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Biochemical Pharmacology (v.75, #10)

Editorial Advisory Board (pp. iii).

Uracil nucleotides: From metabolic intermediates to neuroprotection and neuroinflammation by Davide Lecca; Stefania Ceruti (pp. 1869-1881).

Uracil nucleotides (i.e., UTP and UDP) have been known for years as fundamental intermediates in the de novo synthesis of the other pyrimidine nucleotides, which altogether represent key building blocks for nucleic acid synthesis. In addition, their sugar conjugates (i.e., UDP-glucose and UDP-galactose) enter in several biochemical routes, for example leading to glycogen biosynthesis, and protein and lipid glycosylation, which in turn contribute to the synthesis of essential components of the cellular plasma membrane. More recently, the existence of a “pyrimidinergic transmission” has arisen from the discovery that several purinergic G protein-coupled P2Y receptors can be activated also or exclusively by uracil nucleotides and sugar conjugates. The number of these receptors is continuously growing over years with the discovery that previously “orphan” G protein-coupled receptors are actually responding to this class of molecules. Therefore, new unforeseen effects mediated by uracil derivatives have emerged, in particular in the nervous system, and previously unexplored avenues for the pharmacological manipulation of this system are currently under investigation. In this commentary we shall try to put together our current knowledge on the biochemical and receptor-mediated effects of uracil nucleotide derivatives with a specific focus on the nervous system in order to depict a clearer view of the importance of the pyrimidinergic system in both physiological and pathological conditions.

Keywords: Abbreviations; APP; amyloid precursor protein; BBB; blood–brain barrier; BDNF; brain-derived growth factor; CGRP; calcitonin-gene related peptide; CNS; central nervous system; CNT2; concentrative nucleoside transporter 2; CREB; cAMP-responsive element binding protein; CTP; cytidine triphosphate; CysLTs; cysteinyl-leukotrienes; DRG; dorsal root ganglia; EGF; epidermal growth factor; ER; endoplasmic reticulum; FGF2; fibroblast growth factor 2; GalT-1; UDP-galactose:ceramide glycosyltransferase; GFAP; glial fibrillary acidic protein; Glu1P; glucose-1-phosphate; GS; glycogen-synthase; KA; kainic acid; LPS; lypopolysaccharide; MCAo; middle cerebral artery occlusion; NANA; N; -acetyl-neuraminic acid; NDPK; ecto-nucleoside diphosphokinase; NGF; nerve growth factor; NPCs; neural stem/precursor cells; PDGF; platelet derived growth factor; PP; _i; inorganic phosphate; RGD; arginine-glycine-aspartic acid; ROS; reactive oxygen species; TG; trigeminal ganglia; TNFα; tumor necrosis factor alpha; TRPV1; vanilloid receptor 1; TSP-1; trombospondin 1; UDP-Gal; UDP-galactose; UDP-GlcA; UDP-glucuronic acid; UDP-Glc; UDP-glucose; Urd; uridine; Ura; uracil; VEGF-2; vascular endothelial growth factor 2Pyrimidine release; P2Y receptors; Glycoproteins; Membrane phospholipids; GPR17

The apelin–APJ system in heart failure by Alan G. Japp; David E. Newby (pp. 1882-1892).

Apelin is the endogenous ligand for the previously orphaned G protein-coupled receptor, APJ. This novel peptidic signalling pathway is widely represented in the heart and vasculature, and is emerging as an important regulator of cardiovascular homeostasis. In preclinical models, apelin causes nitric oxide-dependent vasodilatation, reduces ventricular preload and afterload, and increases cardiac contractility in rats with normal and failing hearts. Apelin–APJ signalling also attenuates ischemic myocardial injury and maintains cardiac performance in ageing and chronic pressure overload. Downregulation of apelin and APJ expression coincides with declining cardiac performance raising the possibility that diminished apelin–APJ activity may have pathophysiologic implications. At present, data from human studies is limited but changes in apelin and APJ expression in patients with chronic heart failure parallel those seen in preclinical models. Detailed clinical investigation is now required to establish the role of apelin in human cardiovascular physiology and pathophysiology, and to determine the therapeutic potential of augmenting apelin signalling in patients with heart failure.

Keywords: Apelin; APJ; Inotrope; Heart failure

New players on the center stage: Sphingosine 1-phosphate and its receptors as drug targets by Andrea Huwiler; Josef Pfeilschifter (pp. 1893-1900).

The recent identification of a cellular balance between ceramide and sphingosine 1-phosphate (S1P) as a critical regulator of cell growth and death has stimulated increasing research effort to clarify the role of ceramide and S1P in various diseases associated with dysregulated cell proliferation and apoptosis.S1P acts mainly, but not exclusively, by binding to and activating specific cell surface receptors, the so-called S1P receptors. These receptors belong to the class of G protein-coupled receptors that constitute five subtypes, denoted as S1P₁–S1P₅, and represent attractive pharmacological targets to interfere with S1P action. Whereas classical receptor antagonists will directly block S1P action, S1P receptor agonists have also proven useful, as recently shown for the sphingolipid-like immunomodulatory substance FTY720. When phosphorylated by sphingosine kinase to yield FTY720 phosphate, it acutely acts as an agonist at S1P receptors, but upon prolonged presence, it displays antagonistic activity by specifically desensitizing the S1P₁ receptor subtype.This commentary will cover the most recent developments in the field of S1P receptor pharmacology and highlights the potential therapeutic benefit that can be expected from these novel drug targets in the future.

Keywords: Sphingolipids; Sphingosine 1-phosphate; S1P receptors; Cancer; Drug therapy

Cytotoxic activities of nucleoside and nucleobase analog drugs in malignant mesothelioma: Characterization of a novel nucleobase transport activity by Deepti Damaraju; Vijaya L. Damaraju; Miranda Brun; Delores Mowles; Michelle Kuzma; Richard C. Berendt; Michael B. Sawyer; Carol E. Cass (pp. 1901-1911).

This study was designed to evaluate the cytotoxic activity of several nucleoside and nucleobase analog drugs as possible new agents for treatment of malignant mesothelioma and to identify factors responsible for the clinical variation of nucleoside analog drug response in chemotherapy of mesothelioma. Three human mesothelioma cell lines (MSTO-211H, H2452 and H2052) were tested for gemcitabine sensitivity and nucleoside transport activity. MSTO-211H, H2452 and H2052 exhibited differences in sensitivity to gemcitabine, nucleoside transport rates and hENT1 site densities. In H2052 cells, gemcitabine, 5-fluoro-2′-deoxyuridine, clofarabine and cladribine were most active with IC₅₀ values of 46, 43, 240 and 490nM, respectively, whereas 5-fluorouracil was the least cytotoxic drug tested. In H2052 cells, the combination of gemcitabine and fludarabine or cladribine resulted in synergistic cytotoxic response. In nucleobase transport studies, hypoxanthine and 6-mercaptopurine but not 5-fluorouracil was transported into H2052 cells by a novel purine-specific, sodium-independent nucleobase transport activity. In summary differences in nucleoside analog drug transport activities are likely to contribute to the observed clinical variation in nucleoside analog response in patients and for the first time a correlation between nucleobase drug sensitivities and transport activities was shown. A novel combination of gemcitabine and fludarabine or cladribine had synergistic cytotoxic activity against the least sensitive mesothelioma cell line. These drug combinations merit further evaluation as effective therapeutic regimens in patients with aggressive mesothelioma.

Keywords: Abbreviations; hENT; human equilibrative nucleoside transporter; NBMPR; nitrobenzylmercaptopurine riboside; IC; ₅₀; the concentration of drug that inhibited growth of treated cells by 50% relative to that of untreated cells; Cl-dAdo; cladribine; Fara-A; fludarabine; 5-FU; 5-fluorouracil; 6-MP; 6-mercaptopurine; CI; combination indexMesothelioma; Nucleosides; Nucleobases; Gemcitabine; Synergy; hENT1

Quercetin decreases intracellular GSH content and potentiates the apoptotic action of the antileukemic drug arsenic trioxide in human leukemia cell lines by Adrián M. Ramos; Patricio Aller (pp. 1912-1923).

Arsenic trioxide (ATO) is an effective therapeutic agent for the treatment of acute promyelocytic leukemia, but successful application of this agent may occasionally require the use of sensitizing strategies. The present work demonstrates that the flavonoids quercetin and chrysin cooperate with ATO to induce apoptosis in U937 promonocytes and other human leukemia cell lines (THP-1, HL-60). Co-treatment with ATO plus quercetin caused mitochondrial transmembrane potential dissipation, stimulated the mitochondrial apoptotic pathway, as indicated by cytochrome c and Omi/Htra2 release, XIAP and Bcl-X_L down-regulation, and Bax activation, and caused caspase-8/Bid activation. Bcl-2 over-expression abrogated cytochrome c release and apoptosis, and also blocked caspase-8 activation. Quercetin and chrysin, alone or with ATO, decreased Akt phosphorylation as well as intracellular GSH content. GSH depletion was regulated at the level ofl-buthionine-( S, R)-sulfoximine (BSO)-sensitive enzyme activity, and N-acetyl-l-cysteine failed both to restore GSH content and to prevent apoptosis. Treatment with BSO caused GSH depletion and potentiated ATO-provoked apoptosis, but did not affect apoptosis induction by ara-C and cisplatin. As an exception, ATO plus quercetin failed to elicit Akt de-phosphorylation and GSH depletion in NB4 acute promyelocytic leukemia cells, and correspondingly exhibited low cooperative effect in inducing apoptosis in this cell line. It is concluded that GSH depletion explains at least in part the selective potentiation of ATO toxicity by quercetin, and that this flavonoid might be used to increase the clinical efficacy of the antileukemic drug.

Keywords: Abbreviations; APL; acute promyelocytic leukemia; Ara-C; 1-β-; d; -Arabinofuranosylcytosine; ATO; arsenic trioxide; BSO; l; -Buthionine-(; S; ,; R; )-sulfoximine; Cisplatin, CDDP; cis-platinum(II)-diammine dichloride; DAPI; 4,6-diamino-2-phenylindole; FITC; fluorescein isothiocyanate; γ-GCS; gamma-glutamylcysteine sinthetase, glutamate-cysteine ligase; GSH-OEt; reduced glutathione ethyl ester; HSP; heat-shock protein; JNK; Jun N-terminal kinase; mAb; monoclonal antibody; MAPK; mitogen-activated protein kinase; pAb; polyclonal antibody; MEK/ERK; mitogen-induced extracellular kinase/extracellular signal-regulated kinase; NAC; N; -acetyl-; l; -cysteine; PI3K; phosphatidylinositol 3-kinase; PI; propidium iodide; ROS; reactive oxygen species; z-IETD-fmk; Z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH; ₂; F; z-VAD-fmk; Z-Val-Ala-Asp(OMe)-CH; ₂; FArsenic trioxide; Quercetin; Apoptosis; GSH; Leukemia cells

Fluoxetine mediates G0/G1 arrest by inducing functional inhibition of cyclin dependent kinase subunit (CKS)1 by Anand Krishnan; Ramkumar Hariharan; S. Asha Nair; M. Radhakrishna Pillai (pp. 1924-1934).

Fluoxetine, a well-known antidepressant used clinically for mental depression has gained attention in cancer research owing to its chemosensitizing potential in drug resistant cell lines. Some preliminary reports, however, suggested its independent cytotoxic potential which is not yet well characterized. Our aim in this study was to characterize its antiproliferative activity in tumor cells and to further elucidate the mechanism. We found that fluoxetine sensitized the effect of cyclophosphamide even in drug sensitive MDA MB 231 and SiHa cells. IC₅₀ values of 28 and 32μM were obtained for fluoxetine mediated antiproliferative response in these cells. Further, PARP and caspase 3 cleavage analyses confirmed fluoxetine mediated apoptosis at molecular level. Cell cycle analysis showed that fluoxetine arrested cells at G0/G1 phase in a time dependent manner. The application of bioinformatics tools at this juncture predicted CKS1 as one of the possible targets of fluoxetine, which is of relevance to cell cycle biology. Fluoxetine showed the potential to disrupt skp2–CKS1 assembly required for ubiquitination and proteasomal degradation of p27 and p21. Our in vitro results were in agreement with the predictions made in silico. We found that fluoxetine treatment could accumulate p27 and p21, an immediate outcome characteristic of functional inhibition of CKS1. This was accompanied by the accumulation of cyclin E, another possible target of CKS1. We observed CKS1 downregulation also upon prolonged fluoxetine treatment. Fluoxetine had downregulated cyclin A which confirmed G0/G1 arrest at the molecular level. We conclude that fluoxetine induced cell cycle arrest is CKS1 dependent.

Keywords: CKS1; Fluoxetine; Cell cycle arrest; P27; Docking

Inhibition of the EGF receptor blocks autocrine growth and increases the cytotoxic effects of doxorubicin in rat hepatoma cells by Conrad Ortiz; Laia Caja; Patricia Sancho; Esther Bertran; Isabel Fabregat (pp. 1935-1945).

FaO rat hepatoma cells show increased levels of the epidermal growth factor receptor (EGFR) ligands, when compared with adult normal hepatocytes, and higher activity of the TNF-alpha converting enzyme (TACE/ADAM17), which is required for EGFR ligand proteolysis and activation. In this work we have analysed the consequences of inhibiting the EGFR in FaO rat hepatoma cells, focusing the attention on autocrine growth and protection from apoptosis. Results have indicated that FaO cells show overactivation of the EGFR pathway, which induces basal growth (in the absence of serum) and protection from pro-apoptotic agents, such as doxorubicin, generating drug resistance. Treatment of cells with the combination of doxorubicin and the tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478, a potent and specific inhibitor of EGFR tyrosine kinase) potently inhibits autocrine growth and induces apoptosis. The apoptotic effect correlates with high expression and activation of the pro-apoptotic Bax and decreased transcript and protein levels of the anti-apoptotic Mcl-1 and Bcl-x_L. Furthermore, the combination of AG1478 and doxorubicin induces reactive oxygen species (ROS) production and glutathione depletion in FaO cells, coincident with up-regulation of the NADPH oxidase NOX4 and down-regulation of the gamma-glutamylcysteine synthetase (γ-GCS), a key regulatory enzyme of the glutathione synthesis. Incubation of cells with glutathione ethyl ester attenuates the apoptosis induced by the combination of doxorubicin and AG1478, which indicates that glutathione depletion is required for an efficient cell death. In conclusion, targeting EGFR combined with other conventional pro-apoptotic drugs should potentially be effective in antineoplastic therapy towards liver cancer.

Keywords: Abbreviations; DCFH-DA; 2′,7′-dichlorodihydrofluorescein diacetate; GEE; glutathione ethyl ester; EGF; epidermal growth factor; EGFR; epidermal growth factor receptor; γ-GCS; gamma-glutamylcysteine synthetase; HCC; hepatocellular carcinoma; HB-EGF; heparin-binding epidermal growth factor-like growth factor; ROS; reactive oxygen species; TACE/ADAM17; TNF-alpha converting enzyme; TGF-α; transforming growth factor-alpha; TGF-β; transforming growth factor-betaHepatoma; Epidermal growth factor receptor; Doxorubicin; Apoptosis; Oxidative stress

Quercetin augments TRAIL-induced apoptotic death: Involvement of the ERK signal transduction pathway by Young-Ho Kim; Dae-Hee Lee; Jae-Hoon Jeong; Zong Sheng Guo; Yong J. Lee (pp. 1946-1958).

Combined treatment with quercetin and TRAIL induced cytotoxicity and enhanced annexin V staining and poly (ADP-ribose) polymerase (PARP) cleavage in human prostate cancer cell lines DU-145 and PC-3. These indicators of apoptosis resulted from the activation of caspase-8, -9, and -3. Although the expression levels of FLIPs, cIAP1, cIAP2, and the Bcl-2 family were not changed in quercetin-treated cells, significant downregulation of survivin occurred. Knockdown survivin by siRNA significantly increased TRAIL-induced apoptosis. We hypothesized that quercetin-induced activation of MAPK (ERK, p38, JNK) is responsible for downregulation of survivin gene expression. To test this hypothesis, we selectively inhibited MAPK during treatment with quercetin. Our data demonstrated that inhibitor of ERK (PD98059), but not p38 MAPK (SB203580) or JNK (SP600125), significantly maintained the intracellular level of survivin during treatment with quercetin. Interestingly, PD98059 also prevented quercetin-induced deacetylation of histone H3. Data from survivin promoter activity assay suggest that the Sp1 transcription factor binds to the survivin promoter region and quercetin inhibits its binding activity through deacetylation of histone H3. Quercetin-induced activation of the ERK-MSK1 signal transduction pathway may be responsible for deacetylation of histone H3. Taken together, our findings suggest that quercetin enhances TRAIL induced apoptosis by inhibition of survivin expression, through ERK-MSK1-mediated deacetylation of H3.

Keywords: Abbreviations; DTT; dithiothreitol; FLICE; Fas-associated death domain-like interleukin-1 β-converting enzyme; FLIP; FLICE inhibitory protein; IAP; inhibitor of apoptosis; PAGE; polyacrylamide gel electrophoresis; PARP; poly (ADP-ribose) polymerase; PBS; phosphate-buffered saline; PDK-1; phosphoinositide-dependent kinase-1; PI3K; phosphatidylinositol 3-kinase; PP1; protein phosphatase 1; SDS; sodium dodecyl sulfate; TNF; tumor necrosis factor; TRAIL; tumor necrosis factor-related apoptosis-inducing ligandQuercetin; TRAIL; Apoptosis; Survivin; ERK

Synthesis of bisphosphonate derivatives of ATP by T4 DNA ligase, ubiquitin activating enzyme (E1) and other ligases by María A. Günther Sillero; Anabel de Diego; Francisco J. Pérez-Zúñiga; Antonio Sillero (pp. 1959-1965).

T4 DNA ligase and the ubiquitin activating enzyme (E1), catalyze the synthesis of ATP β,γ-bisphosphonate derivatives. Concerning T4 DNA ligase: (i) etidronate (pC(OH)(CH₃)p) displaced the AMP moiety of the complex E-AMP in a concentration dependent manner; (ii) the K_m values and the rate of synthesis k_cat (s⁻¹), determined for the following compounds were, respectively: etidronate, 0.73±0.09mM and (70±10)×10⁻³s⁻¹; clodronate (pCCl₂p), 0.08±0.01mM and (4.1±0.3)×10⁻³s⁻¹; methylenebisphosphonate (pCH₂p), 0.024±0.001mM and (0.6±0.1)×10⁻³s⁻¹; tripolyphosphate (P₃) (in the synthesis of adenosine 5′-tetraphosphate, p₄A), 1.30±0.30mM and (6.2±1.1)×10⁻³s⁻¹; (iii) in the presence of GTP and ATP, inhibition of the synthesis of Ap₄G was observed with clodronate but not with pamidronate (pC(OH)(CH₂–CH₂–NH₃)p). Concerning the ubiquitin activating enzyme (E1): methylenebisphosphonate was the only bisphosphonate, out of the ones tested, that served as substrate for the synthesis of an ATP derivative ( K_m=0.36±0.09mM and k_cat=0.15±0.02s⁻¹). None of the above bisphosphonates were substrates of the reaction catalyzed by luciferase or by acyl-CoA synthetase. The ability of acetyl-CoA synthetase to use methylenebisphosphonate as substrate depended on the commercial source of the enzyme. In our view this report widens our knowledge of the enzymes able to metabolize bisphosphonates, a therapeutic tool widely used in the treatment of osteoporosis.

Keywords: Abbreviations; Aa; amino acid; Ap; ₄; A; diadenosine 5′,5‴-P; ¹; ,P; ⁴; -tetraphosphate; Ap; ₄; G; adenosine 5′,5‴-P; ¹; ,P; ⁴; -tetraphosphoguanosine; ApppI; triphosphoric acid 1-adenosin-5′-yl ester 3-(3-methylbut-3-enyl) ester; AppCCl; ₂; p; adenosine 5′-(β,γ-dichloromethylenetriphosphate); AppCH; ₂; p; adenosine 5′-(β,γ-methylenetriphosphate); AppC(R; ₁; )(R; ₂; )p; adenosine 5′-triphosphate derivative of any bisphosphonate; BSA; bovine serum albumin; DTT; dithiothreitol; P; ₃; tripolyphosphate; p; ₄; A; adenosine 5′-tetraphosphate; pC(OH)(CH; ₂; –CH; ₂; –NH; ₃; )p; pamidronate, or 3-amino-1-hydroxypropylidene-1,1-bisphosphonate; pC(OH)(CH; ₂; –CH; ₂; –CH; ₂; –NH; ₃; )p; alendronate or 4-amino-1-hydroxybutylidene-1,1-bisphosphonate; pC(OH)(CH; ₃; )p (HEBP); etidronate or ethane-1-hydroxy-1,1-bisphosphonate or hydroxyethylidenebisphosphonate; pCCl; ₂; p (Cl; ₂; MBP); clodronate or methane-1-dichloro-1,1-bisphosphonate or dichloromethylenebisphosphonate; pCH; ₂; p; methylenebisphosphonate or methyleneBP; pC(R; ₁; )(R; ₂; )p; any bisphosphonate; Ub; ubiquitinOsteoporosis; Bisphosphonates; Ligases; Dinucleoside polyphosphates; T4 DNA ligase; Ubiquitin activating enzyme (E1)

Diadenosine polyphosphates Ap3A and Ap4A, but not Ap5A or Ap6A, induce proliferation of vascular smooth muscle cells by Peter Bobbert; Hartmut Schlüter; Heinz Peter Schultheiss; Hans Peter Reusch (pp. 1966-1973).

Depending on the number of phosphate groups, diadenosine polyphosphates (ApnA, Ap3A, Ap4A, Ap5A and Ap6A) differ in properties such as proliferation, apoptosis, vasoconstriction and vasodilatation of vascular smooth muscle cells (VSMCs). Possible signaling pathways leading to effects such as proliferation are still unknown. This study examined the proliferative effects of diadenosine polyphosphates on VSMCs and their intracellular pathways.Proliferation of VSMCs was measured by the cell count and [³H] thymidine incorporation. Phosphorylation of the MAP kinases ERK1/2 was determined by Western blotting. Single-cell [Ca²⁺]_i measurements were done to determine the influence of [Ca²⁺]_i on intracellular signaling. Stress fiber formation was assessed by fluorescence microscopy to detect an influence of Gα₁₂.Ap3A and Ap4A, but not Ap5A or Ap6A, were shown to increase proliferation of VSMCs by activating P2Y receptors, which leads to stimulation of the Ras-Raf-MEK-ERK1/2 cascade. Ap3A- and Ap4A-induced activation of the MAP kinases ERK1/2 was dependent on a signaling pathway that included the EGF receptor, PKC, PLCβ and the increase of [Ca²⁺]_i.In conclusion, Ap3A and Ap4A, but not Ap5A or Ap6A, induce proliferation of VSMCs by a signaling pathway that begins with activation of P2Y receptors and leads to stimulation of the MAP kinases ERK1/2.

Keywords: Diadenosine polyphosphates; Vascular smooth muscle cells; MAP kinases; Signal transduction

Plasmin is involved in inflammation via protease-activated receptor-1 activation in human dental pulp by Naoto Kamio; Hideki Hashizume; Sumi Nakao; Kiyoshi Matsushima; Hiroshi Sugiya (pp. 1974-1980).

Plasmin is a proteolytic enzyme produced from plasminogen by plasminogen activators. We investigated the function of plasmin in human dental pulp fibroblast-like cells. Plasmin induced an increase in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a concentration-dependent manner. Expression of mRNA for protease-activated receptor-1 (PAR-1) was detected, and the PAR-1 activating peptide SFLLRN induced an increase in [Ca²⁺]_i in the cells. The plasmin-induced increase in [Ca²⁺]_i was inhibited in the presence of the PAR-1 antagonist SCH79797. Plasmin stimulated the expression of interleukin-8 (IL-8) mRNA and prostaglandin E₂ release, which are involved in inflammation. These effects of plasmin on expression of IL-8 mRNA and prostaglandin E₂ release were inhibited in the presence of the PAR-1 antagonist SCH79797. These results suggest that plasmin activates PAR-1 and is involved in inflammation in human dental pulp.

Keywords: Plasmin; Protease-activated receptor-1; Ca; ²⁺; mobilization; Inflammation; Dental pulp

Akt activation protects pancreatic beta cells from AMPK-mediated death through stimulation of mTOR by Ying Cai; Qidi Wang; Zhidong Ling; Daniel Pipeleers; Paul McDermott; Mario Pende; Harry Heimberg; Mark Van de Casteele (pp. 1981-1993).

Sustained activation of AMP-activated protein kinase (AMPK) induces apoptosis in several cell types. In pancreatic beta cells this occurs under glucose limitation, or in the presence of the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide-riboside (AICAR). It is unknown whether Akt activation can counteract AMPK-mediated apoptosis, nor whether mTOR activation downstream of Akt mediates any survival signal in these conditions. We report that expression of a constitutively active form of Akt increases mTOR activity and prevents apoptosis upon AMPK activation. Akt-mediated survival was inhibited by rapamycin. Expression of a constitutively active form of the mTOR target ribosomal protein S6 kinase (S6K) or of translation factor eIF4E reduced apoptosis by glucose limitation, and co-expression of S6K and eIF4E protected beta cells to the same extent as active Akt. The protective effects of active Akt and S6K were associated with increased cellular protein synthesis activity. It is concluded that Akt stimulation of mTOR and subsequent activation of the targets by which mTOR affects protein translation are required and sufficient mechanisms for Akt-mediated survival of beta cells undergoing sustained AMPK activation.

Keywords: Abbreviations; AICAR; 5-aminoimidazole-4-carboxamide-riboside; AMPK; AMP-activated protein kinase; Akt; protein kinase B; mTOR; mammalian target of rapamycin; Ad; adenovirus; GFP; green fluorescent proteinDiabetes; Beta cell; Apoptosis; AMPK; Akt; mTOR

Proteasome inhibitors prevent oxidative stress-induced nerve cell death by a novel mechanism by Pamela Maher (pp. 1994-2006).

The role of the proteasome in neurodegenerative diseases is controversial. On the one hand, there is evidence that a dysfunction of proteasome activity can lead to neurodegeneration but there is also data showing that proteasome inhibition can protect nerve cells from a variety of insults. In an attempt to clarify this issue, we studied the effects of four different proteasome inhibitors in a well characterized model of oxidative stress-induced nerve cell death. Consistent with the hypothesis that proteasome inhibition can be neuroprotective, we found that low concentrations of proteasome inhibitors were able to protect nerve cells from oxidative stress-induced death. Surprisingly, the neuroprotective effects of the proteasome inhibitors appeared to be at least partially mediated by the induction of NF-κB since protection was significantly reduced in cells expressing a specific NF-κB repressor. The activation of NF-kB by proteasome inhibitors was mediated by IκBα and IKK and was blocked by antioxidants and inhibitors of mitochondrial reactive oxygen species production. These data suggest that low concentrations of proteasome inhibitors induce a moderate level of mitochondrial oxidative stress which results in the activation of neuroprotective pathways.

Keywords: Reactive oxygen species; NF-κB; Mitochondria; Phosphorylation; Glutathione

Molecular cloning and pharmacological characterization of rat melatonin MT₁ and MT₂ receptors by Valérie Audinot; Anne Bonnaud; Line Grandcolas; Marianne Rodriguez; Nadine Nagel; Jean-Pierre Galizzi; Ales Balik; Sophie Messager; David G. Hazlerigg; Perry Barrett; Philippe Delagrange; Jean A. Boutin (pp. 2007-2019).

In order to interpret the effects of melatonin ligands in rats, we need to determine their activity at the receptor subtype level in the corresponding species. Thus, the rat melatonin rMT₁ receptor was cloned using DNA fragments for exon 1 and 2 amplified from rat genomic DNA followed by screening of a rat genomic library for the full length exon sequences. The rat rMT₂ receptor subtype was cloned in a similar manner with the exception of exon 1 which was identified by screening a rat genomic library with exon 1 of the human hMT₂ receptor. The coding region of these receptors translates proteins of 353 and 364 amino acids, respectively, for rMT₁ and rMT₂. A 55% homology was observed between both rat isoforms. The entire contiguous rat MT₁ and MT₂ receptor coding sequences were cloned, stably expressed in CHO cells and characterized in binding assay using 2-[¹²⁵I]-Iodomelatonin. The dissociation constants ( K_d) for rMT₁ and rMT₂ were 42 and 130 pM, respectively. Chemically diverse compounds previously characterized at human MT₁ and MT₂ receptors were evaluated at rMT₁ and rMT₂ receptors, for their binding affinity and functionality in [³⁵S]-GTPγS binding assay. Some, but not all, compounds shared a similar binding affinity and functionality at both rat and human corresponding subtypes. A different pharmacological profile of the MT₁ subtype has also been observed previously between human and ovine species. These in vitro results obtained with the rat melatonin receptors are thus of importance to understand the physiological roles of each subtype in animal models.

Keywords: Melatonin; Melatonin receptors; Selective agonists; Selective antagonists; Rat

Role of p53, PUMA, and Bax in wogonin-induced apoptosis in human cancer cells by Dae-Hee Lee; Clifford Kim; Lin Zhang; Yong J. Lee (pp. 2020-2033).

We observed that treatment of prostate cancer cells for 24h with wogonin, a naturally occurring monoflavonoid, induced cell death in a dose- and time-dependent manner. Exposure of wogonin to LNCaP cells was associated with increased intracellular levels of p21^Cip-1, p27^Kip-1, p53, and PUMA, oligomerization of Bax, release of cytochrome c from the mitochondria, and activation of caspases. We also confirmed the role of p53 by noting that knock-in in p53 expression by transfecting p53 DNA increased wogonin-induced apoptosis in p53-null PC-3 cells. To study the mechanism of PUMA up-regulation, we determined the activities of PUMA promoter in the wogonin treated and untreated cells. Increase of the intracellular levels of PUMA protein was due to increase in transcriptional activity. Data from chromatin immunoprecipitation (ChIP) analyses revealed that wogonin activated the transcription factor p53 binding activity to the PUMA promoter region. We observed that the up-regulation of PUMA mediated wogonin cytotoxicity. Further characterization of the transcriptional response to wogonin in HCT116 human colon cancer cells demonstrated that PUMA induction was p53-dependent; deficiency in either p53 or PUMA significantly protected HCT116 cells against wogonin-induced apoptosis. Also, wogonin promoted mitochondrial translocation and multimerization of Bax. Interestingly, wogonin (100μM) treatment did not affect the viability of normal human prostate epithelial cells (PrEC). Taken together, these results indicate that p53-dependent transcriptional induction of PUMA and oligomerization of Bax play important roles in the sensitivity of cancer cells to apoptosis induced by caspase activation through wogonin.

Keywords: Abbreviations; ChIP; chromatin immunoprecipitation; CDK; cyclin-dependent kinase; DMSO; dimethyl sulfoxide; PAGE; polyacrylamide gel electrophoresis; PARP-1; poly (ADP-ribose) polymerase; PBS; phosphate-buffered saline; PrEC; normal human prostate epithelial cells; PUMA; p53 up-regulated modulator of apoptosis; SDS; sodium dodecyl sulfateWogonin; Apoptosis; p53; PUMA; Bax

Benzo[ a]pyrene inhibits osteoclastogenesis by affecting RANKL-induced activation of NF-κB by I. Voronov; K. Li; H.C. Tenenbaum; M.F. Manolson (pp. 2034-2044).

Exposure to polycyclic aryl hydrocarbons is linked to cancer, immunosuppression and other numerous health problems. We previously demonstrated that exposure to benzo[ a]pyrene (BaP), an environmental pollutant present in high concentrations in urban smog and cigarette smoke, inhibits osteoclast differentiation and bone resorption. We hypothesized that this inhibition could be due to crosstalk between the receptor activator of NF-κB ligand (RANKL) and AhR signaling cascades competing for NF-κB, a common transcription factor for both pathways. RAW264.7 cells (a mouse macrophage cell line capable of differentiating into osteoclasts in the presence of RANKL) were exposed to different concentrations of RANKL and BaP and the effect on NF-κB activation, nuclear translocation, as well as the effect of NF-κB inhibitors on BaP-mediated CYP1B1 gene expression was measured. The results demonstrated that BaP inhibited both RANKL-induced NF-κB activation and nuclear translocation. At the same time, BaP-induced CYP1B1 gene expression was inhibited by two NF-κB inhibitors in a dose-dependent manner, demonstrating that NF-κB is involved in a BaP-mediated signaling pathway. A reporter gene assay showed that both BaP and RANKL-induced luciferase reporter gene transcription under the control of NF-κB response elements. Co-immunoprecipitation results demonstrated that AhR interacted with NF-κB p65 in RAW cells and BaP appeared to enhance this interaction. However, in the presence of RANKL, we did not observe any interaction between AhR and p65. These results support our hypothesis that BaP-mediated inhibition of osteoclastogenesis is a consequence of crosstalk between AhR and RANKL signaling pathways competing for the common transcription factor NF-κB.

Keywords: Abbreviations; AhR; aryl hydrocarbon receptor; ARNT; aryl hydrocarbon receptor nuclear translocator; BaP; benzo[; a; ]pyrene; CYP1B1; cytochrome P450 1B1; DMSO; dimethyl sulfoxide; IκBα; inhibitor of nuclear factor κB α; NF-κB; nuclear factor κB; RANK; receptor activator of nuclear factor κB; RANKL; receptor activator of nuclear factor κB ligand; PAH; polycyclic aryl hydrocarbonNF-κB; Osteoclasts; RANKL; Benzo[; a; ]pyrene; AhR

Reactions of isodimethoate with human red cell acetylcholinesterase by Peter Eyer; Maria Radtke; Franz Worek (pp. 2045-2053).

Isodimethoate is a thermal decomposition product that is present in usual pesticide formulations of dimethoate. Owing to its PO structure the compound is a direct anticholinesterase agent whose properties, to the best of our knowledge, are presented here for the first time. Isodimethoate shows an inhibition rate constant towards human red blood cell acetylcholinesterase (AChE) of 2.3×10³M⁻¹min⁻¹ (pH 7.4, 37°C), indicating a somewhat higher potency than found with omethoate, the CYP450-mediated active metabolite of pure dimethoate. Isodimethoate-inhibited AChE shows fast spontaneous reactivation and aging kinetics (half-life 2.3 and 25min, respectively). The inhibited, non-aged enzyme is readily reactivated by obidoxime ( k_r=9min⁻¹; K_D=0.1mM) but hardly by pralidoxime at therapeutic concentrations. Interestingly, isodimethoate hydrolyzes readily in buffered solutions at pH 7.4 and 37°C with liberation of methylmercaptan (half-life 16min). Liberation of N-(methyl)mercaptoacetamide, the expected leaving group, was not observed. These properties make isodimethoate a hit-and-run agent that renders part of AChE non-reactivatable within a short period of time. The clinical consequences of exposure to or intentional ingestion of isodimethoate-containing dimethoate formulations are a partly untractable AChE shortly after incorporation. In fact, aging of AChE in dimethoate-poisoned patients on admission was much more advanced than expected from the reaction with omethoate. Manufacturers, researching scientists and clinical toxicologists should be aware of this problem.

Keywords: Abbreviations; AChE; acetylcholinesterase (EC 3.1.1.7); ATCh; acetylthiocholine; AU; absorbance units; DTNB; 5,5′-dithiobis(2-nitrobenzoic acid)Acetylcholinesterase; Dimethoate; Isodimethoate; Obidoxime; Pralidoxime; Pesticides

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Biochemical Pharmacology (v.75, #10)

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Biochemical Pharmacology (v.75, #10)