GENETIC AND EPIGENETIC ANALYSIS OF IMMORTAL AND SENESCENCE ARRESTED LIVER CANCER CELLS A THESIS SUBMITTED TO THE DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS AND THE INSTITUTE OF ENGINEERING AND SCIENCE OF BİLKENT UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY By G. SEVG İ BA ĞIŞLAR August 2009 I certify that I have read this thesis and that in my opinion it is fully adequate, in scope and in quality, as a thesis for the degree of Doctor of Philosophy. ________________________________ Prof. Dr. Mehmet Öztürk (Supervisor) I certify that I have read this thesis and that in my opinion it is fully adequate, in scope and in quality, as a thesis for the degree of Doctor of Philosophy. ________________________________ Asst. Prof. Dr. Özlen Konu I certify that I have read this thesis and that in my opinion it is fully adequate, in scope and in quality, as a thesis for the degree of Doctor of Philosophy. ________________________________ Asst. Prof. Dr. Cengiz Yakıcıer I certify that I have read this thesis and that in my opinion it is fully adequate, in scope and in quality, as a thesis for the degree of Doctor of Philosophy. ________________________________ Prof. Dr. Funda Yılmaz-Barbet I certify that I have read this thesis and that in my opinion it is fully adequate, in scope and in quality, as a thesis for the degree of Doctor of Philosophy. ________________________________ Doç. Dr. Esra Erdal Approved for the Institute of Engineering and Science _______________________________ Prof. Dr. Mehmet Baray Director of Institute of Engineering and Science ii To my mother….. Anneme….. iii ABSTRACT G. SEVG İ BA ĞIŞLAR PhD in Molecular Biology and Genetics Supervisor: Prof. Dr. Mehmet Öztürk August 2009, 123 Pages 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 iv 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. v ÖZET İMMORTAL VE HÜCRE YA ŞLANMASI PROGRAMLI KARAC İĞ ER KANSER HÜCRELER İNDE GENET İK VE EP İGENET İK ANAL İZİ G. SEVG İ BA ĞIŞLAR Doktora Tezi, Moleküler Biyoloji ve Genetik Bölümü Danı şman: Prof. Dr. Mehmet ÖZTÜRK Ağustos 2009, 123 Sayfa Hepatoselüler karsinomda (HCC), hücre ya şlanması ve immortalitesinin genetik ve epigenetik özellikleri henüz zayıf olarak açıklanmıştır. Tezimizin amacı; karaci ğer kanserinde rol alan hücre ya şlanması ve immortalite gen a ğını karakterize etmektir. Ayrıca, hücre ya şlanması ve immotral HCC hücrelerinin epigenetik de ğişikliklerini de incelemek istedik. İlk önce, immortal, erken hücre ya şlanması ve hücre ya şlanması Huh7 klonlarında gen ifade farklılıklarını bulduk. Mikroarray çip çalı şmamız sonucunda 6390 prob seti, bu gruplar arasında de ğişiklik gösterdi. Daha fazlası, bu anlamlı gen seti; hem replikatif hücre ya şlanması hem de oncogen indüklenmi ş hücre ya şlanması modellerini, bunların immortalize edilmi ş kar şılıklarından ba şarı ile ayırdı. Biyolojik fonksiyon gruplama çalı şmaları; E2F1 hedef genlerinin, kök hücre ili şikli genlerin, DNA tamir, RNA splicing ve hücre döngüsü ili şik gen gruplarının özellikle immortal hücrelerde ifadesinin arttı ğını gösterdi. Öte yandan, immün sistem, stres cevabı, elektron ta şınımı, protein modifikasyonu, metabolizma ve kromatin biyogenezi ile ilgili grupları ya şlanmı ş hücre klonlarında ifade gösteriyordu. Ayrıca, immortal ve hücre ya şlanması programlı hücre datasıyla siroz ve HCC doku datasını birle ştirerek, ortak de ğişen “hücre ya şlanması ve immortal gen ağı (SIGN) listesini elde ettik. SIGN gen seti, farklı HCC alt gruplarını birbirinden ayırmayı ba şardı. SIGN gen setinde hücre ya şlanmasından immortale do ğru anlamlı olarak de ğişen gruplar; hepatic de-differensiye genleri, hücre döngüsü, kromozom modifikasyonu ve DNA hasarına cevap veren gen grupları idi. Ayrıca, geni ş bir sayida DNA tamir genlerinin tümor olu şumu ve ilerlemesi ile alakalı oldu ğunu belirledik. Retinoblastoma/E2F yola ğı hücre ya şlanmasında önemli bir rol oynamaktadır, biz de bu yüzden E2F ve DP protein ailesi üyelerinin ya şlanmı ş ve immortal programlı hepatoselüler karsinomlarında analiz ettik. E2F1, E2F5, E2F7, E2F8 ve DP1 genlerinin vi ifadesi immortal hücrelerde artı ş gösterdi. Öte yandan E2F3a ve DP2 genlerinin ifadesi azalmı ştı. DP-2 ifadesinin senesens Huh7 hücrelerde artı şı; DP2 protein artı şı ile ilintilendirildi ve aynı artı ş TGF-beta indüklenmi ş hücre ya şlanmasında da gözlendi. Son olarak, hepatoselüler immortalite ve ya şlanmı ş hücrelerinde önemli epigenetik de ğişiklikler gösterdik. Histone metiltransferaz ve demetilazlar arasında; MLL3, FBXL11, SUV420H1, UTX, SMYD2, SETD2, JMJD2B, JMJD3, JARID1B ve ASH1L genlerinin hücre ya şlanmasında artı ş gösterdi ğini ve EZH2 geninin azalı ş gösterdi ğini gözlemledik. Bu de ğişiklikler e şli ğinde, histon metilasyon seviyeleri de de ğişikli ğe u ğruyordu. Ya şlanmı ş Huh7 ve MRC5 hücrelerde immortal e şde ğerlerine göre, H3K27me1- and 3, H4K20me3, H3R2me2a ve H4R3me2a rezidülerinin artı ş gösterdi ğini gözledik. H3K27me3, H4K20me3, H3K36me3, H3R17me2a, ve H4R3me2a rezidüleri bazı sirotik karaci ğer ve HCC tümörlerinde azalan bir ifade gösterdi. Sonuç olarak, geni ş sayıda immortal ve hücre ya şlanması genlerinin HCC de de de ğişti ğini göstermi ş olduk. Histon modifiye eden genleri ve histon metilasyon seviyesi de de ğişikli ğe u ğruyordu. Yani, hepatositlerin immortalizasyon süreçleri global gen ifade de ğişikli ği ile oluyor ve, büyük ihtimalle, bu de ğişim epigenetik program tarafından yönlendiriliyor. vii ACKNOWLEDGEMENTS I would like to express my gratitude to Prof. Mehmet Öztürk for his supervision, endless support and valuable suggestions throughout the course of my studies. It has always been a privilege for me to be accepted in his team, work with him and being educated by him. I would like to thank to the jury members for evaluating my thesis. I would like to express my special thanks to Assist. Prof. Özlen Konu for sharing her excellent experiences on bioinformatics, her support, her patience, and being such a kind person all the time. I would like to attend my very special thanks to Assist. Prof. Tamer Ya ğcı for his support, for scientific discussions and for paying attention to my scientific ideas, even if they were foolish. I would