O isoprenóide geraniol (GO) apresentou atividade quimiopreventiva quando administrado continuamente durante as fases de iniciação e pós-iniciação em modelo de carcinogênese experimental de cólon por meio da redução do número de focos de criptas aberrantes (FCAs) totais FCAs &#8805; 4 criptas e aumento de apoptose no cólon distal. Dessa forma, optou-se por avaliar os eventuais efeitos de três doses de GO (GO1: 25mg/100g de peso corpóreo [p.c.], G02: 50 mg/100g de p.c. e G03: 100 mg/100g de p.c.) em categorias de lesões pré-neoplásicas (LPNs) induzidas por dimetilhidrazina (DMH) durante a fase de pós-iniciação tardia de modelo de carcinogênese experimental de cólon, caracterizada por apresentar lesões mais avançadas e com alto grau de alterações celulares morfológicas, bioquímicas e moleculares denominadas de displasia. Para isso, analisamos diferentes biomarcadores como: FCAs totais e FCAs < ou &#8805; 4 criptas em cólons corados com azul de metileno; focos depletados ou positivos de mucina (FPMs ou FDMs) em cólons corados com azul de toluidina; FCAs convencionais ou displásicos por meio de análise histopatológica em cortes corados com hematoxilina e eosina (HE) e focos positivos ou negativos para beta-catenina (FPBCs ou FNBCs) citoplasmática e/ou nuclear por meio de imunoistoquímica. Além disso, células apoptóticas foram identificadas utilizando-se critérios morfológicos clássicos em FCAs &#8805; 4 no cólon distaI e a expressão de genes envolvidos na carcinogênese de cólon foi avaliada por meio de RT-PCR: HMGCoA-redutase na mucosa colônica e K-Ras e c-myc em FCAs microdissecados. Em relação ao grupo controle, foi possível observar que o grupo tratado com a maior dose de GO (G03) reduziu a freqüência de FCAs &#8805; 4 criptas e FDMs, além de aumentar a apoptose em FCAs &#8805; 4 displásicos no cólon distaI (p &#8804; 0,05). Já, em relação aos outros biomarcadores e às expressões de HMGCoA-redutase, K-Ras e c-myc não observamos diferenças estatísticas entre os tratamentos (p > 0,05). A partir desses resultados, podemos concluir que a dose de 100 mg/100 g de p.c. de GO mostrou ser mais interessante do ponto de vista quimiopreventivo com efeitos observados principalmente no cólon distaI, onde há maiores relatos de incidência de adenocarcinomas colônicos, tanto em animais quanto em humanos. Assim, a indução da morte celular programada em FCAs &#8805; 4 preferencialmente displásicos poderia representar um mecanismo importante de atuação de G03 na redução da freqüência de FCAs &#8805; 4 criptas e de FDMs (também utilizado como marcador de displasia) durante a fase de pós-iniciação tardia de modelo de carcinogênese experimental de cólon. / The isoprenoid geraniol (GO) showed chemopreventive activity when administered continuously during the initiation and post-initiation phases in an experimental model of colon carcinogenesis by reducing the number of total aberrant crypt foci (ACF) and ACFs &#8805; 4 crypts, as well as increasing apoptosis in the distal colon. We therefore chose to evaluate the effects of three different doses of GO (GO1: 25 mg/100 g body weight [b.w.], GO2: 50 mg/100 g b.w. and GO3: 100 mg/100 g of b.w.) on preneoplastic lesions (PNLs) induced by dimethylhydrazine (DMH) during late post-initiation in an experimental model of colon carcinogenesis that is characterized by more advanced lesions and a higher degree of cellular alterations morphological, biochemical and molecular (dysplasia) than previous models. For this study, we analyzed the following biomarkers: total ACFs, ACFs < 4 crypts, and ACFs &#8805; 4 erypts in colons stained with methylene blue; mucin-depleted or mucin-positive foci (MDFs or MPFs) in colons stained with toluidine blue; ACFs, through conventional or dysplastie histopathological analysis of sections stained with hematoxylin and eosin (HE); and cytoplasmic vs. nuclear foci reactivity for beta-catenin (foci positive for beta-eatenin (FPBC) or foci negative for beta catenin (FNBC)) using immunohistochemistry. Additionally, apoptotic cells were identified using classical morphologic criteria in ACFs &#8805; 4 crypts in the distal colon, and the expression of several genes involved in colon carcinogenesis was assessed by RT-PCR, including HMG-CoA reductase in the colonic mucosa and K-Ras and c-myc in microdissected ACFs. Relative to the control group, we observed that the group receiving the highest dose of GO (GO3 group) had a reduced frequency of both ACFs &#8805; 4 crypts and MDFs and that apoptosis increased in dysplastic ACFs &#8805; 4 crypts in the distal colon (p < 0, 05). Expression of HMG-CoA reductase, K-Ras and c-myc did not differ between treatments (p > 0, 05). Based on these results, we conclude that the 100 mg/100 g b.w. dose of GO is the most promising, as it shows evidence of chemopreventive effects mainly in the distal colon, which is a region that is reported to have a higher incidence of colonic adenocarcinomas, both in animaIs and in humans. lnduction of programmed cell death by GO3 in ACFs &#8805; 4 specifically dysplastic could represent an important mechanism of action in reducing the frequency of both ACFs &#8805; 4 crypts and MDFs during late post-initiation in this experimental model of colon carcinogenesis.

Colorectal cancer (CRC), with an estimated 1.2 million new cases annually, is the third leading cause of cancer incidence and death worldwide. Generally, the majority of CRC patients are diagnosed at the advanced stages with poor prognosis and unfavorable response to multiple therapeutic drugs. In spite of increasing knowledge of the molecular mechanism for the tumorigenesis in CRC patients, the translation from basic science into clinical therapy has been limited for quite a long time. In order to develop novel treatment strategies against CRC, intensive and extensive attempts have been made in the past decades. / The epithelial to mesenchymal transition (EMT) is a multi-step process characterized by the loss of cell polarity, decreased cell-cell adhesion as well as enhanced migration and invasion capacity. It is well documented that EMT is essential for a variety of cellular biological events ranging from embryogenesis to tumor progression. The field of lncRNA is developing rapidly and currently it is one of the most intensively studied fields in the biomedical sciences. Emerging evidence indicates that the majority of human genome encodes thousands of non-protein-coding RNA transcripts, nevertheless, the function of long non-coding RNAs (lncRNAs) in orchestrating EMT progression remains elusive. Historically, the lncRNA H19 was the first identified imprinted non-coding RNA transcript in human, and the H19/IGF2 locus acted as an ideal paradigm for the investigation of genomic imprinting genes. In recent years, the expression profiling and functional characterization of the H19 gene in a variety of human diseases has been extensively studied. / In our studies, H19 was characterized as a novel regulator of EMT in colon cancer. We first observed significant mesenchymal characteristics in the methotrexate-resistant HT-29 cells. Interestingly, significant upregulation of H19 was observed in mesenchymal-like MTX resistant HT-29 cells. We subsequently demonstrated that after treatment of TGF-β1, one of the most widely used EMT inducers, H19 presented dramatic increase during the EMT progression. To further investigate the functional role of H19 in EMT, we generated the stable cell lines overexpressing H19 in colon cancer cells using retroviral infection. Stable overexpression of H19 significantly promoted EMT progression in two epithelial colon cancer cell lines HT-29 and HCT-116. However, overexpression of H19 did not affect cell proliferation as well as cell cycle progression. Further proteomics studies screened out that ectopic expression of H19 upregulated the protein level of Vimentin, a vital biomarker for mesenchymal cells. By using the bioinformatics study in combination with luciferase reporter assays, we demonstrated that H19 potentiated the expression of several core marker genes essential for mesenchymal cells by serving as a competing endogenous RNA(ceRNA), which builds up the missing link between the regulatory miRNA network and EMT progression. According to the results from xenograft tumor model and soft agar assay, stable expression of H19 reinforced the in vitro and in vivo tumor growth. Moreover, the investigation of clinical specimens verified that H19 RNA level was significantly increased in colon cancer tissues compared with corresponding adjacent normal tissues. Taken together, the above observations imply that the lncRNA H19, by acting as a competing endogenous RNA, is an important regulator which tightly modulated the expression of multiple important genes involved in EMT and it could probably serve as a novel therapeutic target against colon cancer. / 大腸癌每年有一百二十萬新增個案，是世界第三大癌症殺手。通常情況下，大部分大腸癌病人發現時已經處於晚期，該時期的癌症病人對多種臨床治療藥物已無法治愈。盡管關於大腸癌發病的分子生物學機制已經不斷完善，但如何從基礎研究轉化為臨床治療手段在很長一段時間內不可實現。為了進一步研究新的抗擊大腸癌治療手段，廣泛且深入的研究已經不斷開展。 / 上皮間充質轉化是一個多步驟的過程，該過程的典型特徵為失去細胞的極性，細胞間粘連減弱以及細胞爬行遷移能力的不斷加強。目前科學家已經知道上皮間充質轉化對於從胚胎發育到腫瘤發展都起著重要的作用。近年來，長非編碼RNA的研究不斷快速發展，已然成為醫學研究中最激烈的領域之一。眾多證據表明人體基因組編碼數以千計不編碼蛋白質的RNA轉錄體。然而，這些RNA轉錄體在上皮間充質轉化中的功能依然所知甚少。長非編碼RNA H19是人體內第一個被鑒別出來參與到基因印記的非編碼RNA。資料表明H19/IGF2位點是一個非常理想的研究基因印記的位點。近年來，H19在眾多癌症中的表達以及功能學研究已不斷湧現，同時也不斷取得令人鼓舞的研究成果。 / 在我們的研究中，H19被鑒定為大腸癌裏上皮間充質轉化過程中一個重要的參與者。通過研究甲氨蝶呤耐藥大腸癌HT-29細胞株，我們發現該HT-29耐藥細胞株有著顯著的間充質細胞特性。有趣的是，H19在該細胞株中有著顯著升高。我們隨後用經典的上皮間充質轉化誘導劑TGF-β1處理兩株大腸癌細胞，處理後H19亦有著顯著升高。為了進一步研究H19在上皮間充質轉化，通過使用逆轉錄病毒，我們建立H19的穩定表達細胞株。穩定表達H19顯著地促進了HT-29以及SW620大腸癌細胞株的上皮間充質轉化。然後，高水平表達(過表達)H19並不影響細胞的生長以及細胞周期的進程。進一步的蛋白質組學研究表明，過表達H19能促進間充質細胞一個重要標記基因Vimentin的表達。通過生物信息學以及熒光素酶報告基因實驗，我們證明了H19通過其競爭內源性RNA的作用，能夠促進間充質細胞所需的幾個重要基因的表達。該發現建立起了miRNA網絡以及上皮間充質轉化進程的交流網絡。通過異位移植以及軟瓊脂實驗，我們發現過表達H19能夠促進腫瘤細胞的生長。而在臨床大腸癌病人組織中，我們更發現H19在大腸癌病人組織中高表達。綜上所述，我們的結果證明H19這一長非編碼RNA，能夠通過其競爭內源性RNA的作用機制，從而調控上皮間充質轉化過程中的關鍵基因。同時H19亦有可能成為治療大腸癌的臨床新靶點。 / Liang, Weicheng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 95-124). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016). / Liang, Weicheng.

Xu, Meng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 112-129). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

La cancérogenèse gastrique est un processus d’origine multifactorielle, incluant des facteurs génétiques de l’hôte, mais aussi d’origine bactérienne et de l’environnent. Les populations humaines peuvent être exposées directement ou indirectement à des composés toxiques/génotoxiques présents dans les plantes, comme la fougère Pteridium aquilinum. Cette plante comprend une toxine, le ptaquiloside associée à des maladies graves et le développement de cancer chez les animaux. Des études épidémiologiques ont démontré une association entre l'exposition à ces fougères et l’incidence du cancer gastrique dans les populations humaines. Cependant, un autre facteur de risque majeur dans le développement du cancer gastrique est la bactérie Helicobacter pylori qui colonise l'estomac humain et induit une réponse génotoxique. Cette étude vise à caractériser l'implication biologique et moléculaire de Pteridium aquilinum et de sa toxine le ptaquiloside dans le processus de cancérogenèse gastrique et d'explorer un effet de synergie potentiel avec l'infection par H. pylori.Nous avons montré que le traitement avec des extraits de Pteridium aquilinum et le ptaquiloside diminue la viabilité cellulaire et favorise l'apoptose des cellules épithéliales gastriques. L'induction de cassures de l'ADN a été observée, exacerbée en présence de l’infection par H. pylori. Dans les cellules traitées, la protéine p53 est induite et associée à l'activation de la voie de signalisation ATR-Chk1. Cette augmentation de p53 est aussi détectée en présence des souches virulentes de H. pylori. L’induction de lésions à l’ADN par le ptaquiloside est en accord avec la dérégulation observée de l’expression d’un certain nombre de gènes impliqués dans la régulation du cycle cellulaire et la réparation de l'ADN. De plus, des souris exposées à Pteridium aquilinum, montrent des modifications histomorphologiques de la muqueuse gastrique ainsi qu’une augmentation de la prolifération cellulaire et l'induction de mutations dans le gène p53 après 7 semaines de traitement. Toutefois, bien qu’une exacerbation de la prolifération cellulaire et des lésions histologiques soient induites par un traitement chronique en association avec l'infection à H. pylori pendant 12 mois, aucune différence significative dans l'expression du gène p53 a été mise en évidence. Cependant, dans ces conditions, une modification du schéma glycophenotypique a été induite dans la muqueuse gastrique des souris. Différentes glycosyltransférases impliquées dans la biosynthèse des antigènes simples de mucines et terminaux antigènes Lewis ont été différentiellement exprimées chez les souris non-infectées et infectées, respectivement. Ces résultats sont également validés par une augmentation de l’expression de Sialyl-LewisX.De plus, des modifications des glycosyltransférases impliquées dans les étapes initiales de O-glycosylation ont été observées. Le ppGalNAcT6 a présenté une expression altérée dans un carcinome gastrique, associée à la présence de l'invasion veineuse.En conclusion, nos données confirment l’activité génotoxique de Pteridium aquilinum et du ptaquiloside sur les cellules gastriques, supportant leur rôle fondamental dans la promotion de la cancérogenèse gastrique. Cette activité est exacerbée en présence de l’infection par H. pylori, soulignant l'importance de l'interaction de ces deux facteurs de risque dans ce processus. / The multifactorial gastric carcinogenesis process encompasses host genetic susceptibility, bacterial and environmental factors. Humans can directly consume or be indirectly exposed to toxic compounds present in plants, such as the bracken fern Pteridium aquilinum. This plant has a carcinogenic toxin, ptaquiloside, and is known to cause severe health problems in animals, including cancer. Epidemiological evidence also demonstrated an association between bracken exposure and gastric cancer development in Humans. Additionally, another major etiological agent is Helicobacter pylori, a bacterium that colonizes the stomach inducing a genotoxic response in gastric cells. This study aimed to characterize the biological and molecular involvement of Pteridium aquilinum and its ptaquiloside toxin in the gastric carcinogenesis process and to evaluate the potential synergistic effect of H. pylori infection.We observed that treatment with Pteridium aquilinum extracts and ptaquiloside toxin decreased cell viability and promoted cell apoptosis in gastric epithelial cells. A genotoxic effect with induction of DNA strand breaks was noted and it was exacerbated in the presence of H. pylori infection. We further demonstrated that in treated cells a p53 accumulation occurs, controlled by the activation of the ATR-Chk1 DNA damage signalling pathway. An increased level of p53 was also detected in the presence of a H. pylori virulent strain. The contribution of ptaquiloside to this genotoxic activity was supported by the deregulation of other genes involved in DNA cell cycle regulation and DNA repair. In addition, using a mouse model exposed to Pteridium aquilinum, we detected histomorphological alterations with increased cell proliferation and induction of frameshift events in the p53 gene. However, a concomitant chronic treatment with Pteridium aquilinum and H. pylori infection did not produce significant differences in p53 gene expression.Moreover, an altered glycophenotypic pattern was induced in the gastric mucosa of mice upon Pteridium aquilinum treatment in the presence of H. pylori infection. Several glycosyltransferases involved in the biosynthesis of simple mucin-type carbohydrate antigens and terminal Lewis antigens were differently expressed in the absence and presence of H. pylori, respectively. These results were also validated by an increased expression of Sialyl-LewisX.Further alterations in glycosyltransferases involved in the initial steps of O-glycosylation were observed. The ppGalNAcT6 was shown to have a heterogeneous expression in human gastric carcinoma, associated with the presence of venous invasion.Overall, our data supports the notion that cell exposure to the genotoxic and carcinogenic Pteridium aquilinum and ptaquiloside has a fundamental role in the promotion of gastric carcinogenesis. The synergistic environment associated to H. pylori infection underlines the importance of risk factor interplay in this process. / A carcinogénese gástrica é um processo multifatorial que engloba fatores genéticos, bacterianos e ambientais. O Homem pode consumir diretamente ou ser exposto de forma indireta a compostos tóxicos presentes em plantas, como é o caso do feto vulgar Pteridium aquilinum. Esta planta tem uma toxina carcinogénica, o ptaquilosídeo, sendo conhecida a sua capacidade natural para induzir lesões neoplásicas em animais. Estudos epidemiológicos também demonstraram a existência de uma associação entre a exposição ao feto e o desenvolvimento de cancro gástrico em humanos. Outro fator etiológico importante é a Helicobacter pylori, uma bactéria que coloniza o estômago, induzindo nas células gástricas uma resposta genotóxica. Este estudo tem por objetivos caracterizar o envolvimento biológico e molecular do Pteridium aquilinum e da sua toxina, ptaquilosídeo, no processo de carcinogénese gástrica e avaliar o potencial efeito sinergístico da infeção por H. pylori.Observámos em células epiteliais gástricas que o tratamento com extratos de Pteridium aquilinum e com a toxina ptaquilosídeo, diminui a viabilidade celular e promove a apoptose. Foi demonstrado um efeito genotóxico com indução de quebras na cadeia de ADN, exacerbado pela presença da infeção por H. pylori. Demonstrámos ainda que, em células tratadas, ocorria uma acumulação de p53, controlada pela ativação da via de sinalização ATR-Chk1. Um aumento nos níveis de p53 foi igualmente detetado na presença de estirpes virulentas de H. pylori. A contribuição do ptaquilosídeo para esta atividade genotóxica foi também apoiada pela desregulação de outros genes envolvidos na regulação do ciclo celular e na reparação do ADN. Adicionalmente, usando um modelo de ratinho exposto ao Pteridium aquilinum, foram detetadas alterações histomorfológicas, bem como um aumento da proliferação celular e indução de mutações no gene p53. Contudo, um tratamento crónico com Pteridium aquilinum e infeção concomitante por H. pylori não produziu diferenças significativas na expressão do gene p53.Um padrão glicofenotípico alterado foi também observado na mucosa gástrica de ratinhos tratados com Pteridium aquilinum na presença de infeção por H. pylori. Várias glicosiltransferases envolvidas na biossíntese de antigénios simples das mucinas e antigénios terminais do tipo Lewis apresentaram uma expressão alterada, respetivamente, na ausência ou presença de H. pylori. Estes resultados foram também validados através de um aumento da expressão de Sialil-LewisX.Foram ainda observadas alterações em glicosiltransferases que estão envolvidas nas etapas iniciais de O-glicosilação. A ppGalNAcT6 apresentou uma expressão alterada em carcinomas gástricos, estando associada à presença de invasão venosa.No geral, estes dados suportam a evidência de que a exposição das células aos genotóxicos e carcinogénicos Pteridium aquilinum e ptaquilosídeo, tem um papel fundamental na promoção da carcinogénese gástrica. O ambiente sinergístico associado à infeção com H. pylori salienta a importância da interação entre os fatores de risco que dão origem a este processo.

BTBD7 is a newly identified candidate gene for HCC using a high-throughput cDNA/EST microassay. This gene encodes for a protein of 410 amino acid residues. This protein was previously named as the function unknown protein 1 (FUP1) because the biological function of this protein was unknown at that time. Bioinformatics analysis revealed that this protein contains two bric-a-brac, tramtrack, broad-complex (BTB) domains located at amino acid positions 143 to 230 and 274 to 342. In order to reflect its structure and functions, and to be consistent with the GeneBank database (Accession No. NM_018167), we rename it as BTBD7 (BTB domain containing 7). / In conclusion, our study demonstrated that BTBD7 is a novel oncogene, which is associated with hepatocellular carcinoma and is essential for the inhibition of cell growth and tumorigenesis. To our knowledge, BTBD7 is the first identified regulator of p16INK4A through inhibiting the promoter activity of p16INK4A. BTBD7 may thus serve as a new tumor marker or as a potential target of treating hepatocellular carcinoma. / In previous studies, the expression of BTBD7 was shown to be tissue-specific as demonstrated by Northern blot. Furthermore, we collected 18-paired HCC samples to further reveal the correlation of BTBD7 gene expression profiles with tumorigenesis. Our data showed that BTBD7 was significantly elevated in 44.4% of the HCC samples. Compared with immortalized hepatocyte cell lines MIHA or LO2, both mRNA level and protein level of BTBD7 were also elevated in the hepatoma cell lines HepG2, BEL7404, Hep3B and Huh7. This gave a due that the expression of BTBD7 may be correlated with carcinogenesis of liver cells. / In the present study, the function of BTBD7 was investigated. We used RNAi approach to silence BTBD7. Compared with the control, siBTBD7 induced cell cycle arrest at G1 phase and later caused obvious cell death. The cell death was further demonstrated to be apoptosis through activation of caspase 3. Furthermore, we carried out candidate gene search using knockdown of BTBD7. The mRNA level of tumor suppresser p16INK4A was upregulated and hTERT was downregulated in BTBD7 knocked down cells. The other key genes involved in cell growth, cell cycle control, cell death and survival (c-myc, c-fos, c-jun, p21CIP1, p27KIP1, p53, Survivin, E2F, NF-kappaB, Bax, p14ARF, p16INK4A and hTERT) did not respond to the reduced BTBD7 levels. On the other hand, double knockdown of p16INK4A and BTBD7 markedly reduced the effects of cell cycle arrest and the death ratio caused by dysfunction of BTBD7 or overexpression of p16INK4A, suggesting that p16 INK4A is a downstream target of BTBD7. We further adopted a dominant negative approach to confirm these results. / Liu, Zheng. / Advisers: C. H. K. Cheng; Mingliang He. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3449. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 120-161). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Tong Hung Man Joanna. / "June 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 137-149). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Li, Sai Kam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 162-179). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.ii / Acknowledgements --- p.iii / Table of Content --- p.iv / Abbreviations --- p.xi / List of Figures --- p.xiv / List of Tables --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Hepatitis B Virus / Chapter 1.1.1 --- General information --- p.1 / Chapter 1.1.2 --- Classification --- p.2 / Chapter 1.1.3 --- Virus life cycle and genome --- p.3 / Chapter 1.1.4 --- Hepatitis B virus X protein (HBx) --- p.7 / Chapter 1.2 --- Enigmatic functions of HB --- p.x / Chapter 1.2.1 --- HBx as a transactivator --- p.10 / Chapter 1.2.2 --- HBx as a cell cycle regulator --- p.12 / Chapter 1.2.3 --- HBx as an apoptosis modulator --- p.13 / Chapter 1.3 --- Etiology of HBV-mediated hepatocarcinogenesis --- p.14 / Chapter 1.4 --- Clinical mutants of HBV --- p.16 / Chapter 1.5 --- Hypothesis and aims of the research --- p.16 / Chapter 1.6 --- Basis of Tet-On system --- p.18 / Chapter CHPATER 2 --- EXPERIMENT MATERIALS / Chapter 2.1 --- Cell culture / Chapter 2.1.1 --- Cell-lines --- p.21 / Chapter 2.1.2 --- Culture medium --- p.22 / Chapter 2.1.3 --- Culture medium supplements --- p.23 / Chapter 2.2 --- Reagents for subcloning / Chapter 2.2.1 --- Reagents for polymerase chain reaction (PCR) --- p.24 / Chapter 2.2.2 --- Reagents for restriction enzyme digestion --- p.24 / Chapter 2.2.3 --- Reagents for ligation --- p.25 / Chapter 2.2.4 --- Reagents for electrophoresis --- p.25 / Chapter 2.2.5 --- Reagents for E. coli DH5a preparation --- p.25 / Chapter 2.2.6 --- Materials for bacterial culture work --- p.27 / Chapter 2.3 --- Reagents for subcellular localization study / Chapter 2.3.1 --- Reagents for cell staining --- p.28 / Chapter 2.3.2 --- Reagents for mounting slides --- p.29 / Chapter 2.3.3 --- Materials for site-directed mutagenesis --- p.29 / Chapter 2.4 --- Reagents for cell cycle analysis and cellular proliferation / Chapter 2.4.1 --- Reagents for cell cycle analysis --- p.29 / Chapter 2.4.2 --- Reagents for cellular proliferation study --- p.30 / Chapter 2.5 --- Reagents for protein expression study / Chapter 2.5.1 --- Cell lysis buffer --- p.30 / Chapter 2.5.2 --- Reagents for SDS-PAGE --- p.30 / Chapter 2.5.3 --- Reagents for Western blot --- p.33 / Chapter 2.5.4 --- Antibodies --- p.34 / Chapter 2.6 --- Reagents for gene expression study / Chapter 2.6.1 --- Reagents for RNA extraction --- p.36 / Chapter 2.6.2 --- Reagents for first strand cDNA synthesis --- p.37 / Chapter 2.6.3 --- Reagents for real-time PCR --- p.37 / Chapter 2.7 --- Reagents for establishment of Tet-On inducible stable cell-lines / Chapter 2.7.1 --- Reagents for MTT assay --- p.38 / Chapter 2.7.2 --- Reagents for selection of stable clones --- p.38 / Chapter 2.8 --- Vectors used in the project / Chapter 2.8.1 --- Vectors for subcellular localization study --- p.39 / Chapter 2.8.2 --- Vectors for establishment of Tet-on inducible cell-lines --- p.39 / Chapter 2.9 --- Primers used in the project / Chapter 2.9.1 --- Primers used for subcloning --- p.42 / Chapter 2.9.2 --- Primers used for site-directed mutagenesis --- p.43 / Chapter 2.9.3 --- Primers used in real-time chain polymerase reaction --- p.43 / Chapter CHAPTER 3 --- RESEARCH METHODS / Chapter 3.1 --- Subcloning of HBx and mutant genes into a green fluorescence protein (GFP) expression vector / Chapter 3.1.1 --- Amplification of HBxWt，HBxΔC44 and HBxAN60 genes --- p.45 / Chapter 3.1.2 --- Purification of PCR products --- p.46 / Chapter 3.1.3 --- Restriction enzyme digestion --- p.47 / Chapter 3.1.4 --- Ligation of gene products with pEGFP-C 1 vector --- p.47 / Chapter 3.1.5 --- Preparation of chemically competent bacterial cells E. coli strain DH5α --- p.47 / Chapter 3.1.6 --- Transformation of the ligation product into competent cells --- p.48 / Chapter 3.1.7 --- PCR confirmation of successful ligation --- p.48 / Chapter 3.1.8 --- Small scale preparation of bacterial plasmid DNA --- p.49 / Chapter 3.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.50 / Chapter 3.1.10 --- Large scale preparation of target recombinant plasmid DNA --- p.50 / Chapter 3.2 --- Subcellular localization pattern study / Chapter 3.2.1 --- Cell transfection --- p.51 / Chapter 3.2.2 --- Mitochondria and nucleus staining --- p.52 / Chapter 3.2.3 --- Epi-fluorescence microscopy --- p.53 / Chapter 3.2.4 --- Analysis of fluorescence images --- p.53 / Chapter 3.2.5 --- In vitro site-directed mutagenesis --- p.53 / Chapter 3.3 --- Cell cycle phase analysis with flow cytometry / Chapter 3.3.1 --- Cell transfection --- p.55 / Chapter 3.3.2 --- Cell staining --- p.55 / Chapter 3.3.3 --- Flow cytometry --- p.55 / Chapter 3.4 --- Cellular proliferation quantification by BrdU proliferation assay / Chapter 3.4.1 --- Cell transfection --- p.57 / Chapter 3.4.2 --- BrdU ELISA measurement --- p.57 / Chapter 3.5 --- Protein expression / Chapter 3.5.1 --- Cell lysate collection --- p.58 / Chapter 3.5.2 --- Quantification of protein samples --- p.59 / Chapter 3.5.3 --- SDS-PAGE --- p.59 / Chapter 3.5.4 --- Western blot --- p.60 / Chapter 3.5.5 --- Western blot luminal detection --- p.60 / Chapter 3.6 --- Gene expression / Chapter 3.6.1 --- Primer design --- p.61 / Chapter 3.6.2 --- Cell transfection --- p.61 / Chapter 3.6.3 --- RNA extraction --- p.61 / Chapter 3.6.4 --- Reverse transcription for first strand complementary DNA (cDNA) --- p.63 / Chapter 3.6.5 --- Quantitative real-time PCR --- p.63 / Chapter 3.7 --- Establishment of Tet-On inducible stable cell-lines / Chapter 3.7.1 --- Subcloning of HBx gene into pTRE2 vector --- p.64 / Chapter 3.7.2 --- Construction of WRL68/Tet-On stable cell-lines --- p.64 / Chapter 3.7.3 --- Construction of WRL68/Tet-On HBx and mutants expression cell-lines --- p.68 / Chapter 3.7.4 --- Characterization of Tet-On gene expression monoclones --- p.69 / Chapter 3.8 --- Statistical analyses --- p.70 / Chapter CHPATER 4 --- STUDY ON MITOCHONDRIA TARGETING / Chapter 4.1 --- Establishment of pEGFP-Cl-HBx and mutants constructs --- p.71 / Chapter 4.2 --- Transactivation C-terminus domain is essential for granular localization --- p.73 / Chapter 4.3 --- Wild-type HBx localizes in mitochondria --- p.76 / Chapter 4.4 --- C-terminal transactivation domain is sufficient for mitochondria targeting --- p.79 / Chapter 4.5 --- Mapping of the HBx region crucial for mitochondria targeting --- p.81 / Chapter 4.6 --- The 111-117 amino acids in HBx do not work as a signal peptide --- p.83 / Chapter 4.7 --- Site-directed mutagenesis identifies the key amino acid at 115 in HBx for mitochondrial targeting --- p.85 / Chapter CHAPTER 5 --- CELL PROLIFERATION AND REGULATION / Chapter 5.1 --- Alteration of S-phase distribution in cell cycle --- p.88 / Chapter 5.2 --- Analysis of DNA synthesis using BrdU proliferation ELISA --- p.92 / Chapter 5.3 --- Differential molecular regulation of cell cycle --- p.94 / Chapter 5.4 --- Regulation of the mRNA expression levels of cyclin-dependent kinases inhibitors p2raf/cipl and p27kipl --- p.98 / Chapter CHAPTER 6 --- TRANSACTIVATION AND RAS/RAF/MAPK PHOSPHORYLATION / Chapter 6.1 --- Determination of p53-dependency of p21、vaf/cipl expression --- p.101 / Chapter 6.2 --- Ras/Raf/MAPK pathway activation by HBx variants / Chapter 6.2.1 --- ERK1/2 phophorylation by HBx variants --- p.104 / Chapter 6.2.2 --- ERK inhibition blocks the regulation effect on p53Wt and p21waf/cipl --- p.107 / Chapter 6.3 --- Transactivation activity on oncogenes/ proto-oncogenes / Chapter 6.3.1 --- Effect on c-myc (NM´ؤ002467) mRNA expression --- p.109 / Chapter 6.3.2 --- Effect on RhoC (NM_017744) and Rabl4 (NM´ؤ016322) mRNA expression --- p.112 / Chapter CHAPTER 7 --- CONSTRUCTION OF TET-ON INDUCIBLE CELL-LINES / Chapter 7.1 --- Establishment of WRL/Tet-On monoclonal cell-lines Page / Chapter 7.1.1 --- Determination of geneticin selection dosage --- p.116 / Chapter 7.1.2 --- Selection of the best WRL/TOn clone using luciferase assay --- p.118 / Chapter 7.2 --- Establishment of inducible WRL/TOn/Gene monoclonal cell-lines / Chapter 7.2.1 --- Determination of hygromycin selection dosage --- p.120 / Chapter 7.2.2 --- Selection of positive WRL/TOn/Gene clones with viral genes --- p.122 / Chapter 7.3 --- Characterization of TOXDC1 cell-line / Chapter 7.3.1 --- Cell morphology --- p.125 / Chapter 7.3.2 --- Growth pattern of TOXDC1 --- p.126 / Chapter 7.3.3 --- HBxAC44 induced p21waf/cipl mRNA expression --- p.127 / Chapter 7.3.4 --- Doxycycline concentration dependent HBxAC44 expression in TOXDC1 --- p.129 / Chapter CHAPTER 8 --- DISCUSSION / Chapter 8.1 --- Selection of cell model / Chapter 8.1.1 --- Selection of cell models --- p.130 / Chapter 8.1.2 --- Selection of truncation mutant --- p.131 / Chapter 8.2 --- Differential sub-cellular localization of HBx and its variants / Chapter 8.2.1 --- Mechanisms of mitochondria targeting --- p.132 / Chapter 8.2.2 --- Mitochondria as site of HBx-induced apoptosis --- p.134 / Chapter 8.2.3 --- Stimulation of calcium release from mitochondria by wild-type HBx --- p.135 / Chapter 8.3 --- Cell cycle distribution profiling and its regulations / Chapter 8.3.1 --- Cell cycle pattern and cell proliferation --- p.136 / Chapter 8.3.2 --- Differential cell cycle molecular pathway activation --- p.138 / Chapter 8.4 --- Ras/Raf/MAPK mediated transactivation by HBxWt and its mutants / Chapter 8.4.1 --- p53-mediated p21waf/cipl expression --- p.142 / Chapter 8.4.2 --- ERK-mediated p21waf/cipl and wild-type p53 mRNA expression --- p.143 / Chapter 8.4.3 --- Regulation of oncogenes/ proto-oncogenes expression --- p.147 / Chapter 8.5 --- General discussions on differential effects of HBxWt and HBxAC44 --- p.149 / Chapter 8.6 --- Establishment of Tet-On/HBxAC44 cell-line TOXDC1 --- p.153 / Chapter 8.7 --- Conclusions --- p.154 / Chapter 8.8 --- Future Prospects / Chapter 8.8.1 --- From mitochondria targeting to calcium signaling --- p.157 / Chapter 8.8.2 --- Construction of a complete cell cycle regulation pathway --- p.158 / Chapter 8.8.3 --- Elucidation of the transcriptional transactivation regulation --- p.159 / Chapter 8.8.4 --- To make the best use of the Tet-on stable cell-line TOXDC1 --- p.159 / Chapter 8.8.5 --- Study with other carboxy-terminal truncation mutants --- p.160 / Chapter 8.8.6 --- In vivo study --- p.160 / REFERENCES --- p.162

Over 2 million adult men in the United States have been diagnosed with prostate cancer, with nearly 200,000 new diagnoses each year. This type of cancer is the leading cause of mortality in U.S. men. One possible risk factor for prostate cancer is a high level of iron in the body, but the association has yet to be confirmed. This study was an investigation of the relationship between serum iron concentration and prostate cancer using data obtained from the 2009-2012 National Health and Nutrition Examination Surveys. This quantitative study involved 1,850 men in the U.S. aged 51 to 70 years. The framework for this research was based on the exposure-disease model. Participants' data were analyzed using chi-squared independence tests and hierarchical logistic regression, while controlling for demographic variables (body mass index, age, ethnicity, poverty-to-income ratio, educational attainment, and hours worked in the last week) to account for potential confounding effects. Serum iron concentration was not found to be significantly associated with prostate cancer diagnosis in this sample. Additional results indicated a significant association between age and prostate cancer, and between ethnicity and prostate cancer, confirming previous research findings. This study contributes to positive social change by confirming the importance of screening for prostate cancer among high-risk populations and by suggesting that it is premature to use serum iron concentration as a screening tool to detect prostate cancer.

Despite significant advances in diagnosis and treatment, breast cancer remains the leading cause of cancer-related deaths in Australian women. Colorectal cancer is the second most common cancer in both males and females; after prostate and breast cancer, respectively, and excluding non-melanocytic skin cancer. Both breast cancer and colorectal cancer follow a common progressive course of illness; presenting (at least initially) with benign symptoms that can be treated by ablation (or removal) of the affected area. Cancer progression is associated with breakdown of tissue barriers (such as basement membranes), leading to the spread of cancer cells (via the vasculature or lymphatic system), and the establishment of secondary metastatic disease at green-field sites. Secondary tumours presenting in the lungs, ovaries, liver, bone, or brain are associated with chronic-debilitating symptoms that are difficult to treat, and will result in death. In the case of breast and colon cancer, effective early therapeutic intervention does have a significant impact upon patient survival. Tumour progression in breast and colon carcinomas is characterised by invasion of the surrounding stroma, and the acquisition of stromal characteristics, by previously epithelial cells. This progression is associated with the expression of extracellular proteases (ECPs) and increased motility. The process of mesenchymal transformation that tumour cells undergo is also referred to as the epithelial to mesenchymal transition (EMT). In general terms the aim of the study, presented in this thesis, was to investigate gene expression in cancer biology; and to characterise changes in breast cancer and colon cancer, with a focus on those genes, and gene products that may play a role in metastasis, including a family of ECPs, the matrix metalloproteinases (MMPs). In our laboratory, we have applied methods in microdissection, differential display polymerase chain reaction amplification (DD-PCR), and array hybridisation analysis to identify gene expression patterns in late stage archival formalin fixed paraffin embedded (FFPE) breast tumour biopsies that may be indicative of the EMT; or the response to the surrounding stroma/interstitium to the presence of the tumour.' The quality of nucleic acid obtainable from FFPE material presents a considerable challenge for gene expression studies. In order to identify tissue specific gene expression patterns, DD-PCR products, amplified from message obtained following segregation of tumour tissue from surrounding stroma, was hybridised to arrayed cDNA libraries created from stromal tumours, or sarcomas. In this way, 21 known genes, or expressed sequence tags (ESTs), were identified. These included the cytoskeletal element and EMT marker, vimentin, the mammary developmental factor and, signal transducer and activator of transcription (STAT)-3, and the cargo selection protein (TIP47). Seventeen genes showed differential expression in either the tumour, or stromal fractions. When applied to transformed breast cancer cell lines (MDA-MB-435 & T47D) DD-array analysis revealed a further 17 genes that were differentially regulated in invasive cells, compared with those displaying a less invasive phenotype. Six of the ESTs identified by DD-PCR array analysis, had no known (or predicted) function. For example, bcaf-2 was identified as the 3'-end of a putative open reading frame (ORF) localised to chromosome 6, while bcaf-10 showed homology with a known ORF. In order to analyse the expression of these bcafs further, a stromal cell culture model, representative of the original osteosarcoma cDNA libraries from which they were obtained, was used. In this model, CD14' (or adherent) peripheral blood mononuclear cells (PBMCs) treated with macrophage colony stimulating factor (M-CSF), can be allowed to differentiate into macrophage-like (ML) cells; while cells treated with M-CSF, and the receptor activator of NF-KB ligand (RANKL) will differentiate into multinucleate osteoclast-like (OCL) multinucleate giant cells. Uniquely, the stromal EST, bcaf-2 was expressed only by RANKL-treated (or OCL) cells. bcaf-2 and other ESTs, identified by DD-PCR analysis (and recently published) are the subject of on going research in our laboratory. The role of RANKL in mammary gland development and bone metastasis suggested that the identification of a RANKL-regulated stromal factor in breast tissue (bcaf-2) was not an artefact. RANKL is a membrane-bound, member of the tumour necrosis factor (TNF)-a cytokine super family. In order to test the hypothesis that RANKL might act as an inflammatory cytokine to regulate clinically significant stromal gene expression in the breast, we employed quantitative real time PCR analysis to examine the relative levels of selected members of a group of metal dependent ECPs, the matrix metalloproteinases (MMPs). RNA was extracted from ML cells and OCL cells, as well as RANK positive breast cancer cell lines (T47D, MDA-MB-435 & MCF-7). When the relative levels of protease mRNA were compared we demonstrated a significant (>20- fold) specific increase in collagenase (collagenase 2lMMP-8 and collagenase 3lMMP-13), and the tissue inhibitor of MMP (TIMP)-2 expression in M-CSF and RANKL treated PBMCs cells. When the assay was applied to RANKL treated breast cancer cell lines (MCF-7, T47D & MDAMB- 231), minor (40-fold) but potentially significant alterations in stromal protease gene expression were observed. The changes observed did not however, support the hypothesis that RANKL might act as an inflammatory cytokine to induce significant alterations in ECP expression in breast cancer cells. To investigate the role of RANKL as a driver of EMT in aberrant breast epithelium, total message (mRNNcDNA) from T47D, MCF-7, MDA-MB-231 cells, and message from the same cell lines treated with RANKL were compared by comparative fluorescent cDNA microarray analysis. Of the 1,700 targets available on the arrays, this study identified 160 that were differentially expressed in RANKL treated cells. The results suggest that RANKL may promote rather than suppress a mammary epithelial phenotype in breast cancer. In fact a putative mesenchymal to epithelial transition (MET) was observed following microscopic analysis, and this finding is the subject of on going research in our laboratory. Sporadic structural alterations in certain mitogenic factors represent important early events in cancer progression, while inherited mutations govern familial susceptibility to disease. In colon cancer, a close link exists between Winglessllnt (WNT) signalling, disease pathology, and the expression of MMPs. To examine the relationship between protease expression and structural genetic alterations in this EMT-linked signalling pathway, and others, we applied combined QPCR analysis of MMP expression and PCR-Single Strand Conformation Analysis (SSCA) to 26 colonic tumours, and patient-matched normal colonic mucosa. In this study, significant correlations between the expression of ECPs, and a key mediator of WNT signalling (p-catenin) were identified. While tumours possessing specific functional mutations in K-Ras, were found to group with phenotypic clustering based on protease gene expression. This result may be due to an interruption of normal interactions between RasIRaf signalling and transforming growth factor (TGF) P signalling, via Sma- and Mad- related protein (SMAD) signalling. These results demonstrate that the already identified link between mutations in kinase signalling, and aspects of gross colon tumour morphology (such as dysplasia) may be due to aberrant MMP expression patterning. The final aim of this research was to utilise methods developed in microdissection and specific Q-PCR analysis, to identify whether tumour-stroma differences in MMP gene expression might be used as markers of disease pathology. Total RNA from tumour, and biopsy-matched adjacent stromal tissue were segregated from 35 FFPE archival breast tumour biopsies. Comparison with stroma identified specific associations between TIMP-2 expression in the stroma and lymph node involvement, as well as stromelysin-3 (MMP-I I ) and TIMP-I expression and calcification of the tumour. Furthermore, a significant correlation was identified in the pattern of gelatinase (gelatinase AIMMP-2 & gelatinaseB1MMP-9) expression; while no significant correlation was identified in tumour-stroma MMP gene expression differences, and tumour grade, or hormone receptor status. These results suggest that coordinated changes within the tumour, and proximal stromal tissues (rather than tissue specific changes per se), regulate pathologically significant changes in breast carcinogenesis. In conclusion, this thesis describes the use of novel techniques in specific and global gene expression analysis that permitted examination of stromal gene expression changes in epithelial tumour progression. Microdissection facilitated localisation of expression to particular tissues, while cell culture models provided material with which to optimise and demonstrate the efficacy of techniques used (where tumour material itself was not abundant). Furthermore, we have identified significant and specific correlations between general stromal protease gene expression changes, a putative mammary epithelial differentiation factor (RANKL), alterations in growth factor signalling, and epithelial tumour pathology in the breast and colon. The combination of techniques developed in this study may assist in improvement of categorisation of tumours in clinical pathology. Specifically, the development of novel grading systems that link underlying molecular genetic changes with changes in tumour pathology. These processes may assist to improve diagnosis and provide more effective patient/tumour-specific drug therapies.

Die Entwicklung von Dickdarmkrebs wird durch eine Reihe von Lebens- und Essgewohnheiten sowie Umweltfaktoren begünstigt. Den letzteren beiden sind Substanzen zuzurechnen, die bei der Zubereitung der Nahrung entstehen und mit ihr aufgenommen werden. Zu diesen Verbindungen gehört das 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridin (PhIP) aus der Substanzklasse der heterozyklischen aromatischen Amine. Es entsteht bei der Erhitzung zahlreicher proteinhaltiger Nahrungsmittel und die Zielorgane in Nagerstudien stimmen mit der Häufung von Krebsinzidenzen in westlichen Industrienationen überein. Dieser Zusammenhang konnte jedoch bis heute nicht endgültig bewiesen werden. Fütterungsversuche mit Ratten wurden mit Konzentrationen der Substanz durchgeführt, die weit über der menschlichen Exposition liegen. Durch das Verfüttern einer humanrelevanten Dosis PhIP sollte geklärt werden, ob auch geringe Konzentrationen dickdarmkrebstypische Mutationen, präneoplastische Läsionen oder Tumore induzierten. Die mit humanrelevanten Dosen gefütterten Tiere wiesen weniger Läsionen als die Hoch-Dosis-PhIP-Gruppe auf, in der allerdings keinerlei maligne Tumoren des Dickdarms auftraten. Hinweise auf dickdarmkrebstypische Mutationen fanden sich ebenfalls in beiden Gruppen, wobei hier keine Dosisabhängigkeit beobachtet werden konnte. Die Sequenzierung ergab ein deutlich von Literaturdaten abweichendes Spektrum. In Bezug auf das verwendete Tiermodell wurden erhebliche Abweichungen in der Empfindlichkeit der Tiere gegenüber der Substanz im Vergleich zu ähnlichen Studien festgestellt. Beide Fütterungsgruppen zeigten deutlich weniger Läsionen; als mögliche Gründe wurden Unterschiede in der Futterzusammensetzung und –zubereitung sowie in der Tierhaltung und –herkunft ausgemacht. Es konnte erstmalig ein Zusammenhang zwischen PhIP in niedrigen Dosen in der Nahrung und der Induktion von Entzündungen gezeigt werden. Diese waren sowohl makroskopisch als auch histologisch sichtbar, der genaue Mechanismus ihrer Entstehung ist jedoch unbekannt.<br><br> Die zusammenfassende Betrachtung aller Ergebnisse lässt vermuten, dass PhIP allein über lange Zeiträume aber in geringen Dosen verabreicht nicht für die hohe Zahl an Krebserkrankungen in westlichen Industrienationen ursächlich ist. / The development of colon cancer is associated with several nutritional, life style, and environmental factors. Among the environmental factors probably involved are substances formed during food processing and taken up with food. One of these substances is the heterocyclic aromatic amine (HAA) 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is formed during the heating of proteinaceous food such as meat and fish. In rodent studies the target organs for HAA-derived cancer development are identical with human organs showing high tumor incidences in western countries. Whether there is an association between exposure to PhIP and high tumor incidences in humans is still uncertain. The amount of PhIP administred to rodents in several studies was far above the levels of human exposure towards HAA. Thus, the aim of this study was to elucidate whether low concentrations of the substance are able to induce finger-print colon cancer gene mutations, preneoplastic lesions or tumors in rats. Animals fed with high amounts of PhIP developed fewer lesions than animals fed with a human-relevant concentration of PhIP. However none of the groups developed tumors of the colon. Both groups showed finger-print mutations for colon cancer, but not in a dose-dependent manner. Sequencing showed that the mutations were different from the known mutation spectum of PhIP. The susceptibility of the F344 rats to PhIP used in this study differed from that in previous feeding studies, with both groups showing much less lesions of the colon. Differences in composition and processing of the animal diets as well as animal maintenance and –origin may explain this discrepancy. For the first time an association between low doses of PhIP in the diet and induction of inflammation was shown. Signs of inflammation were observed macroscopically as well as in histological slices, but the mechanism of its induction remains to be clarified.<br><br> Taken together the results suggest that a chronical exposure to low doses of PhIP alone is not sufficient to explain the high incidences of colon cancer in western countries.