Theses supervised by Prof. Dr. Mehmet Öztürk
31 theses · Muğla Sıtkı Kocman University, Manisa Celal Bayar University, Bolu Abant İzzet Baysal University, İhsan Doğramacı Bilkent University
Ülkemizde yetişen bazı mantarların poliol miktarlarının ve anti-inflamatuvar aktivitelerinin araştırılması
Bu tez çalışmasının amacı, Türkiye'de doğal olarak yetişen 14 mantar türünün bileşenlerinin GC-MS ile araştırılması, içerdikleri poliol miktarlarının tayin edilmesi, anti-inflamatuvar aktivitelerinin belirlenmesidir. Bu çalışmada, Türkiye'nin çeşitli illerinden toplanan 14 farklı tür ve çoğu halk tarafından kullanılmayan ağaç mantarlarına, CHCl3/MeOH çözücü sistemi ile çözücüde oda koşullarında bekletme ve kısa süreli ultrasonik banyoda bekletme şeklinde 2 farklı yöntem kullanılarak eksraksiyon yapıldı. Elde edilen ekstrelerin bileşenleri GC-MS yöntemi ile kalitatif ve kantitatif olarak belirlendi. Tüm ekstrelerin poliol miktarları tayin edildi, içerdikleri fenolikler ve organik asitler tanımlanıp miktarları belirlendi. Mantarların kullanılan yöntemler ile elde edilen ekstreleri ile yapılan anti-inflamatuvar aktivite testlerinde Phaelous schweinitzii (Fr.) Pat.'nin (IC50: 6,6 ± 0,6) yüksek anti-inflamatuvar aktivite gösterdiği, Leptoporus mollis (Pers.) Quél.'in (IC50: 14,5 ± 2,0) orta düzeyde, Hymenochaete rubiginosa (Dicks.) Lév. (IC50: 28,6 ± 0,1) ve Phellinus igniarius (L.) Quél.'un (IC50: 40,2 ± 5,2) düşük seviyede anti-inflamatuvar aktivite gösterdikleri tespit edildi. Mantarlarda bulunan majör poliolün mannitol sonrasında sırasıyla Ksilitol, Gliserol ve Eritritol oldukları görüldü. Yapılan çalışmada kullanılan mantarların yapısında en yüksek ortalama mannitol miktarına sahip mantarın Macrolepiota procera (Scop.) Singer, en yüksek ortalama ksilitol miktarına sahip mantarın Inocutis rheades (Pers.) Fiasson & Niemelä, en yüksek ortalama eritritol miktarına sahip mantarın Macrolepiota procera (Scop.) Singer, en yüksek ortalama gliserol miktarına sahip mantarın Omphalotus olearius (DC.) Singer olduğu tespit edildi. Aynı mantar üzerinde uygulanan çözücüde uzun süreli bekleme ve kısa süreli ultrasonik banyoda bekletme işlemlerinin ekstre ve ekstredeki bileşen miktarlarını etkilediği tespit edildi. Ülkemizde yetişen teze konu olan 14 mantarın önemli miktarda poliol yapılı maddelerin yanı sıra anti-inflamatuvar etki sağlayan birçok organik asit ve fenolik içerdikleri belirlendi.
Ege denizi kıyılarında bulunan bazı makro alg türlerinin antimikrobiyal ve antioksidan aktivitelerinin saptanması
Bu çalışmada; birinci bölümünde, Ayvalık, Çandarlı, Çeşme ve Kuşadası bölgelerinden toplanan 6 farklı makro alg türünün (Cystoseira barbata, Cystoseira compressa, Sargassum acinarium, Codium fragile, Enteromorpha linza, Jania longifurca,) antimikrobiyal ( antibakteriyal ve antifungal) aktiviteleri incelenmiştir. İki farklı ekstraksiyon yöntemine göre kloroform ve metanol çözgenleri kullanılarak elde edilen ekstraktlar, kuyu-difüzyon yöntemine göre antimikrobiyal etki yönünden incelenmiştir. İnhibisyon etki, 10 farklı test mikroorganizmasına karşı saptanmıştır. Kullanılan mikroorganizmalar; Staphylococcus aureus ATCC 6538p, Staphylococcus epidermidis ATCC 12228, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 7064, Salmonella typhimuriumCCM 583, Enterobacter cloacae ATCC 13047D, Escherichia coli , Candida albicans ATCC 10231, Proteus vulgaris ATCC 6897 `dir. Her bir ekstraktın verim hesabı yapılarak, uygun dilüsyonlardan sonra test mikroorganizmalarına karşı denenmiş ve inhibisyon etkisi zon çapları olarak ölçülmüştür. Sonuçlarda tüm alg türlerinin bazı ekstraktlarında belirgin inhibisyon zonları elde edilmiştir, en yüksek inhibisyon etki ise Codium fragile ve Enteromorpha linza türlerinde gözlenmiştir. Bu türlerde antimikrobiyal etki daha çok Bacillus türleri, E.coli, ve Enterococcus faecalis'e karşı gözlenmiştir.Çalışmamızın ikinci bölümünde, yine aynı bölgelerden toplanan 6 farklı makro alg türü (Padina pavonica, Sargassum acinarium, Cystoseira barbata, Jania rubens, Caulerpa racemosa, Laurencia obtusa ) antioksidan etkinliği yönünden incelenmiştir. Antioksidan kapasite spektrofotometrik olarak ve HPLC cihazının kullanılması ile kromotografik olarak ölçülmüştür. Antioksidan enzimlerden glutatyon peroksidaz (GSH-Px) enzimi, canlı sistemlerde reaktif oksijen türlerinden kaynaklanan hasar etkilerinin giderilmesinde önemli roller oynamaktadır. Glutatyon peroksidaz enzim aktivitesi ve hücre membran hasarının göstergesi olan lipid peroksidasyon (LPO) düzeyleri, alg türlerinde ölçülmüştür. Lipid peroksidasyon düzeylerinde anlamlı korelasyon saptanması için, örneklerin toplam protein içeriği de spektrofotometrik olarak ölçülmüş ve sonuçlar değerlendirilmiştir. GSH-Px etkinliği bakımından en yüksek düzey Padina pavonica (13,775 U/L) türünde elde edilirken, en yüksek lipid peroksidasyon ürünü olan malondialdehid (MDA) değeri ise Sargassum acinarium (2,228 mmol/mgr) türünden elde edilmiştir.
Çeşme yarımadası bazı denizel alg türlerinin kimyasal içerikleri ve besin değerleri
Bu çalışmada Çeşme yarımadası kıyılarından toplanan bazı alglerin kimyasal içerikleri ve besin değerleri bir yıl içerisinde her ay saptanmıştır. Örneklenen algler; Yeşil alglerden (Chlorophyeceae) Caulerpa racemosa (Forsskål) J. Agardh., Kahverengi alglerden (Phaeophyceae) Cystoseira barbata (Good et Woodward) C. Agardh 1842, Kırmızı alglerden (Rhodophyceae) Jania rubens (L.) Lamouroux, 1812, Laurencia obtusa (Hudson) Lamouroux türleridir.Bu türlerin toplam su, kuru madde, inorganik madde, organik madde, suda eriyebilir karbonhidrat, toplam azot ve protein içerikleri bir yıl içerisinde her ay saptanmıştır. Topladığımız bu alg türlerinde toplam protein miktarına Bradford ve Kjeldahl yöntemleri ile bakılmıştır. Örnekleme yaptığımız bölgelerde suyun fiziko-kimyasal parametreleri ölçülmüş ve toplanan örneklerin fotoğrafları da çekilerek çalışmaya eklenmiştir.
Çandarlı-Dilek yarımadası (Ege denizi) kıyısal alanlarında yayılış gösteren Posidonia oceanica üzerindeki Chlorophyceae, Phaeophyceae ve Rhodophyceae ait epifit türler
Akdeniz'de ve Ege Deniz'inde geniş yayılım alanlarına sahip deniz çiçekli bitkilerinden Posidonia oceanica (L.) Delile türünün vejetatif yapıları epifitik olarak pek çok türe substrat oluşturmaktadır. Çalışmanın amacı; P. oceanica türünün Ege Denizi sahil bölgelerinde oluşturduğu kommunite üyelerinden yeşil, kahverengi ve kırmızı alglere ait epifitik türlerin ve bu türlerin Ege Denizi'ndeki dağılımının belirlenmesidir. Ege Denizi'nin belirli bölgelerinde (Çandarlı, Yeni Foça, Eski Foça, Urla İskele, Teos ve Kuşadası) sahil kesimine yakın derinliklerde P. oceanica'nın oluşturduğu deniz çayırlıkları tespit edilerek arazi bölgeleri belirlenmiştir. Arazi çalışmaları sırasında düzenli olarak fiziko-kimyasal su parametreleri ölçülmüştür. Arazi bölgelerinde, Ekim 2009-Kasım 2010 tarihleri arasında serbest dalışlar yapılmış, düzenli olarak örneklemeler gerçekleştirilmiş, laboratuvara getirilen örnekler üzerinde epifit kommunite üyelerinden olan, yeşil, kahverengi ve kırmızı alglere ait türlerin tayini ilgili tayin anahtarları ve literatürler kullanılarak gerçekleştirilmiştir. Çalışma sonucunda, 12 yeşil alg, 13 kahverengi alg, ve 49 kırmızı alg üyesi tespit edilmiş ve epifitik alglerin Ege Denizi'ndeki yayılışları aydınlatılmıştır. Sonuçlar P. oceanica türünün, yayılış gösterdiği bölgelerde tür çeşitliliğinin artmasına büyük bir katkıda bulunduğunu kanıtlar niteliktedir. Bununla birlikte, tayini gerçekleştirilen 49 kırmızı alg türünden Polysiphonia atlantica Kapraun & J.N. Norris ve Spermothamnion johannis Feldmann-Mazoyer türleri ile yeşil alglerden Chaetomorpha indica (Kützing) Kützing türü Türkiye denizel alg florası için ilk kez belirtilmektedir.
Kaklık mağarası (Denizli)ve çevresi sulak alanlarda yayılış gösteren algler
Mağaralar özel yaşam koşullarına sahip, eşi bulunmayan doğal ekosistemlerdir. Her mağara sahip olduğu farklılıklar ile çok çeşitli canlı gruplarına yaşam olanağı sağlar. Denizli İli Kaklık Kasabası sınırları içerisinde yer alan Kaklık Mağarası, obruk biçimi ve hidrolojik açıdan zengin bir mağara oluşu ile pek çok canlı grubuna ev sahipliği yapmaktadır. Bu tezin amacı, bu özel ekosistemde yayılış gösteren alg türlerinin belirlenmesi ve yaşam ortamlarının tanımlanmasıdır. Bunun yanında ülkemizde önemli bir eksiklik oluşturan mağaraların biyolojisine de katkıda bulunulması hedeflenmiştir.Çalışma alanımız olan Kaklık Mağarasının termal özellikli Kokarhamam kaynağından çıkan su ile beslenmesi, mağaraya termal özellikler de katmaktadır. Ayrıca mağara, girişinin geniş olmasıyla fazlaca güneş ışığı almakta ve `sıcak mağara' sınıfına girmektedir. Kaklık Mağarası, böylesi farklı özellikler taşıması nedeniyle çalışma alanı olarak seçilmiştir. Mağara içi ve dışı alanların tümünü kapsayacak şekilde toplam 18 istasyon belirlenmiştir. İstasyonlardan Mayıs 2008 ve Ekim 2011 tarihleri arasında ilk 12'si periyodik 4'ü de ön gözlemlere dayalı 16 örnekleme çalışması yapılmıştır. Örneklenen türlerin laboratuarda canlı tayinleri yapılarak gerekli görülenler kültüre alınmıştır. Bunun yanında örnekler %2-4'lük formaldehit eklenerek saklanmıştır. Ayrıca arazi çalışmaları sırasında Kaklık Mağarasının çeşitli atmosferik ölçümleri ile onu besleyen Kokarhamam pınarından kaynaklanan suyun bazı fiziko-kimyasal ölçümleri yapılmıştır. Böylece alınan sonuçlar ile tür kompozisyonları arasında farklı yorumlara gidilebilmiştir. Toplanan türlerin tayini, ilgili tayin anahtarları ve literatür kullanılarak gerçekleştirilmiştir.Çalışma sonucunda, 49 Cyanobacteria, 2 Euglenophyta, 20 Heterokontophyta (18 Bacillariophyceae, 2 Xantophyceae) ve 14 Chlorophyta olmak üzere toplam 85 taksa tanımlanmıştır. Bu türlerden 25 tanesi Türkiye tatlı su ve toprak algleri florası için ilk kez bu çalışma ile belirtilmektedir.
Bakteriyel ekspresyon sisteminde safra tuzu hidrolaz (STH) rekombinant protein sekresyonu için ompa sinyal peptidinin test edilmesi
Proteins are allowed to enter the secretory pathway in both prokaryotic and eukaryotic organisms, only if they are accomplished with a specific signal destination which is called a signal peptide (SP). A typical signal peptide consisting of 25-30 residues is located at the N amino-terminal site of proteins and has the task of protein translocation. Mainly the structure of SP has three main parts: the first one is N-region that has the positive-charged domain, the second one is the H-region which is a hydrophobic core, and the third domain is the C-region that is the cleavage site. Signal peptidases are enzymes that bind to the C-site and release the protein in the secretory pathway. The secreted recombinant protein production ability of Escherichia coli is crucial to the biotechnology industry as it minimizes the production cost and downstream processing. However, the large-scale production of recombinant proteins has various obstructions, among which proteolytic degradation and inclusion body formation via proteases are the dominant ones. This dissertation work aims to investigate the secretory effect of the OmpA SP on a pET-based expression system for the production of recombinant bile salt hydrolase (BSH) in E. coli. To demonstrate that the secretory effect of the OmpA signal peptide, the bsh encoding genes and OmpA SP coding gene were cloned into pET-28b(+) plasmid DNA and obtained pEDE3 construct was overexpressed in E. coli BLR(DE3) cells. While the secretory effect of the OmpA SP was observed by the direct plate assay method, the expression levels of the BSH enzyme was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Direct plate assay results indicated that pEDE3/BLR(DE3) clones containing OmpA SP and bsh gene secreted BSH enzyme into the medium. However, SDS-PAGE results showed that soluble BSH proteins with 35-42 kDa in size were not visible on a polyacrylamide gel. These results demonstrated that the pEDE3 construct containing OmpA SP had the capacity to produce a releasable active BSH protein, albeit a small amount.
Molecular cloning and characterization of bile salt hydrolase from Lactobacillus fermentum
The gastrointestinal normal flora plays a vital role in human physiology, as it controls the host's nutritious, physiological, and immunological activities. In the human intestine, bile salt hydrolase (BSH), produced mainly by intestinal bacteria, catalyzes the hydrolysis of taurine or bile acid bound to glycine binding to amino acid and deconjugated bile acid. Deconjugated bile acids play an important role in the developing certain gastrointestinal diseases, such as gallstone formation, cholestasis, and colon cancer as well as blood cholesterol levels. However, the LAB are a group of microorganisms that mainly essensial for fermentative of dietary food in the market today. Because of the correlation with both BSH and human health, the structural and biochemical properties of BSHs require considerable research in order to understand their role in the regulation bacterial and host metabolism. The catalytic activities and substrate specificities of BSHs vary more. Some amino acids reported to be responsible for catalytic activity were also entirely kept intact across all BSH members of the same family. As an outcome, knowing and ability to understand the mechanism of action of the BSH enzymes of the probiotic microorganisms used in those treatments and fermentation is very vital. Despite the fact that Lactobacillus fermentum is likely one of the most beneficial probiotic bacteria strain on the market in the world today, little information is known about the mechanism of action and substrate preferences of this microorganism's BSH enzyme. In this study, the bsh gene encoding 326-amino acids from L. fermentum strain was cloned, expressed and characterized in Escherichia coli BLR(DE3) strain. An E. coli BLR(DE3)-pET22b expression system was used to produce rcombinant BSH. The recombinant BSH activity of L. fermentum was not determined qualitatively using the Direct Plate Assay with two different bile salts, glydeoxucocholic acid and taurodeoxycholic acid. In addition to this, 37 kDa BSH protein was not visible on SDS-PAGE analysis. Multiple sequence alignment revealed that BSH enzyme of L. fermentum had a one mutation (one codon deletion) on bsh gene. This mutation can affect the stability of BSH enzyme of L. fermentum. In this respect, L. fermentum cannot be a potential probiotic candidate since the capability of BSH activity is one of the criteria for the selection of the probiotics.
Investigation of the probiotic potential of Lactobacillus gasseri ATCC 33323 strain
Lactic acid bacteria (LAB) as a prime part of the human gut microbiota, have gained increasing interest as a research subject for characterization and the potential to be used as a probiotic candidate to promote human and animal health. The introduction of beneficial bacterial species into the gastrointestinal tract (GIT) may be a very appealing option to restore microbial equilibrium and prevent diseases given that the use of antibiotics among other forms of treatment, may change the composition and have an effect on the GIT microbiota. The aim of this study was to investigate and characterize the probiotic potential of the L. gasseri ATCC33323 strain. Probiotic features of the strain were screened using several tests, which included antimicrobial activity, acidifying activity, cell surface properties testing, tolerance to pH, bile salts, and phenol, antibiotic susceptibility, and anti-microbial activity tests including PCR and cloning. Results showed that L. gasseri ATCC33323 had an antagonistic effect against three pathogenic bacteria, S. aureus, E. coli, and L. monocytogenes RSKK. This strain also showed a high percentage of auto-aggregation with the highest value being 97.026% at the 5h mark, and high co-aggregation with all five pathogenic bacteria. L. gasseri ATCC33323 showed low acidifying activity, tolerance to varying pH points including pH 2, tolerance to bile with a growth rate of 50.6% in 0.3% concentration at the 4h mark, and resistance to differing phenol concentrations (0.1%, 0.2%, 0.3%, 0.4%) with 8.515log for 0.4% concentration at 24h mark, and it was susceptible to all four antibiotics which are ampicillin, kanamycin, erythromycin, and neomycin. In-silico analysis for bacteriocin screening results indicated the presence of a small protein obtained using specially designed primers, but further studies are required to identify bacteriocins in this strain. This study demonstrated that the L. gasseri ATCC33323 strain fulfilled the basic desired properties as a probiotic candidate and therefore has considerable potential to be used in the pharmaceutical and food industry.
Investigation of probiotic potential of human-derived Lactobacillus plantarum B14
Başlıca probiyotik mikroorganizmalar ve insan ve diğer memelilerin bağırsak mikrobiyotasının temel bileşenleri olan laktik asit bakterilerinin (LAB), konakçının sağlığı üzerinde önemli olumlu etkileri vardır. Bu nedenle, yeni probiyotik özelliklere sahip yeni probiyotiklerin keşfedilmesi, tıp, bilim ve gıda endüstrileri tarafından son derece takdir edilecektir. Probiyotik suşları çok çeşitli kaynaklardan elde edilebilmesine rağmen, insanlar için uygulanacak probiyotikler için en önemli kriterlerden biri insan kaynaklı olmasıdır. Bu çalışmanın amacı, insan gastro-intestinal sisteminden (GIS) kaynaklanan Lactobacillus Plantarum B14'ün probiyotik potansiyelinin in vitro karakterizasyonudur. Bu suşun probiyotik özellikleri, asit, safra tuzları ve fenol toleransı, antibiyotik duyarlılığı, anti mikrobiyal aktivite, asitleştirme kapasitesi ve agregasyon yeteneği dahil olmak üzere çeşitli testler kullanılarak incelenmiştir. Bakteriyosin geninin taranması için PCR ve gen klonlaması ile moleküler tanımlama da kullanılmıştır Sonuçlar, L. Plantarum B14'ün değişen asit ve safra koşullarına karşı yüksek bir toleransa ve farklı fenol konsantrasyonlarına (%0,1, %0,2, %0,3, %0,4) yüksek toleransa sahip olduğunu gösterdi, ve ampisiline duyarlılık, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes RSKK olan üç patojenik bakteriye karşı antimikrobiyal aktivite göstermiştir. L. Plantarum B14'ün yavaş asitleştirici olduğu ayrıca 5 saatlik inkübasyondan sonra %100'e ulaşan yüksek otomatik agregasyon ve test edilen beş patojenin tümü ile yüksek pıhtılaşma yeteneği gösterdiği bulundu. Klonlanmış PCR ürünü için yapılan in-silico analiz, bakteriyosin kodlayan genin saptanmadığını ve bu suştaki bakteriyosin kodlayan genlerin belirlenmesi için daha fazla araştırma yapılması gerektiğini göstermiştir. Bu testlerin sonuçları, L. Plantarum B14'ün istenen probiyotik özelliklere sahip olduğunu ve yeni bir probiyotik başlatıcı kültür geliştirmeyi amaçlayan daha fazla in vivo araştırma için aday olabileceğini göstermektedir.
Site-directed mutagenesis of Bile Salt Hydrolase (BSH) from Lactobacillus plantarum B14 and substrate specificity analysis of Mutant BSH enzymes
Bile acid (BA) deconjugation is catalysed by the bile salt hydrolase (BSH) enzyme, which is a member of the cholylglycine hydrolase (CGH) family and produced by intestinal bacteria. The clinically significant BSH alters BA-mediated signalling pathways related to lipid absorption, energy homeostasis and glucose metabolism. Nevertheless, BSHs exhibit varied substrate preference to different bile salts across sources. Despite the considerable research on BSH, the investigations of its molecular mechanisms concerning BSH substrate recognition remain limited. This research aims to analyze the correlation between the substrate specificity of Lactobacillus plantarum B14's BSH enzyme (LpBSH) and two residues, V58 and Y65, in its loop II. These residues possess aliphatic-hydrophobic and aromatic-hydrophobic properties, respectively. PCR-based site-directed mutagenesis was utilised to substitute M58, F58, N58, F65, L65 and C65 amino acids for V58 and Y65, respectively. The mutant recombinant LpBSHs (mrLpBSHs) were expressed using the BLR (DE3) strain of E. coli and the activity of mrLpBSHs against six different BAs were detected. The study showed that the V58 and predominantly Y65 residues in loop II could play a vital role in the structural site that is responsible for substrate specificity and catalysis. The results suggest that the Y65 and V58 residues of LpBSH can be involved in substrate specificity. It was also observed that collate group, rather than amino acid moieties, may determine the substrate specificity of BSH. However, further mutagenesis-based research on other CGH family members is necessary to comprehend the structure and substrate specificity relationships of BSHs. Obtained results indicated that BSHs have developed the ability to identify BAs at the steroid nucleus of cholate as well as at the amino acid groups. As a result, it seems likely that the only requirements for binding might be that the cholate moieties and amino acids match and complement the substrate-binding pockets appropriately.
Molecular and biochemical characterization of human originated Lactobacillus reuteri
According to clinical research intestinal probiotics are live microorganisms that provide health benefits when ingested in sufficient quantities and their main functions were related to modulating and maintaining the health of the intestinal tract. Probiotics have several novel applications because of their special ability to produce bioactive molecules including vitamins, bacteriocins, 3-indoleacrylic acid, Indole-3-aldehyde, short-chain fatty acids, exopolysaccharides, peptides, and proteins. These substances are essential in giving the host a variety of health advantages. Although Lactobacillus (L.) species are commonly used as probiotics, only limited data are available for the probiotic properties of human originated Lactobacillus reuteri. This study aims to characterize human originated L. reuteri for its probiotic potential according to the international guidelines for the evaluation of probiotics. In the present study, the probiotic potential of L. reuteri isolated from healthy humans was evaluated. The probiotic properties of L. reuteri were tested for aspects of antimicrobial activity, antibiotic susceptibility, adhesion and aggregation ability, bile salt tolerance low pH resistance and antimicrobial peptide production capacity. While clear zones of inhibition test results indicated that the cell free supernatant and cell lysate of L. reuteri had antagonistic activity against three pathogenic bacteria, Staphylococcus aureus, Yersinia enterocolitica, Salmonella typhimurium, the antibiotic test results showed that L. reuteri was sensitive for Erythromycin, but resistance for Neomycin and Kanamycin. On the other hand, L. reuteri showed a strong ability to adhere to pathogenic bacteria as well as self-binding because it has a high sedimentation rate. Furthermore, L. reuteri showed a high survival rate under acidic and bile salt containing environment that means gastrointestinal tract conditions. While in-silico analysis for BSH screening results revealed the existence of a protein acquired using specifically designed primers, further research is needed to identify BSH in this strain. Obtained results show that such properties of the human originated L. reuteri may be used as probiotics in the healthcare sector and various food products.
p53 proteinin Saccharomyces cerevisiae mayasındaki etkilerini incelemek için deney sisteminin oluşturulması
Establishment of an experimental system to study pS3 effects in Saccharomyces cerevisiae Tuba Dinçer M. S. in Molecular Biology and Genetics Supervisor: Prof. Dr. Mehmet öztürk August 1997, 82 pages The aim of this work was to establish an experimental model to study human wild type and mutant p53 protein effects in yeast Saccharomyces cerevisiae cells. Wild type p53 was previously shown to be a DNA damage response gene that controls the genome stability in mammalian cells. We are interested in using yeast cells to study human p53-mediated cellular events after DNA damage. In this study we established an experimental model as an initial step. For construction of this system pAK31 plasmid expressing a human mutant p53-248W as well as control plasmid pL3 were used to obtain two different yeast cell populations, one expressing the mutant p53 248W protein and the other without p53 expression. Wild type p53 expression vector was avoided because of its known growth inhibitory effects in yeast cells. In this experimental system initially p53 expression at pAK31 transformed yeast cells was shown. Then, the effect of mutant p53-248W expression to growth rate of yeast cells was analysed and no growth rate difference was detected between the cells expressing and non-expressing mutant p53-248W protein. To test the participation of mutant p53-248W protein in DNA damage response in yeast cells, cells were exposed to DNA damaging agents; UVC and cisplatin that were reported to induce wild type p53 protein in mammalian cells. Codon 248 is a common site of 'hot spot'mutation and the arginine residue that corresponds to codon 248 encoded by the wild type p53 sequence is in the DNA interacting face of the p53 protein. Mutant p53-248 (arg->trp) is defective in specific DNA binding and it has lost the ability to act as a transcription factor. Although there are not many reports about mutant p53-248W response to DNA damaging agents, it was shown that mutant p53- 248W exhibit decreased repair of active genes upon UV radiation. In this study cell survival was analysed in mutant p53-248W protein expressing yeast cells in parallel to mutant p53 protein levels following to DNA damage and no effect of mutant p53 248W expression on cell survival upon DNA damage was detected. Also no difference was detected in mutant p53 protein levels following DNA damage. in
İnsan meme kanseri hücre hatlarında ve meme tümörlerinde notch yolağı reseptör ve ligandlarının ekspresyonu
ABSTRACTExpression of Notch Signaling Pathway Receptors and Ligands inHuman Breast Cancer Cell Lines and Human Breast TumorsÖzden YalçınM.Sc. in Molecular Biology and GeneticsSupervisor: Prof. Dr. Mehmet ÖZTÜRKSeptember 2004, 69 pagesBreast cancer is the most common cancer type in women. Traditional therapiestargeting proliferating cells cannot be effective in all cases and recursion is observedin 40% of breast cancers within 10 years. One possible explanation is that the originof breast cancer is `breast cancer stem cells?, which cannot be killed by thesetherapies. Cancer stem cells are thought to be formed due to deregulation of normalstem cells. Breast tissue also contains normal stem cells required for its developmentduring puberty and pregnancy; and putative breast cancer stem cells have recentlybeen isolated. Investigation of pathways used in stem cell regulation is the first stepto understand the contribution of stem cells to tumorgenesis and design newtherapeutic approaches. Notch signaling is involved in stem cell maintenance andmany types of human cancers. Notch activation in mouse mammary glanddevelopment and tumorigenesis lead us to its possible role in human mammary glandtumorigenesis. The expression of Notch receptors and ligands were identified bysemi-quantitative RT-PCR in human breast cancer cell lines and tumor samples. Itwas found that Notch3 expression was strongly upregulated in cancer cells lines andtumors compared to normal cell line, while other receptors and ligands did not havesignificant changes in expression. Depending on the upregulation of Notch3expression in putative breast stem cells, we may hypothesize that its activation keepscells in a stem cell like phenotype, inhibit differentiation and increase cancer risk.ii
Hücre yaşlanmasıyla ilintli FAM134B geni tarafından kodlanan iki endoplasmik retikulum protein izoformunun belirlenmesi ve karakterizasyonu
Liver cancer is the fifth most common cancer in the world. Until recently, tumor cells were known to have the capacity to proliferate indefinitely. In a previous study, we showed the spontaneous induction of replicative senescence in p53-and p16INK4a-deficient HCC (hepatocellular carcinoma) cells. In a follow-up study, we have analyzed the Affymetrix expression profiling of the senescent and immortal HCC clones that we had established. Among the genes with differential expression pattern, in this study, we have focused on a novel gene, FAM134B (family with sequence similarity 134, member B), which is significantly up-regulated (p-value=1.097E-06) in our senescent clones with respect to their immortal counterparts. FAM134B gene is located on human chromosome 5p15.1 near a LOH region, and its protein product has not yet been characterized. To begin with, we confirmed the up-regulation of FAM134B in our senescent clones as compared to our immortal clones by RT-PCR analysis. As a next step, meta-analysis of HCC microarray data indicated that the expression of FAM134B gene is progressively down-regulated in non-metastatic and metastatic HCC as compared to normal liver. Thus, we decided to characterize the protein product of this gene. Two known forms of transcripts were used to construct FLAG-tagged expression plasmids (encoding two isoforms with predicted molecular weights of 30 and 55 kDa). Immuno-staining experiments performed after transient ectopic expression indicated that both short and long isoforms of FAM134B-encoded protein localize to the endoplasmic reticulum (ER). Both protein isoforms co-localized with calnexin, a well known ER-chaperon. Thus, it appears that senescent cells over-express FAM134B-encoded ER protein isoforms, while cancer cells are deficient in their expression. We have also performed gain-of-function studies by stable ectopic expression of these two protein isoforms in an HCC cell line and addressed the potential role(s) of these isoforms in senescence and ER-stress. Our studies indicated that over-expression of these proteins did not have a `causative? role in induction of senescence and did not affect the rate of cell proliferation. We also did not observe any changes in the responses of cells over-expressing these two protein isoforms to ER-stress induced via tunicamycin treatment. Therefore, FAM134B gene may be performing a yet unidentified function in senescent cells. All in all, we have identified two FAM134B-encoded proteins that localize to the ER, the function and the senescence association of which need further investigation.
Hepatoselüler karsinogenez belirteci olarak yaşlanma ve ölümsüzlük genleri
Cellular senescence is a tumor-suppression mechanism, and immortalizationfacilitates neoplastic transformation. Both mechanisms may be highly relevantto hepatocellular carcinoma (HCC) development. We worked on two ma jor as-pects of cellular senescence and immortality in HCC. First, we analyzed the roleof ZEB2 (Smad-interacting protein SIP1, ZFXH1) gene for a senescence-relatedrole in HCC. Then, we extended our work on the identi ? cation and analysis ofa senescence and immortality gene network (SIGN) in relation to hepatocellularcarcinogenesis. ZEB2 is a transcriptional repressor of E-cadherin, and inducesepithelial-mesenchymal transition (EMT), a key process involved in tumor metas-tasis and progression. However, ZEB2 is also a repressor of telomerase reversetranscriptase (TERT) gene, which encodes a key enzyme required for telomeremaintenance and tumor cell immortality. We performed in-vivo, in-silico andin-vitro studies to explore potential implications of ZEB2 in hepatocellular carci-noma (HCC). Tissue expression of ZEB2 transcripts displayed stepwise decreasesin HCC lesions, as compared to liver cirrhosis. This inverse correlation suggestedthat sustained ZEB2 expression is not compatible with HCC progression. Next,we studied in vitro e ? ects of ZEB2 knockdown and overexpression in HCC cells.Huh7 clones stably transfected with a speci ? c ZEB2-shRNA expression vectordisplayed increased colony formation and increased proliferation. Conversely,Hep3B cells stably transfected with ZEB2 expression vector displayed permanentcell cycle arrest associated with increased senescence-associated ß -galactosidaseactivity. ZEB2-induced senescence arrest was correlated with the repression ofTERT expression and concomitant upregulation of cyclin-dependent kinase in-hibitor p21Cip1 . Transient expression of ZEB2 did not induce p21Cip1 expression,suggesting an indirect mechanism. Finally, ZEB2 overexpression was not com-patible with in vitro survival of cancer cells, as ZEB2-overexpressing Hep3B andA431 clones depleted progressively during in vitro culture and expansion. Theseobservations suggest that the ZEB2 gene, aside from its role in EMT, also playsa negative role in HCC cell growth and survival.In the other study, we integrated gene expression data from senescence pro-grammed and immortal HCC cells with the data from cirrhosis and HCC tissuesto generate a SIGN signature. The SIGN signature accurately classi ? ed nor-mal liver, cirrhosis, dysplasia and HCC lesions, and indicated that senescence-to-immortality conversion ? rst occured during dysplasia-to-early HCC transi-tion. Senescence-to-immortality conversion contributed also to tumor progres-sion. This conversion was accompanied by hepatic dedi ? erentiation and increasedexpression of cell proliferation, chromosome modi ? cation and DNA damage re-sponse genes. Thus, HCC immortalization is closely associated with the ac-quisition of stem/progenitor-like features. Finally, we identi ? ed a large set ofupregulated DNA damage checkpoint and DNA repair genes that showed signif-icant associations with tumor initiation and progression. These genes may serveas potential targets for HCC prevention and therapy.
Farklılaşmış ve senesant projenitör oluşturma potansiyeli meme kanserinin heterojen yapısının başlıca belirleyicilerindendir
Breast cancer displays distinct subtypes, such as luminal A, luminal B, and basal-like. The prognosis and therapeutic response of each subtype is different. The mechanisms involved in the generation of these tumor types are poorly understood. Our aim was to test whether the ability to generate senescent progeny contributes to breast cancer heterogeneity. A panel of 12 breast cancer cell lines, 31 isogenic clones, and 12 breast tumors were used. We classified breast cancer cell lines into senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All ER+ cell lines tested and some ER-positive (ER+) breast tumors displayed senescence. Acute loss and tamoxifen-mediated inactivation of ER triggered a robust senescence response in SCP type T47D cell line. In contrast, ER-overexpression, estrogen treatment and p21Cip1 knockdown inhibited senescence. Neutralization of reactive oxygen species also abolished senescence. Breast cancer cell subtypes displayed divergent ability to produce differentiated progeny. The SCP subtype cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some cell lines generated only CD44+/CD24-/ ER-/ASMA- progenitor/stem-like cells, and others only CD24+/ER- luminal-like, but not ASMA+ myoepithelial-like cells. SCP cell lines were less tumorigenic, and they clustered with luminal A/normal like tumors. In contrast, ICP subtypes were more tumorigenic, and they clustered together with basal/luminal B tumors. Our results show that breast cancer cell lines clustering with luminal A/normal-like and basal/luminal B tumors respectively, differ from each other by the ability to generate differentiated and senescence-arrested progeny.
İmmortal ve hücre yaşlanması programlı karaciğer kanser hücrelerinde genetik ve epigenetik analizi
Genetic and epigenetic aspects of cellular senescence and immortality in hepatocellular carcinoma (HCC) are poorly elucidated. The aim of our thesis was to characterize senescence and immortality gene network (SIGN) involved in these cancers. We also wished to explore epigenetic changes associated with senescence and immortality of HCC cells. First, we identified differentially expressed genes in immortal, pre-senescent and senesce-arrested Huh7 clones. Our microarray analysis revealed 6390 probesets significantly changing among groups. Moreover, the significant gene signature could successfully discriminate both replicative senescent cells, and oncogene-induced senescent cells from their immortalized counterparts. E2F1 targets, stem-cell related genes, DNA repair, RNA splicing and cell cycle related gene sets were enriched specifically in immortal cells, whereas immune function, stress response, electron transporter activity, protein modification, metabolism, chromatin biogenesis related gene groups were significantly up-regulated in senescent clones. Next, we integrated gene expression data from senescence-programmed and immortal HCC cells with the data from cirrhosis and HCC tissues to generate a SIGN signature. This signature identified several HCC classes, including one ?normal-like?, and two with increased expression of immortality genes. Senescence-to-immortality transition was accompanied by hepatic dedifferentiation and increased expression of cell proliferation, chromosome modification and DNA damage response genes. Finally, we identified a large set of upregulated DNA damage checkpoint and DNA repair genes that showed significant associations with some SIGN classes of HCC tumors. As retinoblastoma/E2F pathway plays a key role in cellular senescence, we also analyzed E2F and DP family members in senescent and immortal hepatocellular carcinoma cells. E2F1, E2F5, E2F7, E2F8 and DP1 were up-regulated in immortal hepatocellular carcinoma (HCC) cell lines as compared to senescent cells, whereas E2F3a and DP-2 expressions were downregulated. Upregulation of DP2 expression in senescent cells correlated with increased DP2 protein expression, as tested with TGF-beta induced senescence models. Finally, we demonstrated important epigenetic changes associated with hepatocellular immortality and senescence. Among histone methyltransferases and demethylases, MLL3, FBXL11, SUV420H1, UTX, SMYD2, SETD2, JMJD2B, JMJD3, JARID1B and ASH1L genes were up-regulated, and EZH2 was down-regulated in senescent cells. These changes were accompanied with changes in histone methylation patterns. Of particular interest, H3K27me1, H3K27me3, H4K20me3, H3R2me2a and H4R3me2a forms of methylated histones displayed increased expression in both Huh7 and MRC5 senescent cells, as compared to their immortal forms. Finally, H3K27me3, H4K20me3, H3K36me3, H3R17me2a, H4R3me2a also showed decreased expression in some cirrhotic liver and primary HCC tumors. In conclusion, we demonstrated that a large set of senescence and immortailty genes were dysregulated in HCC. This profound change in gene expression was associated with differential expression of histone modifying enzymes, as well as histone methylation status. Thus, the immortalization of hepatocytes during hepatocellular carcinogenesis is accompanied with global gene expression changes probably mediated by a major modification of their epigenetic program via histone demethylation.
Karaciğer kanserinde histon metilasyonu ve metiltransferaz enzimi Set8
Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal cancers worldwide. The epigenetic modifications, which are involved in virtually all cellular processes are also involved in the carcinogenic process, and this is a growing new field of investigation. HCC has also been associated with several epigenetic aberrations which include the ones in histone modifications, histone methyltransferase enzymes, and the epigenetic machinery. The transition from cirrhosis to HCC is related to senescence bypass, and the distinctions between senescence and immortality in HCC cell lines. Global levels of H3K4me3, H3K9me3, H3K27me3, H3K36me3, H3R2me2, H3R17me2 and H4K20me3 histone marks were evaluated in well-differentiated and poorly differentiated HCC cell lines in the presence and absence of TGF-beta induced senescence. No prominent changes in the levels of these histone modifications were indentified in response to TGF-beta induced senescence. However, H4K20me3 levels appeared to correlate with the differentiation status of the cell lines, where a loss of methylation was observed in poorly differentiated cell lines. In order to address the mechanism of this loss, H4K20 specific methyltransferases were analyzed in terms of their transcript levels, and only the expression pattern of monomethyl transferase Set8 was found to correlate with the H4K20me3 methylation patterns. A potential role played by Set8 in HCC development was investigated via overexpression and knockdown studies. But no significant role could be attributed to this enzyme in this study.
Karaciğer kanserinde genetik ve epigenetik hedefler
Hepatocellular carcinoma (HCC) kills nearly 600.000 people each year and the only effective therapy for this cancer is liver transplantation or tumor ablation only when the tumor is small enough. These tumors are surprisingly resistant to conventional therapies such as chemotherapy and radiotherapy. Moreover, as HCC is almost always associated with cirrhosis, the treatment with cytotoxic agents is dangerous as they will also affect hepatic functions of the diseased liver. Therefore, there is urgent need to find novel therapeutic approaches against HCC in order to diminish death toll. Our overall goal is to discover ?druggable target genes? in HCC. In other words, we wish to identify novel genes and novel mechanisms involved in these cancers in order to use them as potential therapeutic targets.During my thesis work, I developed different approaches to find new mechanisms and novel targets:1- Deciphering the role of canonical Wnt signaling in HCC: We classified human HCC cell lines into "well-differentiated" and "poorly differentiated" subtypes, based on the expression of hepatocyte lineage, epithelial and mesenchymal markers. Poorly differentiated cell lines lost epithelial and hepatocyte lineage markers, and overexpressed mesenchymal markers. Also, they were highly motile and invasive. We compared the expression of 45 Wnt pathway genes between two subtypes. Likewise, six Frizzled receptors, and canonical Wnt3 ligand were expressed in both subtypes. In contrast, canonical ligand Wnt8b and noncanonical ligands Wnt4, Wnt5a, Wnt5b and Wnt7b were expressed selectively in well- and poorly differentiated cell lines, respectively. Canonical Wnt signaling activity, as tested by a TCF reporter assay was detected in 80% of well-differentiated, contrary to 14% of poorly differentiated cell lines. TCF activity generated by ectopic mutant b-catenin was weak in poorly differentiated SNU449 cell line, suggesting a repressive mechanism. We tested Wnt5a as a candidate antagonist. It strongly inhibited canonical Wnt signaling that is activated by mutant b-catenin in HCC cell lines.2. Systematic screening of protein kinases and phosphatases as potential therapeutic targets: There is evidence of aberrant activation of several signaling cascades in HCC, and a multikinase inhibitor, sorafenib, has shown survival benefits in patients with advanced HCC. We used siRNAs to screen a large number of kinases and phosphatases to identify related genes involved in HCC cell survival. A total of 7 kinases and 5 phosphatases were identified as strong candidate targets.3-Screening of a set of selected epigenetic regulators as potential therapeutic targets: Recent studies have indicated that senescence arrest or senescence escape could be regulated by epigenetic changes on chromatin. We wanted to identify key histone methylation and acetylation changes associated with senescence or senescence escape, and select key histone modifying enzymes, as potential targets for ?pro-senescence? interventions (therapeutic interventions that allow senescence induction in cancer cells). We identified ATAD2 as an epigenetic target, and found ATAD2 gene overexpressed in HCC compared to normal liver. We also found a stepwise increase of ATAD2 protein expression in late stages with respect to pre-neoplastic and early stage during hepatocellular carcinogenesis. ATAD2 knockdown using siRNAs in cancer cells leads to increase in global histone acetylation and inhibit cell proliferation and induce caspase-3 dependent apoptosis in HCC and induce senescence in MRC5 cells. Its potentiator role (coactivator role with estrogen, androgen and myc targets) indicates ATAD2 as a potential therepeutic target for HCC.
Karaciğer kanserinde başkalaştırıcı büyüme etmeni-beta?ya bağlı yaşlanma yanıtının moleküler mekanizmaları
Hepatocellular carcinoma (HCC) is the fifth most common cancer in theworld. HCC is associated with several etiological factors including infections withhepatitis B and C viruses, heavy alcohol consumption and chronic aflatoxin B1exposure. Due to its multi-step disease hallmark characterized with geneticheterogeneity, liver cancer has very limited therapeutic options. In light of manyprevious findings, cellular senescence acts as a barrier against immortalization andprohibits the proliferation of premalignant cells in various tumors including HCCs.However, implications of this anti-tumor mechanism in hepatic tissues are not wellknown.TGF-ß is a multifunctional cytokine implicated in diverse cellular processesincluding senescence arrest as well as liver physiology and pathophysiology.Although TGF-ß-induced senescence has been described in different cell types, thisissue has never been addressed for hepatic cells. According to our recent data, TGF-ß1 expression pattern in various HCC malignancies closely correlated with reportedfrequencies of SABG activities in these corresponding disease stages. Therefore, wehypothesized that TGF-ß signaling might play key role in hepatocellular senescence.Well-differentiated (WD) five cell lines characterized with epithelial-likemorphology displayed TGF-ß-induced growth inhibition associated with SABGactivity, with lack of evidence of apoptosis induction. Even a brief exposure to TGF-ß was sufficient to trigger a massive senescence response. Senescence arrest in WDcell lines was linked to c-myc down-regulation and a reciprocal increase in p21Cip1and p15Ink4b protein levels. In addition, TGF-ß-induced senescence was correlatedwith Nox4 induction, intracellular accumulation of reactive oxygen species (ROS)and sustained 53BP1 foci formation as a mark of DNA-damage response. Moreover,intratumoral injection of TGF-ß in human HCC tumors, generated subcutaneously inimmunodeficient mice, induced expanded SABG that was associated with a stronganti-tumor response activity.On the other hand, poorly differentiated (PD) HCC cell lines withmesenchymal-like characteristics appeared to be resistant to TGF?ß-inducedsenescence. However, PD cell lines had intact TGF-ß signaling from cell membraneto nucleus. Resistance of PD cell lines was partially due to zeb2 overexpression,homozygous p15Ink4b deletion and lack of pRb expression. Besides, PD cells did notdisplay Nox4 upregulation and also lacked ROS accumulation upon TGF-ßstimulation.In addition, we demonstrated that sustained exposure to TGF-ß establishedresistant Huh7 subclone. The resistance was partially attributed to deregulated Smadsignaling, permanent epithelial-mesenchymal transition-like transformation.Surprisingly enough, removal of TGF-ß from culture medium of continuously treatedHuh7 subclone did not resolve the resistance phenotype in the rescued subclone.Epigenetic regulations mainly histone modifications are considered as candidatemechanisms responsible for irreversible TGF-ß-resistance and maintenance ofmesenchymal-like phenotype. Taken together, our results establish a close linkbetween senescence arrest and anti-tumor activity of TGF-ß signaling pathway inWD cell lines by delineating the mechanisms underlying TGF-ß-induced growtharrest. Moreover, we propose partial explanation for the resistance to TGF-ß-mediated growth arrest in PD cell lines and thoroughly signify the potentialmechanisms of acquired resistance to TGF-ß in continuously treated cultures. Furtherstudies to enlighten our knowledge about implications of TGF-ß signaling in lessdifferentiated HCCs are necessary. As a conclusion, we identify TGF-ß signaling asa potent therapeutic option for well-differentiated early HCCs.
Karaciğer kanserinin moleküler patogenezinde diyet etiyolojik faktörlerin rolü
Hepatocellular carcinoma is ranked third foremost cause of cancer deaths. Dietary factorsplay a crucial role in the molecular pathogenesis of liver cancer. Oxidative stress isusually coupled with the malignancy and progression of HCC since it is considered as acommon factor during inflammation after chronic viral infection. Chemical stress causedby aflatoxin exposure, metabolic stress produced by alcohol abuse and seleniumdeficiency as a risk factor for HCC are associated with oxidative stress. It should beeliminated with an intact antioxidant defense mechanism. It is a major cause ofgenotoxicity endogenously through metabolic stress and exogenously produced bychemical and physical carcinogens. Even though the contribution of dietary factors inHCC progression has been established, the underlying molecular mechanism has not beenfully understood.Cancer cells may respond to genotoxic stress with a cryptic development of survivaladvantage mechanisms. Therefore we wanted to investigate this idea with dietary factorsinvolved in liver cancer. In this work, we studied the implication of Se-deficiency intumorigenesis of hepatocytes and the mechanism underlying the selective selection ofaflatoxins for p53-249 mutation in HCC. Aflatoxins are the most potent naturallyoccurring carcinogens and may play a causative role in 5-28% of hepatocellularcarcinomas, worldwide. Aflatoxins are activated in liver cells and induce principally G->T mutations, including a codon 249 (G->T) hotspot mutation of TP53 gene that isspecifically associated with aflatoxin-related hepatocellular carcinoma.However, our comparative analysis showed that R249S does not provide survivaladvantage at heterozygous state. Thus, the selection could be at the mutation inductionstage. The lack of p53 activation in Aflatoksin B1 exposed HCC cells led us to test DNAdamage response after aflatoxin exposure. Unexpectedly, DNA damage checkpointresponse to aflatoxins has not been studied thoroughly before. Although, DNA damagecheckpoint response acts as an anti-tumor mechanism by protecting genome integrityagainst genotoxic agents, this highly critical aspect of aflatoxin carcinogenicity is poorlyknown.Our findings provide evidence for the contribution of ERK, p38MAPK and PI3K/Aktsurvival pathways under selenium supplementation in some HCC cell lines. Apart fromthe effect of selenium deficiency, our results enlighten the aflatoxin carcinogenicity invitro. Our study pointed out for a negligent G1 and G2/M checkpoint response toaflatoxin B1-induced DNA damage. This defective response may account mostly formutagenic and carcinogenic influences of aflatoxins. It may also associate with thefrequent induction of TP53 hotspot mutation in aflatoxin-related human HCC.
FAM134B?nin karaciğer kanseri ve endoplazmik retikulum stresi konularındaki özelliklerinin belirlenmesi
Family with sequence similarity 134, member B (FAM134B) is a replicative senescence associated gene, previously identified in studies of our group as a result of microarray analysis in spontaneously senescent clones of Huh7 hepatocellular carcinoma cell line and their immortal counterparts. Originating from this finding, this study primarily focused on characterization of FAM134B in the context of hepatocellular carcinoma and endoplasmic reticulum stress. At the beginning, the relationship between senescence and FAM134B was experimented by inducing premature senescence in Huh7 cells. Adriamycin or TGF-ß induced premature senescence did not result in amplification of FAM134B gene expression, suggesting that upregulation of FAM134B expression in spontaneous replicative senescence is not directly associated with a senescence phenotype. Then, FAM134B mRNA and protein levels were analyzed in both well- and poorly-differentiated HCC cell lines. Results showed that FAM134B expression is greater in poorly-differentiated cell lines, which represent advanced and metastatic HCC in vitro. On the other hand, our studies on the relationship between FAM134B and endoplasmic reticulum (ER) stress showed that FAM134B is an ER stress response gene, whose expression is upregulated by induction of ER stress with chemicals, such as thapsigargin, tunicamycin or DTT. Therefore, high protein and mRNA levels of FAM134B in poorly-differentiated cell lines are linked to the presence of a basal level ER stress response in this group of cell lines. Furthermore, overexpression studies in Huh7 cells indicated that FAM134B cannot trigger an ER stress response or autophagic response in these cells. However, FAM134B was detected as an effector in cellular response, when ER stress is artificially induced by thapsigargin or tunicamycin treatments. FAM13B4 overexpression in Huh7 resulted in increased sensitivity to thapsigargin or tunicamycin induced apoptosis. Moreover, increased FAM134B expression was also associated with decreased proliferative capacity in response to ER stress induction with the same chemicals. Consequently, FAM134B was suggested to affect the severity of stress in the ER when ER stress is started with an inducer. In addition, our tissue based experiments revealed that FAM134B is expressed in the brain and liver. Taken together, FAM134B might be an important protein contributing to the liver tissue damage and pathogenesis of HCC.
Konformasyonal ERBB2 epitoplarini hedef alan özgün monoklonal antikorlar
ERBB2 is a tyrosine kinase receptor which can act as homodimers or heterodimers with other members of the ERBB family. Nearly 30% of breast cancers overexpress ERBB2, which can be effectively targeted by anti-ERBB2 monoclonal antibodies. Trastuzumab directed against an epitope on subdomain IV of the extracellular domain (ECD) of ERBB2 is a clinically used therapeutics but the response rate is poor and acquired resistance is frequent. Pertuzumab that binds to subdomain II and inhibits receptor dimerization is another promising therapeutics under clinical trials. Anti-ERBB2 antibodies directed to novel epitopes are potentially useful tools for replacement and combinatorial therapies. We produced five new anti-ERBB2 antibodies, all directed against epitope(s) present only on the native ECD. They performed selective growth inhibitory effects depending on the level of ERBB2 expression and cellular background. When used alone, novel anti-ERBB2 antibodies displayed modest but significant growth inhibition on SK-BR-3, BT-474 and MDA-MB-361 cells with ERBB2 overexpression; while no detectable inhibition was observed on MCF-7 and T47D cells lacking ERBB2 amplification. When the antibodies were tested in combination with TNF-?, they acted synergistically on SK-BR-3 cells, producing upto 80% growth inhibition; but performed antagonistically on BT-474 cells. Detailed investigation of a representative antibody indicated G1-arrest as the main mechanism of the anti-proliferative effects exerted on SK-BR-3 cells. Antibody treatment induced permanent inhibition of DNA synthesis, leading to accumulation of cells at G1-phase; an effect which was accelerated in the presence of TNF-?. In addition, treated SK-BR-3 cells displayed inhibition of Akt and ERK1/2 phosphorylation leading to cyclin D1 accumulation and growth arrest, independently from TNF-?. Novel antibodies against conformational epitopes present on the extracellular domain of ERBB2 receptor may serve as new analytical and diagnostic tools, in addition to being potent anti-cancer bioactive molecules. Cell-dependent synergy and antagonism between anti-ERBB2 antibodies and TNF-? provide evidence for a complex interplay between ERBB2 and TNF-? signaling pathways. Such complexity may drastically affect the outcome of ERBB2-directed therapeutic interventions.
FAM134B'nin karaciğer kanserindeki rolü
The family with sequence similarity 134, member B (FAM134B) protein initially caught attention in our laboratory through demonstrating elevated levels possibly associated with senescent, cirrhotic, and mesenchymal-like states in hepatocellular carcinoma (HCC), and the aim of this thesis work was to identify the role of FAM134B in HCC. The work in this thesis initially demonstrated that the induction of endoplasmic reticulum (ER) stress in normal mice livers with 8 hours of tunicamycin treatment did not significantly alter the levels of FAM134B mRNA or protein. We then demonstrated that induction of ER stress in four HCC cell lines using several ER stress inducers such as thapsigargin, tunicamycin or DTT did not cause a significant increase in the levels of FAM134B mRNA or protein, with the exception of high dose DTT treatment of Snu449 cells which also demonstrated apoptosis. We also observed that FAM134B protein levels may be elevated during epithelial-to-mesenchymal transition (EMT) in PLC cells induced by TGF-ß treatment. Snu449 cells in which FAM134B was silenced demonstrated an altered morphology in cell culture, and appeared to lose their migratory capabilities. Most significantly though, FAM134B-silenced Snu449 cells demonstrated a dramatic loss of resistance to treatment with thapsigargin and adriamycin, which are both known to inhibit the calcium pumps on the ER. The knockdown cells also demonstrated loss of resistance to serum starvation, TGF-ß treatment, tunicamycin and alcohol treatment, but no significant difference was observed in resistance to 5-FU, camptothecin and hydrogen peroxide. These results implicated that FAM134B may play a role that is essential for survival under several forms of cytotoxic threat, especially those that disturb calcium homeostasis.