Eukaryotic initiation factor 4E (eIF4E) is an oncogene over-expressed in a variety of cancers. It is responsible for regulation of synthesis of proteins involved in progression to the malignant phenotype, including cyclin D1, Mcl-1 and VEGF. The oncogenic activity of eIF4E has been shown to be dependent on phosphorylation at serine 209 by the mitogen-activating protein kinase signal integrating kinases (MNKs). This work investigates the role of the MNK proteins in the proliferation and survival of breast and kidney cancer cells. Inhibition of the MNKs with a chemical kinase inhibitor resulted in inhibition of proliferation through cell cycle arrest, with no induction of apoptosis. This is consistent with a reduction in cyclin D1 protein. Combination of mTOR inhibition and inhibition of the MNK proteins in kidney cancer cells resulted in an additive but not synergistic effect on inhibition of proliferation. siRNA knockdown of the MNKs resulted in a reduction in eIF4E phosphorylation but did not inhibit proliferation or induce cell cycle arrest in a renal cancer cell line. The generation of an eIF4E mutant with phospho-mimetic activity induced partial insensitivity to the MNK kinase inhibitor suggesting some specificity of activity of this agent through the MNK proteins. Inhibition of the MNKs resulted in a reduction in cyclin D1 mRNA with no reduction in transcription. An accumulation of cyclin D1 RNA was seen in the nuclear compartment following treatment with MNK inhibitor suggesting inhibition of nuclear export of cyclin D1 RNA and subsequent degradation. Gene expression array analysis revealed regulation of genes with key functions in cellular metabolism. The MNK kinases are thus worthy of further investigation as a therapeutic target in breast and renal cancer. More potent and specific inhibitors of the MNK kinases than CGP57380 are required for this to be clinically relevant.

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DNA fusion vaccines encoding the idiotypic (Id) single chain variable fragments of B-cell malignancies fused to the potato virus X coat protein (PVXCP) were shown to enhance anti Id antibody and T-cell responses which resulted in protection in lymphoma and myeloma models. I further explored the use of PVXCP to enhance induction of anti -tumour immunity. The aims of this study were to generate two types of Id tumour vaccines against the BCL1 lymphoma, a multimeric Id-PVXCP fusion protein and a PVX particle displaying Id antigen, and to compare their performance with the DNA fusion vaccine. The multimeric fusion protein vaccine was produced in plants using the HyperTrans expression system. Three constructs which directed expression to the cytoplasm, for retention in the endoplasmic reticulum (ER) and for secretion were tested. The construct that directed the fusion protein to retain in the ER provided high protein expression and was used for further studies. The fusion protein was purified by antibody affinity chromatography and size exclusion. ELISA confirmed the integrity of the expressed protein and protein multimerisation was shown by transmission electron microscopy (TEM). This vaccine induced both anti-Id and anti-PVXCP antibody responses in mice to the levels comparable to the DNA vaccine and it provided protection against the lymphoma challenge. The plant viral particle (PVP) Id vaccine was produced by linking BCL]IgG to PVX via biotin-streptavidin. The PVP vaccine induced both anti-Id and anti-PVXCP antibodies to significantly higher levels than the DNA vaccine. PVP provided protection to the levels comparable to the DNA vaccine. The PVP vaccine also induced cellular responses as high numbers of PVXCP-specific IFN-y and IL-2 secreting cells were detected. In vivo PVX bound to CD 11 c + dendritic cells (DCs) and induced activation of all CD 11 c + DC subsets as determined by up-regulation of activation markers. PVX-activated DCs also changed their cytokine, chemokine and chemokine receptor expression profiles toward activated DC phenotypes. These data suggest that PVX enhanced immunity to the linked tumour antigen with the involvement of T cells and DCs. The feasibility of genetically linking BCL] scFv to PVX was also determined. BCLI scFv sequence was fused to the N-terminus of PVXCP sequence and inserted into a PVX-based vector to enable expression of the chimeric viral particle (CVP) in plants. CVP was assembled in plants as judged by the ability to infect host plants both locally and systemically. The CVP RNA and proteins (both fusion protein and PVXCP) were detected in all infected leaves. BCLI scFv on the CVP surface was detected by TEM with goldlabelled anti -BCLI antibody. However, low amount of CVP was obtained and therefore this vaccine production strategy requires further optimisation.

n the UK, 86% of lung cancer (LC) patients are diagnosed when curative treatment is not possible. Government guidelines recommend urgent chest X-ray referrals for patients presented with any 1 of 10 suggested LC symptoms. Little evidence currently supports these recommendations. Thus, the need for prospective studies to identify the predictive values of symptoms for LC diagnosis. This study aimed to investigate the feasibility of a prospective study to identify symptoms that predict LC in a secondary care population with high rates of chronic respiratory disease by investigating 1) the content validity and acceptability to this population of a patient-completed questionnaire, and 2) identifying patient-elicited symptoms that predict LC. The Identifying Symptoms that Predict Chest and Respiratory Disease (IPCARD) questionnaire was used to prospectively collect symptom, risk and comorbidity data in a chest clinic population investigated for LC (Patients aged ≥40). LC was identified six months following recruitment. Semi-structured and cognitive interviews explored the acceptability and content validity of the IPCARD questionnaire in this population. Multiple logistic regression was used to identify symptoms independently associated with LC in the clinic population, and in a COPD sub-group; at two levels of entry criteria (p<0.05 and p<0.15). 359 patients (70% of those eligible) completed the IPCARD questionnaire, and 77 (21.4%) were diagnosed with LC. Qualitative research indicated that participants found the IPCARD questionnaire acceptable, and its content validity was established in this secondary care population. Cough and breathing changes first indicated in the last three months, predicted LC in this referred population (p<0.05). In the COPD sub-group, the symptom descriptor, unable to get enough air predicted LC (p<0.05). At the relaxed criteria (p<0.15), five symptoms predicted LC in the full clinic population; a hard/harsh cough without phlegm, increasing chest infections, and weight gain (last 12 months) were added to the previous model. The COPD sub-group at p<0.15, breathing changes experienced (last three months), breathing changes first indicated within the last three months, unable to get enough air, and wheezing sensation, were predictors. Based on the more rigorous entry criteria (p<0.05), the diagnostic criteria for the COPD sub-group (positive likelihood ratio (+LR)=1.91; Area under curve (AUC)=0.739) performed slightly better than the criteria for the full population (+LR=1.49; AUC=0.729) (at optimal cut-off). The better performance of the COPD–specific model supports the need for an adequately powered study to investigate the predictive values of LC symptoms in homogeneous populations with specific respiratory diseases.

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Over the last 10-15 years the medical management of metastatic breast cancer has improved survival, so women are living longer with progressive disease. Little is understood about women’s problems and needs and how they live their everyday lives. This study aimed to explore the experience of women with metastatic breast cancer by applying three phases: a cross-sectional survey exploring quality of life, experience of care and where they turned for support; exploration of the narratives of 30 women considering the social consequences of living with progressive breast cancer on identity; and finally triangulating medical and nursing documentation, a measure of physical functioning and ten women’s narratives to define the illness trajectory of metastatic breast cancer. Phase 1: Quality of life was found to be poor with women experiencing a significant symptom burden. Experience of care was poor with unmet information and support needs. There was little evidence of General Practitioner or palliative care involvement in care. Phase 2: In weathering the oscillations of progressive disease, women faced threats to their social identity. Women sought ways to maintain their social roles and social order to avoid social isolation. To do this they adopted contingent identities: stoicism or absolved responsibility. Women used these contingent identities to mediate any discontinuity between the self, the body and social order. These self-representations were used by women to maintain their social roles and social order and in doing so avoiding being discredited and socially isolated. Phase 3: Mapping women’s illness trajectories identified three typical trajectories. The illness trajectories demonstrated the complexity of living over time with progressive disease, through phases which give definition to a previously ill-defined pathway. Living with metastatic breast cancer challenges the personal resources of the individual and poses interesting questions about how healthcare professionals provide information, effective symptom control, and emotional and practical support to women. Current models of care are not meeting women’s needs and new approaches to care provision need to be considered.

No description available.

Colorectal cancer is a common, age-associated disease with significant comorbidity and mortality. Biomarkers of ageing may have prognostic or predictive value in colorectal cancer. Fetuin A, members of the sirtuin family of proteins and telomeres have shown promise as potential biomarkers of ageing. AIM: To evaluate these potential biomarkers in the context of colorectal cancer. METHODS: Two cohorts of patients were used. Telomere length was measured in peripheral blood leukocytes (PBL), and for a subset of patients, in normal colorectal and colorectal tumour tissue. Serum fetuin A was measured for these patients and data on clinico-pathological factors of accepted significance in colorectal cancer was collected prospectively. Telomere length in the matched samples of leukocytes, normal colorectal and colorectal tumour tissue was compared. Associations between telomere length in the different tissues, serum fetuin A and clinico-pathological factors of accepted significance in colorectal cancer were evaluated. A systematic review of the literature was performed to examine the evidence for correlation between telomere length in different tissues in humans. Tissue from colorectal tumours from the second cohort patients was mounted in a tissue microarray (TMA) and stained for sirtuin proteins (SIRT2-SIRT7). This TMA also contained tissue from a subset of matched samples of adjacent normal colorectal mucosa. Staining of normal colorectal and colorectal tumour tissue was evaluated by the weighted Histoscore method and compared. The effect of staining in tumour tissue on cancer-specific survival was examined. Associations between Histoscores and clinico-pathological factors of accepted significance in colorectal cancer were assessed. RESULTS: Systematic review of the literature did not show robust evidence of correlation between telomere length in different tissues in humans. Telomere length in peripheral blood leukocytes did not show correlation with telomere length in normal colorectal mucosa, or in colorectal tumour tissue. PBL telomere length was potentially related to the presence of distant metastases. Fetuin A was inversely associated with markers of systemic inflammation and with T stage. Novel nuclear localisation was described for SIRT4 and SIRT5. Protein expression of the sirtuins was reduced in tumour tissue in comparison to normal colorectal mucosa, apart from SIRT3 cytoplasmic and nuclear and SIRT6 nuclear stainng. Lowest and highest quartile SIRT2 expression was associated with worse survival. Sirtuin protein expression levels and localisation correlate with increased systemic inflammation and pathological markers of poor prognosis in tumour tissue. Intercorrelations between sirtuin expression levels in normal tissue are not seen in tumour tissue, possibly indicating a breakdown of signalling and control.