„ABC“ o ABC transportérech

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1 „ABC“ o ABC transportérech
Aktivní a pasivní transport živin ABC transportéry a jejich detekce Fyziologická role Kmenové buňky a regenerace Nádorové bujení Multiléková rezistence Regulace exprese ABC transportérů

2 Zdroj energie

3 Hydrophobické molekuly
Permeabilita živin Hydrophobické molekuly bez náboje Malé polární molekuly bez náboje Velké molekuly Ionty O2, CO2, N2 H2O, glycerol Glukóza, sacharóza H+,Na+,NCO3–, Ca2+,CL-,Mg2+,K+ Fosfolipidová dvouvrstva Nízká permeabilita Vysoká permeabilita 100 10-2 10-4 10-6 10-8 10-10 10-12 H2O Glycerol Glukóza Cl– K+ Na+ Pasivní transport Figure: 4.7b Caption: (b) This diagram summarizes the relative permeabilities of various molecules and ions, based on data like those presented in part (a).  Question About how fast does water cross the lipid bilayer?

4 Transport přes membránu
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.

5 Typy aktivního transportu
Uniport - a carrier protein that only transports one molecule across a membrane Symports - a carrier protein that carries two molecules in the same direction Antiports - a carrier protein that carries two molecules in the opposite direction Many facilitated diffusion transport proteins are symports When considering direction of molecule flow, the relative concentrations of both molecules must be considered together This may allow one molecule type to move against its concentration gradient However, when the overall concentration gradient is considered, you'll see that both are still flowing down a net gradient This is more fully explained in the The H+ / Sucrose Pump below Transportér specifický pro jednu molekulu Transportér pro dvě molekuly ve stejném směru (H+/sacharóza) Transportér pro dvě molekuly v opačném směru

6 ATP-binding cassette ABC transportéry
ATP-binding cassette Za spotřeby ATP pumpují toxické látky/metabolické produkty VEN z buňky (výjimka CFTR) Fyziologická funkce – sekrece látek produkovaných buňkou + obrana proti xenobiotikám

7 Sarkadi et al., Physiol Rev • VOL 86 • 2006
Struktura NBD = ABC Sarkadi et al., Physiol Rev • VOL 86 • 2006 FULL Transporter MDR1, MRP1 HALF Transporter BCRP

8 Krystalografická struktura
Transmembránové domény Kaspar P. Locher, Allen T. Lee and Douglas C. Rees, Caltech Transport proteins, embedded in lipid membranes, facilitate the import of nutrients into cells or the release of toxic products into the surrounding medium. The largest and arguably the most important family of membrane transport proteins are the ABC transporters. They are ubiquitous in biology and power the translocation of substrates across the membrane, often against a concentration gradient, by hydrolyzing ATP (Higgins, 1992).      Figure 1. Ribbon diagram of the BtuCD protein structure. The transporter is assembled from two membrane-spanning BtuC subunits (red and yellow) and two ABC cassettes BtuD (green and blue). At the ATP binding sites, cyclotetravanadate molecules are bound to the transporter (ball and stick models at the BtuD interface. Vitamin B12 is delivered to the periplasmic side of the transporter by a binding protein (BtuF, not shown), then translocated through a pathway provided at the interface of the two membrane-spanning BtuC subunits. It finally exits into the cytoplasm at the large gap between the four subunits (arrows). This transport cycle is powered by the hydrolysis of ATP by the ABC cassettes BtuD.  Several human ABC transporters are medically relevant. For example, mutations in the CFTR (cystic fibrosis transmembrane conductance regulator) protein cause cystic fibrosis. A separate subclass of ABC transporters are associated with multidrug resistance in tumors, i.e. the ability of certain cancer cells to extrude cytotoxic agents used in chemotherapy. Yet another ABC transporter, the TAP protein, is critical for the proper functioning of the cellular immune response, as it pumps antigenic polypeptides from the cytoplasm into the endoplasmic reticulum, where they are being loaded onto MHC class I molecules for subsequent presentation on the cell surface. In bacteria, ABC transporters are predominantly involved in nutrient uptake, although they also participate in the export of bacterial toxins and harmful substances, contributing to bacterial multidrug resistance.  Despite the immense amount of biochemical studies, and recent advances in the visualization of ABC transporters, answers to critical questions about their translocation mechanisms have remained elusive. The structure of a bacterial ABC transporter facilitating vitamin B12 import into E. coli, the BtuCD protein, was recently solved from data collected at SSRL. It is the first complete ABC transporter to have its high resolution (3.2 Å) structure determined in the physiological assembly, and it has yielded valuable insight into how ABC transporters work (Locher et al. 2002).  All ABC transporters contain two membrane-spanning domains that harbor a translocation pathway for a specific substrate. Attached are two cytoplasmic adenosine triphosphate-binding cassettes (hence ABC). As the ABC cassettes bind and hydrolyze ATP, conformational changes occur that are transmitted to the membrane-spanning domains, where they induce rearrangements that translocate the substrate from one side of the membrane to the other. The initial motion of the ABC cassettes has been dubbed the power stroke, and it is generally assumed that this rearrangement is similar in all ABC transporters, irrespective of the size of the substrate to be transported or the directionality of the translocation (import or export).  The structure of the BtuCD protein provides insight at just how ABC transporters may carry out their tasks. In particular, three critical elements were visualized for the first time in an intact transporter:  Transport pathway: Accessible from the outside (periplasm), but sealed from the cytoplasm of the cell, a large cavity is buried at the interface of the two membrane-spanning BtuC subunits. The cavity is big enough to accommodate the corrin ring of the substrate, vitamin B12, and therefore likely represents the transport pathway through the membrane. Power source: The arrangement of the two BtuD subunits places the binding sites for two ATP molecules at the subunit interface, with each ABC cassette contributing to the binding site. Well-conserved motifs form the contact interface of the two ABC cassettes, suggesting that this arrangement is universal among ABC transporters. The placement of the ATP binding sites wedged between two opposing ABC cassettes provides an explanation for the cooperativity in ATP binding and hydrolysis observed for ABC transporters. Relay station: The interface between the ABC cassette BtuD and the membrane-spanning subunit BtuC is where the mechanical energy produced by the hydrolysis of ATP is transmitted to the membrane-spanning domain. A prominent cytoplasmic loop of BtuC, forming two short helices, makes extensive contact with BtuD. Alignment with the protein sequences of other ABC transporters reveals that a comparable interface to that observed in BtuCD may exist in other transporters. The functionality and assembly of transporters with mutations in these critical interface residues is severely affected. As a striking example, 70% of cystic fibrosis patients have a single residue deleted that corresponds to a BtuD residue at the interface of the ABC cassette and the membrane-spanning domain. While much remains to be learned about substrate binding as well as the detailed conformational changes concomitant with substrate translocation through ABC transporters, the BtuCD structure provides a framework for addressing these central mechanistic issues.  References: Higgins, C. F. (1992) ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol Locher, K. P., Lee, A. T., Rees, D. C. (2002) The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism. Science Substrát ABC domény

9 Filogenetický strom ABC transportérů
48 různých proteinů u lidí 7 rodin (A-G) podle homologie

10 Výskyt ABC transportérů
Šipky označují výskyt a směr transportu

11 Substráty ABC transportérů - výživa
Živina Membránový Transportér Efekty Fytochemikálie z výživy Ø       P-glycoprotein (MDR1) Inhibice Flavonoidy (quercetin,) flavonoid glycosidy (genistein-7-glucosid) a flavonoid glucuronidy – ovoce, zelenina Ø       Multidrug Resistant Protein 1 (MRP1) Ø       Multidrug Resistant Protein 2 (MRP 2) Ø       Breast Cancer Resistance Protein BCRP (MXR) Akumulace, transport a potenciální efekty živiny Flavonoid-like molecules (polyphenol phloretin) Extrakty z hořkého melounu (1-monopalmitin), grape fruitu (bergamottin and quercetin), sójii Extrakt z hroznových jader Steroly (e.g. Cholesterol) Ø       ABCA1, ABCG1, ABCG5 and ABCG8 Substrát Sezamové semínko (lsophosphatidylcholine, linoleoyl) Ø       Některé transportéry ve střevu Mono-, di-, and triglutamáty folátů Rostlinné výtažky Inhibice (Curcumin, ginsenosides, piperine, some catechins from green  tea, silymarin from milk thistle); Some catechins from green tea (modulation); Hyperforin, kava (Activation of pregnane X receptor, an orphan nuclear receptor acting as a key regulator of MDR1) 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).

12 Antigeny - lipopolysacharidy
Syntéza antigenu 0 Antigeny - lipopolysacharidy Lipid fukóza N-acetyl galaktóza glukosamin Polysacharid 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. Lipidové jádro

13 Dědičné choroby 16 ABC genů bylo asociováno s dědičnými poruchami Tangierova choroba (ABCA1) – poruchy sekrece cholesterolu a fosfolipidů (nadměrná hladina v buňkách, narušena homeostáza) Dubin Johnson syndrom (ABCC2) – neschopnost jater sekretovat konjugovaný bilirubin do žluče Pseudoxanthoma elasticum (ABCC6) – mineralizace a fragmentace elastinových vláken, problém s vitamínem K Cystická fibróza (ABCC7) – poruchy sekrece působků pankreatu a dalších exokrinních žláz

14 „Dye exclussion assays“
Kompetitivní substráty Inhibice funkce transportéru Detekce ABC transp. Inhibitory „Dye exclussion assays“ MDR1 - JC1, rhodamin x Cyclosporin D, reversin MRP1 - Calcein AM x MK571, NSAID BCRP - Hoechst x fumitremorgin C - Bodipy-prazosin - Pheophorbid A Obecné inhibitory transportu – verapamil, cyclosporin A Imunodetekce

15 Calcein AM je substrátem MRP1, MDR1
Calcein AM je substrátem MRP1, MDR1 Inhibitory - MK571, NSAID, cyclosporin D, reversin

16 Kmenové buňky V průběhu diferenciace klesá exprese ABC transportérů
V průběhu diferenciace klesá exprese ABC transportérů

17 ABC v kmenových buňkách
Barvení Calcein AM MDR1/Pgp << MRP1

18 „SP“ buňky Hoechst 33342 - substrát BCRP, MDR1 Buňky C2C12 - myoblasty
- inhibitor verapamil Buňky C2C12 - myoblasty

19 Linie SK-N-SH Důkaz „kmenovosti“ SP buňky Majoritní populace
Hirschmann-Jax, C. et al. (2004) Proc. Natl. Acad. Sci. USA 101, SP buňky Majoritní populace 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

20 Játra Rozhraní krev vs. Hepatocyty Regenerace – MDR + BCRP
ABC (ATP-binding cassette) transporters are a large superfamily of integral membrane proteins involved in the cellular export or import of a wide variety of different substances,including ions,lipids,cyclic nucleotides,peptides,and proteins. ABC transporters are systemically classified into eight subfamilies by sequence similarity,i.e., ABCA (ABC1),ABCB (MDR/TAP),ABCC (MRP/CFTR),ABCD (ALD),ABCE (RNAseLI/OABP),ABCF (GCN20),ABCG (White) and ANSA subclass. In general,the transmembrane part of ABC transporters contains a polar channel formed by two homologous domains,each usually consisting of five (uptake transporters) or six (efflux transporters) transmembrane alpha-helices (Ref.1 & 2). 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).

21 BCRP Transporter Na+ Regenerace ledvin glomerulus
Immunohistochemical image of human renal tissue (colours indicate transporter proteins). Red: BCRP in the membrane of the proximal tubulus. Green: the sodium transporter Na+,K+-ATPase in the basal membrane. G: glomerulus. Magnification 200x. (Credit: Image courtesy of Dutch Kidney Foundation) cienceDaily (Jan. 28, 2009) — In a study funded by the Dutch Kidney Foundation (DKF) a research group at Radboud University Nijmegen Medical Centre in the Netherlands, found that stem cells and ABC transporter proteins are indispensable for tubular regeneration after acute kidney injury. See also: Health & Medicine Kidney Disease Stem Cells Leukemia Osteoporosis Prostate Cancer Skin Cancer Reference Healing Astrocyte Transplant rejection Dialysis Said project leader Dr. Rosalinde Masereeuw: 'To our surprise, our knockout mice for the ABC transporters P-gp and BCRP, P-gycoprotein and breast cancer resistance protein, were protected against acute kidney damage. This was the opposite of what we expected since the transporters usually have a protective function in excreting potentially toxic compounds, while these mice lack expression. Moreover, when we cross transplanted bone marrow between normal mice and the knockouts it turned out that bone marrow from the knockouts was the source of protection.' Regeneration Acute kidney injury is an important cause for the need of acute hemodialysis and a source of kidney failure. On the other hand, the kidney has a remarkable capacity for recovery. Stem cells seemed to have a limited share in the repair process, but now this study suggests otherwise. 'It was known that stem cells from the bone marrow express P-gp and BCRP abundantly but will downregulate them at differentiation. Repair of tubular damage in the kidney depends primarily on local cells but stem cells are involved as well. Further, we observed an upregulation in the expression of the transporters during ischemic injury. .So we thought they might be important in renal regeneration.' Transporter Proteins ABC transporters (ATP binding cassette transporters) form a superfamily of highly conserved transporter proteins whose functions are not yet well understood. However, BCRP and especially P-gp have been studied in more detail in man. These cell membrane pumps are responsible for the transport of many substances, for instance drug molecules in the intestine. P-gp plays an important role in drug resistance of tumour cells. Masereeuw: 'Our new hypothesis claims a bigger role for bone marrow derived stem cells in kidney regeneration. A possible mechanism is the infiltration of macrophages. These large immune cells have subgroups one of which increases damage but another supports tissue regeneration.' Also, the study showed that mice without P-gp expression lose renal tubular function in a way comparable to Fanconi syndrome in man. BCRP knockouts, on the other hand, have a normal kidney function. Blocking P-gp and BCRP There is a great need for novel therapies that limit kidney damage after acute injury by toxic substances or shortage of oxygen, as in transplant kidneys which have no blood supply during transport. The results from this DKF study are pointing at inhibition of the transporters in kidney or bone marrow to strengthen the regenerative power of stem cells. 'Next, we will try to discover the mechanism by which stem cells and ABC transporters contribute to kidney repair', concludes Dr. Masereeuw, 'and we will test the effect of transporter blockers in our mouse models. We are convinced there are good opportunities here for new drug targets.' BCRP Transporter Na+ glomerulus

22 Teorie vzniku metastáz
Model klonální selekce Model paralelní evoluce nádoru Integrující model evoluce nádoru A normální kožní tkáň B primární nádor C subkutánní metastázy D metastázy v lymfatické uzlině E metastázy v plicích Models of cancer evolution. (A) The 'clonal selection model' (blue arrows) is the prevailing view to explain the successive steps of mutation and selection from normal tissue to primary tumour and metastasis. However, metastasis-generating cells can emerge relatively early in the tumorigenic process and 'seed' distant tissues, thereby evolving in parallel with the primary tumour and delineating the 'parallel evolution' model (red arrows). Finally, these two models can occur simultaneously and metastatic deposits can act as sites from which additional metastases can be generated, therefore leading to an integrated model of cancer evolution (green arrows). (B) Microphotographs provide a histological snapshot of normal skin tissue (a), primary tumour (superficial b1 and deep b2, macroscopic appearance inset in b), subcutaneous metastasis (c), metastasis in the lymph node (d) and metastasis in the lung (e), and are shown in correspondence with the cancer-evolution models. This melanoma—which originates from the transformation of pigmented skin cells—provides a visual example of the modelling paradigms, illustrating the gap between ideal models and actual observations.

23 Nádorová kmenová buňka
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

24 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 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

25 (multidrug rezistance, multiléková rezistence)
MDR a rakovina (multidrug rezistance, multiléková rezistence) Selhání chemoterapie (nádorové b. jsou obvykle citlivější než „zdravé“ buňky) De novo/získaný fenotyp MDR (cytotoxic in vitro MTT-assay) Mechanizmy: 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 Nádorová b. se vymyká vlivu svého prostředí (obsahujícího chemoterapeutikum), získává „evoluční“ výhodu 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 )

26 ABC-B1 – MDR1; ABC-C1 – MRP1; ABC-G2 - BCRP
! Různé substráty -> MULTIléková rezistence! ABC-B1 – MDR1; ABC-C1 – MRP1; ABC-G2 - BCRP Dean et al.: THE ROLE OF THE ABC TRANSPORTERS DRUG RESISTANCE AND PHARMACOLOGY Mitoxantron - substrát BCRP a MDR1 senzitivní rezistentní

27 Imunocytochemie Gen Substráty - chemoterapeutika Inhibitory
ABCB1/PGP colchicine, doxorubicin, VP16, Verapamil, PSC833 adriamycin, vinblastine, digoxin ABCC1/MRP doxorubicin, daunorubicin, VP16, Cyclosporin A, V-104 colchicines, etoposide, rhodamine ABCG2/BCRP Mitoxantrone, topotecan, CPT-11, Fumitremorgin C, rhodamine GF120918

28 Podle:The Medical Letter
Pgp substráty ve vstupní terapii: Podle:The Medical Letter Lung: Breast: Prostate: Colorectal: Pancreas: Ovarian: Uterus: Lymphoma: Leukemia: Bladder: Myeloma: SCLC: C + G or C + Taxol or C + Vinorelbine NSCLC: Taxol + P Adriamycin + C ± Taxol or C + Adriamycin + F or CMF GnRH analogue 5-Fu + L G C + Taxol M or Adriamycin + P + C HD:Adriamycin +B + Vinblastine + D or M + Vincristine +PP/ABV NHL: C + Adriamycin + Vincristine + P or C + Vincristine + P High grade: C + Adriamycin + Vincristine + P + M ALL: Vincristine +P + L-asp + Adriamycin / Daunomycin AML: Ara-C + Daunomycin / Idarubicin CLL: C + Vincristine + P or Fludarabine CML: Gleevec M + Vinblastine + Adriamycin + C Vincristine + Adriamycin + D or MP

29 ABC transportéry MDR1 enhacesom: Scotto KW, Johnson RA. Transcription of the multidrug resistance gene MDR1: a therapeutic target. Mol Interv Jun;1(2):117-25 Stres (teplo, zánět, hypoxie, UV záření, diferenciační činidla) vedou k zvýšení jejich exprese – enhacesom Rychlé zvýšení exprese po inkubaci s jejich substráty (doxorubicin, vinca alkaloidy, etoposid, taxely) = jak zamezit zvýšení exprese dřív než nastane??? Studium signálních drah vedoucích k zvýšené expresi ABC transportérů ve stresových podmínkách – role p38 MAP kinázy 29

30 MAPK

31 Modelová buněčná linie
Metody Modelová buněčná linie A549 epiteliální buňky NSCLC mESC wt, mESC p38-/- HL-60 transfektanti Modelový treatment IFNa (prozánětlivý cytokin, terapie CML, melanomů…, kombinace s imanitibem, cytarabinem) Experimentální design Dye exclussion assays (průtoková cytometrie) MDR1: JC1 / Cyclosporin D MRP1: Calcein AM / MK571 BCRP: bodipy-prazosin, pheophorbid A / Fumitremorgin C Exprese studovaných proteinů Western blotting Vysetí buněk (SB203580) / IFNa Sběr buněk 24 h h

32 „Dye exclussion assays“
HL-60 transfektanti „Dye exclussion assays“ JC1(MDR1/CSD) Calcein AM (MRP1/MK571) Bodipy-prazosin (BCRP/Fumitremorgin C) Pheophorbid A HL60 transfektant + inhibitor HL60 2

33 IFNa způsobuje fosforylaci kinázy p38
x1000 U/ml 1.5x103 U/ml Krejčová et al. 2009, General physiology and biophysics, in print

34 A549 IFNa vede k zvýšení exprese ABC transportérů
„Western Blotting“ Krejčová et al. 2009, General physiology and biophysics, in print

35 „Dye exclussion assays“
IFNa snižuje funkčnost MRP1 a BCRP, inhibice p38 tento efekt zvrátí „Dye exclussion assays“ JC1(MDR1) Calcein AM (MRP1) Pheophorbid A (BCRP) JC Calc. Pheo. Kontrola 55,7 111,4 56,3 IFNa 50,9 73,6 45,7 SB ,7 148,5 99,1 IFNa + SB 42,9 87,4 85,8

36 „Dye exclussion assays“
mESC Inhibice p38 vede k snížení funkčnosti všech ABC tr. „Dye exclussion assays“ JC1(MDR1) Calcein AM (MRP1) median median p38+/+ p38-/- Bodipy-prazosin (BCRP) Pheophorbid A median median

37 IFNa indukuje stabilní zvýšení aktivity p38 MAPK
Shrnutí IFNa indukuje stabilní zvýšení aktivity p38 MAPK IFNa vede k zvýšení exprese ABC transportérů Inhibice p38 sníží expresi ABC transportérů Mechanismus účinku p38 Ostatní MAPK?

38 Děkuji za pozornost ABC transporters' by Vicky Summersby, inspired by the Review on p218