Check out our New Publishers' Select for Free Articles

Biochemical Pharmacology (v.77, #12)

Editorial Advisory Board (pp. iii).

Graphical Abstract contents list (pp. e1-e4).

Editorial Advisory Board (pp. iii).

Graphical Abstract contents list (pp. e1-e4).

Mechanisms of interferon-β effects on bone homeostasis by Anson K. Abraham; Murali Ramanathan; Bianca Weinstock-Guttman; Donald E. Mager (pp. 1757-1762).

The interplay between interferon-β (IFN-β), bone tissue, and bone-related diseases is complex. This commentary highlights new mechanisms of bone homeostatic regulation mediated through the induction of endogenous IFN-β.Restoration of dysregulated bone homeostasis is a therapeutic goal in many diseases including osteoporosis, rheumatoid arthritis and metastatic cancer. The molecular pathways regulating bone remodeling are major therapeutic targets, and studies continue to reveal endogenous factors that may be pathologically up- or down-regulated and lead to an uncoupling of bone formation and resorption. The purpose of this commentary is to highlight new mechanisms of bone homeostatic regulation mediated through the induction of endogenous interferon-β (IFN-β). The receptor activator of nuclear factor-κB (RANK) ligand (RANKL) is an important factor in the bone resorption cascade, and the RANK–RANKL interaction has been shown to induce IFN-β and osteoclastogenesis via induction of the c-fos gene. Subsequent binding of IFN-β to its biological receptor initiates a signal transduction cascade through the classic JAK/STAT pathway, causing an inhibition of c-fos protein production and osteoclast proliferation and differentiation (negative feedback). Another mechanism pertinent to the anti-resorptive effect of IFN-β is the induction of nitric oxide which has been shown to inhibit osteoclast formation. The role of IFN-β in bone metabolism could warrant its systematic evaluation as a potential adjunct to therapeutic regimens of osteolytic diseases. Here we also provide discussion of the potential challenges to optimizing IFN-β pharmacotherapy for such purposes.

Keywords: Abbreviations; IFN-β; interferon-beta; iNOS; inducible NO synthase; MAPK; mitogen-activated protein kinase; NO; nitric oxide; OPG; osteoprotegrin; PD; pharmacodynamics; PK; pharmacokinetics; PTH; parathyroid hormone; RANK; receptor activator of nuclear factor-κB; RANKL; RANK ligand; SOCS; suppressors of cytokine signaling; TNF; tumor necrosis factor; TRAF; TNF receptor associated factorc-Fos; Nitric oxide; Osteoblasts; Osteoclasts; Receptor activator of nuclear factor-κB

Mechanisms of interferon-β effects on bone homeostasis by Anson K. Abraham; Murali Ramanathan; Bianca Weinstock-Guttman; Donald E. Mager (pp. 1757-1762).

Regulators of endothelial and epithelial barrier integrity and function in acute lung injury by Rudolf Lucas; Alexander D. Verin; Stephen M. Black; John D. Catravas (pp. 1763-1772).

Permeability edema is a life-threatening complication accompanying acute lung injury (ALI), severe pneumonia and the acute respiratory distress syndrome (ARDS), which can be associated with a reduced alveolar liquid clearance (ALC) capacity, a disruption of the alveolar epithelial barrier, and an increased capillary endothelial permeability. Bacterial and viral infections can directly promote pulmonary endothelial hyperpermeability and indirectly decrease the function and/or expression of ion transporters regulating ALC in type II alveolar epithelial cells, by means of inducing a strong inflammatory and oxidative stress response in the infected lungs. Apart from ventilation strategies, no standard treatment exists for permeability edema, making the search for novel regulators of endothelial and epithelial hyperpermeability and dysfunction important. Here, we present an overview of recently identified substances that inhibit and/or reverse endothelial barrier disruption and permeability or alveolar epithelial dysfunction: (1) zinc chelators, which were shown to attenuate the effects of oxidative stress on the pulmonary endothelium; (2) peroxisome proliferator activated receptor (PPAR) ligands, which have been shown to exert anti-inflammatory effects, by decreasing the expression of pro-inflammatory genes; (3) extracellular ATP, produced during inflammation, which induces a rapid and dose-dependent increase in transendothelial electrical resistance (TER) across pulmonary endothelial cells; (4) the lectin-like domain of TNF, which is spatially distinct from the receptor binding sites and which protects from hydrostatic and permeability edema and (5) Hsp90 inhibitors, which prevent and repair toxin-induced hyperpermeability. Unraveling the mechanism of action of these agents could contribute to the development of novel therapeutic strategies to combat permeability edema.

Keywords: Abbreviations; ALC; alveolar liquid clearance; ALI; acute lung injury; ARDS; acute respiratory distress syndrome; BALF; broncholaveolar lavage fluid; DISC; death inducing signaling complex; Hsp90; heat shock protein 90; iNOS; inducible nitric oxide synthase; MLC; myosin light chain; MT; metallothioneins; NF-AT; nuclear factor of activated T cells; PPAR; peroxisome proliferator-activated receptor; STATs; signal transducers and activators of transcription; TACE; TNF-alpha converting enzyme; TER; transendothelial electrical resistance; TRAF2; TNF receptor associated factor 2Permeability edema; PPAR agonists; ATP; hsp90 inhibitors; Lectin-like domain of TNF

Regulators of endothelial and epithelial barrier integrity and function in acute lung injury by Rudolf Lucas; Alexander D. Verin; Stephen M. Black; John D. Catravas (pp. 1763-1772).

7-Diethylamino-3(2′-benzoxazolyl)-coumarin is a novel microtubule inhibitor with antimitotic activity in multidrug resistant cancer cells by Su-Nam Kim; Nam Hyun Kim; Yeon Sook Park; Hanna Kim; Seokjoon Lee; Qian Wang; Yong Kee Kim (pp. 1773-1779).

A novel synthetic microtubule inhibitor 7-diethylamino-3(2′-benzoxazolyl)-coumarin for treatment of multidrug resistant cancer.Microtubules are a proven target for anticancer drug development because they are critical for mitotic spindle formation and the separation of chromosomes at mitosis. We here report a novel synthetic microtubule inhibitor 7-diethylamino-3(2′-benzoxazolyl)-coumarin (DBC). DBC causes destabilization of microtubules, leading to a cell cycle arrest at G₂/M stage. In addition, human cancer cells are more sensitive to DBC (IC₅₀ 44.8–475.2nM) than human normal fibroblast (IC₅₀ 7.9μM), and DBC induces apoptotic cell death of cancer cells. Furthermore, our data show that DBC is a poor substrate of drug efflux pumps and effective against multidrug resistant (MDR) cancer cells. Taken together, these results describe a novel pharmacological property of DBC as a microtubule inhibitor, which may make it an attractive new agent for treatment of MDR cancer.

Keywords: 7-Diethylamino-3(2′-benzoxazolyl)-coumarin; Microtubule; Multidrug resistance

Intracellular cytarabine triphosphate production correlates to deoxycytidine kinase/cytosolic 5′-nucleotidase II expression ratio in primary acute myeloid leukemia cells by Takahiro Yamauchi; Eiju Negoro; Shinji Kishi; Kazutaka Takagi; Akira Yoshida; Yoshimasa Urasaki; Hiromichi Iwasaki; Takanori Ueda (pp. 1780-1786).

Cytarabine (ara-C) is the key agent for treating acute myeloid leukemia (AML). After being transported into leukemic cells by human equilibrative nucleoside transporter 1 (hENT1), ara-C is phosphorylated to ara-C triphosphate (ara-CTP), an active metabolite, and then incorporated into DNA, thereby inhibiting DNA synthesis. Deoxycytidine kinase (dCK) and cytosolic 5′-nucleotidase II (cN-II) are associated with the production of ara-CTP. Because ara-C's cytotoxicity depends on ara-CTP production, parameters that are most related to ara-CTP formation would predict ara-C sensitivity and the clinical outcome of ara-C therapy. The present study focused on finding any correlation between the capacity to produce ara-CTP and ara-C-metabolizing factors. In vitro ara-CTP production, mRNA levels of hENT1, dCK, and cN-II, and ara-C sensitivity were evaluated in 34 blast samples from 33 leukemic patients including 26 with AML. A large degree of heterogeneity was seen in the capacity to produce ara-CTP and in mRNA levels of hENT1, dCK, and cN-II. Despite the lack of any association between each of the transcript levels and ara-CTP production, the ratio of dCK/cN-II transcript levels correlated significantly with the amount of ara-CTP among AML samples. The HL-60 cultured leukemia cell line and its three ara-C-resistant variants (HL-60/R1, HL-60/R2, HL-60/R3), which were 8-, 10-, and 500-fold more resistant than HL-60, respectively, were evaluated similarly. The dCK/cN-II ratio was again proportional to ara-CTP production and to ara-C sensitivity. The dCK/cN-II ratio may thus predict the capacity for ara-CTP production and ultimately, ara-C sensitivity in AML.

Keywords: Abbreviations; ara-C; cytarabine or 1-β-; d; -arabinofuranosylcytosine; ara-CTP; cytarabine triphosphate or 1-β-; d; -arabinofuranosylcytosine triphosphate; AML; acute myeloid leukemia; hENT1; human equilibrative nucleoside transporter 1; IC; ₅₀; 50% growth-inhibitory concentration; dCK; deoxycytidine kinase; cN-II; cytosolic 5′-nucleotidase II; XTT; sodium 3′-(1-[(phenylamino)-carbonyl-3,4-tetrazolium])-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrateCytarabine; Cytarabine triphosphate; Deoxycytidine kinase; Cytosolic 5′-nucleotidase II; Acute myeloid leukemia

A COX-2 inhibitor nimesulide analog selectively induces apoptosis in Her2 overexpressing breast cancer cells via cytochrome c dependent mechanisms by Bin Chen; Bin Su; Shiuan Chen (pp. 1787-1794).

Compound 76 selectively suppressed Her2 positive SKBR-3, BT474 and MDA-MB-453 breast cancer cell proliferation with IC₅₀ of 0.9μM, 2.2μM and 4.0μM respectively via inducing cell apoptosis.Epidemiological and animal model studies have suggested that non-steroidal anti-inflammatory drugs (NSAIDs) can act as chemopreventive agents. The cyclooxygenase-2 (COX-2) inhibitor nimesulide shows anti-cancer effect in different type of cancers. In the current study, five breast carcinoma cell lines were used to explore the anti-cancer mechanisms of a nimesulide derivative compound 76. The compound dose dependently suppressed SKBR-3, BT474 and MDA-MB-453 breast cancer cell proliferation with IC₅₀ of 0.9μM, 2.2μM and 4.0μM, respectively. However, it needs much higher concentrations to inhibit MCF-7 and MDA-MB-231 breast cancer cell growth with IC₅₀ at 22.1μM and 19.6μM, respectively. Further investigation reveals that compound 76 induced apoptosis in SKBR-3 and BT474 cells. Since these cells are Her2 overexpressing cells, the Her2 intracellular signaling pathways were examined after the treatment. There was no significant changing of kinase activity. However, the cytochrome c release assay indicated that the apoptosis induced by the compound was mediated by the mitochondria. These results suggest that compound 76 selectively induce apoptosis in Her2 overexpressing breast cancer cells through the mitochondria, and could be used as a lead to design more potent derivatives.

Keywords: Abbreviations; PI3K; phosphatidylinositol 3-kinase; AKT; protein kinase B; PDK; phosphoinositide-dependent kinase; MAPK; mitogen activated protein kinase; COX-2; cyclooxygenase 2; NSAIDs; non-steroidal anti-inflammatory drugs; PGE2; prostaglandin E2; Her2; human epidermal growth factor receptorBreast cancer; Her2; COX-2 inhibitor; Apoptosis

A COX-2 inhibitor nimesulide analog selectively induces apoptosis in Her2 overexpressing breast cancer cells via cytochrome c dependent mechanisms by Bin Chen; Bin Su; Shiuan Chen (pp. 1787-1794).

Poly(ADP-ribose) polymerase-1 (PARP-1) transcriptionally regulates angiotensin AT2 receptor (AT2R) and AT2R binding protein (ATBP) genes by Jana Reinemund; Kerstin Seidel; Ulrike M. Steckelings; Daniela Zaade; Sabrina Klare; Franziska Rompe; Marlen Katerbaum; Jens Schacherl; Yaosi Li; Mario Menk; Jan H. Schefe; Petra Goldin-Lang; Csaba Szabo; Gabor Olah; Thomas Unger; Heiko Funke-Kaiser (pp. 1795-1805).

The renin–angiotensin system (RAS) plays a crucial role in cardiovascular and neuronal (patho-)physiology. The angiotensin AT2 receptor (AT2R) seems to counteract the proinflammatory, prohypertrophic and profibrotic actions of the AT1 receptor. Recently, we identified a novel protein, termed “AT2R binding protein” (ATBP/ATIP) which seems essential for AT2R-mediated growth inhibition. Poly(ADP-ribose) polymerase-1 (PARP-1) can act as a nuclear integrator of angiotensin II-mediated cell signalling, and has been implicated in the pathogenesis of cardiovascular and neuronal disease.In this study, promoters of human AT2R and ATIP1 were cloned and two transcriptional start sites in the ATIP1 promoter were identified whereas only one was detected in the AT2R promoter. Promoter assays indicated that the exon 1–intron 1 region of AT2R is necessary and sufficient for AT2R promoter activity. Inverse cloning experiments indicated that this regulatory region is a promoter but not an enhancer element implicating (a) further start site(s) in this region. Consistently, the exon 1–intron 1 region of AT2R was shown to tether the basal transcriptional machinery. Overexpression, pharmacological inhibition and ablation of PARP demonstrated that PARP-1 activates the ATIP1 gene but represses the AT2R on promoter and mRNA levels in vitro, and in brain tissue in vivo. Additional experiments indicated that AT2R activation does not modulate PARP-1 transcript levels but increases AT2R promoter activity, thereby creating a positive feedback mechanism.Our results demonstrate that PARP-1 acts as novel node within the RAS network based on its ability to regulate downstream targets such as AT2R and its adapter protein ATBP.

Keywords: Abbreviations; RAS; renin–angiotensin system; AT1R; angiotensin AT1 receptor; AT2R; angiotensin AT2 receptor; PARP-1; poly(ADP-ribose) polymerase-1; SHP-1; SH2 domain-containing phosphatase 1; PLZF; promyelocytic zinc finger protein; ATBP; AT2R binding protein; ATIP; AT2 receptor interacting protein; MTSG; mitochondrial tumor suppressor gene 1; PAR; poly(ADP-ribose); MEFs; mouse embryonic fibroblasts; RLA; relative luciferase activity; ChIP; chromatin-immunoprecipitation; TBP; TATA box-binding proteinRenin–angiotensin system (RAS); Promoter; PARP-1; Angiotensin AT2 receptor; ATBP genes; Cardiovascular pathophysiology

Heme oxygenase-1 induction modulates microsomal prostaglandin E synthase-1 expression and prostaglandin E₂ production in osteoarthritic chondrocytes by Javier Megías; María Isabel Guillén; Victoria Clérigues; Ana I. Rojo; Antonio Cuadrado; Miguel Angel Castejón; Francisco Gomar; María José Alcaraz (pp. 1806-1813).

Pro-inflammatory cytokines such as interleukin-1β (IL-1β) may participate in the pathogenesis of cartilage damage in osteoarthritis (OA) through the production of catabolic enzymes and inflammatory mediators. Induction of heme oxygenase-1 (HO-1) has previously been shown to exert anti-inflammatory effects in different cell types. We have investigated whether HO-1 induction may modify chondrocyte viability and the production of relevant mediators such as oxidative stress and prostaglandin E₂ (PGE₂) elicited by IL-1β in OA chondrocytes. Chondrocytes were isolated from OA cartilage and used in primary culture. Cells were stimulated with IL-1β in the absence or presence of the HO-1 inducer cobalt protoporphyrin IX (CoPP). Gene expression was assessed by quantitative real-time PCR, protein levels by ELISA and Western blot, apoptosis by laser scanning cytometry using annexin V–FITC and TUNEL assays, and oxidative stress by LSC with dihydrorhodamine 123. HO-1 induction by CoPP enhanced chondrocyte viability and aggrecan content while inhibiting apoptosis and oxidative stress generation. PGE₂ is produced in OA chondrocytes stimulated by IL-1β by the coordinated induction of cyclooxygenase-2 and microsomal PGE synthase 1 (mPGES-1). The production of PGE₂ was decreased by HO-1 induction as a result of diminished mPGES-1 protein and mRNA expression. Transfection with HO-1 small interfering RNA counteracted CoPP effects. In addition, the activation of nuclear factor-κB and early growth response-1 was significantly reduced by CoPP providing a basis for its anti-inflammatory effects. These results confirm the protective role of HO-1 induction in OA chondrocytes and suggest the potential interest of this strategy in degenerative joint diseases.

Keywords: Heme oxygenase-1; Chondrocyte; Osteoarthritis; Interleukin-1β; Prostaglandin E; ₂; Microsomal PGE synthase-1

Cromoglycate drugs suppress eicosanoid generation in U937 cells by promoting the release of Anx-A1 by Samia Yazid; Egle Solito; Helen Christian; Simon McArthur; Nicolas Goulding; Roderick Flower (pp. 1814-1826).

Using biochemical, epifluorescence and electron microscopic techniques in a U937 model system, we investigated the effect of anti-allergic drugs di-sodium cromoglycate and sodium nedocromil on the trafficking and release of the anti-inflammatory protein Annexin-A1 (Anx-A1) when this was triggered by glucocorticoid (GC) treatment. GCs alone produced a rapid (within 5min) concentration-dependent activation of PKCα/β (Protein Kinase C; EC 2.7.11.13) and phosphorylation of Anx-A1 on Ser²⁷. Both phosphoproteins accumulated at the plasma membrane and Anx-A1 was subsequently externalised thereby inhibiting thromboxane (Tx) B₂ generation. When administered alone, cromoglycate or nedocromil had little effect on this pathway however, in the presence of a fixed sub-maximal concentration of GCs, increasing amounts of the cromoglycate-like drugs caused a striking concentration-dependent enhancement of Anx-A1 and PKCα/β phosphorylation, membrane recruitment and Anx-A1 release from cells resulting in greatly enhanced inhibition of TxB₂ generation. GCs also stimulated phosphatase accumulation at the plasma membrane of U937 cells. Both cromoglycate and nedocromil inhibited this enzymatic activity as well as that of a highly purified PP2A phosphatase preparation. We conclude that stimulation by the cromoglycate-like drugs of intracellular Anx-A1 trafficking and release (hence inhibition of eicosanoid release) is secondary to inhibition of a phosphatase PP2A (phosphoprotein phosphatase; EC 3.1.3.16), which probably forms part of a control loop to limit Anx-A1 release. These experiments provide a basis for a novel mechanism of action for the cromolyns, a group of drugs that have long puzzled investigators.

Keywords: Sodium nedocromil; Glucocorticoids; Okadaic acid; PKC; PP2A phosphatase

Inhibition of the MAP kinase ERK protects from lipopolysaccharide-induced lung injury by Katrin Schuh; Andreas Pahl (pp. 1827-1834).

Inhibition of the MAP kinase ERK reduces inflammation in a murine model of acute lung injury.The pathogenesis of chronic obstructive pulmonary disease (COPD) is characterized by pulmonary inflammation associated with lung neutrophilia and elevated levels of pro-inflammatory mediators in the bronchoalveolar lavage fluid or sputum of patients. Recent findings revealed that mitogen-activated protein kinase (MAPK) signaling cascade is involved in the inflammatory response of lung injury. In the present study we could elucidate the role of extracellular signal-related MAPK in the murine model of LPS-induced acute lung injury by using U0126, a specific inhibitor of MEK1/2, upstream kinases of ERK. Phosphorylation of ERK was inhibited by U0126 in vivo as well as in vitro. In freshly isolated human peripheral blood mononuclear cells U0126 dose-dependently blocked the release of IL-2 and TNF-α. For in vivo studies mice were exposed to aerosolized LPS to induce an acute lung injury mimicking some aspects of COPD. This led to a recruitment of neutrophils to the lung and to the release of pro-inflammatory cytokines into bronchoalveolar lavage. Pretreatment of mice with U0126 significantly reduced lung neutrophilia and diminished levels of TNF-α and chemotactic MIP-2 and KC in bronchoalveolar fluid. U0126 also decreased albumin levels in BAL fluid, a marker of vascular leakage. Histological examination of lung tissues revealed that ERK MAPK inhibition using U0126 efficiently attenuated LPS-induced pulmonary inflammatory responses. These data suggest that ERK signaling plays an important role in acute lung injury and pharmacologic inhibition of ERK provides a promising new therapeutic strategy for lung inflammatory diseases and in particular COPD.

Keywords: Abbreviations; COPD; chronic obstructive pulmonary disease; MAPKK; mitogen-activated protein kinase kinase; MAPKKK; mitogen-activated protein kinase kinase kinase; LPS; lipopolysaccharide; ERK; extracellular signal-related kinase; MEK; extracellular signal-regulated kinase kinase; TNF; tumor necrosis factor; PBMC; peripheral blood mononuclear cells; MIP; macrophage inflammatory protein; KC; keratinocyte-derived chemokine; BAL; bronchoalveolar lavage; MPO; myeloperoxidaseLung; Inflammation; Lipopolysaccharide; ERK; MEK-inhibitor; COPD

Inhibition of the MAP kinase ERK protects from lipopolysaccharide-induced lung injury by Katrin Schuh; Andreas Pahl (pp. 1827-1834).

Interleukin-17A inhibits adipocyte differentiation in human mesenchymal stem cells and regulates pro-inflammatory responses in adipocytes by Jennifer H. Shin; Dong Wook Shin; Minsoo Noh (pp. 1835-1844).

IL-17A inhibits adipocyte differentiation in hBM-MSCs. IL-17A also stimulates the secretion of pro-inflammatory PGE₂, IL-6, and IL-8 from adipocytes. This implicates that IL-17A may play a role in the crosstalk between adipose tissue and Th17 cell-associated immune responses. It is still unclear whether adipocyte-derived IL-6 contributes to the development of Th17 cells.The immune system is closely linked to human metabolic diseases. Serum levels of IL-6 increase with obesity and insulin resistance. Not only does IL-6 decrease the insulin sensitivity of human cells such as adipocytes, but it also regulates the lineage commitment of naïve T cells into interleukin (IL)-17A-producing CD4(+) T (Th17) cells. Although IL-17A exerts a variety of effects on somatic tissues, its functional role in human adipocytes has not been identified. In this work, we show that IL-17A inhibits adipocyte differentiation in human bone marrow mesenchymal stem cells (hBM-MSCs), while promoting lipolysis of differentiated adipocytes. We find that IL-17A increases both mRNA and protein secretion of IL-6 and IL-8 during adipocyte differentiation in hBM-MSCs. IL-17A up-regulates cyclooxygenase (COX)-2 gene expression and thereby increases the level of prostaglandin (PG) E₂ in differentiated adipocyes. The suppression of anti-adipogenic PGE₂ by COX inhibitors such as aspirin and NS-398 partially blocked the effect of IL-17A on adipocyte differentiation in hBM-MSCs. Therefore, IL-17A exhibits its inhibitory effect in part via the COX-2 induction in differentiated adipocytes. In addition, treatment with anti-IL-17A antibody neutralizes IL-17A-mediated effects on adipocyte differentiation and function. These results suggest that IL-17A plays a regulatory role in both the metabolic and inflammatory processes of human adipocytes, similar to other pro-inflammatory cytokines such as IL-1, IFNγ, and TNFα.

Keywords: Abbreviations; ADIPOQ; adiponectin; FABP4; fatty acid binding protein 4; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; hBM-MSCs; human bone marrow mesenchymal stem cells; PPARγ; peroxisome proliferator-activated receptor gamma; Q-RT-PCR; quantitative real-time reverse transcription polymerase chain reactionIL-17A; Metabolic syndromes; Inflammation; Adipocyte differentiation; Human bone marrow mesenchymal stem cells

S-adenosylmethionine regulates thiopurine methyltransferase activity and decreases 6-mercaptopurine cytotoxicity in MOLT lymphoblasts by Miha Milek; Natasa Karas Kuzelicki; Alenka Smid; Irena Mlinaric-Rascan (pp. 1845-1853).

Six-mercaptopurine (6-MP) is a pro-drug widely used in treatment of various diseases, including acute lymphoblastic leukaemia (ALL). Side-effects of thiopurine therapy have been correlated with thiopurine methyltransferase (TPMT) activity.We propose a novel TPMT-mediated mechanism of S-adenosylmethionine (SAM)-specific effects on 6-mercaptopurine (6-MP) induced cytotoxicity in a model cell line for acute lymphoblastic leukemia (MOLT). Our results show that exogenous SAM (10–50μM) rescues cells from the toxic effects of 6-MP (5μM) by delaying the onset of apoptosis. We prove that the extent of methylthioinosine monophosphate (MeTIMP) induced inhibition of de novo purine synthesis (DNPS) determines the concentrations of intracellular ATP, and consequently SAM, which acts as a positive modulator of TPMT activity. This leads to a greater conversion of 6-MP to inactive 6-methylmercaptopurine, and thus lower availability of thioinosine monophosphate for the biotransformation to cytotoxic thioguanine nucleotides (TGNs) and MeTIMP. We further show that the addition of exogenous SAM to 6-MP treated cells maintains intracellular SAM levels, TPMT activity and protein levels, all of which are diminished in cells incubated with 6-MP. Since TPMT mRNA levels remained unaltered, the effect of SAM appears to be restricted to protein stabilisation rather than an increase of TPMT expression. We thus propose that SAM reverses the extent of 6-MP cytotoxicity, by acting as a TPMT-stabilizing factor.This study provides new insights into the pharmacogenetics of thiopurine drugs. Identification of SAM as critical modulator of TPMT activity and consequently thiopurine toxicity may set novel grounds for the rationalization of thiopurine therapy.

Keywords: S-adenosylmethionine; Thiopurine methyltransferase; Cytotoxicity; Lymphoblasts; 6-Mercaptopurine

Sections

Personal tools

Biochemical Pharmacology (v.77, #12)

Sections

Personal tools

Local resources

Biochemical Pharmacology (v.77, #12)