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Biochemical Pharmacology (v.69, #12)

Editorial Advisory Board (pp. iii).

Novel regulatory roles for protein phosphatase-2A in the islet β cell by Anjaneyulu Kowluru (pp. 1681-1691).

Protein phosphorylation constitutes one of the key signaling steps in physiological insulin secretion. The phosphorylation status of a given protein represents the balance of the activities of protein kinases and phosphatases, which induce the addition and removal of phosphate from that protein, respectively. Although several extant studies were focused on the identification and characterization of protein kinases in islets, relatively little information is available on the localization and regulation of protein phosphatases in β cells. Emerging evidence implicates protein phosphatase 2A (PP2A) in the phenomenon of insulin secretion. The three principal objectives of this commentary are to: (i) review the existing evidence, which suggests regulation, by glucose and other insulin secretagogues, of PP2A in the β cell; (ii) discuss the experimental evidence, which implicates PP2A-like enzymes in the dephosphorylation and inactivation of key β cell phosphoprotein substrates (e.g., Akt and Bcl-2), which may be necessary for β cell proliferation and survival, culminating in the loss of the β cell mass; and (iii) highlight potential avenues for future research, including the development of specific pharmacological and therapeutic interventional modalities for the inhibition of specific PP2A-like phosphatases for the prevention of loss of β cell mass leading to the onset of diabetes.

Keywords: Protein phosphatase-2A; Pancreatic islet; Insulin secretion; Ceramide; Apoptosis; Diabetes mellitusAbbreviations; ACC; acetyl CoA carboxylase; CAPP; ceramide-activated protein phosphatase; CML; carboxylmethylation; GAPP; glutamate- and magnesium-activated protein phosphatase; IHP; inositol hexakiphosphate; mTOR; mammalian target of rapamycin; OKA; okadaic acid; PPases; protein phosphatases; PP2A; protein phosphatase 2A; PP2Ac; catalytic subunit of protein phosphatase 2A; PPM1; protein phosphatase methyl transferase 1

Anti-tumoral effect of native and immobilized bovine serum amine oxidase in a mouse melanoma model by Diana A. Averill-Bates; Anissa Ch�rif; Enzo Agostinelli; Andr� Tanel; Guy Fortier (pp. 1693-1704).

Bovine serum amine oxidase (BSAO) oxidatively deaminates polyamines containing primary amine groups, spermidine and spermine, to form the cytotoxic products hydrogen peroxide and aldehyde(s). Polyamines are present at elevated levels in many tumor tissues. The aims of the study were to evaluate the anti-tumoral activities of native and immobilized BSAO in mouse melanoma and also to determine the mechanism of tumor cell death. C57BL mice received a subcutaneous injection of B16 melanoma cells to induce formation of tumors, prior to antitumor treatments with native and immobilized BSAO. The enzyme was immobilized in a poly(ethylene glycol) (PEG) biocompatible matrix. Antitumor treatments consisted of a single injection of enzyme into the tumor. When immobilized BSAO (2.5mU) was injected into the tumor, there was a marked decrease of 70% of the tumor growth. This was compared with a decrease of only 32% of tumor size when the same amount of native BSAO was administered. The type of cell death was analysed in tumors that were treated with native or immobilized BSAO. When tumors were treated with immobilized BSAO, there was induction of a high level of apoptosis (around 70%), compared to less than 10% with the native enzyme. Apoptotic cell death was assessed by nuclear chromatin condensation using Hoechst staining and labelling of externalized phosphatidylserine using Annexin V. However, native BSAO, probably due to a burst of cytotoxic products, induced a high level of necrosis of about 40%, compared to less than 10% with immobilized BSAO. In conclusion, the advantage is that immobilized BSAO can act by allowing the slow release of cytotoxic products, which induces tumor cell death by apoptosis rather than necrosis.

Keywords: Abbreviations; BSAO; bovine serum amine oxidase; DAH; 1,6-diaminohexane; DFMO; α-difluoromethylornithine; DMEM; Dulbecco's modified Eagle medium; GPx; glutathione peroxidase; GSH; reduced glutathione; GST; glutathione S-transferase; IU; international unit; PEG; polyethylene glycol; PBS; phosphate buffered saline; PI; propidium iodide; SD; standard deviation; S.E.M.; standard error of the meanAmine oxidase; Apoptosis; Immobilized; Mouse melanoma; Necrosis; Polyamine

Differential CB1 and CB2 cannabinoid receptor-inotropic response of rat isolated atria: Endogenous signal transduction pathways by Leonor Sterin-Borda; Claudia F. Del Zar; Enri Borda (pp. 1705-1713).

In this study, we have determined the contractile effects of CB1 and CB2 cannabinoid receptor activation on rat isolated atria and the different signaling pathways involved. Anandamide did not has significantly effect on atria contractility, however, the treatment with both CB1 (AM251) or CB2 (AM630) receptor antagonists, the endocannabinoids triggered stimulation or inhibition on contractility respectively. The ACEA stimulation of CB1 receptor exerted decrease on contractility, that significantly correlated with the decrement of cAMP and the stimulation of nitric oxide synthase (NOS) and the accumulation of cyclic GMP (cGMP). On the contrary, JWH 015 stimulation of CB2 receptor triggered positive contractile response that significantly correlated with the increase cAMP production. The inhibiton of adenylate cyclase activity impaired the JWH 015 activation of CB1 receptor induced positive contractile effect, while inhibitors of phospholipase C (PLC), NOS and soluble nitric oxide (NO)-sensitive guanylate cyclase blocked the dose-response curves of ACEA on conntractility. Those inhibitors also attenuated the CB1 receptor-dependent increase in activation of NOS and cGMP accumulation. These results suggest that CB2 receptor agonist mediated positive contractile effect associated with increased production on cAMP while CB1 receptor agonist mediated decrease on contractility associated with decreased cAMP accumulation and increase production of NO and cGMP; that occur secondarily to stimulation of PLC, NOS and soluble guanylate cyclase. Data give pharmacological evidence for the existence of functional CB1 and CB2 cannabinoid receptors in rat isolated atria and may contribute to a better understanding the effects of cannabinoids in the cardiovascular system.

Keywords: Cannabinoid receptors; Atria contractility; NOS; cGMP; Camp

Luteolin prevents PDGF-BB-induced proliferation of vascular smooth muscle cells by inhibition of PDGF β-receptor phosphorylation by Jin-Ho Kim; Yong-Ri Jin; Beoung-Soo Park; Tack-Joong Kim; Soo-Yeon Kim; Yong Lim; Jin-Tae Hong; Hwan-Soo Yoo; Yeo-Pyo Yun (pp. 1715-1721).

Luteolin occurs as glycosylated forms in celery, green pepper, perilla leaf and camomile tea, and has been shown to possess antimutagenic, antitumorigenic, antioxidant and antiinflammatory properties. In this study, we have investigated the antiproliferable effect and its mechanism of luteolin on platelet-derived growth factor (PDGF)-BB-induced proliferation of rat aortic vascular smooth muscle cells (VSMCs). Luteolin significantly inhibited PDGF-BB-induced proliferation and DNA synthesis of rat aortic VSMCs in a concentration-dependent manner. In addition, flow cytometry analysis of DNA content revealed blocking of the PDGF-BB-inducible cell cycle progression by luteolin. Pre-incubation of rat aortic VSMCs with luteolin significantly inhibited the PDGF-BB-induced extracellular signal-regulated kinase 1/2 (ERK1/2), Akt and phospholipase C (PLC)-γ1 activation as well as c-fos gene expression. Consisted with these findings, luteolin inhibited PDGF-Rβ phosphorylation induced by PDGF-BB in a concentration-dependent manner. These results suggest that the inhibitory effect of luteolin on the PDGF-BB-induced proliferation of rat aortic VSMCs may be mediated by blocking phosphorylation of PDGF-Rβ.

Keywords: Abbreviations; DMEM; Dulbecco's modified Eagle's medium; ERK; extracellular signal-regulated kinase; FBS; fetal bovine serum; FACS; fluorescence activated cell sorter; MAP; mitogen-activated protein; PDGF; platelet-derived growth factor; RASMC; rat aortic smooth muscle cellLuteolin; Vascular smooth muscle cell; Proliferation; Cell cycle; PDGF-Rβ

Real-time measurement in living cells of insulin-like growth factor activity using bioluminescence resonance energy transfer by Lisbeth S. Laursen; Claus Oxvig (pp. 1723-1732).

Insulin-like growth factor (IGF)-I and -II function in normal physiology to control growth, development, and differentiation, but are also important in pathophysiological conditions, particularly in cancer. The biological effects of the IGFs are mediated by the IGF-I receptor (IGFR), a covalent homodimer composed of two alpha and two beta chains, similar in structure to the insulin receptor (IR). To allow measurement of the stimulation of IGFR in living cells, we developed an assay based on bioluminescence resonance energy transfer (BRET) between a donor molecule, Renilla luciferase, and an acceptor fluorophore, enhanced yellow fluorescent protein (EYFP). Initial attempts based on fusion of the luciferase to IGFR, and EYFP to IGFR, or to downstream signaling molecules, insulin receptor substrate-1 (IRS1) or protein tyrosine phosphatases-1B (PTP-1B), failed. However, similar experiments with IR, carried our in parallel, proved successful. We therefore, constructed assays based on chimeric IGFR/IR proteins, in which the ligand binding site was derived from IGFR. With the most efficient assay, in which the luciferase is fused to a chimeric receptor with the entire intracellular portion derived from IR, and EYFP fused to PTP-1B, IGF activity was measured specifically with sensitivity similar to the corresponding assay for insulin, based on IR. The established system allows efficient evaluation of candidate ligand- or receptor-directed molecules for the modulation of IGF activities. Furthermore, we demonstrate that a set of inhibitory IGF binding proteins (IGFBPs) or activating IGFBP-specific proteinases, unique to the IGF system, may serve as potential targets. In addition to screening, real-time measurement of IGFR stimulation may be important in efforts to understand the kinetics of receptor stimulation, in particular differences between IGFR and IR.

Keywords: Insulin-like growth factor; Insulin; Receptor; Bioluminescence resonance energy transfer; Insulin-like growth factor binding proteins

Rosiglitazone, an agonist of peroxisome proliferator-activated receptor gamma, reduces chronic colonic inflammation in rats by M. S�nchez-Hidalgo; A.R. Mart�n; I. Villegas; C. Alarc�n De La Lastra (pp. 1733-1744).

Recent studies have shown the implication of the peroxisome proliferator-activated receptor gamma (PPARγ) in control of inflammation, immune and apoptotic responses during early experimental colitis. However, there is little information about the effects of these agents on colonic mucosa under chronic inflammatory conditions. In this study, we have evaluated the effects of rosiglitazone, a PPAR-γ agonist, on the chronic injury caused by intra-colonic administration of trinitrobenzensulfonic acid (TNBS) in rats. Rosiglitazone (1 and 5mg/kg p.o.) was administered by oral gavage, 24h after TNBS instillation and daily during 2 weeks before killing the rats. Colons were removed for histological and biochemical analysis. Administration of rosiglitazone corrected the disorders in morphology associated to lesions, significantly reduced the ulceration index, the rise of myeloperoxidase (MPO) and the levels of tumour necrosis factor alpha (TNF-α). In addition, rosiglitazone treatment increased prostaglandin (PG)E₂ production and returned PGD₂ to basal levels. Also, reduced cyclooxygenase (COX)-2 and nuclear transcription factor NF-kappa B (NF-κB) p65 proteins expression. Furthermore, treatment of rats with rosiglitazone caused a significant increase of TNBS-induced apoptosis. In summary, rosiglitazone exerts protective effects in chronic experimental colitis. The anti-inflammatory effects seem to be related to impairment of neutrophil function, absence of up-regulation of TNF-α and decrease of nuclear NF-κB p65 expression. Our results also suggest that the activation of the PPARγ pathway reduces COX-2 overexpression, returns the increased PGD₂ values to basal levels and induces a significant increase of TNBS-induced apoptosis. We conclude that rosiglitazone represents a novel approach to the treatment of ulcerative colitis.

Keywords: Abbreviations; COX; cyclooxigenase; IBD; inflammatory bowel disease; UC; ulcerative colitis; MPO; myeloperoxidase; NF-κB; nuclear transcription factor NF-kappa B; PG; prostaglandin; PPARγ; peroxisome proliferator-activated receptor gamma; ROS; rosiglitazone; TNBS; trinitrobenzensulfonic acid; TNF-α; tumour necrosis factor alphaPPAR-γ; Colitis; COX-2; Prostaglandins; NF-κB; Apoptosis

Intestinal inflammation induces adaptation of P-glycoprotein expression and activity by Marion Buyse; Genia Radeva; Andr� Bado; Robert Farinotti (pp. 1745-1754).

Recent studies suggest that P-glycoprotein (Pgp) encoded by MDR1 gene, may be an important factor in the pathogenesis of inflammatory bowel disease (IBD). In this study, we investigated intestinal Pgp expression and activity: (1) in IL10 deficient (IL10^−/−) mice which spontaneously develop intestinal inflammation affecting the small and large intestine and (2) in DSS (dextran sodium sulfate)-induced rat colitis.In IL10^−/− enterocolitis mice, rhodamine 123 efflux was reduced by two to three-fold along the small and large intestine. This decrease was associated with a reduction in membrane's Pgp protein levels. A similar three-fold decrease in Pgps activity and expression was observed in the proximal colon in DSS-induced colitis in rats. However, in the non-inflamed ileum in DSS-induced rat colitis, epithelial cell's Pgp activity and protein levels were unexpectedly increased. This effect was specific to local inflammation since LPS induced systemic inflammation did affect neither the intestinal rho 123 efflux transport nor the abundance of the Pgp protein.These data demonstrate for the first time, an impaired function of epithelial Pgp in IL10 deficient enterocolitis mice. They also show an increase in Pgps activity in the non-inflamed ileum in the DSS-induced rat colitis, which may represent an adaptive mechanism to compensate the impaired activity of Pgp in the colon.

Keywords: Pgp; Colitis; DSS; IL10; ^−/−; mice

Regulation of ileal bile acid-binding protein expression in Caco-2 cells by ursodeoxycholic acid: Role of the farnesoid X receptor by Gabriele Campana; Patrizia Pasini; Aldo Roda; Santi Spampinato (pp. 1755-1763).

Ursodeoxycholic acid (UDCA) is beneficial in cholestatic diseases but its molecular mechanisms of action remain to be clearly elucidated. Other bile acids, such as chenodeoxycholic (CDCA), are agonists for the nuclear farnesoid X receptor (FXR) and regulate the expression of genes relevant for bile acid and cholesterol homeostasis. In ileal cells CDCA, through the FXR, up-regulates the expression of the ileal bile acid-binding protein (IBABP), implicated in the enterohepatic circulation of bile acids. We report that UDCA (100 and 200μM) induced a moderate increase of IBABP mRNA (≈10% of the effect elicited by 50μM CDCA) in enterocyte-like Caco-2 cells and approximately halved the potent effect of CDCA (50μM). On the contrary, UDCA reduced by 80–90% CDCA-induced IBABP transcription in hepatocarcinoma derived HepG2 cells. We confirmed that these effects on IBABP transcription required the FXR by employing a cell-based transactivation assay. Finally, in a receptor binding assay, we found that UDCA binds to FXR expressed in CHO-K1 cells ( K_d=37.7μM). Thus, UDCA may regulate IBABP in Caco-2 cells, which express it constitutively, by acting as a partial agonist through a FXR mediated mechanism. The observation that in HepG2 cells, which do not express constitutively IBABP, UDCA was able to almost completely prevent CDCA-induced activation of IBABP promoter, suggests that tissue-specific factors, other than FXR, may be required for bile acid regulation of FXR target genes.

Keywords: Abbreviations; CDCA; chenodeoxycholic acid; Cyp7a; cholesterol 7α-hydroxylase; DCA; deoxycholic acid; FCS; fetal calf serum; FXR; farnesoid X receptor; IBABP; ileal bile-acid binding protein; LDH; lactate dehydrogenase; RLU; relative light units; UDCA; ursodeoxycholic acidBile acids; Ursodeoxycholic acid; Chenodeoxycholic acid; Caco-2 cells; Ileal bile acid-binding protein; mRNA; Farnesoid X receptor

The role of thioredoxin reductase activity in selenium-induced cytotoxicity by Zbigniew Madeja; Jolanta Sroka; Christina Nystr�m; Linda Bj�rkhem-Bergman; Tomas Nordman; Anastasios Damdimopoulos; Ivan Nalvarte; Lennart C. Eriksson; Giannis Spyrou; Jerker M. Olsson; Mikael Bj�rnstedt (pp. 1765-1772).

The selenoprotein thioredoxin reductase is a key enzyme in selenium metabolism, reducing selenium compounds and thereby providing selenide to synthesis of all selenoproteins. We evaluated the importance of active TrxR1 in selenium-induced cytotoxicity using transfected TrxR1 over-expressing stable Human Embryo Kidney (HEK-293) cells and modulation of activity by pretreatment with low concentration of selenite. Treatment with sodium selenite induced cytotoxity in a dose-dependent manner in both TrxR1 over-expressing and control cells. However, TrxR1 over-expressing cells, which were preincubated for 72h with 0.1μM selenite, were significantly more resistant to selenite cytotoxicity than control cells. To demonstrate the early effects of selenite on behaviour of HEK-293 cells, we also investigated the influence of this compound on cell motility. We observed inhibition of cell motility by 50μM selenite immediately after administration. Moreover, TrxR1 over-expressing cells preincubated with a low concentration of selenite were more resistant to the inhibitory effect of 50μM selenite than those not preincubated. It was also observed that the TrxR over-expressing cells showed higher TrxR1 activity than control cells and the preincubation of over-expressing cells with 0.1μM selenite induced further significant increase in the activity of TrxR1. On the other hand, we demonstrated that TrxR1 over-expressing cells showed decreased glutathione peroxidase activity compared to control cells. These data strongly suggest that TrxR1 may be a crucial enzyme responsible for cell resistance against selenium cytotoxicity.

Keywords: Abbreviations; Trx; thioredoxin; TrxR1; thioredoxin reductase 1; HEK-293; human embryo kidney cells; GR; glutathione reductase; GPx; glutathione peroxidase; DTNB; 5,5′dithiobis(nitrobenzoic acid)Selenium; Thioredoxin reductase; Cytotoxicity; Glutathione peroxidase; Cell motility; Oxidative stress

Involvement of Ras/extracellular signal-regulated kinase, but not Akt pathway in risedronate-induced apoptosis of U937 cells and its suppression by cytochalasin B by Hirofumi Fujita; Toshihiko Utsumi; Shikibu Muranaka; Tetsuya Ogino; Hiromi Yano; Jitsuo Akiyama; Tatsuji Yasuda; Kozo Utsumi (pp. 1773-1784).

Although risedronate, a nitrogen containing bisphosphonate (BPs), strongly inhibits bone resorption by enhanced apoptosis of osteoclasts, its mechanism remained unclear. In this study, we investigated the molecular mechanism of risedronate-induced apoptosis of U937 cells, with a focus on extracellular signal-regulated kinase 1/2 (ERK 1/2) and protein kinase B (Akt) pathways, mitochondria-mediated apoptosis, and the effect of disruption of the actin cytoskeleton. Risedronate facilitated the relocation of Ras from membrane to cytosol through inhibited isoprenylation. Accordingly, risedronate suppressed the phosphorylation of ERK 1/2, a downstream survival signaling kinase of Ras, affected the intracellular distribution of Bcl-xL, and induced the mitochondrial membrane depolarization, cytochrome c release, activated caspase cascade and DNA fragmentation. The risedronate-induced apoptosis was effectively suppressed with cyclosporine A plus trifluoperazine, potent inhibitors of mitochondrial membrane permeability transition (MPT). The risedronate-induced apoptosis was independent of Akt, another cAMP-dependent survival signaling kinase. Risedronate facilitated dephosphorylation of Bad at Ser112, an ERK phosphorylation site, but not at Ser136, an Akt phosphorylation site. All of these apoptosis-related changes induced by risedronate were strongly suppressed by cytochalasin B, an inhibitor of actin filament polymerization. These results indicate that risedronate-induced apoptosis in U937 cells involves Ras/ERK, but not Akt signaling pathway, and is dependent on MPT, and that disruption of the actin cytoskeleton inhibits the risedronate-induced apoptosis at its early step.

Keywords: Abbreviations; ERK; extracellular signal-regulated kinase; PS; phosphatidylserine; BPs; bisphosphonates; AMC; 7-amino-4-methyl-coumarin; Ac-IETD-CHO; acetyl-Ile-Glu-Thr-Asp-CHO; JC-1; 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol carbocyanine iodide; FPP; farnesyl pyrophosphate; GGOH; geranylgeraniol; s-GTP-bp; small GTP-binding proteins; MPT; membrane permeability transition; pCPT-cAMP; 8-(4-chlorophenylthio) adenosine 3′:5′-cyclic monophosphateApoptosis; Bisphosphonate; Cytochalasin B; ERK1/2; Bad; Membrane permeability transition

The role of epidermal growth factor receptor in ethanol-mediated inhibition of activator protein-1 transactivation by Cuiling Ma; Kimberly A. Bower; Hong Lin; Gang Chen; Chuanshu Huang; Xianglin Shi; Jia Luo (pp. 1785-1794).

A potential mechanism underlying ethanol-induced alterations in gene expression is the disruption of transcription factor activity. Growth factor receptors, particularly receptor tyrosine kinases, play an important role in modulating many biological effects of ethanol. We demonstrated here that the expression of epidermal growth factor receptor (EGFR) mediated the effect of ethanol on the activity of transcription factor activator protein-1 (AP-1). Ethanol had little effect on AP-1 activity in the fibroblast cells devoid of EGFR (B82); however, it significantly suppressed AP-1 activity in B82 cells that were stably transfected with either a wild-type EGFR (B82L) or a kinase-deficient receptor (B82M721) in a concentration-dependent manner. EGF activated AP-1 only in B82L cells; the activation was mediated primarily by Akt and ERK. Ethanol inhibited EGF-induced EGFR autophosphorylation, phosphorylation of ERK as well as Akt and its substrate GSK-3β, and subsequently blocked EGF-stimulated AP-1 activation in B82L cells. On the other hand, ethanol had little effect on EGF-stimulated JNK activation. Phorbol ester 12- O-teradecanoyl-phorbol-13-acetate (TPA) activated AP-1 in B82L and B82M721 cells, but not B82 cells. TPA-induced activation of ERK and PKCδ was dependent on the expression of EGFR although the intrinsic kinase activity of EGFR was not required. In contrast, TPA-induced phosphorylation of p38 MAPK, JNKs and other PKC isoforms was independent of EGFR. Ethanol selectively inhibited TPA-induced phosphorylation of ERK and PKCδ, and modestly suppressed TPA-stimulated AP-1 activation in B82L and B82M721 cells. Thus, EGFR plays a critical role in the interaction between ethanol and AP-1.

Keywords: Alcohol; Cell signaling; Gene transcription; Receptor tyrosine kinases

A sensitive new method for clinically monitoring cytarabine concentrations at the DNA level in leukemic cells by Takahiro Yamauchi; Takanori Ueda (pp. 1795-1803).

Cytarabine (ara-C), a major antileukemic agent, is phosphorylated in the cell to cytarabine triphosphate (ara-CTP), which is then partly incorporated into DNA. The drug incorporation into DNA poisons the extending primer against further incorporation of deoxyribonucleotides including dCTP, ultimately inhibiting DNA synthesis. While intracellular ara-CTP concentration has been found to predict clinical outcome, cytotoxicity in vitro is determined primarily by the extent of drug incorporation into DNA. However, clinically appropriate quantitation methods for ara-C at the DNA level have not been available. We developed a sensitive new method for monitoring ara-C incorporated into DNA in vivo. After DNA from leukemic cells was fractionated using the Schmidt-Thannhauser-Schneider method, it was degraded to constituent nucleosides to release ara-C, which was isolated from the nucleosides using HPLC and then measured by radioimmunoassay. Recovery for DNA fractionation, ara-C release by degradation, and ara-C isolation were 92.0�6.4%, 90.7�9.4%, and 98.5�1.4%, respectively. The method was found to determine ara-C incorporation into DNA of ara-C-treated HL 60 cells in vitro with minimal interassay variation. The values determined were compatible with those determined by scintillation counting in parallel experiments using tritiated ara-C. Our method could be used to monitor DNA-incorporated ara-C concentrations during ara-C therapy, together with plasma ara-C and intracellular ara-CTP concentrations. ara-C incorporation into DNA appeared to be associated with intracellular retention of ara-CTP or persistence of plasma ara-C. Thus, the present method is sensitive, accurate, precise, and may permit therapeutic drug monitoring at the DNA level for better individualization of antileukemic regimens.

Keywords: Abbreviations; ara-C; cytarabine or 1-β-; d; -arabinofuranosylcytosine; ara-CTP; cytarabine triphosphate or 1-β-; d; -arabinofuranosylcytosine triphosphate; TDM; therapeutic drug monitoring; STS; Schmidt-Thannhauser-SchneiderCytarabine; DNA incorporation; Cytarabine triphosphate; Acute leukemia; TDM; Individualization

Transfection of adult primary rat hepatocytes in culture by C. Gardmo; P. Kotokorpi; H. Helander; A. Mode (pp. 1805-1813).

The use of adult primary hepatocytes in culture is of importance for the understanding of hepatic processes at the cellular and molecular levels, and the possibility to employ transient transfection of reporter constructs is invaluable for mechanistic studies on hepatic gene regulation. Although frequently used, there is a lack of reports addressing optimization and characterization of transfection of primary rodent hepatocytes. Here, we have shown that the efficiency of biochemical transfection reagents varies significantly and that Lipofectamine2000 was a superior transfection reagent for adult primary rat hepatocytes when using luciferase reporter vectors. The efficiency increased when the cells were allowed ample time to adapt to the in vitro milieu. Cotransfection of a second reporter gene indicated a risk for promoter competition, and we found that relating reporter activity to total cellular protein content gave consistent and reliable results. Differentiation of the cells, achieved by including biomatrix from the Engelbreth–Holm–Swarm mouse sarcoma in the culture system, was to a larger extent required for hormonal/drug responses of transfected constructs than for responses of endogenous genes and assured responses of transfected constructs. Dexamethasone (Dex) is most often included in hepatocyte culture media, but we could not demonstrate a general beneficial effect of Dex on expression of luciferease reporter contructs. Using the established protocol, we have demonstrated responses of transfected constructs to growth hormone, glucocorticoid and LXR stimuli.

Keywords: Rat liver; Primary hepatocytes; Transfection; Lipofection; Luciferase reporter; Extracellular matrix

Chrysin induces G1 phase cell cycle arrest in C6 glioma cells through inducing p21^Waf1/Cip1 expression: Involvement of p38 mitogen-activated protein kinase by Meng-Shih Weng; Yuan-Soon Ho; Jen-Kun Lin (pp. 1815-1827).

Flavonoids are a broadly distributed class of plant pigments, universally present in plants. They are strong anti-oxidants that can inhibit carcinogenesis in rodents. Chrysin (5,7-dihydroxyflavone) is a natural and biologically active compound extracted from many plants, honey, and propolis. It possesses potent anti-inflammatory, anti-oxidant properties, promotes cell death, and perturbing cell cycle progression. However, the mechanism by which chrysin inhibits cancer cell growth remains poorly understood. Therefore, we developed an interest in the relationship between MAPK signaling pathways and cell growth inhibition after chrysin treatment in rat C6 glioma cells. Cell viability assay and flow cytometric analysis suggested that chrysin exhibited a dose-dependent and time-dependent ability to block rat C6 glioma cell line cell cycle progression at the G1 phase. Western blotting analysis showed that the levels of Rb phosphorylation in C6 glioma cells exposed to 30μM chrysin for 24h decreased significantly. We demonstrated the expression of cyclin-dependent kinase inhibitor, p21^Waf1/Cip1, to be significantly increased, but the p53 protein level did not change in chrysin-treated cells. Both cyclin-dependent kinase 2 (CDK2) and 4 (CDK4) kinase activities were reduced by chrysin in a dose-dependent manner. Furthermore, chrysin also inhibited proteasome activity. We further showed that chrysin induced p38-MAPK activation, and using a specific p38-MAPK inhibitor, SB203580, attenuated chrysin-induced p21^Waf1/Cip1 expression. These results suggest that chrysin exerts its growth-inhibitory effects either through activating p38-MAPK leading to the accumulation of p21^Waf1/Cip1 protein or mediating the inhibition of proteasome activity.

Keywords: Chrysin; Cell cycle; p21; ^Waf1/Cip1; p38-MAPK; Proteasome activity

Spontaneous apoptosis, necrosis, energy status, glutathione levels and biotransformation capacities of isolated rat hepatocytes in suspension: Effect of the incubation medium by Greetje Elaut; Tamara Vanhaecke; Yvan Vander Heyden; Vera Rogiers (pp. 1829-1838).

Isolated hepatocytes in suspension express most of the functional activities of the intact liver and offer an easy-to-handle in vitro system for investigating both the biotransformation and damaging effects induced after a single exposure to xenobiotics upto 3–4h. There is, however, a general lack of consensus with respect to the choice of a suitable suspension medium. This motivated us to perform a comparative study of the effects of five frequently used bicarbonate-based media (Ca²⁺-containing Krebs–Henseleit buffer (KHB) with or without 25mM HEPES, 10mM glucose and 2% (g/v) BSA supplements, and Williams’ E culture medium) on the viability (LDH leakage, caspase-3 processing and activity, Bid/Bax expression) and functionality (energy status, glutathione content, phases I and II biotransformation) of freshly isolated rat hepatocytes in suspension upto 3h. Also included was the bicarbonate-free HEPES buffer that does not require carbogen gassing, and is therefore handled more easily. The results clearly demonstrated that the type of incubation medium profoundly affected the functionality of the suspended hepatocytes, changing their sensitivity and response to exogenous damaging effects. While HEPES buffer and Williams’ E medium offered the lowest background of spontaneous cell death, bicarbonate-based buffers and media seemed more suitable for obtaining both phases I and II biotransformation. Williams’ E medium ensured a constant glutathione content of the cells and a lower level of oxidative stress.

Keywords: Abbreviations; BSA; bovine serum albumin; CYP; cytochrome P450-dependent monooxygenase; 7-EC; 7-ethoxycoumarin; GSH; reduced glutathione; GSSG; oxidized glutathione; 7-HC; 7-hydroxycoumarin; HEPES; N; -2-hydroxyethylpiperazine-; N; ′-2-ethanesulfonic acid; KHB; Krebs–Henseleit buffer; LDH; lactate dehydrogenase; PBS; phosphate-buffered saline solutionIsolated rat hepatocytes in suspension; Necrosis; Apoptosis; Cellular energy status; Glutathione content; Phase I and II xenobiotic biotransformation capacities

Quercetin, but not rutin and quercitrin, prevention of H₂O₂-induced apoptosis via anti-oxidant activity and heme oxygenase 1 gene expression in macrophages by Jyh-Ming Chow; Shing-Chuan Shen; Steven K. Huan; Hui-Yi Lin; Yen-Chou Chen (pp. 1839-1851).

In the present study, we examine the protective mechanism of quercetin (QE) on oxidative stress-induced cytotoxic effect in RAW264.7 macrophages. Results of Western blotting show that QE but not its glycoside rutin (RUT) and quicitrin-induced HO-1 protein expression in a time- and dose-dependent manner, and HO-1 protein induced by QE was blocked by an addition of cycloheximide or actinomycin D. Induction of HO-1 gene expression by QE was accompanied by inducing ERKs, but not JNKs or p38, proteins phosphorylation. Addition of PD98059, but not SB203580 or SP600125, significantly attenuates QE-induced HO-1 protein and mRNA expression associated with blocking the expression of phosphorylated ERKs proteins. H₂O₂ addition reduces the viability of cells by MTT assay, and appearance of DNA ladders, hypodiploid cells, and an increase in intracellular peroxide level was detected. Addition of QE, but not QI or RUT, significantly reduced the cytotoxic effect induced by H₂O₂ associated with blocking the production of intracellular peroxide, DNA ladders, and hypodiploid cells. QE protection of cells from H₂O₂-induced apoptosis was significantly suppressed by adding HO inhibitor SnPP or ERKs inhibitor PD98059. Additionally, QE protects cells from H₂O₂-induced a decrease in the mitochondrial membrane potential and a release of cytochrome c from mitochondria to cytosol by DiOC6 and Western blotting assay, respectively. Activation of apoptotic proteins including the caspase 3, caspase 9, PARP, D4-GDI proteins was identified in H₂O₂-treated cells by Western blotting and enzyme activity assay, and that was significantly blocked by an addition of QE, but not RUT and QI. Furthermore, HO-1 catalytic metabolites carbon monoxide (CO), but not Fe²⁺, Fe³⁺, biliverdin or bilirubin, performed protective effect on cells from H₂O₂-induced cell death with an increase in HO-1 protein expression and ERKs protein phosphorylation. These data suggest that induction of HO-1 protein may participate in the protective mechanism of QE on oxidative stress (H₂O₂)-induced apoptosis, and reduction of intracellular ROS production and mitochondria dysfunction with blocking apoptotic events were involved. Differential anti-apoptotic effect between QE and its glycosides RUT and QI via distinct HO-1 protein induction was also delineated.

Keywords: Abbreviations; HO-1; heme oxygenase 1; SnPP; tin protoporphyrin; CO; carbon monoxide; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NBT; nitroblue tetrazolium; BCIP; 5-bromo-4-chloro-3-indolyl phosphate; RT-PCR; reverse transcriptase-polymerase chain reaction; GAPDH; glutaldehyde-3-phosphate dehydrogenase; H; ₂; O; ₂; hydrogen peroxide; ERKs; extracellular regulatory kinases; MAPKs; mitogen activated protein kinases; JNKs; c-Jun N-terminal kinasesFlavonoids; Quercetin; Heme oxygenase 1; Reactive oxygen species; ERKs

Formation and disposition of diethylphosphoryl-obidoxime, a potent anticholinesterase that is hydrolyzed by human paraoxonase (PON1) by D. Kiderlen; P. Eyer; F. Worek (pp. 1853-1867).

The potential of pyridinium-4-aldoximes, such as obidoxime, to reactivate diethylphosphorylated acetylcholinesterases is not fully exploited due to the inevitable formation of phosphoryloximes (POX) with high anticholinesterase activity. Mono(diethylphosphoryl) obidoxime (DEP-obidoxime) was isolated for the first time showing remarkable stability under physiological conditions (half-life 13.5min; pH 7.1; 37°C). The half-life was considerably extended to 20h at 0°C, which facilitated the preparation and allowed isolation by HPLC. The structure was confirmed by mass spectrometry and the degradation pattern. DEP-obidoxime decomposed by an elimination reaction forming the intermediate nitrile that hydrolyzed mainly into the pyridone and cyanide. The intermediates were prepared and confirmed by mass spectroscopy. DEP-Obidoxime was an extremely potent inhibitor of human acetylcholinesterase approaching a second-order rate constant of 10⁹M⁻¹min⁻¹ (pH 7.4; 37°C). The nitrile and the pyridone were still good reactivators. In the presence of human plasma DEP-obidoxime was hydrolyzed into parent obidoxime. Calcium-dependence and sensitivity towards chelators, substitution pattern by other divalent cations and protein-modifying agents all pointed to human paraoxonase (hPON1) as the responsible protein with POX-hydrolase activity. Subjects, probably belonging to the homozygous₁₉₂arginine subtype, were virtually devoid of POX-hydrolase activity while a highly purified hPON1 of the homozygous₁₉₂glutamine subtype exhibited particularly high POX-hydrolase activity. Two parathion-poisoned patients with high and low POX-hydrolase activity responded well and poorly, respectively, to obidoxime treatment although the former patient had higher plasma paraoxon levels than the poor responder. Hence, the POX-hydrolase associated PON1 subtype may be another contributor that modulates pyridinium-4-aldoxime effectiveness.

Keywords: Abbreviations; AChE; acetylcholinesterase (E.C.3.1.1.7); DEP-obidoxime; (; O; -(; O; ,; O; -diethylphosphoryl)-obidoxime; DTNB; 5,5′-dithiobis(2-nitrobenzoic acid); Ery-AChE; erythrocyte AChE; HLö7; 1-[[[4-(aminocarbonyl)-pyridinio]methoxy]methyl]-2,4-bis[(hydroxyimino)methyl]pyridinium dimethanesulfonate; MOPS; 3-(morpholino)propane sulfonate; p; -OHMB; p; -(hydroxymercuri)benzoate; PB; phosphate buffer; PMA; phenylmercury acetate; POX; phosphoryloxime; TMB-4; (1,1′-trimethylene bis[4-(hydroxyimino)methyl]pyridinium dibromidePhosphoryl oximes; Acetylcholinesterase; Paraoxonase; Obidoxime; Paraoxon; Species

Role of P-glycoprotein in transplacental transfer of methadone by Tatiana Nanovskaya; Ilona Nekhayeva; Nedra Karunaratne; Kenneth Audus; Gary D.V. Hankins; Mahmoud S. Ahmed (pp. 1869-1878).

Methadone is the therapeutic agent of choice for treatment of the pregnant opiate addict. However, little is known on the factors affecting its concentration in the fetal circulation during pregnancy and how it might relate to neonatal outcome. Therefore, a better understanding of the function of placental metabolic enzymes and transporters should add to the knowledge of the role of the tissue in the disposition of methadone and its relation to neonatal outcome. We hypothesized that the expression and activity of the placental efflux transporter P-glycoprotein (P-gp) would affect the transfer of methadone to the fetal circulation. Data obtained utilizing dual perfusion of placental lobule and monolayers of Be–Wo cell line indicated that methadone is extruded by P-gp. Transfer of methadone to the fetal circuit was increased by 30% in the presence of the P-gp inhibitor GF120918 while the transfer of paclitaxel, a typical substrate of the glycoprotein, was increased by 50%. In the Be–Wo cell line, methadone and paclitaxel uptake was also increased in the presence of the P-gp inhibitor cyclosporin A. Moreover, the expression of P-gp in placental brush–border membranes varied between term placentas. Taken together, these data strongly suggest that the concentration of methadone in the fetal circulation is affected by the expression and activity of P-gp. It is reasonable to speculate that placental disposition of methadone affects its concentration in the fetal circulation. If true, this may also be directly related to the incidence and intensity of neonatal abstinence syndrome (NAS).

Keywords: P-Glycoprotein; Human placenta; Methadone; Dual perfusion; Transplacental transfer

Inhibition of multidrug resistance proteins MRP1 and MRP2 by a series of α,β-unsaturated carbonyl compounds by Heleen M. Wortelboer; Mustafa Usta; Jelmer J. van Zanden; Peter J. van Bladeren; Ivonne M.C.M. Rietjens; Nicole H.P. Cnubben (pp. 1879-1890).

To study the possible interplay between glutathione metabolism of and MRP inhibition by thiol reactive compounds, the interactions of a series of α,β-unsaturated carbonyl compounds with multidrug resistance proteins 1 and 2 (MRP1/ABCC1 and MRP2/ABCC2) were studied. α,β-Unsaturated carbonyl compounds react with glutathione, and therefore either their parent compound or their intracellularly formed glutathione metabolite(s) can modulate MRP-activity. Inhibition was studied in Madin-Darby canine kidney cells stably expressing MRP1 or MRP2, and isolated Sf9-MRP1 or Sf9-MRP2 membrane vesicles. In the latter model system metabolism is not an issue. Of the series tested, three distinct groups could be discriminated based on differences in interplay of glutathione metabolism with MRP1 inhibition. Curcumin inhibited MRP1 transport only in the vesicle model pointing at inhibition by the parent compound. The glutathione conjugates of curcumin also inhibit MRP1 mediated transport, but to a much lesser extent than the parent compound curcumin. In the cellular model system, it was demonstrated that glutathione conjugation of curcumin leads to inactivation of its inhibitory potential. Demethoxycurcumin and bisdemethoxycurcumin inhibited MRP1 in both the vesicle and cellular model pointing at inhibitory potency of at least the parent compound and possibly their metabolites. A second group, including caffeic acid phenethyl ester inhibited MRP1-mediated calcein transport only in the MDCKII-MRP1 cells, and not in the vesicle model indicating that metabolism appeared a prerequisite to generate the active inhibitor. Finally cinnamaldehyde, crotonaldehyde, trans-2-hexanal, citral, and acrolein did not inhibit MRP1. For MRP2, inhibition was much less in both model systems, with the three curcuminoids being the most effective. The results of this study show the importance to study the complex interplay between MRP-inhibitors and their cellular metabolism, the latter affecting the ultimate potential of a compound for cellular MRP-inhibition.

Keywords: Abbreviations; ACRO; acrolein; BDCUR; bisdemethoxycurcumin; Calcein-AM; calcein acetoxymethylester; CAPE; caffeic acid phenethyl ester; CINN; cinnamaldehyde; CITR; citral; CROT; crotonaldehyde; CsA; cyclosporin A; CUR; curcumin; DCUR; demethoxycurcumin; EA; ethacrynic acid; EASG; glutathionylconjugate of ethacrynic acid; GST; glutathione; S; -transferase; HBSS; Hanks’ balanced salt solution; HEX; trans; -2-hexenal; MDCK cells; Madin-Darby canine kidney cells; MDR; multidrug resistance protein; Pgp; P-glycoprotein; MRP; multidrug resistance protein; Sf; 9; Spodoptera frugiperda; insect cellsMRP1; MRP2; α,β-Unsaturated carbonyl compounds; Inhibition

Author Index Volume 69 (pp. 1891-1896).

Subject Index Volume 69 (pp. 1897-1922).

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Biochemical Pharmacology (v.69, #12)