Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-09-27T14:19:27Z No. of bitstreams: 1 KELLEN ROCHA.pdf: 1430079 bytes, checksum: ba3882603d46f2a9824b73319d20cd73 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-09-27T14:19:44Z (GMT) No. of bitstreams: 1 KELLEN ROCHA.pdf: 1430079 bytes, checksum: ba3882603d46f2a9824b73319d20cd73 (MD5) / Made available in DSpace on 2018-09-27T14:19:44Z (GMT). No. of bitstreams: 1 KELLEN ROCHA.pdf: 1430079 bytes, checksum: ba3882603d46f2a9824b73319d20cd73 (MD5) Previous issue date: 2017-07-14 / A doença de Alzheimer (DA) é uma desordem neurodegenerativa crônica caracterizada clinicamente pela perda progressiva de função cognitiva, distúrbios neuropsiquiátricos e comportamentais. Patologicamente esta doença caracteriza-se pelo acúmulo anormal do peptídeo β-amilóide (Aβ) no córtex e no hipocampo, emaranhados neurofibrilares intracelulares formados por tau hiperfosforilada, disfunção progressiva sináptica e, posteriormente perda neuronal. As opções terapêuticas disponíveis melhoram os sintomas, mas não impedem a progressão da doença, portanto, ainda está faltando uma estratégia terapêutica efetiva para DA. Há estudos relacionados à utilização de terapia epigenética para o tratamento da DA, a terapêutica mais desenvolvida é a que envolve a classe dos inibidores das deacetilases (HDACs). Assim, este trabalho tem por objetivo investigar o efeito protetor do inibidor da HDAC ácido hidroxâmico de suberoilanilida (SAHA) em um modelo de DA em camundongos. Para isso, foram utilizados 50 camundongos Swiss adultos, pesando entre 30-35 g, divididos em dois experimentos. No primeiro, os camundongos foram divididos em 6 grupos que receberam uma injeção de Aβ1-42 via intracerebroventricular (i.c.v.) no início da experiência (exceto o grupo Sham que foi utilizado como controle) para investigar a atividade das histonas   acetiltransferase (HATs) e HDAC, determinação dos níveis do fator neurotrófico derivado do cérebro (BDNF), expressão do mRNA de BDNF e modulação da via (cAMP/PKA/CREB) em uma curva de tempo (6 horas, 1, 3, 7 e 21 dias). Ao final de cada tempo, os animais foram submetidos ao teste cognitivo e foram eutanasiados. O córtex pré-frontal e o hipocampo foram removidos para posteriores análises. No segundo experimento, os camundongos foram dividos em 4 grupos: Grupo Controle (sham+veículo); Grupo Aβ1-42 (Aβ1-42 + veículo); Grupo SAHA (25 mg/kg, via intraperitoneal) (sham + SAHA); Grupo Interação (Aβ1-42 + SAHA). O peptídeo Aβ1-42 ou o veículo foram infundidos por injeção i.c.v. e, um dia depois, iniciou-se o tratamento, por via i.p., durante 21 dias. Ao final do experimento os animais foram submetidos ao teste cognitivo, eutanásiados para retirada das estruturas cerebrais. As amostras foram utilizadas para a determinação dos níveis de BDNF, expressão do mRNA de BDNF, atividade enzimática das histonas (HDAC e HATs) e regulação da via cAMP/PKA/CREB. O presente estudo observou deficiências significativas causadas pela Aβ1-42 na memória (Labirinto Aquático de Morris), bem como causou desequilíbrio das enzimas HAT/HDAC, redução de cAMP, PKA e CREB e BDNF no córtex pré-frontal e hipocampo de camundongos. A inibição de HDAC, com SAHA demostrou neuroproteção nas alterações comportamentais e neuroquímicas induzidas por Aβ1-42. Estes dados mostram que a acetilação através da inibição do HDAC, desempenha um papel fundamental na mediação da memória e demonstra que SAHA poderá ser uma ferramenta médica promissora na abordagem terapêutica para o tratamento da DA. / Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized clinically by the progressive loss of cognitive function, neuropsychiatric and behavioral disorders. Pathologically this disease is characterized by the abnormal accumulation of β-amyloid peptide (Aβ) in the cortex and hippocampus, intracellular neurofibrillary tangles formed by hyperphosphorylated tau, progressive synaptic dysfunction and, later, neuronal loss. The available therapeutic options improve the symptoms, but they do not prevent the progression of the disease, therefore, an effective therapeutic strategy for AD is still lacking. There are studies related to the use of epigenetic therapy for the treatment of AD, the most developed therapy is that involving the class of deacetylase inhibitors (HDACs). Thus, this work aims to investigate the protective effect of the HDAC inhibitor hydroxamic acid suberoilanilide (SAHA) in an AD model in mice. For this, 50 Swiss adult mice weighing between 30-35 g were used, divided in two experiments. In the first, the mice were divided into 6 groups that received an injection of Aβ1-42 via the intracerebroventricular (i.c.v.) at the beginning of the experiment (except the Sham group that was used as control) to investigate histone activity acetyltransferase (HATs) and HDAC, determination of brain derived neurotrophic factor (BDNF) levels, expression of BDNF mRNA and modulation of the pathway (cAMP / PKA / CREB) in a time curve (6 hours, 1, 3, 7 and 21 days). At the end of each time, the animals were submitted to the cognitive test and were euthanized. The prefrontal cortex and hippocampus were removed for further analysis. In the second experiment, the mice were divided into 4 groups: Control Group (sham + vehicle); Group Aβ1-42 (Aβ1-42 + vehicle); SAHA group (25 mg / kg, intraperitoneal route) (sham + SAHA); Interaction Group (Aβ1-42 + SAHA). The Aβ1-42 peptide or vehicle was infused by i.c.v. and one day later the treatment was started i.p. for 21 days. At the end of the experiment the animals were submitted to the cognitive test, euthanasia for removal of the cerebral structures. The samples were used for the determination of BDNF levels, expression of BDNF mRNA, histone enzymatic activity (HDAC and HATs) and regulation of the cAMP / PKA / CREB pathway. The present study observed significant deficiencies caused by Aβ1-42 in memory (Morris Aquatic Labyrinth), as well as caused imbalance of HAT / HDAC enzymes, cAMP, PKA and CREB and BDNF reduction in the prefrontal cortex and hippocampus of mice. Inhibition of HDAC with SAHA demonstrated neuroprotection in behavioral and neurochemical changes induced by Aβ1-42. These data show that acetylation through inhibition of HDAC plays a key role in memory mediation and demonstrates that SAHA may be a promising medical tool in the therapeutic approach to AD.

Wong Yue Hang Albert. / Thesis submitted in: December 2005. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 108-119). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.v / List of Figures --- p.x / List of Tables --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter Chapter 2 --- Literature Review --- p.4 / Chapter 2.1 --- Nasopharyngeal Carcinoma (NPC) --- p.4 / Chapter 2.1.1 --- Anatomy of Nasopharynx --- p.4 / Chapter 2.1.2 --- Histopathology of Nasopharyngeal Carcinoma --- p.5 / Chapter 2.1.3 --- Epidemiology and Etiology of Nasopharyngeal Carcinoma --- p.5 / Chapter 2.1.3.1 --- Endemic Regions of Nasopharyngeal Carcinoma --- p.5 / Chapter 2.1.3.2 --- Gender and Age Bias --- p.6 / Chapter 2.1.3.3 --- Nasopharyngeal Carcinoma in Hong Kong --- p.6 / Chapter 2.1.3.4 --- Environmental Factors and Diet --- p.7 / Chapter 2.1.3.5 --- HLA Haplotypes and Nasopharyngeal Carcinoma --- p.9 / Chapter 2.1.4 --- Epstein-Barr Virus (EBV) and Nasopharyngeal Carcinoma --- p.10 / Chapter 2.1.4.1 --- EBV and Human Cacners --- p.10 / Chapter 2.1.4.2 --- EBV Infection --- p.10 / Chapter 2.1.4.3 --- "Latent, Clonal EBV Infection" --- p.11 / Chapter 2.1.4.4 --- EBV Latency Form --- p.11 / Chapter 2.1.4.5 --- Reactivation of EBV --- p.12 / Chapter 2.1.5 --- Molecular Pathogenesis of Nasopharyngeal Carcinoma --- p.13 / Chapter 2.1.5.1 --- Genetic Changes --- p.13 / Chapter 2.1.5.2 --- Epigenetic Changes --- p.13 / Chapter 2.1.6 --- Therapy of Nasopharyngeal Carcinoma and its Deficiency --- p.14 / Chapter 2.1.6.1 --- Radiotherapy --- p.14 / Chapter 2.1.6.2 --- Concurrent Chemoradiotherapy --- p.16 / Chapter 2.1.6.3 --- Adjuvant and Neo-adjuvant Chemotherapy --- p.17 / Chapter 2.1.6.4 --- Chemotherapy in Metastatic Nasopharyngeal Carcinoma --- p.18 / Chapter 2.1.6.5 --- Novel Therapeutic Agents and Approach --- p.19 / Chapter 2.2 --- Histone Modification and Cancer --- p.20 / Chapter 2.2.1 --- Histone Modification and Transcription Regulation --- p.20 / Chapter 2.2.2 --- Carcinogenic Effect of Aberrant HAT and HDAC Activities --- p.21 / Chapter 2.2.3 --- Structural Classes of HDAC Inhibitors --- p.24 / Chapter 2.2.4 --- Anti-Cancer Mechanisms of HDAC Inhibitors --- p.25 / Chapter 2.3 --- Suberoylanilide Hydroxamic Acid (SAHA) --- p.27 / Chapter 2.3.1 --- Anti-tumor Effect of SAHA in Various Cancer Cell Lines --- p.27 / Chapter 2.3.2 --- SAHA Mediated Non-apoptotic Programmed Cell Death --- p.29 / Chapter 2.3.3 --- Anti-tumor and Preventive Effect of SAHA in Animal Model --- p.29 / Chapter 2.3.4 --- Clinical Trials of SAHA --- p.30 / Chapter 2.4 --- FK228 (Depsipeptide or FR901228) --- p.31 / Chapter 2.4.1 --- Anti-malignancy mechanism of FK228 --- p.31 / Chapter 2.4.2 --- Anti-angiogenesis --- p.32 / Chapter 2.4.3 --- Drug Resistance and FK228 --- p.33 / Chapter 2.4.4 --- Studies of FK228 on Animal Models --- p.33 / Chapter 2.4.5 --- Clinical Trials --- p.34 / Chapter 2.5 --- Histone Modification and Nasopharyngeal Carcinoma --- p.34 / Chapter Chapter 3 --- Materials and Methods --- p.36 / Chapter 3.1 --- Cell Lines --- p.36 / Chapter 3.2 --- EBER ish Hybridization (EBER ISH) --- p.37 / Chapter 3.3 --- HDAC Inhibitors --- p.38 / Chapter 3.4 --- Cellular Sensitivity of NPC Cell Lines to HDAC Inhibitors --- p.38 / Chapter 3.4.1 --- Drug Treatment --- p.38 / Chapter 3.4.2 --- Determining Relative Amount of Survival Cells (WST-1 Assay) --- p.39 / Chapter 3.5 --- Flow Cytometry Analysis --- p.40 / Chapter 3.5.1 --- Collecting Cells and Fixation --- p.40 / Chapter 3.5.2 --- Staining --- p.41 / Chapter 3.5.3 --- Flow Cytometry Analysis --- p.41 / Chapter 3.6 --- Protein Extraction --- p.41 / Chapter 3.6.1 --- Harvesting Samples --- p.41 / Chapter 3.6.2 --- Protein Extraction --- p.42 / Chapter 3.6.3 --- Protein Quantification --- p.42 / Chapter 3.7 --- Western Blotting --- p.43 / Chapter 3.7.1 --- SDS-Polyarcylamide Gel Electrophoresis (PAGE) (SDS-PAGE) --- p.43 / Chapter 3.7.2 --- Wet Transfer of Proteins --- p.43 / Chapter 3.7.3 --- Immunoblotting --- p.44 / Chapter 3.7.4 --- Signal Detection --- p.44 / Chapter 3.8 --- CodeLin´kёØ Oligonucleotide Microarray --- p.45 / Chapter 3.8.1 --- HDAC Inhibitor Treatment --- p.45 / Chapter 3.8.2 --- RNA Extraction --- p.45 / Chapter 3.8.3 --- Quality and Quantity Assessment of Total RNA Extracted --- p.46 / Chapter 3.8.4 --- CodeLinkIM Expression Bioarray System --- p.46 / Chapter 3.8.5 --- Data Analysis --- p.48 / Chapter 3.9 --- Real-time Reverse Transcription PCR (Real-time RT-PCR) --- p.48 / Chapter Chapter 4 --- Results --- p.50 / Chapter 4.1 --- Presence of EBV --- p.50 / Chapter 4.2 --- Anti-prolirative Effect of HDAC Inhibitors --- p.52 / Chapter 4.3 --- Histone Acetylation --- p.56 / Chapter 4.4 --- Induction of p21 Expression in NPC Cell Lines --- p.58 / Chapter 4.5 --- HDAC Inhibitors Induced Cell Cycle Arrest and Polyploidy Formation --- p.60 / Chapter 4.5.1 --- Trichostatin A Induced G2/M Arrest --- p.60 / Chapter 4.5.2 --- Suberoylanilide Hydroxamic Acid Induced G1 Arrest --- p.62 / Chapter 4.5.3 --- FK228 Mediated G2/M Arrest --- p.64 / Chapter 4.6 --- HDAC Inhibitors Altered the Expression of Cell Cycle Regulatory Proteins --- p.66 / Chapter 4.6.1 --- TSA Down-regulated Cyclin A and B --- p.66 / Chapter 4.6.2 --- Suppressed Expression of Cyclin D1 and B by SAHA --- p.69 / Chapter 4.6.3 --- Effect of FK228 on Expression of Different Cyclins in NPC Cell Lines --- p.71 / Chapter 4.7 --- Effect of HDAC Inhibitors on EBV Proteins --- p.73 / Chapter 4.8 --- HDAC Inhibitors Modulated Gene Expression Profile --- p.76 / Chapter 4.8.1 --- SAHA and FK228-Induced Gene Expression Profile --- p.76 / Chapter 4.8.2 --- Validation of Expression Profile of Selected Genes by Real-time RT-PCR --- p.83 / Chapter Chapter 5 --- Discussion --- p.87 / Chapter 5.1 --- Anti-proliferative Effect of SAHA and FK228 on NPC Cell Lines --- p.88 / Chapter 5.2 --- Resistance of SAHA or FK228 in NPC --- p.93 / Chapter 5.3 --- Growth Inhibitory Mechanism of SAHA and FK228 in NPC Cells --- p.94 / Chapter 5.4 --- Induction of Polyploidy Cells in NPC Cell Lines --- p.98 / Chapter 5.5 --- Does EBV play a Role in HDAC Inhibiotrs Induced Growth Arrest in NPC Cell Lines? --- p.99 / Chapter 5.6 --- Transcriptional Signature of SAHA and FK228 in NPC Cell Lines --- p.100 / Chapter 5.7 --- Combining SAHA or FK228 with other Anti-tumor Agents --- p.104 / Chapter 5.8 --- Future Prospectus --- p.105 / Chapter Chapter 6 --- Summary --- p.106 / References --- p.108 / Appendix 1 --- p.120 / Appendix 2 --- p.121

Salicylic Acid Binding Protein2 (SABP2) is an enzyme known to play important role in SA mediated pathway. SBIP-428 (SABP2 Interacting Protein-428), a SIR2 like deacetylase, has been found to interact with SABP2. We demonstrate that SBIP-428 functions as a Sirtuin deacetylase. We show that SBIP-428 itself is lysine acetylated. Interactions of a SBIP-428 with SABP2 also raised the possibility of SABP2 itself being lysine acetylated. The recombinant purified SABP2 or native partially purified SABP2 displayed no acetylation. In response to TMV infection, the expression of SBIP-428 was down regulated at 48 hpi. In addition, SBIP-428 was up regulated in plant known to accumulate less SA. Taken together expression of SBIP-428 is negatively correlated to the levels of SA in plants. The AtSRT2 plants exhibit no altered growth phenotype but exhibit a higher pathogen resistance against bacterial pathogen. Our results indicate that SBIP-428 is an important regulator in plant defense pathway.

Human adenovirus (HAdV) mainly causes minor illnesses, but can lead to severe disease and death in both immunocompromised and immunocompetent patients. In such cases, the current standards of treatment often do not improve disease outcome and no approved antiviral therapy against HAdV exists. Since HAdV relies on cellular machinery to assist in the progression of the virus lifecycle, we hypothesized that small molecules targeting certain cellular proteins/pathways, without severely affecting cell health, may serve as effective anti-HAdV compounds. Thus, we aimed to identify novel inhibitors of HAdV, and investigate the molecular mechanism to determine new therapeutic targets for intervention in HAdV infection. We first examined the antiviral properties of pan-histone deacetylase (HDAC) inhibitor SAHA and found that the drug affects multiple stages of the HAdV lifecycle, resulting in significant reductions in virus yield. SAHA was effective in decreasing gene expression from clinically relevant HAdV serotypes. Subsequent investigations on the role of HDACs in HAdV infection led us to determine that class I HDAC activity, mainly HDAC2, is necessary for optimal viral gene expression. Using a wildtype-like HAdV reporter construct that allows us to monitor virus replication by fluorescence microscopy, we then designed an efficient system for screening small molecules to identify novel HAdV inhibitors. We screened over 1300 small molecules, and the screen was sensitive enough to detect compounds with both robust and modest antiviral activity. Several positive hits were validated to reduce HAdV gene expression and yield from infected cells. Further investigation on the efficacy of these compounds and the mechanism behind their inhibition of HAdV can lead to the discovery of new pharmacological targets and the development of more effective antivirals.

<p>Heparan sulfate (HS) is a glycosaminoglycan present in all cell types covalently attached to core proteins forming proteoglycans. HS interacts with different proteins and thereby affects a variety of processes. The biosynthesis of HS takes place in the Golgi network where a complex of the enzymes EXT1 and EXT2 adds N-acetyl glucosamine and glucuronic acid units to the growing chain. The HS chain is <i>N</i>-sulfated by the enzyme <i>N</i>-deacetylase <i>N</i>-sulfotransferase (NDST). <i>N</i>-Sulfation occurs in domains where further modifications (including <i>O</i>-sulfations) take place, giving the chain a complex sulfation pattern.</p><p>In this thesis, new data about the regulation of NDST enzyme activity is presented. By studying NDST1 with active site mutations overexpressed in HEK 293 cells we show that <i>N</i>-deacetylation is the rate-limiting step in HS <i>N</i>-sulfation and that two different NDST molecules can work on the same GlcN unit.</p><p>By analyzing recombinant forms of NDST1 and NDST2 we determined the smallest substrate for <i>N</i>-deacetylation to be an octasaccharide. Importantly, the sulfate donor PAPS was shown to regulate the NDST enzymes to modify the HS chain in domains and that binding of PAPS had a stimulating effect on <i>N</i>-deacetylase activity. </p><p>We could also show that increased levels of NDST1 were obtained when NDST1 was coexpressed with EXT2, while coexpression with EXT1 had the opposite effect. We suggest that EXT2 binds to NDST1, promoting the transport of functional NDST1 to the Golgi network and that EXT1 competes for binding to EXT2. </p><p>Using cell lines overexpressing EXT proteins, it was demonstrated that overexpression of EXT1 increases HS chain length and coexpression of EXT2 results in even longer chains. The enhancing effect of EXT2 was lost when EXT2 was carrying mutations identical to those found in patients with hereditary multiple exostoses, a syndrome characterized by cartilage-capped bony outgrowths at the long bones.</p><p>.</p>

Heparan sulfate (HS) is a glycosaminoglycan present in all cell types covalently attached to core proteins forming proteoglycans. HS interacts with different proteins and thereby affects a variety of processes. The biosynthesis of HS takes place in the Golgi network where a complex of the enzymes EXT1 and EXT2 adds N-acetyl glucosamine and glucuronic acid units to the growing chain. The HS chain is N-sulfated by the enzyme N-deacetylase N-sulfotransferase (NDST). N-Sulfation occurs in domains where further modifications (including O-sulfations) take place, giving the chain a complex sulfation pattern. In this thesis, new data about the regulation of NDST enzyme activity is presented. By studying NDST1 with active site mutations overexpressed in HEK 293 cells we show that N-deacetylation is the rate-limiting step in HS N-sulfation and that two different NDST molecules can work on the same GlcN unit. By analyzing recombinant forms of NDST1 and NDST2 we determined the smallest substrate for N-deacetylation to be an octasaccharide. Importantly, the sulfate donor PAPS was shown to regulate the NDST enzymes to modify the HS chain in domains and that binding of PAPS had a stimulating effect on N-deacetylase activity. We could also show that increased levels of NDST1 were obtained when NDST1 was coexpressed with EXT2, while coexpression with EXT1 had the opposite effect. We suggest that EXT2 binds to NDST1, promoting the transport of functional NDST1 to the Golgi network and that EXT1 competes for binding to EXT2. Using cell lines overexpressing EXT proteins, it was demonstrated that overexpression of EXT1 increases HS chain length and coexpression of EXT2 results in even longer chains. The enhancing effect of EXT2 was lost when EXT2 was carrying mutations identical to those found in patients with hereditary multiple exostoses, a syndrome characterized by cartilage-capped bony outgrowths at the long bones. .

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Burkitt lymphoma (BL) was the first tumor found to be strongly associated with Epstein-Barr virus (EBV). Almost 100% of the lymphoma cells are cycling, necessitating dose- and time-intense multi-agent chemotherapy regimens to achieve a cure of the disease. Whilst standard risk BL can be cured with this approach, high risk BL with leukaemic and CNS disease has significantly inferior survival. The intensive chemotherapy regimen causes considerable toxicity to the patients and relapse of BL is largely incurable. Thus, novel therapeutic approaches for high risk and relapsed BL are needed. Histone deacetylase inhibitors (HDACis) represent a novel class of drugs with potent anti-cancer effect in a wide range of malignancies. In the first part of this study, we tested HDACis of different classes for their ability to inhibit cell proliferation and activate the lytic cycle of EBV in a panel of EBV-positive BL cells of different latent viral gene expression patterns (type I, Wp-restricted and type III latency with highly restrictive, partial and full spectrum of EBV latent gene expression, respectively). Different HDACis could inhibit proliferation of EBV-positive BL cells in a time- and dose-dependent manner but only weakly activate the viral lytic cycle indicating that the drugs’ cytotoxic effect is independent of the EBV lytic cycle. Of note, BL cells of Wp-restricted or type III latency were more resistant to killing by HDACis than those of latency I, suggesting a possible link between relative resistance to the drug and expression of the latent viral genes. Bortezomib, a proteasome inhibitor, may have synergistic action with HDACis on lymphoid malignancies. We hypothesized that Bortezomib could potentiate the killing of EBV-positive BL cells by HDACis. In the second part, we tested the effect of combination of a FDA-approved HDACi, suberoylanilide hydroxamic acid (SAHA) and Bortezomib in the same panel of BL cells and also EBV-transformed lymphoblastoid cell lines (LCLs) which represent an in-vitro model of EBV-associated post-transplant lymphoproliferative disorder (PTLD). Interestingly, combination of SAHA and Bortezomib significantly enhanced the killing of BL cells of Wp-restricted or type III latency. Furthermore, the resistance to either SAHA or Bortezomib alone in contrast to synergistic killing by the combination of the two drugs could be observed in LCLs which also have the type III latency pattern. Compared with either drug alone, combination of SAHA and Bortezomib induced enhanced apoptosis in Wp-restricetd BL cells and LCLs as shown by the increase in the percentage of annexin V-positive cell, sub-G1 population and the proteolytic cleavage of apoptotic markers including PARP, caspase-3 and -9. The drug combination hyper-acetylated histone and induced cell cycle arrest. Combination of SAHA and Bortezomib was further shown to suppress the growth of BL xenograft in nude mice. In conclusion, our data indicated that expression of partial or full spectrum of viral latent genes in EBV-positive BL cells of Wp-restricted or type III latency confers resistance of the tumor cells to cytotoxic effect of HDACis. Bortezomib could potentiate SAHA-induced apoptosis of both BL cells and LCLs and might overcome mechanism of drug resistance. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

Proteoglycans (PGs) are composed of highly sulfated glycosaminoglycans chains (GAGs) attached to specific core proteins. They are present in extracellular matrices, on the cell surface and in storage granules of hematopoietic cells. Heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS) GAGs play indispensable roles in a wide range of biological processes, where they can serve as protein carriers, be involved in growth factor or morphogen gradient formation and act as co-receptors in signaling processes. Protein binding abilities of GAGs are believed to be predominantly dependent on the arrangement of the sugar modifications, sulfation and epimerization, into specific oligosaccharide sequences. Although the process of HS and CS/DS assembly and modification is not fully understood, a set of GAG biosynthetic enzymes have been fairly well studied and several mutations in genes encoding for this Golgi machinery have been linked to human genetic disorders. This thesis focuses on the zebrafish N-deacetylase/N-sulfotransferase gene family, encoding key enzymes in HS chain modification, as well as glycosyltransferases responsible for chondroitin/dermatan sulfate elongation present in zebrafish. Our data illustrates the strict spatio-temporal expression of both the NDST enzymes (Paper I) and CS/DS glycosyltransferases (Paper II) in the developing zebrafish embryo. In Paper III we took advantage of the four preexisting zebrafish mutants with defective GAG biosynthesis. We could demonstrate a relation between HS content and the severity of the pectoral fin defects, and additionally correlate impaired HS biosynthesis with altered chondrocyte intercalation. Interestingly, altered CS biosynthesis resulted in loss of the chondrocyte extracellular matrix. One of the main findings was the demonstration of the ratio between the HS biosynthesis enzyme Extl3 and the Csgalnact1/Csgalnact2 proteins, as a main factor influencing the HS/CS ratio. In Paper IV we used the newly developed CRISPR/Cas9 technique to create a collection of zebrafish mutants with defective GAG biosynthetic machineries. Lack of phenotypes linked to null-mutations of most of the investigated genes is striking in this study.

Today, cancer is understood as an epigenetic as well as a genetic disease. The main epigenetic hallmarks of the cancer cell are DNA methylation and histone modifications. The latter changes may be an optimal target for novel anticancer agents. The main goal of using histone deacetylase inhibitors (HDACIs) would be restoration of gene expression of those tumor-suppressor genes that have been transcriptionally silenced by promoter-associated histone deacetylation. However, HDACIs have pleiotropic effects that we are only just starting to understand. These may also be responsible for the induction of differentiation, cell-cycle arrest and pro-apoptotic effects. There are now so many HDACIs available, with such different chemical structures and biological and biochemical properties, that it is hopeful that at least some of them will succeed, probably in combination with other agents or therapies. In our studies we focussed ourselves on studies some new HDACIs, that can be useful for treating cancers, including leukemia and epithelial cancer. To do that, we used novel HDACIs, like BML-210, and their combination with the differentiation inducer all-trans retinoic acid (ATRA). Cell differentiation and proliferation in general, and specific gene expression require de novo protein synthesis and/or post-translational protein modifications. So, we tried to identify proteins in general and specifically the proteins that could be important for the cell differentiation process, and when and where in the cell the proteins appear. We delineated that HDACIs inhibited leukemia (NB4 and HL-60) cell growth in a time- and dose-dependent way. Moreover, BML-210 blocked HeLa cell growth and promoted apoptosis in a time-dependent way. Combining of BML-210 with ATRA induced a differentiation process in leukemia cell lines that lead to apoptosis. This correlated with cell cycle arrest in G0/G1 stage and changes in expression of cell cycle proteins (p21, p53), transcription factors (NF-κB, Sp1) and their binding activity to consensus or specific promoter sequences. We also assessed histone modifications, i.e. H3 phosphorylation and H4 hyperacetylation due to HDACI, leading to chromatin remodeling and changes in gene transcriptions. We have also studied changes in protein maps caused by HDACIs and differentiation agents, identifying differences for a few proteins due to growth inhibition and induction of differentiation in NB4 cells using BML-210 alone or in combination with ATRA. These proteins are involved in cell proliferation and signal transduction, like Rab, actin and calpain. One of them was alpha-dystrobrevin (α-DB). To further study possible roles of the latter, we determined changes of α-DB protein isoform expression that correlated with induction of differentiation. We thus identified a novel ensemble of α-DB interacting proteins in promyelocytic leukemia cells, including tropomyosin 3, actin, tubulin, RIBA, STAT and others, being important in cytoskeleton reorganization and signal transduction. Using confocal microscopy, we determined that α-DB co-localizes with HSP90 and F-actin in NB4 and HeLa cells. We also revealed that it changes sub-cellular compartment after treatment with ATRA and/or BML-210. α-DB silencing affected F-actin expression in HeLa cells, further supporting the idea that α-DB is involved in cytoskeleton reorganization in cells. Altogether, our results suggest that α−DB may work as a structural protein during proliferation and differentiation processes of human cancer cells. Based on our findings, we suggest that HDACIs, like BML-210, can be promising anticancer agents, especially in leukemia treatment, by inducing apoptosis and regulating proliferation and differentiation through the modulation of histone acetylations and gene expression.