No description available.

No description available.

The primary function of the mismatch repair (MMR) pathway is to protect cells against the mutagenic and cytotoxic effects of mismatches incorporated by polymerases during replication. Accordingly, mutations in the MMR genes are commonly identified in patients with hereditary non-polyposis colon cancer (HNPCC) and somatic colorectal cancers. MMR function is of particular importance in cells exposed to DNA-damaging agents, as aberrant processing of damaged bases and erroneous bypass of bulky lesions increase the tendency for mismatch incorporation. The precise role of the MMR proteins in the processing of CDDP-induced DNA damage is, however, poorly understood.

No description available.

Platinum-based anticancer drugs are believed to exert their action through chemical reactions with genomic DNA, forming adducts with DNA bases. Although the pharmacology of such adducts has been widely studied, the cytotoxic mechanism remains unclear. The possibility that non-DNA molecules have the potential to alter the types of adducts formed has received very little attention, and limited information is available on the levels of adducts formed in clinical tumours. Further understanding of platinum-DNA adduct formation may be important in explaining the efficacy of platinum-based drugs in different tumour types, providing insights into both the cytotoxic mechanism and the development of clinical resistance. The aims of the work described in this thesis were: a) to analyse the nature of DNA adducts formed by three clinically used platinum-based anticancer drugs and to investigate the potential intracellular formation of additional types of adducts to those previously characterised on pure DNA; b) to determine platinum-DNA adduct levels formed in solid ovarian cancer tissue following treatment of patients with carboplatin and test the hypothesis that these levels are comparable to the levels of DNA adducts formed in blood cells; and c) to determine whether sodium thiosulfate (STS), which is currently in clinical trials to protect against cisplatin-induced normal tissue toxicity, impacts on DNA adduct formation. Analysis of the properties of all DNA adducts formed in cells was made possible by analysing enzymatically digested DNA using anion exchange chromatography together with inductively-coupled plasma mass spectrometry (ICP-MS). Putative adducts involving deoxyguanosine monophosphate cross-linked via cisplatin to glutathione were prepared and the chromatographic properties determined. Studies were carried out to characterise the types of adducts formed following incubations of cisplatin with four cancer cell lines. No additional types of adducts were observed compared to those formed by the reaction of cisplatin with pure DNA. The chromatographic behaviour of adducts formed in cells incubated with carboplatin and oxaliplatin were comparable to those formed by cisplatin. This study is the first to investigate carboplatin-DNA adduct levels induced in solid tumours during therapy in patients. Total DNA adduct levels in tumour biopsies and blood cells were measured using ICP-MS with thallium as an internal standard. Tumour biopsies from all four patients studied showed clearly detectable levels of treatment-induced DNA adducts ranging from 1.9 - 4.2 nmoles Pt/g DNA. Blood cell adduct levels ranged from 0.15 – 3.5 nmoles Pt/g DNA. Both tumour and blood cell adduct levels were significantly above background measurements. No correlation was observed between adduct levels in DNA from biopsies and levels in DNA from peripheral blood cells. Concurrent incubation of four human tumour cell lines with cisplatin and STS caused greater than 2-fold decreases in total DNA adducts. Delayed administration of STS had no effect of adducts levels. STS did not appear to affect the chromatographic behaviour of DNA adducts formed in cells following incubation with cisplatin.

T(4;11) acute lymphoblastic leukaemia (ALL) presenting the fusion gene MLL/AF4 is a hallmark of infant ALL. The 5 year event free survival is less than 40%. It was shown that MLL/AF4 is important for cell cycle progression, proliferation, clonogenicity, engraftment in mice and repression of apoptosis. However, in order to improve treatment MLL/AF4 needs to be examined in contexts closer to the leukaemic situation in vivo. To investigate the fusion gene, MLL/AF4 positive SEM, generated from an ALL patient, were depleted for MLL/AF4 by RNAi. The consequential changes in gene expression patterns were analyzed using cDNA- and Oligo- arrays. These gene expression patterns were assigned to biological functions and pathways using the Ingenuity platform. A set of significantly differently expressed genes such as N-CADHERIN and FGFR1, both described for the adherence and regulation of haematopoietic stem cells (HSC), was identified and validated by qRT-PCR. The HSC niche context was further investigated by establishment of a leukaemic - bone marrow feeder cell-to-cell interaction assay. Increased cell death, cell cycle arrest and prolonged growth curves caused by MLL/AF4 depletion in SEM could also be shown on feeders. Additionally, culturing of MLL/AF4 positive patient material on feeders allowed for long-term culture. An imaging method using fluorescent MLL/AF4 positive cells in mouse xenograft models was employed to monitor the distribution and biology of MLL/AF4 positive cells, showing colonisation of bone marrow rich spinal, femoral and cranial bones. Finally, in order to analyze the function of identified target genes of MLL/AF4 by overexpression, a novel lentiviral expression system allowing transduction of stem cells and tightly regulating induction of expression, was initiated. In conclusion, these data indicate a central role of MLL/AF4 in leukaemogenisis while the systems established in this work are of relevance for drug development assays.

Prostate cancer is the leading cause of cancer-related death in western men and results in approximately 10,000 deaths in the UK per year (Cancer Research UK). The androgen receptor (AR) plays a prominent role in both androgen-dependent (AD) and androgen-independent (AI) disease, but treatments that attempt to inactivate the receptor are in-effective. There is a requirement therefore to develop new therapies that permanently disrupt AR function and attenuate disease progression. Hence, identification of new targets within the AR signalling cascade is vital. Numerous AR co-regulators have been identified that regulate AR activity and several of these proteins have been suggested to play a role in the progression of AD and AI disease. In this project, using CaP cell lines, different aspects of the histone methyltransferase enzyme SET9 were studied including its phenotypic influence, expression dynamics and also the molecular mechanisms it mediates in CaP cells. Our previous data demonstrated that SET9 enhances AR activity by directly methylating the receptor at lysine (K) residue 632 in the KLKK motif within the hinge domain of the receptor, and this affected co-activator-AR interaction in LNCaP prostate cancer cells. To assess the physiological role of SET9 in CaP cells, SET9 expression in LNCaP cells was reduced by siRNA interference and the effects on proliferation and apoptosis were investigated. Interestingly, SET9 knockdown reduced LNCaP cell proliferation and up-regulated apoptosis, implicating a role for SET9 in driving CaP progression. Moreover, a combination of SET9 knockdown and treatment with the DNA-damaging agent Doxorubicin in LNCaP cells synergised to increase apoptosis suggesting SET9 may be a potential therapeutic target for advanced CaP. Using a GFP-SET9 fusion protein and immunofluorescence, incorporating an anti-SET9 antibody, SET9 was demonstrated to be predominantly cytoplasmic in LNCaP and U2OS cells, suggesting additional, non-nuclear roles for SET9. To address this issue, and also to explore novel mechanisms of SET9 regulation, the enzyme was immunoprecipitated from LNCaP cells and the immunoprecipitated material was subjected to in solution based protein separation (OFF-GEL fractionation) followed by LC-MS/MS analysis to identify novel SET9 interacting proteins. Amongst several SET9 interacting partners, FXR1 was identified as a pro-apoptotic protein that facilitates SET9 knockdown mediated apoptosis in response to Doxorubicin. A predominantly cytoplasmic co-localisation pattern was confirmed for FXR1 using confocal microscopy, which was consistent with the data obtained from prostate clinical specimen using immunohistochemistry. Moreover, FXR1 was shown to function through AR to repress SET9 mediated co-activation of AR in reporter assays. More surprisingly, FXR1 displayed potent repressive effects on AR without the induction of SET9. In summary, this data highlights SET9 as a novel AR co-regulator that is important for prostate cancer cell growth. Further characterisation of SET9- interacting proteins including FXR1 may also provide novel protein targets for CaP therapy.

Ewing's sarcoma is the second most common bone tumour in children and adolescents. The prognosis is mainly influenced by the occurrence of primary metastasis. Although great improvement in treatment has been achieved, still only 2/3 of patients with localized disease can be cured. Furthermore, the 3-year event free survival in patients with lung metastases is only ~50%, and is less than 20% in patients with bony metastases. Metastatic models of Ewing’s sarcoma developed in this study using cell lines in immunocompromised mice show a pattern of disease spread similar to that found in patients, providing a suitable system for studying the metastatic process likely occurring in the course of Ewing’s sarcoma. The comparison of microarray gene expression patterns revealed interesting candidate genes for diagnosis and identified putative metastasis-specific targets that might be exploited in the development of new treatment approaches. However, it will be necessary to additionally analyse these patterns in primary material. One gene that formerly has been shown to play a role in the metastasis to bones in a variety of cancer types is CXCR4, which encodes for the cytokine receptor of CXCL12 (SDF-1), and which plays a role in the metastasis to bones in a variety of other cancer types. As Ewing’s sarcoma cells express CXCR4, a shRNA vector was constructed, transduced and stably expressed to investigate the role of the CXCR4/CXCL12 axis in Ewing’s sarcoma cells via RNA interference. This stability provides the possibility of an in vitro and furthermore an in vivo use for investigations. In order to investigate the biology of bone malignancy and especially the interaction of tumour cells with cells of the microenvironment of the bone directly, an orthotopic model for Ewing’s sarcoma was developed. Additionally, osteosarcoma as a further primary bone sarcoma and prostate carcinoma as a cancer type with frequent bone metastases were tested in this model. The previously described technique of intrafemoral transplantation was used in this model. Using small animal imaging techniques such as nano computed tomography and magnetic resonance imaging in combination with histology it could be shown that the transplanted cells led to the development of orthotopic tumours presenting a comparable picture to the clinical situation. This model will be further used for research projects performed in the Northern Institute for Cancer Research on the effectiveness of drugs targeting Ewing’s sarcoma cells.

The pro-inflammatory cytokine tumour necrosis factor (TNF) exerts its bioactivity via two plasma membrane receptors, TNF receptor (TNFR)1 and TNFR2. Both receptors are fully activated by membrane-bound TNF, while soluble TNF (sTNF) activates only TNFR1 efficiently. Preliminary data from our group suggest that the membrane proximal extracellular region (stalk region) and/or the transmembrane region of the TNFR control the differential responsiveness to sTNF. The aims of this project were to ascertain the region determining sTNF responsiveness and to investigate the underlying molecular mechanism(s). Fibroblasts from tnfr1-/-/tnfr2-/- double knockout mice were stably transfected with chimaeras consisting of the extracellular and transmembrane domain of TNFR and the intracellular portion of Fas (TNFR-Fas). Using this cellular system, I could show that 42 amino acids within the TNFR2 stalk region control responsiveness to sTNF. Replacement of the stalk regions of TNFR1-Fas and TNFR2-Fas with artificial linkers proved that stalk region length does not determine differential responsiveness. Furthermore, responsiveness to sTNF was not affected when either conserved proline residues or O-glycosylation sites in the TNFR2 stalk region were mutated. Moreover, partial replacement of the TNFR2 stalk region with overlapping artificial linkers also left sTNF responsiveness unaltered. Therefore, the underlying molecular mechanism controlling responsiveness to sTNF appears to be more complex and remains to be elucidated. Importantly, the critical role of the TNFR stalk region in sTNF responsiveness could also be confirmed for wild type TNFR2 at the level of signalling complex formation. Data from chemical crosslinking and confocal microscopy studies revealed that the stalk region controls ligand-independent receptor pre-assembly and formation of larger receptor clusters. Taken together, data obtained in this PhD thesis indicate that the TNFR2 stalk region is a major determinant of differential sTNF responsiveness and ligand-independent receptor-receptor interactions, the latter being a potential prerequisite for the formation of larger ligand/receptor clusters and signal initiation.

Tumour growth and metastasis depend on the vascularization of tumours by angiogenesis. This is regulated by the combined action of several growth factors (e.g. vascular endothelial growth factor, VEGF) that are secreted by the growing tumour, and activate VEGF receptors (VEGFR) expressed on the surface of endothelial cells to stimulate new blood vessel formation. Therapies that target VEGF/VEGFR signalling have indicated that anti-angiogenic therapy may be a useful supplementary anti-cancer treatment in the clinic. In addition to VEGF, malignant cells secrete transforming growth factor (TGF)-β, which is thought to stimulate new blood vessel formation by interacting with endoglin, an endothelial co-receptor for TGF-β that regulates angiogenesis. However, it is not yet clear whether this property could also be utilised to inhibit angiogenesis and metastasis, consistent with endoglin acting as a therapeutic target in a clinical setting. Therefore, the aim of my project was to investigate the role of endoglin in tumour angiogenesis and metastasis and its potential as an anti-angiogenic therapeutic target. I used a conditional endoglin knockout mouse model, that was generated by combining a floxed endoglin allele with a tamoxifen inducible vascular specific Cre (Cdh5(PAC)Cre-ERT2). Angiogenesis was tested using the matrigel subdermal plug assay and was significantly less in endoglin-deficient adult mice compared with tamoxifen treated control mice. Subsequently, angiogenesis and metastasis were investigated using a subdermal lewis lung carcinoma (LLC) model. The growth of the primary tumours was initially reduced, suggesting that targeting endoglin may delay tumour progression at an early stage. However, there was no significant effect of endoglin loss on primary tumour growth at later stages of tumour progression. Furthermore, loss of endoglin was associated with a significant increase in metastases, in a similar way to recent findings for other anti-angiogenesis treatments. The reasons for this are not yet clear. iii In terms of animal health, endothelial specific loss of endoglin alone did not appear to cause any major adverse effects. Endoglin inducible knockout (Eng-iKOe) mice did not lose weight and appeared healthy (over two months). However, Eng-iKOe mice did exhibit abnormal venous enlargement close to matrigel plugs supplemented with angiogenic growth factors compared to control mice. There was no evidence for a similar response in the peritumoral vasculature. In parallel to the in vivo studies, I took advantage of combining the conditional Eng- iKO line and the „immortomouse‟ line to create conditionally immortalised Eng-iKO mouse lung endothelial cell lines (MLECs) to investigate the role of endoglin in regulating endothelial cell viability, proliferation and migration. In standard media, MLECs showed normal cell viability, proliferation and migration in the absence of endoglin. However, titration of the growth factor supplements did result in significant reduction in viability in the absence of endoglin, suggesting endoglin is important for maintaining endothelial cell viability. Although the exact mechanisms regulating the role of endoglin in angiogenesis are still unclear, this study has increased our understanding of the endothelial cell phenotype in pathophysiological conditions in the absence of endoglin. In particular, the finding that endoglin depletion delays tumour progression in the early stage but is associated with increased metastatic risk is important when considering appropriate utilisation of anti-endoglin therapy, which is already being given to cancer patients in phase I/II clinical trials.