In this study, I used the serum-free MCDB 153 culture system to investigate calcium-induced keratinocyte differentiation. Treating normal human keratinocytes with high extracellular calcium concentrations (1mM) increased the proportion of cells expressing differentiation-specific proteins. I showed that this was not caused by calcium-induced cell-cycle arrest, nor was it a consequence of stratification. However, the expression of differentiation-specific proteins was preceded by the formation of cadherin-mediated cell-cell adhesions. The likely importance of the cadherin-mediated adhesions in initiation the differentiation program was confirmed in two ways. Firstly, clustering cell-surface E-cadherin in low extracellular calcium using monoclonal antibodies increased the proportion of keratinocytes expressing differentiation-specific proteins. Secondly, suppressing the formation of cadherin-mediated cell-cell adhesions using synthetic peptide analogous to the cadherin recognition domain attenuated the calcium-induced expression of differentiation-specific proteins. These data are consistent with a role for the cadherin-mediated cell-cell adhesions in initiating the keratinocyte differentiation program in response to calcium in vitro. A second aspect of this project involved an investigation of the role played by the Src-family of protein tyrosine kinases at calcium-induced cadherin-mediated adherens junctions. The ubiquitously expressed members of this family, c-Src, Fyn and c-Yes were localised to the cadherin-mediated adhesions formed in response to high extracellular calcium. Treating adherent keratinocytes maintained in low extracellular calcium with a specific Src kinase inhibitor, PD162531, induced the assembly of cadherin-mediated cell-cell adhesions leading to the formation of contiguous groups of cells similar to those seen in response to high extracellular calcium. The data presented are consistent with a role for the Src kinases in regulating adherens junction turnover but do not exclude a role also in modulating aspects of differentiation.

Tyrosine kinase inhibitors (TKIs) have revolutionised the treatment of chronic myeloid leukemia (CML), however, fail to cure the disease due to the persistence of a refractory fraction of stem/progenitor cells. Autophagy is a recycling mechanism utilised by the cell as a survival mechanism under stressful conditions, and its induction has been suggested to have a cytoprotective role in cancer cells. In this study we demonstrate that autophagy is triggered in CML upon TKI-mediated inhibition of BCR-ABL, and protects from cell death. In order to evaluate if specific autophagy inhibition enhances TKI effects, we stably transduced primary CML stem/progenitor cells with a vector carrying a short-hairpin against the key autophagy gene ATG7. Knock-down of basal ATG7/autophagy levels in CML stem/progenitor cells inhibited by approximately 50% the survival of the cells in a clonogenic assay, and reduced by 75% their erythroid differentiation potential. Furthermore, ATG7 knock-down enhanced the effects of TKIs imatinib (IM; 1st), dasatinib (DAS; 2nd), nilotinib (NIL; 2nd) and ponatinib (PON; 3rd generation), reducing by 92-98% the survival of these cells in a clonogenic assay. In contrast, ATG7 knock-down in normal stem cells, with or without TKI treatment, did not have a significant effect on survival and proliferation. ATG7 was also knocked-down in final disease stage, blast crisis (BC), patient-derived K562 and KCL22 cell lines. Both cell lines appeared to depend significantly on autophagy for survival as indicated by high apoptosis levels (70-100%) after ATG7 knock-down. Interestingly, ATG7 knock-down cells appeared to be more differentiated compared to the control (scrambled shRNA). Our findings suggest a role for basal autophagy in the survival, differentiation decisions and clonogenicity of CML cells, and support the combined use of autophagy inhibition with TKIs for the eradication of CML stem/progenitor cells. This could be partially attributed to a bypass of the differentiation block upon autophagy inhibition, which facilitates TKI-targeting. We underline the necessity for the development of specific autophagy inhibitors that in combination with TKIs could potentially eradicate the fraction of persistent CML stem/progenitor cells and offer a curative option for CML patients.

In spite of extensive research there is little information about apoptosis or programmed cell death in the genesis and progression of cancers of the lung. In our project we have investigated the role of apoptosis and two of the genes controlling apoptosis (bcl2 and C040) in squamous cell carcinoma of the lung. Also we have tried to formulate an accurate way of measuring apoptotic rate in tumour specimens. We counted apoptotic cells in Haematoxylin and Eosin stained histological sections of squamous cell carcinoma of the lung. The apoptotic indices we obtained were very reliable showing remarkable reproducibility and strong correlation with apoptosis measured by monoclonal antibody to apoptosis specific protein (ASP). In our series apoptotic index did not correlate with survival, disease stage, differentiation, AgNOR or DNA ploidy. Histological sections were stained with monoclonal antibodies to Bc12 and CD40. In all, 32% of squamous cell carcinoma of lung were Bc12 positive (i.e. more than 50% of the tumour cells contained Bc12 protein) and 22% were positive for C040. The expression of Bc12 correlated positively with the apoptotic indices. Patients with Bcl2 positive squamous cell lung cancers survived significantly longer although Bcl2 expression did not correlate with any marker of disease severity e.g. stage, grade, AgNOR or DNA ploidy. C040 expression in squamous cell lung cancer had no effect on apoptosis, survival or any of the other previously mentioned markers of disease severity. The expression of C040 in our series showed a tendency to correlate inversely with the expression of Bcl2. We devised a way to measure apoptotie rate in prinwy cultures of tumour cells by serial estimation of sub-diploid fractions in cell suspensions double stained with PI and BerEP4-FITC. We believe that the apoptotic rate measured in this fashion is biologically more relevant, and therefore could be more useful in predicting prognosis, than apoptosis measured from histological sections.

In the UK, more than two million people are alive following a cancer diagnosis and people with cancer live an average six times longer than they did forty years ago. There have been dramatic survival improvements in some cancers with six now having median survival expectations of over ten years. This is remarkable but cancer consists of more than two hundred ‘types’ and, for some types, predicted survival is still only weeks. Furthermore, some issues related to long term survival are only just emerging, many remain underresearched and studies that exist have been criticised for being drawn from limited cancer sites and ignoring the coping strategies and social contexts of those diagnosed with cancer. The aim of this work is to explore the experience of living with and beyond the diagnosis of a ‘poor prognostic’ cancer in contemporary society and from a sociological perspective. The work is informed by a literature review which explores lay understanding of cancer, a theoretically driven investigation designed to produce a sociological understanding of what it is like to live with cancer, a feasibility study and a full empirical study, which were both supported by users. Data for the principal study was generated by ‘photovoice’; a novel participatory method in which participants created and discussed photographs to illustrate and describe their experience in depth. This study revealed that living with and beyond cancer was an ongoing disruptive experience for participants and their constant fear of recurrence impacted on future plans. Furthermore, society’s stigmatising perception of cancer bestowed certain responsibilities and obligations on the participants. Photographs added a power and richness to the data. This work adds to the very limited understanding of the experience of cancer and ‘survivorship’ for this group and will hopefully guide appropriate communication, service provision and future research.

No description available.

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder arising in a haemopoietic stem cell (HSC) as a result of the reciprocal translocation between the long arms of chromosomes 9 and 22 (t9;22), leading to the formation of the fusion oncogene BCR-ABL. BCR-ABL has constitutive tyrosine kinase (TK) activity which drives, at least during the chronic phase (CP) of the disease, myeloid progenitor cells expansion through terminally differentiated cells and is necessary for the transformed phenotype. The introduction at the end of the last century of BCR-ABL TK inhibitors (TKI) has dramatically changed the management of newly diagnosed CP CML patients as the vast majority achieve deep molecular responses while enjoying good quality of life when treated with TKI. However about 20% of patients still show various degree of resistance to all currently available TKI while in those achieving deep responses, there is compelling evidence of persistent minimal residual disease demanding lifelong treatment which has obvious implications in terms of compliance, adverse events and costs. It is now known that the main reason for disease persistence in CML patients treated with TKI lies in the insensitivity of the most primitive CML leukaemia stem cell (LSC). More recent evidence has demonstrated that, in contrast to more mature leukaemic progenitor cells, CML LSC are not addicted to BCR-ABL kinase activity but rather rely on other stem cell intrinsic pathways for their survival. The main focus in the CML field is therefore to identify these pathways while also trying to exploit them therapeutically to achieve CML LSC eradication and as a result disease cure. Growth factor (GF) signals are known to provide survival cues to CML stem and progenitor cells (SPC) and potentially support their survival even in the presence of TKI. Moreover CML SPC are also known to produce higher levels of some GFs via an autocrine loop and support their survival and proliferation through this mechanism. In this thesis, the characterisation of the autocrine GF production by CML SPC was extended while also investigating the role of several GFs and downstream signals in survival, proliferation and self-renewal of CML SPC. Whenever possible, the consequences of therapeutic targeting of these signals on CML SPC survival and proliferation were also assessed in vitro. In particular the role of the intracellular janus kinase (JAK) 2, which relays several myeloid GF signals, such as those from interleukin (IL)-3 and granulocyte macrophage colony-stimulating factor (GM-CSF), in CML SPC survival and proliferation was investigated mainly because higher levels of autocrine expression of GM-CSF by CML SPC relative to normal were demonstrated, while autocrine IL-3 production by CML SPC had already been shown. Moreover the cognate receptor of both GM-CSF and IL-3 (CSF2RB) was also shown to be expressed at higher levels in CML SPC relative to their normal counterparts, further supporting investigations on the role of JAK2 in CML SPC biology. Indeed targeting JAK2 with small molecule inhibitors in CML SPC in vitro, particularly in the presence of maximal BCR-ABL TK inhibition, resulted in increased apoptosis, reduced proliferation and colony output of CML SPC. The JAK2 inhibitor plus TKI combination treatment, compared to either single agent, further reduced survival of the more primitive quiescent LSCs in vitro, while also reducing engraftment of primary CML CD34+ cells in vivo in immunocompromised hosts. Although a degree of toxicity to normal haemopoietic stem and progenitor cells (HSPC) was observed, this was not as great as seen in CML SPC, thus suggesting that a therapeutic window for using JAK2 inhibitors in CML patients might be present when a carefully selected concentration of these compounds is chosen. Tumour necrosis factor (TNF)-α was another GF shown to be produced in an autocrine fashion at higher levels by CML SPC relative to normal HSPC. Moreover its levels of production by CML SPC were not modulated by BCR-ABL TK activity. Using a small molecule TNF-α inhibitor and exogenous TNF-α, it was shown that autocrine TNF-α acts as a survival and proliferative signal in CML SPC. Moreover its role became even more important in the presence of TKI, as combining TNF-α inhibition with TKI led to high levels of apoptosis in CML CD34+ cells, including the more primitive quiescent population, while also causing increased apoptosis in a population enriched for CML LSCs based on its surface marker expression (CD34+ CD38-). Finally given the known importance of quiescence and self-renewal pathways in CML LSC persistence following TKI treatment, the role of transforming growth factor (TGF)-β1 and novel neurotransmitter mediated pathways in CML LSC quiescence and self-renewal was investigated based on the findings of a genome and epigenome-wide screen of primary CML LSCs and normal HSCs carried out in our laboratory. Using in vitro assays the putative role of the neuromediators norepinephrine and acethylcoline in CML LSC self-renewal was demonstrated. Moreover the role of TGF-β1 in inducing primary CML LSC quiescence mainly by modulating the AKT pathway was also demonstrated. Overall the work presented in this thesis has furthered our understanding of the role of both autocrine and paracrine known and novel regulators of haemopoiesis in several aspects of CML SPC biology such as their survival, proliferation and self-renewal. Furthermore the efficacy in eradicating CML SPC of therapeutic strategies targeting some of these GF signals has been explored in vitro, thus providing evidence supporting their subsequent testing in in vivo assays and in due course in clinical studies. It is hoped therefore that the work presented will contribute to devise novel therapeutic strategies to eradicate CML LSC and in turn lead to a cure for CML patients.

The aim of this body of work was to characterise a novel cisplatin drug delivery system and to develop new tools based on biophotonic imaging that could be used to enhance studies of drug delivery in vivo. Cucurbiturils (CB) are macrocycles which are formed by acid catalysed condensation of glycoluril and formaldehyde. The internal cavity of CB[7] encapsulates a single molecule of cisplatin and the hypothesis was that encapsulation would reduce thiol degradation of the drug. Drug sensitivity studies in vitro with the cisplatin-sensitive human ovarian cancer cell line, A2780, and a cisplatin-resistant derivative, A2780/cp70, showed that the CB[7] encapsulated cisplatin retained activity but that this encapsulation drug delivery system was not able to overcome resistance to platinum. However, when these cell lines were grown as subcutaneous xenografts in nu/nu mice, the encapsulated cisplatin was able to reduce the growth of A2780/cp70 tumours which are resistant to the maximum tolerated dose of cisplatin in vivo. One possible explanation of this observation is that encapsulation might alter the pharmacokinetics of cisplatin and a method for the detection of platinum in biological samples by ICP-MS was established and validated. This assay was sufficiently sensitive to detect the low levels of platinum present in mouse plasma 24 hours after administration of either free or encapsulated cisplatin. Plasma and tissue pharmacokinetics show that encapsulation had no effect on the peak plasma concentration of cisplatin but did reduce the rate at which cisplatin was cleared from the plasma. The increased plasma AUC of cisplatin resulted in a non-selective increase in the delivery of cisplatin to both tumour and normal tissues. However, there was no apparent increase in toxicity which could be explained by the fact that encapsulation, unlike an increase in the dose of free cisplatin, had no effect on the peak plasma concentration. Subcutaneous xenografts lack critical features of human tumours. The development of more complex models for use in drug development has been limited due to lack of a method for monitoring tumour growth. Biophotonic imaging was, therefore, investigated to determine whether it is sufficiently sensitive and reproducible to be able to evaluate growth of disseminated tumours in mice. The bioluminescent signal is dependent on the metabolism of luciferin by luciferase. Subcutaneous injection of luciferin was shown to produce a consistent signal in all injected mice. The bioluminescent signal was transient but reached a maximum intensity 6 minutes after injection and remained stable for about 4 minutes which defined the window during which measurements were taken. Sensitivity was shown to be dependent on the level of expression of luciferase by the cells. Injection of commercially available HCT116Luc cells, where the luciferase gene was inserted by a lentiviral system, was shown to allow detection of 10,000 cells in the lungs of mice. This sensitivity was about 10 fold greater than was obtained by lipofectamine based gene transfection. When HCT116Luc cells were grown as subcutaneous xenografts in mice, an exponential growth pattern was easily detected by bioluminescence imaging and the reproducibility between mice was comparable to that routinely obtained by calliper measurements. Activity of encapsulated cisplatin was determined in a model of disseminated ovarian cancer. Rab25, a member of the RAS oncoprotein superfamily, is up-regulated in around 80% of ovarian cancer samples compared to normal ovarian epithelium. Rab25 contributes to tumour progression by enabling the tumour cells to invade the extracellular matrix by altering the trafficking of integrin. Transfection of Rab25 into A2780 cells results in cells that can grow in the peritoneal cavity of mice. A2780-Rab25 cells were 4 fold resistant to cisplatin in vitro which confirms a previous observation that Rab25 expression in A2780 makes them less sensitive to the induction of apoptosis in response to stress. A2780-Rab25 cells that express the luciferase gene (A2780-Rab25Luc) were injected into the peritoneal cavity of mice and growth was measured by biophotonic imaging. Exponential growth was clearly apparent at a stage at which no obvious abdominal distension was apparent. The disseminated A2780-Rab25Luc tumour xenografts were less sensitive to cisplatin than are subcutaneous xenografts of A2780. This is the first study that suggests that Rab25 over-expression results in reduced drug sensitivity in vivo. In contrast, a very significant growth inhibition was observed when mice were treated with an equivalent dose of encapsulated cisplatin regardless of whether it was administered by the intraperitoneal or subcutaneous route. These results are very encouraging since they confirm the enhanced activity of encapsulated cisplatin and also demonstrate the value of biophotonic imaging for measurement of tumour growth in vivo. Pharmacodynamic measures of drug activity in vivo in animal models are often based either on measures of surrogate tissue response or on single measures on tumour tissue removed at the end of the experiment. Biophotonic imaging in vivo allows the translation of reporter assays used in cell lines in vitro to studies of tumour response in vivo. A plasmid was prepared that links the p53 transcriptional response element to the luciferase gene and it was then transfected in to A2780 cells which express wild type p53. Stable transfectants of A2780p53Luc were treated with cisplatin, doxorubicin and paclitaxel and induction of p53 determined by bioluminescence and confirmed by Western blotting. A very low bioluminescent signal was present in untreated cells and a clear dose dependent increase in bioluminescence was seen in response to all three drugs. When A2780p53Luc cells were grown as subcutaneous xenografts the bioluminescent signal was significant in untreated tumours but was markedly increased 24 hours after treatment of the mice with cisplatin. Induction of p53 in the tumours was confirmed by immunohistochemistry and this also confirmed significant expression of p53 in untreated tumours. The possible implications of these findings for the improved delivery of cisplatin are discussed.

Protein tyrosine phosphatases (PTPs) have been identified that mediate a range of physiological and pathological processes, such as proliferation and tumour metastasis. PTPRK and PTPRM belong to the same subfamily of PTPs. This study aims to investigate the role of PTPRK and PTPRM in cancer development and progression. Knockdown of PTPRK expression was performed in PC-3 and DU-145 cells. Functional assays were then carried out on these cells in order to determine any changes in their biological properties. Knockdown of PTPRK significantly reduced the growth and adhesion of both PC-3 and DU-145 cells. The experimental results suggested that reduction of cell growth is potential involvement of p53 and/or caspase-3 and -8 and its up-stream molecule JNK. The decreased expression of PTPRK and PTPRM are associated with poor prognosis and reduced survival. Knockdown of PTPRK resulted in increased adhesive and invasive abilities, and promoted cell proliferation and motility of breast cancer cells. Moreover, PTPRM knockdown resulted in elevated adhesion, invasion, and proliferation of breast cancer cells. Activation of ERK and JNK by tyrosine phosphorylation and consequent elevated MMP9 activity is involved in increased cell migration and invasion by PTPRM knockdown. These results suggested that PTPRK and PTPRM are involved in the disease progression of prostate and breast cancer by regulating a complex network of pathways and molecules. This provides further proof of the importance of the R2B subfamily, a subgroup of PTP superfamily, in cancer. In addition, it sheds some light on the use of PTPs as prognostic indicators of disease, aiding in diagnosis and treatment. The major effect is the promotion of motility and invasiveness of cancer cells via ERK and JNK pathways. However it can also impair the apoptosis mediated by JNK pathways in certain cancer cells, such as prostate cancer cells. Such contrasting effects on survival and motility require further investigation, and should also be considered when treating cancers by targeting these molecules.

CLL is an incurable B-cell malignancy associated with profound tumour cell-mediated immune dysfunction. It therefore represents a challenging disease for the successful application of immunotherapeutic strategies aimed at promoting anti-tumour T-cell responses. In this study, extensive immunophenotypic analysis of T-cells from the blood of CLL patients was performed, in order to better characterise their dysfunctional status within the disease. Analysis of CLL patient blood samples revealed a skewing of T-cells towards a highly differentiated effector memory phenotype as well as the expression of markers associated with exhaustion/senescence (CD28- and CD57+) and immunosuppressive molecules (PD-1 and CD200). In addition this study revealed the expansion of CD8+ T-cells in a subset of CLL patients leading to an inversion in the normal CD4:CD8 ratio. The presence of an inverted CD4:CD8 ratio was subsequently shown to be associated with a shorter time to first treatment and reduced progression-free survival. Characterisation of T-cells identified several molecules that could be targeted therapeutically in order to break T-cell tolerance in CLL patients and potentially restore normal immune responses. Investigation of the immunosuppressive molecules PD-1 and CD200 showed that they are over expressed in CLL patients, suggesting that they may be involved in maintaining T-cell tolerance in the disease. However, blockade of PD-1-PDL-1 and CD200-CD200R signalling pathways failed to enhance T-cell responses from CLL patients in vitro. Investigation of an alternative approach to enhance T-cell responses in CLL involved the use of a bi-specific antibody targeting CD19 and CD3 called blinatumomab. In vitro testing showed that blinatumomab can induce T-cell activation, promoting the release of pro-inflammatory cytokines and granzyme B secretion from both CD4+ and CD8+ T-cells. In addition, blinatumomab was shown to mediate T-cell dependent killing of CLL cells requiring the formation of T-cell:CLL cell conjugates. Finally this study provided clear evidence that blinatumomab can break T-cell tolerance in CLL and strongly advocates the progression of blinatumomab into clinical trials as a novel therapeutic agent in CLL.

CD8+ T-cells recognise pathogens and cancer through a specific interaction between the T-cell receptor (TCR) and a 8-14 amino-acid residue peptide presented by class I major histocompatibility complex (pMHCI) molecules expressed on the target cell surface. The first structures of murine and human TCR/pMHC complexes, published in 1996, revealed a number of important features of the TCR/pMHC interface. Currently, <25 unique human TCR/pMHC complexes are reported in the literature. This is a relatively low number compared with the number of antibody or unligated pMHC structures. The lack of structural information regarding human TCR/pMHC complexes has compromised the determination of a comprehensive and accepted set of rules that govern T-cell antigen recognition. Difficulties in generating TCR/pMHC complex crystals partly explain the low number of these structures. The first part of this thesis reports the development of a new crystallization screen (TOPS) designed specifically for the generation of such protein crystals. I also had access to MART-1-specific TCRs, the MART-1 protein being expressed by virtually all fresh melanoma tumour specimens. Different human leukocyte antigen (HLA)- A*0201-restricted peptides from this protein are presented at the melanoma cell surface. As TCRs are known to bind to cancer-derived “self” peptides with weak affinity, there is considerable interest in designing enhanced affinity TCRs for the recognition of HLA-A*0201-MART-1. My work concentrated on the MART-1- specific TCR MEL5 and its affinity-enhanced variant selected by phage display, α24β17. I analysed the biophysical properties of α24β17 and determined that it bound HLA-A*0201-MART-1 with >30,000-fold enhanced affinity and distinct thermodynamics. Comparison of TCR/HLA-A*0201-MART-1 complex structures solved with TOPS and binding biophysics showed that: (i) TCR affinity can be enhanced by increasing interactions between the TCR and the MHC surface; (ii) soluble α24β17 retains the peptide specificity by a novel mechanism involving interactions with solvent molecules; and, (iii) MEL5 interaction with the physiologically relevant MART-127-35 nonameric antigen led to a peptide anchor residue switch, a TCR-induced modification that has never been observed before. I also initiated a preliminary study on the generation of genetically modified Jurkat cells and CD8+ T-cells expressing a range of affinity-enhanced TCRs directed against melanoma for adoptive cell therapy. These results suggested that melanoma specificity is retained after MEL5 transduction and that there is no need to optimize beyond a TCR affinity threshold to obtain optimal T-cell activation. Collectively, these data shed light on the complex and unpredictable nature of T-cell antigen recognition.