide reduces polysomes vs. monosomes ratio. (B) HEK293 cells overexpressing PKCβII were permeabilized in presence of R7 or R9 peptide and then stimulated as in (A). Fractions from polysomal profile were immunoblotted as indicated. Note that R7 peptide prevents PKCβII loading on ribosomes. (C) Methionine incorporation in HEK293 cells. Data are from independent experiments carried out in triplicate. ANOVA test for statistic significance is indicated. (D) Same as in Figure1C, using peptides R7 and R9, where indicated. R7 peptide impairs translation without affecting the phosphorylation pattern of mTOR targets. (E) Cells transfected with RACK1 siRNA or control. Densitometric analysis confirms reduction of RACK1 levels. PKCβII expression is not modified by RACK1 downregulation. (F) Cells transfected with RACK1 siRNA or control as in (E), were PMA-insulin stimulated or left untreated and metabolically labeled. Data from triplicate independent samples.

Fig. 5 .

 5 Fig. 5. Exogenous PKCβII associates with ribosomes. (A) Levels of endogenous and transfected PKCβII in HeLa cells. (B) RACK1 co-purifies with PMA-activated PKCβII. Input is 10% of IP. (C-D) Polysomal profiles from unstimulated (C) and PMA-stimulated (D) HeLa cells prepared as in Figure 1A. Fractions were probed for PKCβII and RACK1. The asterisk marks a PMA-dependent PKCβII degradation product. (E) PKCβII pull-downs were immunoprobed for PKCβII and for rpS14 as a ribosomal marker. Input is 10% of IP.

Fig. 6 .

 6 Fig. 6. Exogenous PKCβII affects translation through RACK1 binding. (A) Methionine incorporation in either mock-transfected or PKCβII-transfected HeLa cells. (B) As in (A) but with transfected PKCι. PKCβII transfection increases translational rate, whereas PKCι does not. (C) R7 reduces translation as compared to R9 (control peptide). Experiment carried out as in Fig. 3C. Grey bars: PMA-stimulation; white bars: basal. Data were obtained from triplicate independent samples.

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 16 Jun 2008 as manuscript BJ20080463 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080463 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

AKNOWLEDGEMENTS

This work was supported by grants AICR n. 05-360 and AIRC 306 to SB, and State of California for Medical Research on Alcohol and Substance Abuse through the University of California, San Francisco to D.R.. We thank for reagents: J.W.Hershey, F. Loreni, A. Newton, P. Parker, T-A. Sato, J-W. Soh, N. Sonenberg, T. Pestova, A.Willis. We thank C. Gorrini for preliminary observations and L. Magri for the setup of RNAi experiments. We thank A. Mondino and N. Offenhaeuser for helpful discussions.


Dennis Wegener

Christian Hildmann
email: christian.hildmann@tu-ilmenau.de

Daniel Riester

Andreas Schober

Franz-Josef Meyer-Almes

Hedwig E Deubzer

Ina Oehme

Olaf Witt

Siegmund Lang

Martina Jaensch

Franz- Josef Meyer-Almes

Vadim Makarov

Corinna Lange

Benedikt Busse

Andreas Schwienhorst

Identification of novel small-molecule histone deacetylase inhibitors

Keywords: throughput screening, trifluoromethylketone, pyran-4one, p-benzochinone, chromone

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Histone deacetylases (HDACs) are important enzymes in the regulation of gene expression in eukaryotic cells [START_REF] Hildmann | Histone deacetylases -an important class of cellular regulators with a variety of functions[END_REF]. Together with histone acetyltransferases (HATs) HDACs maintain the delicate equilibrium between acetylated and deacetylated ε-amino groups of lysines in the amino terminal parts of histone proteins. In general, increased levels of histone acetylation are associated with increased transcriptional activity, whereas decreased levels of acetylation are associated with repression of gene expression [START_REF] Davie | Signal transduction pathways and the modification of chromatin structure[END_REF]. Although histone deacetylation appears to have a fundamental role in regulating gene expression, HDAC inhibitors seem to directly affect transcription of only a relatively small number of genes [START_REF] Mariadason | Genetic reprogramming in pathways of colonic cell maturation induced by short chain fatty acids: comparison with trichostatin A, sulindac, and curcumin and implications for chemoprevention of colon cancer[END_REF][START_REF] Peart | Identification and functional significance of genes regulated by structurally different histone deacetylase inhibitors[END_REF]. Most of the genes regulated by HDAC inhibitors participate in the control of cell growth and survival, providing a mechanistic explanation for the anticancer properties of these inhibitors. Particularly, activation of differentiation programs, inhibition of the cell cycle and induction of apoptosis are key antitumor activities that make HDAC inhibitors promising anticancer agents. Several synthetic compounds and natural products have been reported to inhibit HDACs (reviewed in: [START_REF] Monneret | Histone deacetylase inhibitors[END_REF][6][START_REF] Minucci | Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer[END_REF]. Furthermore, a small number of drug candidates are currently in phase I -III clinical trials (reviewed in: [8] with the first drug, vorinostat (Zolinza, Merck & Co., Inc.) recently approved by the FDA. However, many currently available HDAC inhibitors are disadvantageous e.g. in regard of their limited metabolic stability [START_REF] Veale | Orally active trifluoromethyl ketone inhibitors of human leukocyte elastase[END_REF][START_REF] Frey | Trifluoromethyl ketones as inhibitors of histone deacetylase[END_REF] or their crossreactivity against other enzymes such as esterases [START_REF] Moreth | An active site tyrosine residue is essential for amidohydrolase but not for esterase activity of a class 2 histone deacetylase-like bacterial enzyme[END_REF]. Thus, there is still a need for new HDAC inhibitors belonging to yet unexplored structural classes of compounds. Here, we report the identification of novel small-molecule HDAC inhibitors through mediumthroughput screening of a compound library using a bacterial histone deacetylase-like amidohydrolase (HDAH; [12-14]) as a model system. Several of the compounds specifically inhibit the bacterial enzyme. Other compounds proved to be pan-inhibitors that also inhibit eukaryotic HDACs and induce hyperacetylation in a neuroblastoma cell line (SHEP) and bladder carcinoma cells (T24). The novel inhibitor structures provide new lead structures for further development of improved anticancer drugs.

EXPERIMENTAL

Reagents FB188-HDAH was expressed in E. coli and purified by affinity chromatography (Zn 2+ -IMAC) as described [12]. The enzyme was stable for weeks as 150 µg/ml solution in 10 mM potassium phosphate, pH 8.0 at 4°C. Rat liver HDAC was purchased from Merk Biosciences (Bad Soden, Germany), whereas human HDAC8 was expressed in E. coli and purified as described [START_REF] Riester | Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates[END_REF]. Trypsin from porcine pancreas (Type IX-S, 13,000-20,000 BAEE units/mg) was purchased from Sigma (Taufkirchen, Germany) and after solvation in trypsin buffer (50 mM Tris-HCl, pH 8.5, 100 mM NaCl, 0.1% PEG8000) to a solution of 10 mg/ml was stable for several days at 4 °C. Boc-Lys(Ac)-MCA (Bachem, Weil am Rhein, Germany) and Tos-GPR-MCA (Sigma, Taufkirchen, Germany) were dissolved to 30 mM in DMSO. Tos-GPK(Ac)-MCA was synthesized as described [16]. Sodium valproate (Sigma, Taufkirchen, Germany) was dissolved to 1 M in phosphate-buffered saline (PBS) and stored at -20 °C. Apart from RPMI 1640 medium and trypsin/EDTA (Cambrex, Nottingham, UK), all cell culture reagents were purchased from Sigma (Taufkirchen Germany).

Library Screening

The compound collection was obtained from the Hans-Knöll-Institute (HKI, Jena) as solutions of approximately 10 µg/ml and stored at -80 °C. The thawed substances were diluted 1:25 in assay buffer (10 mM potassium phosphate, pH 8.0). From each dilution 20 µl were transferred into cavities of black 96-well screening plates (Fluotrac200, Greiner, Frickenhausen, Germany). Screening of the compound library for inhibitors of the target enzyme FB188-HDAH was performed using the 1-step version of the standard fluorogenic assay [16,[START_REF] Wegener | A fluorogenic histone deacetylase assay well suited for high-throughput activity screening[END_REF]. A robotic screening device (CyBi-Screen-Machine, CyBio, Jena, Germany) equipped with a Polarstar MTP reader (BMG Labtech, Offenburg, Germany) was used (see also supplementary material). To exclude false-positive candidates, control experiments were conducted with all hits from primary screening. For detection of compound autofluorescence, reactions without enzymes or substrate but with the same concentration of each compound as used for screening were measured. To discriminate real hits from substances predominantly inhibiting the reporter enzyme, trypsin inhibition assays were performed for all primary hits at the same compound concentrations as used in primary screening. 150 µl of the mock reactions used for autofluorescence measurements were transferred in black 96 well MTP and 10 µl trypsin (10 ng/ml diluted in trypsin buffer) were added. After 10 min at 30 °C, the reaction was started with 50 µl trypsin substrate (200 µM Tos-GPR-MCA in trypsin buffer). Trypsin activity was recorded for 10 min as increasing MCA fluorescence at 30°C in the plate reader at 460 nm after excitation at 390 nm.

Determination of Z´-factor

For determination of the Z´-factor, a quality parameter used to judge assay performance control enzyme inhibition experiments were carried out using the known HDAH inhibitor CYPX [14]. In principle, the screening protocol was used except that deacetylase activity was measured in the presence (48 wells) or absence (48 wells) of 10 µM model inhibitor instead of library compounds, respectively. The Z´-factor was calculated as described previously [18].

Determination of IC50 values

To assess the potency of hits 8 point dose-response curves were measured in triplicates. For HDAH, the same protocol was applied as for primary screening. Reactions were monitored in the presence of 20 µl of hit compounds diluted in assay buffer to final concentrations of 1 nM to 100 µM. Obtained slopes of the activity curves were corrected against blank reactions without HDAH. Dose-response curves for primary hits with rat liver HDAC and human HDAC8 were generated using a two-step HDAC assay protocol as described [START_REF] Riester | Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates[END_REF]16]. IC50 values were calculated with the help of GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, USA).

Cell culture

SHEP neuroblastoma cells were cultured in RPMI 1640 medium (Cambrex) supplemented with 10% FCS (Sigma) at 37 °C, 5% CO 2 [19]. Primary human fibroblasts (PHFs) derived from a healthy donor were grown in DMEM (10% FCS, 1% NEAA) at 37 °C, 5% CO 2 as described previously [19].

Cellular metabolic activity

Metabolic activity was assessed with the tetrazolium derivative WST-1, which is reduced to a soluble coloured formazan product by cellular mitochondrial dehydrogenases. The metabolic activity measured corresponds to the number of living cells and is used as a screening assay for cell viability [20]. Cells were plated at 2 x 10 3 cells/ well into 96-well plates and exposed to different concentrations of compounds 46F08 and 50B09 for 48 h. Compound 46F08 was dissolved in MeOH p.a. to give a stock solution of 100 mM, compound 50B09 was dissolved in DMSO to give a stock solution of 10 mM. Compounds were further diluted in cell culture medium to give the final concentrations (Fig. 2). In control experiments, cells were treated with MeOH (0.1 ‰ v/v) and DMSO (0.25% v/v) corresponding to the highest solvent concentration applied in the compound treated cells. WST-1 reagent was added to a final dilution of 1:10 to the wells with those with medium only serving as blank control. After 60 min at 37 °C, absorbance at 450 nm was determined with a plate reader (BMG Labtech, Offenburg, Germany) and corrected for absorbance at 650 nm. Blank control values were subtracted from all results and values for untreated cells were set as 100%.

Western Blot Analysis

The level of acetylated histone H4 was assessed as previously described [21]. Protein concentrations of the cell lysates were determined using the BCA protein assay (Pierce, Rockford, USA). Briefly, 5µl of cell lysates were incubated for 1h at 37°C with 200 µl of BCA-Mix. Absorbance was measured with a plate reader (BMG Labtech, Offenburg, Germany). The following primary antibodies were used: anti-β-actin mouse monoclonal (clone AC-15, Sigma) and anti-acetyl-histone 4 rabbit polyclonal (Upstate, Lake Placid, USA). Secondary antibodies were HRP-conjugated goat anti-mouse (Dianova, Hamburg, Germany) and donkey anti-rabbit IgG (Promega, Mannheim, Germany).

RESULTS

Medium-throughput screening and hit confirmation

For medium-throughput screening the 1-step version of the standard fluorogenic HDAC assay was used [16,[START_REF] Wegener | A fluorogenic histone deacetylase assay well suited for high-throughput activity screening[END_REF] because the enzyme FB188 HDAH is resistant against trypsin digest. For the assay conditions used, a Z´-factor of 0.75 was determined as part of the overall screening program. A total of 3719 compounds containing mostly natural products were tested at a concentration of 0.04 µg/ml. Thirty-eight compounds (1 % of total tested samples) that displayed more than 75 % inhibition were selected as primary hits (see also supplementary material). From the selected primary hits 22 were confirmed to be FB188 HDAH inhibitors and do not show autofluorescence or trypsin inhibition. Unfortunately, 6 out of the 22 hits were no longer available from the supplier. The results of all 16 remaining primary hits were validated by repeated measurements in the screening assay and subsequently by doseresponse curves using final inhibitor concentrations ranging from 1 nM to 100 µM. Based on the selection criterion of IC50 < 10 µM for the FB188 HDAH assay, a total of 10 compounds (0.26 % of total tested samples) were selected as confirmed hits for follow-up studies (Table 1). The most efficient HDAH inhibitors exhibited IC50 values of 610 nM (45E09) and 550 nM (46F08).

Selectivity of hit compounds

To determine whether the confirmed hit compounds selectively inhibit only FB188 HDAH, two other HDAC preparations were tested: rat liver HDAC (Calbiochem, Bad Soden, Germany; this preparation mainly contains HDAC1, 2 and 3 [START_REF] Riester | Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates[END_REF]) and recombinant human HDAC8 [START_REF] Riester | Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates[END_REF]. Interestingly, seven compounds (21E08, 23F10, 26H05, 33D02, 33C08, 40H03, 45E09) showed a marked selectivity for FB188 HDAH (Table 1). Compound 22G03 exhibited a clear preference for HDAH but also inhibited HDAC8 with a 5-fold increased IC50 value. Two compounds (46F08; 50B09) proved to be rather unspecific pan-inhibitors. Both of these compounds showed a marked preference for human HDAC8.

Competition assay with selected compounds

For several hit compounds sufficient material was available to perform competition binding assays as published previously [START_REF] Riester | Histone deacetylase inhibitor assay based on fluorescence resonance energy transfer[END_REF]. Briefly, a fluorescent HDAC inhibitor (FAHA), which shows fluorescence resonance energy transfer (FRET) with tryptophans of the enzyme was used. Upon competition with other HDAC inhibitors the displacement of the fluorescent inhibitor is accompanied by a decrease in FRET. In our experiments, compounds 21E08, 22G03, 33D02 and 33C08 were able to displace fluorescent inhibitor FAHA from the enzyme complex (Fig. 1).

Structure classes of inhibitors

The 10 hit compounds listed in table 1 fall into different structure classes. Surprisingly, only half of these inhibitors match the typical structure of many HDAC inhibitors, i.e. a cap and a Zn-binding moiety linked by a long spacer group [START_REF] Jung | Inhibitors of histone deacetylase as new anticancer agents[END_REF]. Examples of this type are methylketones 45E09 and 23F10, trifluoromethyl ketone 46F08 and carboxylic acids 22G03 and 33C08 (Table 1). All other hit compounds belong to structural classes that have not been described as HDAC inhibitors so far. The HDAH-selective inhibitors 45E09 and 23F10 (Table 1) showed IC50 vlaues of 0.61 µM and 1.12 µM and comprise a pyran-4-one or a p-benzochinone ring as cap group, a C 9-10 aliphatic spacer and a methylketone moiety. The latter of which in principle may interact with the active site zinc ion as this has been demonstrated for the trifluoromethyl ketone moiety [START_REF] Nielsen | Complex structure of a bacterial class 2 histone deacetylase homologue with a trifluoromethylketone inhibitor[END_REF]. Because an additional series of related compounds was available, we were able to identify structural elements that were important for activity of this inhibitor class (Table 2). Simultaneous changes in the substituents of the pyran-4-one ring, i.e. replacement of the ethyl groups in 3-and 5-position by methyl groups and the introduction of a hydroxyl group replacing the 6-methoxy group lead to a 7-fold increase in the IC50 value for An2 as compared to 23F10. The favourable effect of at least the ethyl group in 3-position as compared to a methyl group was also observed in the context of the structural scaffold shared by compounds An3, An4 and An5. Changing the pyran-4-one ring (23F10) into a pbenzochinone ring (45E09) obviously does not influence the inhibitory activity significantly. The opposite seems to be true for the length of the spacer. An inhibitor with a C 6 -spacer like An1 appeared to be inactive, whereas inhibitors like An2, 23F10 or 45E09 with a C 9-10 aliphatic spacers show significant activity. Compound 46F08 (Table 1; [25] is a pan-inhibitor with IC50 values for HDAH, rat liver HDAC and human HDAC8 of 0.55 µM, 0.64 µM and 0.10 µM, respectively. It belongs to the group of trifluoromethyl ketone-based HDAC-inhibitors that have been described previously [START_REF] Frey | Trifluoromethyl ketones as inhibitors of histone deacetylase[END_REF]. From a recent crystallographic study it is known that the oxygen atom of the trifluoromethyl ketone moiety binds to the zinc atom of the enzyme and also the fluoric atoms participate in various interactions with the enzyme [START_REF] Nielsen | Complex structure of a bacterial class 2 histone deacetylase homologue with a trifluoromethylketone inhibitor[END_REF]. In our screening, it was the most active inhibitor of FB188 HDAH with an IC50 of 0.55 µM for FB188 HDAH. For the rat liver HDAC preparation, the IC50 was similar to that of HDAH. In contrast, the IC50 for human HDAC8 showed a 5-fold decrease, i.e. a value of 0.10 µM. Thus, the potency of 46F08 is at least similar if not slightly improved as compared to previously published trifluoromethyl ketone-based HDAC-inhibitors [START_REF] Frey | Trifluoromethyl ketones as inhibitors of histone deacetylase[END_REF], which already were the result of subsequent cycles of optimization. In addition, resembling the more extended type of HDAC inhibitors, compounds 22G03 and 33C08 show a high selectivity for HDAH with IC50 values of 9.24 µM and 3.66 µM, respectively. As shown in table 1, compounds 22G03 and 33C08 (isolated from yeast Candida bombicola ATCC 22214; [START_REF] Asmer | Microbial production, structure elucidation and bioconversion of sophorose lipids[END_REF] are marked by the presence of carboxylic acid moiety at the end of a long spacer, suggesting that this moiety may contact the active site zinc ion. 22G03 contains an epoxide group in the middle of an aliphatic chain reminiscent of depudecin [START_REF] Matsumoto | Depudecin: a novel compound inducing the flat phenotype of NIH3T3 cells doubly transformed by rasand src-oncogene, produced by Alternaria brassicicola[END_REF]28], whereas 33C08 belongs to the class of sophorose lipids. The view that both compounds directly bind to the active site pocket of the enzyme is supported by the finding that 22G03 as well as 33C08 are active in the FAHA competition assay (Figure 1). However, the structural similarity between 33C08 and 40H03 (isolated from yeast Candida bombicola ATCC 22214; [START_REF] Brakemeier | Novel sophorose lipids from microbial conversion of 2-alkanols[END_REF] argues against the importance of a direct binding to the active site zinc ion (see Table 1). Compound 40H03 shows an IC50 value of 6.18 µM for HDAH and contains a lipid chain without a polar end group, such as carboxylic acid functionality. In agreement with the view that a possible direct contact to the zinc ion in 33C08 does not significantly contribute to binding, the IC50 of 40H03 (6.18 µM) shows only an increase by a factor of 2 as compared to that of 33C08 (3.66 µM). Further support of this view comes from the fact that a cyclic "lactonic" derivative of 33C08 termed 33D02 (isolated from yeast Candida bombicola ATCC 22214; [START_REF] Asmer | Microbial production, structure elucidation and bioconversion of sophorose lipids[END_REF] was also among the most active hits. In this molecule, no free carboxylic acid end is present. In addition, it shows a second acetylation of the sophorose moiety. Similar to the open chain derivatives it proved to be a selective HDAH inhibitor with an IC50 value of 2.71 µM. Reexamination of the compound library indicated that it contained several compounds similar to 33C08 and 40H03, which appeared to be inactive under screening conditions (data not shown). Inactive variants of 40H03 included (i) derivatives with altered lipid chain length (± C 2 ), and (ii) derivatives combining a second sophorose acetylation with either a longer lipid chain(± C 2 ) or hydroxylation at position 12 of the lipid chain. Three types of inactive derivatives of 33C08 were identified, (i) a derivative with glucose replacing sophorose, (ii) a derivative with trehalose replacing sophorose and at the same time missing the carboxylic acid moiety, and (iii) derivatives containing a saturated lipid chain in which the carboxyl acid moiety was replaced by butyl amide or higher amide moieties. In summary, it appears that the nature of the sugar moiety as well as the length and the nature but not necessarily the cyclization of the lipid chain have a significant influence on inhibitory activity. Compound 26H05 (Table 1) is a chromone also termed pilopleurin originally isolated from the roots of Pilopleura kozo-poljanskii Schischk [START_REF] Avramenko | The structure of pilopleurin -A new chromone from Pilopleura kozo-poljanskii[END_REF]. In our experiments compound 26H05 proved to be rather selective for FB188 HDAH (IC50 = 1.61 µM), not inhibiting eukaryotic HDACs. Unfortunately, no closely related compounds were available for structure-activityrelationship studies. Actinomycin X (compound 21E08; originally isolated by H. Zahner from Streptomyces michiganensis ATCC 14970; Table 1) also was among the most potent hit compounds with an IC50 value of 1.08 µM for HDAH. Basically, no inhibitory activity was observed for rat liver HDAC and human HDAC8. Actinomycin X is a close relative of actinomycin D, an approved antitumor agent (Lyovac-Cosmegen®, Cosmegen®) [START_REF] Tan | The effect of actinomycin D on cancer in childhood[END_REF][START_REF] Shiba | Studies on the therapy of reticulosarcoma(-tosis) with actinomycin[END_REF]. In contrast to actinomycin X, actinomycin D carries an additional amino group in the phenoxazone system and a L-proline replacing one sarcosine to give to identical pentapeptidic cycles. Our results with actinomycin X prompted us to also test actinomycin D for a possible HDAC inhibitory activity. As a result, it appeared that actinomycin D inhibited FB188 HDAH with an IC50 of 81 µM and only weakly blocked rat liver HDAC (IC50 Hit compound 50B09 (Table 1) belongs to the class of pyrazolopyrimidinones. It has been identified previously as a potent inhibitor of coxsackievirus B3 replication [START_REF] Makarov | Anti-coxsackievirus B3 activity of 2-amino-3nitropyrazolo[1,5-a]pyrimidines and their analogs[END_REF]. In our study, it was among the two inhibitors of FB188 HDAH, which also blocked eukaryotic HDAC preparations. For HDAH, rat liver HDAC and human HDAC8 IC50 values of 2.77 µM, 4.00 µM and 0.31 µM were measured.

Effects of hit compounds on tumor cells

Because 46F08 and 50B09 showed significant activity toward eukaryotic HDACs, we next focused our attention on the inhibitory activity of both compounds in cell culture models. Treatment of SHEP neuroblastoma cells with 46F08 (0.5 -4µM) indeed generated an increase in acetylation of histone 4, indicating HDAC-inhibition. Similar results were obtained for T24 cells (see supplementary material). Acetylation, however, was weaker compared to that induced by TSA (Figure 2A). In contrast, treatment with compound 50B09 did not show an increase in histone 4 acetylation at concentrations of 25 -100 µM. Here, it rather seems that 50B09 suppressed the increased level of histone H4 acetylation caused by DMSO [34,[START_REF] Marks | Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug[END_REF], which was used as solvent. HDAC inhibitors are known to have anti-cancer effects. Therefore, the influence of hit compounds 46F08 and 50B09 on the metabolic activity of SHEP neuroblastoma cells was investigated using the WST-1 assay. This assay quantifies the enzymatic activity of mitochondrial dehydrogenases in the sample and correlates with the number of viable cells [20]. Treatment of cells with compound 46F08 resulted in a concentration-dependent, pronounced reduction of cell metabolic activity even at low concentrations of 0.1 -4 µM. Similar results were obtained for T24 cells (see supplementary material). Compared to the well established HDAC inhibitor valproic acid (VPA) already used in clinical trials, 46F08 was approximately 10 4 fold more active (figure 2). Because tumor cell selective activity of HDAC inhibitors has been described [36], the effect of 46F08 on the metabolic activity of normal, non-transformed human skin fibroblasts was investigated. 46F08 did not obviously influence fibroblast metabolic activity even at the highest concentration applied suggesting tumor cell selective activity of the compound (figure 2). In contrast, 50B09 elicited no effect on the metabolic activity of SHEP cells at concentrations up to 10 µM (figure 2B).

DISCUSSION

We have successfully confirmed the feasibility of our screening assay to efficiently screen a large compound library. Using a robotic workstation (CyBiScreen, CyBio, Jena, Germany) the assay exhibited a very high Z′-factor of 0.75 indicating an excellent assay. The screening identified several compounds capable of inhibiting HDAH reporter enzyme activity. Here, 10 out of 3719 compounds showed IC50 values < 10 µM. The identification of inhibitors 46F08 and 50B09, both of which are highly potent inhibitors of human HDAC8, proved that FB188 HDAH indeed can be used as a model system to also find inhibitors of human HDAC. Altogether, hit compounds fall into different structural classes. Only half of the inhibitors identified followed the simple HDAC inhibitor architecture, realized e.g. in SAHA, in which a cap and a Zn-binding moiety are linked by an extended spacer. For the group of ketone-based inhibitors with pyran-4-one or p-benzochinone rings as cap moieties, a preliminary structureactivity study showed that structure elements like alkyl substituents in the ring system, linker length and the potential zinc-binding moiety are important for inhibitory activity. However, based on the relatively small number of derivatives examined so far it is not justified to draw more specific conclusions. One of our hit compounds, 46F08 belongs to the class of trifluoromethyl ketones. It has been shown that electrophilic ketones such as trifluoromethyl ketones are potent inhibitors of zincdependent enzymes [START_REF] Frey | Trifluoromethyl ketones as inhibitors of histone deacetylase[END_REF]37,[START_REF] Christians | Novel human DNA alkyltransferases obtained by random substitution and genetic selection in bacteria[END_REF]. In contrast to simple methylketones, trifluoromethyl ketones do not only show an increase in electrophilicity at the carbonyl carbon with favourable effects on hydrogen bonding capabilities of the carbonyl oxygen. The recently solved crystallographic structure of a complex of FB188 HDAH with a trifluoromethyl ketone inhibitor [START_REF] Nielsen | Complex structure of a bacterial class 2 histone deacetylase homologue with a trifluoromethylketone inhibitor[END_REF] also revealed that the fluoric atoms actively participate in direct protein binding. Compound 46F08 not only proved to be a potent inhibitor of FB188 HDAH but also blocked rat liver HDAC and human HDAC8 with submicromolar IC50 values. Furthermore, it significantly reduced the metabolic acitivity of SHEP neuroblastoma cells, which correlates with a decrease in the number of viable cells. Interestingly, normal human fibroblasts were not affected by the compound suggesting tumor cell selective activity of 46F08 as has been described for other HDAC inhibitors [36]. In T24 bladder carcinoma cells, a similar effect was observed. Thus, 46F08 is an interesting candidate for further development. However, previously developed trifluoromethyl ketones had exhibited an unfavourably short half-life in animals [START_REF] Frey | Trifluoromethyl ketones as inhibitors of histone deacetylase[END_REF]. It remains to be shown, if compound 46F08 also is readily metabolized. In this regard, it will be interesting to test derivatives of 46F08 with substituents at the CH 2 -moiety next to the carbonyl group. Compounds 40H03, 33C08 and 33D02 all belong to the class of sophorose lipids, a group of microbial glycolipids produced by yeasts [START_REF] Cameotra | Recent applications of biosurfactants as biological and immunological molecules[END_REF]. Comparison with structurally similar but inactive derivatives of the compound library suggested that these molecules do not directly [START_REF] Scholz | Bioactivity of extracellular glycolipids -investigation of potential anti-cancer activity of sophorolipids and sophorolipid-derivatives[END_REF]43] had already demonstrated that certain sophorolipids induce cell differentiation of human leukemia cells by an "unknown mechanism". We may now hypothesize that the anti-cancer activity of sophorolipids and maybe also of other glycolipids [40, [START_REF] Sudo | Induction of the differentiation of human HL-60 promyelocytic leukemia cell line by succinoyl trehalose lipids[END_REF][START_REF] Isoda | Succinoyl trehalose lipid induced differentiation of human monocytoid leukemic cell line U937 into monocyte-macrophages[END_REF] is due to their inhibition of certain histone deacetylases, particularly those with similarities to FB188 HDAH. The same may be true for hit compound 26H05. In our experiments, it did show selectivity for HDAH and not for eukaryotic HDACs. However, several chromones have already been described as anticancer agents [46] with proposed mechanisms as diverse as inhibition of breast cancer resistance protein (ABCG2) [47] or inhibition of phosphatidylinositol 3'-kinase [48]. It cannot be excluded, though, that chromones may also work as inhibitors of certain histone deacetylases, particularly those with similarities to FB188 HDAH. Neither actinomycin X nor actinomycin D have been described as HDAC inhibitors so far. On the contrary, actinomycin D has been shown to intercalate into DNA by virtue of its planar phenoxazone ring system [START_REF] Liu | Solution structure of actinomycin-DNA complexes: Drug intercalation at isolated G-C sites[END_REF][START_REF] Yu | NMR study of the solution conformation of actinomycin D[END_REF]. As a consequence, it is believed that actinomycin D blocks DNA-dependent RNA polymerase [START_REF] Yu | Selective inhibition of rat liver nuclear RNA polymerase II by actinomycin D in vivo[END_REF] and poisons topoisomerase II, which results in double-strand DNA breaks [START_REF] Ross | DNA double-stranded breaks in mammalian cells after exposure to intercalating agents[END_REF]. In principle, the action of actinomycin D should not be restricted to a certain phase in the cell cycle. However, there are experimental evidences that it is by far most active in the S-and G2-phases, indicating that it also may act in a different way [START_REF] Wu | Cell cycle phase-dependent cytotoxicity of actinomycin D in HeLa cells[END_REF]. In the light of our data, it can be speculated that compounds such as actinomycin X and D may also work as inhibitors of certain histone deacetylases. With compound 50B09 another new class of HDAC inhibitors has been discovered in our screening program. Previously 50B09 and related pyrazolopyrimidinones had been identified as potent inhibitors of coxsackie virus B3 replication [START_REF] Makarov | Anti-coxsackievirus B3 activity of 2-amino-3nitropyrazolo[1,5-a]pyrimidines and their analogs[END_REF]. In this previous study, cytotoxicity was also determined. Whereas compound 50B09 did not show a significant cytotoxicity against HeLa cells or a pronounced growth inhibition in L-929 and K-562 cells, derivatives of 50B09 did show inhibitory activity on these cell lines [START_REF] Makarov | Anti-coxsackievirus B3 activity of 2-amino-3nitropyrazolo[1,5-a]pyrimidines and their analogs[END_REF]. In our study, we also measured the effect of 50B09 on eukaryotic cells. Despite a good inhibitory activity against rat liver and human HDAC8, we neither detected a pronounced effect on the metabolic activity of SHEP neuroblastoma cells nor an increase in histone H4 acetylation. Structure-activity-relationship studies are on the way to identify derivatives with improved anti-tumor cell activities. In summary, several inhibitors have been discovered that block enzymes belonging to the HDAC family of proteins. Most of the identified inhibitor structures have not been described as HDAC inhibitors so far and therefore may be a good starting point for further development toward future generations of HDAC inhibitors with possible applications in the fields of malignant and other diseases.

Biochemical

  Journal Immediate Publication. Published on 25 Mar 2008 as manuscript BJ20080536 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080536 S t a g e 2 ( a ) P O S T -P R I N T

Biochemical

  Journal Immediate Publication. Published on 25 Mar 2008 as manuscript BJ20080536 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080536 S t a g e 2 ( a ) P O S T -P R I N T bind to the active site zinc ion. Interestingly, earlier work by Isoda et al. [40, 41] and Scholz et al.

Table 1 : 31 *Fig. 1 .

 1311 FIGURE CAPTIONS

Fig. 2 .

 2 Fig. 2. Cellular effects produced by hit compounds 46F08 and 50B09. (A) Influence on cell metabolic activity. Upper panel: SHEP neuroblastoma cells and normal primary human fibroblasts (PHF) were treated with compound 46F08 for 48 h with the indicated concentrations. Cellular metabolic activity was determined using WST-1 assay. Vehicle: cells were treated with solvent only (MeOH, 0.1 ‰ v/v), corresponding to the MeOH concentration applied in 10µM 46F08 treated cells. Values for untreated cells were set to 100 %. Lower panel: SHEP neuroblastoma cells were treated with compound 50B09 for 48 h with the indicated concentrations. Vehicle: cells were treated with solvent only (DMSO, 0.1 % v/v), corresponding to the DMSO concentration applied in 10 µM 50B09 treated cells. (B) Influence on histone H4 acetylation. SHEP cells were treated for 4 h with indicated compounds and harvested for Western analysis using an anti-histone H4-acetyl antibody. MeOH (0.04 ‰ v/v) was used as a solvent control for 4 µM 46F08 treated cells. DMSO (0.25, 0.5, 1% v/v) were used as solvent controls for 25, 50, 100 µM 50B09 treated cells, respectively. Note that DMSO, in contrast to MeOH, harbors HDAC-inhibiting activity. βactin served as loading control.

Figure 1 Biochemical

 1 Figure 1

Figure 2 Biochemical

 2 Figure 2

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 25 Mar 2008 as manuscript BJ20080536 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080536 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

This research was in part supported by grant BioFuture 0311852 from the Bundesministerium für Forschung und Technologie, Germany to A.S. and by a grant of the Nationales Genomforschungsnetzwerk (NGFN2) to O.W. A.S. would like to thank R. Jansen (Braunschweig), H.-P. Fiedler (Tübingen), G.-V. Roeschenthaler and D.V. Sevenard (Bremen) for providing hit compounds and for valuable discussions.