Dissertations / Theses on the topic 'Gastrointestinal system – Cancer'

Tumour hypoxia has been linked to increased resistance to both radiotherapy and chemotherapy, especially in solid metastatic GI tumours. Under hypoxic conditions, genes that promote tumour growth and survival are up-regulated, via the transcription factor hypoxia-inducible factor-1 (HIF-1). The digestive hormone gastrin, which is often over-expressed in GI cancers, has also been shown to act as a pro-survival factor, up-regulating processes such as tumour proliferation, angiogenesis and migration, and down-regulating apoptosis. Due to the high level of similarity between the downstream events mediated by the two proteins, the relationship between gastrin and HIF-1 was investigated. HIF-1α nuclear protein expression was inducible under hypoxic conditions, which led to an expected increase in VEGF gene expression, followed by a 12-50 fold increase in hypoxic gastrin mRNA expression. HIF-1α expression and transcriptional activity were not consistently affected by exogenous gastrin. RNA-interference-mediated knockdown of HIF-1α resulted in a 40-60% down-regulation of gastrin gene expression under hypoxic conditions suggesting that HIF-1α is partially responsible for gastrin up-regulation in hypoxia. Potential hypoxia-response elements (HREs) were identified within the gastrin promoter, but were only partially responsive to hypoxic incubation in GI carcinoma cells in luciferase-reporter assays. Other possible mechanisms that may account for the increased gastrin gene expression induced under hypoxic conditions include interactions of gastrin with other transcriptional regulators, either in synergy with or independent from HIF-1, or the sequestration of gastrin within the cell by ‘P’-bodies or RNA-binding proteins. These findings may indicate that the addition of anti-gastrin agents such as CCK-2 receptor antagonists or gastrin immunogens to the treatment regime of patients with solid GI tumours may be clinically beneficial, especially if combined with agents used to reduce radiotherapy and chemotherapy resistance.

Purpose: Hypoxia in colorectal cancer (CRC) and gastroesophageal cancer (GEC) decreases tumour responsiveness to radio and chemotherapy leading to cancer progression and poor prognosis. This is the first study to utilise [18F]FAZA hypoxia radiotracer in patients with CRC and GEC. Methods: Six patients (mean age 68±8 years, 2 males and 4 females) with CRC and 4 patients diagnosed with GEC (mean age 65 years, 3 males and 1 female) were included in the study. [18F]FAZA was synthesised at the John Mallard Scottish PET Centre. After injection with 370 MBq of [18F]FAZA, PET/CT images with 60 min dynamic scan were acquired. In addition, 15 min static scans 2 hr post injection were performed. 3D PET images were reconstructed iteratively using an ordered subset expectation maximization (OSEM) method and fused to the corresponding low-dose CT images. [18F]FAZA uptake parameters including maximum standard uptake value (SUVmax), tumour-to-muscle ratio (T/M), tumour-to-bowel ratio (T/B) and volume of interest (VOI) were measured. Results: 4 out 6 patients with CRC (66%) showed clear uptake of [18F]FAZA in the primary tumour. The mean tumour SUVmax was 2.2±0.91 (range 1.12 - 3.71). The tumour SUVmax was significantly higher compared with muscle and bowel (t(5) =3.11, P=0.03), (t(5) =3.08, P=0.03), respectively. However, tumour SUVmean didn't differ significantly compared with muscle and bowel (t(5) =2.41 , P=0.06), (t(5) =2.46 , P=0.06) respectively. The mean tumour to muscle ratio (T/M) ratio was 1.89±0.64 (range 1.10 - 2.87), while the mean tumour to normal bowel (T/B) was 1.92±0.64 (range 1.08 - 2.74). However, [18F]FAZA did not accumulate in any of the tumours found in patients with GEC. Conclusions: [18F]FAZA PET/CT imaging is suitable and feasible for detecting CRC hypoxic tumour regions with image quality that can be used in clinical practice.

Pancreatic cancer (PC) is the fourth leading cause of cancer death in Western societies. Despite significant progress in understanding the molecular pathology of PC and its precursor lesion: pancreatic intraepithelial neoplasia (PanIN), there remain no molecules with proven clinical utility. Affymetrix Genechipfi oligonucleotide microarrays were used to interrogate mRNA expression of PC and normal pancreas to identify molecular pathways dysregulated in PC. Analysis of these data identified altered expression of numerous components of the S100 Calcium Binding Protein Family, Retinoic Acid signalling pathway and the HOX transcriptional network in PC compared to normal pancreas. These pathways were assessed using immunohistochemistry (IHC) and in-situ hybridisation (ISH) in a cohort of patients with PC. Increased protein expression, of S100A2, S100A6 and S100P was observed in 43%, 60% and 48% of PC respectively. Expression of S100A2 was associated with a poor outcome (p = 0.009), whilst increased expression of S100A6 (p = 0.0008) and S100P (p = 0.0005) were associated with an improved outcome. Additionally, S100A2 expression was identified as an independent marker of outcome in resected tumours. Aberrant expression of retinoic acid signalling components was demonstrated in PC cell lines using semi-quantitative RT-PCR. ISH demonstrated expression of Retinoic Acid Induced 3 (RAI3), an orphan G protein coupled receptor normally expressed in the fetal lung, in 68% of PC, and this co-segregated with an improved overall survival (p = 0.026).Ectopic protein expression of HOXB2, a transcription factor normally expressed in the developing hindbrain and modulated by retinoic acid, was observed in 15% of early PanIN lesions and 38% of PC specimens. Expression of HOXB2 was associated with non-resectable tumours and was an independent predictor of poor survival in resected tumours. Suppression of HOXB2 protein expression using small interfering RNA, resulted in epithelioid trans-differentiation in the Panc-1 PC cell line, however no alteration in proliferation rates were observed compared to controls. This thesis has shown that transcript profiling and tissue validation has identified potential markers of early diagnosis and outcome in PC. Furthermore, pathways and molecules previously thought to be associated with normal human development have been implicated to play a role in the development and progression of PC. Further analyses of these markers will determine any potential role in future diagnostic and therapeutic strategies.

Thesis (MSc(R)) - University of Glasgow, 2008.
Submitted to the University of Glasgow for the degree of Master of Science (Medical Science) in the Faculty of Medicine, 2008. Includes bibliographical references. Print version also available.

Thesis (MSc)--Stellenbosch University, 2004.
ENGLISH ABSTRACT: The work presented in this thesis deals with the association between cancers of the upper gastro-intestinal tract and the tumor suppressor gene, TP53, and can be divided into three parts: (i) the analysis of the mutational spectrum of TP53 with respect to laryngeal cancer, (ii) the analysis of the mutational spectrum of TP53 with respect to esophageal cancer and (iii) the analysis of TP53 transcriptional levels in esophageal cancer. Laryngeal cancer (LC) is the 6th most common cancer in the world and the 2nd most common respiratory cancer, with approximately 500 000 new cases per annum detected worldwide. Over the last few years, LC has become increasingly prevalent within the Coloured Community of the Western Cape. The mechanisms of tumorigenesis in LC remain unknown, although smoking and alcohol consumption are considered to be major risk factors. Mutations within the gene TP53 have been strongly implicated as playing a role in cancer development, as they are frequently found in several cancer types. We therefore screened exons 5 - 8 of TP53 for mutations in DNA from tumor biopsies (n=44) and blood samples (n=42) from Coloured LC patients, using polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. Blood samples from a healthy, matched control group (n=40) were included in the study as controls. Significant correlations were found between the occurrence of LC and age and smoking, whereas daily meat consumption was a possible protective factor. In tumor-derived samples, mutations were found in 3 of the exons under investigation, representing 25% of the samples. The mutations were unique to the tumor biopsies, indicating a somatic origin for mutations. The data confirms that the region between codons 175 and 273 of TP53 is a mutational hotspot for cancers in general. This study reports 6 novel mutations within this same region. Esophageal cancer (EC) has a very high incidence in South Africa, relative to the rest of the world, and is particularly common amongst the Black Transkei population. The goal of this study was to determine whether there are differences in the TP53 mutational pattern observed in the Coloured Western Cape community as compared to that observed in the Black Transkei community. This required the analysis of the molecular structure of TP53, specifically exons 5 - 8, in a group of Coloured EC patients (n=44) treated at Tygerberg Hospital, Cape Town, South Africa. DNA obtained from tumor biopsies and blood (from patients) as well as from apparently healthy surrounding tissue was screened via PCR-SSCP and direct sequencing analysis. Only 4 nucleotide changes were observed from a total of 124 sequences obtained, of which two were novel to esophageal squamous cell carcinoma. These 4 nucleotide alterations were found only within the tumor biopsy sample set, representing 9% of the tumors investigated. This study revealed that the mutational spectrum of TP53 within the Coloured population of the Western Cape greatly differs from that of the Black community of the Transkei. This suggests that a different set of etiological factors are involved in the tumorigenic process for each of these distinct geographical communities, which is the subject of an epidemiological study undertaken by the MRC. The final part of this thesis deals with the quantification and comparison of TP53 transcription levels in esophageal cancer tumor tissue to the TP53 levels in healthy esophageal tissue obtained from patients from a unique geographical and ethnic background. The cohort used in this study consisted of Coloured patients (n=2) treated at Tygerberg Hospital. The LightCycler system was implemented in order to try to accurately quantify TP53 mRNA levels. Unfortunately, the desired results were unattainable due to unforeseen difficulties encountered during the study. These difficulties included the insufficient preservation of samples for RNA based studies. Several recommendations were made concerning future similar studies, including an improved planning strategy as well as the employment of an RNA stabilizing agent. Additionally, a few important contributions were made through this study, including the design and optimization of TP53 primers specifically intended for future RNA studies. These primers would enable the identification of the presence of TP53 RNA species as well as the absence of DNA contamination in a single PCR amplification step. Other contributions include the development of a well-optimized RNA extraction method for the extraction of RNA from tough tissues (such as the human esophageal tissue used in this study). This method makes the extraction of large quantities of RNA from small amounts of tough tissue types possible. In conclusion, this study has made a significant contribution to the field of cancer research, by shedding light on the TP53 mutational spectrum with regards to laryngeal as well as esophageal cancer in a population unique to the Western Cape. The first part of this thesis has been published in Cancer Genetics and Cytogenetics (Barnard, D., K. Lehmann, E.G. Haal, P.O. van Heiden, and l.C. Victor. 2003. The spectrum of mutations in TP53 in laryngeal cancer patients from a high-incidence population shows similarities to many of the known mutational hotspots. Cancer Genetics and Cytogenetics 145:126-132), of which a copy can be found in Appendix I. This work has also been presented (by D. Barnard) at an international conference entitled "Cancer of the Esophagus and Gastric Cardia: From Gene to Cure", held in Amsterdam, the Netherlands during the period 13 - 15 December 2002.
AFRIKAANSE OPSOMMING: Die werk wat in hierdie tesis voorgelê word handel oor die assosiasie tussen kankers van die boonste gastrointestinale weg en die tumor suppressor geen, TP53, en kan in 3 dele gedeel word, (i) die analise van die mutasiespektrum van TP53 in laringiale kanker (LK), (ii) die analise van die mutasiespektrum van TP53 in slukderm kanker (SK) en (iii) die analise van die transkripsievlakke van TP53 in SK. Laringeal kanker (LK) is die 6de algemeenste kanker in die wêreld en die 2de algemeenste respiratoriese kanker, met "n benaderde 500 000 nuwe gevalle jaarliks wêreldwyd. Oor die afgelope paar jare het LK "n toenemende probleem geraak, veral in die Kleurling gemeenskap van die Wes Kaap. Die meganismes van die tumorvorming in LK is onbekend, alhoewel rook-en alkoholgebruik vername risiko faktore is. Die voorkoms van mutasies in TP53 is verskeie kere aangetoon in verskillende kanker tipes en daar word vermoed dat dit "n rol speel in tumorvorming. In hierdie studie is dus na mutasies in eksons 5 - 8 van TP53 gesoek in tumor biopsie weefsel (n=44) en bloed isolate (n=42) van Kleurling LK pasiënte d.m.v. polimerase ketting reaksie - enkelstring konformasie polimorfisme (PKR-ESKP) analisering en direkte volgorde bepaling. Bloed monsters van "n vergelykbare groep (n=40) is ook in die studie ingesluit as "n kontrole. Betekenisvolle positiewe korrelasies is gevind tussen die voorkoms van LK en ouderdom sowel as rook. Daarmee saam is daaglikse vleisinname as potensiële beskermende faktor gevind. In tumor biopsies is mutasies in 3 van die ondersoekte eksons gevind, wat 25% van die biopsie monsters verteenwoordig. Hierdie mutasies is uniek aan die tumor biopsie weefsels en dui op "n somatiese oorsprong van mutasies. Hierdie bevindinge bevestig dat die gedeelte tussen kodons 173 - 273 van TP53 "n hipermuteerbare gebied geassosieer met kankers is. Hierdie studie bevestig 6 nuwe mutasies. Daar is 'n hoë insidensie van slukderm kanker (SK) in Suid Afrika relatief tot die res van die wêreld. Hierdie soort kanker word veral gevind by die Swart populasie van die Transkei. Die doel van hierdie studie was om verskille tussen die TP53 mutasie patroon van die Kleurling gemeenskap van die Wes Kaap en die Swart gemeenskap van die Transkei te vergelyk. Hiervoor is die molekulêre struktuur van TP53, veral eksons 5 - 8, in 'n groep Kleurling SK pasiënte (n=42) wat behandel is by Tygerberg Hospitaal, Kaapstad, Suid Afrika, geanaliseer. Analisering is gedoen deur DNS van tumor, bloed en ook oënskynlike gesonde aangrensende weefsel van dieselfde pasiënte te onderwerp aan PKR-ESKP analise en direkte volgorde bepaling. Slegs 4 nukleotied veranderings is gevind in 124 volgorde bepalings, waarvan 2 nuwe veranderings is in SK. Hierdie 4 nukleotied veranderinge verteenwoordig 9% van al die tumors wat ondersoek is in die studie. Hierdie studie bewys dat die mutasiespektrum van TP53 in die Kleurling gemeenskap van die Wes Kaap grootliks verskil van die Swart gemeenskap van die Transkei. Dit impliseer dat verskillende etiologiese faktore moontlik 'n rol mag speel op die tumorvormingsproses in die 2 afsonderlike geografiese gemeenskappe. Hierdie is die onderwerp van 'n epidemiologiese studie wat deur die MNR onderneem word. Die laaste deel van hierdie tesis handel oor die kwantifisering en vergelyking van TP53 transkripsievlakke in SK tumor weefsel teenoor TP53 vlakke in gesonde slukderm weefsel van pasiënte in 'n unieke geografiese en etniese agtergrond. Die studie populasie in hierdie projek het bestaan uit Kleurling pasiënte (n=2) wat by Tygerberg hospitaal behandel is. Die "LightCycler" sisteem is gebruik vir die akkurate kwantifisering van TP53 boodskapper RNS vlakke. Ongelukkig is die verlangde resultate nie gekry nie as gevolg van onvoorsiene probleme wat ondervind is tydens die studie. Hierdie probleme sluit in die onvoldoende preserv RNS studies. Hierdie inleiers maak dit nou moontlik om die teenwoordigheid van TP53 RNS spesies sowel as die afwesigheid van DNS kontaminasie in een PKR amplifikasie stap te kan identifiseer. 'n Ander belangrike bydrae is die ontwikkeling van 'n goed geoptimaliseerde RNS ekstraksie metode vir moeilike starre weelfsel tipes (soos menslike slukderm weefsel in hierdie studie) en maak die ekstraksie van groot hoeveelhede RNS uit klein hoeveelhede van moeilik hanteerbare weefsel tipes moontlik. Om saam te vat, hierdie studie het betekenisvolle bydraes gemaak tot die veld van kankernavorsing deur die ontrafeling van die TP53 mutasiespektrum in beide laringeale sowel as slukderm kanker, in 'n populasie uniek aan die Wes Kaap. Die eerste deel van hierdie tesis is gepubliseer in Cancer Geneties and Cytogenetics (Barnard, D., K. Lehmann, E. G. Hoal, P. D. van Heiden, and T. C. Victor. 2003. The spectrum of mutations in TP53 in laryngeal cancer patients from a high-incidence population shows similarites to many of the known mutational hotspots. Cancer Genetics and Cytogenetics 145: 126-132) en 'n afskrif van die artikel is ingesluit in Appendix I. Hierdie werk is ook voorgedra (deur D. Barnard) by 'n internasionale kongres getiteld "Cancer of the Esophagus and Gastric Cardia: From Gene to Cure", wat in Amsterdam, Nederland gehou is gedurende 13 - 15 Desember 2002

Ample studies have shown that perturbation of metabolic phenotype is correlated with gene mutation and pathogenesis of colorectal cancer (CRC) and rheumatoid arthritis (RA). Mass spectrometry (MS)-based metabolomics as a powerful and stable approach is widely applied to bridge the gap from genotype/metabolites to phenotype. In CRC suffers, KRAS mutation accounts for 35%-45%. In previous study, SLC25A22 that encodes the mitochondrial glutamate transporter was found to be overexpressed in CRC tumor and thus to be essential for the proliferation of CRC cells harboring KRAS mutations. However, the role of SLC25A22 on metabolic regulation in KRAS-mutant CRC cells has not been comprehensively characterized. We performed non-targeted metabolomics, targeted metabolomics and isotope kinetic analysis of KRAS-mutant DLD1 cells with or without SLC25A22 knockdown using ultra-high performance liquid chromatography (UHPLC) coupled to Orbitrap MS and tandem MS (MS/MS). In global metabolomics analysis, 35 differentially regulated metabolites were identified, which were primarily involved in alanine, aspartate and glutamate metabolism, urea cycle and polyamine metabolism. Then targeted metabolomics analysis on intracellular metabolites, including tricarboxylic acid (TCA) cycle intermediates, amino acids and polyamines, was established by using LC-MS/MS coupled with an Amide BEH column. Targeted metabolomics analysis revealed that most TCA cycle intermediates, aspartate (Asp)-derived asparagine, alanine and ornithine (Orn)-derived polyamines were strongly down-regulated in SLC25A22 knockdown cells. Moreover, the targeted kinetic isotope analysis using [U-13C5]-glutamine as isotope tracer showed that most of the 13C-labeled TCA cycle intermediates were down-regulated in SLC25A22-silencing cells. Orn-derived polyamines were significantly decreased in SLC25A22 knockdown cells and culture medium. Meanwhile, accumulation of Asp in knockdown of GOT1 cells indicated that oxaloacetate (OAA) was majorly converted from Asp through GOT1. Exogenous addition of polyamines could significantly promote cell proliferation in DLD1 cells, highlighting their potential role as oncogenic metabolites that function downstream of SLC25A22-mediated glutamine metabolism. SLC25A22 acts as an essential metabolic regulator during CRC progression as promotes the synthesis of TCA cycle intermediates, Asp-derived amino acids and polyamines in KRAS-mutant CRC cells. Moreover, OAA and polyamine could promote KRAS-mutant CRC cell growth and survival. Rheumatoid arthritis (RA) is a chronic, inflammatory and symmetric autoimmune disease and a major cause of disability. However, there is insufficient pathological evidence in term of metabolic signatures of rheumatoid arthritis, especially the metabolic perturbation associated with gut microbiota (GM). Based on consistent criteria without special diet and therapeutic intervention to GM, we enrolled 50 RA patients and 50 healthy controls. On basis of the platform of UHPLC-MS and GC-MS, were performed for the non-targeted metabolomics to investigate alterations of endogenous metabolites in response to RA inflammation and interaction with GM. 32 and 34 significantly changed metabolites were identified in urine and serum of patients with RA, respectively. The altered metabolites were identified by HMDB, METLIN database or authentic standards, and mostly metabolites were attributed into tryptophan and phenylalanine metabolism, valine, leucine and isoleucine biosynthesis, aminoacyl-tRNA biosynthesis and citrate cycle. To obtain alterations of more components in tryptophan and phenylalanine metabolism, we developed and validated a targeted metabolomics method of 19 metabolites by using LC-QqQ MS. Combining the results of targeted metabolomics with global metabolomics, significantly up-regulated kynurenine (KYN), anthranilic acid (AA) and 5-hydroxylindoleacetic acid (HIAA) simultaneously in urine and serum was found to implicate the activation of tryptophan metabolism under the condition of RA, which acted pro-inflammatory roles in inflammation and was closely correlated with GM. IDO/TDO functioned as a pro-inflammation mediator was overexpressed in RA patients. Urinary kynurenic acid and serum serotonin that have impacts on anti-inflammation in immune system were down-regulated in RA patients. The levels of phenylacetic acid and phenyllactic acid serving as a pro-inflammatory and an anti-inflammatory agent, respectively, increased in serum of patients with RA. Moreover, certain essential amino acids (EAAs), and mostly conditional EAAs were decreased in RA patients, which have been reported to inhibit cell proliferation of immune cells. In particular, deficiency of branched chain amino acids (BCAAs, valine and isoleucine) was observed in serum of patients with RA, which may lead to muscle loss and cartilage damage. The specificity of all altered metabolites resulted from RA was considerably contributed through the GM-derived metabolites. The findings revealed that GM-modulated RA inflammation was mainly resulted from tryptophan and phenylalanine metabolism, and amino acid biosynthesis, which may provide more information for better understanding the RA mechanism.

Changes in the human gastrointestinal microbiome are associated with several diseases. To infer causality, experiments in representative models are essential, but widely used animal models exhibit limitations. Here we present a modular, microfluidics-based model (HuMiX, human-microbial crosstalk), which allows co-culture of human and microbial cells under conditions representative of the gastrointestinal human-microbe interface. We demonstrate the ability of HuMiX to recapitulate in vivo transcriptional, metabolic and immunological responses in human intestinal epithelial cells following their co-culture with the commensal Lactobacillus rhamnosus GG (LGG) grown under anaerobic conditions. In addition, we show that the co-culture of human epithelial cells with the obligate anaerobe Bacteroides caccae and LGG results in a transcriptional response, which is distinct from that of a co-culture solely comprising LGG. HuMiX facilitates investigations of host-microbe molecular interactions and provides insights into a range of fundamental research questions linking the gastrointestinal microbiome to human health and disease.

Gastric and oesophageal cancers were responsible for more than one million deaths in 2002. Although global incidence of gastric cancer is decreasing, this malignancy is still the fourth most common cause of cancer worldwide. The incidence of oesophageal adenocarcinoma is rising rapidly, three-fold in the last two decades. The incidence of adenocarcinoma of gastric cardia is stable. In the pathogenesis of both gastric and oesophageal adenocarcinomas, the state of the gastric mucosa and its secretory function plays a central role. Non-cardia adenocarcinoma develops in subjects with H.pylori associated atrophic gastritis and hypochlorhydria. Little is known about the gastric phenotype in patients with adenocarcinoma of the cardia and gastroesophageal junction. Another important but poorly understood risk factor for upper GI adenocarcinoma is male gender. In the first study we aimed to investigate the association between the pattern of H.pylori gastritis and gastric secretory function in 255 H.pylori-infected patients with dyspepsia showing normal endoscopy. Our findings showed that maximal acid output correlates inversely with severity of corpus gastritis, corpus atrophy, and positively related to male gender and serum pepsinogen I. In the second study we compared cancers at the cardia and non-cardia subsites with respect to pre-morbid gastric mucosal atrophy and acid secretion. In a nested case-control study comprising 101,601 men and women enrolled in the Norwegian JANUS cohort, 230 cases of gastric cancer were identified. 173 cases including 144 non-cardia and 44 cardia cancer were enrolled to study. Three controls were matched to each case. Serum pepsinogen I, pepsinogen II, anti-H.pylori IgG antibody and gastrin were measured using serums which had been collected a median of 11.9 years before cancer diagnosis radioimmunoassay method. Non-cardia cancer was positively associated with H.pylori and gastric atrophy. The diffuse and intestinal histological subtypes of non-cardia cancer were of similar proportions and both showed a positive association with H.pylori and atrophy. Cardia cancer was negatively associated with H.pylori, but H.pylori positive cardia cancer showed a positive association with gastric atrophy. The predominant histological subtype of cardia cancer was intestinal and it was not associated with gastric atrophy compared to the diffuse subtype. Cardia cancer in atrophic patients had an intestinal: diffuse ratio similar to non-cardia cancer, whereas cardia cancers in persons without atrophy were predominantly intestinal. These findings indicate two aetiologies of cardia cancer, one associated with H.pylori atrophic gastritis, resembling non-cardia cancer, and the other associated with non-atrophic gastric mucosa, resembling oesophageal adenocarcinoma. Serological markers of gastric atrophy may provide the key to determining gastric versus oesophageal origin of cardia cancer. In the third study we extended our investigation of the aetiology of cardia cancer by examining the association of both serological evidences of gastric atrophy and gastroesophageal reflux disease (GORD) symptoms with adenocarcinoma of the oesophagus, cardia and non-cardia regions of the stomach. This has been performed for the different histological subtypes of the cancer. We have also included H.pylori status and smoking history which are other well established risk factors for upper GI cancer. This has been undertaken in a population in Northwest Iran with a high incidence of upper gastrointestinal cancer. Serum pepsinogen I/II was used as a marker of atrophic gastritis and categorised to five quintiles. History of GORD symptoms, smoking and H.pylori infection was incorporated in logistic regression analysis. Lauren classification was used to subtype gastric and oesophageal adenocarcinoma. Non-cardia cancer was associated with atrophic gastritis but not with GORD symptoms; 55% of these cancers were intestinal subtype. Oesophageal adenocarcinoma was associated with GORD symptoms, but not with atrophic gastritis; 84% were intestinal subtype. Cardia cancer was positively associated with both severe gastric atrophy and with frequent GORD symptoms though the latter was only apparent in the non-atrophic subgroup and in the intestinal subtype. The association of cardia cancer with atrophy was stronger for the diffuse versus intestinal subtype and this was the converse of the association observed with non-cardia cancer. These findings indicate two distinct aetiologies of cardia cancer, one arising from severe atrophic gastritis and being of intestinal or diffuse subtype similar to non-cardia cancer, and one related to GORD and intestinal in subtype, similar to oesophageal adenocarcinoma. Gastric atrophy, GORD symptoms and histological subtype may distinguish between gastric versus oesophageal origin of cardia cancer. In the fourth study we investigated the relationship between gender and upper gastrointestinal adenocarcinoma. Male gender is a well-established risk factor for oesophageal adenocarcinoma. Male predominance of gastric cancer is related to the histological subtype of the tumour being more marked in the intestinal versus diffuse histological subtype. In addition, global data suggests that the male predominance of upper gastrointestinal cancer is related to the anatomical location, being higher for proximal and lower for distal tumours. However, the proportion of the intestinal histological subtype differs according to anatomical site and it is unclear whether it is the anatomical site or the histological subtype which is associated with the gender phenomenon. We have conducted a population-based study to investigate this. The study was based upon 3270 gastric and oesophageal cancers recorded in West of Scotland Cancer Registry between 1998 and 2002. The Lauren subtype of adenocarcinoma was determined by reviewing 1204 reports and 3241 slides in a sample of 812 cases. Logistic regression models were used to estimate relationship between male predominance and histological subtype, tumour location and age. We found that the crude incidence rate of intestinal subtype was higher in males (23.86/ 100,000 person-years) versus females (9.00/ 100,000 person-years), giving M/F of 2.65. M/F ratio of intestinal subtype cancer was 3.41 at age <50, reached a peak of 7.86 at age 50-59, and then showed a progressive decrease throughout the life. In contrast, the incidence rate of diffuse subtype adenocarcinoma was similar in both sexes (5.58 vs. 5.20 /100,000 person-years) yielding M/F of 1.07. Multivariate analyses including histological subtype, tumour location and age indicated that the male predominance was related to the histological type rather than anatomical location. Intestinal type tumour showed similar male predominance of incidence irrespective of its anatomical location (OR, 95% CI: 2.6, 1.78 – 3.9). Further analysis of the age-specific incidence curves indicated that the male predominance of intestinal subtype was due to a 17.2-year delay of development of this cancer in females.

Die Rationale für immuntherapeutische Ansätze zur Behandlung maligner Neoplasien geht davon aus, daß Tumore über spezifische Tumorantigene verfügen. Dendritische Zellen als die wichtigsten antigenpräsentierenden Zellen sind in der Lage, Tumorantigene naiven T-Zellen zu präsentieren und spezifische zytotoxische T-Zellen zu stimulieren. In der vorliegenden Arbeit wurden dendritische Zellen durch Stimulation mit Interleukin-4 (IL-4) und Granulozyten/ Makrophagen Koloniestimulierender Faktor (GM-CSF) aus peripheren mononukleären Blutzellen gesunder Spender und an Tumoren des gastroenteropankreatischen Systems erkrankter Patienten generiert. Mit den dendritischen Zellen cokultivierte immunologische Effektorzellen (Zytokin-induzierte Killerzellen, CIK-Zellen) wurden im Zytotoxizitätstest gegen kolorektale und pankreatische Karzinomzellen eingesetzt. CIK-Zellen sind zytototoxische Zellen, die durch Stimulation mit Zytokinen aus peripheren Blutlymphozyten erzeugt werden. Durch die Cokultivierung der Effektorzellen mit dendritischen Zellen konnte eine signifikante Steigerung der unspezifischen zytotoxischen Wirkung der CIK-Zellen bewirkt werden. Zur Steigerung der spezifischen Zytotoxizität wurden dendritische Zellen mit dem Gesamtprotein der tumor-assoziierten Antigene cancer associated antigen (CA 19-9) und carcinoembryonic antigen (CEA) gepulst. Effektorzellen zeigten nach der Cokultur mit gepulsten dendritischen Zellen zytotoxische Wirkung gegen Targetzellen, die das zum Pulsen verwendete Tumorantigen auf der Zelloberfläche exprimieren. Die Antigenspezifität der zytotoxischen Wirkung konnte durch eine signifikant verminderte Zellyse nach Blockade des Tumorantigens auf den Targetzellen belegt werden. Erstmals beschrieben ist hier das Pulsen dendritischer Zellen mit sowohl autologen als auch allogenen Seren von Patienten mit erhöhten Tumormarkerspiegeln. Eine Kultivierung dendritischer Zellen in tumormarkerhaltigem Serum bewirkte dosisabhängig eine verstärkte zytotoxische Wirkung cokultivierter Effektorzellen gegen Tumorzellen. Die verstärkte Zellyse zeigte sich unabhängig vom allogenem oder autologem Charakter des Serums. Der immunstimulierende Effekt des Patientenserums konnte durch eine vorhergehende Hitzeinaktivierung des Serums neutralisiert werden. Die höchsten Zellysen wurden durch eine Kultivierung dendritischer Zellen in tumormarkerhaltigem Serum und zusätzlichem Pulsen mit exogenem Tumorantigen erreicht. Unte rsuchungen an komplett autologen Systemen reproduzierten die an Zellkulturen erhobenen Befunde. Hierfür wurden erfolgreich Primärkulturen kolorektaler Tumore etabliert. Aus dem Blut von Tumorpatienten wurden dendritische Zellen generiert, die mit autologem Serum kultiviert wurden. Die cokultivierten autologen Effektorzellen erwiesen sich im Zytotoxizitätstest gegen autologe Tumorzellen als zytotoxisch. Die Cokultivierung der Effektorzellen mit den dendritischen Zellen bewirkte bei beiden Zellpopulationen Veränderungen. Dendritische Zellen zeigten nach der Cokultur eine verstärkte Expression antigenpräsentierender und costimulatorischer Moleküle. Bei den CIK-Zellen kam es zu einem Anstieg der Proliferationsrate. Bei Untersuchungen zur Antigenspezifität von T-Zellrezeptoren konnte vermehrt antigenspezifischer T-Zellrezeptor nachgewiesen werden. Des weiteren stieg das Verhältnis zwischen zytotoxischen T-Zellen und T-Helferzellen zugunsten der zytotoxischen T-Zellen. In ELISpot-Untersuchungen wurde eine Zunahme Interferon-gamma sezernierender CIK-Zellen nachgewiesen. Dendritische Zellen ließen sich erfolgreich mit inaktiviertem Adenovirus, an das kovalent Poly-L-Lysin gekoppelt ist, transfizieren. Die für den adenoviralen Gentransfer benötigten Oberflächenstrukturen konnten auf dendritischen Zellen nachgewiesen werden. Zur Verbesserung der Zytotoxizität wurden dendritische Zellen erfolgreich mit dem Gen für den Transaktivator CIITA transfiziert. CIITA- transfizierte dendritische Zellen exprimierten vermehrt MHC Klasse II-Moleküle. Die transduzierten dendritischen Zellen induzierten bei cokultivierten Effektorzellen eine erhöhte unspezifische Zytotoxizität. Mit Tumorantigen gepulste dendritische Zellen können bei der Entwicklung immuntherapeutischer Protokolle bei malignen Neoplasien von Bedeutung sein.
The immunotherapeutic approach against malignant neoplasias appreciates that tumours encode tumour rejection antigens, that enable them to induce protective immunity. Dendritic cells are major antigen-presenting cells and are able to present tumour antigens to naive T-cells and stimulate cytotoxic T-cells in a specific manner. In the present graduation-manuscript dendritic cells were generated in the presence of Interleukin-4 and granulocyte/macrophage colony-stimulating factor (GM-CSF) from peripheral mononuclear blood cells of healthy donors and tumour- patients. Immunological effector cells termed cytokine- induced killer cells (CIK cells) were co-cultured with dendritic cells and tested for their cytotoxic capacity against colorectal and pancreatic cancer cell-lines in a LDH-release assay. CIK cells are cytotoxic lymphocytes generated by incubation of peripheral blood lymphocytes with different cytokines. Co-culture of effector cells with dendritic cells led to a significant increase of the cytotoxic effect of CIK cells. For a further increase of specific cytotoxicity dendritic cells were pulsed with total protein of the tumour-associated antigens cancer associated antigen CA 19-9 and carcinoembryonic antigen (CEA). Co-cultured effector cells showed an increase in cytotoxicity against tumour-antigen expressing target cells, after co-culture with pulsed dendritic cells. The specificity of the cytotoxic effect could be shown by blocking the tumour-antigens with a monoclonal antibody. Autologous and allogenec untreated serums from patients with elevated tumour-marker levels were also used for pulsing of dendritic cells. Similar to the results when using total protein for pulsing, a cultivation in serum of patients with elevated tumour marker levels caused an intensified cytotoxic effect of effector cells against tumour cells in a dose-dependent manner. The intensified cytotoxicity was seen independent of the allogenec or autologous character of the serum. The immuno-stimulating effect of the patient serum could be neutralized by preceding heat inactivating. The highest cytotoxicity was achieved by a cultivation of dendritic cells in serum from patients with elevated tumour marker levels and additional pulsing with exogenous tumour antigen. Experiments with completely autologous systems reproduced the results made with cell-lines. Primary cultures of colorectal tumours were established. Dendritic cells were generated from the blood of tumour patients and were cultivated in autologous serum. Co-cultured autologous effector cells showed cytotoxicity when used against autologous tumour cells. Co-culturing of effector cells with dendritic cells caused modifications at both cell populations. Dendritic cells showed an increase expression of antigen-presenting and co-stimulatory molecules. CIK cells showed a higher proliferation-rate when co-cultured. They express more antigen-specific T-cell receptor, and the cytotoxic T-cells to T-helper cells ratio increased. ELISpot-assays showed an increase of interferon gamma producing cells. Dendritic cells were successfully transduced by using an inactivated adenovirus, which covalently binds poly-L- lysine. Dendritic cells express the molecules that enables adenoviral gene delivery on their surface. For the improvement of cytotoxicity dendritic cells were transduced with the gene encoding for the transactivator CIITA. CIITA transduced dendritic cells increases expression of MHC class II molecules. Cytotoxicity experiments with transduced dendritic cells resulted in an increased induction of non-specific cytolysis from co-cultured effector cells. DC pulsed with tumour-antigens may have a major impact on immunotherapeutic protocols for cancer patients.

In this thesis, we develop and apply top-down, quantitative approaches to gain novel insights into various complex biological systems. Beginning at the multicellular level, we study human gut microbiome dynamics from an ecological perspective. We develop computational frameworks to enable a global understanding of the spatiotemporal variability of gut bacterial abundances. We demonstrate the utility of our frameworks to elucidate the ecological processes governing abundance changes of gut microbiota. We then shift our focus to the intracellular level by investigating the metabolic limitations of cancer cell growth. We use coarse-grained mathematical modeling to identify a major growth limitation of cancer cells associated with electron acceptor deficiency, which we then experimentally validate. Collectively, these set of approaches help to decipher the organizing principles of complex biological systems at both the individual and multicellular levels.

Introduction: Gastric cancer (GC) is ranked as the second most common fatal malignancy worldwide. Although Helicobacter pylori is recognized as a major predisposing factor for non-cardia GC, infection alone is not sufficient to cause cancer. This thesis aimed to determine the variation in host genetic polymorphisms in subjects from Malaysia and Singapore and to examine the role of H. pylori infection, host genetic factors and dietary factors in the etiology of non-cardia GC in Chinese subjects resident in Malaysia. Methods: Functional dyspepsia (FD) controls from three ethnic groups in Malaysia, Chinese (123), Indian (110) and Malay (84) and Singaporean Chinese (127) plus Malaysian Chinese gastric cancer cases (55)were examined. Polymorphisms in IL-1B-511, IL-1RN, IL-10 cluster, TNFA-308 and TLR5+1174 were determined by PCR-RFLP or PCR; H. pylori status by serology, dietary intake by questionnaire and gastric IL-1b levels by real time PCR. Results: 1) Significant differences existed in the frequency of all polymorphisms, except IL-1B-1473 and TNFA-308, in the three Malaysian ethnic groups and in the IL-1B-511 polymorphism in Malaysian and Singaporean Chinese FD 2) Globally, two distinct patterns of IL-1B-511, IL-1RN, IL-10-1082, IL-10-592 and TNFA-308 exist, Western and East-Asian 3) In Malaysian Malays, the IL-10 ATA haplotype was associated with H. pylori susceptibility 4) In Malaysian Chinese an increased risk of GC was associated with carriage of the IL-1B-1473 G allele {OR=4.4(1.3-15.3)} and the IL-1B-511 C allele {OR=1.8(0.8-4.1)} 5) Increased levels of IL-1b were observed in Singaporean and Malaysian Chinese FD subjects carrying the IL-1-511C and IL-1-1473G alleles 6) Malaysian Chinese not consuming fresh fruit and vegetables had the highest risk of GC {OR=10.2 (3.4-30.6)} 7) The highest risk of GC {OR=37.3(3.3-424.8)} was observed in H. pylori positive Malaysian Chinese who carried both the IL-1B-511C and IL-1B-1473G alleles and did not consume fresh fruit and vegetables. Conclusions: In Malaysian Chinese, H. pylori infection, host genetic and dietary factors all contribute to the risk of GC. However the significant difference observed in the frequency of host genetic polymorphisms within and between ethnic groups suggests that a single group of risk factors cannot be used to determine GC risk across all populations.

Thesis (M.Sc.)--University of Alberta, 2009.
A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Oncology. Title from pdf file main screen (viewed on October 24, 2009). Includes bibliographical references.

Indiana University-Purdue University Indianapolis (IUPUI)
The class IV alcohol dehydrogenase (ADH7, µ-ADH, σ-ADH) efficiently metabolizes ethanol and retinol. ADH7 is expressed mainly in the upper gastrointestinal tract with no expression in the liver unlike the other ADHs, and is implicated in various diseases including alcoholism, cancer and fetal alcohol syndrome. Genome wide studies have identified significant associations between ADH7 variants and alcoholism and cancer, but the causative variants have not been identified. Due to its association with two important metabolic pathways and various diseases, this dissertation is focused on studying ADH7 regulation and the effects of variants on this regulation using cell systems that replicate endogenous ADH7 expression. We identified elements regulating ADH7 transcription and observed differences in the effects of variants on gene expression. A7P-G and A7P-A, two promoter haplotypes differing in a single nucleotide at rs2851028, had different transcriptional activities and interacted with variants further upstream. A sequence located 12.5 kb upstream (7P10) can function as an enhancer. These complex interactions indicate that the effects of variants in the ADH7 regulatory elements depend on both sequence and cellular context, and should be considered in interpretation of the association of variants with alcoholism and cancer. The mechanisms governing the tissue-specific expression of ADH7 remain unexplained however. We identified an intergenic region (iA1C), located between ADH7 and ADH1C, having enhancer blocking activity in liver-derived HepG2 cells. This enhancer blocking function was cell- and position- dependent with no activity seen in CP-A esophageal cells. iA1C had a similar effect on the ectopic SV40 enhancer. The CCCTC-binding factor (CTCF) bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of downstream ADH enhancers and thereby contributes to the cell specificity of ADH7 expression. Thus, while genetic factors determine level of ADH7 transcriptional activity, iA1C helps determine the cell specificity of transcription.