Tesis sobre el tema "Carcinogenesis"

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Toxicam
Um ensaio de média-duração em múltiplos órgãos de roedores, baseado no paradigma iniciação-promoção da carcinogênese, foi proposto por pesquisadores japoneses como alternativa ao ensaio convencional de longaduração para detecção de cancerígenos químicos. Esse ensaio alternativo, denominado DMBDD (acrônimo para os 5 agentes iniciadores da carcingênese neste protocolo), foi originalmente padronizado com a linhagem de ratos Fischer 344. Em 1996, o IBAMA adotou oficialmente uma variação (DMBDDb), proposta por nosso laboratório, como fonte de evidência do potencial cancerígeno de praguicidas agrícolas. O protocolo adotado pelo IBAMA tem algumas particularidades, como o uso de ambos os gêneros de ratos da linhagem Wistar e dois grupos controle positivo (tratados com fenobarbital - FB ou com 2’-acetoaminofluoreno -2’-AAF). Este protocolo foi utilizado ao longo de seis anos em nosso laboratório (TOXICAN) para a realização de cinco bioensaios sob contratos com empresas do setor agroquímico. O presente estudo consiste da revisão dos diagnósticos de três órgãos desses ensaios - o fígado, rins e intestinos - escolhidos porque foram os que apresentaram mais lesões na análise de cada um daqueles ensaios. A capacidade indutora enzimática dos agentes do protocolo foi avaliada pela expressão imunohistoquímica das enzimas hepáticas CYP 2B1/2B2 e 1A2. Os resultados indicam atividade promotora do FB, embora menos evidente que a do 2’-AAF, particularmente nos ratos machos. Apesar da alta variabilidade da linhagem de rato Wistar , este estudo permitiu estabelecer um banco de informações sobre as lesões que caracteristicamente são encontradas naqueles órgãos dos animais Wistar submetidos ao protocolo DMBDDb.
A medium-term multi-organ rat bioassay based on the initiation-promotion carcinogenesis paradigm has been proposed by Japanese researchers as an alternative to the conventional long-term assay for chemical carcinogenesis detection. This alternative bioassay, designated DMBDD (after the five carcinogenic initiators of this protocol), was originally standardized with the male Fischer 344 strain of rats. In 1996, the Brazilian Agency for the Environment (IBAMA) officially recognized a variation (DMBDDb), proposed by our laboratory, as a valid source of evidence of the carcinogenic potential of agrochemicals. The protocol adopted by IBAMA has some modifications, such as the use of both sexes of the Wistar strain of rats and two positive controls (Phenobarbital – PB, 2’-acetoaminofluorene - 2’-AAF). During six years, five different bioassays managed under contract with agrochemical companies were developed by our laboratory (TOXICAN). This study presents the revised results obtained from three organs of this protocol – liver, kidney and intestines –, chosen because they most frequently presented lesions through those assays analyses. Besides, the induction of the CYP 2B1/2B2, 1A2 isoforms was also immunohistochemically evaluated in the liver. Our results document the promoting activity of PB, otherwise less evident than 2’-AAF, especially in male rats. Although a high variability of the Wistar rat strain tested was evident, this study allowed building up a data bank of characteristic lesions in those selected organs of Wistar rats under the DMBDDb protocol treatment.

Tumour cells which lack DNA mismatch repair are resistant to the cytotoxic effects of DNA methylating agents and Cisplatin. To address whether this resistance is mediated through loss of an MSH2-dependent apoptotic pathway, the apoptotic response of the murine small intestine to DNA methylating agents and Cisplatin was studied. MSH2 was found to play a significant role in the initiation of apoptosis in vivo. The immediate apoptotic response to these agents was p53-dependent in the first 24 hours, however a smaller p53-independent apoptotic response was observed beyond this point. Mice doubly null for both MSH2 and p53 revealed that this delayed p53-indepdnent response was entirely MSH2-dependent. These results demonstrate the existence of a pathway to apoptosis following DNA methylation which is dependent upon both MSH2 and p53. The DNA repair enzyme O⁶-Alkylguanine-DNA-alkyltransferase (AGT), which removes potentially mutagenic methyl groups from guanine residues, was found to play no role in modulating the MSH2-dependeent apoptotic response of intestinal cells to methylating agents or Cisplatin, indicating that the rate of removal of methylated bases is not a major factor in the decision to enter this pathway. O⁶-Benzylguanine, a competitive inhibitor of AGT, prevented the metabolic activation of Dacarbazine probably through the inhibition of cytochrome P450 enzymes. This novel finding has adverse implications towards the potential clinical use of O⁶-Benzylguanine. The data presented in this thesis demonstrate that MSH2 plays a pivotal role in determining both the apoptotic response and mutation frequency of the murine intestine to methylating DNA damage and suggests that the consequences of MSH2-deficiency may be more significant to the initial stages of carcinogenesis that loss of p53.

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Universidade Estadual Paulista (UNESP)
O consumo de mate em altas temperaturas tem sido considerado um fator de risco para o desenvolvimento do carcinoma epidermóide de esôfago (CE) na América do Sul. Desta forma, os efeitos da ingestão de mate sobre danos de DNA e a carcinogênese de esôfago, induzidos pela dietilnitrosamina (DEN) e injúria térmica, foram avaliados em ratos Wistar machos. Na fase de iniciação, os animais foram iniciados com injeções i.p. da DEN (8 x 80 mg/Kg p.c.) e submetidos a injúria térmica (água a 65°C, lmIlrato, instilado no interior do esôfago) e receberam, concomitantemente, mate (2.0% p/v, grupo teste) ou chá-verde (2.0% p/v, grupo controle positivo) como única fonte de líquidos por oito semanas. Nenhum tratamento adicional foi introduzido durante a fase de pós-iniciação (nona a vigésima semana do experimento). Amostras de sangue periférico foram coletadas quatro horas após a última administração da DEN para o teste do cometa na oitava semana e amostras de esôfago e fígado foram coletadas na oitava e vigésima semanas do experimento. Na oitava semana, a ingestão de mate e chá-verde por si não foi genotóxica e reduziu de forma significativa os níveis de danos no DNA de leucócitos de sangue periférico nos animais tratados com a DEN. Além disso, uma redução significativa nos níveis de proliferação celular no epitélio do esôfago e no parênquima hepático e no número de lesões hepáticas pré-neoplásicas foram também observadas nos grupos iniciados e que receberam mate ou chá-verde. Na vigésima semana, uma menor incidência de neoplasias de esôfago e fígado foi observada nos grupos que receberam previamente mate e chá-verde quando comparado ao grupo iniciado pela DEN e submetido à injúria térmica. Os resultados do presente estudo indicam que a ingestão de mate se mostrou benéfica contra danos no DNA e a carcinogênese de esôfago e fígado induzidos pela DEN.
Drinking hot mate has been associated with risk for esophageal squamous cell carcinoma m South America. Thus, the modifying effects of mate tea intake on DNA damage and esophageal carcinogenesis induced by diethylnitrosamine (DEN) plus thermal injury were evaluated in male Wistar rats. In the initiation phase, rats were treated with DEN injections (8 x 80 mg/Kg b.w.) plus thermal injury (water 65°C, lml/rat, instilled into the esophagus) and concomitantly received mate tea (2.0% w/v, test group) or green tea (2.0% w/v, positive control group) as the sole source of drinking fluid for 8 weeks. Any additional treatment was introduced at post-initiation until week 20. Peripheral blood was collected 4 hr after the last DEN application for comet assay at week 8 and samples from esophagus and liver were collected at weeks 8 and 20. At week 8, mate or green tea intake itselfwere non-genotoxic and significantly decreased DNA damage levels in peripheral blood leucocytes from DEN-treated animaIs. Also, a significant reduction of cell proliferation rates in both esophageal epithelium and liver parenchyma and on the number of putative preneoplastic liver lesions were observed in initiated and mate or green tea-treated animaIs at week 8. A significant lower incidence of esophageal and liver neoplasms and tumor multiplicity was observed in the groups previously treated with mate or green tea when compared to the DEN initiated/thermal injury group at week 20. These data indicate that mate tea presented protective effects against DNA damage and esophageal and liver carcinogenesis induced by DEN.

Abstract Colorectal cancer is the third most common cancer in the developed countries. Originally, development of CRC was thought to proceed by a sequence of steps known as an adenoma-carcinoma sequence. At present CRC is recognized as a disease developing through diverse pathways. Serrated adenocarcinoma represents an endpoint of tumors developing from serrated pathway. This thesis focuses on studying the molecular alterations in serrated adenocarcinoma. Microsatellite instability, hypermethylation of promoter region in DNA repair genes hMLH1 and MGMT, frequency of KRAS and BRAF mutations and mutation spectrum of PTCH1 was determined in serrated adenocarcinomas (n=42) and compared to non-serrated adenocarcinomas (n=75). MSI, particularly low level of MSI (p=0.02) and methylation of both hMLH1 and MGMT promoters (p=0.004, p=0.026) were found to be more prevalent for serrated CRC. BRAF mutation was frequent and specific to serrated adenocarcinomas (p<0.001) and KRAS mutations were more frequent in serrated adenocarcinomas than in non-serrated cancers (p=0.002). A significant association between BRAF mutation, hMLH1 and MGMT methylation and MSI-H phenotype was found in serrated carcinomas. KRAS mutation was seen in association with MSS/MSI-L phenotype; in fact, if serrated adenocarcinoma presents with MSI-L there always seems to be a KRAS mutation as well. Negative immunohistochemical staining of the hMLH1 enzyme was in association with methylation of the gene and proved reliable in the detection of MSI-H phenotype (p<0.0001). Sequencing analysis of the whole coding regions of the PTCH1 gene did not reveal any truncating mutation to explain the previously detected downregulation of the gene in serrated CRCs. In conclusion, serrated adenocarcinomas proved to be an independent, but heterogeneous subtype of CRCs. High combined mutation rate (79–82%) of KRAS and BRAF in serrated adenomas and adenocarcinomas indicates that MAPK activation is a crucial part of the serrated pathway. BRAF mutations are specific for serrated adenocarcinoma, and identify a subset of serrated adenocarcinomas with gene methylation and a tendency for MSI-H. High frequency of KRAS mutations in serrated adenocarcinomas suggests that a significant proportion of KRAS-mutated CRCs originate from serrated precursors
Tiivistelmä Paksu- ja peräsuolisyöpä eli kolorektaalisyöpä on Suomessa kolmanneksi yleisin syöpätyyppi. Syöpää edeltävien muutosten tunnistaminen on tärkeää, jotta sen ehkäisy ja seuranta olisi tehokasta. Tavallisia adenoomapolyyppeja on pidetty tärkeimpinä kolorektaalisyövän esiastemuutoksina. 2000-luvulla on havaittu, että nk. sahalaitapolyypit edustavat tärkeää osaa esiastemuutoksista, ja näistä kehittyvää syöpää kutsutaan sahalaitaiseksi syöväksi. Sahalaitaisen syövän kehittymismekanismit eroavat huomattavasti tavallisesta kolorektaalisyövästä. Tässä väitöskirjassa keskityttiin tutkimaan sahalaitaiselle syövälle tyypillisiä morfologisia piirteitä sekä geneettisiä muutoksia. Työssä selvitettiin DNA mikrosatelliitti-instabiliteetin sekä DNA korjausgeenien hMLH1 ja MGMT promoottorialueiden hypermetylaation esiintyminen, nk. MAPK –signaalinsiirtoreitin komponenttien, KRAS ja BRAF -geenien, mutaatioiden yleisyys sekä PTCH1 geenin mutaatiokirjo sahalaitaisissa (n=42) ja tavallisissa kolorektaalisyövissä (n=75). DNA:n mikrosatelliitti-instabiliteetti, erityisesti matala-asteisena (MSI-L) (p=0.02) sekä MLH1 ja hMGMT -geenien metylaatio (p=0.004, p=0.026) olivat yleisempiä sahalaitaisissa syövissä. BRAF mutaatio oli yleinen sekä spesifinen sahalaitasyöville (p<0.001). Myös KRAS -mutaatiot olivat yleisempiä sahalaitaisissa syövissä (p=0.002). BRAF mutaatio, hMLH1 sekä MGMT metylaatio ja korkea-asteinen mikrosatelliitti-instabiliteetti (MSI-H) esiintyivät hyvin usein yhdessä sahalaitaisissa syövissä. Sahalaitaisissa syövissä KRAS –mutaatiot liittyivät MSI-L fenotyyppiin. hMLH1 geenin ilmentyminen tutkittiin myös immunohistokemiallisesti. Sahalaitaisissa syövissä MLH1 –proteiinin häviäiminen oli yhteydessä metylaatioon ja liittyi spesifisesti MSI-H:n esiintymiseen (p < 0.0001). PTCH1 geenin sekvensointi ei paljastanut proteiinin toimintaa vahingoittavia muutoksia, eikä tuloksen perusteella pystytä selittämään aikaisemmin havaittua geenin ilmentymisen häviämistä sahalaitaisessa syövässä. Tulosten perusteella sahalaitainen syöpä on oma, mutta heterogeeninen kolorektaalisyövän alatyyppi. KRAS ja BRAF –geenien aktivoivien mutaatioiden yleisyys (79–82%) osoittaa, että MAPK -reitin aktivaatio on tärkeää sahalaitaisen syövän kehityksessä. BRAF -mutaatiot ovat spesifisiä sahalaitaisille syöville, ja yhdessä metylaation sekä MSI-H:n kanssa identifioi osan sahalaitasyövistä omaksi ryhmäkseen. KRAS –mutaatioiden yleisyys sahalaitaisissa syövissä antaa aiheen epäillä, että merkittävä osa KRAS –mutaation sisältävistä kolorektaalisyövistä kehittyy sahalaitapolyypeista

The work was undertaken to examine the effects of nutritional deficiencies on cancer induction. Two of the most common and widely distributed nutrients, iron and folic acid, were examined to evaluate the effects of their deficiency on animals. The Syrian golden hamster was the animal model for all experimental work. In the first part of the study an attempt was made to induce iron deficiency in young adult male hamsters by feeding iron deficient diet coupled with repeated venesection of 1.5ml every two weeks. Following twelve weeks on this regime a superficial biopsy was taken, on week 13, from the medial wall of one pouch in each hamster in order to evaluate the effect of iron depletion on the epithelial compartment thicknesses. After allowing the biopsy sites to heal for two weeks, a solution of 0.25% DMBA in acetone was painted, three times per week, for eight weeks, on a defined one square centimetre area in each pouch of each hamster of the experimental and control groups. The hamsters were then maintained on the same dietary regimes for twelve weeks before being killed at the beginning of week 37 for analysis. Iron deficiency anaemia could not be induced in the experimental animals of this study. The effect of the iron deficient diet on epithelial compartment thicknesses at the stage of the biopsy was not clear. However, restriction of iron intake did cause animals to develop significantly fewer grossly seen tumours and histologically identified carcinomas than control animals. In the second part of this thesis an attempt was made to investigate alternative hamster dietary components that have less iron contamination than the diet given in the first part of this thesis. Casein and calcium lactate were the main contributers to iron in the hamster diet. Casein could not be substituted by another source of protein for hamsters. However, other sources for calcium with less iron contamination were available and therefore investigated in this part of the study. Three groups of young adult male and female hamsters were given the fully nourishing powdered diet used in previous studies. However, calcium lactate was substituted for by either calcium acetate, calcium chloride or calcium sulphate in each group. None of the three diets was accepted by the animals and many of them died of starvation. When calcium salts were replaced by calcium lactate the surviving animals accepted the diet and recovered quickly afterwards. This study proved that calcium lactate could not be substituted by any other calcium salt with less iron content and therefore iron contamination in the hamster diet could not be further reduced by this method. In the third part of this thesis the effect of nutritional folate deficiency on cancer induction was studied. A group of young adult female hamsters was given folate deficient diet for four weeks. On week 5, DMBA in acetone at a concentration of 0.25% was painted on a defined one square centimetre area of the medial wall of each pouch in each hamster in folate deficient and control groups. The carcinogen was applied three times per week for eight weeks following which animals were maintained on the same dietary regimes for a further 13 weeks before being killed at the beginning of week 27 for the final analysis of the study. It was found that nutritional folate deficiency had significantly reduced the number of animals developing grossly counted tumours and histologically identified carcinomas. The folate deficient animals also developed significantly less tumours and carcinomas compared to control groups. In the last part of this thesis, the effect of combined iron and folate deficiency was examined for its role in carcinogenesis of the hamster cheek pouch. Two groups of young adult male hamsters were fed powdered diet lacking iron and folic acid and a third group was fed diet lacking iron only. One of the combined deficiency groups and the iron deficiency group were bled 1.0-1.3ml every week. On week 6 of the study DMBA in acetone at a concentration of 0.25% was painted three times per week for eight weeks on the same area of the pouch used in the previous studies. The animals were then maintained on the same experimental regimes for a further eleven weeks before being sacrificed, on week 25, for the final analysis of the study. In this study, iron deficiency anaemia was induced in animals of the bleeding groups. Animals in the group with combined iron and folate deficiency without bleeding showed low normal folate levels and normal haemoglobin levels. The two groups that were bled repeatedly showed iron deficiency anaemia. In all groups, the numbers of tumours counted grossly and the numbers of carcinomas identified histologically were significantly reduced compared to control animals in the previous studies. The folate deficient diet did not appear to influence the induction of iron deficiency. The studies reported in this thesis proved that nutritional folate deficiency not only reduces the incidence, but it also reduces the numbers of tumours and carcinomas in the hamster cheek pouch. Iron deficiency anaemia was also found to significantly reduce the numbers of tumours and carcinomas of the hamster cheek pouch. It was not possible to produce combined iron and folate deficiency under the conditions of these studies. However, animals fed on a diet lacking iron and folic acid had significantly reduced numbers of grossly seen tumours and histologically identified carcinomas in the cheek pouch in response to DMBA applications. In each of the reported studies, the nutritional deficiency of iron and folic acid, whether individually or combined was found to significantly reduce the growth rate of affected animals.

Introduction: Barrett’s oesophagus (BO) is a metaplastic premalignant disease which can undergo a metaplasia-­‐dysplasia-­‐adenocarcinoma pathway. It represents an example of field cancerization by which an area occupied by BO can undergo molecular and genetic changes associated with carcinogenesis without being phenotypically cancerous. Previous work suggested that non-­‐cancerous BO contains a monoclonal population. More recent work demonstrated that premalignant Barrett’s fields are polyclonal suggesting that clonal interactions may be important in carcinogenesis. It is the aim of this thesis to further investigate clonal interactions in BO by understanding the effects of therapy in altering the relationships of clonal populations in BO, by assessing the relationship of clonal populations in dysplasia as compared with the associated cancer, and by attempting to elucidate a potential molecular mechanism of clonal interactions. Results: The overall results can be summarised as follows: 1.Premalignant clonal populations are well mixed allowing for clonal interactions. However, the adenocarcinoma associated with high grade dysplasia is monoclonal and derived from clonal populations found in the dysplasia, indicating possible clonal interactions during carcinogenesis. 2. Patients with persistent disease after endoscopy retain the same clonal populations. However, the clonal populations of recurrent disease changes such that new clonal populations arise or may benefit from the extinction of others. 3. These clonal populations may be derived from deep submucosal glands or may be found in phenotypically normal squamous epithelium indicating a common stem cell origin. 4. A possible mechanism of clonal interaction may be the senescence associated secretory phenotype: senescence is abundant in BO and can cause proliferation in neighbouring cells in vitro. Conclusion: This thesis has investigated the implications of clonal interactions in BO. The demonstration of temporal clonal heterogeneity as a result of endoscopic therapy, as well as spatial clonal heterogeneity possibly resulting in carcinogenesis, asks for a mechanistic explanation of clonal interactions. The consequences of senescence may well provide one such mechanism.

The work outlined in this thesis is concerned with the use of chemical agents in the prevention and cure of cancer. Under this generally broad brief, we examined three topics of considerable interest in this field at the present time. Firstly we investigated the role of three dietary indoles which have been shown to inhibit the induction of cancer by certain procarcinogens, in particular benzo[a]pyrene. In this work we prepared analogues of the dietary indoles with a view to ascertaining their mode of action and increasing their activity. Next we devised a synthesis of dendrodoine, a natural product extracted in small quantities from the marine tunicate, Dendroda Grossular. This compound was shown in preliminary tests to be of potential use as a cytotoxic anti-cancer drug, but because of its inaccessibility its chemical synthesis was required if its properties were to be fully investigated. Finally, we devised a new strategy in the synthesis of 6H-pyrido-[4,3-b]carbazoles. Although there has been a great volume of work carried out on these compounds over the last 25 years, there has yet to be devised a truly general synthetic route, which allows the function- alisation of these compounds at key points in their structure.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Lemongrass (Cymbopogon citratus STAPF) has been described as a potential chemopreventive agent. Thus, the present study objectives were evaluate the protective effects of oral treatment with lemongrass essential oil (LGEO) on carcinogenesis initiation phase with N-methyl-N-nitrosurea (MNU) and post-initiation carcinogenesis phase in multipleorgans model, through 7,12-dimethylbenz(a)antracene (DMBA), 1,2-dimethylhidrazine (DMH), and N-buthyl-N-(4-hidroxibuthyl)nitrosamine (BBN) administrations in Balb/C female mice. The animals were distributed into 2 experimental protocols. Experiment 1: the animals were allocated into 3 experimental groups: G1A group (negative control), G2A group (treated with LGEO 500 mg/kg b.wt., i.g., during 5 weeks and, at the end of the 3rd and 5th weeks, received one 30 mg/kg MNU i.p. application) and G3A group (treated with the LGEO vehicle, and MNU at the end of the 3rd and 5th weeks). After 4 hours of each MNU application, blood samples were collected to perform the comet assay, and, at the end of the 5th week, all animals were euthanatized and the urinary bladder, mammary glands and colon were collected for histological analysis, apoptosis and cellular proliferation counting. Experiment 2: the animals were allocated into 3 experimental groups: G1B group (positive control, DDB-treated animals), initiated with DMBA (5x1 mg i.g. applications), DMH (4x30 mg/kg s.c. applications) and BBN (8x7.5 mg/kg i.g. applications) and treated with the LGEO vehicle, and G2B and G3B groups, similarly DDB-treated, and treated with 125 mg/kg or 500 mg/kg LGEO respectively (5x/week during 6 weeks). At the end of the experimental period, all animals were euthanatized and urinary bladder, mammary glands and colon were collected for preneoplastic and neoplastic lesions analysis. The LGEO treatment reduced DNA damage in peripheral blood leukocytes as well as mammary gland cellular proliferation index.

The initiating potential of a range of 4 MeV X-rays was studied using the mouse skin two-stage model of carcinogenesis. A single dose of radiation was followed by promotion with 12-O-tetradecanoyl phorbol-13-acetate (TPA). The effect of TPA on tumor incidence when applied as a single dose 24 hours prior to irradiation was examined. Studies were also designed to investigate the effect of promotion duration on tumor incidence. Animals were promoted with TPA for 10 or 60 weeks. Evidence presented here indicates that ionizing radiation can act as an initiator in this model system. All animals that were promoted with TPA for the same duration had a similar incidence of papillomas (pap) regardless of radiation or TPA pretreatment. However, squamous cell carcinomas (scc) arose only in animals that were initiated with ionizing radiation followed by TPA promotion. Increasing the promotion duration enhanced the incidence of scc at the lower initiation dose. TPA pretreatment at the higher irradiation dose resulted in an overall decrease in tumor incidence. At the lower dose of radiation, TPA pretreatment resulted in an increase in the incidence of scc. The incidence of basal cell carcinomas (bcc) was dose dependent and appeared to be independent of TPA promotion. Although ionizing radiation acts as a weak initiator in mouse skin, the conversion of pap to scc was higher than that reported for chemical initiators. To test this further animals were initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) followed by biweekly promotion with TPA. After 20 weeks of promotion, the animals were treated with either acetone, TPA or 8 fractions of 1 MeV electrons. Data indicate that the dose and fractionation protocol used in this study enhanced the progression of pre-existing pap. To examine the role of oncogene activation in radiation induced mouse skin tumors, DNA from various tumors (pap, bcc, scc) were examined for the presence of dominant transforming activity by the NIH3T3 and Rat-2 focus assays. Dominant transforming activity was observed in all tumor types but not in normal or treated epidermis or corresponding liver. The transformed phenotype was further confirmed by growth in soft agar and tumorigenicity in Nude mice. Southern blot hybridization to ras (Ha, Ki, N), raf, neu, erbB and β-lym indicate that these genes are not responsible for the observed transforming activity. These data suggest that the oncogenic sequences activated in these tumors are unique. The work presented here also provides evidence for novel c-myc transcripts and corresponding genomic rearrangements in a few of the tumors studied.

Prostate cancer (PC) is the most common cancer in men. In the UK alone, there are over 30,000 men diagnosed with PC every year. Loss of SPRY2 and activation of receptor tyrosine kinases are common events in PC. However, the molecular basis of their interaction and clinical impact remains to be fully examined. SPRY2 loss may functionally synergise with aberrant cellular signalling to drive PC and to promote treatment resistant disease. Using a combination of in vitro, pre-clinical in vivo models and clinical PC, this thesis shows the impact of SPRY2 loss upon activation of the ErbB signalling system via a positive feedback regulation of the ErbB-PI3K/AKT cascade. Loss of SPRY2 resulted in hyper-activation of PI3K/AKT signalling to drive proliferation and invasion by enhanced internalisation of EGFR/HER2 and their sustained localisation and signalling at the early endosome in a PTEN-dependent manner. This involves activation of p38 MAPK by PI3K to facilitate clathrin-mediated ErbB receptor endocytosis. Furthermore, this thesis suggests a critical role of PI3K/AKT in PC whereby in vitro and in vivo inhibition of PI3K suppresses proliferation and invasion, supporting PI3K/AKT as a target for therapy particularly in patients with PTEN-haploinsufficiency, low SPRY2 and ErbB expressing tumours. In conclusion, SPRY2 is an important tumour suppressor in PC; its loss drives the PI3K/AKT pathway via functional interaction with the ErbB system.

Prostate cancer (PC) is characterised by dependence upon androgen receptor (AR) as its driving oncogene. When organ-confined, radical treatment can be curative, however there is no cure for advanced, castration-resistant prostate cancer (CRPC). There is therefore a need to better understand the biology of PC, and how influencing AR can modify disease progression. Estrogen is essential for prostate carcinogenesis with evidence from epidemiological, in vitro, human tissue and animal studies. Most suggests that estrogen receptor beta (ERβ) is tumour-suppressive, but trials of ERβ-selective agents have not improved clinical outcomes. ERβ has also been implicated as an oncogene, therefore its role remains unclear. Additional evidence suggests interplay between ERβ and AR, the mechanisms of which are uncertain. The study hypothesis ‘ERβ is an important modulator of prostate carcinogenesis’ was developed to establish whether targeting ERβ could affect PC progression. Much of the confusion around ERβ stems from use of inadequately validated antibodies and cell line models. The first phase of this work was to test ERβ antibodies using an ERβ-inducible cell system. Eight ERβ antibodies were assessed by multiple techniques, showing that commonly used antibodies are either non-specific or only specific in one modality. Two reliable antibodies were identified. Next, cell lines previously used to study ERβ were assessed using validated antibodies and independent approaches. No ERβ expression was detected; an important finding that casts doubt on previously published ERβ biology. Subsequently, a PC cell line with inducible ERβ expression (LNCaP-ERβ) was developed and validated to enable controlled experiments on the effects of ERβ on proliferation, gene expression and ERβ/AR genomic cross-talk. Phase three of this work focused on ERβ biology in PC and its relationship to AR. Interrogation of clinical datasets showed that greater ERβ expression associated with favourable prognosis. Gene expression data from men treated with androgen deprivation therapy revealed that AR represses ERβ. This was confirmed in vitro. The LNCaP-ERβ cell line was treated with androgen and/or ERβ-selective estrogen. Activated ERβ in the presence of androgen-stimulated AR inhibited cell proliferation and down-regulated androgen-dependent genes. Genome-wide mapping of ERβ binding sites reveals that ERβ antagonises AR through competition for shared DNA binding sites. In conclusion, ERβ expression is down-regulated by AR during malignant transformation of prostate epithelium. We reveal an antagonistic relationship between ERβ and AR whereby sustaining or replacing ERβ may inhibit tumour growth through down-regulation of AR-target genes. In future, an ERβ-selective compound may be used to slow or abrogate PC progression.

The urothelium, the epithelial lining of the bladder, is exposed to urinary-excreted carcinogens from environmental, occupational and dietary sources. These carcinogens include heavy metals such as cadmium. As cadmium is a weak mutagen, this suggests that genetic mechanisms are not responsible for cadmium-induced carcinogenesis. Non-genotoxic carcinogenesis is relatively poorly understood, however recent advances show that epigenetic dysregulation of gene expression may play an important role. The aim of my research was to investigate epigenetic dysregulation as the candidate mechanism underlying cadmium carcinogenesis of human urothelial cells. Normal human urothelial (NHU) cells were cultured as finite cell lines following isolation from surgical specimens. When established in vitro, NHU cells have a highly proliferative phenotype and can be induced to differentiate using two published methods: either through PPARγ-mediated differentiation or serum-mediated differentiation. Exposure of NHU cells to cadmium inhibited expression of the tumour suppressor genes, p16, APC and RASSF1A. As dysplasia is axiomatic of carcinoma in situ, the precursor to muscle invasive urothelial carcinoma, the potential of NHU cells to differentiate in the presence of cadmium was investigated. Following exposure to cadmium, there was a failure to upregulate archetypical differentiation-associated genes, including uroplakin 1A and 2, and cytokeratins 13 and 20. Trichostatin A, a histone deacetylase inhibitor was able to reverse some of these changes. Mass spectrometry and immunoblotting were utilised to investigate post-translational histone modification changes caused by cadmium exposure. Data showed that there was a change in histone modification marks present in NHU cell cultures exposed to cadmium that failed to upregulate differentiation markers. An increase in repressive histone marks such as methylation at H3K9 was found alongside a decrease in active marks such as acetylation at H3K18 and H3K23. This study presents evidence that cadmium exposure changes the epigenome of NHU cells and leads to compromised urothelial differentiation and downregulation of tumour suppressor genes.

The skin is a vital organ for life; whose primary function is to act as a defensive barrier. Dysfunction in the epidermal barrier of the skin is commonly observed in autosomal recessive congenital ichthyosis (ARCI). The 12R-Lipoxygenase (12R-L) and epidermis-type Lipoxygenase 3 (eLOX-3) pathway plays a key role in the process of epidermal barrier acquisition by affecting lipid metabolism, as well as protein processing. 12R-L KO mice exhibited differential expression of filaggrin, glucosyl ceramide, and ceramide markers, suggesting that they are essential in the proper formation of the epidermis. The skin disorder ARCI, provides perspective into how disruption of the normal skin physiology may contribute to pre-cancerous phenotypes like hyperplasia and dysplasia. The roles of liver kinase B1 (LKB1), adenosine monophosphate-activated protein kinase (AMPK), and human apurinic (apyrimidinic) endonuclease/redox-factor 1 (Ape1/Ref-1) are addressed to highlight some of the molecular pathways associated with ultra violet (UV)-induced skin carcinogenesis. We used UVA-340 as an energy source because it comes closest to mimicking the natural effects of sunlight exposure by containing biologically relevant amounts of UVA and UVB radiation. UVA-340 exerts carcinogenic effects, by reducing AMPK phosphorylation and the activity of its upstream kinase LKB1. Additionally, we demonstrated that UVA-340 stimulates mammalian target of rapamycin (mTOR) activation. Finally, we found that UVA-340 solar simulated light increases the abundance of Ape1/Ref-1 protein in epidermal keratinocytes. The Ape1/Ref-1 protein is involved in activating AP-1 that subsequently activates a transcriptional program associated with cellular proliferation. The natural compound resveratrol was found to inhibit AP-1 DNA binding, although at high concentrations.

Gastric cancer (GC) is a heterogeneous disease with two major histological subtypes “intestinal” (IGC) and “diffuse” (DGC). 1-3% of GCs are truly hereditary and are dominated by the Hereditary Diffuse Gastric Cancer (HDGC) syndrome, predisposing to DGC. HDGC is mainly explained by germline alterations in the CDH1 tumor suppressor gene; however, at least 50% of cases remain without a known genetic determinant. It has recently been reported that intron 2 of the CDH1 gene gives rise to a number of non-canonical transcripts, none of which have been investigated in GC patients. Moreover, the application of exome sequencing has lately unraveled germline lesions in diverse unexpected genes predisposing to the disease. Therefore, to better characterize a cohort of 30 Italian HDGC patients already proven to be CDH1 mutation-negative, I analyzed the expression patterns of the CDH1 canonical transcript and the CDH1a and CDH1j non-canonical transcripts, arising from intron 2, in the blood of HDGC patients and normal controls by reverse transcription quantitative PCR (RT-qPCR). In addition, as part of a larger collaborative study, 7 cases underwent exome sequencing of a panel of 94 cancer-related genes and I performed an in silico analysis on all resulting sub-polymorphic and rare non-synonymous variants. In all 30 cases no expression defects in CDH1, CDH1a and CDH1j could be detected, thus dismissing the involvement of CDH1 defective splicing in those patients. Regarding exome sequencing, upon combining my in silico analysis with supportive literature findings, two genes, CDKN2A and SDHC, were found to explain disease manifestation in 2 out of the 7 patients, respectively. These genes could be put forth as novel candidates for HDGC. The CDH1 gene encodes E-cadherin, a cell-cell adhesion protein. CDH1 inactivating mutations, leading to loss of protein expression, are common in DGC, while alternative mechanisms that can subtly modulate E-cadherin expression characterize the more common IGC. These mechanisms are still poorly understood and may include small regulatory RNAs (micro-RNAs). Moreover, non-canonical transcripts arising from CDH1 intron 2, which has recently emerged as a cis-modulator of E-cadherin expression, can also be involved. Among those transcripts CDH1a proved to be expressed in GC cell lines, while being absent in the normal stomach; however its presence in GC tissue is yet to be investigated. Hence, I evaluated by digital PCR (dPCR) the expression of CDH1 and CDH1a transcripts in cancer and normal tissue samples from 32 IGC patients. Following the application of the fine-tuned dPCR, I found a significant decrease in CDH1 expression in tumors compared to the normal counterparts (P=0.001), which was especially evident in 76% of cases. Furthermore, CDH1a was detected at extremely low levels in 47% of tumors, but not in normal mucosa. A trend was additionally observed of having more CDH1 in tumors lacking the CDH1a transcript. To determine whether miRNAs could have a contributing role, I applied an in silico and literature driven approach to select potential CDH1-regulating miRNAs in IGC. A list of 16 miRNAs was produced, among which miR-92a and miR-101, have been quantified thus far by RT-qPCR. While neither miRNA correlated with the expression of CDH1, miR-101 was found to be significantly lower in tumors compared to normal mucosa (P=1.565x10-05), which points towards its implication in gastric carcinogenesis in ways that surpass CDH1 regulation. On the whole, the dPCR results support the notion that abnormal isoforms and transcripts’ imbalance resulting from cryptic abnormalities along the CDH1 locus, although subtly modulating E-cadherin expression, can still contribute to the carcinogenic process of the intestinal type.
Gastric cancer (GC) is a heterogeneous disease with two major histological subtypes “intestinal” (IGC) and “diffuse” (DGC). 1-3% of GCs are truly hereditary and are dominated by the Hereditary Diffuse Gastric Cancer (HDGC) syndrome, predisposing to DGC. HDGC is mainly explained by germline alterations in the CDH1 tumor suppressor gene; however, at least 50% of cases remain without a known genetic determinant. It has recently been reported that intron 2 of the CDH1 gene gives rise to a number of non-canonical transcripts, none of which have been investigated in GC patients. Moreover, the application of exome sequencing has lately unraveled germline lesions in diverse unexpected genes predisposing to the disease. Therefore, to better characterize a cohort of 30 Italian HDGC patients already proven to be CDH1 mutation-negative, I analyzed the expression patterns of the CDH1 canonical transcript and the CDH1a and CDH1j non-canonical transcripts, arising from intron 2, in the blood of HDGC patients and normal controls by reverse transcription quantitative PCR (RT-qPCR). In addition, as part of a larger collaborative study, 7 cases underwent exome sequencing of a panel of 94 cancer-related genes and I performed an in silico analysis on all resulting sub-polymorphic and rare non-synonymous variants. In all 30 cases no expression defects in CDH1, CDH1a and CDH1j could be detected, thus dismissing the involvement of CDH1 defective splicing in those patients. Regarding exome sequencing, upon combining my in silico analysis with supportive literature findings, two genes, CDKN2A and SDHC, were found to explain disease manifestation in 2 out of the 7 patients, respectively. These genes could be put forth as novel candidates for HDGC. The CDH1 gene encodes E-cadherin, a cell-cell adhesion protein. CDH1 inactivating mutations, leading to loss of protein expression, are common in DGC, while alternative mechanisms that can subtly modulate E-cadherin expression characterize the more common IGC. These mechanisms are still poorly understood and may include small regulatory RNAs (micro-RNAs). Moreover, non-canonical transcripts arising from CDH1 intron 2, which has recently emerged as a cis-modulator of E-cadherin expression, can also be involved. Among those transcripts CDH1a proved to be expressed in GC cell lines, while being absent in the normal stomach; however its presence in GC tissue is yet to be investigated. Hence, I evaluated by digital PCR (dPCR) the expression of CDH1 and CDH1a transcripts in cancer and normal tissue samples from 32 IGC patients. Following the application of the fine-tuned dPCR, I found a significant decrease in CDH1 expression in tumors compared to the normal counterparts (P=0.001), which was especially evident in 76% of cases. Furthermore, CDH1a was detected at extremely low levels in 47% of tumors, but not in normal mucosa. A trend was additionally observed of having more CDH1 in tumors lacking the CDH1a transcript. To determine whether miRNAs could have a contributing role, I applied an in silico and literature driven approach to select potential CDH1-regulating miRNAs in IGC. A list of 16 miRNAs was produced, among which miR-92a and miR-101, have been quantified thus far by RT-qPCR. While neither miRNA correlated with the expression of CDH1, miR-101 was found to be significantly lower in tumors compared to normal mucosa (P=1.565x10-05), which points towards its implication in gastric carcinogenesis in ways that surpass CDH1 regulation. On the whole, the dPCR results support the notion that abnormal isoforms and transcripts’ imbalance resulting from cryptic abnormalities along the CDH1 locus, although subtly modulating E-cadherin expression, can still contribute to the carcinogenic process of the intestinal type.

Gastro-esophageal reflux disease (GERD) is a relevant health problem worldwide. It impairs patients' quality of life and predisposes to intestinal-like esophageal metaplasia (i.e. Barrett's esophagus, BE), that is recognized as the major risk factor for the development of esophageal adenocarcinoma (Eac). The incidence of Eac has dramatically increased since the mid of the 1970s in the USA and Western countries for unknown reasons, while the prognosis of Eac has only slightly been improved. During the same time potent acid suppressors have been introduced for the treatment of GERD. Nowadays these drugs lead the list of the world best seller drugs with a US$ 8 billion market per year. Some authors have linked the increase of the incidence of Eac with the wide diffusion of acid suppression in the general population and among patients with GERD(1). The risk of Eac in patients with GERD is too low to justify endoscopic surveillance. On the other hand endoscopic surveillance for patients with BE is generally accepted. Clinical evidence is still lacking on the best treatment for BE, in order to minimize the risk for neoplastic progression(2). Pharmacological, surgical and endoscopic therapies have been used, without a clear evidence about the benefit of a treatment on the others (chapter 1). The experimental surgical models of reflux-induced esophageal carcinogenesis can reproduce in laboratory animals the stepwise progression from inflammation to Eac, through BE(3,4). In Chapter 2 we provide a detailed description of the microsurgical technique we used for the reflux induced esophageal carcinogenesis model, in order to increase its reproducibility and minimize the number of animals needed to set up the experiments. Chapter 3 shows the results of a study about the effect of chronic GERD on animal welfare. The main short and long-term clinical complications are analyzed, as well as the significance and prognostic value of two different scoring systems based on clinical parameters. Using these methods, humane endpoints can be defined. A time-course experiment of long-lasting GERD in the rat is presented in chapter 4 with both the histological findings and Cdx2 immunostaining(5). Two types of metaplastic lesions are described: intestinal metaplasia (BE) and multi-layered epithelium (MLE). MLE consists of four to seven layers of cells that appear as basaloid squamous cells in the basal part and columnar cells in the superficial layer. Therefore MLE is a hybrid epithelium in which both squamous and columnar epithelia coexist and is considered a "protometaplasia" (i.e. a precursor of BE). Consistently with its phenotype, MLE expresses markers of both squamous and columnar differentiation6. The presence of MLE has been associated with reflux(1). Cdx2 is a transcription factor that regulates the expression of differentiation-related molecules and it is specifically involved in intestinal cells commitment. The prevalence of Cdx2 expression (i.e. the prevalence of BE and MLE) increases significantly with time in the study, suggesting a time-dependent relationship between the "chemical" injury and the severity of the lesions. De novo Cdx2 expression is shown to be an early event among the morphological changes caused by the refluxate, consistently with the results by Pera and collaborators(7), who described Cdx2 immunostaining in the basal cell layer close to esophageal ulcers 16 weeks after surgery. Chapter 5 provides evidence of both esophagitis cystica profunda, metaplasia and Eac in the model in use. Esophagitis cystica profunda has been defined as a highly differentiated mucinous lesion commonly found in the submucosa at the site of surgical anastomosis(8). This entity has to be considered an inflammatory reaction, caused by the surgical insult. On the other side we present an external validation that both intestinal metaplasia (i.e. BE) and true Eac can be obtained using our surgically-induced reflux model. Chapter 6 is an experimental study on the effect of long-term proton pump inhibitor (PPI) treatment in the rat model of reflux-induced esophageal carcinogenesis. Consistently with the literature, describing PPIs as very effective drugs in ulcer healing, ulcers resulted more severe in the placebo group, compared with the PPI group, in our study. Surprisingly, esophagitis cystica profunda was more common among PPI-treated animals. This mucous-producing lesion has been generally described as well differentiated mucinous adenocarcinoma. However, we consider these tumours as inflammatory reactions, consistently with Ten Kate and collaborators(8). Of note, the misinterpretation of those lesions as adenocarcinomas could lead to the false belief that PPI treatment had increased the incidence of adenocarcinoma in the present study. On the contrary, an effect of the drug on the incidence of carcinomas was not demonstrated by our study. Surgical anti-reflux treatments and acid-suppressors in humans aim primarily to relieve symptoms of GERD. Anti-reflux surgery offers the advantage of reducing both acid and bile reflux, which has been shown to act synergistically in the pathogenesis of Barrett’s esophagus(9). On the other hand, PPIs are acid suppressors. The effect of PPIs in preventing or inducing Eac progression in patients with GERD or BE is controversial. In vivo experimental studies of reflux treated with proton pump inhibitor have not revealed a reduction in adenocarcinoma risk(10-12). However using the esophagoduodenostomy model for esophageal reflux in the rat a recent study comparing refluxates of different pH found that non-acidic refluxate increases the occurrence of intestinal metaplasia with dysplasia and EAC while the low-pH gastric juice exerts a protective effect in the presence of duodenal juice(13). Acid has been recently shown to have antiproliferative effects in nonneoplastic Barrett's epithelial cells cultured in vitro and it has been suggested that the prescription of acid suppressors in dosages beyond those required to control GERD symptoms could be detrimental(14). Gastric acid secretion is a complex, tightly regulated, physiological mechanism, with neural, hormonal, paracrine, and intracellular pathways. Gastrin, histamine, acetylcholine are the major stimuli for acid secretion, that is primarily inhibited by somatostatin, and to a lesser extent by cholecystokinin, atrial natriuretic peptide, and nitric oxide(15). PPIs act on the final common pathway of gastric acid secretion, permanently inactivating the H+/K+ ATPase (proton pump) in the parietal cell. The consequent increase in gastric pH removes the negative feedback for gastrin production by G cells. As a consequence, hypergastrinemia develops in patients with GERD treated with PPIs chronically or life-long. Concerns have been expressed about the potential role of gastrin on esophageal carcinogenesis. In vitro studies suggested that BE is sensitive to the proliferative effects of gastrin via its cholecystokinin-type 2/gastrin receptor (CCK-2R)(16). An antiapoptotic role for gastrin through up-regulation of PKB/Akt in BE samples has been recently suggested and the treatment with a CCK-2R antagonist has been shown to reduce the levels of activated PKB/Akt(17). A better understanding of the effect of pathways regulating gastric secretions could lead to new pharmacological strategies to treat gastroesophageal reflux disease.
La malattia da reflusso gastroesofageo (MRGE) è un problema clinico di rilevanza mondiale. Influisce negativamente sulla qualità di vita dei pazienti e predispone alla metaplasia esofagea di tipo intestinale (Esofago di Barrett, EB), che è riconosciuta essere il principale fattore di rischio per lo sviluppo di adenocarcinoma esofageo (ACE). L'incidenza di ACE è aumentata drasticamente negli USA e paesi occidentali dalla metà degli anni Settanta per ragioni sconosciute, mentre la prognosi di ACE rimane infausta. Nello stesso periodo sono stati introdotti efficaci soppressori acidi per il trattamento della MRGE. Allo stato attuale queste terapie guidano la classifica dei farmaci più venduti al mondo con un mercato annuo di 8 miliardi di dollari. Alcuni Autori hanno collegato l'aumento nell'incidenza di ACE con l'ampia diffusione di soppressori acidi nella popolazione generale e tra i pazienti con MRGE(1). Il rischio di ACE nei pazienti con MRGE è troppo basso per giustificare una sorveglianza endoscopica. D'altro canto il follow up endoscopico per i pazienti con EB è generalmente accettato. Tuttora manca evidenza clinica sul miglior trattamento per BE al fine di rendere minimo il rischio di progressione neoplastica(2). Le varie terapie in uso, farmacologiche, chirurgiche ed endoscopiche, non hanno ancora dimostrato una chiara evidenza di beneficio di un trattamento sugli altri (capitolo 1). I modelli chirurgici sperimentali di carcinogenesi esofagea indotta da reflusso possono riprodurre negli animali da laboratorio la progressione a tappe dall'infiammazione all'ACE, attraverso il BE(3,4). Nel capitolo 2 viene fornita una descrizione dettagliata della tecnica microchirurgica in uso per il modello di carcinogenesi esofagea indotta da reflusso, al fine di aumentare la riproducibilità dei dati e minimizzare il numero di animali necessari per il set up sperimentale. Il capitolo 3 riporta i risultati di uno studio sugli effetti della MRGE cronica sul benessere animale. Le principali complicanze a breve e lungo termine vengono analizzate, così come l'importanza e il valore prognostico di due sistemi di valutazione del benessere basati su parametri clinici. Un esperimento time-course di MRGE cronica nel ratto viene presentato nel capitolo 4 con i risultati istologici e immunoistochimici per Cdx2(5)). Vengono descritti 2 tipi di lesioni metaplastiche: la metaplasia intestinale (EB) e il multi-layered epithelium (MLE). MLE consiste di diversi strati di cellule, da 4 a 7, che appaiono squamose basaloidi nella parte basale e colonnari nello strato superficiale. Per questo MLE è un epitelio ibrido nel quale sia l'epitelio squamoso che il colonnare coesistono e viene considerato un precursore di EB. Coerentemente con il proprio fenotipo, MLE esprime marcatori sia di differenziazione squamosa che colonnare(6). La presenza di MLE è stata associata a reflusso(1). Cdx2 è un fattore di trascrizione che regola l'espressione di molecole collegate alla differenziazione ed è coinvolto specificamente nel commitment delle cellule intestinali. La prevalenza dell'espressione di Cdx2 (vale a dire di EB e MLE) aumenta significativamente con il tempo, ad indicare una relazione tempo-dipendente tra l'insulto “chimico” e la gravità delle lesioni. L'espressione di Cdx2 de novo risulta essere un evento precoce nelle modifiche morfologiche secondarie a reflusso, in accordo con i risultati del gruppo di Pera(7), che descrive positività per Cdx2 nello strato di cellule basali in vicinanza di ulcere esofagee già dalla sedicesima settimana dopo l'intervento. Il capitolo 5 dimostra la presenza sia di esophagitis cystica profunda, che di metaplasia e ACE nel modello in uso. L'esophagitis cystica profunda è stata definita come una lesione mucinosa altamente differenziata di comune riscontro a livello dell'anastomosi chirurgica(8). Questa lesione deve essere considerata di natura infiammatoria, secondaria all'atto chirurgico. Il capitolo fornisce un autorevole parere esterno che sia la metaplasia intestinale (EB) che veri ACE possono essere ottenuti utilizzando il nostro modello di reflusso indotto chirurgicamente. Il capitolo 6 è uno studio sperimentale sugli effetti del trattamento con inibitore di pompa protonica (PPI) nel modello sperimentale di carcinogenesi esofagea. Coerentemente con i dati di letteratura, che riconoscono i PPI come farmaci molto efficaci nella guarigione delle ulcere, nel nostro studio la gravità delle ulcere è risultata inferiore nel gruppo trattato con il farmaco rispetto al placebo. Al contrario l'esophagitis cystica profunda è risultata più frequente tra gli animali trattati. L'interpretazione di queste lesioni come carcinomatose ci avrebbe portato a ritenere erroneamente che l'incidenza di cancro fosse più alta tra i trattati, mentre un effetto del farmaco sull'incidenza di carcinomi non è dimostrato nel nostro studio. I trattamenti chirurgici antireflusso e i farmaci soppressori acidi hanno l'indicazione clinica principale di controllare i sintomi nei pazienti con MRGE. La chirurgia antireflusso offre inoltre il vantaggio di ridurre sia il reflusso acido sia quello biliare, che hanno mostrato azione sinergistica nello sviluppo di EB(9). Al contrario i PPI sono soppressori acidi. L'effetto dei PPI nella prevenzione o nell'induzione di ACE nei pazienti con MRGE o EB è controverso. Esperimenti in vivo di reflusso trattato con PPI non hanno rilevato una riduzione nel rischio di adenocarcinoma(10-12). Tuttavia uno studio recente che utilizzava un modello di esofagoduodenostomia nel ratto e confrontava i pH del reflussato ha dimostrato che il reflusso alcalino aumenta il rischio di EB, displasia e EAC, mentre un pH basso esercita un effetto protettivo in presenza di succo duodenale(13). L'acido ha dimostrato avere effetti antiproliferativi in celllule epiteliali di Barrett non neoplastico coltivate in vitro ed è stato suggerito che la prescrizione di soppressori acidi non dovrebbe superare i dosaggi necessari per il controllo dei sintomi di MRGE(14). La secrezione acida gastrica è un meccanismo fisiologico complesso e finemente regolato da vie nervose, ormonali, paracrine e intracellulari. La gastrina, l'istamina e l'acetilcolina costituiscono i maggiori stimoli per la secrezione acida, che viene principalmente inibita dalla somatostatina e in misura minore dalla colecistochinina, dal peptide natriuretico atriale e dall'ossido nitrico(15). I PPI agiscono a livello della tappa finale della secrezione acida gastrica, inattivando la pompa protonica (H+/K+ ATPasi) nella cellula parietale. Di conseguenza, l'aumento del pH intragastrico rimuove il feedback negativo per la produzione di gastrina dalle cellule G. Nei pazienti con MRGE trattati cronicamente con PPI si sviluppa perciò un quadro di ipergastrinemia. Preoccupazione è stata espressa su un potenziale ruolo della gastrina nella carcinogenesi esofagea. Studi in vitro hanno suggerito che EB sia sensibile agli effetti proliferativi della gastrina attraverso il suo recettore CCK-2R(16). Recentemente è stato proposto un ruolo antiapoptotico per la gastrina nell'EB attraverso l'up-regulation di PKB/Akt in BE e il trattamento di campioni di EB con un antagonista per CCK-2R ha dimostrato di ridurre il livello di attivazione di PKB/Akt(17). Una più profonda comprensione degli effetti dei regolatori della secrezione acida potrebbe portare allo sviluppo di nuove strategie farmacologiche per trattare la malattia da reflusso.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
A carcinogênese consiste em um processo de alterações genéticas após contato celular com agentes físicos, químicos ou biológicos. Esta interação pode culminar em manifestações de fenótipos malignos celulares. No estudo experimental da carcinogênese em glândulas salivares animais, os autores são unânimes em apontar os hidrocarbonetos policíclicos aromáticos (HPA) como potentes agentes carcinogênicos. O 7,12 - dimetilbenzantraceno (DMBA), pertencente ao grupo dos HPA, é considerado o carcinógeno químico de eleição para a tumorigênese de glândulas salivares animais. Este trabalho visou o estudo de DMBA injetado em glândulas salivares submandibulares de ratos. Foram utilizados 28 ratos (Rattus norvegicus), com três meses de idade e peso aproximado de 300g. Os resultados revelaram, na 5ª semana, sete casos de sialadenite crônica. Na 10ª semana, um caso com atipia celular ductal, dois casos de carcinoma epidermóide e quatro de sialadenite crônica. Entre a 15ª e 20ª semanas, foram observados três casos de hiperemia, três casos de carcinoma epidermóide, um caso de sarcoma e sete casos de carcinossarcoma. A análise dos dados, em porcentagem, revelou: 3,6% de atipia celular, 3,6% de sarcoma, 10,7% de hiperemia, 17,9% de carcinoma epidermóide, 25% de carcinossarcoma e 39,4% de sialadenite crônica. Conclusão: Os dados obtidos permitiram o estudo da história natural da carcinogênese glandular por DMBA desde os processos inflamatórios iniciais até à formação de neoplasias mesenquimais, epiteliais e mistas.
The carcinogenesis consists in a process in which the direct contact between cells and some physical, chemical or biological agents results in cell malignization. In the scope of experimental salivary gland carcinogenesis there is a consensus in the use of polycyclic aromatic hydrocarbons (PAH) as carcinogens. The 7,12 - dimethylbenzanthracene (DMBA) is the most used PAH. This paper aims the investigations of the DMBA injected in rats submandibular glands. For this purpose, 28 rats (Rattus norvegicus), three months old (300 gr. weight, approximately) were used. The animals were divided into four groups of seven each. All animals were anesthetized and shaved in the neck. After antisepsis, one ventral neck incision, followed by dissection was performed in each animal. The left submandibular gland was injected with 0.1 ml of 2% DMBA in acetone. The skin was closed with 3-Ø silk suture. By the end of the 5th, 10th, 15th and 20th weeks the animals were sacrificed by lethal doses of anesthetics. The results in the 5th week presented seven cases of chronic sialadenitis. After 10 weeks one case of ductal cell atypia was evident, two cases of squamous cell carcinoma and four cases of chronic sialadenitis were also seen. Between the 15th and 20th weeks the cases were diagnosed as follows: three cases of hyperemia, three cases of squamous cell carcinoma, one case of sarcoma and seven cases of carcinosarcoma. The data analysis showed 3.6% of cellular atypia, 3.6% of sarcoma, 10.7% of hyperemia, 17.9% of squamous cell carcinoma, 25% of carcinosarcoma and 39.4% of chronic sialadenitis. Conclusion: the results allowed the investigation of the glandular carcinogenesis natural history after DMBA injection, from the beginning of inflammatory changes to the neoplastic manifestation of mesenquimal, epithelial and mixed tumours.

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O teste de carcinogênese experimental em língua de rato Wistar que utiliza a 4- nitroquinoline 1-oxide (4NQO) como agente cancerígeno, é um excelente modelo para se estudar o carcinoma espinocelular em todas as suas fases, além das lesões pré-neoplásicas e neoplásicas induzidas serem semelhantes àquelas da cavidade bucal de seres humanos. O objetivo do presente estudo foi investigar o papel da proteína p53 e de mutações no gene supressor tumoral TP53 exons 5 a 8 durante a carcinogênese induzida pela 4NQO em língua de rato Wistar. Para isso, 30 animais foram tratados com o cancerígeno na concentração de 50 ppm, por via oral (água de beber), e sacrificados 4, 12 ou 20 semanas após o tratamento. Dez animais foram utilizados como controle negativo. Os resultados mostraram diferenças estatisticamente significativas (p<0,05) com a aumento na expressão da proteína p53 nos grupos de animais sacrificados 12 e 20 semanas após a exposição ao cancerígeno, que também apresentaram lesões pré-neoplásicas e carcinomas espinocelulares, respectivamente. Fraca expressão da proteína p53 foi encontrada nos grupos controle e de 4 semanas de exposição ao carcinógeno. O sequenciamento genético dos exons 5 a 8 do gene TP53 não indicou a presença de mutações. Concluindo, a expressão anômala da proteína p53 nas fases intermediária e final da carcinogênese bucal não pode ser relacionada à presença de mutações nos exons 5 a 8 do respectivo gene.
The medium-term tongue carcinogenesis assay is a useful model for studying oral squamous cell carcinomas phase by phase. The aim of the present study was to investigate the expressivity of p53, as well as mutations in exons 5-8 of TP53 gene during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO) using immunohistochemistry and DNA sequencing, respectively. A total of 30 male Wistar rats were treated with 4- nitroquinoline 1-oxide in drinking water for 4, 12, and 20 weeks.Ten animals were used as negative control. Statistically differences (p<0.05) were found in p53 expression 12 and 20 weeks after treatment,i.e., pre-neoplastic lesions and squamous cell carcinomas, respectively. A weak immunoexpression was observed in the negative control and in ‘normal’ oral mucosa following 4 weeks after exposure to 4NQO. Regarding DNA sequencing, no mutation was found in all of the exons evaluated at all experimental periods. Taken together, our results suggest that abnormal p53 expression was present in pre-neoplastic lesions and squamous cell carcinomas of the oral cavity. However, no mutations were detected during oral cancer progression.

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Intestinal tract cancer is one of the more common cancers in the United States. While in some individuals a genetic component causes the cancer, the rate of cancer in the remainder of the population is believed to be affected by diet. Since cancer usually develops slowly, the amount of oxidative damage to DNA can be used as a cancer biomarker. This dissertation examines effective ways of analyzing FLARE assay data, which quantifies oxidative damage. The statistical methods will be implemented on data from a FLARE assay experiment, which examines cells from the duodenum and the colon to see if there is a difference in the risk of cancer due to corn or fish oil diets. Treatments of the oxidizing agent dextran sodium sulfate (DSS), DSS with a recovery period, as well as a control will also be used. Previous methods presented in the literature examined the FLARE data by summarizing the DNA damage of each cell with a single number, such as the relative tail moment (RTM). Variable skewness is proposed as an alternative measure, and shown to be as effective as the RTM in detecting diet and treatment differences in the standard analysis. The RTM and skewness data is then analyzed using a hierarchical model, with both the skewness and RTM showing diet/treatment differences. Simulated data for this model is also considered, and shows that a Bayes Factor (BF) for higher dimensional models does not follow guidelines presented by Kass and Raftery (1995). It is hypothesized that more information is obtained by describing the DNA damage functions, instead of summarizing them with a single number. From each function, seven points are picked. First, they are modeled independently, and only diet effects are found. However, when the correlation between points at the cell and rat level is modeled, much stronger diet and treatment differences are shown both in the colon and the duodenum than for any of the previous methods. These results are also easier to interpret and represent graphically, showing that the latter is an effective method of analyzing the FLARE data.

Abstract Cancer is the leading cause of death worldwide. Despite the significant research effort, underlying mechanisms of carcinogenic processes are still poorly understood. In recent decades, a group of extremely reactive oxygen metabolites, reactive oxygen species (ROS), have been linked closely to carcinogenesis. Levels of ROS are constantly controlled by antioxidants to ensure stable redox balance in our cells. An aberrant cellular redox balance is thought to be connected to carcinogenesis by inflicting damage to cellular macromolecules and disturbing normal cellular signalling. In this work, the role of ROS in carcinogenesis was studied by observing the ROS-derived DNA damage marker 8-hydroxydeoxyguanosine (8-OHdG) in breast cancer and endometriosis-associated ovarian cancer. This marker was also measured in connection with endometriosis and PCOS to study the early stages of the carcinogenic process. In addition, peroxiredoxin antioxidant enzymes were studied in endometriosis-associated ovarian cancer to explore their impact on the carcinogenic process and relationship with ROS-derived DNA damage. There seems to be a decreasing trend in the expression of 8-OHdG in the development of breast cancer and endometriosis-associated ovarian cancer. In breast cancer, low levels of 8-OHdG in serum and in tumour tissue were found to be associated with more aggressive disease. In endometriosis-associated ovarian cancer, 8-OHdG and Prx II expressions in tissue decreased with malignant transformation from benign endometriosis tissue to ovarian cancer. Patients with PCOS were found to have lower levels of 8-OHdG in serum compared with healthy controls and metformin treatment further decreased 8-OHdG levels in obese patients. These results, together with observations is previous studies indicate that in breast cancer and endometriosis-associated ovarian cancer, a high level of ROS-derived DNA damage could be significant factor in the initiation stage of carcinogenesis, whereas in later stages carcinomas benefit from lower ROS levels that support tumour growth and survival via cellular signalling. In endometriosis, there seem to be high amounts of ROS-derived DNA damage, which could explain the increased ovarian cancer risk, while in PCOS, aberrant ROS levels could contribute to the pathogenesis of the disease itself and also to possible cancer incidence by inducing abnormal cellular signalling
Tiivistelmä Syöpä on nykyisin maailman yleisin kuolinsyy. Vaikka syöpätutkimukseen kohdistetaan maailmanlaajuisesti huomattavia resursseja, syövän kehittymisen perimmäinen syy on edelleen heikosti tunnettu. Yhä kasvavan todistusaineiston perusteella happiradikaalien epäillään liittyvän läheisesti syövän kehittymiseen. Nämä erittäin reaktiiviset hapen aineenvaihduntatuotteet ovat välttämättömiä solujemme normaalille toiminnalle, mutta liian suurina määrinä ne voivat vaurioittaa solun rakenteita ja häiritä solun normaalia viestintää. Solut sisältävät useita antioksidantteja, joiden tärkeimpänä tehtävänä on kontrolloida happiradikaalien määrää ja näin ylläpitää solun hapetus-pelkistys -tasapaino. Tässä väitöskirjatutkimuksessa tutkittiin happiradikaalien yhteyttä syövän kehittymiseen tarkastelemalla niiden aiheuttaman DNA-vaurion merkkiainetta, 8-hydroksideoksiguanosiinia (8-OHdG), rintasyövässä ja endometrioosiin liittyvässä munasarjasyövässä sekä peroksiredoksiiniperheen antioksidanttientsyymejä endometrioosiin liittyvässä munasarjasyövässä. 8-OHdG:n avulla selvitettiin myös munasarjojen monirakkulaoireyhtymän (PCOS) ja endometrioosin yhteyttä syövän kehittymiseen. Lisäksi tutkittiin metformiinin vaikutusta happiradikaalien aiheuttamaan DNA-vaurioon. Väitöskirjatutkimuksen tulosten perusteella 8-OHdG:n määrä vähentyy rintasyövän edetessä ja endometrioosiin liittyvän munasarjasyövän kehittyessä. Matalat 8-OHdG -tasot syöpäkudoksessa ja verinäytteissä ovat yhteydessä aggressiivisempaan taudinkuvaan rintasyövässä. Endometrioosiin liittyvässä munasarjasyövässä kudoksen ilmentämä 8-OHdG ja Prx II vähentyi asteittain siirryttäessä hyvänlaatuisesta endometrioosista munasarjasyöpään. Munasarjojen monirakkulaoireyhtymässä potilaiden verinäytteiden 8-OHdG -tasot olivat merkittävästi matalammat terveisiin verrokkeihin verrattuna. Korkeilla happiradikaalipitoisuuksilla ja niistä aiheutuvalla DNA-vauriolla on väitöskirjatutkimuksen tulosten perusteella tärkeä rooli syövän syntyvaiheessa, kun taas syövän myöhemmissä kehitysvaiheissa matalat happiradikaalipitoisuudet tukevat syövän kasvua ja selviytymistä soluviestinnän avulla. Endometrioosipotilaiden kohonnut munasarjasyöpäriski vaikuttaisi olevan seurausta runsaasta happiradikaalien aiheuttamasta DNA-vauriosta endometrioosikudoksessa. Munasarjojen monirakkulaoireyhtymässä poikkeavien happiradikaalitasojen aiheuttama puutteellinen soluviestintä liittyy mahdollisesti taudin patogeneesiin ja syöpäriskiin

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.

The aim of this research is to investigate whether there is any evidence for a role of RA in the development of Barrett’s metaplasia and subsequent carcinogenesis. First, an ex vivo organ culture system was established in which columnar metaplasia could be induced by exogenous all-trans RA (ATRA) from a squamous tissue, and conversely, squamous metaplasia could be induced from a Barrett’s tissue. Molecular characterisation of these induced-phenotypes was performed with a variety of markers. In addition, the factors that may have contributed to the activation of endogenous RA signalling in vivo were explored. The results demonstrated that bile acid, lithocholic acid (LCA) and Cdx2 over-expression can increase RA bioactivity. On the other hand, a relative depletion of RA activity was noted in Barrett’s cancerous cells comparing to non-dysplastic Barrett’s cells. I went on to demonstrate that reduced RA may be due to the increased expression of a RA catabolising enzyme – CYP26A1. Using serial functional assays, CYP26A1 gene over-expression was demonstrated to be able to enhance Barrett’s cell proliferation, invasion, and drive the cells into an undifferentiated state. The pattern of gene expression was in keeping with the functional changes in that there was induction of genes involved in cell survival and reduced expression of cell death genes. In addition, there was induction of the oncogenes c-Myc and EGFR (epidermal growth factor receptor). The role of CYP26a1 gene activation in Barrett’s carcinogenesis introduces the possibility of a wider role for this gene in cancer. It is hoped that works arsing from this thesis can help further understanding of Barrett’s oesophagus and associated adenocarcinoma so that future developments can be made in its treatment and prevention.

Carcinogenesis is a multistep process, and epithelial cancers are preceded by a series of morphologically recognisable pre-invasive lesions that develop over a period of time. Lung cancers arise from morphological steps involving hyperplasia, metaplasia, dysplasia, carcinoma in situ, invasive carcinoma and metastatic carcinoma. Chromosome 3 and p53 gene damage are two most common genetic abnormalities found in lung cancer. Loss of chromosome 3p and mutation of p53 gene occurred in severe dysplasia, a lesion believed to immediately precede invasive tumour. However, little is known about their involvement in early bronchial pre-invasive lesions. By studying different grades of pre-invasive lesions and tumours obtained from the same patient (parallel study), it was found that chromosome 3p loss and mutation of p53 gene occurred in early dysplastic lesions, suggesting that they were early events in the development of lung cancer. Moreover, damage to chromosome 3 was an earlier event than mutation of p53 gene. Sequential loss within chromosome 3p was observed. Chromosome 3p loss in invasive tumours was more frequent and more extensive than that in pre-invasive lesions. A study of pre-invasive lesions obtained over a period of nine months (longitudinal study) from a patient without tumour showed that there was a sequential damage of 3p, confirming the findings of the parallel study. In addition, the longitudinal study showed that there was a clonal expansion of p53 mutant cells over the nine-month period. Using microsatellite markers within a homozygously deleted region in a small lung cancer cell line, U2020, an interstitial deletion on 3p in dysplastic lesions was delineated which only partly overlaps the U2020 deletion. This suggested that allele loss study in pre-invasive lesions can potentially provide refinement to the location of tumour suppressor genes.

Cancer is a group of diseases characterized by autonomous, uncontrollable cell proliferation, evasion of cell death, self-construction of oxygen and nutrient supply and spreading of cancerous cells through metastasis. It is vital to elucidate pathways that underlie the cancer process. Mechanistic cancer models, to this extent, attempt to relate the occurrence: of malignant neoplasm to diverse risk factors such as genetic alterations, susceptibility of individuals and exogenous and endogenous carcinogenic exposures. The main objectives of this thesis are to examine the validity of the two-hit hypothesis for retinoblastoma (Knudson, 1971, PNAS (68) 820-23), to unify multiple types of genomic instability and to further assess the role of genomic instability played in the process of carcinogenesis. I shall also explore characteristics of existing mechanistic cancer models. This thesis begins with a survey' of basic cancer biology and existing mechanistic models. Utilizing a fully-stochastic two-stage clonal expansion model, the thesis specifically assesses the validity of the two-hit theory for retinoblastoma-(RB), a childhood ocular malignancy. Comparison of fits of a variety of models (in particular those with up to three mutations) to a population-based RB dfltaset demonstrates the superior fit of the two-stage model to others. This result strongly suggests both the necessity and sufficiency of the two RBI mutations to initiate RB and hence validates the two-hit theory. The thesis goes on to develop a comprehensive,. framework to incorporate multiple types of genomic instability, characterized by-numerous numerical and structural damages exhibiting in the cancer cell genome. This generalized model embraces���·;most, if not all, of the existing MVK-type models. Specific forms of the model are fitted to U.S. white American colon cancer incidence data. Based on comparison of fits to the population-based data, there is little evidence to support the hypothesis that models with more than one type of genomic instability fits better than those with a single type of genomic instability. Since the age-specific incidence data may not possess sufficient information for model discrimination, further investigation is required. The remainder ofthis thesis is concerned with two theoretical aspects. To facilitate a Bayesian implementation for data fitting, a flexible blocking algorithm is developed. In the presence of parameter correlation, the algorithm considerably improves the performance of the Markov chain Monte Carlo simulations. In addition, following a similar approach of Heidenreich et al. (1997, Risk Anal. (17) 391-399), the maximum number of identifiable parameters in the proposed cancer model WIth r types of genomIc InstabIlIty IS r +1 less than the number of biologically-based parameters.

Background: Cutaneous squamous cell carcinoma (SCC) is the second most common cancer to affect populations of European descent. It is potentially fatal and causes substantial morbidity. Up to 70% of SCC arise from clinically identifiable precancerous lesions, known as actinic keratoses (AK), permitting early diagnosis and intervention. Keratinocyte Intra-epidermal Neoplasia (KIN) are the histological representation of AK. Immunosuppressed organ transplant recipients have 60 – 200 fold higher rates of SCC that are characteristically multiple, aggressive, recurrent and have higher metastatic tendency. The molecular pathogenesis of SCC involves the accumulation of multiple genetic and epigenetic aberrations, but in spite of the magnitude of the problem, the cellular basis for SCC has received little scientific attention compared with other epithelial cancers. Methodology: Skin samples representing sequential keratinocyte carcinogenesis (from non sun-exposed, sun-exposed and AK/KIN skin) were collected from 53 individuals (31 immunosuppressed and 22 immunocompetent), 30 of whom underwent an additional biopsy after prospective treatment with one of three commercially available topical agents. Genome-wide single nucleotide polymorphism (SNP) profiling was performed on all samples and transcriptional profiling was performed on 7 series from each treatment group. Results: The mean number of SNP changes was 3.57 and 3.48 in NSE and SE skin, respectively, increasing to 6.93 in KIN. Several recurrent areas of loss or gain were identified in KIN, specifically deletion at 3p, 9p, 12q, 16q, 18q and X and amplification of 8q, 9p and 17p. Expression profiling identified 428 genes (false discovery rate 0.05) with altered expression in KIN skin compared to paired normal skin. Of these genes, 31% displayed concordant change with regions of copy number change. Several gene networks of interest were identified including F- and -actin, NFB, PPAR and TGF suggesting that they may be key pathways in KIN evolution. Several candidate genes in KIN skin continue to be dysregulated in SCC, some of which, such as ANG, S100A9, ACVR2A, FHL1 and SFN resolve after topical chemoprevention and others, WIF1, CCL27 and LEPR persist. Conclusions: This is the first systematic, in-depth, in vivo study of molecular events characterising KIN and provides a detailed profile of the critical events contributing to malignant progression in keratinocyte neoplasia and the molecular effects of widely used topical chemopreventive agents Together, these data provide clinically relevant insights into cutaneous squamous tumour biology and identify biomarkers with diagnostic, prognostic and therapeutic potential. Acknowledgements

Cytochrome P450 enzymes (CYP) are key oxidative enzymes that are crucial in several biological processes, such as metabolism of exogenous and endogenous substances, the biological transformation of drugs and xenobiotics and biosynthesis of steroids and fatty acid. Several CYP have been identified in extra hepatic tissues implying that these enzymes exert other biological functions, which might explain their association with a number of diseases including diabetes, obesity and cancer. Understanding of these functions may provide the platform for the development of new therapeutic approaches and this is the aim of this investigation, namely to delineate the role of CYP in breast carcinogenesis. Cancer cells exhibit high levels of glycolysis even in the presence of high oxygen concentration. Cancer cells have very high proliferating rates so they need more biosynthesis materials like nucleic acids, phospholipids, fatty acids and glycolysis is the main source of biosynthetic precursors. Energy metabolism has recently attracted the interest of several laboratories as targeting the pathways for energy production in cancer cells could be an efficient anticancer treatment. Previous studies have shown that reactive oxygen species (ROS) regulate the energy metabolism in cancer cells. CYP are one of the ROS source. Expression of CYP in extrahepatic implies that these enzymes exert other biological functions which have not yet been elucidated. These findings led us to hypothesise that cytochrome P450 enzymes might be involved in the determination of the pathway of cellular energy metabolism in breast cancer cells and in particular in directing tumour cells to produce energy through glycolysis rather than Oxidative phosphorylation (OXPHOS). To investigate the role of CYP in breast carcinogenesis, we followed the protein levels of CYP1B1, CYP1A1, CYP2E1, CYP2C8, CYP2C9 and CYP3A4 in MCF-7 (Michigan Cancer Foundation-7), T47-D, MDA-MB-231 (MD Anderson series 231 cell line) and MDA-MB-468 (MD Anderson series 468 cell line) breast cancer cells treated with glycolytic inhibitors 3-Bromopyruvate and 2-Deoxyglucose (3BP and 2DG). CYP were differentially expressed in breast cancer cells upon treatment with the glycolytic inhibitors (2DG and 3BP) in breast cancer cell lines bearing different genetic background and migratory capacity. The CYP mediated ROS generation was followed in breast cancer cells overexpressing CYP1B1, CYP2C8, CYP2C9 and CYP2E1 or treated with 3BP, 2DG and CYP1B1 specific inhibitor 2,3',4,5'-Tetramethoxystilbene (TMS) by H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) staining. The functional significance of the CYP1B1, CYP2C8, CYP2C9, CYP2E1 mediated modulation of the cellular redox state was investigated by recording changes of indicators of biological pathways known to be affected by the cellular redox state such as cell cycle, adenosine triphosphate (ATP) level, lactate level, mitochondrial potential, autophagy and endoplasmic reticulum (ER) stress. Furthermore, the effect of CYP1B1 and CYP2E1 induction by their inducers (Benzopyrene and Acetaminophen respectively) and inhibition by their specific inhibitors (TMS and chlormethiazole (CMZ) respectively) on cell survival was investigated. Migratory potential of breast cancer cells was investigated under the treatment of glycolytic inhibitors, CYP1B1 inducer and inhibitors. The results obtained provide evidence that CYP are potentially involved in the regulation of ROS, cell cycle, ATP level, lactate level, mitochondrial potential, autophagy, ER stress and migratory potential in a manner dependent on the genetic background of the cells and the stage of the breast cancer, supporting the notion that CYP are potential breast cancer biomarkers.