The two mains projects are the development of innovative skin models reproducing cutaneous microenvironment and/or specific epithelia and the study of skin aging mechanism to build new strategies for its prevention/treatment.
Development of innovative skin models
Our studies on physioxia have highlighted the importance of reconstituting physiological skin microenvironment for cutaneous biology studies. The characterization of pathways affected by oxygen level is investigated at the molecular level by studying transcriptome and miRnome with the aim to define optimized 3D models to replace animal use. We intend to use skin models grown in physioxia to determine more pertinent molecular targets for dermatology and cosmetology purposes in collaboration with cosmetic industries.
We develop vulvar tissue 3D models including Langerhans cells and endothelial cells. Transcriptome including miRNome mapping of vulvar tissue established from biopsies is used to validate them. The impact of the microenvironment (mucin production, commensal bacterial microflora), mechanical stress, inflammation and aging physiology will be assessed on these models. Better understanding of the cellular and molecular mechanisms involved in the pathology will contributed to the development of cosmetics and pharmaceutics accurate for this unique tissue.
Skin aging biology and regeneration
Studies are conducted on the role of microRNA in skin aging biology by evaluating their impact on the regulation of pigmentation, extracellular matrix production and anti-oxidant defenses. We also decipher whether such exosomes containing microRNA could play a role in cell-cell communications regulating skin homeostasis. To prevent or treat skin aging/injury, novel strategies are set up to exploit microRNAs regulation, glycosylated compounds, bioactive molecules. Most of these studies involved private partners. New project aims to evaluate the potential effects of non-thermal cold plasma to treat skin aging damages.
- Nadim M., Hassanaly S., Dubannet L. and Grillon C.
Physioxia and microRNAs as key factors in the skin microenvironmentarticle. International Federation of Societies of Cosmetic Chemists (2015) 18 (1) 35-43
- Nadim, M. Auriol, D. Lamerant-Faye, L. N. Lefevre, F. Dubanet, L. Redziniak, G. Kieda and C. Grillon, C.
Improvement of polyphenol properties upon glucosylation in a UV-induced skin cell ageing modelarticle. International journal of cosmetic science (2014) 36 (6) 579-587
- Grillon C., Matejuk A., Nadim M., Lamerant-Fayel N. and Kieda C.
News on microenvironmental physioxia to revisit skin cell targeting approachesarticle. Exp Dermatology (2012) 21 (10) 723-728
- Carreau, A., El Hafny-Rahbi, B., Matejuk, A., Grillon, C. and Kieda, C.
Why is the partial oxygen pressure of human tissues a crucial parameter ? J. Cell. Molecular Medecine (2011) 15 (6) 1239-1253
- Carreau A., Kieda C. and Grillon C.
Nitric oxide modulates endothelial cell adhesion molecules involved in angiogenesis and leukocyte recruitment. Exptl. Cell (2011) 317 (1) 29-41
Collet, G. Szade, K. Nowak, W. Klimkiewicz, K. El Hafny-Rahbi, B. Szczepanek, K. Sugiyama, D. Weglarczyk, K. Foucault-Collet, A. Guichard, A. Mazan, A. Nadim, M. Fasani, F. Lamerant-Fayel, N. Grillon, C. Petoud, S. Beloeil, J. C. Jozkowicz, A. Dulak, J. and Kieda, C. (2016)
Endothelial precursor cell-based therapy to target the pathologic angiogenesis and compensate tumor hypoxiaCancer Letters (2016) 370 (2) 345-357 - doi : 10.1016/j.canlet.2015.11.008
Hypoxia-inducing pathologies as cancer develop pathologic and inefficient angiogenesis which rules tumor facilitating microenvironment, a key target for therapy. As such, the putative ability of endothelial precursor cells (EPCs) to specifically home to hypoxic sites of neovascularization prompted to design optimized, site-specific, cell-mediated, drug-/gene-targeting approach. Thus, EPC lines were established from aorta-gonad-mesonephros (AGM) of murine 10.5 dpc and 11.5 dpc embryo when endothelial repertoire is completed. Lines representing early endothelial differentiation steps were selected : MAgEC10.5 and MagEC11.5. Distinct in maturation, they differently express VEGF receptors, VE-cadherin and chemokine/receptors. MAgEC11.5, more differentiated than MAgEC 10.5, displayed faster angiogenesis in vitro, different response to hypoxia and chemokines. Both MAgEC lines cooperated to tube-like formation with mature endothelial cells and invaded tumor spheroids through a vasculogenesis-like process. In vivo, both MAgEC-formed vessels established blood flow. Intravenously injected, both MAgECs invaded Matrigel(TM)-plugs and targeted tumors. Here we show that EPCs (MAgEC11.5) target tumor angiogenesis and allow local overexpression of hypoxia-driven soluble VEGF-receptor2 enabling drastic tumor growth reduction. We propose that such EPCs, able to target tumor angiogenesis, could act as therapeutic gene vehicles to inhibit tumor growth by vessel normalization resulting from tumor hypoxia alleviation.
Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetiteScience signaling (2016) 9 (424) ra39 - doi : 10.1126/scisignal.aae0374
The ability of the gut hormone ghrelin to promote positive energy balance is mediated by the growth hormone secretagogue receptor (GHSR). GHSR is a G protein-coupled receptor (GPCR) that is found centrally and peripherally and that can signal in a ligand-independent manner basally or when heterodimerized with other GPCRs. However, currentGhsrknockout models cannot dissect ghrelin-dependent and ghrelin-independent signaling, precluding assessment of the physiological importance of these signaling pathways. An animal model carrying aGhsrmutation that preserves GHSR cell surface abundance, but selectively alters GHSR signaling, would be a useful tool to decipher GHSR signaling in vivo. We used rats with theGhsr(Q343X)mutation (Ghsr(M/M)), which is predicted to delete the distal part of the GHSR carboxyl-terminal tail, a domain critical for the signal termination processes of receptor internalization and beta-arrestin recruitment. In cells, the GHSR-Q343X mutant showed enhanced ligand-induced G protein-dependent signaling and blunted activity of processes involved in GPCR signal termination.Ghsr(M/M)rats displayed enhanced responses to submaximal doses of ghrelin or GHSR agonist. Moreover,Ghsr(M/M)rats had a more stable body weight under caloric restriction, a condition that increases endogenous ghrelin tone, whereas under standard housing conditions,Ghsr(M/M)rats showed increased body weight and adiposity and reduced glucose tolerance. Overall, our data stress the physiological role of the distal domain of GHSR carboxyl terminus as a suppressor of ghrelin sensitivity, and we propose using theGhsr(M/M)rat as a physiological model of gain of function inGhsrto identify treatments for obesity-related conditions.
Alteration of cathepsin D trafficking induced by hypoxia and extracellular acidification in MCF-7 breast cancer cellsBiochimie (2016) 121, 123-130 - doi : 10.1016/j.biochi.2015.11.007
The microenvironment that surrounds tumor cells is characterized by hypoxic conditions and extracellular acidity. These hostile conditions induce crucial changes in cell behavior and can promote the secretion of many soluble factors such as growth factors, cytokines and enzymes. The lysosomal aspartyl-endopeptidase cathepsin D (CD) is a marker of poor prognosis in breast cancer and is associated with a metastatic risk. In this study, the transport of CD was investigated in a model of breast cancer cells line (MCF-7) cultivated under hypoxia and acidification of media. CD secretion was assessed using Western blot analysis and protease activity was measured in conditioned culture media. We demonstrate that cultured MCF-7 cells secrete an active 52 kDa pCD precursor and report that under hypoxia there was an increased amount of pCD secreted. More surprisingly, extracellular acidification (pH 6 and 5.6) induced the secretion of the fully-mature and active (34 kDa + 14 kDa) double chain CD. Our findings reflect the fact that chemical anomalies influence the secretion path of CD in a breast cancer cell model, resulting in altered trafficking of the mature form. This important result may provide new arguments in favor of the role of extracellular CD in the degradation of the matrix proteins that constitute the breast tumor microenvironment.
Noman M. Z., Hasmim M., Messai Y., Terry S., Kieda C., Janji B. and Chouaib S. (2015)
Hypoxia : a key player in antitumor immune response. A Review in the Theme : Cellular Responses to HypoxiaAmerican journal of physiology. Cell physiology (2015) 309 (9) C569-C579 - doi : 10.1152/ajpcell.00207.2015
The tumor microenvironment is a complex system, playing an important role in tumor development and progression. Besides cellular stromal components, extracellular matrix fibers, cytokines, and other metabolic mediators are also involved. In this review we outline the potential role of hypoxia, a major feature of most solid tumors, within the tumor microenvironment and how it contributes to immune resistance and immune suppression/tolerance and can be detrimental to antitumor effector cell functions. We also outline how hypoxic stress influences immunosuppressive pathways involving macrophages, myeloid-derived suppressor cells, T regulatory cells, and immune checkpoints and how it may confer tumor resistance. Finally, we discuss how microenvironmental hypoxia poses both obstacles and opportunities for new therapeutic immune interventions.
The cancer stem cell (CSC) model has recently been approached also in renal cell carcinoma (RCC). A few populations of putative renal tumor-initiating cells (TICs) were identified, but they are indifferently understood ; however, the first and most thoroughly investigated are CD105-positive CSCs. The article presents a detailed comparison of all renal CSC-like populations identified by now as well as their presumable origin. Hypoxic activation of hypoxia-inducible factors (HIFs) contributes to tumor aggressiveness by multiple molecular pathways, including the governance of immature stem cell-like phenotype and related epithelial-to-mesenchymal transition (EMT)/de-differentiation, and, as a result, poor prognosis. Due to intrinsic von Hippel-Lindau protein (pVHL) loss of function, clear-cell RCC (ccRCC) develops unique pathological intra-cellular pseudo-hypoxic phenotype with a constant HIF activation, regardless of oxygen level. Despite satisfactory evidence concerning pseudo-hypoxia importance in RCC biology, its influence on putative renal CSC-like largely remains unknown. Thus, the article discusses a current knowledge of HIF-1 alpha/2 alpha signaling pathways in the promotion of undifferentiated tumor phenotype in general, including some experimental findings specific for pseudo-hypoxic ccRCC, mostly dependent from HIF-2 alpha oncogenic functions. Existing gaps in understanding both putative renal CSCs and their potential connection with hypoxia need to be filled in order to propose breakthrough strategies for RCC treatment.
The role of FGF2 in migration and tubulogenesis of endothelial progenitor cells in relation to pro-angiogenic growth factor productionMolecular and Cellular Biochemistry (2015) 410 (1-2) 131-142 - doi : 10.1007/s11010-015-2545-5
In recent years, special attention has been paid to finding new pro-angiogenic factors which could be used in gene therapy of vascular diseases such as critical limb ischaemia (CLI). Angiogenesis, the formation of new blood vessels, is a complex process dependent on different cytokines, matrix proteins, growth factors and other pro- or anti-angiogenic stimuli. Numerous lines of evidence suggest that key mediators of angiogenesis, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) together with fibroblast growth factor2 (FGF2) are involved in regulation of the normal and pathological process of angiogenesis. However, less information is available on the complex interactions between these and other angiogenic factors. The aim of this study was to characterise the effect of fibroblast growth factor2 on biological properties of human endothelial progenitor cells with respect to the expression level of other regulatory cytokines. Ectopic expression of FGF2 in EP cells stimulates their pro-angiogenic behaviour, leading to increased proliferation, migration and tube formation abilities. Moreover, we show that the expression profile of VEGF and other pro-angiogenic cytokines, such as HGF, MCP2, and interleukins, is affected differently by FGF2 in EPC. In conclusion, we provide evidence that FGF2 directly affects not only the biological properties of EP cells but also the expression pattern and secretion of numerous chemocytokines. Our results suggest that FGF2 could be applied in therapeutic approaches for CLI and other ischaemic diseases of the vascular system in vivo.
Rational Design, Pharmacomodulation, and Synthesis of Dual 5-Hydroxytryptamine 7 (5-HT7)/5-Hydroxytryptamine 2A (5-HT2A) Receptor Antagonists and Evaluation by F-18 -PET Imaging in a Primate BrainJournal of Medicinal Chemistry (2015) 58 (20) 8066-8096 - doi : 10.1021/acs.jmedchem.5b00874
We report the synthesis of 46 tertiary amine-bearing N-alkylated benzo[d]imidazol-2(3H)-ones, imidazo-[4,5-b]pyridin-2(3H)-ones, irnidazo[4,5-c]pyridin-2(3H)-ones, benzo [d] oxazol-2 (3H) -ones, oxazolo [4,5- b]pyridin-2(3H)-ones and N,N’-dialkylate d benzo [d] imidazol-2 (3H)-ones. These compounds were evaluated against 5-HT7R, 5-HT2AR, 5-HT1AR, and 5-HT6R as potent dual 5-HT7/5-HT2A serotonin receptors ligands. A thorough study of the structure-activity relationship of the aromatic rings and their substituents, the alkyl chain length and the tertiary amine was conducted. 1-(4-(4-(4-Fluorobenzoyl)piperidin-1-yl)butyl)-1H-benzo[d]imidazol-2(3H)-one (79) and 1-(6-(4-(4-fluorobenzoyDpiperidin-1-yOhexyl)-1H-benzo[d]imidazol-2(3H)-one (81) were identified as full antagonist ligands on cyclic adenosine monophosphate (cAMP, K-B = 4.9 and 5.9 nM, respectively) and inositol monophosphate (IP1, K-B = 0.6 and 16 nM, respectively) signaling pathways of 5-HT7R and 5-HT2AR. Both antagonists crossed the blood-brain barrier as evaluated with [F-18] radiolabeled compounds [F-18]79 and [F-18]81 in a primate’s central nervous system using positron emission tomography. Both radioligands showed standard uptake values ranging from 0.8 to 1.1, a good plasmatic stability, and a distribution consistent with 5-HT7R and 5-HT2AR in the CNS.
The biological effect of the nitroimidazole derivative of a polypyridyl ruthenium complex on cancer and endothelial cellsThe Royal Society of Chemistry (2015) 7 (3) 553-566 - doi : 10.1039/C5MT00037H
The ruthenium polypyridyl complexes [Ru(dip)2(bpy/bpy-2-nitroIm)]2+ (dip = 4,7-diphenyl-1,10-phenanthroline, bpy = 2,2[prime or minute]-bipyridine, bpy-2-nitroIm = 4-[3-(2-nitro-1H-imidazol-1-yl)propyl]) were found to be ca. ten times more cytotoxic against breast cancer (4T1) and human lung adenocarcinoma epithelial cells (A549) than a well-known anticancer drug, cisplatin. Even though the Ru complexes were quite cytotoxic towards FVB mouse lung microvascular endothelial cells (MLuMEC FVB) their efflux from these non transformed cells was much more efficient than from cancer ones. Both Ru complexes accumulated in cells. The cellular uptake of both Ru complexes occurs through passive diffusion while the nitroimidazole derivative is also endocytosed. They arrest cell growth in the S-phase and induce apoptosis. Such cell response can result from activation of oxidative stress by Ru complexes. The modulation of the mRNA expression profile for genes which might be involved in metastasis and angiogenesis processes by Ru complexes was analyzed for both cancer (4T1) and endothelial (MLuMEC FVB) cells. Ru complexes appeared to have a distinct impact on cell adhesion and migration as well as they affect endothelial cell vasculature. They are not only cytotoxic but are also potentially invasive and anti-metastatic agents. This work illustrates the putative future development of polypyridyl ruthenium.
The tumour microenvironment, long considered as determining cancer development, still offers research fields to define hallmarks of cancer. An early key-step, the “angiogenic switch”, allows tumour growth. Pathologic angiogenesis is a cancer hallmark as it features results of tumour-specific properties that can be summarised as a response to hypoxia. The hypoxic state occurs when the tumour mass reaches a volume sufficient not to permit oxygen diffusion inside the tumour centre. Thus tumour cells turn on adaptation mechanisms to the low pO(2) level, inducing biochemical responses in terms of cytokines/chemokines/receptors and consequently recruitment of specific cell types, as well as cell-selection inside the tumour. Moreover, these changes are orchestrated by the microRNA balance strongly reflecting the hypoxic milieu and mediating the cross-talk between endothelial and tumour cells. MicroRNAs control of the endothelial precursor-vascular settings shapes the niche for selection of cancer stem cells.
The skin microenvironment is characterized by the extracellular matrix components, soluble molecules produced by neighboring cells but also physicochemical parameters and particularly a very low oxygen level (called physioxia) compared to other organs and the atmosphere. This condition is not taken into account in classical in vitro experiments, which are now replacing animal models for dermocosmetic evaluations. It is now well known that the oxygen level controls the expression of various proteins, either by induction of hypoxia inducible factors (HIFs), or modulation of microRNAs, which are small molecules regulating mRNA transcription. In this review, we focus on both microRNAs regulated by oxygen level and microRNAs involved in the regulation of various skin functions. We highlight the importance of this new concept of skin hysioxia and its consequences on microRNA regulation.
Background and Purpose The transmembrane protein LINGO-1 is a negative regulator in the nervous system mainly affecting axonal regeneration, neuronal survival, oligodendrocyte differentiation and myelination. However, the molecular mechanisms regulating its functions are poorly understood. In the present study, we investigated the formation and the role of LINGO-1 cis-dimers in the regulation of its biological activity. Experimental Approach LINGO-1 homodimers were identified in both HEK293 and SH-SY5Y cells using co-immunoprecipitation experiments and BRET saturation analysis. We performed a hypothesis-driven screen for identification of small-molecule protein–protein interaction modulators of LINGO-1 using a BRET-based assay, adapted for screening. The compound identified was further assessed for effects on LINGO-1 downstream signalling pathways using Western blotting analysis and AlphaScreen technology. Key Results LINGO-1 was present as homodimers in primary neuronal cultures. LINGO-1 interacted homotypically in cis-orientation and LINGO-1 cis-dimers were formed early during LINGO-1 biosynthesis. A BRET-based assay allowed us to identify phenoxybenzamine as the first conformational modulator of LINGO-1 dimers. In HEK-293 cells, phenoxybenzamine was a positive modulator of LINGO-1 function, increasing the LINGO-1-mediated inhibition of EGF receptor signalling and Erk phosphorylation. Conclusions and Implications Our data suggest that LINGO-1 forms constitutive cis-dimers at the plasma membrane and that low MW compounds affecting the conformational state of these dimers can regulate LINGO-1 downstream signalling pathways. We propose that targeting the LINGO-1 dimerization interface opens a new pharmacological approach to the modulation of its function and provides a new strategy for drug discovery.
Mazuryk O., Magiera K., Rys B., Suzenet F., Kieda, C. and Brindell M. (2014)
Multifaceted interplay between lipophilicity, protein interaction and luminescence parameters of non-intercalative ruthenium(II) polypyridyl complexes controlling cellular imaging and cytotoxic propertiesJournal of biological inorganic chemistry (2014) 19 (8) 1305-1316 - doi : 10.1007/s00775-014-1187-5
Here, we examine the photophysical properties of five ruthenium(II) complexes comprising two 4,7-diphenyl-1,10-phenanthroline (dip) ligands and functionalized bipyridine (R(1)bpy-R(2), where R(1)= H or CH3, R(2)= H, CH(3), COO(-),4-[3-(2-nitro-1H-imidazol-1-yl)propyl] or 1,3-dicyclohexyl-1-carbonyl-urea) towards development of luminescence probes for cellular imaging. These complexes have been shown to interact with albumin and the formed adducts exhibited up to eightfold increase in the luminescence quantum yield as well as the average lifetime of emission. It was demonstrated that they cannot bind to DNA through the intercalation mode and its luminescence in the presence of DNA is quenching. Cell viability experiments indicated that all complexes possess significant dose-dependent cytotoxicity (with IC(5)(0) 5-19 muM) on 4T1 breast cancer cell line and their anti-proliferative activity correlates very well with their lipophilicity. Cellular uptake was studied by measuring the ruthenium content in cells using ICP-MS technique. As expected, the better uptake is directly related to higher lipophilicity of doubly charged ruthenium complexes while uptake of monocationic one is much lower in spite of the highest lipophilicity. Additionally staining properties were assessed using flow cytometry and fluorescence microscopy. These experiments showed that complex with 1,3-dicyclohexyl-1-carbonyl-urea substituent exhibits the best staining properties in spite of the lowest luminescence quantum yield in buffered solution (pH 7.4). Our results point out that both the imaging and cytotoxic properties of the studied ruthenium complexes are strongly influence by the level of internalization and protein interaction.
Polyphenols are strong antioxidant molecules allowing prevention of skin photo-ageing damages, but their use is limited due to low solubility and toxicity towards skin cells. We postulated that enzymatic glucosylation could improve their solubility, stability and, consequently, their efficacy. The aim of this work was to study changes induced by addition of a glucose moiety on two polyphenols displaying very different chemical structures [caffeic acid (CA), epigallocatechin-3-gallate (EGCG) and there glucosylated form, Glc-CA and Glc-EGCG] by assessing their cytotoxic properties and their antioxidant and anti-inflammatory activities.
Their antioxidant effect was assessed first by the classical DPPH radical-scavenging method. Then, a panel of human skin cells (keratinocytes, melanocytes, fibroblasts and endothelial cells) was used to evaluate their effect on cell toxicity and their antioxidant activities. With this aim, a photo-ageing model based on UV irradiation of skin cells was established. Molecule activity was assessed on reactive oxygen species (ROS) production, on superoxide dismutase (SOD) and catalase activities and, finally, on inflammatory factor production IL-6, IL-8 and IL-1β.
In an acellular model, antioxidant activity assessed by DPPH method was strongly reduced for Glc-CA compared to CA, whereas it remained the same for Glc-EGCG compared to EGCG. Glucosylated derivatives did not display more toxic effect on various skin cells. Moreover, toxicity was even strongly reduced for caffeic acid upon glucosylation. The efficacy of glucosyl-compounds against UV-induced ROS production was preserved, both with pre- and post-UV treatments. Particularly, a better antioxidant efficacy was shown by Glc-EGCG, vs. EGCG, on keratinocytes. In addition, an induction of SOD and catalase activity was clearly observed for Glc-CA. Both glucosyl-polyphenols display the same activity as their parent molecule in decreasing inflammatory factor production.
Our results demonstrated that enzymatic glucosylation of CA and EGCG led to an improved or preserved antioxidant activity in a cellular model of UV-induced skin ageing, despite the decrease in instantaneous antioxidant properties observed for Glc-CA. Glc-EGCG is specifically more active on keratinocytes, suggesting a specific targeting. Such glucosylated polyphenols displaying improved physicochemical and biological properties should be better candidates than natural ones for use in food additives and cosmetics.
Regulation and Novel Action of Thymidine Phosphorylase in Non-Small Cell Lung Cancer : Crosstalk with Nrf2 and HO-1PloS One (2014) 9 (5) e97070 - doi : 10.1371/journal.pone.0097070
Proangiogenic enzyme thymidine phosphorylase (TP) is a promising target for anticancer therapy, yet its action in non-small cell lung carcinoma (NSCLC) is not fully understood. To elucidate its role in NSCLC tumor growth, NCI-H292 lung mucoepidermoid carcinoma cells and endothelial cells were engineered to overexpress TP by viral vector transduction. NSCLC cells with altered expression of transcription factor Nrf2 or its target gene heme oxygenase-1 (HO-1) were used to study the regulation of TP and the findings from pre-clinical models were related to gene expression data from clinical NSCLC specimens. Overexpression of Nrf2 or HO-1 resulted in upregulation of TP in NCI-H292 cells, an effect mimicked by treatment with an antioxidant N-acetylcysteine and partially reversed by HO-1 knockdown. Overexpression of TP attenuated cell prolifera\tion and migration in vitro, but simultaneously enhanced angiogenic potential of cancer cells supplemented with thymidine. The latter was also observed for SK-MES-1 squamous cell carcinoma and NCI-H460 large cell carcinoma cells. TP-overexpressing NCI-H292 tumors in vivo exhibited better oxygenation and higher expression of IL-8, IL-1 beta and IL-6. TP overexpression in endothelial cells augmented their angiogenic properties which was associated with enhanced generation of HO-1 and VEGF. Correlation of TP with the expression of HO-1 and inflammatory cytokines was confirmed in clinical samples of NSCLC. Altogether, the increased expression of IL-1 beta and IL-6 together with proangiogenic effects of TP-expressing NSCLC on endothelium can contribute to tumor growth, implying TP as a target for antiangiogenesis in NSCLC.
The serotonin6 receptor (5-HT6R) is a promising target for treating cognitive deficits of schizophrenia often linked to alterations of neuronal development. This receptor controls neurodevelopmental processes, but the signaling mechanisms involved remain poorly understood. Using a proteomic strategy, we show that 5-HT6Rs constitutively interact with cyclin-dependent kinase 5 (Cdk5). Expression of 5-HT6Rs in NG108-15 cells induced neurite growth and expression of voltage-gated Ca2+ channels, two hallmarks of neuronal differentiation. 5-HT6R–elicited neurite growth was agonist independent and prevented by the 5-HT6R antagonist SB258585, which behaved as an inverse agonist. Moreover, it required receptor phosphorylation at Ser350 by Cdk5 and Cdc42 activity. Supporting a role of native 5-HT6Rs in neuronal differentiation, neurite growth of primary neurons was reduced by SB258585, by silencing 5-HT6R expression or by mutating Ser350 into alanine. These results reveal a functional interplay between Cdk5 and a G protein–coupled receptor to control neuronal differentiation.
Lethal and sublethal effects of imidacloprid, after chronic exposure, on the insect model Drosophila melanogasterEnvironmental science and technology (201) 48 (7) 4096-4102 - doi : 10.1021/es405331c
Neonicotinoids are subjected to vigilance because of environmental contaminations and deleterious effects on bees. Imidacloprid (IMI) is one of the most representative insecticides of this family. At chronic exposure, concentration-effect relationships are non linear. An insect model should allow a better description of this toxicity. We compared the lethal concentration 50% (LC50) of IMI for a Drosophila-field strain, after acute and chronic exposure. Relative to the acute LC50, the chronic LC50 was lowered by a factor of 29 for males (1.3 mM/45 muM), 52 for larvae (157 muM/3 muM) and more than 172 for females (>3.1 mM/18 muM). Chronic exposure also revealed significant lethal and sublethal effects, at concentrations 3-5 orders of magnitude lower than the chronic LC50. Mean mortalities reached 28% (at 3.91 nM) and 27% (at 39.1 nM) for females and males, respectively. Fecundity decreased of 16% at 1.96 nM. Mating increased of 30% at 0.391 nM. The LOEC (lowest observed effect concentration : 0.391 nM) was 46 000 times lower than the chronic LC50 for males ; it was 115 000 times lower than the chronic LC50 for females. This study illuminates effects that neonicotinoids can induce at very low concentrations. This is of particular interest for nontarget insects and for insect dependent species.
The lack of oxygen is a major reason for the resistance of tumor cells to treatments such as radiotherapies. A large number of recent publications on non-thermal plasma applications in medicine report cell behavior modifications and modulation of soluble factors. This in vivo study tested whether such modifications can lead to vascular changes in response to plasma application. Two in situ optical-based methods were used simultaneously, in real time, to assess the effect of non-thermal plasma on tissue vasculature. Tissue oxygen partial pressure (pO(2)) was measured using a time-resolved luminescence-based optical probe, and the microvascular erythrocyte flow was determined by laser Doppler flowmetry. When plasma treatment was applied on mouse skin, a rapid pO(2) increase (up to 4 times) was subcutaneously measured and correlated with blood flow improvement. Such short duration, i.e. 5 min, plasma-induced effects were shown to be locally restricted to the treated area and lasted over 120 min. Further investigations should elucidate the molecular mechanisms of these processes. However, improvement of oxygenation and perfusion open new opportunities for tumor treatments in combination with radiotherapy, and for tumor blood vessel normalization based strategies.
VEGFs are found at high levels in hypoxic tumors. As major components directing pathologic neovascularization, they regulate stromal reactions. Consequently, novel strategies targeting and inhibiting VEGF overproduction upon hypoxia offer considerable potential for modern anticancer therapies controlling rather than destroying tumor angiogenesis. Here, we report the design of a vector expressing the soluble form of VEGF receptor-2 (sVEGFR2) driven by a hypoxia-responsive element (HRE)-regulated promoter. To enable in vivo imaging by infrared visualization, mCherry and IFP1.4 coding sequences were built into the vector. Plasmid construction was validated through transfection into embryonic human kidney HEK293 and murine B16F10 melanoma cells. sVEGFR2 was expressed in hypoxic conditions only, confirming that the gene was regulated by the HRE promoter. sVEGFR2 was found to bind efficiently and specifically to murine and human VEGF-A, reducing the growth of tumor and endothelial cells as well as impacting angiogenesis in vitro. The hypoxia-conditioned sVEGFR2 expression was shown to be functional in vivo : Tumor angiogenesis was inhibited and, on stable transfection of B16F10 melanoma cells, tumor growth was reduced. Enhanced expression of sVEGFR2 was accompanied by a modulation in levels of VEGF-A. The resulting balance reflected the effect on tumor growth and on control of angiogenesis. A concomitant increase of intratumor oxygen tension also suggested an influence on vessel normalization. The possibility to express an angiogenesis regulator as sVEGFR2, in a hypoxia-conditioned manner, significantly opens new strategies for tumor vessel–controlled normalization and the design of adjuvants for combined cancer therapies. Mol Cancer Ther ; 13(1) ; 165–78. ©2013 AACR.
Robert, E., Vandamme, M., Brullé, L., Lerondel, S., Le Pape, A., Sarron, V., Riès, D., Darny, T., Dozias, S., Collet, G., Kieda, C., Pouvesle, J.-M. (2013)
The developmentofthePlasmaGun,generatingPulsedAtmospheric-pressurePlasmaStreamsinside long andsmalldiametertubes flushed withlowgas flow rates,isreportedwithaspecialemphasisonthe first demonstrationandperspectivesfor in vivo endoscopic applications.Thesafedeliveryandantitumor action ofplasmahavebeenachievedfor in vivo colorectal andpancreaticcancers.Plasmadeliveryand plasma triggeredinflammation insidemouselungswereperformedthroughanendoscopicprotocol. The studyofplasmasplittingortransferacrossmetallicsectionsordielectricbarriersisdocumentedand open upopportunitiesforselfguidedandlowinvasiveplasmaendoscopy.
Interplay between heme oxygenase-1 and miR-378 affects non-small cell lung carcinoma growth, vascularization, and metastasisAntioxidants & redox signaling (2013) 19 (7) 644-660 - doi : 10.1089/ars.2013.5184
AIMS : Heme oxygenase-1 (HO-1, HMOX1) can prevent tumor initiation ; while in various tumors, it has been demonstrated to promote growth, angiogenesis, and metastasis. Here, we investigated whether HMOX1 can modulate microRNAs (miRNAs) and regulate human non-small cell lung carcinoma (NSCLC) development. RESULTS : Stable HMOX1 overexpression in NSCLC NCI-H292 cells up-regulated tumor-suppressive miRNAs, whereas it significantly diminished the expression of oncomirs and angiomirs. The most potently down-regulated was miR-378. HMOX1 also up-regulated p53, down-regulated angiopoietin-1 (Ang-1) and mucin-5AC (MUC5AC), reduced proliferation, migration, and diminished angiogenic potential. Carbon monoxide was a mediator of HMOX1 effects on proliferation, migration, and miR-378 expression. In contrast, stable miR-378 overexpression decreased HMOX1 and p53 ; while enhanced expression of MUC5AC, vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and Ang-1, and consequently increased proliferation, migration, and stimulation of endothelial cells. Adenoviral delivery of HMOX1 reversed miR-378 effect on the proliferation and migration of cancer cells. In vivo, HMOX1 overexpressing tumors were smaller, less vascularized and oxygenated, and less metastatic. Overexpression of miR-378 exerted opposite effects. Accordingly, in patients with NSCLC, HMOX1 expression was lower in metastases to lymph nodes than in primary tumors. INNOVATION AND CONCLUSION : In vitro and in vivo data indicate that the interplay between HMOX1 and miR-378 significantly modulates NSCLC progression and angiogenesis, suggesting miR-378 as a new therapeutic target. REBOUND TRACK : This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16, 293-296, 2012) with the following serving as open reviewers : James F. George, Mahin D. Maines, Justin C. Mason, and Yasufumi Sato.
Heterogeneity of endothelial cells—role in vessel specialization and cooperation in vasculogenic mimicryPostepy biochemii (2013) 59 (4) 372-378
Among endothelial cells (ECs), subpopulations are mainly distinguished in terms of maturation, tissue specialization and functions. Heterogeneity is an important characteristic of endothelial cells responsible for organ-specific cell and molecule delivery. Endothelial cell heterogeneity is a key to embryonic development as well as cell recruitment in adult organism during the immune response ; it determines also the pathologic spreading of diseases, such as cancer invasion and infectious disease progression among species. Heterogeneity is also a feature of intra-organ specialization of endothelial cells. ECs are highly reactive to the microenvironment and their condition reflects healthy vs. diseased states. They respond to tissue hypoxia which brings a new parameter to endothelial heterogeneity. Hypoxia changes the phenotype and biology of ECs by turning on angiogenesis to restore physioxia. Highly responsive to hypoxia are the endothelial precursor cells (EPCs) and the selected cancer stem cell (CSC) populations, the most aggressive dedifferentiated tumor cells. They cooperate with one another and contribute to the vascular mimicry process of angiogenesis. This has a most effective impact on tumor cells escape and spreading.
Constitutive expression of ligand for natural killer cell NKp44 receptor (NKp44L) by normal human articular chondrocytesCellular immunology (2013) 285 (1-2) 6-9 - doi : 10.1016/j.cellimm.2013.08.005
Normal chondrocytes display susceptibility to lysis by natural killer (NK) cells and this phenomenon may play a role in some inflammatory cartilage disorders. The mechanisms of chondrocyte recognition and killing by NK cells remain unclear. Using flow cytometry and immunohistochemical staining we found that normal human articular chondrocytes constitutively express a ligand for NKp44, one of stimulatory NK cell receptors involved in recognition and killing of target cells. Expression of NKp44 ligand by normal articular chondrocytes is not involved in their killing by unstimulated NK cells ; however, it is responsible for anti-chondrocyte cytotoxicity mediated by long-term activated NK cells. Thus, expression of NKp44 ligand may play a role in chondrocyte destruction in course of chronic inflammatory cartilage disorders.
This chapter describes a short historical overview of the progress in endothelium research and point the importance of organ-selective characteristics according to the present knowledge about endothelium biology. Uncovering the advantages that the endothelial cell properties and characteristics provide for the development of future targeted therapies, the review describes why mature endothelial cells due to their organ-specificity can be useful to target diseased organs.
In the same line, endothelium properties will be exploited to make the endothelial cells a disease marker, e.g., in diabetes, stroke, cancer, inflammation, or ischemia and to provide a potential diagnostic indicator for the estimation of metastatic progression. New perspectives are thus opened by endothelial cells that can be considered both as a reporter and a target. These features can be combined with new cell-mediated and cell-targeted therapeutics designed to correct angiogenesis. Examples of such possible applications are detailed in the repair of tumor angiogenesis with help of endothelial cell precursors through their ability to target the pathologic angiogenesis and participate to normalization of the pathologic vasculature. The hypothesis that normalized angiogenesis may provide an efficient treatment, working as adjuvant to classical therapies, is being developed. The objective is to reach a mechanical stabilization that should result in an advantageous change of the tumor microenvironment.
We have created unique near-infrared (NIR)–emitting nanoscale metal-organic frameworks (nano-MOFs) incorporating a high density of Yb3+ lanthanide cations and sensitizers derived from phenylene. We establish here that these nano-MOFs can be incorporated into living cells for NIR imaging. Specifically, we introduce bulk and nano-Yb-phenylenevinylenedicarboxylate-3 (nano-Yb-PVDC-3), a unique MOF based on a PVDC sensitizer-ligand and Yb3+ NIR-emitting lanthanide cations. This material has been structurally characterized, its stability in various media has been assessed, and its luminescent properties have been studied. We demonstrate that it is stable in certain specific biological media, does not photobleach, and has an IC50 of 100 μg/mL, which is sufficient to allow live cell imaging. Confocal microscopy and inductively coupled plasma measurements reveal that nano-Yb-PVDC-3 can be internalized by cells with a cytoplasmic localization. Despite its relatively low quantum yield, nano-Yb-PVDC-3 emits a sufficient number of photons per unit volume to serve as a NIR-emitting reporter for imaging living HeLa and NIH 3T3 cells. NIR microscopy allows for highly efficient discrimination between the nano-MOF emission signal and the cellular autofluorescence arising from biological material. This work represents a demonstration of the possibility of using NIR lanthanide emission for biological imaging applications in living cells with single-photon excitation.
Inefficient immune response is a major glitch during tumor growth and progression. Chaotic and leaky blood vessels created in the process of angiogenesis allow tumor cells to escape and extricate anti-cancer immunity. Proangiogenic characteristics of hypoxic tumor microenvironment maintained by low oxygen tension attract endothelial progenitor cells, drive expansion of cancer stem cells, and deviantly differentiate monocyte descendants. Such cellular milieu further boosts immune tolerance and eventually appoint immunity for cancer advantage. Blood vessel normalization strategies that equilibrate oxygen levels within tumor and fix abnormal vasculature bring exciting promises to future anticancer therapies especially when combined with conventional chemotherapy. Recently, a new group of microRNAs (miRs) engaged in angiogenesis, called angiomiRs and hypoxamiRs, emerged as new therapeutic targets in cancer. Some of those miRs were found to efficiently regulate cancer immunity and their dysregulation efficiently programs aberrant angiogenesis and cancer metastasis. The present review highlights new findings in the field of miRs proficiency to normalize aberrant angiogenesis and to restore anti-tumor immune responses.
Among innovative strategies developed for cancer treatments, gene therapies stand of great interest despite their well-known limitations in targeting, delivery, toxicity or stability. The success of any given genetherapy is highly dependent on the carrier efficiency. New approaches are often revisiting the mythic trojan horse concept to carry therapeutic nucleic acid, i.e. DNAs, RNAs or small interfering RNAs, to pathologic tumor site. Recent investigations are focusing on engineering carrying modalities to overtake the above limitations bringing new promise to cancer patients. This review describes recent advances and perspectives for gene therapies devoted to tumor treatment, taking advantage of available knowledge in biotechnology and medicine.
Stable tumor vessel normalization with pO increase and endothelial PTEN activation by inositol trispyrophosphate brings novel tumor treatmentJ Mol Med (Berl) 91 (7) 883-899
Tumor hypoxia is a characteristic of cancer cell growth and invasion, promoting angiogenesis, which facilitates metastasis. Oxygen delivery remains impaired because tumor vessels are anarchic and leaky, contributing to tumor cell dissemination. Counteracting hypoxia by normalizing tumor vessels in order to improve drug and radio therapy efficacy and avoid cancer stem-like cell selection is a highly challenging issue. We show here that inositol trispyrophosphate (ITPP) treatment stably increases oxygen tension and blood flow in melanoma and breast cancer syngeneic models. It suppresses hypoxia-inducible factors (HIFs) and proangiogenic/glycolysis genes and proteins cascade. It selectively activates the tumor suppressor phosphatase and tensin homolog (PTEN) in vitro and in vivo at the endothelial cell (EC) level thus inhibiting PI3K and reducing tumor AKT phosphorylation. These mechanisms normalize tumor vessels by EC reorganization, maturation, pericytes attraction, and lowering progenitor cells recruitment in the tumor. It strongly reduces vascular leakage, tumor growth, drug resistance, and metastasis. ITPP treatment avoids cancer stem-like cell selection, multidrug resistance (MDR) activation and efficiently enhances chemotherapeutic drugs activity. These data show that counteracting tumor hypoxia by stably restoring healthy vasculature is achieved by ITPP treatment, which opens new therapeutic options overcoming hypoxia-related limitations of antiangiogenesis-restricted therapies. By achieving long-term vessels normalization, ITPP should provide the adjuvant treatment required in order to overcome the subtle definition of therapeutic windows for in vivo treatments aimed by the current strategies against angiogenesis-dependent tumors.
In preclinical research, genetic studies have made considerable progress as a result of the development of transgenic animal models of human diseases. Consequently, there is now a need for higher resolution MRI to provide finer details for studies of small animals (rats, mice) or very small animals (insects). One way to address this issue is to work with high-magnetic-field spectrometers (dedicated to small animal imaging) with strong magnetic field gradients. It is also necessary to develop a complete methodology (transmit/receive coil, pulse sequence, fixing system, air supply, anesthesia capabilities, etc.). In this study, we developed noninvasive protocols, both in vitro and in vivo (from coil construction to image generation), for drosophila MRI at 9.4 T. The 10*10*80-μm resolution makes it possible to visualize whole drosophila (head, thorax, abdomen) and internal organs (ovaries, longitudinal and transverse muscles, bowel, proboscis, antennae and optical lobes). We also provide some results obtained with a Drosophila model of muscle degeneration. This opens the way for new applications of structural genetic modification studies using MRI of drosophila.
Grillon C, Matejuk A, Nadim M, Lamerant-Fayel N, Kieda C. (2012)
The skin is a multifunctional organ and a first line of defense actively protecting from environmental stress caused by injury, microbial treat, UV irradiation and environmental toxins. Diverse cutaneous cell types together with extracellular matrix elements and factors create a dynamic scene for cellular communication crucial in vital processes such as wound healing, inflammation, angiogenesis, immune response. Direct functional success of skin equilibrium depends on its microenvironment settings and particularly the local oxygen tension. Indeed, skin entire milieu is characterized by and highly dependent on its low oxygen tension called physioxia as emphasized in this review. In the context of skin physioxia, we review and propose here new approaches to minimize age-related changes in skin state and function. We particularly emphasize carbohydrate-mediated interactions and new 3D models of engineered skin substitutes. We highlight newly emerged tools and targets including stem cells, miRNAs, matrix metalloproteinases, mitochondria and natural antioxidants that are promising in prevention of skin ageing and disease restraint. In the era of advanced dermatology, new attempts are bringing us closer to ’well being’ perception.
Hypoxia control to normalize pathologic angiogenesis : Potential role for endothelial precursor cells and miRNAs regulationVascular Pharmacology 56 (5-6) 252-261
Tumor microenvironment is a complex and highly dynamic milieu that provides very important clues on tumor development and progression mechanisms. Tumor-associated endothelial cells play a key role in stroma organization. They achieve tumor angiogenesis, a formation of tumor-associated (angiogenic) vessels mainly through sprouting from locally preexisting vessels and/or recruitment of bone marrow-derived endothelial progenitor cells. This process participates to supply nutritional support and oxygen to the growing tumor.
Endothelial cells constitute the interface between circulating blood cells, tumor cells and the extracellular matrix, thereby controlling leukocyte recruitment, tumor cell behavior and metastasis formation.
Hypoxia, a critical parameter of the tumor microenvironment, controls endothelial/tumor cell interactions and is the key to tumor angiogenesis development. Under hypoxic stress, tumor cells produce factors that promote angiogenesis, vasculogenesis, tumor cell motility, metastasis and cancer stem cell selection.
Targeting tumor vessels is a therapeutic strategy that has lately been fast evolving from antiangiogenesis to vessel normalization as discussed in this review. We shall focus on the pivotal role of endothelial cells within the tumor microenvironment, the specific features and the part played by circulating endothelial precursors cells. Attention is stressed on their recruitment to the tumor site and their role in tumor angiogenesis where they are submitted to miRNAs-mediated de/regulation. Here the compensation of the tumor deregulated angiogenic miRNAs – angiomiRs - is emphasized as a potential therapeutic approach. The strategy is to over express anti-angiomiRs in the tumor angiogenesis site upon selective delivery by precursor endothelial cells as miRs carriers.
Non-thermal plasma (NTP) is generated by ionizing neutral gas molecules/atoms leading to a highly reactive gas at ambient temperature containing excited molecules, reactive species and generating transient electric fields. Given its potential to interact with tissue or cells without a significant temperature increase, NTP appears as a promising approach for the treatment of various diseases including cancer. The aim of our study was to evaluate the interest of NTP both in vitro and in vivo. To this end, we evaluated the antitumor activity of NTP in vitro on two human cancer cell lines (glioblastoma U87MG and colorectal carcinoma HCT-116). Our data showed that NTP generated a large amount of reactive oxygen species (ROS), leading to the formation of DNA damages. This resulted in a multiphase cell cycle arrest and a subsequent apoptosis induction. In addition, in vivo experiments on U87MG bearing mice showed that NTP induced a reduction of bioluminescence and tumor volume as compared to nontreated mice. An induction of apoptosis was also observed together with an accumulation of cells in S phase of the cell cycle suggesting an arrest of tumor proliferation. In conclusion, we demonstrated here that the potential of NTP to generate ROS renders this strategy particularly promising in the context of tumor treatment.
A fraction of neurofibromin interacts with PML bodies in the nucleus of the CCF astrocytoma cell lineBiochemical and Biophysical Research Communications 418 (4) 689-694
Neurofibromatosis type 1 is a common genetic disease that causes nervous system tumors, and cognitive deficits. It is due to mutations within the NF1 gene, which encodes the Nf1 protein. Nf1 has been shown to be involved in the regulation of Ras, cAMP and actin cytoskeleton dynamics. In this study, using immunofluorescence experiments, we have shown a partial nuclear localization of Nf1 in the astrocytoma cell line : CCF and we have demonstrated that Nf1 partially colocalizes with PML (promyelocytic leukemia) nuclear bodies. A direct interaction between Nf1 and the multiprotein complex has further been demonstrated using ‘‘in situ’’ proximity ligation assay (PLA).
Paprocka, M., Krawczenko, A., Dus, D., Kantor, A., Carreau, A., Grillon, C. and Kieda, C. (2011)
Endothelial progenitor cells (EPCs) modulate postnatal vascularization and contribute to vessel regeneration in adults. Stem cells and progenitor cells were found in umbilical cord blood, bone marrow, and mobilized peripheral blood cells, from where they were isolated and cultured. However, the yield of progenitor cells is usually not sufficient for clinical application and the quality of progenitor cells varies. The aim of the study was the immortalization of early progenitor cells with high proliferative potential, capable to differentiate to EPCs and, further, toward endothelial cells. Two cell lines, namely HEPC-CB. 1 and HEPC-CB. 2 (human endothelial progenitor cells-cord blood) were isolated. As assessed by specific antibody labeling and flow cytometric analysis, they express a panel of stem cell markers : CD133, CD13, CD271, CD90 and also endothelial cell markers : CD202b, CD309 (VEGFR2), CD146, CD105, and CD143 but they do not present markers of finally differentiated endothelial cells : CD31, vWf, nor CD45 which is a specific hematopoietic cell marker. Using the multiplex Cytometric Bead Assay, the simultaneous production of proangiogenic cytokines IL8, angiogenin, and VEGF was demonstrated in normoxia and was shown to be increased by hypoxia. Both cell lines, similarly as mature endothelial cells, underwent in vitro pre-angiogenic process, formed pseudovessel structures and present an accelerated angiogenesis in hypoxic conditions. To date, these are the first CD133 positive established cell lines from human cord blood cells. (C) 2011 International Society for Advancement of Cytometry.
The MAP kinase ERK and its scaffold protein MP1 interact with the chromatin regulator Corto during Drosophila wing tissue development.Bmc Developmental Biology 11 (1) 17
Background : Mitogen-activated protein kinase (MAPK) cascades (p38, JNK, ERK pathways) are involved in cell fate acquisition during development. These kinase modules are associated with scaffold proteins that control their activity. In Drosophila, dMP1, that encodes an ERK scaffold protein, regulates ERK signaling during wing development and contributes to intervein and vein cell differentiation. Functional relationships during wing development between a chromatin regulator, the Enhancer of Trithorax and Polycomb Corto, ERK and its scaffold protein dMP1, are examined here.
Results : Genetic interactions show that corto and dMP1 act together to antagonize rolled (which encodes ERK) in the future intervein cells, thus promoting intervein fate. Although Corto, ERK and dMP1 are present in both cytoplasmic and nucleus compartments, they interact exclusively in nucleus extracts. Furthermore, Corto, ERK and dMP1 co localize on several sites on polytene chromosomes, suggesting that they regulate gene expression directly on chromatin. Finally, Corto is phosphorylated. Interestingly, its phosphorylation pattern differs between cytoplasm and nucleus and changes upon ERK activation.
Differential Effects of Bartonella henselae on Human and Feline Macro- and Micro-Vascular Endothelial CellsPLOS One - 6 (5) e20204 Carreau, A., El Hafny-Rahbi, B., Matejuk, A., Grillon, C. & Kieda, C. (2011)
Nitric oxide modulates endothelial cell adhesion molecules involved in angiogenesis and leukocyte recruitment.Exptl. Cell. - 317 (1) 29-41
Brutovsky, B., Horvath, D. (2010)
Any process in which competing solutions replicate with errors and numbers of their copies depend on their respective fitnesses is the evolutionary optimization process. As during carcinogenesis mutated genomes replicate according to their respective qualities, carcinogenesis obviously qualifies as the evolutionary optimization process and conforms to common mathematical basis. The optimization view accents statistical nature of carcinogenesis proposing that during it the crucial role is actually played by the allocation of trials. Optimal allocation of trials requires reliable schemas’ fitnesses estimations which necessitate appropriate, fitness landscape dependent, statistics of population. In the spirit of the applied conceptual framework, features which are known to decrease efficiency of any evolutionary optimization procedure (or inhibit it completely) are anticipated as "therapies" and reviewed. Strict adherence to the evolutionary optimization framework leads us to some counterintuitive implications which are, however, in agreement with recent experimental findings, such as sometimes observed more aggressive and malignant growth of therapy surviving cancer cells.
Characterisation of a GroEL Single-Ring Mutant that Supports Growth of Escherichia coli and Has GroES-Dependent ATPase ActivityJournal of Molecular Biology (2010) 396 (5) 1271-1283 - doi : 10.1016/j.jmb.2009.11.074
Binding and folding of substrate proteins by the molecular chaperone GroEL alternates between its two seven-membered rings in an ATP-regulated manner. The association of ATP and GroES to a polypeptide-bound ring of GroEL encapsulates the folding proteins in the central cavity of that ring (cis ring) and allows it to fold in a protected environment where the risk of aggregation is reduced. ATP hydrolysis in the cis ring changes the potentials within the system such that ATP binding to the opposite (trans) ring triggers the release of all ligands from the cis ring of GroEL through a complex network of allosteric communication between the rings. Inter-ring allosteric communication thus appears indispensable for the function of GroEL, and an engineered single-ring version (SR1) cannot substitute for GroEL in vivo. We describe here the isolation and characterisation of an active single-ring form of the GroEL protein (SR-A92T), which has an exceptionally low ATPase activity that is strongly stimulated by the addition of GroES. Dissection of the kinetic pathway of the ATP-induced structural changes in this active single ring can be explained by the fact that the mutation effectively blocks progression through the full allosteric pathway of the GroEL reaction cycle, thus trapping an early allosteric intermediate. Addition of GroES is able to overcome this block by binding this intermediate and pulling the allosteric pathway to completion via mass action, explaining how bacterial cells expressing this protein as their only chaperonin are viable.
A novel pyridine derivative, 8-4-[(6-methoxy-2,3-dihydro-[1,4]dioxino[2,3-b]pyridine-3-ylmethyl)-amino]-butyl -8-aza-spiro[4.5]decane-7,9-dione hydrochloride, termed JB-788, was designed to selectively target 5-HT(1A) receptors. In the present study, the pharmacological profile of JB-788 was characterized in vitro using radioligands binding tests and in vivo using neurochemical and behavioural experiments. JB-788 bound tightly to human 5-HT(1A) receptor expressed in human embryonic kidney 293 (HEK-293) cells with a K(i) value of 0.8 nM. Its binding affinity is in the same range as that observed for the (+/-)8-OH-DPAT, a reference 5HT(1A) agonist compound. Notably, JB-788 only bound weakly to 5-HT(1B) or 5-HT(2A) receptors and moreover the drug displayed only weak or indetectable binding to muscarinic, alpha(2), beta(1) and beta(2) adrenergic receptors, or dopaminergic D(1) receptors. JB-788 was found to display substantial binding affinity for dopaminergic D(2) receptors and, to a lesser extend to alpha(1) adrenoreceptors. JB-788 dose-dependently decreased forskolin-induced cAMP accumulation in HEK cells expressing human 5-HT(1A), thus acting as a potent 5-HT(1A) receptor agonist (E(max.) 75%, EC(50) 3.5 nM). JB-788 did not exhibit any D(2) receptor agonism but progressively inhibited the effects of quinpirole, a D(2) receptor agonist, in the cAMP accumulation test with a K(i) value of 250 nM. JB-788 induced a weak change in cAMP levels in mouse brain but, like some antipsychotics, transiently increased glycogen contents in various brain regions. Behavioral effects were investigated in mice using the elevated plus-maze. JB-788 was found to increase the time duration spent by animals in anxiogenic situations. Locomotor hyperactivity induced by methamphetamine in mouse, a model of antipsychotic activity, was dose-dependently inhibited by JB-788. Altogether, these results suggest that JB-788 displays pharmacological properties, which could be of interest in the area of anxiolytic and antipsychotic drugs.
Le système histaminergique : une cible pour de nouveaux traitements des déficits cognitifs - The Histaminergic System : a Target for Innovative Treatments of Cognitive DeficitsThérapie 65 (5) 415-522 - doi : 10.2515/therapie/2010058
L’histamine exerce ses effets centraux en activant des récepteurs H1,H2 et H3. Le récepteur H3 inhibe la libération de l’histamine cérébrale. Ainsi, les agonistes inverses H3, en levant ce frein, augmentent l’activité des neurones à histamine. Le système histaminergique est un système majeur de la cognition et les agonistes inverses H3 sont pressentis comme thérapeutique potentielle des déficits cognitifs de la maladie d’Alzheimer (AD). Ils sont d’autant plus attendus que d’autres traitements de la maladie, tels que la tacrine ou la mémantine, augmentent aussi, mais par d’autres mécanismes, la neurotransmission histaminergique. Il existe une perte importante de neurones histaminergiques dans l’AD, mais la mesure des taux du métabolite de l’histamine dans le LCR de patients atteints d’AD montre que leur activité globale n’est diminuée que de 25 %. Ces données montrent qu’il devrait bien être possible d’activer les neurones histaminergiques dans l’AD.
The central effects of histamine are mediated by H1, H2 and H3 receptors. The H3 receptor inhibits histamine release in brain. Therefore, H3 receptor inverse agonists, by suppressing this brake, enhance histamine neuron activity. The histaminergic system plays a major role in cognition and H3 receptor inverse agonists are expected to be a potential therapeutics for cognitive deficits of Alzheimer’s disease (AD). They are eagerly awaited inasmuch as other treatments of the disease, such as tacrine or memantine, also enhance, through different mechanisms, histaminergic neurotransmission. An important loss of histaminergic neurons has been observed in AD. In contrast, levels of the histamine metabolite in the CSF of AD patients show that their global activity is decreased by only 25%. This indicates that activating histamine neurons in AD can be envisaged.
Effects of betahistine at histamine H3 receptors : mixed inverse agonism/agonism in vitro and partial inverse agonism in vivoJournal of Pharmacology and Experimental Therapeutics 334 (3) 945-954 - doi : 10.1124/jpet.110.168633
We previously suggested that therapeutic effects of betahistine in vestibular disorders result from its antagonist properties at histamine H(3) receptors (H(3)Rs). However, H(3)Rs exhibit constitutive activity, and most H(3)R antagonists act as inverse agonists. Here, we have investigated the effects of betahistine at recombinant H(3)R isoforms. On inhibition of cAMP formation and [(3)H]arachidonic acid release, betahistine behaved as a nanomolar inverse agonist and a micromolar agonist. Both effects were suppressed by pertussis toxin, were found at all isoforms tested, and were not detected in mock cells, confirming interactions at H(3)Rs. The inverse agonist potency of betahistine and its affinity on [(125)I]iodoproxyfan binding were similar in rat and human. We then investigated the effects of betahistine on histamine neuron activity by measuring tele-methylhistamine (t-MeHA) levels in the brains of mice. Its acute intraperitoneal administration increased t-MeHA levels with an ED(50) of 0.4 mg/kg, indicating inverse agonism. At higher doses, t-MeHA levels gradually returned to basal levels, a profile probably resulting from agonism. After acute oral administration, betahistine increased t-MeHA levels with an ED(50) of 2 mg/kg, a rightward shift probably caused by almost complete first-pass metabolism. In each case, the maximal effect of betahistine was lower than that of ciproxifan, indicating partial inverse agonism. After an oral 8-day treatment, the only effective dose of betahistine was 30 mg/kg, indicating that a tolerance had developed. These data strongly suggest that therapeutic effects of betahistine result from an enhancement of histamine neuron activity induced by inverse agonism at H(3) autoreceptors.
Endothelial cells as key determinants of the tumor microenvironment : interaction with tumor cells, extracellular matrix and immune killer cells.Crit. Rev. Immunol. - 30 (6) 529-545 Li, S., Starckx, S., Martens, E., Dillen, C., Lamerant-Fayel, N., Berghmans, N., Gouwy, M., van Pel, M., Heremans, H., Kieda, C., Fibbe, W.E., Billiau, A., Van Damme, J.& Opdenakker, G. (2010)
Myeloid cells are tunable by a polyanionic polysaccharide derivative and co-determine host rescue from lethal virus infection.J. Leukoc. Biol. 88 (5) 1017-1029 Brauer, R., Wang, L-C S., Woon, S.T., Bridewell, D., Henare, K., Malinger, D., Palmer, B.D., Vogel, S.N. Kieda, C. & Ching, L.-M. (2010)
Preferential labelling of oxidisable proteins with a photoactivatable analogue of the anti-tumor agent DMXAA and evidence for redox signalling in its mode of action.Neoplasia 12, 755-765 Lamiable, O., Rabhi, M., Peronnet, F., Locker, D. & Decoville, M. (2010)
Benlalam, H., Jalil, A., Hasmim, M., Pang, B., Tamouza, R., Mitterrand, M., Godet, Y., Lamerant, N., Robert, C., Avril, M.F., Neefjes, J., Tursz, T., Mami-Chouaib, F., Kieda, C. & Chouaib, S. (2009)
Gap Junction Communication between Autologous Endothelial and Tumor Cells Induce Cross-Recognition and Elimination by Specific CTLJ. Immunol. 182, 2654-2664. Rautureau, G., Jouvensal, L., Vovelle, F., Schoentgen, F., Locker, D. & Decoville, M. (2009)
An optimized extended DNA kappa B site that enhances plasmid DNA nuclear import and gene expression.J. Gene Med. 11, 401-411. Crola Da Silva, C., Lamerant-Fayel, N., Paprocka, M., Mitterrand, M., Gosset, D., Dus, D. & Kieda, (2009)
Serotonin binds to purified neuronal nitric oxide synthase : A possible explanation for ROS production induced by 5HT in the presence of nNOS.Free Radic. Res. 43, 206-213. Bialoszewska, A., Niderla-Bielinska, J., Hyc, A., Osiecka-Iwan, A., Radomska-Lesniewska, D.M., Kieda, C. & Malejczyk, J. (2009)
Szczepanek, K., Kieda, C. & Cichy, J. (2008)
Flow cytometric assay for quantitative and qualitative evaluation of adhesive interactions of tumor cells with endothelial cells.Microvasc. Res. 76, 134-138. Abouzahr-Rifai, S., Hasmim, M., Boukerche, H., Hamelin, J., Janji, B., Jalil, A., Kieda, C., Mami-Chouaib, F., Bertoglio, J. & Chouaib, S. (2008)
Resistance of Tumor Cells to Cytolytic T Lymphocytes Involves Rho-GTPases and Focal Adhesion Kinase Activation.J. Biol. Chem. 283, 31665-31672. Davenas E, Rouleau A, Morisset S, and Arrang JM. (2008)
Palmer, BD ; Henare, K ; Woon, ST ; Sutherland, R ; Reddy, C ; Wang, LCS ; Kieda, C ; Ching, LM (2007)
Synthesis and biological activity of azido analogues of 5,6-dimethylxanthenone-4-acetic acid for use in photoaffinity labelingJournal of Medicinal Chemistry 50 3757-3764
5,6-Dimethylxanthenone-4- acetic acid (1) is scheduled for phase III clinical trials as a vascular disrupting agent. However, its biochemical receptor(s) have yet to be identified. In this report, the synthesis of azido analogues of I that could be used for photoaffinity labeling of proteins as an approach toward identifying its molecular targets is described. While 5-azidoxantfienone-4-acetic acid (2) and 5-azido-6-methylxantheone-4-acetic acid (3) were found to have biological activities similar to that of 1, 6-azido-5-methylxanthenone-4-acetic acid (4) was unstable and could not be evaluated.
Dual role of melanoma cell adhesion molecule (MCAM)/CD146 in lymphocyte endothelium interaction : MCAM/CD146 promotes rolling via microvilli induction in lymphocyte and is an endothelial adhesion receptorJournal of Immunology 179 6673-6685
The melanoma cell adhesion molecule (MCAM)/CD146 is expressed as two isoforms differing by their cytoplasmic domain (MCAM long (MCAM-1) and MCAM short (MCAM-s)). MCAM being expressed by endothelial cells and activated T cells, we analyzed its involvement in lymphocyte trafficking. The NK cell line NKL1 was transfected by MCAM isoforms and submitted to adhesion on both the endothelial cell monolayer and recombinant molecules under shear stress. MCAM-1 transfection reduced rolling velocity and increased NKL1 adhesion on the endothelial cell monolayer and VCAM-1.
Serotonin, an important neurotransmitter, is colocalized with neuronal nitric oxide synthase (nNOS), a homodimeric enzyme which catalyzes the production of nitric oxide (NO center dot) and/or oxygen species. As many interactions have been reported between the nitrergic and serotoninergic systems, we studied the effect of serotonin on nNOS activities. Our results reveal that nNOS is activated by serotonin as both NADPH consumption and oxyhemoglobin (OxyHb) oxidation were enhanced. The generation of L-citrulline from L-arginine (L-Arg) was not affected by serotonin in the range of 0-200 mu M, suggesting an additional production of oxygen-derived species. But 5-hydroxytryptamine (5HT) induced the formation of both O-2(center dot-) and H2O2 by nNOS, as evidenced by electron paramagnetic resonance (EPR) and by using specific spin traps. Overall, these results demonstrate that serotonin is able to activate nNOS, leading to the generation of reactive oxygen species (ROS) in addition to the NO center dot production. Such a property must be considered in vivo as various nNOS-derived products mediate different signaling pathways.
Histamine H3 and dopamine D2 receptor-mediated [35S]GTPg[S]binding in rat striatum : evidence for additive effects but lack of interactionsBiochem Pharmacol (2007), 73 (8) : 1172-1181 Arrang J, Morisset S and Gbahou F. (2007)
Kieda, C., Greferath, R., Da Silva, C.C., Fylaktakidou, K.C., Lehn, J.M. & Nicolau, C. (2006)
Suppression of hypoxia-induced HIF-1 alpha and of angiogenesis in endothelial cells by myo-inositol trispyrophosphate-treated erythrocytes.Proc. Nat.l Acad. Sci. USA 103, 15576-15581. Mennesson, E ; Erbacher, P ; Kuzak, M ; Kieda, C ; Midoux, P ; Pichon, C (2006)
DNA/cationic polymer complex attachment on a human vascular endothelial cell monolayer exposed to a steady laminar flowJournal of Controlled Release 114 (3) 389-397
This study evaluated for the first time the binding of pDNA/polymer complexes (polyplexes) on a human lung microvascular endothelial cell (HLMEC) monolayer under flow conditions. A slide of a HLMEC monolayer was mounted on a parallel flow chamber connected to an open flow system from a reservoir containing fluorescent polyplexes to a syringe.
Corto and DSP1 interact and bind to a maintenance element of the Scr Hox gene : understanding the role of Enhancers of trithorax and PolycombBmc Biology 4 Art No. 9
Background : Polycomb-group genes (PcG) encode proteins that maintain homeotic (Hox) gene repression throughout development. Conversely, trithorax-group (trxG) genes encode positive factors required for maintenance of long term Hox gene activation. Both kinds of factors bind chromatin regions called maintenance elements ( ME). Our previous work has shown that corto, which codes for a chromodomain protein, and dsp1, which codes for an HMGB protein, belong to a class of genes called the Enhancers of trithorax and Polycomb (ETP) that interact with both PcG and trxG. Moreover, dsp1 interacts with the Hox gene Scr, the DSP1 protein is present on a Scr ME in S2 cells but not in embryos.
Cloning, high yield over-expression, purification, and characterization of CG18594, a new PEBP/RKIP family member from Drosophila melanogasterProtein Expression and Purification 48 (1) 90-97
The phosphatidylethanolamine-binding protein (PEBP) family is widely distributed in various species, from bacteria to mammals. These proteins seem to modulate important cell mechanisms : they control heterotrimeric G-proteins, inhibit the MAP-kinase and NF kappa B signaling pathways, and also serine proteases (thrombin, neuropsin, and chymotrypsin). In order to establish structure-function relationships for this family of proteins, our study focuses on PEBPs expressed within a single organism : Drosophila melanogaster, which constitutes a model system that lends itself well to establishing links between genes’ expression and the corresponding proteins’ functions, and to studying physiological mechanisms such as development.
Overexpression of heme oxygenase-1 in murine melanoma - Increased proliferation and viability of tumor cells, decreased survival of miceAmerican Journal of Pathology 169 (6) 2181-2198
Heme oxygenase-1 (HO-1), a cytoprotective enzyme, can be induced in tumors in response to anti-cancer therapies. We investigated the role of HO-1 in B16(F10), S91, and Sk-mel188 melanoma cells. Overexpression of HO-1 after transduction with adenoviral vectors increased cell proliferation, resistance to oxidative stress generated by H2O2, and angiogenic potential as determined by induction of endothelial cell divisions. Likewise, cells stably transfected with HO-1 cDNA (B16-HO-1) showed higher proliferation, stress resistance, and angiogenic activity than the wild-type line (B16-WT).
Mennesson, E ; Erbacher, P ; Piller, W ; Kieda, C ; Midoux, P ; Pichon C (2005)
Transfection efficiency and uptake process of polyplexes in human lung endothelial cells : a comparative study in non-polarized and polarized cellsJournal of Gene Medicine 7 (6) 729-738
Background Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (TM) (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells. Methods After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles.
Transfection efficiency and uptake process of polyplexes in human lung endothetial cells : A comparative study in non-polarized and polarized cellsJournal of Gene Medicine 7 (6) 729-738
Background : Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (TM) (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells.
Polycomb and trithorax group (PcG and trxG) proteins maintain silent and active transcriptional states, respectively, throughout development(1). In Drosophila, PcG and trxG proteins associate with DNA regions named Polycomb and trithorax response elements (PRE and TRE), but the mechanisms of recruitment are unknown. We previously characterized a minimal element from the regulatory region of the Abdominal-B gene, termed Ab-Fab. Ab-Fab contains a PRE and a TRE and is able to maintain repressed or active chromatin states during development(2).
DSP1, a Drosophila HMG protein, is involved in spatiotemporal expression of the homoeotic gene Sex combs reducedBiology of The Cell 97 (10) 779-785
Background information. The Pc-G (Polycomb group) and trx-G (trithorax group) genes play a key role in the regulation of the homoeotic genes. The homoeotic gene Scr (Sex combs reduced) contained in the Antennapedia complex specifies segmental identity of the labial and prothoracic segments in Drosophila. Regulation of Scr requires the action of different enhancer elements spread over several kilobases. We previously identified an HMGB (high mobility group)-like protein DSP1 (dorsal switch protein 1), which works like a trx-G protein for the normal Scr expression. Results. In the present study, we attempted to characterize the regulatory sequences involved in the maintenance of the Scr activation by DSP1.
Tumor and host endothelial cell selective interactions and modulation by microenvironmental chemokines : Tumor-endothelial cell cross talk specificityIntegration/Interaction of Oncologic Growth 15 219-231 - Editor(s) : Meadows, GG ; Meadows, GG Krawczenko, A ; Kieda, C ; Dus, D (2005)
Interleukin (lL)-7 is a pleiotropic, non-redundant cytokine necessary for the development of B and T lymphocytes, in particular ? d T cell receptor-positive cell differentiation. The cytokine can function as a cofactor during myelopoiesis and the generation of cytotoxic T cells and natural killer cells, can activate monocytes/macrophages, and support the survival of mature T cells.
Human NK cells lyse organ-specific endothelial cells : Analysis of adhesion and cytotoxic mechanismsJournal of Immunology 174 (9) 5573-5582
Human organ-specific microvascular endothelial cells (ECs) were established and used in the present study to investigate their susceptibility to natural killer cell line (NKL)-induced lysis. Our data indicate that although IL-2-stimulated NKL (NKL2) cells adhered to the human peripheral (HPLNEC.B3), mesenteric lymph node (HNILNEQ, brain (HBrMEC), and lung (HLMEC) and skin (HSkMEC.2) ECs, they significantly killed these cells quite differently. A more pronounced lysis of OSECs was also observed when IL-2-stimulated, purified peripheral blood NK cells were used as effector cells. In line with the correlation observed between adhesion pattern and the susceptibility to NKL2-mediated killing, we demonstrated using different chelators that the necessary adhesion step was governed by an Mg2+-dependent, but Ca2+-independent, mechanism as opposed to the subsequent Ca2+- dependent killing.
Numerous adhesion molecules have been described, and the molecular mechanisms of lymphocyte trafficking across the endothelium is starting to be elucidated. Identification of the molecules involved in the organoselectivity of this process would help in the targeting of drug therapy to specific tissues. Adhesion-regulating molecule-1 (ARM-1) is an adhesion-regulating molecule previously identified on T cells. It does not belong to any known families of adhesion molecules. In this study, we show the presence of ARM-1 in endothelial cells, the adhesion partners of lymphocytes.
A comparison of the ability of DMXAA and xanthenone analogues to activate NF-kappa B in murine and human cell linesOncology Research 15 (7-8) 351-364
DMXAA (5,6-dimethylxanthenone-4-acetic acid), the most potent of a series of xanthenone (XAA) analogues developed in this laboratory, is currently undergoing combination clinical trials as an antivascular agent for cancer treatment. XAAs have a complex mode of action, and in vitro assays that are predictive of in vivo antitumor activity have been difficult to develop. In this study, we have utilized a series including XAA, DMXAA, and mono-substituted XAA derivatives to determine firstly whether in vitro NF-kappa B activation of mouse cell lines predicts for the in vivo antitumor potential of this class of agents, and secondly whether the relative activity of these analogues is similar in murine and human cell lines.
Pharmacokinetics of 1-nitrosomelatonin and detection by EPR using iron dithiocarbamate complex in miceBiochemical Journal 387 473-478 Part 2
The N-nitroso-derivative of tnelatonin, NOM (1-nitrosomelatonin), which has been demonstrated to be a NOcenter dot [oxidonitrogen(center dot)] donor in buffered solutions, is a new potential drug particularly in neurological diseases. The advantage of NOM, a very lipophilic drug. is its ability to release both melatonin and NOcenter dot, an easily diffusible free radical. In order to evaluate the distribution and the pharmacokinetics of NOM, [O-methyl-H-3]NOM was administered to and followed in mice. A complementary method for monitoring NOM, EPR, was performed in vitro and ex vivo with (MGD)(2)-Fe2+ (iron-N-methyl-D-glucamine dithiocarbamate) complex as a spin trap.
Lewandowicz-Uszynska A., Paprocka M., Kieda, C. and Dus D. (2004)
Efficiency of lymphocytes adhesion to endothelial cells of distinct tissue origin from children with asthmaPolskiego Towarzystwa Lekarskiego (2004) 16 (92) 104-107
Lymphocytes extravasation is determined by their adhesion to the endothelial cells. The process goes along with high degree selectivity and is limited to lymph nodes and lymphatic tissue. It comes to uncontrolled extravasation to the tissues of involved organs in diseases with allergic origin as well in inflammation. Most information concerning adhesive interactions comes from researches dealing with normal lymphocytes. However, this phenomenon plays also crucial role in inflammation, metastasis and other pathologies. The aim of the work was to search for the possible distinct efficacy of adhesive interactions between peripheral blood lymphocytes from children with asthma and endothelial cells isolated from lung, skin and intestine. Isolated peripheral blood leukocytes were overlayered into endothelial cell monolayer. After washing the unbound cells, adhering lymphocytes were collected with endothelial cells. For quantifying the percentage of the particular cell populations cytofluorometric method was applied. The results are presented as a number of adhering lymphocytes per one endothelial cell. It has been shown that lymphocytes from asthmatic children have significantly greater adhesive potential towards endothelial cell lines from lungs and skin as compared with adhesion to endothelium of intestine origin. However, their B lymphocytes subpopulation demonstrated significantly higher percentage of cells adhering to all endothelial cell lines tested, as compared with B lymphocytes from normal controls. There were not statistically different adhesion efficiencies of T lymphocytes and NK cells. These findings indicate that local, tissue specific adhesive leukocyte-endothelial interactions may be of some importance in pathogenesis of allergic diseases.
Martin, D ; Daulny, A ; Decoville, M ; Locker, D (2003)
Mutagenesis analysis of the interaction between the dorsal Rel homology domain and HMG boxes of DSP1 proteinJournal of Biochemistry 134 (4) 583-589
DSP1 is an HMG-like protein of Drosophila melanogaster consisting of 386 amino acids with two HMG domains at the C-terminal end. It was shown to interact with Dorsal protein through the HMG domains and to enhance its DNA binding. Each HMG domain consists of approximately 80 amino acid residues, forming three alpha helices folded into an L-shaped structure. We have compared the interaction of various truncated and mutated forms of DSP1 with the dorsal Rel homology domain (RHD). In particular, we have mutated the conserved tryptophan residue 212 or 302 in A or B boxes or the lysine-rich region ((KKRK256)-K-253) of the A/B linker. Analysis by circular dichroism revealed that the protein tertiary structure is affected in these mutants. However, these mutations do not abolish the DSP1 binding to Dorsal, except if the two HMG boxes are altered, i.e., in a double mutant or in mutant isolated domain. Finally, studies on the enhancement of Dorsal DNA binding by DSP1 revealed that the DNA affinity is maximum in the presence of wild-type DSP1, is dramatically reduced when box A is altered, and is completely abolished when box B is altered.
DSP1 is an HIVIG-box protein which has been implicated in the regulation of homeotic genes in Drosophila melanogaster. Here we report that DSP1 is also involved in the regulation of the kni gap gene. Analysis of the phenotype of a null mutation of dsp1 (dspl(1)) reveals that the absence of maternal DSP1 results in A4 segmentation defects that are correlated with a diminution of the kni expression domain. Genetic interaction studies demonstrate that a bcd mutation enhances the A4 defect of dsp1(1). We present in vitro and in vivo evidences for a direct interaction between DSP1 and Bicoid, mediated by the BCD homeodomain and the HMG box of DSP1. Finally, we show by immunoprecipitation of cross-linked chromatin the association of DSP1 with the kni-regulating region and discuss the potential mechanism of DSP1-mediated activation of kni. (C) 2003 Wiley-Liss, Inc.
Drosophila DSP1 and rat HMGB1 have equivalent DNA binding properties and share a similar secondary foldJournal of Biochemistry 133 (4) 533-539
The protein DSP1 belongs to the group of HMG-box proteins, which share the common structural feature of the HMG-box. This approximately 80 amino acid long motif binds DNA via the minor groove. DSP1 was discovered as a,transcriptional co-repressor of Dorsal in Drosophila melanogaster and then was shown to participate to the remodeling of chromatin. By means of sequence alignment and gene organization, DSP1 was classified as the fly homologue of the vertebrate proteins HMGB1/2. DSP1 contains two HMG boxes flanked by two glutamine-rich domains at the N-terminus. In addition, the HMG domain of DSP1 displays two differences in its primary sequence as compared to the vertebrate HMGB1 : a shorter acidic tail and a linker between the two boxes longer by 6 amino acids. By comparing several functional parameters of DSP1 with those of HMGB1, the present study establishes the functional equivalence of both proteins in terms of DNA recognition. The major structural difference between the two proteins, the glutamine-rich N-terminal tail of DSP1, which does not exist in HMGB1, did not interfere with any of the studied DNA-binding properties of the proteins.
Interleukin-7 (IL-7) is a pleiotropic, non-redundant cytokine crucial for development of B and T lymphocytes. The cellular response to IL-7 is triggered by binding of the cytokine to its receptor, IL-7R. Until now the expression of the receptor was evidenced only in lymphoid and myeloid cell lineages. The receptor consists of two chains : IL-7 specific a chain (CD127) and the common 7. chain (CD132) which is a component of several other cytokine receptors : IL-2, IL-4, IL-9 and IL-15. The former observation that exogenous IL-7 is biologically active towards murine endothelial cell lines from secondary lymphoid organs was the starting point of our studies. This observation has prompted us to search for the presence of IL-7 receptor in human microvascular endothelial cells. We used in our studies a set of human endothelial cell lines established from various organs.
Normal and transformed cells home into tissues from the circulation in a very selective way thanks to highly complex molecular mechanisms that govern cell-to-cell interactions and drive the homing of circulating cells so that it is achieved properly. Because this is characterized by a resulting high selectivity, it constitutes a template for targeted drug-, gene- or cell-therapy strategies. Designing a mimetic-based therapy requires the identification of the responsible selective molecules, but also their mechanisms of action and interactions with their ligands together with their biological modulation and regulation. This homing/invasion event is decisive at the level of the endothelium that lines the vessel walls.
Therapeutic implications of constitutive activity of receptors : the example of the histamine H3 receptorJ Neural Transm (2003) Suppl:1-16 Ilien B., Franchet C., Bernard P., Morisset S., Weill C. Bourguignon J., Hibert M. and Galzi J.L. (2003)
Fluorescence resonance energy transfer to probe human muscarinic M1 receptor structure and drug binding propertiesJ. Neurochem. (2003) 85:768-778 Gbahou F, Rouleau A, Morisset S, Parmentier R, Crochet S, Lin JS, Ligneau X, Tardivel-Lacombe J, Stark H, Schunack W, Ganellin CR, Schwartz JC and Arrang JM. (2003)
Kieda, C ; Paprocka, M ; Krawczenko, A ; Zalecki, P ; Dupuis, P ; Monsigny, M ; Radzikowski, C ; Dus, D (2002)
Vascular endothelial cells recognize blood-borne circulating cells and allow them to extravasate in a tissue-specific manner. Because this property determines the selectivity of lymphocyte homing, it is fundamental in physiological as well as pathological processes (inflammation, autoimmune diseases, metastasis). As a tool to assess the molecular basis of endothelium selectivity, microvascular endothelial cell lines of distinct tissue origin were established. Endothelial cells, isolated from lymphoid tissues (lymph nodes and appendix) and from nonlymphoid immune sites-intestine, lung, and skin-were immortalized in vitro. Their general endothelial characteristics, such as the presence of von Willebrand factor (wWf), angiotensin-converting enzyme (ACE), VE-cadherin, and the intracellular E-selectin, were preserved.
Induction of endothelial cell apoptosis by the antivascular agent 5,6-dimethylxanthenone-4-acetic acidBritish Journal of Cancer 86 (12) 1937-1942
5,6-Dimethylxanthenone-4-acetic acid, synthesised in this laboratory, reduces tumour blood flow, both in mice and in patients on Phase I trial. We used TUNEL (TdT-mediated dUTP nick end labelling) assays to investigate whether apoptosis induction was involved in its antivascular effect. 5,6-Dimethylxanthenone-4-acetic acid induced dose-dependent apoptosis in vitro in HECPP murine endothelial cells in the absence of up-regulation of mRNA for tumour necrosis factor. Selective apoptosis of endothelial cells was detected in vivo in sections of Colon 38 tumours in mice within 30 min of administration of 5,6-Dimethylxanthenone-4-acetic acid (25 mg kg(-1)). TUNEL staining intensified with time and after 3 h, necrosis of adjacent tumour tissue was observed.
Histamine H3-receptor-mediated [35S]GTP gamma[S] binding : evidence for constitutive activity of the recombinant and native rat and human H3 receptorsBr J Pharmacol. (2002) 135, 383-392 Morisset S., Pilon C., Tardivel-Lacombe J., Weinstein D., Rostene W., Betancur C., Sokoloff P., Schwartz J.C. and Arrang J.M. (2002)
Acute and chronic effects of methamphetamine on tele-methylhistamine levels in mouse brain : selective involvement of the D(2) and not D(3) receptorJ. Pharmacol. Exp. Ther. (2002) 300, 621-628 Chotard C., Ouimet T., Morisset S., Sahm U., Schwartz J.C. and Tottier S. (2002)
Effects of histamine H3 receptor agonist and antagonist on histamine co-transmitter expression in rat brainJ. Neural. Transm. (2002) 109, 293-306
Decoville, M ; Giacomello, E ; Leng, M ; Locker, D (2001)
The Drosophila dsp1 gene, which encodes an HMG-like protein, was originally identified in a screen for corepressors of Dorsal. Here we report that loss of dsp1 function causes homeotic transformations resembling those associated with loss of function in the homeotic genes Sex combs,reduced (Scr), Ultrabithorax (Ubx), and Abdominal-B. The expression pattern of Scr is altered in dsp1 mutant imaginal discs, indicating that dsp1 is required for normal expression of this gene. Genetic interaction studies reveal that a null allele of dsp1 enhances trithorax-group gene (trx-G) mutations and partially suppresses Polycomb-group gene (Pc-G) mutations. On the contrary, overexpression of dsp1 induces an enhancement of the transformation of wings into halteres and of the extra sex comb phenotype of Pc. In addition, dsp1 male mutants exhibit a mild transformation of A4 into A5. Comparison of the chromatin structure at the Mcp locus in wild-type and dsp1 mutant embryos reveals that the 300-bp DNase I hypersensitive region is absent in a dsp1 mutant context. We propose that DSP1 protein is a chromatin remodeling factor, acting as a trx-G or a Pc-G protein depending on the considered function.
KDR promoter can transcriptionally target cytosine deaminase suicide gene to cancer cells of nonendothelial originAnticancer Research 21 (5) 3471-3475
The KDR/flk-1 gene promoter is considered to be endothelial cell-specific. We show its activity in two cancer cell lines of non-endothelial origin : in murine L1 sarcoma and OVP-10 human ovarian carcinoma cell lines. KDR promoter-driven cytosine deaminase gene can be efficiently expressed in these cells leading to sensitization to 5-fluorocytosine, as demonstrated both in vitro and in vivo. Our results indicated that KDR promoter activity is not endothelial cell-exclusive and that this promoter can also be used to obtain specific expression of therapeutic genes in certain cancer cells.
An analytical procedure has been developed for the analysis of intracellular didanosine triphosphate (ddATP). An electrospray ionization tandem mass spectrometer (ESI-MS) was interfaced to liquid chromatography (LC) using a mobile phase CH3OH/H2O (25/75) containing 1% formic acid for the analysis of the 5’-triphosphate metabolite of the antiviral didanosine. In this procedure. ddATP was extracted from CEM-T4 cells, isolated using an exchange anion solid phase extraction procedure, enzymatically dephosphorylated and then analyzed by LC-MS/MS within a I min run time.
Analysis of anti-HIV nucleoside inhibitors by capillary electrophoresis-electrospray ionization mass spectrometryNucleosides Nucleotides & Nucleic Acids 20 (4-7) 375-381
A method employing capillary electrophoresis (CE) with tandem mass spectrometry (MS) has been developed for the simultaneous determination, on one hand, of zidovudine (AZT) with stavudine (d4T), and on the other hand, of lamivudine (3TC) with a didanosine metabolite (ddA), four potent human immunodeficiency virus reverse transcriptase (RT-HIV) inhibitors. The influence of several parameters (pH and ionic strength of volatile formic acid-ammonia buffer) as well as the influence of magnesium cation upon electroosmotic flow, electrophoretic mobility and peak efficiency has been studied. The limit of detection (LOD) by this method is 2.5 ppb for AZT and 20 ppb for d4T, 2 ppb for ddA and 5 ppb for 3TC, respectively. This paper illustrates the current importance in CE-ESI/MS/MS technique as a complementary or substituted method to measure levels (at ng/mL) of anti-HIV drugs alone or in combination.
Synthesis and biological evaluation of analogues of the tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP), an inhibitor of primitive haematopoietic cell proliferationJournal of Peptide Science 7 (5) 284-293
The tetrapeptide IV-Acetyl-Ser-Asp-Lys-Pro (AcSDKP), an inhibitor of hacmatopoletic stem cell proliferation, reduces in vivo and in vitro the damage to the stein cell compartment resulting from treatment with chemotherapeutic agents or ionizing radiations. In order to provide new molecules likely to improve the myeloprotection displayed by this tetrapeptide, we have prepared a set of analogues of AcSDKP. These compounds are derived from the parent peptide by substitution or modification of the N- or of the C-terminus, or substitution of side chains.
The histamine H3 receptor : gene organization, multiple isoforms, constitutive activity and molecular pharmacologyDans ‘’Histamine Research in the New Millenium’’. Edité par T. Watanabe, H. Timmermann et K. Yanai. Elsevier, Amsterdam, (2001), 9-21
Decoville, M ; Giraud-Panis, MJ ; Mosrin-Huaman, C ; Leng, M ; Locker, D (2000)
Formation of the dorsoventral axis in Drosophila melanogaster is mediated through control of the expression of several genes by the morphogen Dorsal. In the ventral part of the embryo Dorsal activates twist and represses ren amongst others, Recently, several proteins have been shown to assist Dorsal in the repression of ten, one of which is DSP1, a HMG box protein that was isolated as a putative co-repressor of Dorsal. In this report we used a DSP1 null mutant to ascertain in vivo the involvement of DSP1 in Dorsal-mediated repression of ten but not in the activation of twist.