„ABC“ o ABC transportérech lAktivní a pasivní transport živin lABC transportéry – historie, filogeneze, struktura, evoluce, SNP lFyziologická role lKmenové buňky lNádorové bujení lMultiléková rezistence lNeurodegenerativní choroby lDetekce ABC transportérů lAktuální projekty Jiřina Procházková Kotlářská 2, Brno jipro@sci.muni.cz Transport přes membránu facilitated_diffusion active_transport passive_diffusion http://www.solvo.hu Nutrients can be transported through the cell membrane in 3 ways (Figure 2): Passive diffusion consists of the transport of water and water-soluble substances and small lipids through the lipid bilayer with a concentration gradient. In the case of a facilitated diffusion, transporter proteins create a water-filled pore through which ions and small hydrophilic molecules can pass by diffusion. Fructose, riboflavin and vitamin B12 (in combination with intrinsic factor) are among the substances absorbed by facilitated diffusion. During active transport, transmembrane proteins, called transporters, use the energy of ATP to force ions or small molecules through the membrane against their concentration gradient. These active transport mechanisms have been identified for intestinal absorption of many substances including glucose, galactose, amino acids, calcium, iron, folic acid, ascorbic acid, thiamin and bile acids. Transport proteins, embedded in lipid membranes, facilitate the import of nutrients into cells or the release of toxic products into the surrounding medium. The most important family of membrane transport proteins are the ATP-binding cassette (ABC) transporters. These ABC proteins play a central role in all living cells in the nutrient uptake, protein, drug and antibiotic excretion, osmoregulation, antigen presentation, signal transduction and other important cellular functions. ABC transportéry lATP-binding cassette l = NBD doména l lZa spotřeby ATP pumpují toxické látky/metabolické produkty VEN z buňky (výjimka CFTR) l lFyziologická funkce – sekrece látek produkovaných buňkou + obrana proti xenobiotikám l l http://publications.nigms.nih.gov/medbydesign/images/ch1_mdr.jpg mimobuněčný prostor cytosol substrát membrána Historie l50. léta – jak jednobuněčný plankton přežije ve slané vodě? l60. léta – iontové pumpy závislé na ATP l70. léta – transportéry větších molekul závislé na ATP l80. léta – nadměrná exprese ABC transportérů způsobuje rezistenci nádorových buněk vůči chemoterapii lAž dosud – hledání látek, které inhibují funkci nebo expresi ABC transportérů l http://oceanicdefense.blogspot.com/2009_07_01_archive.html http://www.dailymail.co.uk/health/article-1294676/ Filogenetický strom ABC transportérů 100 proteinů celkem 48 proteinů u lidí 7 rodin (A-G) podle sekvenční homologie http://www.nature.com/onc/journal/v22/n47/fig_tab/1206938f1.html#figure-title Struktura NBD … nucleotid (ATP)-vázající doména TMD … transmembránová doména R … regulační doména Full transportéry Half transportéry ABC transporters consist mainly of transmembrane domains (TMDs) that form the “pore” and have affinity for particular substrates, and cytosolic domains binding the ATP nucleotide (nucleotide-binding domain, NBD). The domains are connected by cytosolic and extracellular loops, the later being often glycosylated. The cytosolic regions of the TMDs are thought to coordinate ATP coupling with substrate binding and translocation. The NBDs contain the characteristic Walker A, B motifs and the “signature motif”, participating to create the “pocket” for ATP binding. All those sequences are either involved directly in ATP binding and hydrolysis or they facilitate interfaces in the assembled transporter. All the conserved domains form together a hydrophilic pore closed on the internal cytosolic side and thus creating and aqueous compartment inside the hydrophobic membrane bilayer (for more details see [9]). Generally, we can classify ABC transporters either according to their sequence homology to the families (ABC A – G) or structurally to half- and full-transporters. The full transporters (typical example is ABC B1 protein) have two TMDs and NBDs. As the name indicates the half-transporters (represented by ABC G2) consist of only one TMD and NBD (reviewed in [11]). For the proper assembly of transporting “pores” the typical number of involved TMD is twelve. The half transporters have therefore to homo- or heterodimerize. Evoluce lNekovalentně spojený transmembránový protein a ATPáza (bakterie) lNejprve vznikly half transportéry lFull transportéry vznikly jejich duplikací později l lABC B5 lVíce sestřihových variant – dlouhá a krátká forma lKrátká forma vznikla delecí části TMD z dlouhé formy lZ původního half transportéru vznikla dlouhá forma a z ní krátká forma l lABC G2 vs ABC B1 lABC B1: N konec – TMD1 – NBD1 – TMD2 – NBD2 – C konec lABC G2: N konec – NBD – TMD – C konec –TMD ABC G2 podobnější kvasinkovým ABC transportérům –Spojovací oblast je velmi homologická http://www.theologylived.com/creation-and-human-origins/ Výskyt ABC transportérů lŠipky označují výskyt a směr transportu http://www.bio.davidson.edu/courses/Immunology/Students/spring2000/buxton/a Polymorfismy lPolymorfismus (SNP) – mutace, kterou nese více než 1 % populace lABC transportéry jsou velmi polymorfické lNapř. ABC B1 – více než 50 SNP, často synonymní, často více SNP ve vazbě l! i synonymní SNP může změnit afinitu substrátu! lZkoumá se, zda právě SNP mohou ovlivnit výsledek léčby lJediná pozitivní korelace – lnádor ovária ABC B1 G2677T/A l– nižší pravděpodobnost relapsu http://bio.mq.edu.au Knock-outs lDelece obou alel genu u myší lTestováno 32 z 48 savčích ABC lJen 5 z nich je embryonálně nebo postnatálně letálních –ABC B7 – kolaps respiračních procesů –ABC E5 – selhání recyklace ribosomů –ABC A3 – respirační selhání (nedostatek surfaktantu) –ABC A12 – defekty plic a kůže –ABC C9 – srdeční selhání lJsou evolučně konzervované, ale flexibilní, a tak se mohou vzájemně zastoupit a také exportovat nové člověkem vyrobené látky l By now were knockouts of 32 from the 48 mammalian ABC transporters produced and only five of them resulted in embryonic (ABC B7 and ABC E5) or early postnatal (ABC A3, ABC A12 ABC C9) lethality. ABC B7 knockout embryos die due to collapse of mitochondrial respiration process [25]. Embryonic lethality was described also in ABC E5 mice, which is understandable as this transporter plays an important role in ribosome binding and recycling. ABC A3 newborn knockout mice die due to lack of surfactant in lung and following respiratory failure [26]. ABC A12 knockouts are also lethal because of severe lung and skin defects [27]. Mice lacking ABC C9/Sur2 regulating sulfonylurea metabolism die of cardiac failure soon after birth [28]. The current status of scientific progress in knockouts could be monitored at website Mouse genome informatics (http://www.informatics.jax.org). Fyziologická role ABC transportérů Substráty Kanál Fyziologické substráty MDR1 Konjugáty estrogenu, endorfin, glutamát, beta-amyloid, steroidy, PAF MRP1 Konjugáty glutationu a glukuronidu s leukotrienem C4, bilirubinem, žlučovými solemi BCRP Vitamíny (riboflavin, biotin), porfyriny, konjugáty estrogenu Substráty ABC transportérů - výživa Živina Membránový Transportér Efekty Flavonoidy (quercetin) jejich glukosidy a glukuronidy – ovoce, zelenina Ø Multidrug Resistant Protein 1 (MRP1) Ø Multidrug Resistant Protein 2 (MRP 2) Ø Breast Cancer Resistance Protein BCRP (MXR) Ø P-glycoprotein (MDR1) Substrát Extrakty z hořkého melounu (1-monopalmitin), grapefruitu (bergamottin and quercetin), sóji Ø P-glycoprotein (MDR1) Inhibice Extrakt z hroznových jader Ø P-glycoprotein (MDR1) Inhibice Steroly (e.g. Cholesterol) Ø ABCA1, ABCG1, ABCG5 and ABCG8 Substrát Sezamové semínko (lsophosphatidylcholine, linoleoyl) Ø Některé transportéry ve střevu Inhibice Mono-, di-, and triglutamáty folátů Ø Breast Cancer Resistance Protein BCRP (MXR) Ø Multidrug Resistant Protein 1 (MRP1) Substrát Rostlinné výtažky Curcumin, ginsenosidy, piperin, katechiny ze zeleného čaje, silymarin, hyperforin z kávy Ø P-glycoprotein (MDR1) Substrát/inhibitor http://www.bio.davidson.edu/courses/Immunology/Students/spring2000/buxton/a Importance of ABC transporters in nutrition {1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19} Some studies suggest that dietary constituents regulate the expression of ABC Transporters. Changes in ABC Transporter expression may represent an important physiological response to foods containing toxins and an important component of the acute phase immune response. It has been shown that dietary phytochemicals have inhibitory effects on P-glycoprotein (MDR1) and potencies to cause drug-food interactions. Moreover, the observations in a number of studies demonstrated important roles of membrane transporters, i.e. Multidrug Resistant Protein 1 (MRP1), Multidrug Resistant Protein 2 (MRP 2), Breast Cancer Resistance Protein BCRP (MXR) and P-glycoprotein (MDR1) in the cellular accumulation, transport and potential effects of many nutrients. Since some of these nutrients are found in fruits and vegetables, their effect on MRP1, MRP2, MXR and MDR1 may be a mechanism relevant to carcinogenesis and the observed lowered cancer risk in humans with higher dietary intake of fruits and vegetables (Table 1). Játra http://solvo.jp/Solvo%20Solutions/img/lumen.jpg krev žluč Distribuce živin a metabolitů, zbavování se toxických látek Ledviny, střevo… Typically,ABC proteins are relatively specific for a particular set of substrates (except ABCB1). Substrates can be amino acids,sugars,inorganic ions,peptides,proteins,lipids and various organic and inorganic compounds. Various family members are attractive candidates for Flippases that translocate lipids from the inner to the outer leaflet of the plasma membrane. The canalicular membrane in the hepatocytes contains several ATP-dependent export pumps: MDR1 (Multidrug-Resistance-1 P-Glycoprotein,also known as ABCB1),the phospholipid transporter MDR3 (ABCB4),the canalicular MRP2 (Multispecific-Organic-Anion Transporter or cMOAT),and the canalicular BSEP (Bile Salt Export Pump or SPGP). In addition,the canalicular membrane contains several ATP-independent transport systems,including ClCn (Chloride Channel),a chloride-bicarbonate AE2 (Anion Exchanger isoform-2) for secretion of bicarbonate,and a Gsh (Glutathione) transporter (Ref.3). The liver-specific ABC transporter MDR3 specifically transports phosphatidylcholine across the canalicular membrane during bile formation. By contrast,MDR1 expels a variety of short-chain lipids and amphiphilic drugs from the cell. It mediates outward transport of natural lipids such as PAF (Platelet-Activating Factor),phosphatidylserine,sphingomyelin and glucosylceramide. The glutathione-dependent multidrug transporter MRP1 (Multispecific Organic Anion Transporter),transport short-chain phosphatidylcholine,phosphatidylserine,sphingomyelin and GlcCer analogs,and helps to maintain the outward orientation of natural choline phospholipids in the plasma membrane (Ref.4). ABCA1 controls the extrusion of membrane phospholipids (mostly hosphatidylcholine) and cholesterol to cell surface-bound apolipoproteins. The ABCA1-dependent control on the lipid content of the membrane dramatically influences the plasticity and fluidity of the membrane itself and,as a result,affects the lateral mobility of membrane proteins and/or their association with membrane domains of special lipid composition. There are two sinusoidal systems for bile-salt uptake in hepatocytes-NTCP (Sodium-Taurocholate Cotransporter) and a sodium-independent OATP (Organic Anion-Transporting Polypeptides). Sodium-dependent uptake of bile salts through the NTCP is driven by an inwardly directed sodium gradient generated by Na+/K+-ATPase and the membrane potential generated in part by a KCn (Potassium Channel). In addition,the basolateral membrane contains a Na+-H+ (Sodium-Hydrogen Exchanger) and a Na+-HCO3- (Sodium-Bicarbonate) symporter. In addition,Na+/K+-ATPase,together with a KCn,helps to generate a transmembrane electrical potential (Ref.5). These chemical and electrical potentials are used for the maintenance of intracellular ion and pH homeostasis. They provide the driving forces for proton extrusion by a mechanism of Na+-H+ exchange and for HCO3- entry,as well as for the electrogenic Na+-dependent uptake of conjugated bile salts (or bile acids). In contrast to conjugated bile salts,the unconjugated bile salt cholate,the organic anion sulfobromophthalein,and numerous other lipophilic albumin-bound compounds are transported from plasma into hepatocytes by Na+-independent transport systems,including the OATP. ABC transporters are probably the most common as well as the most wide-spread active transport systems. They have been widely implicated in disease processes,such as Stargardt macular degeneration,cholestasis of pregnancy,cystic fibrosis,and confer resistance of bacterial and eukaryotic cells to antibiotics and numerous drugs applied for the treatment of infectious diseases,cancer,malaria,AIDS,etc (Ref.6 & 7). Krevně mozková bariéra http://www.medscape.org/viewarticle/711587 Ochrana mozku před toxickými látkami z krve bbb Exprese a lokalizace lExprese je vysoká v „bariérových“ tkáních, v epitelech vystavených vnějšímu prostředí, v tkáních které redistribuují metabolity l!Specifická apikální/bazolaterální lokalizace ! Syntéza antigenu 0 http://www.uoguelph.ca/~cwhitfie/images/abc_transporter.gif Antigeny - lipopolysacharidy Lipidové jádro Polysacharid Lipid fukóza N-acetyl galaktóza glukosamin Lipopolysaccharides (LPS) are unique and abundant glycolipids found in the outer leaflet of the outer membrane in most Gram-negative bacteria. In Escherichia coli, there are approximately 106 LPS molecules per cell and these constitute 75% of outer membrane surface area. In the Enterobacteriaceae (e.g. E. coli, Salmonella), LPS is comprised of three regions: the lipid anchor (lipid A), a core oligosaccharide, and the O-antigenic polysaccharide. The O antigen is a variable chain length repeat-unit polysaccharide that provides protection from complement-mediated killing. Structural organization of E. coli LPSs O antigens are assembled by pathways that resemble those involved in capsule biosynthesis. The systems are differentiated by the addition of the newly synthesized O antigen to the lipid A-core, and by distinct pathways for translocation of the completed products across the periplasm and outer membrane to the cell surface (Raetz and Whitfield, 2002). The Wzy-dependent O-antigen biosynthesis system is very similar to that forming group 1 capsules.The assembly of other O antigens and group 2 capsules involves an ATP-binding cassette (ABC)-transporter-dependent pathway. Members of the large ABC-transporter superfamily are widely distributed in both prokaryotes and eukaryotes, where they participate in nutrient uptake and membrane trafficking of proteins, drugs, lipids and polysaccharides. Their transport activity is energized by ATP hydrolysis. ABC transporters are comprised of transmembrane domains and nucleotide-binding domains. For O antigens, the corresponding domains are formed by the Wzm and Wzt polypeptides, respectively. Elucidating the structure and mechanism of the ABC-transporter-dependent pathway for O-antigen biosynthesis represents a major research initiative in the lab. O-antigen assembly in an ABC-transporter-dependent pathway The biosynthesis steps are: 1-2 glycosyltransferase enzymes sequentially add the glycose residues to a lipid carrier (undecaprenol-P) to form the polymer. 3-4 the newly synthesized O antigen is exported across the inner membrane by the ABC transporter (comprising Wzm and Wzt) beginning with the non-reducing terminus. However, it is not yet known which end of the newly formed polymer is exported first, so an export could be initiated at the reducing terminus. 5 the O antigen is ligated to lipid A-core by a reaction involving WaaL, the putative “ligase”. The completed LPS molecule is then translocated to the cell surface by an unknown process. Kmenové buňky http://community.livinglakecountry.com/blogs/eagles_eye/Stem%20cells%20diagram%5B1%5D.jpg lV průběhu l diferenciace l klesá l exprese l ABC l transportérů Výjimka: zvyšování exprese v bariérových a metabolických tkáních Detekce ABC transp. „Dye exclussion assays“ MDR1 - JC1, rhodamin 123 x Cyclosporin D, reversin MRP1 - Calcein AM x MK571, NSAID BCRP - Hoechst 33342 x fumitremorgin C - Bodipy-prazosin - Pheophorbid A Obecné inhibitory transportu – verapamil, cyclosporin A Imunodetekce qRT-PCR Inhibitory Kompetitivní substráty Inhibice funkce transportéru „SP“ buňky http://www.bu.edu/cms/www.bumc.bu.edu/leukemia-lymphoma-laboratory/files/Images/sidepop.jpg Hoechst 33342 - substrát BCRP, MDR1 - inhibitor verapamil lBuňky C2C12 - myoblasty Důkaz „kmenovosti“ Hirschmann-Jax, C. et al. (2004) Proc. Natl. Acad. Sci. USA 101, 14228-14233 A: V linii se vyskytují SP buňky a majoritní populace B: SP buňky z neuroblastomové buněčné linie produkují SP a ne-SP C: Buňky majoritní populace zůstávají jen majoritními Linie SK-N-SH SP buňky Majoritní populace Patologický výskyt Dědičné choroby 16 ABC genů bylo asociováno s dědičnými poruchami Tangierova choroba (ABC A1) – poruchy sekrece cholesterolu a fosfolipidů (nadměrná hladina v buňkách, narušena homeostáza) Dubin Johnson syndrom (ABC C2) – neschopnost jater sekretovat konjugovaný bilirubin do žluče Pseudoxanthoma elasticum (ABC C6) – mineralizace a fragmentace elastinových vláken, problém s vitamínem K Cystická fibróza (ABC C7) – poruchy sekrece působků pankreatu a dalších exokrinních žláz Asociace ABC trans- portérů a chorob CNS V mozku je HODNĚ ABC Wolf et al. 2012. Frontiers in psychiatry.3(54): 1-14 Picture ABC A1 a Alzheimerova choroba • Amyloid precursor protein (APP) – Amyloid β 40, 42 • AD pacienti – 100x zvýšená hladina Aβ plaků • ABC A1 v CNS – substrát cholesterol a fosfolipidipidy(ne Aβ) eflux z neuronů, astrocytů, microglií – lipidace ApoE • ABC A1 malfunkce – špatně lipidované ApoE částice – přímá interakce s Aβ – agregace a tvoření plaků • ABC A1 – exprese zvýšená pomocí LXR aktivátorů – zvýšená sekrece Aβ 40, 42 • myši – zvýšená exprese ABC A1 po LXR – zlepšení paměti x • AD pacienti – zvýšená hladina ABC A1 mRNA a proteinu (+ ApoE) – SNPs(?) 8 z 14 studií ukazuje spojení mezi polymorfismem a zvýšeným rizikem vzniku AD ThephysiologicalfunctionofAβ is stillunclear, but inhealthyindividualsAβ brainlevelsarelow,whereasinAD patients Aβ levelscanbeincreasedupto100-foldinthebrain, whereitaggregatestoplaques(Gravinaetal.,1995; Hardyand Selkoe,2002). ABCA1, alsoknownascholesteroleffluxregulatoryprotein,isa plasma membraneproteinthatishighlyexpressedintheadrenal glandanduterus.ABCA1hasalsobeendetectedintheCNSin neurons,astrocytes,microglia,andinepithelialcellsofthechoroid plexus(Figure1;Koldamovaetal.,2003;Kimetal.,2006; Fujiyoshi et al.,2007) In the periphery, ABCA1 mediates cholesterol and phospholipid efflux from cells for transfer onto lipid-free or lipid-poor apoprotein A-1 (ApoA-1) particles, the main component of high density lipoproteins (HDL; Lawn et al., 1999). In the CNS, ABCA1 mediates cholesterol and phospholipid efflux from astrocytes and microglia thereby facilitating lipidation of ApoE, which is the major apoprotein in the brain (Panzen- boeck et al., 2002; Hirsch-Reinshagen et al., 2004). Consequently, ABCA1-deficiency results in poorly lipidated ApoE particles, but also in a thus far unexplained decrease in ApoE brain levels (Hirsch-Reinshagen et al., 2004;Wahrle et al., 2004). important factor for brain cholesterol homeostasis is ApoE, which supplies brain cells with cholesterol and lipids for development, repair, and nerve growth. IntheADfield,thisincreased riskisthoughttobedueto ApoE-ε4 directly interacting with Aβ, thereby favoring Aβ aggregationandplaqueformation(Holtzman et al.,2000). Inthisregard, Hirsch-Reinshagenetal.(2004) found that Abca1-deficiency inmiceleadstoreducedlipideffluxtoApoEand, for anunknownreason,alsoinadramaticdecreaseofApoEbrain levels.ThesamegroupalsoshowedintwohAPP-overexpressing AD mousemodels(Tg-SwDI/B;APP/PS1)that Abca1-deficiency reducesApoEbrainlevels,buthasnoeffectonAβ brainlevels (Hirsch-Reinshagenetal.,2005). Incontrast,twootherstudies demonstratethatalackof Abca1 in hAPP-overexpressingmice (APP23 andPDAPPmodels)decreasesbothApoElipidationand ApoEbrainlevels,andalsoincreasesbrainlevelsofinsolubleAβ40 and Aβ42 (Koldamovaetal.,2005a; Wahrleetal.,2005). From these observations,theauthorsconcludedthatpoorlylipidated ApoEhasahighaffinitytoAβ and promotesAβ fibrillogenesis. UsingmouseNeuro2Acells,theauthorsshowedthat upregulationofAbca1proteinlevelsbyvariousLXRactivators increasedsecretionofbothAβ40 andAβ42. Thiseffectwasreduced when Abca1expressionwasblockedwithRNAi(Fukumotoetal., 2002). Thisobservationisconsistentwithfind- ingsfromin vivo studies usingLXRagonistsinADmousemodels. Koldamovaetal.(2005b) showedthattreatmentof11-week-old APP23 micewithT0901317,a synthetic LXR ligand,increased Abca1 protein expression and significantly decreased brain levels of Aβ40 and Aβ42. Vanmierloetal.(2009) who also demonstrated that LXR activation improved memory function in aged APP/PS1 mice without reducingAβ deposition. ABCA1 does not directly transportAβ (Akanumaetal.,2008), Interestingly, low ApoE brain levels per se are associated with decreased Aβ deposition, while poorly lipidated ApoE has been discussed to have an amyloidogenic effect in vivo (Koldamovaetal.,2005a;Wahrleetal., 2005). Akram et al. found increased ABCA1 mRNA and protein expres- sion in hippocampal brain tissue from AD patients compared to age-matched control subjects. 8 ze 14 studies show there is a link between SNP of ABC A1 and increased risk of AD activating LXR also increases expression levels of ABCG1, another lipid transporter of the ABC superfamily, and ApoE. Regulace funkce ABC A1 FIG. 5. Oram, Heinecke Physiol Rev October 1, 2005 vol. 85 no. 4 1343-1372 Fosforylace oblasti: PEST – degradace PKA – optimální funkce PKCK2 – oslabení funkce Known phosphorylation sites in ABCA1. Approximate sights of phosphorylated threonines in the PEST sequence that mediate ABCA1 degradation by calpain (triangles), of serines phosphorylated by protein kinase A in the NBDs that are required for optimum lipid translocase activity (diamonds), and of threonines and serine phosphorylated by protein kinase CK2 that attenuate lipid transport and apolipoprotein binding acitivities (pentagons). There is growing evidence that ABCA1 activity is modulated by the interaction of a diverse group of proteins (Table 2). ABCA1 was coimmunoprecipitated with Cdc42 (270), a member of the RhoGTPase family that forms complexes with proteins that control the cytoskeletal architecture and vesicular transport (262). This interaction may cause the changes in cell morphology associated with under- or overexpressing ABCA1 (57, 270). Moreover, forced expression of Cdc42 was shown to enhance apoA-I-mediated cholesterol efflux, whereas expression of dominant negative Cdc42 impaired this efflux, consistent with a role of Cdc42 in modulating ABCA1 function (108, 197). Immunoprecipitation studies also identified Fas-associated death domain protein (FADD) as an ABCA1 interacting protein (30). Blocking this interaction impaired apolipoprotein-mediated phospholipid efflux. The biological significance of the FADD/ABCA1 association is unclear, as FADD is mainly involved in death receptor-induced apoptosis. These findings raise the possibility that ABCA1 has an antiapoptotic function, perhaps related to its ability to extrude lipophilic molecules from cells. It is likely that additional proteins will be discovered that interact with ABCA1 and modulate activity. The COOH-terminal region of ABCA1 contains a highly conserved VFVNFA motif that is required for activity (74). Mutations in this motif have no effect on ABCA1 trafficking but severely impair its apoA-I interactions and lipid efflux. Peptide competition experiments suggest that this motif promotes the interaction of ABCA1 with an unknown cellular protein that is a component of the active complex (74). lABC B1 exportuje Aβ (luminální část kapiláry) lABC B1a,b -/- myši – snížené odstraňování Aβ z mozku lDysfunkční ABC B1 na BBB – akumulace Aβ lAD pacienti – snížená hladina ABC B1 v cévách kolem Aβ plaků lAsociace mezi SNPs a rizikem vzniku AD nebyla nalezena lABC B1 exprese je zvýšena aktivací PXR – pomalejší l pokles ztráty mozkových funkcí (klinická studie) lAβ může regulovat (snižovat) expresi ABC B1 l ABC B1 a Alzheimerova choroba shapeimage_2 Odstraňování Ab Wolf et al. 2012. Frontiers in psychiatry.3(54): 1-14 MDR a rakovina lSelhání chemoterapie (nádorové b. jsou obvykle citlivější než „zdravé“ buňky) lDe novo/získaný fenotyp MDR (cytotoxic in vitro MTT-assay) lMechanizmy: –Změny v metabolických drahách, které se podílejí na detoxifikaci látek –Změny v reakci organizmu na poškození DNA –Změny v aktivitě topoizomerázy II –Změny v drahách regulujících apoptózu –Zvýšená produkce ABC transportérů lNádorová b. se vymyká vlivu svého prostředí (obsahujícího chemoterapeutikum), získává růstovou výhodu (multidrug rezistance, multiléková rezistence) The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo II alpha gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Beck et al. Cytotechnology. 1993;11(2):115-9. DNA topoisomerase II (topoII) is a nuclear enzyme that resolves DNA supercoiling and catenation by the breakage, strand-passage, and rejoining of double-stranded DNA (Champoux, 1990 ), thereby relieving topological constraints that occur during essential cellular processes such as DNA replication, transcription, cell division, and repair (Nelson et al., 1986 ; Brill et al., 1987 ). TopoII can also serve as a structural component of mitotic chromosome scaffolding (Uemura et al., 1987 ), playing an essential role in chromatin condensation during prometaphase and in sister chromatid segregation during anaphase (Adachi et al., 1991 ). DNA topoII is a target for a number of clinically useful antitumor agents, in part because it is essential for cell survival. To date, there are two general classes of topoII inhibitors that interfere with enzyme catalysis at distinct points of the enzyme reaction. DNA topoII inhibitors, such as teniposide (VM-26), etoposide (VP-16), and the anthracyclines (daunorubicin and doxorubicin), stabilize cleaved DNA-topoII complexes (Chen et al., 1984 ; Robinson and Osheroff, 1991 ) Nádorová kmenová buňka http://www.gnf.org/assets/001/23050.jpg Venkateshwar Reddy, Ph.D. Group Leader Our aim is to characterize cancer stem cells from various tumors and to develop therapeutics capable of specifically eliminating them. The traditional model of cancer development considers that tumors arise from a series of sequential mutations resulting from genetic instability and/or environmental factors effecting normal cells. A major argument against this model is the prolonged period required to develop the first mutation that subsequently leads to malignant tumor formation. In many tissues in which tumors arise, mature cells have a short lifespan and a limited opportunity to accumulate the multiple mutations required for tumor development. More recently, a new model has been proposed, which considers that tissue stem cells undergo mutations that deregulate normal self-renewal pathways, leading to tumor formation. Since stem cells are immortal or have a longer lifespan, they can more easily accumulate mutations. This latter model, supported by recent studies, suggests that tumor formation may result from the deregulation of normal self-renewal pathways of tissue stem cells. The cancer stem cell hypothesis would have profound implications for cancer therapy. Cancer stem cells, like normal stem cells, are more resistant to conventional chemotherapies than other more differentiated cancer cells; hence, to cure cancer, it is important to target not only proliferating cells but also stem cells. Developing therapies that are selectively toxic to cancer stem cells while sparing normal stem cells may lead to more effective treatment options. And understanding cancer stem cell biology will help in the development of predictive markers and of targeted therapeutic strategies. At GNF, we are using our technology infrastructure to characterize the cancer stem cells from various tumors and to develop therapeutics capable of eliminating them. Our efforts include understanding the processes that normal stem cells employ and identifying the defects that lead to the development of cancer stem cells. Within our group, we are: Isolating and characterizing cancer stem cells from a range of solid tumors and identifying the pathways required for their self-renewal Developing relevant self-renewal assays that are compatible with high throughput screening (HTS) format for functional genomic and small molecule screens in order to identify biological networks essential for cancer stem cell's function Determining the frequency of non-tumorigenic cancer cells acquiring properties of cancer stem cells Functional genomic and proteomic characterization of cancer stem cells and normal stem cells Creating a new generation of in vitro assays for the validation of identified oncology targets with a focus on cancer stem cells Developing in vivo efficacy models that are capable of interrogating the effect of therapeutics on cancer stem cells. Copyright © 2006 Genomics Institute of the Novartis Research Strategie léčby Figure 5: Conventional therapies may shrink tumor mass, but spare cancer stem cells (CSC). CSC are resistant and remain viable and after some time re-establish the tumor. By contrast if therapies can be targeted against CSC, then they might render tumors unable to maintain themselves to grow. Thus, therapies that target CSC should not shrink the tumor immediately but may eventually lead to tumor degeneration. Combination of conventional therapies with drugs that specifically target CSC should lead to fast and long lasting cures. Cancer stem cells (CSCs) are cancer cells (found within tumors or hematological cancers) that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. These cells are therefore tumorigenic (tumor-forming), perhaps in contrast to other non-tumorigenic cancer cells. CSCs may generate tumors through the stem cell processes of self-renewal and differentiation into multiple cell types. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors. Therefore, development of specific therapies targeted at CSCs holds hope for improvement of survival and quality of life of cancer patients, especially for sufferers of metastatic disease. Existing cancer treatments were mostly developed on animal models, where therapies able to promote tumor shrinkage were deemed effective. However, animals could not provide a complete model of human disease. In particular, in mice, whose life spans do not exceed two years, tumor relapse is exceptionally difficult to study. The efficacy of cancer treatments are, in the initial stages of testing, often measured by the amount of tumor mass they kill off. As CSCs would form a very small proportion of the tumor, this may not necessarily select for drugs that act specifically on the stem cells. The theory suggests that conventional chemotherapies kill differentiated or differentiating cells, which form the bulk of the tumor but are unable to generate new cells. A population of CSCs, which gave rise to it, could remain untouched and cause a relapse of the disease. Contents [hide] 1 Evidence for CSCs 1.1 Importance of stem cells 1.2 Mechanistic and mathematical models 2 Origins 3 Implications for cancer treatment 4 Cancer stem cell pathways 4.1 Bmi-1 4.2 Notch 4.3 Sonic hedgehog and Wnt 5 External links 6 References [edit] Evidence for CSCs Opponents of the paradigm do not deny the existence of CSCs as such. Cancer cells must be capable of continuous proliferation and self-renewal in order to retain the many mutations required for carcinogenesis, and to sustain the growth of a tumor since differentiated cells cannot divide indefinitely (constrained by the Hayflick Limit). However, it is debated whether such cells represent a minority. If most cells of the tumor are endowed with stem cell properties there is no incentive to focus on a specific subpopulation. There is also debate on the cell of origin of these CSCs - whether they originate from stem cells that have lost the ability to regulate proliferation, or from more differentiated population of progenitor cells that have acquired abilities to self-renew (which is related to the issue of stem cell plasticity). The first conclusive evidence for CSCs was published in 1997 in Nature Medicine. Bonnet and Dick[1] isolated a subpopulation of leukaemic cells that express a specific surface marker CD34, but lacks the CD38 marker. The authors established that the CD34+/CD38- subpopulation is capable of initiating tumors in NOD/SCID mice that is histologically similar to the donor.[2] In cancer research experiments, tumor cells are sometimes injected into an experimental animal to establish a tumor. Disease progression is then followed in time and novel drugs can be tested for their ability to inhibit it. However, efficient tumor formation requires thousands or tens of thousands of cells to be introduced. Classically, this has been explained by poor methodology (i.e. the tumor cells lose their viability during transfer) or the critical importance of the microenvironment, the particular biochemical surroundings of the injected cells. Supporters of the cancer stem cell paradigm argue that only a small fraction of the injected cells, the CSCs, have the potential to generate a tumor. In human acute myeloid leukemia the frequency of these cells is less than 1 in 10,000.[1] Further evidence comes from histology, the study of tissue structure of tumors. Many tumors are very heterogeneous and contain multiple cell types native to the host organ. Heterogeneity is commonly retained by tumor metastases. This implies that the cell that produced them had the capacity to generate multiple cell types. In other words, it possessed multidifferentiative potential, a classical hallmark of stem cells.[1] The existence of leukaemic stem cells prompted further research into other types of cancer. CSCs have recently been identified in several solid tumors, including cancers of the: Brain[3] Breast[4] Colon[5] Ovary[6] Pancreas[7] Prostate[8][9] [edit] Importance of stem cells Not only is finding the source of cancer cells necessary for successful treatments, but if current treatments of cancer do not properly destroy enough CSCs, the tumor will reappear. Including the possibility that the treatment of for instance, chemotherapy, will leave only chemotherapy-resistant CSCs, then the ensuing tumor will most likely also be resistant to chemotherapy. If the cancer tumor is detected early enough, enough of the tumor can be killed off and marginalized with traditional treatment. But as the tumor size increases, it becomes more and more difficult to remove the tumor without conferring resistance and leaving enough behind for the tumor to reappear. Some treatments with chemotherapy, such as paclitaxel in ovarian cancer (a cancer usually discovered in late stages), may actually serve to promote certain cancer growth (55-75% relapse <2 years[10]). It potentially does this by destroying only the cancer cells susceptible to the drug (targeting those that are CD44-positive, a trait which has been associated with increased survival time in some ovarian cancers), and allowing the cells which are unaffected by paclitaxel (CD44-negative) to regrow, even after a reduction in over a third of the total tumor size.[11] There are studies, though, which show how paclitaxel can be used in combination with other ligands to affect the CD44-positive cells.[12] While paclitaxel alone, as of late, does not cure the cancer, it is effective at extending the survival time of the patients.[10] [edit] Mechanistic and mathematical models Once the pathways to cancer are hypothesized, it is possible to develop predictive mathematical biology models,[13]e.g., based on the cell compartment method. For instance, the growths of the abnormal cells from their normal counterparts can be denoted with specific mutation probabilities. Such a model has been employed to predict that repeated insult to mature cells increases the formation of abnormal progeny, and hence the risk of cancer.[14] Considerable work needs to be done, however, before the clinical efficacy of such models is established. [edit] Origins This is still an area of ongoing research. Logically, the smallest change (and hence the most likely mutation) to produce a cancer stem cell would be a mutation from a normal stem cell. Also, in tissues with a high rate of cell turnover (such as the skin or GI epithelium, where cancers are common), it can be argued that stem cells are the only cells that live long enough to acquire enough genetic abnormalities to become cancerous. However it is still possible that more differentiated cancer cells (in which the genome is less stable) could acquire properties of 'stemness'. It is likely that in a tumor there are several lines of stem cells, with new ones being created and others dying off as a tumor grows and adapts to its surroundings.[15] Hence, tumor stem cells can constitute a 'moving target', making them even harder to treat. [edit] Implications for cancer treatment The existence of CSCs have several implications in terms of future cancer treatment and therapies. These include disease identification, selective drug targets, prevention of metastasis, and development of new strategies in fighting cancer. Normal somatic stem cells are naturally resistant to chemotherapeutic agents - they have various pumps (such as MDR) that pump out drugs, DNA repair proteins and they also have a slow rate of cell turnover (chemotherapeutic agents naturally target rapidly replicating cells). CSCs, if they are the mutated counterparts of normal stem cells, may also have similar functions which allows them to survive therapy. These surviving CSCs then repopulate the tumor, causing relapse. By selectively targeting CSCs, it would be possible to treat patients with aggressive, non-resectable tumors, as well as preventing the tumor from metastasizing. The hypothesis implies that if the CSCs are eliminated, the cancer would simply regress due to differentiation and cell death. There has also been a lot of research into finding specific markers that may distinguish CSCs from the bulk of the tumor (as well as from normal stem cells), with some success.[4] Proteomic and genomic signatures of tumors are also being investigated. Success in these area would enable faster identification of tumor subtypes as well as personalized medicine in cancer treatments by using the right combination of drugs and/or treatments to efficiently eliminate the tumor. [edit] Cancer stem cell pathways A normal stem cell may be transformed into a cancer stem cell through disregulation of the proliferation and differentiation pathways controlling it. Scientists working on CSCs hope to design new drugs targeting these cellular mechanisms. The first findings in this area were made using haematopoietic stem cells (HSCs) and their transformed counterparts in leukemia, the disease whose stem cell origin is most strongly established. However, these pathways appear to be shared by stem cells of all organs. [edit] Bmi-1 The Polycomb group transcriptional repressor Bmi-1 was discovered as a common oncogene activated in lymphoma[16] and later shown to specifically regulate HSCs.[17] The role of Bmi-1 has also been illustrated in neural stem cells.[18] The pathway appears to be active in CSCs of pediatric brain tumors.[19] [edit] Notch The Notch pathway has been known to developmental biologists for decades. Its role in control of stem cell proliferation has now been demonstrated for several cell types including haematopoietic, neural and mammary[20] stem cells. Components of the Notch pathway have been proposed to act as oncogenes in mammary[21] and other tumors. [edit] Sonic hedgehog and Wnt These developmental pathways are also strongly implicated as stem cell regulators.[22] Both Sonic hedgehog(SHH) and Wnt pathways are commonly hyperactivated in tumors and are required to sustain tumor growth. However, the Gli transcription factors that are regulated by SHH take their name from gliomas, where they are commonly expressed at high levels. A degree of crosstalk exists between the two pathways and their activation commonly goes hand-in-hand.[23] This is a trend rather than a rule. For instance, in colon cancer hedgehog signalling appears to antagonise Wnt.[24] Sonic hedgehog blockers are available, such as cyclopamine. There is also a new water soluble cyclopamine that may be more effective in cancer treatment. There is also DMAPT, a water soluble derivative of parthenolide that targets AML (leukemia) stem cells, and possibly other CSCs as in myeloma or prostate cancer. A clinical trial of DMAPT is to start in England in late 2007 or 2008. Furthermore, GRN163L was recently started in trials to target myeloma stem cells. If it is possible to eliminate the cancer stem cell, than a potential cure may be achieved if there are no more CSCs to repopulate a cancer. [edit] External links Cancer Stem Cell News A blog of news items related to cancer stem cells, with an emphasis on recent research and articles that are openly accessible Chemoterapeutika Protein Substráty – chemoterapeutika ABCB1/MDR1 colchicine, doxorubicin, etoposid, adriamycin, vinblastine, digoxin ABCC1/MRP1 doxorubicin, daunorubicin, etoposid, colchicines, etoposide, rhodamine ABCG2/BCRP Mitoxantrone, topotecan, CPT-11, rhodamine ! Jeden transportér má různé substráty -> MULTIléková rezistence ! MXR fluorescence Dean et al.: the role of the abc transporters drug resistance and pharmacology Mitoxantron - fluoreskující substrát BCRP a MDR1 senzitivní rezistentní Zkřížená rezistence Rezistence buněk S1-M1-80 získána kultivací s Doxorubicinem (substrát MDR1 a BCRP) Pgp substráty ve vstupní terapii: Podle:The Medical Letter Aktuální projekty HL-60 buňky s vysokou expresí jednotlivých ABC Buněčné modely qRT-PCR WB Nádorové buňky ROS Produkce ROS Většina barviv používaných na detekci ROS jsou substráty ABC transportérů fig3 Dye Exclusion Assays ABC transportéry ovlivňují výsledky klasických metod Falešně negativní výsledek je způsoben nevhodnou kombinací barviva a linie PMA indukuje produkci ROS Regulace transkripce lStres (teplo, zánět, hypoxie, UV záření, diferenciační činidla) vedou k zvýšení transkripce ABC transportérů Studium promotoru + enhaceosomu l lRychlé zvýšení exprese po inkubaci s jejich substráty (doxorubicin, vinca alkaloidy, etoposid, taxely) l Jak zamezit zvýšení exprese dřív než nastane??? l lStudium signálních drah vedoucích lk zvýšené expresi ABC transportérů – p38 MAPK l MDR1 enhacesom: Scotto KW, Johnson RA. Transcription of the multidrug resistance gene MDR1: a therapeutic target. Mol Interv. 2001 Jun;1(2):117-25 ENHANCEOSOM PROMOTOR ABC gen IFNa induces fosforylation of p38 kinase lKrejčová et al. 2009, General physiology and biophysics, in print x1000 U/ml A549 A549 Krejčová et al. 2009, General physiology and biophysics, in print IFNa increases expression of ABC transporters mESC „Dye exclussion assays“ median 45.3 67.9 median 37.5 57.2 median 74.3 90.5 median 107.1 164.5 JC1(MDR1) Calcein AM (MRP1) Bodipy-prazosin (BCRP) Pheophorbid A Knock-down of p38 results in lesser function of ABCs p38+/+ p38-/- Hledání vazebného místa pro STAT3 lZvýšená exprese MDR1 je doprovázena aktivací STAT3 lInhibice STAT3 vedla ke snížení rezistence nádorových buněk k chemoterapeutikám lKo-transfekce STAT3 a sady reportérů pro MDR1 sramkova fig1 STAT3… transkripční faktor ENHANCEOSOM PROMOTOR Increased activity of STAT3 was found to correlate with the increased expression of MDR1 (e.g. Bourguignon 2008, Zhang 2010). Recent studies have reported that the Signal Transducers and Activators of Transcription3 (STAT3) inhibition in tumor cell lines is associated with increasing sensitivity of cells to chemoterapeutics (Zhang 2010). Melanomy a ABC B5 Role PI3K inhibitoru LY Produkce melaninu Magn.x20 Role ABC B5 v melanogenezi Shrnutí lABC transportéry přenášejí látky aktivně VEN l z buňky lDůležité pro obranu organizmu lJejich nadměrná exprese v rakovinných buňkách vede k multilékové rezistenci lSubstrátem ABC transportérů jsou i fluorescenční próby pro stanovení ROS, mitochondriální funkce Do čeho se můžete zapojit? lAnalýza vazebného místa STAT3 v MDR1 enhanceosomu lRezistence melanomů způsobená MDR5 lVliv základních kinázových kaskád na transkripci ABC transportérů v nádorových a ES buňkách lRole ABC transportérů v buněčném cyklu lBiokompatibilita materiálů s povrchy upravenými plazmou l Děkuji za pozornost ABC transporters' by Vicky Summersby, inspired by the Review on p218 jipro@sci.muni.cz