Browsing by Subject "Liver neoplasms."

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Open Access
Analysis of members of the SLIT-ROBO pathway as diagnostic and prognostic tools in hepatocellular carcinoma with special focus on ROBO2-associated cellular phenotype
(2009) Avcı, Mehmet Ender
Hepatocellular carcinoma is the sixth most common cancer in the world, with an annual incidence exceeding half a million. It is associated mainly with hepatitis B and C viral infections; and is the main cause of death among cirrhotic patients. Aflatoxin B1 exposure, chronic alcohol consumption and virtually all cirrhosis-inducing conditions are of the other etiologies. For early diagnosis of HCC, surveillance of the risk groups is a crucial task requiring the development of novel markers for HCC with stronger sensitivity and specificity. In addition, description of biomarkers specific to hepatocellular carcinoma subtypes could identify novel targets for therapy. In this study, we analyzed members of the SLIT-ROBO gene families as novel diagnostic and prognostic markers in hepatocellular carcinoma. We defined an expression signature for members of the SLIT-ROBO gene families in HCC cell lines and tissues by real-time quantitative RT-PCR analysis. We showed that ROBO1 was overexpressed as stage and differentiation of the HCC proceeds. Furthermore, ROBO4 downregulation and SLIT2 overexpression marked late stage and poorly differentiated HCCs. Our results suggest that the expression of ROBO1 and ROBO4 can be used in early diagnosis of HCC. As another focus, we stably knockdowned ROBO2 expression in a model AFP positive cell line Huh7 and characterized the associated cellular phenotype. ROBO2 downregulation caused a significant decrease in proliferation rate whereas in wound-healing assay no significant difference in migration rate was observed. In addition, we performed a microarray experiment and found the differentially expressed genes between stable ROBO2 knockdown and negative clones. In this analysis, we found an overexpression of CK19, CD44, ABCG2/ BCRP1 hepatic progenitor cell markers and CD133 that is also a putative cancer stem cell marker of HCC, in stable ROBO2 knockdown clones. In addition KLF4 expression was augmented in these ROBO2 knockdown clones. We propose a genetic association between SLIT-ROBO pathway and CD133 at transcriptional level.
Open Access
Genetic analysis of Smad4 gene in TGF-Beta signalling pathway in human liver cancer
(1998) Irmak, Meliha Burcu
HCC is a multistep genetic disease in which many genomic changes occur as a result of uncontrolled proliferation of hepatocytes. Molecular events leading to HCC is still unclear. Until now, neither an oncogene nor a tumor suppressor gene has been shown to be prefentially altered in HCC. Genetic alterations other than p53, pl6, BRCA2 (Breast Carcinoma Associated Protein), M6P/IGFIIR (lyiannose 6 Phosphate/ Insulin Like Growth Factor II Receptor), Rb (Retinoblastoma), PRLTS (Platelet Derived Growth Factor Receptor-|3-Like Tumor Suppressor Gene), and Tg737 (Candidate polycyctic kidney disease gene) genes remain unknown. TGF-P is a strong inhibitor of hepatocyte proliferation. In HCC and cirrhosis increased levels of TGF-P is observed, so this shows that the presence of high levels of TGF-3 does not avoid hepatocyte proliferation. Thus, there may be a disruption in the signalling pathway of TGF-p. The common mediator Smad4 gene, which is among the genes located in TGF-P signalling pathway, is found to be mutated in many cancer types. We decided to do the mutational analysis of Smad4 gene, which is located in the signalling pathway of the hepatocyte antiproliferative factor, TGF-p. Exons 8, 9, 10, and 11 which are in MH2 region, and exon 2 which is in MHl region of Smad4 is mutationally analysed by SSCP for 35 HCC cases. In the 35 HCC tumors, 5 alterations were observed (14%), 3 of them being in exon 8, one of them being in exon 9a, and the last one being in exon 10 of Smad4 gene. In the samples we tested, no big deletions were observed, but the alterations observed are probably single base changes. Also HCC cell lines namely, HepG2, Hep3B, Huh-7, FOCUS, Mahlavu, and PLC/PRF/5 were checked for their mutations and cell lines other then PLC/PRF/5 were analysed for their mRNA transcription. There were no big deletions or alterations in Nand C- terminals of the cell lines and we have shown mRNA transcription for all cell lines except Hep3B in which PCR has revealed very weak amplification. Our results suggest that Smad4 might be involved in at least a part of primary HCC tumor development.
Open Access
Genetic and epigenetic analysis of immortal and senescence arrested liver cancer cells
(2009) Bağışlar, G. Sevgi
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.
Open Access
Senescence and immortality genes as markers of hepatocellular carcinogenesis
(2009) Arslan Ergül, Ayça
Cellular senescence is a tumor-suppression mechanism, and immortalization facilitates neoplastic transformation. Both mechanisms may be highly relevant to hepatocellular carcinoma (HCC) development. We worked on two major aspects of cellular senescence and immortality in HCC. First, we analyzed the role of ZEB2 (Smad-interacting protein SIP1, ZFXH1B) gene for a senescence-related role in HCC. Then, we extended our work on the identification and analysis of a senescence and immortality gene network (SIGN) in relation to hepatocellular carcinogenesis. ZEB2 is a transcriptional repressor of E-cadherin, and induces epithelial-mesenchymal transition (EMT), a key process involved in tumor metastasis and progression. However, ZEB2 is also a repressor of telomerase reverse transcriptase (TERT) gene, which encodes a key enzyme required for telomere maintenance and tumor cell immortality. We performed in-vivo, in-silico and in-vitro studies to explore potential implications of ZEB2 in hepatocellular carcinoma (HCC). Tissue expression of ZEB2 transcripts displayed stepwise decreases in HCC lesions, as compared to liver cirrhosis. This inverse correlation suggested that sustained ZEB2 expression is not compatible with HCC progression. Next, vekt¨or¨u ile transfekte edilen Hep3B h¨ucrelerinin, ya¸slanma ili¸skili !-galaktozidaz aktivitesi ile, kalıcı h¨ucre d¨ong¨us¨u hapsine girdiklerini g¨ord¨uk. ZEB2 ile ind¨uklenen ya¸slanma hapsi, TERT ifadesinin baskılanması ve e¸slik eden siklin-ba˘gımlı kinaz engelleyici p21Cip1’in ifadesindeki artı¸s ile ba˘gıntılı idi. ZEB2’nin ge¸cici ifadesi, p21Cip1 ifadesindeki artı¸sı ind¨uklemedi. Son olarak, ZEB2’yi a¸sırı ifade eden Hep3B ve A431 klonlarının, in vitro k¨ult¨urde dereceli olarak azalması ile ZEB2 a¸sırı ifadesinin, kanser h¨ucrelerinin in vitro ya¸samı ile uyumlu olmadı˘gı sonucuna varıldı. Bu g¨ozlemler, ZEB2 geninin, EMT’deki rol¨un¨un dı¸sında, HCC h¨ucre b¨uy¨umesi ve ya¸samasında negatif rol oynadı˘gını d¨u¸s¨und¨urd¨u. Di˘ger ¸calı¸smamızda SIGN imzasını olu¸sturmak i¸cin, ya¸slanmaya programlanmı ¸s ve ¨ol¨ums¨uz HCC h¨ucrelerinden gelen gen ifade datasını, siroz ve HCC dokularından gelen data ile birle¸stirdik. SIGN imzası normal karaci˘ger, siroz, displazi ve HCC lezyonlarını do˘grulukla sınıflandırdı ve ya¸slanmadan ¨ol¨ums¨uzl¨u˘ge d¨on¨u¸s¨um¨un, ilk olarak displaziden erken HCC’ye ge¸ci¸ste ger¸cekle¸sti˘gini belirledi. Bu d¨on¨u¸s¨um, t¨um¨or ilerlemesine de katkıda bulunur. Ya¸slanmadan ¨ol¨ums¨uzl¨u˘ge d¨on¨u¸s¨ume, hepatik geri ba¸skala¸sım ile ve h¨ucre ¸co˘galması, kromozom de˘gi¸simi ve DNA hasar yanıtı genlerindeki ifade artı¸sı e¸slik etti. Bu nedenle, HCC ¨ol¨ums¨uzl¨u˘g¨u, k¨ok h¨ucre/¨onc¨u h¨ucre benzeri ¨ozellikler ile yakından ili¸skilidir. Son olarak, DNA hasar kontrol noktası ve DNA tamir genlerindeki artı¸s ile, t¨um¨or ba¸slangıcı ve ilerlemesi arasında ili¸ski bulduk. Bu genler, HCC’nin engellenmesinde ve terapisinde potansiyel hedefler olabilirler.