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Biochemical Pharmacology (v.82, #10)

Editorial Board (pp. iii).

Integrated cellular pathology—Systems biology of human diseases by Cyril Sobolewski; Elodie Viry; Noémie Legrand; Florian Muller; Franck Morceau; Marc Diederich (pp. 1253-1255).

p63 in tooth development by Alessandro Rufini; Alberto Barlattani; Raffaella Docimo; Tania Velletri; Maria Victoria Niklison-Chirou; Massimiliano Agostini; Gerry Melino (pp. 1256-1261).

Recent findings have shown that the development of teeth involves a complex sequence of molecular events in which the p53 family member p63 is involved. Indeed, mice lacking p63 do not have teeth and humans bearing mutations in p63 suffer developmental syndromes that affect tooth morphology and number. Several isoforms of p63 have been described: the use of two different promoters produces longer TAp63 isoforms, or shorter, 5′ truncated isoforms known as ΔNp63. The 3′ end of primary transcripts is then subject to alternative splicing resulting in three additional isoforms: alpha (α), beta (β) and gamma (γ). Tooth development relies mainly on the activity of the N-terminally truncated ΔNp63 isoforms. Here we review the experimental evidence for the involvement of ΔNp63 in tooth development through its ability to sustain the molecular signalling that orchestrates epithelial–mesenchymal interaction.

Keywords: Abbreviations; BMP; bone morphogenetic protein; FGF; fibroblast growth factor; FGFR; fibroblast growth factor receptor; SHH; sonic hedgehog; TNF; tumor necrosis factor; Apc; adenomatous polyposis coli; SAM; sterile alpha motif; TID; transactivation inhibitory domain; ABBP1; apobec-1-binding protein-1p63; Tooth development; Epidermis; FGF; Notch

The sterile alpha-motif (SAM) domain of p63 binds in vitro monoasialoganglioside (GM1) micelles by Stefano Rufini; Anna Maria Lena; Bruno Cadot; Sonia Mele; Ivano Amelio; Alessandro Terrinoni; Alessandro Desideri; Gerry Melino; Eleonora Candi (pp. 1262-1268).

We have performed a biochemical analysis of p63-SAM domain preferences in lipid binding. We have identified the ganglioside GM1 as a high affinity interactor, capable of modulating p63 transcriptional ability.The transcription factor p63 plays pivotal roles in epidermal barrier formation and in embryonic development. The protein structures of TAp63 and ΔNp63α isoforms include a C-terminal steril alpha-motif (SAM) involved in protein–protein interaction. Identification of p63 SAM domain interactors could lead to the explanation of novel mechanisms of regulation of p63 activity, possibly relevant in the physiological role of p63 and in genetic disorders associated with mutations of the p63 gene. In this work, we have performed a biochemical analysis of p63 SAM domain preferences in lipid binding. We have identified the ganglioside GM1 as a high affinity interactor, capable of modulating p63 transcriptional ability exclusively on epidermal target genes. In agreement with these data we report a consistent expression profile and localization analysis of p63 and GM1 in primary keratinocytes and in human epidermal biopsies. Therefore, we propose a potential biological role of p63–GM1 interaction in regulation of p63 during epidermal differentiation.

Keywords: Abbreviations; SAM; sterile alpha motif; PA; phosphatidic acid; PC; phosphatidylcholine; GM; ganglioside; PS; phosphatidylserine; SM; sphingomyelin; HEKn; human epithelial keratinocytes normalp63; Ganglioside; Keratinocytes; Skin; SAM domain

DNA damage response: The emerging role of c-Abl as a regulatory switch? by Emiliano Maiani; Marc Diederich; Stefania Gonfloni (pp. 1269-1276).

A complex regulatory network of signaling pathways safeguards genome integrity following DNA damage. When double strand breaks occur several enzymes and mediators are recruited to the sites of lesion to release a network of DNA repair processes referred to as DNA damage response (DDR). c-Abl interacts in the nucleus with several proteins implicated in distinct aspects of DNA repair. This suggests that c-Abl may be involved in the regulation of double strand break repair. The involvement of c-Abl in DNA repair mechanisms came into the spotlight in female germ cells under genotoxic stress. Recent findings have implicated c-Abl in a cisplatin-induced signaling pathway eliciting death of immature oocytes. Pharmacological inhibition of c-Abl by Imatinib (STI571) protects the ovarian reserve from the toxic effect of cisplatin. This implies that the extent of c-Abl catalytic outcomes may tip the balance between survival (likely through DNA repair) and activation of a death response. Many observations indicate that timely ubiquitin-modifications and signal decoding are implicated in regulating DNA repair. Here, we discuss some connections between phosphorylation- and ubiquitin-mediated signaling at the damaged sites. We speculate about multiple interactions that may occur between c-Abl (and ‘sensor’ kinases) with ubiquitin-related proteins involved in DDR. Additional work is required to understand the complexity of the physiological outcomes of c-Abl in DDR. However, a fine-tuning of nuclear outcomes, through pharmacological inhibition of c-Abl, may provide novel paradigms for DDR and, potentially, therapeutic strategies for cancer treatment.

Keywords: Abbreviations; 53BP1; Tumor suppressor p53-binding protein 1; ATM; Ataxia telangiectasia mutated; ATR; Ataxia telangiectasia and Rad3-related protein; BARD; BRCA1-associated RING domain protein 1; BER; Base excision repair; BRCA1; Breast cancer type 1 susceptibility protein; BRCC36; BRCA1–BRCA2 containing complex subunit 36; CtIP; CtBP-interacting protein; DDB1; DNA damage-binding protein 1; DDB2; DNA damage-binding protein 2; DNAPK; DNA-dependent protein kinase; DSBR; Double-strand break repair; ERCC6; Excision repair cross-complementing 6; HERC2; HECT domain and RCC1-like domain-containing protein 2; MDM2; Double minute 2 protein; MMR; Mismatch repair; MRN; Mre11/Nbs1/Rad50 complex; MSH5; DNA Mismatch repair protein 5; NER; Nucleotide excision repair; PIAS; Protein inhibitor of activated STAT; RAP 80; Receptor-associated protein 80; RNF8; RING finger protein 8; RNF20; RING finger protein 20; RNF40; RING finger protein 40; RNF168; RING finger protein 168; TopBP1; DNA topoisomerase 2-binding protein 1; UBC13; Ubiquitin-conjugating enzyme E2 13; USP1; Ubiquitin-specific-processing protease 1; WRN; Werner syndrome ATP-dependent helicase; YAP1; Yes-associated proteinDNA damage; c-Abl; Histone modifications; DNA repair; Germ cells; Chemotherapy

COX-2 inhibitors block chemotherapeutic agent-induced apoptosis prior to commitment in hematopoietic cancer cells by Claudia Cerella; Cyril Sobolewski; Sébastien Chateauvieux; Estelle Henry; Michael Schnekenburger; Jenny Ghelfi; Mario Dicato; Marc Diederich (pp. 1277-1290).

Enzymatic inhibitors of pro-inflammatory cyclooxygenase-2 (COX-2) possess multiple anti-cancer effects, including chemosensitization. These effects are not always linked to the inhibition of the COX-2 enzyme. Here we analyze the effects of three COX-2 enzyme inhibitors (nimesulide, NS-398 and celecoxib) on apoptosis in different hematopoietic cancer models. Surprisingly, COX-2 inhibitors strongly prevent apoptosis induced by a panel of chemotherapeutic agents. We selected U937 cells as a model of sensitive cells for further studies. Here, we provide evidence that the protective effect is COX-independent. No suppression of the low basal prostaglandin (PG)E₂ production may be observed upon treatment by COX-2 inhibitors. Besides, the non-active celecoxib analog 2,5-dimethyl-celecoxib is able to protect from apoptosis as well. We demonstrate early prevention of the stress-induced apoptotic signaling, prior to Bax/Bak activation. This preventive effect fits with an impairment of the ability of chemotherapeutic agents to trigger apoptogenic stress. Accordingly, etoposide-induced DNA damage is strongly attenuated in the presence of COX-2 inhibitors. In contrast, COX-2 inhibitors do not exert any anti-apoptotic activity when cells are challenged with physiological stimuli (anti-Fas, TNFα or Trail) or with hydrogen peroxide, which do not require internalization and/or are not targeted by chemoresistance proteins. Altogether, our findings show a differential off-target anti-apoptotic effect of COX-2 inhibitors on intrinsic vs. extrinsic apoptosis at the very early steps of intracellular signaling, prior to commitment. The results imply that an exacerbation of the chemoresistance phenomena may be implicated.

Keywords: Abbreviations; Bak; Bcl-2 homologous antagonist killer; Bax; Bcl-2-associated X protein; Bcl-2; B-cell lymphoma 2; Bcl-Xl; B-cell lymphoma-extra large; CPT; camptothecin; CISP; cisplatin; COX-2; cyclooxygenase-2; CTR-1; copper transporter 1; CYT; cytarabine; DMC; 2,5-dimethyl-celecoxib; DOXO; doxorubicin; Fas; apoptosis stimulating fragment; γ-H2A.x; phosphorylated histone H2A.x; H; ₂; O; ₂; hydrogen peroxide; IAP; inhibitor of apoptosis; IRINO; irinotecan; MDR-1; multidrug resistance protein 1; MRP-1; multidrug resistance-associated protein 1; MRP-6; multidrug resistance-associated protein 6; MTX; methotrexate; PMC; puromycin; NSAIDs; non-steroidal anti-inflammatory drugs; Rh 123; rhodamine 1,2,3; TNFα; tumour necrosis factor alpha; TRAIL; TNF-related apoptosis inducing ligand; VP16; etoposide; XIAP; X-linked inhibitor of apoptosis proteinNimesulide; NS-398; Celecoxib; Chemoresistance; Mitochondrial pathway

Erythropoietin, erythropoiesis and beyond by S. Chateauvieux; C. Grigorakaki; F. Morceau; M. Dicato; M. Diederich (pp. 1291-1303).

Erythropoietin (EPO) is a glycoprotein that is mainly produced in the adult kidney, and it was initially highlighted for its action on the hematopoietic system. Moreover, EPO is also expressed in several non-hematopoietic tissues, where it plays a role in the protection from apoptosis and inflammation due to hypoxia, toxicity or injury. These protective effects are mainly known and studied in cardioprotection and neuroprotection but are also reported in retina degeneration, auditory injury and pancreatic-related diseases. The tissue protective effect of EPO is mainly mediated through the interaction with the heterodimeric receptor EPOR/βcR. Human recombinant EPO (HuREPO), which has been developed to treat anemia, is not adequate for tissue protection. The low affinity of the alternative receptor for EPO involves the injection of excessive concentration of erythropoiesis-stimulating agents (ESAs), implicating side effects due to the cross-talk with hematopoietic activity. For these reasons, EPO derivatives with less affinity for the EPO homodimeric receptor are under development. In this review, we provide an overview of the erythroid and non-erythroid functions of EPO by detailing the molecular mechanisms activated by the binding of EPO to its receptors in different tissues.

Keywords: EPO; EPOR; EPO derivative; Erythropoiesis; Cardioprotection; Neuroprotection

Killing of tumor cells: A drama in two acts by Vincenzo Giansanti; Micol Tillhon; Giuliano Mazzini; Ennio Prosperi; Paolo Lombardi; A. Ivana Scovassi (pp. 1304-1310).

Cancer still represents a major health problem worldwide, which urges the development of more effective strategies. Resistance to chemotherapy, a major obstacle for cancer eradication, is mainly related to an intrinsic failure to activate the apoptotic pathways. However, a protective effect of autophagy toward cancer cells has been recently observed, thus adding further complexity to the development of an effective approach counteracting cancer cell growth and improving the response to therapy.

Keywords: Apoptosis; Autophagy; Cancer; Chemotherapy

Yellow submarine of the Wnt/Frizzled signaling: Submerging from the G protein harbor to the targets by Alexey Koval; Vladimir Purvanov; Diane Egger-Adam; Vladimir L. Katanaev (pp. 1311-1319).

The Wnt/Frizzled signaling pathway plays multiple functions in animal development and, when deregulated, in human disease. The G-protein coupled receptor (GPCR) Frizzled and its cognate heterotrimeric Gi/o proteins initiate the intracellular signaling cascades resulting in cell fate determination and polarization. In this review, we summarize the knowledge on the ligand recognition, biochemistry, modifications and interacting partners of the Frizzled proteins viewed as GPCRs. We also discuss the effectors of the heterotrimeric Go protein in Frizzled signaling. One group of these effectors is represented by small GTPases of the Rab family, which amplify the initial Wnt/Frizzled signal. Another effector is the negative regulator of Wnt signaling Axin, which becomes deactivated in response to Go action. The discovery of the GPCR properties of Frizzled receptors not only provides mechanistic understanding to their signaling pathways, but also paves new avenues for the drug discovery efforts.

Keywords: Wnt; Frizzled; GPCR; Heterotrimeric G protein; Rab5

Enzymatic and non-enzymatic activities of SHIP-1 in signal transduction and cancer by Claude Condé; Geoffrey Gloire; Jacques Piette (pp. 1320-1334).

PI3K cascade is a central signaling pathway regulating cell proliferation, growth, differentiation, and survival. Tight regulation of the PI3K signaling pathway is necessary to avoid aberrant cell proliferation and cancer development. Together with SHIP-1, the inositol phosphatases PTEN and SHIP-2 are the gatekeepers of this pathway. In this review, we will focus on SHIP-1 functions. Negative regulation of immune cell activation by SHIP-1 is well characterized. Besides its catalytic activity, SHIP-1 also displays non-enzymatic activity playing role in several immune pathways. Indeed, SHIP-1 exhibits several domains that mediate protein-protein interaction. This review emphasizes the negative regulation of immune cell activation by SHIP-1 that is mediated by its protein-protein interaction.

Keywords: Abbreviations; AA; amino acid; Ag; antigen; APC; antigen presenting cell; Btk; burton tyrosine kinase; DAG; diacylglycerol; Dok; downstream of kinase; Grb2; growth factor receptor-bound protein 2; IFN-β; interferon β; Ig; immunoglobulin; IKKα/ɛ; IκB kinaseα/ɛ; IL-1; interleukin 1; IP3; inositol triphosphate; IRAK; interleukin-1 receptor-associated kinase; IRF3; interferon regulatory factor 3; LAT; linker of activated T cells; MAPK; mitogen-activated protein kinase; MHC; major histocompatibility complex; NFAT; nuclear factor of activated T cells; NF-κB; nuclear factor κB; PI3K; phophoinositide 3 kinase; PIP3; phosphatidyl-inositol triphosphate; PKC; protein kinase C; PLCγ; phospholypase C gamma; PTEN; phosphatase and tensin homolog; RasGAP; Ras GTPase activating protein; Shc; src homology and collagen; SOS; Son of sevenless; TBK-1; tank binding kinase1; TLR; toll like receptor; TNF-α; tumor necrosis factor α; TRAF; TNF receptor-associated factorSHIP-1; Grb2; Shc; Dok-1/2; immune response

15-Deoxy-Δ^12,14-prostaglandin J₂, an electrophilic lipid mediator of anti-inflammatory and pro-resolving signaling by Young-Joon Surh; Hye-Kyung Na; Jong-Min Park; Ha-Na Lee; Wonki Kim; In-Soo Yoon; Dae-Duk Kim (pp. 1335-1351).

15-deoxy-Δ^12,14-prostagandin J₂ (15d-PGJ2) is produced in the inflamed cells and tissues as a consequence of upregulation of cyclooxygenase-2 (COX-2). 15d-PGJ2 is known to be the endogenous ligand of peroxisome proliferator-activated receptor gamma (PPARγ) with multiple physiological properties. Though one of the terminal products of the COX-2-catalyzed reactions, this cyclopentenone prostaglandin exerts potent anti-inflammatory actions, in part, by antagonizing the activities of pro-inflammatory transcription factors, such as NF-κB, STAT3, and AP-1, while stimulating the anti-inflammatory transcription factor Nrf2. These effects are not necessarily dependent on its activation of PPARγ, but often involves direct interaction with the above signaling molecules and their regulators. The locally produced 15d-PGJ2 is also involved in the resolution of inflammatory responses. Thus, 15d-PGJ2, especially formed during the late phase of inflammation, might inhibit cytokine secretion and other events by antigen-presenting cells like dendritic cells or macrophages. 15d-PGJ2 can also affect the priming and effector functions of T lymphocytes and induce their apoptotic cell death. These represent a negative feedback explaining how once-initiated immunologic and inflammatory responses are switched off and terminated. In this context, 15d-PGJ2 and its synthetic derivatives have therapeutic potential for the treatment of inflammatory disorders.

Keywords: Abbreviations; 15d-PGJ2; 15-deoxy-Δ; ^12,14; -prostaglandin J; ₂; COX-2; cyclooxygenase-2; PPARγ; proliferator-activated receptor gamma; PGH2; prostaglandin H; ₂; PGD2; prostaglandin D; ₂; PGJ2; prostaglandin J; ₂; NF-κB; nuclear factor-kappaB; STAT3; signal transducers and activators of transcription 3; Nrf2; nuclear factor-erythroid 2p45 (NF-E2)-related factor; AP-1; activator protein-1; GSH; reduced glutathione; CNS; central nervous system; PGE2; prostaglandin E; ₂; MMP; matrix metalloproteinases; TNF-α; tumor necrosis factor-alpha; iNOS; inducible nitric oxide synthase; NO; nitric oxide; HO-1; heme oxygenase-1; I/R; ischemia/reperfusion; LPS; lipopolysaccharide; EAE; encephalomyelitis; COPD; chronic obstructive pulmonary disease; IFN-γ; interferon-γ; MPO; myeloperoxidase; PGA1; prostaglandin A; ₁; ICAM-1; intercellular adhesion molecule-1; MCP-1; macrophage chemotactic protein 1; α-SMA; α-smooth muscle actin; IL; interleukine; HAT; histone acetyltransferase; HDAC; histone deacetylase; NAC; N; -acetylcysteine; PGA2; prostaglandin A; ₂; BSO; buthionine sulfoximine; IκBα; inhibitory protein κB; IKK; IκB kinase; JNK; c-Jun N-terminals kinase; MAPK; mitogen activated protein kinase; TF; tissue factor; ERK; extracellular-regulated protein kinase; MEKK1; MAPK kinase; mPGES; microsomal prostaglandin E synthase; TLR; toll-like receptor; ox-LDL; oxidized low-density lipoprotein; HUVECs; human vascular endothelial cells; HMEC-1; human microvascular endothelial cell line; SOCS; suppressor of cytokine signaling; JAK; Janus kinase; TGF; transforming growth factor; EGF; epidermal growth factor; ES; embryonic stem; LIF; leukemia inhibitory factor; NQO1; NAD(P)H:quinone oxidoreductase-1; Keap1; Kelch-like erythroid cell-derived protein with ‘capn’collar homology associated protein; ARE; antioxidant response elements; EpRE; electrophile response element; VSMCs; vascular smooth muscle cells; CO; carbon monoxide; SIN-1; 3-morpholinosydnonimine hydrochloride; GCL; glutamate cysteine ligase; PMNs; polymorphonuclear leukocytes; HSA; human serum albumin15-deoxy-Δ; ^12,14; -prostagandin J; ₂; Cyclopentenone prostaglandin; Inflammation; Resolution; Anti-inflammation

15-Hydroxyprostaglandin dehydrogenase as a novel molecular target for cancer chemoprevention and therapy by Hye-Kyung Na; Jong-Min Park; Hee Geum Lee; Ha -Na Lee; Seung-Jae Myung; Young-Joon Surh (pp. 1352-1360).

Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in arachidonic acid cascade, plays a key role in the biosynthesis of prostaglandin E₂ (PGE₂) upon inflammatory insults. Overproduction of PGE₂ stimulates proliferation of various cancer cells, confers resistance to apoptosis of cancerous or transformed cells, and accelerates metastasis and angiogenesis. Excess PGE₂ undergoes metabolic inactivation which is catalyzed by NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). In this context, 15-PGDH has been speculated as a physiological antagonist of COX-2 and a tumor suppressor. Thus, overexpression of 15-PGDH has been known to protect against experimentally induced carcinogenesis and renders the cancerous or transformed cells susceptible to apoptosis by counteracting oncogenic action of PGE₂. In contrast, silence of 15- PGDH is observed in some cancer cells, which is associated with epigenetic modification, such as DNA methylation and histone deacetylation, in the promoter region of 15-PGDH. A variety of compounds capable of inducing the expression of 15-PGDH have been reported, which include the histone deacetylase inhibitors, nonsteroidal anti-inflammatory drugs, and peroxisome proliferator-activated receptor-gamma agonists. Therefore, 15-PGDH may be considered as a novel molecular target for cancer chemoprevention and therapy. This review highlights the role of 15-PGDH in carcinogenesis and its regulation.

Keywords: 15-Hydroxyprostaglandin dehydrogenase (15-PGDH); Tumor suppressor; Chemoprevention; Cyclooxygenase-2 (COX-2); Prostaglandin E; ₂; (PGE; ₂; )

Epigenetics and senescence: Learning from the INK4-ARF locus by Elisabeth Simboeck; Joana D. Ribeiro; Sophia Teichmann; Luciano Di Croce (pp. 1361-1370).

Cellular senescence is the biological consequence of aging. However, the same mechanisms that provoke senescence during aging have been proven to act in tumor suppression and thus to occur in premalignant cells. All the diverse aspects of the senescent phenotype, as are observed for many other cell fates, arise from alterations of the chromatin architecture. Relatively little is known overall about the changes in chromatin structure, and which regulatory networks are implicated in these. Major insight into the epigenetic contributions to senescence has been gained by studying the regulation of the INK4-ARF locus. Activation of the tumor suppressors encoded by this locus leads to an irreversible cell cycle exit. Importantly, epigenetic alterations at this locus have been associated with the onset of cancer. Here we discuss the recent findings that link epigenetics to the senescence pathway.

Keywords: Transcription; Gene regulation; Epigenetics; Senescence; Polycomb

Importance of PIKKs in NF-κB activation by genotoxic stress by Hélène Sabatel; Céline Pirlot; Jacques Piette; Yvette Habraken (pp. 1371-1383).

Alteration of the genome integrity leads to the activation of a vast network of cellular responses named “DNA damage response”. Three kinases from the phosphoinositide 3-kinase-like protein kinase family regulate this network; ATM and DNA-PK both activated by DNA double-strand breaks and ATR activated by replication blocks. “DNA damage response” pathway coordinates cell cycle arrest, DNA repair, and the activation of transcription factors such as p53 and NF-κB. It controls senescence/apoptosis/survival of the damaged cells. Cell death or survival result from a tightly regulated balance between antagonist pro- and anti-apoptotic signals. NF-κB is a key transcription factor involved in immunity, inflammation and cell transformation. When activated by DNA double-strand breaks, NF-κB has most often a pro-survival effect and thereof interferes with chemotherapy treatments that often rely on DNA damage to induce tumor cell death (i.e. topoisomerase inhibitors and ionizing radiation). NF-κB is thus an important pharmaceutical target. Agents leading to replication stress induce a pro-apoptotic NF-κB. The molecular mechanisms initiated by DNA lesions leading to NF-κB nuclear translocation have been extensively studied these last years. In this review, we will focus on ATM, ATR and DNA-PK functions both in the IKKα/IKKβ/NEMO-dependent or -independent signaling pathways and on the regulation they can exercise at the promoter level of NF-κB regulated genes.

Keywords: Abbreviations; aNHEJ; alternate non-homologous end joining; A-T; ataxia telangiectasia; ATM; ataxia telangiectasia mutated; ATR; ataxia telangiectasia and Rad3-related kinase; cIAP-1; cellular inhibitor of apoptosis protein 1; cNHEJ; classical non-homologous end joining; CPT; camptothecin; DNA-PK; DNA-dependent protein kinase; DNA-PKcs; DNA-dependent protein kinase catalytic subunit; DSB; double-strand breaks; Etp; etoposide; HR; homologous recombination; HU; hydroxyurea; IAP; inhibitors of apoptosis protein; IκB; inhibitor of κB; IKK; IκB kinase; IR; ionizing radiation; IRIF; ionizing radiation induced foci; NCS; neocarzinostatin; NEMO; NF-κB essential modulator; NF-κB; nuclear factor-κB; MRN; Mre11/Rad50/NBS; PARBM; poly(ADP-ribose) binding motif; PARP-1; poly(ADP-ribose) polymerase 1; PIASy; protein inhibitor of activated STAT y; PIDD; p53-inducible-death-domain containing protein; PIKK; phosphoinositide 3-kinase-like protein kinase; PP5; protein phosphatase 5; RPA; replication protein A; RIP1; receptor-interacting protein1; ROS; reactive oxygen species; SSB; single-strand breaks; TAK-1; TGF-beta activated kinase 1; WIP1; wild-type p53-induced regulator 1; XIAP; X-linked inhibitor of apoptosis proteinDNA double-strand breaks; Replication stress; ATM; ATR; NF-κB

Catalase overexpression in mammary cancer cells leads to a less aggressive phenotype and an altered response to chemotherapy by Christophe Glorieux; Nicolas Dejeans; Brice Sid; Raphaël Beck; Pedro Buc Calderon; Julien Verrax (pp. 1384-1390).

Because reactive oxygen species (ROS) are naturally produced as a consequence of aerobic metabolism, cells have developed a sophisticated set of antioxidant molecules to prevent the toxic accumulation of these species. However, compared with normal cells, malignant cells often exhibit increased levels of intracellular ROS and altered levels of antioxidant molecules. The resulting endogenous oxidative stress favors tumor growth by promoting genetic instability, cell proliferation and angiogenesis. In this context, we assessed the influence of catalase overexpression on the sensitivity of breast cancer cells towards various anticancer treatments. Our data show that catalase overexpression in MCF-7 cells leads to a 7-fold increase in catalase activity but provokes a 40% decrease in the expression of both glutathione peroxidase and peroxiredoxin II. Interestingly, proliferation and migration capacities of MCF-7 cells were impaired by the overexpression of catalase, as compared to parental cells. Regarding their sensitivity to anticancer treatments, we observed that cells overexpressing catalase were more sensitive to paclitaxel, etoposide and arsenic trioxide. However, no effect was observed on the cytotoxic response to ionizing radiations, 5-fluorouracil, cisplatin or doxorubicin. Finally, we observed that catalase overexpression protects cancer cells against the pro-oxidant combination of ascorbate and menadione, suggesting that changes in the expression of antioxidant enzymes could be a mechanism of resistance of cancer cells towards redox-based chemotherapeutic drugs.

Keywords: Cancer; Catalase; Oxidative stress; Chemotherapy; Ionizing radiations

The adaptor Lnk (SH2B3): An emerging regulator in vascular cells and a link between immune and inflammatory signaling by Julie Devallière; Béatrice Charreau (pp. 1391-1402).

Lnk (SH2B3) emerges as a key regulator in hematopoeitic and vascular cells moderating growth factor, integrin and cytokine receptor-mediated signaling.A better knowledge of the process by which inflammatory extracellular signals are relayed from the plasma membrane to specific intracellular sites is a key step to understand how inflammation develops and how it is regulated. This review focuses on Lnk (SH2B3) a member, with SH2B1 and SH2B2, of the SH2B family of adaptor proteins that influences a variety of signaling pathways mediated by Janus kinase and receptor tyrosine kinases. SH2B adaptor proteins contain conserved dimerization, pleckstrin homology, and SH2 domains. Initially described as a regulator of hematopoiesis and lymphocyte differentiation, Lnk now emerges as a key regulator in hematopoeitic and non hematopoeitic cells such as endothelial cells (EC) moderating growth factor and cytokine receptor-mediated signaling. In EC, Lnk is a negative regulator of TNF signaling that reduce proinflammatory phenotype and prevent EC from apoptosis. Lnk is a modulator in integrin signaling and actin cytoskeleton organization in both platelets and EC with an impact on cell adhesion, migration and thrombosis. In this review, we discuss some recent insights proposing Lnk as a key regulator of bone marrow-endothelial progenitor cell kinetics, including the ability to cell growth, endothelial commitment, mobilization, and recruitment for vascular regeneration. Finally, novel findings also provided evidences that mutations in Lnk gene are strongly linked to myeloproliferative disorders but also autoimmune and inflammatory syndromes where both immune and vascular cells display a role. Overall, these studies emphasize the importance of the Lnk adaptor molecule not only as prognostic marker but also as potential therapeutic target.

Keywords: Abbreviations; AGM; aorta–gonad–mesonephros; APS; adaptor protein with PH and SH2 domains; BM; bone marrow; CAM; cell adhesion molecules; CEBPα; CCAAT/enhancer-binding protein-α; CD; coeliac disease; c-Fms; macrophage colony stimulating factor receptor (M-CSFR); CH; calponin homology; DD; dimerization domain; EC; endothelial cell; eNOS; endothelial nitric oxide synthase; EPC; endothelial progenitor cell; EPO; erythropoietin; EPOR; erythropoietin receptor; ERK1/2; extracellular signal-regulated kinases1/2; ESC; embryonic stem cells; Gab2; Grb2-associated-binding protein 2; GH; growth hormone; Grb2; growth factor receptor bound-2; GSK3b; glycogen synthase kinase 3 beta; GWAS; genome-wide association study; HO-1; heme oxygenase-1; HPC; hematopoietic progenitor cells; HUVEC; human umbilical vein EC; HSC; hematopoietic stem cells; ICAM-1; intercellular adhesion molecule 1; IFNγ; interferon-γ; JAK2; Janus kinase 2; JNK; c-jun N-terminal kinase; KSL; c-Kit-positive, Sca-1-positive, lineage marker-negative cells; MAPK; mitogen-activated protein kinase; MHC; major histocompatibility; MPL; thrombopoietin receptor; MPN; myeloproliferative neoplasms; NFkB; nuclear factor kappa-B; PDGFR; platelet-derived growth factor receptor; PDZ; postsynaptic density 95: PSD-85, discs large: Dlg, zonula occludens-1: ZO-1; PH; pleckstrin homology; PI3 K; phosphatidylinositol 3 kinase; RA; rheumatoid arthritis; RNA; ribonucleic acid; SCF; stem cell factor; SH2,3; Src homology 2,3; SMC; smooth muscle cells; SNP; single nucleotide polymorphisms; STAT; signal transducers and activators of transcription; TCR; T-cell receptor; T1D; type 1 diabetes; TA; transplant arteriosclerosis; TNF; tumor necrosis factor alpha; TPO; thrombopoietin; Trk; neurotrophic tyrosine kinase receptor; VCAM-1; vascular cell adhesion molecule-1; VLA; very late antigen; WT; wild-typeSignaling; Adaptor protein; Lnk (SH2B3); Inflammation; Endothelial cells

Identification of the human testis protein phosphatase 1 interactome by Margarida Fardilha; Sara L.C. Esteves; Luís Korrodi-Gregório; Ana Paula Vintém; Sara C. Domingues; Sandra Rebelo; Nick Morrice; Patricia T.W. Cohen; Odete A.B. da Cruz e Silva; Edgar F. da Cruz e Silva (pp. 1403-1415).

Protein phosphorylation is a critical regulatory mechanism in cellular signalling. To this end, PP1 is a major eukaryotic serine/threonine-specific phosphatase whose cellular functions, in turn, depend on complexes it forms with PP1 interacting proteins—PIPs. The importance of the testis/sperm-enriched variant, PP1γ2, in sperm motility and spermatogenesis has previously been shown. Given the key role of PIPs, it is imperative to identify the physiologically relevant PIPs in testis and sperm. Hence, we performed Yeast Two-Hybrid screens of a human testis cDNA library using as baits the different PP1 isoforms and also a proteomic approach aimed at identifying PP1γ2 binding proteins. To the best of our knowledge this is the largest data set of the human testis PP1 interactome. We report the identification of 77 proteins in human testis and 7 proteins in human sperm that bind PP1. The data obtained increased the known PP1 interactome by reporting 72 novel interactions. Confirmation of the interaction of PP1 with 5 different proteins was also further validated by co-immunoprecipitation or protein overlays. The data here presented provides important insights towards the function of these proteins and opens new possibilities for future research. In fact, such diversity in PP1 regulators makes them excellent targets for pharmacological intervention.

Keywords: Key words; PP1; Human testis; Yeast Two-Hybrid; PP1 interacting proteins

Targeting microRNAs involved in human diseases: A novel approach for modification of gene expression and drug development by Roberto Gambari; Enrica Fabbri; Monica Borgatti; Ilaria Lampronti; Alessia Finotti; Eleonora Brognara; Nicoletta Bianchi; Alex Manicardi; Rosangela Marchelli; Roberto Corradini (pp. 1416-1429).

MicroRNAs regulate gene expression and anti-sense microRNA molecules and microRNA mimics are proposed in diagnostics and therapy. Peptide nucleic acids (PNAs) efficiently target microRNAs, leading to therapeutic effects.The identification of all epigenetic modifications (i.e. DNA methylation, histone modifications and expression of noncoding RNAs such as microRNAs) involved in gene regulation is one of the major steps forward for understanding human biology in both normal and pathological conditions and for development of novel drugs. In this context, microRNAs play a pivotal role. This review article focuses on the involvement of microRNAs in the regulation of gene expression, on the possible role of microRNAs in the onset and development of human pathologies, and on the pharmacological alteration of the biological activity of microRNAs. RNA and DNA analogs, which can selectively target microRNAs using Watson–Crick base pairing schemes, provide a rational and efficient way to modulate gene expression. These compounds, termed antago-miR or anti-miR have been described in many examples in the recent literature and have proved to be able to perform regulatory as well as therapeutic functions. Among these, a still not fully exploited class is that of peptide nucleic acids (PNAs), promising tools for the inhibition of miRNA activity, with important applications in gene therapy and in drug development. PNAs targeting miR-122, miR-155 and miR-210 have already been developed and their biological effects studied both in vitro and in vivo.

Keywords: Abbreviations; miRNA (miR); microRNA; pri-miRNA; primary miRNA; pre-miRNA; precursor miRNA; RISC; RNA-induced silencing complex; ODN; oligodeoxyribonucleotide; PNA; peptide nucleic acid; LNA; locked nucleic acid; RNA Pol II; RNA polymerase II; TF; transcription factor; mitron; intron containing miRNA sequences; hESC; human embryonic stem cells; EB; embryoid body; UCB; umbilical cord blood; HbF; fetal hemoglobin; ErPCs; erythroid precursor cells; MTH; mithramycin; HPFH; high persistence of fetal hemoglobin; EPO; erythropoietinMicroRNAs; Erythroid differentiation; Epigenetics; Gene transcription; Gene regulation; Peptide nucleic acids

Epigenetic regulation of CIITA expression in human T-cells by Marja C.J.A. van Eggermond; Daniël R. Boom; Petra Klous; Erik Schooten; Victor E. Marquez; Rutger J. Wierda; Tjadine M. Holling; Peter J. van den Elsen (pp. 1430-1437).

In humans, T-cells accomplish expression of MHC-II molecules through induction of CIITA upon activation. Here we show that CIITA promoter accessibility in T-cells is epigenetically regulated. In unstimulated T-cells, CIITA-PIII chromatin displays relative high levels of repressive histone methylation marks (3Me-K27-H3 and 3Me-K20-H4) and low levels of acetylated histones H3 (Ac-H3) and H4 (Ac-H4). These repressive histone marks are replaced by histone methylation marks associated with transcriptional active genes (3Me-K4-H3) and high levels of Ac-H3 and Ac-H4 in activated T-cells. This is associated with concomitant recruitment of RNA polymerase II. In T-leukemia cells, devoid of CIITA expression, similar repressive histone methylation marks and low levels of acetylated histone H3 correlated with lack of CIITA expression. This in contrast to CIITA expressing T-lymphoma cells, which display high levels of Ac-H3 and 3Me-K4-H3, and relative low levels of the 3Me-K27-H3 and 3Me-K20-H4 marks. Of interest was the observation that the levels of histone acetylation and methylation modifications in histones H3 and H4 were also noted in chromatin of the downstream CIITA-PIV promoter as well as the upstream CIITA-PI and CIITA-PII promoters both in normal T-cells and in malignant T-cells. Together our data show that CIITA chromatin in T-cells expressing CIITA display similar histone acetylation and methylation characteristics associated with an open chromatin structure. The opposite is true for T-cells lacking CIITA expression, which display histone modifications characteristic of condensed chromatin.

Keywords: Class II TransActivator (CIITA); MHC class II (MHC-II); Epigenetic regulation; T-cells; T-leukemia; T-lymphoma

Engineering and screening the N-terminus of chemokines for drug discovery by Andy Chevigné; Virginie Fievez; Jean-Claude Schmit; Sabrina Deroo (pp. 1438-1456).

Chemokines are small chemoattractive proteins involved in many important physiological and pathological processes such as leukocyte mobilisation, inflammation, cancer and HIV-1 infection. The N-terminus of chemokines was shown to be crucial for interaction and activation with their cognate receptors. Therefore, multiple strategies including elongation, truncation, mutagenesis or chemical modifications of chemokine N-terminus were developed to identify analogues with modified selectivity displaying antagonist or enhanced agonist activities. Library approaches allowed fast screening of a large number of such chemokine variants and led to the identification of promising therapeutic candidates.Additional studies were able to reduce the chemokine to the size of a peptide while retaining its receptor affinity and selectivity. In analogy to full length chemokines, peptides derived from the chemokine N-terminal sequence were improved by mutagenesis, elongation and truncation approaches to develop potential therapeutic molecules used in various clinical trials.Altogether these studies demonstrated the pharmacophore potential of the chemokine N-terminus and its vast modulation properties to develop analogues with great therapeutic value for a large set of pathologies.

Keywords: Chemokine analogues; Chemokine receptors; Antagonists; Therapeutic peptides; CXCR4; CCR5

Tyrosine kinase inhibitors for the treatment of acute myeloid leukemia: Delineation of anti-leukemic mechanisms of action by Elodie Lainey; Sylvain Thépot; Cyrielle Bouteloup; Marie Sébert; Lionel Adès; Maximilien Tailler; Claude Gardin; Stéphane de Botton; André Baruchel; Pierre Fenaux; Guido Kroemer; Simone Boehrer (pp. 1457-1466).

Here we describe the results of a side-by-side comparison of the anti-leukemic efficacy of four different TKI (erlotinib, lapatinib, sorafenib and dasatinib) in MDS and AML.Initially, tyrosine kinase inhibitors (TKIs) were developed as targeted therapies that would solely interfere with aberrant tyrosine kinase activation in malignant cells. Nevertheless, preclinical and clinical studies demonstrated that TKI also exhibit “off-target” effects, that is effects not mediated by the assumed mechanisms of action. We and others showed that the epidermal growth factor receptor (EGFR) inhibitors erlotinib and gefitinib exert potent antineoplastic effects on EGFR-negative myeloblasts from patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Here, we undertook a side-by-side comparison of the anti-leukemic efficacy of four different TKI in MDS and AML. Besides the EGFR inhibitor erlotinib, which served as a point of reference, we employed the dual EGFR/HER2 TKI lapatinib, as well as the multikinase inhibitors dasatinib and sorafenib. All four drugs had anti-leukemic effects on cell line models of MDS/AML in vitro as well as on malignant blasts from MDS/AML patients ex vivo. We explored the biological phenomena underlying this anti-leukemic efficacy. Since it is established that a therapeutic benefit in MDS/AML can be conveyed by induction of cell cycle arrest, apoptosis and/or differentiation, we deciphered the individual contribution of these three phenomena to the anti-leukemic action of each of the four TKI. The concomitant assessment of the panel of TKI enables us thus to define (and quantify) their differential capacity to impact on the three biological phenomena, and provide further evidence that these mechanisms are not solely explained by on-target effects.

Keywords: Apoptosis; Differentiation; Epidermal growth factor receptor; Tyrosine kinases; Erlotinib; Lapatinib

Receptor tyrosine kinase inhibitors and cytotoxic drugs affect pleural mesothelioma cell proliferation: insight into EGFR and ERK1/2 as antitumor targets by Federica Barbieri; Roberto Würth; Roberto E. Favoni; Alessandra Pattarozzi; Monica Gatti; Alessandra Ratto; Angelo Ferrari; Adriana Bajetto; Tullio Florio (pp. 1467-1477).

Malignant pleural mesothelioma (MPM) is an aggressive chemotherapy-resistant cancer. Up-regulation of epidermal growth factor receptor (EGFR) plays an important role in MPM development and EGFR-tyrosine kinase inhibitors (TKIs) may represent novel therapeutic options. We tested the effects of the EGFR TKIs gefitinib and erlotinib and TKIs targeted to other growth factors (VEGFR and PDGFR), in comparison to standard antineoplastic agents, in two human MPM cell lines, IST-Mes2 and ZL55. All drugs showed IC₅₀ values in the micromolar range: TKIs induced cytostatic effects at concentrations up to the IC_50, while conventional drug growth-inhibitory activity was mainly cytotoxic. Moreover, the treatment of IST-Mes2 with TKIs (gefitinib and imatinib mesylate) in combination with cisplatin and gemcitabine did not show additivity. Focusing on the molecular mechanisms underlying the antiproliferative and pro-apoptotic effects of EGFR-TKIs, we observed that gefitinib induced the formation and stabilization of inactive EGFR homodimers, even in absence of EGF, as demonstrated by EGFR B_max and number of sites/cell. The analysis of downstream effectors of EGFR signaling demonstrated that EGF-induced proliferation, reverted by gefitinib, involved ERK1/2 activation, independently from Akt pathway. Gefitinib inhibits MPM cell growth and survival, preventing EGF-dependent activation of ERK1/2 pathway by blocking EGFR-TK phosphorylation and stabilizing inactive EGFR dimers. Along with the molecular definition of TKIs pharmacological efficacy in vitro, these results may contribute to delve deep into the promising but still controversial role for targeted and conventional drugs in the therapy of MPM.

Keywords: EGFR; Mesothelioma; Proliferation; Tyrosine-kinase inhibitors

Administration of carnosine in the treatment of acute spinal cord injury by Rosanna Di Paola; Daniela Impellizzeri; Angela Trovato Salinaro; Emanuela Mazzon; Francesco Bellia; Monia Cavallaro; Carolin Cornelius; Graziella Vecchio; Vittorio Calabrese; Enrico Rizzarelli; Salvatore Cuzzocrea (pp. 1478-1489).

l-Carnosine is an endogenously synthesized dipeptide composed of beta-alanine andl-histidine. It acts as a free radical scavenger and possesses antioxidant properties.l-Carnosine reduces proinflammatory and profibrotic cytokines such as transforming growth factor-beta (TGF-beta), interleukin (IL)-1, and tumor necrosis factor (TNF)-alpha in different experimental settings. In the present study, we investigated the efficacy ofl andd-carnosine on the animal model of spinal cord injury (SCI). The spinal cord was exposed via a four-level T5–T8 laminectomy and SCI was produced by extradural compression of the spinal cord at level T6–T7 using an aneurysm clip with a closing force of 24g. Treatment withd-carnosine (150mg/kg administered i.p., 1h and 6h, after SCI), but notl-carnosine significantly decreased (a) the degree of spinal cord inflammation and tissue injury (histological score), (b) neutrophil infiltration (myeloperoxidase activity), (c) nitrotyrosine formation, inducible NO synthase (iNOS) and Hsp70 expression, (d) proinflammatory cytokines, and (e) apoptosis (TUNEL staining, Fas ligand, Bax, and Bcl-2 expression). Furthermore,d-carnosine (150mg/kg administered i.p., 1h and 6h, after SCI) significantly ameliorated the loss of limb function (evaluated by motor recovery score). Taken together, our results demonstrate the strong difference betweenl-carnosine andd-carnosine. The result strongly suggests thatd-carnosine treatment reduces the development of inflammation and tissue injury associated with spinal cord trauma.

Keywords: Carnosine; Spinal cord injury; Inflammation; Apoptosis

Redox regulation of cellular stress response in multiple sclerosis by G. Pennisi; C. Cornelius; M.M. Cavallaro; A. Trovato Salinaro; M.T. Cambria; M. Pennisi; R. Bella; P. Milone; B. Ventimiglia; M.R. Migliore; L. Di Renzo; A. De Lorenzo; V. Calabrese (pp. 1490-1499).

Multiple sclerosis (MS) is an autoimmune-mediated neurodegenerative disease with characteristic foci of inflammatory demyelination in the brain, spinal cord, and optic nerves. Recent studies have demonstrated not only that axonal damage and neuronal loss are significant pathologic components of MS, but that this neuronal damage is thought to cause the permanent neurologic disability often seen in MS patients. Emerging finding suggests that altered redox homeostasis and increased oxidative stress, primarily implicated in the pathogenesis of MS, are a trigger for activation of a brain stress response. Relevant to maintenance of redox homeostasis, integrated mechanisms controlled by vitagenes operate in brain in preserving neuronal survival during stressful conditions. Vitagenes encode for heat shock proteins (Hsp) Hsp32, Hsp70, the thioredoxin and the sirtuin protein systems. In the present study we assess stress response mechanisms in the CSF, plasma and lymphocytes of control patients compared to MS patients. We found that the levels of vitagenes Hsp72, Hsc70, HO-1, as well as oxidative stress markers carbonyls and hydroxynonenals were significantly higher in the blood and CSF of MS patients than in control patients. In addition, an increased expression of Trx and sirtuin 1, together with a decrease in the expression of TrxR were observed. Our data strongly support a pivotal role for redox homeostasis disruption in the pathogenesis of MS and, consistently with the notion that new therapies that prevent neurodegeneration through nonimmunomodulatory mechanisms can have a tremendous potential to work synergistically with current MS therapies, unravel important targets for new cytoprotective strategies.

Keywords: Multiple sclerosis; Oxidative stress; Vitagenes; Heat shock proteins; Sirtuins

Roles of p75NTR in the pathogenesis of Alzheimer's disease: A novel therapeutic target by Fan Zeng; Jian-Jun Lu; Xin-Fu Zhou; Yan-Jiang Wang (pp. 1500-1509).

Alzheimer's disease (AD), the most common form of dementia, is characterized by the deposition of amyloid plaques, accumulation of fibrillary tangles in neurons, neurite degeneration, loss of neurons, and a progressive loss of cognitive function. The pathogenesis of AD is not fully understood, and no strong disease-modifying therapies are currently available. Recent studies suggest that the pan-neurotrophin receptor, p75NTR, is a critical factor involved in the pathogenesis of AD. In this review, we have discussed the roles of p75NTR in the production of amyloid-beta (Aβ), neuronal death, neurite degeneration, tau hyperphosphorylation, cell cycle re-entry and cognition decline in AD, and proposed that p75NTR is a potential target for the development of therapeutic drugs for AD. Finally we provide perspectives in developing various therapeutic strategies targeting different aspects of AD hallmarks which relate to p75NTR functions and breaking the p75NTR-mediated positive feedback loop which promotes the cascades in the pathogenesis of AD.

Keywords: Alzheimer's disease; Amyloid-beta; p75NTR; Degeneration; Tau; Cell cycle

Heteroplasmic mitochondrial disease in Dictyostelium discoideum by Lisa M. Francione; Paul R. Fisher (pp. 1510-1520).

The bewildering complexity of the relationship between genotype and phenotype in human mitochondrial diseases has delayed an understanding of the related cytopathological mechanisms. To explore the relationship between mitochondrial dysfunction in Dictyostelium discoideum and the related cytopathologies, we determined whether the phenotypic outcomes were similar regardless of which D. discoideum mitochondrial gene was targeted for disruption. The disruption of the mitochondrial genes resulted in a similar pattern of phenotypes to those caused by other mitochondrial defects. These include impairment of phototaxis, multicellular development and growth on plates and in liquid medium. As the reduced growth rates could have been due to defective phagocytic or macropinocytic nutrient uptake, these processes were tested but found to be unaffected. Since mitochondria have been associated with Legionella pathogenesis of human macrophages, it was also determined if mitochondrially diseased Dictyostelium strains were better or worse than healthy cells at supporting the growth of Legionella pneumophila. The results revealed that the mitochondrially diseased strains supported greater L. pneumophila growth than the wild type Dictyostelium strain (AX2). Quantitative Northern blotting showed a significant reduction in the level of expression of the entire mitochondrial genome, regardless of which mitochondrial gene was targeted for disruption, suggesting a generalized deficiency in mitochondrial gene expression and function. The phenotypic outcomes were the same as those shown previously to result from chronic hyperactivity of the energy-sensing protein kinase, AMPK, after knockdown of mitochondrial chaperonin 60.

Keywords: Dictyostelium; Phototaxis; Cell growth; Legionella; Mitochondrial disease

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Biochemical Pharmacology (v.82, #10)

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Biochemical Pharmacology (v.82, #10)