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Università degli Studi di Trieste Graduate School in MOLECULAR BIOMEDICINE PhD Thesis Is bilirubin able to affect the cell cycle in Gunn rat brain? -An in vivo and in vitro study- María Celeste Robert

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Università degli Studi di Trieste Graduate School in MOLECULAR BIOMEDICINE PhD Thesis Is bilirubin able to affect the cell cycle in Gunn rat brain? -An in vivo and in vitro study- María Celeste Robert XXIV ciclo Anno Accademico 2011 UNIVERSITA DEGLI STUDI DI TRIESTE DOTTORATO DI RICERCA IN BIOMEDICINA MOLECOLARE Ciclo XXIV Is bilirubin able to affect the cell cycle in Gunn rat brain? -An in vivo and in vitro study- Settore Scientifico - disciplinare: Biologia Molecolare (Bio/11) PhD Student: María Celeste Robert Doctoral program Coordinator: Prof. Giannino Del Sal Università degli Studi di Trieste Thesis Supervisor: Prof. Claudio Tiribelli Università degli Studi di Trieste Thesis Tutor: Dr. Silvia Gazzin Fondazione Italiana Fegato Anno accademico 2010/2011 Supervisor: Prof. Claudio Tiribelli Università degli Studi di Trieste Tutor: Dr. Silvia Gazzin Fondazione Italiana Fegato External Supervisor: Prof. Stefano Gustincich Scuola Internazionale Superiore di Studi Avanzati- SISSA Opponent: Dr. Germana Meroni CBM s.c.r.l Thesis Committee: Prof. Quattrone Alessandro Università di Trento Prof. Cordenonsi Michelangelo Università di Padova Prof. Pucillo Carlo Ennio Michele Università di Udine Prof. Brancolini Claudio Università di Udine Dr. Benetti Roberta Università di Udine Prof. Claudio Santoro Università di Novara This study was supported by a fellowship from the Italian Ministry of Foreign Affairs (MAE) in Rome, Italy. In particular, I wish to thank Dr. Paola Ranocchia. A Mariano, Susana, Gustavo, Constanza y Augusto Que me ayudaron a recorrer este camino. CONTENTS ABSTRACT... xi PUBLICATIONS... xiii ABBREVIATIONS... xiv 1. INTRODUCTION Bilirubin Bilirubin metabolism Bilirubin Characteristics The free bilirubin theory Disorders of bilirubin metabolism Disorders leading to unconjugated hyperbilirubinemia...6 Neonatal hyperbilirubinemia...6 Bilirubin encephalopathy...7 Kernicterus...8 Crigler - Najjar syndrome...8 Gilbert syndrome Disorders leading to conjugated hyperbilirubinemia...9 Dubin - Johnson syndrome...9 Rotor syndrome Bilirubin neurotoxicity The Gunn rat The cerebellum The cell cycle Cell cycle regulation Cell cycle and apoptosis Cryopreservation AIMS OF THE STUDY MATERIALS & METHODS Materials, Chemicals and Reagents in vivo and in vitro bilirubin effect on cell cycle Animals vii CONTENTS Rat cerebella dissection Primary cell cultures Primary astrocyte cultures Primary cerebellar granule cultures UCB solutions and Bf measurements Culture and treatment conditions Cell viability after UCB treatment Gene expression analysis RNA extraction mrna Quantification by Real-Time RT-PCR Protein expression analysis Protein extraction Western blot Cell cycle analysis by FACS Cell dispersal and fixation Staining and analysis in vitro analysis of apoptosis/ necrosis by FACS Neural cells cryopreservation Equipment for sample freezing Primary cerebellar granule cells cultures Cryopreservation Freezing conditions Rewarming conditions Viability after rewarming Statistical analysis RESULTS in vivo bilirubin effect on cell cycle Gene expression Selection of reference genes mrna expression of cell cycle regulators Protein expression Protein expression of cell cycle regulators Parp-1 cleavage analysis viii CONTENTS Cell cycle analysis by FACS Cell cycle progression during development Bilirubin effect on cell cycle progression in vitro bilirubin effect on cell cycle Bf and UCB solutions Cell viability after UCB treatment Primary astrocytes culture Primary cerebellar granule culture Gene expression analysis Primary astrocytes culture Primary cerebellar granule culture Cell cycle analysis Primary astrocytes culture Primary cerebellar granule culture Apoptosis/ necrosis analysis in astrocyte primary culture Neural cells cryopreservation Proportion of cryoprotective agent Freezing and rewarming conditions DISCUSSION Bilirubin effect on cell cycle in vivo in vitro in vivo- in vitro Cryopreservation of primary granule neurons CONCLUSION ACKNOWLEDGEMENTS REFERENCES ix x ABSTRACT The hyperbilirubinemic jj Gunn rat is a well established animal model for Crigler- Najjar type I Syndrome and neonatal jaundice. Similarly to humans, they present neurological deficits and what is more a marked cerebellar hypoplasia with a prominent loss and degeneration of Purkinje cells and granule neurons. Since high levels of bilirubin have been proven to arrest the cell cycle progression, we addressed the question if the cerebellar hypoplasia observed in the hyperbilirubinemic Gunn rat could be somehow linked to a cell cycle arrest, and if this cell cycle arrest was affecting selectively primary cultures of astrocytes and cerebellar granule neurons. In the in vivo study we report that the high levels of bilirubin present in the cerebellum of hyperbilirubinemic Gunn rat cause a cell cycle arrest in the late G0/G1 phase, characterized by a decrease in the protein expression of Cyclin D1, Cyclin A, Cyclin A1 and most importantly Cdk2. Meanwhile an increase in protein expression of total Cyclin E, due to a rose in the levels of low molecular weight Cyclin E forms (a supposed attempt to bypass the cell cycle arrest), was in vain. Furthermore, we observed an increment in the 18 kda fragment of Cyclin E (implicated in the amplification of the apoptotic pathway) suggesting us the presence of an increased apoptosis. Consistent with this speculation, the levels of the cleaved form of Poly (ADP-ribose) Polymerase (PARP-1) were increased. In the in vitro study we support the selectivity of bilirubin to damage specific cells as cerebellar granule neurons. Cerebellar granule cells viability was more affected respect to astrocytes in the same treatment conditions. The cell cycle was affected by high concentration of bilirubin only in cerebellar granule cells. We hypothesised that the characteristic cerebellar hypoplasia of hyperbilirubinemic Gunn rat may be due to the conjunction between cell cycle arrest and apoptosis, and that these two processes are intimately connected. Furthermore, only granule neurons cell cycle was affected. xi ABSTRACT Cryopreservation has been used routinely in prolonged storage of many mammalian tissues. The cryopreservation of neural cells/ tissue started to be interesting after the successful transplantation of such tissues, mainly for research. Several studies have been performed to achieve cryopreservation of granule cells, however, for these cell types there is no defined protocol for cryopreservation with sufficient success to enable it to be incorporated into routine clinical practice. As we were thinking to perform cerebellar granule cells transplantation as a way to treat Gunn rats cerebellar hypoplasia, we started to set up a protocol for cerebellar granule cells cryopreservation using the slow-freezing methodology. Cerebellar granule cells were successfully cryopreserved with a protocol that involves the use of 10 % of DMSO as cryoprotective agent, a freezing rate of 2.1 C/min, and a fast (154.4 C/ min) rewarming at 39 C. The cells cryopreserved in this way had a good cell viability and were kept in culture for 7 days. More experiments have to be made to standardize this protocol. xii PUBLICATIONS List of publications Robert MC, Furlan G, Rosso N, Tiribelli C, and Gazzin S. Alterations in the cell cycle and apoptosis account for the cerebellar atrophy of hyperbilirubinemic Gunn rat pups. -In preparation- Gazzin S, Zelenka J, Zdrahalova L, Konickova R, Zabetta CC, Giraudi PJ, Berengeno AL, Raseni A, Robert MC, Vitek L, Tiribelli C. Bilirubin accumulation and Cyp mrna expression in selected brain regions of jaundiced Gunn rat pups Pediatric Research, Feb 15 doi: /pr Robert MC, Gazzin S., Tiribelli, C. Bilirubin arrests the cell cycle in the Cerebellum of developing hyperbilirubinemic Gunn rat. FEBS Journal 2011, 278 (Suppl. 1), 274. Robert MC, Gazzin S, Berengeno A, Bellarosa C, Tiribelli C. Bilirubin modulates cell cycle in rat cerebella. Medicina 2010, Vol. 70 (Supl. II), 84. Berengeno A, Gazzin S, Bellarosa C, Robert MC, Tiribelli C. Does Bilirubin affect the ABCc (Mrp1) expression in the brain of the Gunn rat?. Pediatric Research Nov (Supl. I), 300. Congress presentations Gambaro SE, Gazzin S, Robert MC, Tiribelli C. Study on the role of cytochrome P 450 in bilirubininduced encephalopathy. In abstract of poster for International Symposium on Biology and Translational Aspects of Neurodegeneration. Venice, Italy. March 12-14, Robert MC, Gazzin S, Tiribelli C. Bilirubin arrests the cell cycle in the cerebellum of developing hyperbilirubinemic Gunn rat. In abstract of poster for 36 th FEBS Congress Biochemistry for Tomorrow s Medicine, Torino, Italy. June 25-30, Robert MC, Gazzin S, Tiribelli C. Cell cycle alterations in hyperbilirubinemic Gunn rat cerebella. In abstract of poster for 7th Seminar Frontiers in Molecular Biology, Società Italiana di Biofisica e Biologia Molecolare (SIBBM), Trieste, Italy. May 26-28, Robert MC, Gazzin S, Berengeno A, Bellarosa C, Tiribelli C. Bilirubin modulates cell cycle in rat cerebella. In abstract of poster for LV Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica (SAIC) Mar del Plata, Argentina. November 17-20, xiii ABBREVIATIONS List of Abbreviations Ara-C Bf BME BSA CB CGC Cll CNS Ct div DMEM DMSO FACS FBS FL FSC jj JJ LMW LN 2 MTT P Pi RT SSC UCB UGT1A1 Cytosine-β-D-arabino-furanoside Free bilirubin Basal Medium Eagle with Earle s salts, without L-glutamine Bovine Serum Albumin Conjugated bilirubin Cerebellar Granule Cell Cerebella Crigler- Najjar syndrome Cycle Threshold Days in vitro Dulbecco s Modified Eagle Medium Dimethyl sulfoxide Fluorescence activated cell sorter Foetal bovine serum Full length Forward-scattered light Homozygous hyperbilirubinemic Gunn rats Homozygous normobilirubinemic Gunn rats Low molecular weight forms Liquid nitrogen 3(4,5-dimethiltiazolil-2)-2,5 diphenil tetrazolium Post-natal age in days Propidium iodide Room Temperature Side-scattered light Unconjugated bilirubin Uridine diphosphoglucuronate glucuronosyltransferase xiv 1. INTRODUCTION 1.1 Bilirubin Bilirubin metabolism Bilirubin is produced as end product of the degradation of the protoporphyrin portion of the heme group present in haemoglobin, myoglobin, and some enzymes. This breakdown generates 250 to 400 mg/day of bilirubin in humans (LONDON et al., 1950). More than 80 % of the bilirubin produced in human body derives from heme catabolism liberated from senescent red cells, % from the turnover of myoglobin, cytochromes and other hemoproteins, and less than 3 % from destruction of immature red blood cells in the bone marrow (LONDON et al., 1950; Ostrow et al., 1962). The physiological degradation of heme group is mediated by the microsomal heme oxygenase enzyme, which is particularly abundant in spleen and liver, principal site of red cell breakdown. This enzyme directs the stereospecific cleavage of the α-methene bridge of heme group to form the linear tetrapyrrole, biliverdin, carbon monoxide, and iron (Tenhunen et al., 1968). This reaction is energy requiring, as the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) and molecular oxygen (O 2 ) are metabolized by cytochrome cp450 reductase to the oxidized form NADP (Yoshida and Kikuchi, 1978; Wilks and Ortiz de Montellano, 1993). Once synthesized biliverdin is converted to bilirubin (UnConjugated Bilirubin, UCB) by the cytosolic enzyme biliverdin reductase, in the presence of NADPH (Figure 1). As soon as bilirubin is released in the blood, and due to its lipophilicity, it must bind a carrier molecule to be transported to the liver, where it will be modified for its subsequent excretion. This function is accomplished by albumin that posses two binding sites for bilirubin. Almost all the bilirubin (99.9 %) present in plasma binds tightly but reversibly to serum albumin (Wennberg et al., 1979; Weisiger et al., 2001; Ahlfors, 2001). 1 INTRODUCTION Figure 1: Bilirubin metabolism. Bilirubin derives from heme metabolism by heme oxygenase and biliverdin reductase. When bilirubin-albumin complex reaches the liver, bilirubin is rapidly transferred from plasma into the liver. At the sinusoidal surface of the hepatocyte, bilirubin dissociates from albumin and is internalized, through mechanism not fully elucidated. It has been proposed that bilirubin is able to diffuse through cellular membranes (Zucker et al., 1999). There is also evidence that the hepatic uptake is saturable and occurs against a concentration gradient, supporting the idea of a protein-mediated transport mechanism (Cui et al., 2001). Within the aqueous environment of the hepatocyte, bilirubin is again bound to another group of carrier proteins, mainly to glutathione- S- transferases class alpha (ligandins) (Zucker et al., 1995). The binding to these enzymes, increases the net uptake of bilirubin by reducing efflux from the cell. In the endoplasmic reticulum, a specific form of uridine diphosphoglucuronate glucuronosyltransferase (UGT1A1) catalyzes the transfer of either one or two molecules of glucuronic acid to bilirubin, yielding mono and diglucuronides, collectively known as conjugated bilirubin (CB) (Hauser et al., 1984; Bosma et al., 1994). This conversion is critical since renders bilirubin water soluble and unable to diffuse across membranes. 2 INTRODUCTION Finally, the conjugated bilirubin is excreted across the bile canaliculus by the membrane transporter multidrug resistance protein 2 (MRP2 or ABCC2) (Kamisako et al., 1999) in the intestinal lumen (Figure 2). Bilirubin passes through the small intestine without significant absorption. In the colon, the glucuronide residues are released by bacterial hydrolases, and bilirubin degraded to a large family of reduction- oxidation products, collectively known as urobilinoids, which are mostly excreted by faeces. The conversion of bilirubin conjugates to urobilinoids is an important natural detoxification mechanism because it blocks the intestinal absorption of bilirubin known as enterohepatic circulation (Poland and Odell, 1971; Lester and Schmid, 1963). Only a small proportion of unconjugated bilirubin is reabsorbed by the intestine, returned to the liver and secreted again to the bile. Figure 2: Hepatic metabolism of bilirubin. (1) bilirubin is transported bound to albumin. (2) Bilirubin is dissociated from albumin and enters hepatocytes by facilitated diffusion. (3) Binding to glutathione-s-transferases (GSts). (4) Bilirubin is converted to mono- and diglucuronide by the action of UGT1A1. (5) Bilirubin glucuronides are actively transported into bile by ATP dependent pump ABCC2 (MRP2). (Adapted from Roy Chowdhury et al., 2001) Bilirubin Characteristics Unconjugated bilirubin is a nearly symmetrical tetrapyrrole, consisting of two rigid, planar dipyrrole units, joined by a methylene (-CH 2 -) bridge at carbon 10. The structure thus resembles a two bladed propellor, in which the blades could theoretically be joined 3 INTRODUCTION at different angles and each blade could rotate its bond to the methylene bridge, reviewed by Ostrow (Ostrow et al., 1994). In the preferred ridge-tile conformation, the two dipyrrinones are synperiplanar as in a partially opened book, and the angle (ϴ) between the two planes is about 95. The rigid biplanar structure of bilirubin IXα, the most naturally occurring isomer (Figure 3), with its internal hydrogen bonds, was first demonstrated in the crystalline state by X-ray diffraction (Bonnett et al., 1978), is also the preferred conformation in solution of UCB in water, alcohols, and chloroform. Figure 3: The structure of bilirubin IXα-Z,Z, diacid (H 2 B), which consist of two slightly asymmetrical, rigid, planar dipyrrinone chromophores, connected by a central methylene bridge. (Adapted from Pu et al. Tetrahedron 47: ). At physiological ph values in plasma (7.40), tissues (7.60) and bile (6.00 to 8.00) there is a significant ionization of the COOH groups of the natural IXα isomer of UCB (Hahm et al., 1992), so in addition to the diacid (H 2 B), a proportion of UCB is present as monoanion (HB - ) and dianion (B 2- ) as shown in Figure 4. The pk a values of the COOH groups on the two carboxymethyl (propionyl) side chains determine the proportion of the free UCB species at any given ph. At ph 7.40 (e.g. plasma) the preponderant form of 4 INTRODUCTION UCB is H 2 B (83 %), HB - represent the 15 % and B 2- less than 1.5 % of total UCB (Hahm et al., 1992) The free bilirubin theory In blood, the total amount of bilirubin is formed by three principal forms: albuminbound unconjugated bilirubin (UCB-A), conjugated bilirubin (CB) and unbound unconjugated bilirubin (free bilirubin, Bf). The Bf represents less than 0.1 % of the pigment found in blood (Ahlfors, 2001; Weisiger et al., 2001). Figure 4: Proportions of unbound species of unconjugated bilirubin at ph 6.00 to 8.00, derived from partitions of UCB from chloroform into buffered NaCl at ionic strength (Adapted from Hahm et al., J. Lipid Res. 33: ). In 1959 Odell (Odell, 1959) proposed for the first time that only non-albumin-bound bilirubin (free bilirubin, Bf) is available for transport into brain and recommended that the measurement of Bf level would be better than total bilirubin in predicting risk for kernicterus. However it took additional 15 years before Wennberg reports a successful method to assess Bf in serum (Jacobsen and Wennberg, 1974). This proposal has been successfully popularized as the free bilirubin theory (Wennberg, 2000; Calligaris et al., 2007). 5 INTRODUCTION The proportion of free UCB depends on the presence of albumin and its binding affinity constants. If the quantity of bilirubin does not exceed the albumin binding capacity (bilirubin/ albumin ratio is lower than 1) the maximal amount of UCB circulates in blood bound to human serum albumin (HSA), being 435 μm (25 mg/dl) the highest quantity of UCB transported bound to HSA (Brito, 2006). On the contrary, if the bilirubin/ albumin ratio is more than 1, the albumin binding capacity for UCB is exceeded and the amount of Bf sharply rises (Diamond and Schmid, 1966). In these conditions, the Bf, which is mainly in the non-ionised (diacid, H 2 B) form at physiological ph, can passively diffuse across membranes and enters into tissues (Gourley, 1997; Zucker et al., 1999). 1.2 Disorders of bilirubin metabolism Hepatic transport of bilirubin, as described previously, involves four distinct but related stages: uptake from circulation, intracellular binding or storage, conjugation and biliary excretion. In pathological situations, abnormalities in the bilirubin metabolic pathway may cause hyperbilirubinemia. In severe inheritable disorders, the transfer of bilirubin from blood to bile is disrupted at a specific stage, depending on which stage abnormality occurs, hyperbilirubinemia of unconjugated or conjugated type will be produced (Strassburg, 2010; Roy-Chowdhury, 2001) Disorders leading to unconjugated hyperbilirubinemia Neonatal hyperbilirubinemia During the foetal development, the maternal placenta is responsible for cleaning the bilirubin from the foetal circulation. Upon birth, this placental protection is suddenly lost, and bilirubin clearance is totally accomplished by the infants. Just at that moment a sharp increase in the production of unconjugated bilirubin occurs. 6 INTRODUCTION The daily production of bilirubin is of 6-8 mg/kg in healthy term infants and 3-4 mg/kg in adults. This increase is principally due to: the shorter red blood cells life span of newborns (70-90 vs. 120 days in adults), the immaturity of the hepatic bilirubin conjugation (Rubaltelli, 1993). In addition, the absence of the intestinal flora in the newborn infant lead to more non-metabolized UCB available for intestinal absorption, increasing the enterohepatic circulation of UCB (Vitek et al., 2000). Therefore, a significant retention of UCB occurs in almost all healthy ter
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