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Biochemical Pharmacology (v.70, #6)

Editorial Advisory Board (pp. iii).

Redox regulation: A new challenge for pharmacology by Daniel Frein; Stefan Schildknecht; Markus Bachschmid; Volker Ullrich (pp. 811-823).

Redox signaling is evolving as a new field of biochemical and pharmacological research. Unlike oxidative stress which is characterized by a macroscopic shift in cellular redox potentials and usually accompanied by oxygen radical induced damage, redox regulation involves subtle and more chemically defined oxidations of short duration. Most important is the reductive component as a necessary part of a reversible regulatory process. Examples of redox regulation occur during early stages of the immune response, in hypoxia or in endothelial dysfunction. Persistent oxidative events together with a decline in the cellular reduction potential lead to oxidative stress as is seen in the pathophysiology of sepsis, reperfusion damage, atherosclerosis and diabetes. Oxidative signals involve superoxide and nitric oxide as the main players which form a system of oxidizing, nitrating or nitrosating species leading to posttranslational modifications of proteins. Modern techniques of immunohistochemistry and mass spectrometry allow a correlation of protein modification, e.g., disulfide, S-oxide, S-nitroso or nitrotyrosine formation, with enzyme activities and cellular responses. In this commentary, examples of the control of prostanoid synthesis by theNO/O₂⁻ system are described. Redox regulation represents an interesting challenge for the development of drugs that modulate the oxidative trigger mechanisms or enforce the reductive pathways.

Keywords: Redox regulation; Nitric oxide; Superoxide; Peroxynitrite; Oxidative stress; Vascular inflammation

Effects of the cyclin-dependent kinase inhibitor CYC202 ( R-roscovitine) on the physiology of cultured human keratinocytes by Ganka Atanasova; Ralph Jans; Nikolai Zhelev; Vanio Mitev; Yves Poumay (pp. 824-836).

CYC202 ( R-roscovitine) is a potent cyclin-dependent kinase inhibitor, investigated as a potential anti-cancer agent. The knowledge of the action of this pharmacological agent on normal human cells is still limited. In this study, we have explored the effects of the cyclin-dependent kinase inhibitor CYC202 on normal human epidermal keratinocytes. The loss of cell viability induced by this compound was strongly dependent on the rate of keratinocyte proliferation. At slightly cytotoxic doses, CYC202 inhibited the proliferation of subconfluent keratinocytes in a dose-dependent manner, and at higher concentrations induction of early apoptosis was observed, evidenced by caspase-3 activation. The signal transduction pathways in subconfluent keratinocytes were altered, as CYC202 increased the phosphorylation of p38 MAP kinase. The activation of this kinase was confirmed by the increased phosphorylation of p38 MAPK substrate, the small heat shock protein HSP27. Prolonged inhibition of highly proliferative cells with CYC202 for 48 and 72h altered the expression of epidermal differentiation markers. The use of the selective p38 kinase inhibitor PD169316 demonstrated that involucrin mRNA was upregulated by CYC202 via p38 MAPK pathway. These effects were strongly dependent on cell density and were observed only in highly proliferative keratinocytes. We concluded that CYC202 although highly potent against cancer cells inhibits also the proliferation and induces early apoptotic events in autocrine culture of normal human keratinocytes, activates p38 MAP kinase pathway and alters the expression of the epidermal differentiation markers. These results suggest that despite this potency against tumour cells, CYC202 must be used attentively in the clinical practice.

Keywords: Abbreviations; CDK; cyclin-dependent protein kinases; EGF; epidermal growth factor; ERK; extracellular signal-regulated kinase; MAPK; mitogen-activated protein kinase; HSP27; heat shock protein 27Roscovitine; Cell cycle inhibition; p38 Signaling; Gene expression; Keratinocytes; Involucrin

Bcl-2 mediated inhibition of erucylphosphocholine-induced apoptosis depends on its subcellular localisation by R. Handrick; J. Rudner; I. M�ller; H. Eibl; C. Belka; V. Jendrossek (pp. 837-850).

The synthetic phospholipid derivative erucylphosphocholine (ErPC) is a potent inducer of apoptosis in human tumor cell lines. This membrane-targeted drug induces apoptosis independently from death receptor signaling through a mitochondrial pathway that is inhibited by over-expression of Bcl-2.Within the cell, Bcl-2 resides in membranes of mitochondria, endoplasmic reticulum (ER) and the nucleus. However, the importance of its subcellular localisation in distinct organelles for protection against apoptosis is not completely understood.To investigate the impact of Bcl-2 localised at defined subcellular compartments on its protective effects against ErPC-induced apoptosis, Bcl-2 expression was directed to the outer membrane of the mitochondria or the ER of Jurkat T Lymphoma cells, using Bcl-2 mutants with modified membrane anchors. The mitochondrial insertion sequence of ActA directed Bcl-2 to the mitochondria (Bcl-2/MT), the ER-specific sequence of cytochrome b5 to the ER (Bcl-2/ER). Additionally, Jurkat cells expressing wild-type Bcl-2 (Bcl-2/WT) or a transmembrane domain-lacking mutant (Bcl-2/ΔTM) were employed.While restricted expression of Bcl-2 either at membranes of the mitochondria or the ER strongly interfered with ErPC-induced mitochondrial damage and apoptosis, cytosolic Bcl-2/ΔTM exhibited only reduced protection. Thus, membrane localisation of Bcl-2 is a prerequisite for substantial protection against ErPC-induced apoptosis. For efficient long-term inhibition of ErPC-induced apoptosis Bcl-2 had to be present in the membranes of both compartments, the ER and the mitochondria.The finding that ER-targeted Bcl-2 interferes with ErPC-induced mitochondrial damage points to an involvement of the ER in apoptosis signaling upstream of the mitochondria and to a crosstalk between both compartments.

Keywords: Abbreviations; APC; alkylphosphocholine; Bcl-2/ER; Bcl-2 with targeted expression at the endoplasmic reticulum; Bcl-2/ΔTM; Bcl-2 mutant lacking the transmembrane domain; Bcl-2/MT; Bcl-2 with targeted expression at the mitochondria; Bcl-2/WT; Bcl-2 with wild type subcellular distribution; DISC; death inducing signaling complex; ErPC; erucylphosphocholine; ER; endoplasmic reticulum; FADD; Fas associated protein with death domain; HePC; hexadecylphosphocholineErucylphosphocholine; Mitochondria; ER; Bcl-2; Apoptosis

Effect of the digitoxigenin derivative, INCICH-D7, on Na⁺, K⁺-ATPase by Margarita Ramirez; Leonardo Del Valle; Alicia Sanchez-Mendoza; Fermin Alejandro Tenorio; Gabriela Zarco; Gustavo Pastelin (pp. 851-857).

Compound 14β,17β-cycloketoester-3β-OH androstane (INCICH-D7) is a semisynthetic product of a structural modification of the digitoxigenin molecule. INCICH-D7 has a heterocyclic ketoester type fusion between positions C14 and C17 of the steroid nucleus, which confers this molecule stronger electronegativity than that of digitoxigenin. INCICH-D7 retained positive inotropic effect, with a greater safety margin, when compared to digitoxigenin and ouabain. In this study we have examinated the INCICH-D7 effect on Na⁺, K⁺-dependent adenosinetriphosphatase (Na⁺, K⁺-ATPase) and compared these results with the ones observed with digitoxigenin and ouabain. The inhibitory effect of INCICH-D7 on Na⁺, K⁺-ATPase was five times lower (IC₅₀=4μM) than that of ouabain (IC₅₀=0.8μM) and 70 times lower than that of digitoxigenin (IC₅₀=0.06μM). The inhibitory effect of INCICH-D7 and ouabain on the enzyme was irreversible while digitoxigenin's one was reversible in up to an 80%. Our results indicate that inclusion of the heterocycle between positions C14 and C17 in the digitoxigenin molecule lowers significantly the inhibitory effect on Na⁺, K⁺-ATPase and renders the interaction between INCICH-D7 and enzyme irreversible under the studied reaction conditions.

Keywords: Na; ⁺; , K; ⁺; -ATPase; Digitoxigenin derivates; Digitalis; Cardioactive steroid; Ouabain; Cardiac glycosides

Regulation of testis-specific carnitine transporter (octn3) gene by proximal cis-acting elements Sp1 in mice by Tomoji Maeda; Masamichi Hirayama; Daisuke Kobayashi; Ikumi Tamai (pp. 858-868).

The mouse octn transporter family consists of three genes, octn1, octn2 and octn3. The gene products octn2 and octn3, which transport carnitine with high affinity, are both expressed in testis, where carnitine is required to maintain sperm cell motility. Here, we focused on the regulatory mechanism of the expression of octn3 in an attempt to determine whether the differential tissue expression profiles of the octn2 and octn3 genes reflect distinct physiological roles of octn2 and octn3. The promoter activity of the octn3 gene was examined by luciferase assay and gel mobility shift assay using the mouse Sertoli cell line TM4 as host cells. Deletion-mutant assay demonstrated that a gene segment of the 5′-untranslated region located at about −500bp relative to the transcription start site is required for constitutive octn3 transcription. Deletion of the Sp1-binding site within the region resulted in loss of transcriptional activity. In addition, overexpression of Sp1 in TM4 cells led to a further increase of transcription of octn3. These results demonstrated that Sp1-binding sites are necessary and sufficient for constitutive octn3 gene transcription. Furthermore, the expressions of both of octn2 and octn3 genes in TM4 cells were up-regulated by palmitic acid, whereas carnitine increased only the expression of octn2 without any change in octn3 expression. Accordingly, the expressions of octn2 and 3 are regulated by distinct mechanisms, suggesting distinct roles of octn2 and octn3 in carnitine transport.

Keywords: Abbreviations; AP-1; activator protein 1; CPT I; carnitine palmitoyl transferase I; DMEM; Dulbecco's modified Eagle's medium; G3PDH; glyceraldehyde-3-phosphate dehydrogenase; PBS; phosphate-buffered saline; PPAR-α; peroxisome proliferator activating receptor α; octn; organic cation/carnitine transporteroctn3; Promoter; Carnitine; Testis; Sp1

Ellagic acid blocks activation of pancreatic stellate cells by Atsushi Masamune; Masahiro Satoh; Kazuhiro Kikuta; Noriaki Suzuki; Kennichi Satoh; Tooru Shimosegawa (pp. 869-878).

Activated pancreatic stellate cells (PSCs) play a pivotal role in the pathogenesis of pancreatic fibrosis and inflammation. Ellagic acid is a plant-derived polyphenol found in fruits and nuts, and has anti-oxidant and anti-inflammatory properties. But, little is known about the effects of ellagic acid on PSCs as well as on the activation of signal transduction pathways. We here evaluated the effects of ellagic acid on the activation and cell functions of PSCs. PSCs were isolated from rat pancreas tissue and used in their culture-activated, myofibroblast-like phenotype unless otherwise stated. Ellagic acid inhibited platelet-derived growth factor (PDGF)-BB-induced proliferation and migration, interleukin (IL)-1β- and tumor necrosis factor (TNF)-α-induced monocyte chemoattractant protein-1 production, and expression of α-smooth muscle actin and collagen genes. Ellagic acid inhibited PDGF-BB-induced tyrosine phosphorylation of PDGF β-receptor and the downstream activation of extracellular signal-regulated kinase and Akt. Ellagic acid inhibited IL-1β- and TNF-α-induced activation of activator protein-1 and mitogen-activated protein kinases (extracellular signal-regulated kinase, c-Jun N-terminal kinase and p38 mitogen-activated protein kinase), but not of nuclear factor-κB. In addition, ellagic acid inhibited transformation of freshly isolated cells to an activated, myofibroblast-like phenotype. In conclusion, ellagic acid inhibited key cell functions and activation of PSCs.

Keywords: Abbreviations; AP-1; activator protein-1; BrdU; bromo-2′-deoxyuridine; EGCG; epigallocatechin-3-gallate; GFAP; glial fibrillary acidic protein; IκB; inhibitor of NF-κB; MCP-1; monocyte chemoattractant protein-1; PPAR; peroxisome proliferator-activated receptor; PSCs; pancreatic stellate cells; α-SMA; α-smooth muscle actinPancreatitis; Pancreatic fibrosis; Pancreatic stellate cells; MAP kinase; Polyphenol; Platelet-derived growth factor

Motilin and erythromycin-A share a common binding site in the third transmembrane segment of the motilin receptor by Luo Xu; Inge Depoortere; Pascale Vertongen; Magali Waelbroeck; Patrick Robberecht; Theo L. Peeters (pp. 879-887).

The motilin receptor (MTLR) represents a clinically useful pharmacological target, as agonists binding to the MTLR have gastroprokinetic properties. In order to compare the molecular basis for interaction of the MTLR with motilin and with the non-peptide motilin agonist, erythromycin-A (EM-A), the negatively charged E¹¹⁹ located in the third transmembrane (TM₃) region was mutated to D (E119D) and Q (E119Q), respectively, and changes in activity of the mutant receptors were verified.Each mutant receptor was stably transfected in CHO-cells containing the Ca²⁺ indicator apo-aequorin. Receptor activation in response to motilin, EM-A and their analogues was assessed by Ca²⁺-luminescense.In the E119Q mutant, the Ca²⁺ response to motilin and EM-A was abolished while in the E119D mutant it was reduced with 62% (motilin) and 81% (EM-A). The pEC₅₀ values were shifted from 9.65±0.03 to 7.41±0.09 (motilin) and from 6.63±0.12 to 4.60±0.07 (EM-A). Acetylation of the N-terminal amine group as in [ N-acetyl-Phe]¹ mot (1–14), decreased the potency 6.3-fold (WT-MTLR) and 148-fold (E119D). Acetylation of EM-A enol ether induced a more pronounced shift in potency: 7943-fold (WT-MTLR) and 1413-fold (E119D).The comparable loss of affinity of the mutant receptors for motilin and EM-A indicate that these agonists both interact with the TM₃ domain of the MTLR. The results with acetylated derivatives support an ionic interaction between E¹¹⁹ of the MTLR with the N⁺ of the desosamine sugar in EM-A, but not with the N⁺ of the free amine group in motilin.

Keywords: Abbreviations; CHO-cell line; Chinese hamster ovary cell line; EM-A; erythromycin-A; GHS-R; growth hormone secretagogue receptor; GPCR; G-protein coupled receptors; MTLR; motilin receptor; PCR; polymerase chain reaction; TM; transmembrane; TRH-R; thyrotropin releasing hormone receptor; WT; wild typeMotilin receptor; Mutagenesis; Erythromycin-A; Ca; ²⁺; luminescence; Motilin fragments

Salicylic acid modulates oxidative stress and glutathione peroxidase activity in the rat colon by Janice E. Drew; John R. Arthur; Andrew J. Farquharson; Wendy R. Russell; Philip C. Morrice; Garry G. Duthie (pp. 888-893).

Oxidative stress is a characteristic of cancerous colon tissue and inflammatory bowel diseases that increase colon cancer risk. Epidemiological evidence supports a protective effect of plant-derived compounds. Aspirin is also protective against colon cancer. The mechanism of action is unclear although salicylic acid, the main metabolite of aspirin, has been shown to decrease the synthesis of pro-inflammatory and potentially neo-plastic prostaglandins. Salicylic acid is found in significant quantities in a plant-based diet. However, in plants salicylic acid is also reported to modulate the expression of numerous enzymes with antioxidant activity. The aim of this study was to assess whether salicylic acid can modulate pro-cancerous biological pathways in the colon. Oxidative stress, prostaglandins and cytosolic glutathione peroxidase (cyGPX) were analysed in proximal, transverse and distal colon from a rat model of diet-induced oxidative stress. Elevated plasma pyruvate kinase activity (1293�206U/ml) and increased indices of lipid peroxidation in colon (proximal 6.4�0.84nM MDA/mg protein; transverse 6.9�0.97nM MDA/mg protein; distal 5.2�0.62nM MDA/mg protein) from rats fed a Vitamin E deficient diet were significantly decreased on supplementation with salicylic acid (plasma pyruvate 546�43U/ml; salicylic acid proximal 3.6�0.39nM MDA/mg protein; transverse 4.5�0.61nM MDA/mg protein; distal 4.4�0.27nM MDA/mg protein). Reductions in oxidative stress and prostaglandin production on supplementation with salicylic acid were associated with an elevation in glutathione peroxidase activity (Vitamin E deficient proximal 0.056�0.013U/mg protein; transverse 0.073�0.008U/mg protein; distal 0.088�0.010U/mg protein; Vitamin E deficient with salicylic acid proximal 0.17�0.01U/mg protein; transverse 0.23�0.016U/mg protein; distal 0.16�0.020U/mg protein). Gpx1 and Gpx2 gene transcripts were not elevated in association with increased activity of the soluble glutathione peroxidase activity. Glutathione peroxidases are key antioxidant enzymes, catalysing the decomposition of potentially toxic lipid peroxides. Gpx activity and regulation of Gpx gene transcription has been shown previously to be complex with activity not necessarily mirrored by a corresponding elevation in gene transcription. By supplementing the diet of Vitamin E deficient rats with salicylic acid (1g/kg diet), this study assessed effects of salicylic acid on cytosolic glutathione peroxidase activity in the colon. The ability of salicylic acid to modulate antioxidant enzymes in colon tissue may be an important mechanism in inhibiting colon cancer development.

Keywords: Glutathione peroxidase; Colon cancer; Vitamin E; Prostaglandins

Inhibition of human telomerase by diphosphates of acyclic nucleoside phosphonates by Miroslav Hájek; Naděžda Matulová; Ivan Votruba; Antonín Holý; Eva Tloušt’ová (pp. 894-900).

Diphosphates of the antiviral acyclic nucleoside phosphonates (ANPs) were evaluated in telomeric repeat amplification protocol (TRAP) for their ability to inhibit the extension of telomeres by human telomerase. Extracts from human leukaemia HL-60 cells were used as a source of the enzyme. Data show that the most effective compound studied was the guanine derivative PMEGpp (IC₅₀ 12.7±0.5μmolL⁻¹ at 125μmolL⁻¹ deoxynucleoside triphosphates (dNTPs)). The inhibitory effects of other PME, PMP and HPMP diphosphates on telomerase reverse transcriptase decreased in the order: ( R)-PMPGpp>( R)-HPMPGpp>PMEDAPpp>( S)-PMPGpp>( S)-HPMPApp>PMEO-DAPypp>( R)-6-cyprPMPDAPpp>( R)-PMPApp>( R)-PMPDAPpp≥PMEApp≥PMECpp>PMETpp>( S)-PMPApp∼6-Me₂PMEDAPpp. These results are consistent with the observed antineoplastic activities of the parental guanine (PMEG) and 2,6-diaminopurine (PMEDAP) PME-derivatives. Moreover, structure–activity relationship indicates enantioselectivity of some of these human telomerase inhibitors: ( R)-isomers of the PMP-derivatives possess stronger inhibitory potency towards the enzyme than ( S)-isomers. The data may contribute to the rational design of telomerase inhibitors based on the structure of acyclic nucleotide analogues.

Keywords: Abbreviations; ANP; acyclic nucleoside phosphonate; ANPpp; ANP diphosphate; araGTP; 9-β-; d; -arabinofuranosylguanine 5′-triphosphate; AZT-TP; 3′-azido-3′-deoxythymidine 5′-triphosphate; CBV-TP; 2′,3′-didehydro-2′,3′-dideoxyguanosine 5′-triphosphate; CdG-TP; carbocyclic 2′-deoxyguanosine 5′-triphosphate; CHAPS; 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; d4TTP; 2′,3′-didehydro-2′,3′-dideoxythymidine 5′-triphosphate; ddC; 2′,3′-dideoxycytidine; deazadATP; 7-deaza-2′-deoxyadenosine 5′-triphosphate; deazadGTP; 7-deaza-2′-deoxyguanosine 5′-triphosphate; FaraTTP; 2′-fluoro-2′-deoxy-β-arabinofuranosylthymine 5′-triphosphate; GI; ₅₀; growth inhibition concentration; hTERT; human telomerase reverse transcriptase; 6-Me; ₂; PMEDAP; 2-amino-6-(dimethylamino)-9-[2-(phosphonomethoxy)ethyl]purine; PMEA; 9-[2-(phosphonomethoxy)ethyl]adenine; PMEC; 1-[2-(phosphonomethoxy)ethyl]cytosine; PMEDAP; 2,6-diamino-9-[2-(phosphonomethoxy)ethyl]purine; PMEG; 9-[2-(phosphonomethoxy)ethyl]guanine; PMEO-DAPy; 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidine; PMET; 1-[2-(phosphonomethoxy)ethyl]thymine; (; R; )-6-cyprPMPDAP; (; R; )-2-amino-6-(cyclopropylamino)-9-[2-(phosphonomethoxy)propyl]purine; (; R; )-HPMPG; (; R; )-9-[(3-hydroxy-2-phosphonomethoxy)propyl]guanine; (; R; )-PMPA; (; R; )-9-[2-(phosphonomethoxy)propyl]adenine; (; R; )-PMPDAP; (; R; )-2,6-diamino-9-[2-(phosphonomethoxy)propyl]purine; (; S; )-HPMPA; (; S; )-9-[(3-hydroxy-2-phosphonomethoxy)propyl]adenine; (; S; )-PMPA; (; S; )-9-[2-(phosphonomethoxy)propyl]adenine; (; S; )-PMPG; (; S; )-9-[2-(phosphonomethoxy)propyl]guanine; thiodGTP; 6-thio-2′-deoxyguanosine 5′-triphosphate; TRAP; telomeric repeat amplification protocol; TS; telomerase substrate (nontelomeric oligonucleotide)Acyclic nucleoside phosphonates; Human telomerase; Telomerase inhibition; Reverse transcriptase; TRAP assay; Repeat addition processivity

Inhibition of phospholipase D activation by CYL-26z in formyl peptide-stimulated neutrophils involves the blockade of RhoA activation by Yu-Hsiang Kuan; Ruey-Hseng Lin; Lo-Ti Tsao; Yeh-Long Chen; Cherng-Chyi Tzeng; Jih-Pyang Wang (pp. 901-910).

5-[4-Acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one (CYL-26z) inhibited the formyl-Met-Leu-Phe (fMLP)-stimulated phospholipase D (PLD) activity, which was assessed by the production of phosphatidylethanol (PEt) in the presence of ethanol, in rat neutrophils (IC₅₀ 1.2±0.2μM). CYL-26z caused a slight but significant attenuation of the global protein tyrosine phosphorylation stimulated by fMLP only at concentrations of CYL-26z up to 30μM. CYL-26z blocked the membrane recruitment of protein kinase C-α (PKC-α) at concentrations of CYL-26z ≥3μM, but failed to affect the membrane association of PKC-βI and -βII. The translocation of RhoA to the membrane was attenuated by CYL-26z (IC₅₀ 3.8±0.8μM) in fMLP-stimulated neutrophils, whereas CYL-26z caused no significant inhibition of the membrane recruitment of ADP-ribosylation factor (Arf). CYL-26z inhibited the activation of RhoA and dissociation of the RhoA-Rho guanine nucleotide dissociation inhibitor (GDI) complex in fMLP-stimulated neutrophils (IC₅₀ 1.8±1.0μM and 1.8±0.9μM, respectively). In a cell-free system, CYL-26z effectively attenuated the membrane association of RhoA in response to GTPγS (IC₅₀ 1.3±0.5μM). In contrast, the GTPγS-stimulated translocation of Arf to membrane was suppressed only at concentrations of CYL-26z up to 30μM. CYL-26z inhibited the fMLP-stimulated membrane expression of CD11b, CD45 and CD63, and the release of lysozyme and β-glucuronidase. These results indicate that CYL-26z inhibited the fMLP-stimulated PLD activity, mainly through the blockade of RhoA activation, and degranulation in rat neutrophils.

Keywords: Abbreviations; Arf; ADP-ribosylation factor; CYL-26z; 5-[4-acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one; dhCB; dihydrocytochalasin B; DMSO; dimethyl sulfoxide; fMLP; formyl-Met-Leu-Phe; GDI; guanine nucleotide dissociation inhibitor; GEF; guanine nucleotide exchange factor; HBSS; Hanks’ balanced salt solution; PA; phosphatidic acid; PEt; phosphatidylethanol; PKC; protein kinase C; PLD; phospholipase D; PMA; phorbol 12-myristate 13-acetate; TK; tyrosine kinaseCYL-26z; Phospholipase D; RhoA; RhoGDI; Degranulation; Neutrophils

A novel Gcn5p inhibitor represses cell growth, gene transcription and histone acetylation in budding yeast by Prisca Ornaghi; Dante Rotili; Gianluca Sbardella; Antonello Mai; Patrizia Filetici (pp. 911-917).

Histone acetyltransferases are key chromatin regulators responsible for transcriptional activation and cell cycle progression. We propose a simple yeast-based assay to determine the specificity and targets of novel Gcn5p inhibitors. Here, we report the finding of a novel, small molecule, MC1626, which is able to inhibit yeast cell growth, Gcn5p-dependent gene transcription and acetylation of the histone H3 N-terminal tail in vivo. Because HATs misregulation is invariably associated with human diseases, the identification of MC1626 as a novel cell-permeable Gcn5p inhibitor suggests that it may be a very useful starting tool for the further development of new molecules to be applied to expression profiling of genes regulated by histone H3 acetylation. In addition, our results demonstrate that MC1626 is a Gcn5p-dependent yeast growth inhibitor.

Keywords: Gcn5p; HAT inhibitor; Histone acetyltransferase; MC1626; Chromatin; Yeast

A novel adenosine analog, thio-Cl-IB-MECA, induces G₀/G₁ cell cycle arrest and apoptosis in human promyelocytic leukemia HL-60 cells by Eun-Jin Lee; Hye-Young Min; Hwa-Jin Chung; Eun-Jung Park; Dae-Hong Shin; Lak Shin Jeong; Sang Kook Lee (pp. 918-924).

Human A₃ adenosine receptor (A₃AR) agonists have been shown to play important roles in several physiological and pathological processes, including growth inhibition of human cancer cells. On this line, we recently found that a novel adenosine analog, 2-chloro- N⁶-(3-iodobenzyl)-4′-thioadenosine-5′- N-methyluronamide (thio-Cl-IB-MECA) was a potent human A₃AR agonist, and is superior to a known agonist Cl-IB-MECA [Jeong LS, Jin DZ, Kim HO, Shin DH, Moon HR, Gunaga P, et al. J Med Chem 2003;46:3775]. Here, we report that a novel A₃AR agonist, thio-Cl-IB-MECA inhibited the growth of human promyelocytic leukemia HL-60 cells by arresting cell cycle and induction of apoptosis. Thio-Cl-IB-MECA induced the cell cycle arrest of G₀/G₁ in the early time and at lower concentration (up to 25μM). At higher concentration (50μM), the apoptotic cell deaths were manifested by observation of the increase of sub-G₀ phase of cell cycle distribution, DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage. In addition, the down-regulation of checkpoint protein cyclin D1 and c-myc by thio-Cl-IB-MECA was well correlated with the arrest of cell cycle transition of G₁ to S phase. Further study revealed that the growth inhibitory activity of thio-Cl-IB-MECA is also related with the modulation of Wnt signaling pathway. The levels of β-catenin, phosphorylated forms of GSK-β and Akt were down-regulated by the treatment of thio-Cl-IB-MECA (10nM) in a time-dependent manner, providing one of plausible mechanistic evidence for the involvement of the Wnt signaling pathway in the HL-60 cell growth inhibitory effects by thio-Cl-IB-MECA. These results suggest that a novel A₃AR agonist, thio-Cl-IB-MECA can down-regulate Wnt signaling, inhibit proliferation and induce apoptosis in HL-60 leukemia cells, and thus provide the possibility of this compound in the potential therapeutic value of the treatment of leukemia.

Keywords: Abbreviations; A; ₃; AR; A; ₃; adenosine receptor; thio-Cl-IB-MECA; 2-chloro-; N; ⁶; -(3-iodobenzyl)-4′-thioadenosine-5′-; N; -methyluronamideA; ₃; adenosine receptor agonist; Thio-Cl-IB-MECA; HL-60 cells; Cell cycle arrest; Apoptosis; Wnt signaling

Activation of the aryl hydrocarbon receptor by berberine in HepG2 and H4IIE cells: Biphasic effect on CYP1A1 by Radim Vrzal; Adéla Zdařilová; Jitka Ulrichová; Luděk Bláha; John P. Giesy; Zdeněk Dvořák (pp. 925-936).

Berberine has long been considered a candidate for an antimalarial drug. It exerts a plethora of biological activities and has been used in the treatment of diarrhea and gastro-enteritis for centuries. Here we provide evidence that berberine activates the aryl hydrocarbon receptor (AhR) in human hepatoma (HepG2) and rat hepatoma cells stably transfected with a dioxin responsive element fused to the luciferase gene (H4IIE. luc). AhR was activated by high doses of berberine (10–50μM) after 6 and 24h of incubation as revealed by CYP1A1 mRNA expression (HepG2) and AhR-dependent luciferase activity (H4IIE. luc). Berberine induced nuclear translocation of AhR-GFP chimera transiently transfected to Hepa1c1c7 cells. In contrast, low doses of berberine (<1μM) and prolonged times of the treatments (48h) failed to produce any activation of AhR in H4IIE. luc cell line. HPLC analysis ruled out the hypothesis that the loss of berberine capacity to activate AhR in H4IIE. luc cells is due to metabolic inactivation of the alkaloid. We demonstrate that berberine is a potent inhibitor (IC₅₀=2.5μM) of CYP1A1 catalytic activity (EROD) in HepG2 cell culture and in recombinant CYP1A1 protein. Collectively, our results imply that while berberine activates the Ah receptor, it is accompanied by inactivation of the catalytic activity of CYP1A1 and occurs at concentrations that exceed those predicted to occur in vivo. Given these data, it appears that activation of the AhR pathway by berberine has a low toxicological potential.

Keywords: Abbreviations; AhR; aryl hydrocarbon receptor; AhR-GFP; chimera aryl hydrocarbon receptor-green fluorescent protein; ARNT; AhR nuclear translocator; CAR; constitutive androstane receptor; CDK; cyclin-dependent kinase; COX-2; prostaglandin H synthase; DRE; dioxin-responsive element; ERK; extracellular signal-regulated kinase; EROD; 7-ethoxyresorufin-; O; -deethylase; GR; glucocorticoid receptor; JNK; Jun-N-terminal kinase; LDH; lactate dehydrogenase; MAPK; mitogen activated protein kinase; PXR; pregnane X receptor; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxin; VDR; Vitamin D receptorBerberine; Metabolism; AhR; Cytochrome P450

Role of benoxaprofen and flunoxaprofen acyl glucuronides in covalent binding to rat plasma and liver proteins in vivo by Jennifer Q. Dong; Jianhua Liu; Philip C. Smith (pp. 937-948).

Benoxaprofen (BNX) has been implicated in rare but serious hepatotoxicity which led to its withdrawal from the world market. Flunoxaprofen (FLX), a structural analog, appears to be less toxic. It has been postulated that the nonsteroidal antiinflammatory drugs associated toxicity may be related to covalent modification of proteins by their reactive acyl glucuronides, and the extent of covalent protein binding depends on both reactivity of the acyl glucuronide and the exposure to the reactive metabolite. The disposition of BNX and FLX in rats were compared upon intravenous administration of 20mg/kg of BNX, FLX or their metabolites. Covalent binding of BNX and FLX to plasma and liver proteins were also determined, and an immunochemical approach was used to detect their hepatic targets. Similar concentrations of plasma protein adducts for BNX and FLX were detected even though the AUC of BNX-glucuronide (BNX-G) was almost twice that of FLX-glucuronide (FLX-G). Similar concentrations of liver protein adducts for BNX and FLX were also detected at 8h, however, the hepatobiliary exposure of BNX-G was only 1/3rd that of FLX-G indicating that BNX-G was more reactive than FLX-G, which was in agreement with in vitro data. Proteins of 110 and 70kDa were the major liver protein targets modified by covalent attachment of BNX and FLX. In conclusion, measuring covalent binding to tissue proteins in animals in addition to plasma adducts should be considered when evaluating and comparing carboxylic acid analogs that form reactive acyl glucuronides.

Keywords: Abbreviations; A(m); e,bile,0→8; _h; cumulative biliary excretion of metabolite up to 8; h; AUC; area under the plasma concentration curve; BNX; benoxaprofen; BNX-G; benoxaprofen glucuronide; CL; systemic clearance; Cl; _f,bile; biliary formation clearance; Cl; _e,bile; biliary excretion clearance; DMF; N; ,; N; -dimethylformamide; ECL; enhanced chemiluminescence; EDC; 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; ELISA; enzyme-linked immunoabsorbance assay; FCS; fetal calf serum; FLX; flunoxaprofen; FLX-G; flunoxaprofen glucuronide; HPLC; high performance liquid chromatography; HSA; human serum albumin; IBP; ibuprofen; KLH; keyhole limpet hemocyanine; NSAIDs; nonsteroidal antiinflammatory drugs; PBS; phosphate buffered saline; RSA; rat serum albumin; SDS-PAGE; sodiumdodecyl sulfate polyacrylamide gel electrophoresis; t; _1/2; half lifeReactive acyl glucuronide; Covalent protein binding; NSAIDs; Reversible metabolism; Immunoblot; Liver protein targets

Expression of the drug transporters MDR1/ ABCB1, MRP1/ ABCC1, MRP2/ ABCC2, BCRP/ ABCG2, and PXR in peripheral blood mononuclear cells and their relationship with the expression in intestine and liver by Nadine Albermann; Friedrich Hubertus Schmitz-Winnenthal; Kaspar Z’graggen; Christine Volk; Michael Marcus Hoffmann; Walter Emil Haefeli; Johanna Weiss (pp. 949-958).

ATP binding cassette (ABC)-transporters like P-glycoprotein (multidrug resistance ( MDR) 1/ ABCB1), the multidrug resistance associated proteins 1 and 2 ( MRP1/ ABCC1 and MRP2/ ABCC2), and the breast cancer resistance protein ( BCRP/ ABCG2) have a large impact on the pharmacokinetics of numerous drugs and may also modulate the effectiveness of drug therapy. Prediction of a patient's susceptibility to xenobiotics and individualization of drug therapy would become possible, if a simple test were available for an easy screening of transporter expression. This study quantified the mRNA expression of the four ABC-transporters and of the pregnane X receptor (PXR), a key regulator in drug metabolism and efflux, in peripheral blood mononuclear cells (PBMCs), and corresponding liver or small intestine samples of humans by real-time reverse transcription-polymerase chain reaction (RT-PCR). The results obtained prove the absence of a correlation between the expression of four major ABC-transporters in PBMCs and in the intestine or liver. For all transporters (except MRP1/ ABCC1 in the intestine), mRNA amount of the ABC-transporters was positively correlated with PXR expression in PBMCs and intestine. In conclusion, the study suggests that basal expression levels of the transporters are directly influenced by PXR expression in liver and PBMCs and demonstrates that PBMCs do not qualify as surrogate tissue for the expression of the four ABC-transporters in small intestine and liver. However, the transporter status in PBMCs remains important for drugs, whose primary site of therapeutic action is the lymphocyte and which are known substrates of the transporters.

Keywords: Abbreviations; ABC; ATP binding cassette; BCRP; breast cancer resistance protein; FCS; fetal calf serum; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HBSS; Hanks balanced salt solution; HHBSS; with HEPES supplemented HBSS; HIV; human immunodeficiency virus; LY335979; zosuquidar; MDR; multidrug resistance; β2mg; β2-microglobulin; MRP; multidrug resistance associated protein; PBMCs; peripheral blood mononuclear cells; PCR; polymerase chain reaction; PE; phycoerythrin; P-gp; P-glycoprotein; PXR; pregnane X receptor; RFLP; restriction fragment length polymorphism; RT-PCR; reverse transcription PCR; S.D.; standard deviationMDR1/ABCB1; MRP1/ABCC1; MRP2/ABCC2; ABCG2/BCRP; PXR; PBMC

Thrombin induces DNA synthesis and phosphoinositide hydrolysis in airway smooth muscle by activation of distinct receptors by Trevor R. Walker; Karen A. Cadwallader; Alison MacKinnon; Edwin R. Chilvers (pp. 959-967).

Chronic airway inflammation induces numerous structural changes of the airways involving hypertrophy and hyperplasia of airway smooth muscle (ASM). Thrombin has been identified in the bronchoalveolar lavage fluid of asthmatic subjects and displays potent bronchoconstrictor and mitogenic activity towards ASM. This study has addressed which proteinase-activated receptors (PARs) and signalling pathways are involved in mediating distinct effects of thrombin. Using cultured bovine tracheal smooth muscle (BTSM) cells as a model system, thrombin stimulated a marked increase in [³H]inositol phosphate ([³H]InsPs) accumulation, which was fully mimicked by a selective PAR1 activating peptide. In contrast, PAR1, PAR2, PAR3 and PAR4 activating peptides were unable to replicate the ability of thrombin to stimulate DNA synthesis as assessed by [³H]thymidine incorporation. Further investigation demonstrated that the mitogenic effect of thrombin did not involve stimulation of PDGF secretion but did involve activation of PDGF or EGF receptors and a G_i/o-dependent activation of phosphoinositide 3-kinase. Thrombin, but not the PAR1, PAR2, PAR3 or PAR4 activating peptides was able to stimulate PtdIns(3,4,5)P₃ mass accumulation. PAR3 antisense oligonucleotides substantially inhibit thrombin-stimulated [³H]thymidine incorporation and PtdIns(3,4,5)P₃ generation but had no effect on thrombin-induced phosphoinositide hydrolysis. These data indicate that while PI hydrolysis and Ca²⁺ mobilisation induced by thrombin operates via PAR1-dependent activation of phospholipase C, phosphoinositide 3-kinase activation and DNA synthesis occurs via a distinct proteinase-activated receptor pathway, possibly involving PAR3.

Keywords: Abbreviations; ASM; airway smooth muscle; BTSM; bovine tracheal smooth muscle; DMEM; Dulbecco's modified Eagle's medium; ECL; enhanced chemiluminescence; EGFR; epidermal growth factor receptor; GPCR; G protein-coupled receptor; InsPs; inositol phosphates; PAGE; polyacrylamide gel electrophoresis; PAR; proteinase-activated receptor; PBS; phosphate buffered saline; PDGFRα; platelet-derived growth factor receptor α; PDGFRβ; platelet-derived growth factor receptor β; PMSF; phenylmethylsulfonyl fluoride; PPACK; d; -phenylalanyl-; l; -prolyly-; l; -arginine chloro-methyl ketone; PVDF; polyvinylidene difluoride; RTK; receptor tyrosine kinase; RT-PCR; reverse transcriptase-polymerase chain reaction; TLC; thin layer chromatographyThrombin; Proteinase-activated receptors; PAR; Airway smooth muscle

Corrigendum to “Adenosine induces apoptosis in the human gastric cancer cells via an intrinsic pathway relevant to activation of AMP-activated protein kinase�? [Biochem. Pharmacol. 67/10 (2004) 2005–2011] by Masaru Saitoh; Kaoru Nagai; Kazuhiko Nakagawa; Takehira Yamamura; Satoshi Yamamoto; Tomoyuki Nishizaki (pp. 968-968).

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Biochemical Pharmacology (v.70, #6)