A population-based study of quality of life in patients with lung cancer cases and chronic respiratory disease controls was carried out at Stobhill Hospital in Glasgow between January 1995 and April 1996. The main results may be summarised as follows: There were no significant differences between quality of life in cases and controls except for pain and loss of appetite. Patients with different socio-economic status had different quality of life. The poorer reported a lower level of quality of life. Social support systems, social networks, and socio-demographic status of the patients were found to predict baseline quality of life prior to diagnosis. Non-medical factors (Deprivation Category and marital status) were found to be significant predictors of patients' global quality of life at follow-up, whereas medical factors (cell type and treatment modalities) were not. Global quality of life prior to diagnosis was a clear predictor of survival. Treatment regimens were found to be ineffective regardless of cell types and stage of disease when comparing baseline and follow-up assessments of quality of life in patients with lung cancer. Patients' reactions to the study indicated that they did not find the study intrusive. Patients' perceptions of quality of life were found to differ from those of health professionals. In the light of study findings it is concluded that conducting a robust epidemiological study of quality of life in patients with lung cancer is feasible. It is essential that such an assessment be carried out in the context of their socio-economic status. The results suggest that quality of life is a real and useful prognostic factor. It predicts survival and it is important to include quality of life measures in future studies of outcomes in lung cancer care. The above forms the basis of recommendations to improve lung cancer care and to provide guidelines for further work.

Limited oxygen availability (hypoxia) influences cell migration and invasion, but the underlying mechanisms are poorly understood. Much of the cellular response to hypoxia is regulated by a family of Hypoxia Inducible Factor (HIF) prolyl hydroxylases (PHD1-3), each of which is thought to regulate specific pathways.Their activity is dependent on the availability of oxygen and alpha-ketoglutarate but despite intensive studies their activity in vivo and their substrates are poorly defined. In this study we performed a quantitative proteomic screen to identify new substrates of PHDs. Co-immunoprecipitations using FLAG-tagged PHDs were performed under hypoxia to trap the enzyme-substrate interactions, and binding partners were identified by mass spectrometry. Actin was identified to interact with PHD3 specifically under hypoxia. Subsequently two defined prolyl residues in beta-actin were shown to be hydroxylated. Hypoxia-induced rearrangement of the actin cytoskeleton was shown to be dependent on PHD3 activity as a knockdown of PHD3 was sufficient to increase the intracellular G- to F-actin ratio. An increase in cell migration and invasion was also found to be dependent on PHD3 activity. Mutation of both hydroxylated prolyl residues led to a similar phenotype regarding actin rearrangement and cell migration. Using constantly active HIF-mutants, we could show that these PHD3-dependent pathways are independent of HIF. All together, this study shows a pro-invasive pathway linking HIF-independent oxygen-sensing pathways and actin signalling. However, the mechanism of how hypoxia-induced actin rearrangement leads to increased migration and invasion remains to be elucidated.

This research has been carried out to study the UAE population structure, particularly as the UAE has an unusual population composition. Transient workers from Indian sub-continental outnumber the native Arabic population by approximately three (60%) to one (20%). Other Arab populations make up approximately 15% of the population, the majority are from Egypt, Yemen, Palestine and Sudan. The relatively high levels of consanguineous marriages (between close relatives such as first cousin) between UAE native population added more complexity to the population. Therefore, it was important to analysis the population before applying any new tools to forensic analysis and paternity testing in the UAE. Two polymorphic systems were chosen (VNTRs and STRs) to investigate the population substructure. Five single loci VNTR/HinfI probes MS1, MS31, MS43A, YNH24 and G3 were used to profile 173 individuals from the UAE native Arabic population, 154 individuals from Indian and 112 individuals from Pakistani populations. The FST was calculated by comparing the UAE Arabic population to both Indian and Pakistani populations. The highest value of (0.0062) was observed between the UAE Arabic and Indian populations. No evidence of substructure was observed when Indian population compared to Pakistani population (FST = -0.003). In addition, eight sample STR loci D5S818, D7S820, D13S317, D16S539, vWA, THO1, TPOX and CSF1PO (GenePrintTM PowerPlexTM 1.2 System) were used to profile 229 UAE native Arab (100 from Sharjah and 129 from Abu Dhabi), 194 Indian, 197 Pakistani and 121 Egyptian individuals. The data were analysed to estimate a number of forensic and paternity parameters, including matching probability, discrimination power, probability of paternal exclusion and typical paternity index in order to assess the application of these two systems for forensic and paternity tests in the UAE. The five VNTR loci and eight STR loci together were proved to be very powerful tool for forensic analysis and determining paternity within the UAE.

Oral cancer has caused great concern in all of the western countries over the past two decades because of its progressively increasing incidence, mainly in young males, and its consistently low 5-year survival rate. Oral premalignant lesions (dysplasias) are thought to precede the development of cancer, but no clinical or histological criteria is at present available to predict their potential for malignant transformation. Therefore, it would be of diagnostic and therapeutic relevance to understand the molecular event, involved at different stages of oral cancer. Using a unique series of primary cultures from biopsies of normal oral mucosa, dysplasias and squamous cell carcinomas we have identified molecular changes characteristic of early oral cancer progression. Our group reported previously that acquisition of the immortal phenotype is an early event in oral cancer development (McGregor et al. 1997): data obtained during the course of this thesis project indicate that about half of oral dysplasia cultures are immortal and this is associated with loss of expression of retinoic acid receptor (RAR)-b and the cell cycle inhibitor, p16INK4a, p53 mutations and increased levels of telomerase/hTERT mRNA. In contrast, increased expression of the EGF receptor (EGF-R), known to be a characteristic of oral cancer, does not occur until after the dysplasia stage in squamous cell carcinomas (SCCs). Interestingly, one atypical mortal dysplasia with a considerably extended lifespan has lost expression of RAR-b and p16INK4a, but it still expresses wild-type p53 (albeit at higher level than normal) and has not activated telomerase. Further experiments demonstrate that retroviral transduction of hTERT immortalises D17 and this is associated with telomere lengthening but p53 remains wild-type. In contrast, transduction of hTERT does not extend the lifespan of two other typical mortal dyplasia cultures (that retain RAR-b and p16INK4a expression), even though telomeres are lengthened. Thus, telomerase activation/telomere maintenance is not sufficient per se to immortalise mortal dysplasias without concomitant loss of RAR-b and/or p16INK4a, but p53 mutation is not required if telomerase is activated exogenously. However, further work is required to clarify whether the molecular changes associated with the acquisition of an immortal phenotype in culture have any clinical or prognostic significance.

An investigation was carried out to examine the production of the inflammatory cytokines IL1, IL1, IL6, TNF, and TNF in two tumour cell lines, the MCF-7 breast cell line and the T-24 bladder cell line, and in samples of breast, bladder, lung and ovarian tumours. Two methods were used to investigate cytokine production. These were the polymerase chain reaction method (PCR) to examine cytokine mRNA production and immuno-staining of frozen or paraffin-embedded tissue sections to demonstrate the presence of the cytokine polypeptide directly. In the PCR experiments, the most frequently found cytokine was IL6, followed by IL1. Only a few tumours of any type displayed TNF, and even fewer produced TNF. In the immunostaining experiments performed on frozen sections, IL1 and IL6 proteins were detected in sections of tumours which gave positive results with PCR. Cell phenotyping indicated that the IL1 and IL6 were probably being synthesised by the tumour cells themselves although there was lymphocyte infiltration in every section examined. In the immuno-histology study performed on the paraffin-embedded sections, a new collection of tumours was used. These tumours were not subjected to parallel PCR due to size of tumour samples being too small. The results obtained from these experiments conflicted with the results observed in the PCR study. IL1 was detected in all of the breast tumours used for immuno-histology but in none of the breast tumours in the PCR experiments. While the conflict could not definately be resolved, it was thought that the results of the immuno-histology experiments were more accurate as they detected expression of cytokine protein on a cellular scale. The immuno-histology experiments demonstrated that some tumour cells produced IL1 in breast and bladder carcinomas, and some produced IL6 in breast and lung carcinomas.

Aberrant methylation of CpG islands (CGIs) is associated with transcriptional silencing of key tumour suppressor genes in cancer and is a frequent epigenetic event in epithelial ovarian cancer (EOC). The methylation status of 24 CGIs in a retrospective group of 142 EOCs and 16 non-tumour adjacent tissues were analysed using methylation-specific PCR (MSP) and Combined Bisulphite Restriction Analysis (COBRA) methods. CGI methylation of at least one of these loci was a frequent event in both early (78%) and late stage (60%) disease. A group of loci were identified as being methylated in 64% of early stage tumours (CGIs linked to the OPCML, RASSFIA and HIC1genes). The HIC1 CGI was frequently methylated in matched non-tumour adjacent tissues, but not in normal ovarian surface epithelium, potentially representing an early epigenetic event in the carcinogenic process present even before apparent morphological change. Differential methylation hybridisation (DMH) of a 12K CGI microarray using ovarian cell lines identified methylation of a CGI located at the LMX1A gene. This CGI was shown by MSP to be a potential early epigenetic marker methylated in 75% of early stage ovarian tumours. 87.5% of the early stage tumours examined were methylated in at least one of four loci (LMX1A, OPCML, RASSFIA or HIC1). The clinical application of this group of methylated CGIs was examined in matched plasma from chemonaive patients with EOC for similar methylation changes. Methylation of LMX1A was detected in 43.3% of all plasma samples and in 48.2% of those patients with methylated LMXIA in their tumour. When methylation was detected in plasma, it was always detectable in the corresponding tumour. Therefore, detection of LMX1A methylation in plasma has a sensitivity of 48.2% and a specificity of 100%.

Epigenetic mechanisms have been implicated in the regulation of telomerase gene expression and here we show that specific modifications within the chromatin environment of the hTR and hTERT promoters correlate with expression of hTR and hTERT in ALT, normal and telomerase-positive tumour cell lines. Lack of expression of hTR and hTERT is associated with repressive histone modification, while, hTR and hTERT expression is associated permissive histone modifications. Methylation of lysine 20 H4 was not linked to gene expression but instead was specific to the hTR and hTERT promoters of ALT cells providing an insight into the differences between ALT and telomerase-positive cells as well as a novel marker for the ALT phenotype. Basal transcription machinery dynamics were also shown to be different between normal and cancer cells at the telomerase gene promoters. Modulation of the chromatin environment was also shown to cause re-expression or increased expression of hTR and hTERT further supporting the role of the chromatin environment in controlling telomerase gene expression. Epigenetic mechanisms are also shown to be involved in the repression of hTERT transcription in human mesenchymal stem cell (hMSCs) and modulation of the chromatin environment is shown to allow re-expression of hTERT expression, while the disruption of telomerase gene expression in human haematopoietic stem cells (hHSCs) in chronic myeloid leukaemia (CML) and the role of the chromatin environment was also studied. These data establishes how epigenetic mechanisms can contribute to transcriptional regulation of telomerase and also highlights the potential importance of epigenetics in senescence and tumourigenicity.

Radiotherapy is commonly used in the treatment of head and neck cancer. For early stage tumours, conventional radiotherapy techniques have a high cure rate and low levels of long-term complications. Patients with more advanced cancers have much lower cure rates and high levels of treatment-related complications. Intensity modulated radiotherapy (IMRT) is a new form of focussed radiation therapy. It has been used to reduce the radiation dose to normal tissue structures and increase the dose delivered to tumour bearing tissues. This potentially allows reduced side effects and increased tumour control compared to conventional radiotherapy. The rationale of this thesis was to test whether these twin goals could be achieved in head and neck cancer patients. The first part of the thesis describes improvements in patient immobilisation, optimisation of techniques for neck irradiation, and evaluation of the technique in a busy radiotherapy department. It includes pre-clinical evaluation of IMRT for different tumour sites, the development of quality assurance programs and the conduct of a national randomised controlled trial of parotid-sparing IMRT. This trial concluded that IMRT significantly reduced patient-reported xerostomia, allowed recovery of saliva production and improved quality of life. The second part of the thesis describes pre-clinical evaluation of techniques to escalate radiation dose in patients with larynx and hypopharynx tumours. A phase I/II clinical trial showed that higher doses of radiation can be delivered at the expense of an increase in acute radiation toxicity but without a measurable increase in late radiation side effects. In the larynx and hypopharynx groups, a possible increase in local control was observed. This thesis describes the process of evaluation of a new radiotherapy technology and could be used as a template for testing other new technologies in the future.

The purpose of this research was to enhance the targeted radiotherapy of two metastatic malignant diseases: neuroblastoma and prostatic carcinoma. By virtue of its high affinity for the norepinephrine transporter (NET), [131I]meta-iodobenzylguanidine ([131I]MIBG) has been used for the therapy of tumours of neuroectodermal origin for more than 25 years. Although not yet universally adopted, [131I]MIBG targeted radiotherapy remains a highly promising means of management of neuroblastoma. MIP-1095, a glutamate-urea-lysine dipeptide has high affinity for prostate-specific membrane antigen (PSMA) and has recently demonstrated exquisite specificity for PSMA-expressing, metastatic prostatic carcinoma. Preliminary imaging studies in patients, using [123I]MIP-1095, revealed tumour-selective binding and prolonged retention only in malignant sites. This indicates the therapeutic potential of this agent when labeled with Iodine-131. Our aim is to make the most effective use of [131I]MIBG and [131I]MIP-1095 for the treatment of neuroblastoma and prostatic carcinoma by combining the [131I]-labelled radiopharmaceutical with radiosensitiser drugs. The thiol-containing molecule disulfiram was selected for combination with targeted radiotherapy because of its reported inhibition of the 26S proteasome and NF-kB activity, its ability to chelate copper and its pro-oxidative effects. The copper-dependence of the cytotoxicity and radiosensitising activity of disulfiram was established in neuroblastoma cell models. Radiation dose enhancement values at the 50% toxicity level were 4.24 and 2.00 in SK-N-BE(2c) and UVW/NAT cells, respectively. The radiosensitising mechanism of disulfiram-copper was shown to involve the inhibition of cell cycle arrest in G2. The enhancement of the cytotoxicity of [131I]mIBG and of [131I]MIP-1095 by disulfiram-copper was demonstrated by delay of the growth of multicellular tumour spheroids. Finally, the screening of the enhancing effect of chemotherapeutic agents on the spheroid growth delay induced by [131I]MIP-1095 indicated that combinations with topotecan, nutlin-3, bortezomib or olaparib have good prospects for therapy of metastatic prostate carcinoma. In conclusion, it is expected that DSF:Cu will enhance the outcome of patients undergoing targeted radiotherapy due to its radiosensitising properties.

One of the major problems associated with colorectal cancer is resistance to cytotoxic chemotherapeutic agents. New strategies are therefore required to inhibit colon cancer proliferation and survival. Here I use modulators of cAMP pathways, including inhibitors of phosphodiesterase 4 (PDE4) enzymes, which are under clinical development for other disease states, to inhibit the breakdown of cAMP and to assess the effects of raising intracellular cAMP on colon cancer proliferation and survival. I found that some chemo-resistant cancer cells are addicted to keeping low cAMP in PDE4 regulated compartments, and modulation of this pool causes G1/S-phase arrest and apoptosis. I also show that PDE4 controlled cAMP negatively regulates the PI 3-Kinase/Akt pathway, which some cells are addicted to for survival. Furthermore, I investigated the expression and role of PDE4 enzymes in metastatic colon cancer cells and assessed the effects of modulating their expression on survival. Also, I used a clinically relevant analogue of forskolin, an agonist of adenylyl cyclase, to examine the general effect on growth of epithelial cancer cell lines. This work might provide new strategies for the treatment of advanced colon cancer.