THE THYMIDINE KINASE-1(TK-1) AS A POTENTIAL TUMOR MARKER: SERUM LEVELS IN SERUM OF PATIENTS WITH SOLID AND SYSTEM MALIGNANCE NEOPLASMS

The review summarizes the results of studies of levels and/or activity in the blood serum of a metabolic marker thymidine kinase-1 (TK-1) of proliferating cells in patients with lymphoproliferative diseases (LPD) and malignant neopasms (NM).Comparison of the data in the literature in some cases have been difficult due to the fundamentally different methods of detection the activity or concentration of TK-1, used by authors, even despite the presence of relatively high (but not absolute) correlation between these parameters (maximum 0.8).Many clinical and laboratory studies have shown levels of correlation and/or TK-1 activity with clinical stages and different types of LPD and solid MN and can serve as a prognostic factor for overall and recurrence-free survival of patients. When solid MN shown that the activity of TK-1 accurately reflects the proliferative status of tumor.A comparison of the dynamics of TC-1 in the process of chemotherapy and its clinical efficacy, different authors have received fundamentally different results: in some cases the marker reduction was associated with treatment efficacy, and in part of publications they show that the clinically relevant effects of the treatment observed increase in the marker after the first chemotherapy.The entire set of received data demonstrates the relevance of the further development of the algorithm use of TK-1 in oncology practice

Investigation of the effect of disulfiram on the chemoresistance and invasiveness in pancreatic cancer cells

A thesis submitted in partial fulfilment for the degree of Doctor of Philosophy in Molecular Oncology.Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide with a mortality to incidence ratio of 94%. It is the 10th most common cancer in the UK with a 5-year survival less than 7%. In contrast to the improved therapeutic outcomes in many other cancers, the prognosis of PDAC remains dismal. One reason for this is because most PDAC patients are asymptomatic and end up being diagnosed after the cancer has advanced to a late stage. Another major obstacle in PDAC management is that PDAC cells are highly resistant to currently available anticancer drugs and the resistant cells metastasize to vital organs leading to a high rate of fatalities. Cancer stem cells (CSCs) are responsible for chemoresistance, relapse and metastasis. It is widely accepted that CSCs are located in the hypoxic niche which is responsible for maintaining stemness and epithelial to mesenchymal transition (EMT). The stemness of cancer cells is a reversible state mediated by the hypoxic tumour microenvironment. Hypoxia initiates stemness in cancer cells by activating genes which inhibit apoptosis, modify glucose metabolism, increase cell proliferation and enhance cell pluripotency. Therefore, development of new drugs to target hypoxia-induced CSCs will be of clinical urgency in PDAC treatment. Due to the time and costs for new drug development, repositioning of old drugs for new ailments is an emerging drug R&D strategy in recent years. Disulfiram (DS) is an anti-alcoholism drug used in clinic for over 60 years. It demonstrates excellent activity against a wide range of cancers such as glioblastoma, non-small cell lung cancer and, head and neck squamous cell carcinoma without toxicity to normal cells. Whereas, its effect on PDAC cells is still largely unknown. In this study, the in vitro effect of hypoxia on the stemness, chemosensitivity and invasiveness of Panc-1, a PDAC cell line, and a panel of patient-derived PDAC primary cultures was investigated. The sphere-cultured PDAC cells contained high hypoxic population which demonstrated CSC/EMT traits and were resistant to the first line anti-PDAC drugs; gemcitabine and paclitaxel. The study manifested that the hypoxia-cultured monolayer PDAC cell line and primary cells also expressed CSC markers, ‘ALDH, CD133, ABCG2’ and EMT markers, ‘Vimentin, Snail1, N-cadherin, Snail2’. The hypoxia-cultured cells were highly resistant to gemcitabine and paclitaxel. Significantly higher migration and invasion activities were detected in the hypoxia-cultured PDAC cells compared to the normoxic cultures. Our previous studies demonstrated that copper is essential for the anticancer activity of DS. In this study, the effect of cyclodextrin encapsulated DS and copper (CycDex DS/Cu) on PDAC cells was examined. In line with previous studies, CycDex DS/Cu showed strong cytotoxicity in sphere- and hypoxia-cultured PDAC cells. It blocked hypoxia-induced CSC/EMT traits and reversed hypoxia-induced chemoresistance to gemcitabine and paclitaxel in PDAC cells. DS is an FDA approved medicine. The study suggests that further studies may translate it into PDAC clinic application in a fast track. Many hypotheses claim that hypoxia activates NFкB which in turn activates a cascade of genes that promote metastasis and chemoresistance in cancer. Our previous results indicate that NFкB plays a key role in chemoresistance and invasiveness in some types of cancer. For these reasons, the effect of NFкB on PDAC cells was investigated, NFкBp65 was genetically overexpressed and knocked out in Panc-1 PDAC cell line. The NFкBp65 overexpressed clones showed significantly higher migration rate but failed to induce chemoresistance. In contrast to our previous findings, the NFкBp65 overexpression and knockout did not influence the expression of CSC/EMT markers. These results suggest that we still need to set up further studies to elucidate the molecular anti-PDAC mechanisms of cyclodextrin encapsulated DS/Cu in PDAC cells.PCU

The role of the Kinin-Kallikrein System in chronic lymphocytic leukaemia

Background:Chronic lymphocytic leukaemia (CLL) is an incurable heterogeneous disease. The identification of reliable and cost effective biomarkers is therefore imperative. A comparative proteomic approach was previously employed to study protein expression changes associated with in vitro BCR ligation. Kininogen, a critical protein of Kinin-Kallikrein System (KKS) was found to be upregulated (p≥2) in 3/3 “high risk” clinical samples upon BCR stimulation. Both High and Low Molecular Weight Kininogens (HMWK and LMWK, respectively) serve as a substrate from which Plasma and Tissue Kallikreins liberate Kinins, which in turn act upon B1 and B2 kinin receptors. This project aimed to investigate the role of the KKS in CLL and to identify novel proteins which may have clinical relevance in this disease.Materials and Methods:KKS was investigated using CLL clinical samples and a range of methods such as immunoblotting, reverse transcription polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and flow cytometry as appropriate.Results:The up regulation of LMWK upon in vitro BCR stimulation was confirmed by immunoblotting in 3/3 CLL samples previously used in a discovery phase proteomics. In a pilot series of 52 unselected CLL samples, 71% demonstrated basal LMWK expression. A total of 18 of these samples were also analysed for LMWK transcript, which was not detected in any of these samples. The expression of Kallikrein 6 was confirmed in 25 CLL samples. The B1 and B2 receptors were identified in 15 and 3 CLL samples, respectively. An elevated Bradykinin level was demonstrated in 27/36 (75%) plasma samples from CLL patients and was found to be associated with untreated (p=0.039) stage A (p=0.03) CLL and increased Plasma Kallikrein (p=0.001).Discussion:It has been demonstrated for the first time that CLL cells express the components for KKS signalling pathway, which can be further investigated for clinical relevance

Multiplexed affinity peptidomic assays: multiplexing and applications for testing protein biomarkers

Biomarkers are increasingly used in a wide range of areas such as sports and clinical diagnostics, biometric applications, forensic analysis and population screening. Testing for such biomarkers requires substantial resources and has traditionally involved centralised laboratory testing. From cancer diagnosis to COVID testing, there is an increasing demand for protein based assays that are portable, easy to use and ideally multiplexed, so that more than one biomarker can be tested at the same time, thus increasing the throughput and reducing time of the analysis and potentially the costs. Events in recent years, not least the ongoing investigations into claims of widespread state-sponsored doping schemes in sport and the COVID-19 pandemic of 2020 highlight the ever-growing requirement and importance of such tests across multiple frontiers. The project evaluated the feasibility of new antipeptide affinity reagents and suitable technologies for application to multiplexed affinity assays geared towards quantitatively analysing a range of analytes. In the first part of this project, key protein biomarkers available from blood serum and covering a range of conditions including cancer, inflammation, and various behavioural traits were chosen from the literature. Peptide antigens for the development of antipeptide polyclonal antibodies for each protein were selected following in silico proteolysis and ranking of the peptides using an algorithm devised as part of this research. A microarray format was used to achieve spatial multiplexing and increase throughput of the assays. The arrays were evaluated experimentally and were tested for their usability for studying up/down regulation of the target biomarkers in human sera samples. Another protein assay format tested for compatibility with affinity peptidomics approach was a gold nanoparticle based lateral flow test. An affinity-based lateral flow test device was built and used for the detection of the benzodiazepine Valium. Here spectral multiplexing of detection was considered. The principle was tested using quantum dot nanoparticles instead of traditionally used gold nanoparticles. The spectral deconvolution was achieved for mixtures containing up to six differently sized quantum dots. In the final part of this project, a search for novel peptide affinity reagents against insulin growth-like factor 1 (IGF-1) was conducted using phage display. Four peptides were identified after screening a phage display library, and the binding of these peptides to IGF-1 was compared to that of traditional antibody

The isolation and characterisation of MHC-presented peptides from CML-derived cell-lines, with a focus on post-translational modification

Phosphorylation is a key regulator of protein function and activity, and aberrant kinase activity is implicated in a wide range of malignancies, of which the bcr:abl fusion kinase found in chronic myeloid leukaemia is a classic example. As phosphopeptides are known to be presented by both the MHC class-I and class- II pathways, against which specific CD4+ and CD8+ T cell responses may be generated, study of MHC-presented phosphopeptides may reveal unique cancer antigens with direct links to the neoplastic state. Mild acid cell-surface elution is a rapid and effective method for MHC class-I peptide capture, though complicated by contamination with non-MHC peptides and poor downstream compatibility, especially with IMAC, a popular method for phosphopeptide enrichment. As an alternative to the citrate-phosphate elution buffer, a TMA-formate elution buffer is proposed. This was developed for IMAC compatibility, and osmotically balanced and supplemented to minimise cell lysis, (assessed by several assays) and used with a pH 5.5 prewash to reduce non- MHC peptide contamination. MALDI-MS/MS of MHC class-I peptides from K562- A3 cells found a sequence with high homology to a known cancer antigen as the common peak for both citrate-phosphate and TMA-formate eluted cells

Role of microRNAs in Jaagsiekte sheep retrovirus infection

Ovine pulmonary adenocarcinoma (OPA) is a lung cancer that affects sheep, caused by jaagsiekte sheep retrovirus (JSRV). OPA is present in most sheeprearing countries of the world, but, at present, there are no reliable early-stage tests to diagnose the disease, and OPA continues to pose an animal welfare threat and cause substantial economic losses. In addition, OPA is a valuable animal model to study early oncogenic events in human lung cancer. Specifically, OPA and some types of human lung cancer present similarities in activated signalling pathways (Ras-MEK-ERK1/2 and PI3K-AKT-mTOR) and their association with type II pneumocytes. Nevertheless, study of the molecular pathogenesis of OPA has been hindered due to the lack of a permissive cell line for JSRV replication. JSRV encodes an unusual envelope protein (Env) which is actively oncogenic and sufficient to drive transformation in vivo and in vitro. Despite the lack of a permissive cell line, early oncogenic events induced by JSRV can be studied by transfection of cell lines with plasmids encoding JSRV Env. MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression and essential cell processes such as cell proliferation and apoptosis. miRNAs are being extensively studied as biomarkers of several diseases, including cancer. The aims of this project were to investigate the role of miRNAs in the early oncogenic events induced by JSRV and to investigate their potential as OPA biomarkers. miRNA expression levels were investigated using small RNA sequencing in lung tissue from cases of experimentally induced OPA. No evidence of JSRV-encoded miRNAs was found, but levels of 40 miRNAs were found differentially expressed between affected and control sheep. Of those, upregulation of nine microRNAs (miR-135b, miR-182, miR-183, miR-21, miR-200b, miR-205, miR-31, miR-503 and miR-96) was confirmed by RT-qPCR in experimental and natural cases of OPA, suggesting that increased levels of these miRNAs were characteristic of OPA affected lung tissue. To investigate miRNAs as potential biomarkers, miRNA expression was measured in serum and bronchoalveolar lavage fluid (BALF) macrophages of OPA affected sheep. small RNA sequencing revealed 74 microRNAs and 85 miRNAs differentially expressed in serum and BALF macrophages, respectively. Interestingly, BALF macrophage microRNA expression was found to resemble more closely that of OPA affected sheep lungs. In addition, miRNA expression levels varied at different stages of the disease and no miRNAs were found to be consistently dysregulated in serum of OPA affected animals. Discordances in miRNA signatures in lung tissue and serum are not entirely unexpected. Lung tissue miRNAs might represent the tumour microenvironment and localised response to it, whereas miRNAs in serum may represent the global state of the animal, and tumour miRNAs might be released into circulation at low levels, making them difficult to detect. Expression of the nine upregulated miRNAs was then investigated in in vitro models to study their involvement in transformation. Lentiviral vectors encoding green fluorescent protein (GFP) or a GFP-2A-Env fusion protein were produced and used to transduce cell lines. Transformation was verified by immunocytochemical detection of the transformation markers P-Akt and PERK1/2. Nevertheless, miRNA expression levels in culture did not resemble those observed in lung tissue of OPA-affected sheep. These differences might be due to species variation, upregulation of miRNAs late in the transformation process, or involvement of other cell types in tissue besides the transformed cells. To study these questions further, JSRV and the GFP-2A-Env encoding lentiviral vectors were used to infect lung slices in culture. Expression levels of miRNAs did not, in any of the cases, resemble lung tissue findings. Fewer than 5% of cells in lung slices were found to be infected, suggesting that changes in miRNA expression could be masked by the background of normal cells. Nevertheless, increasing JSRV21 concentration did not yield higher infection levels, indicating that those might be more dependent on the availability of JSRV’s target cells, dividing type II pneumocytes, than viral concentration. Taken together, this study has revealed new information on miRNA expression in OPA-affected sheep, including expression patterns in lung and serum. Future work should focus on developing a permissive replication system to allow the study of miRNAs in early JSRV-induced transformation events

Identifying new genes in mitochondrial disease

PhD ThesisMendelian mitochondrial disease presents vast clinical and genetic heterogeneity, which provides challenges in attaining genetic diagnoses for affected patients. With approximately 1,200 proteins encoded by the nuclear genome that tightly coordinate mitochondrial function, targeted screening of a subset of genes provides a low diagnostic yield. Clinicians and researchers have increasingly turned to whole exome sequencing (WES), a targeted next-generation sequencing technology for the identification of all variants in the exons (coding regions) of all known genes for variant discovery and prioritisation, constituting approximately 1% of the human genome. WES was utilised to provide genetic diagnoses in two patients cohorts with clinically well-defined, genetically undetermined mitochondrial disease: (i) adult-onset progressive external ophthalmoplegia (PEO) (n=19), a mitochondrial DNA (mtDNA) maintenance disorder characterised by extraocular paresis and skeletal muscle restricted multiple mtDNA deletions, with broad phenotypes ranging from indolent PEO and fatal multisystem PEO-plus; (ii) early-onset mitochondrial respiratory chain complex (RC) deficiency (n=20), typically affecting patients in the first decade of life and are associated with heterogeneous phenotypes. Pathogenic mtDNA mutations were previously excluded in all patients. Adult-onset PEO with multiple mtDNA deletions patients were recruited following negative genetic testing of commonly associated nuclear genes, while early-onset RC deficiency patients were negative genetic testing in a small selection of nuclear genes. Using custom variant filtering strategies for each cohort, confirmed diagnoses were attained in 10% of adult-onset PEO with multiple mtDNA deletions patients and 35% of early-onset RC deficiency patients. This was comprised of known mitochondrial disease associated genes in both cohorts (AARS2, EARS2, MRPS22, PDHA1, SCO1, TK2, TRMU, TWNK), while novel candidate genes involved in mitochondrial translation, mtDNA replication, nucleotide metabolism and mitochondrial dynamics were also proposed. The pathological mechanism of a novel heterozygous GMPR1 variant was characterised as a potential new candidate gene associated with late-onset PEO due to impaired nucleotide balance for mtDNA maintenance. GTPBP3 was identified as a novel locus causing defective tRNA modification with multiple RC deficiency and the clinical, molecular and genetic features of AARS2, EARS2 and YARS2 mutations were expanded. Finally, this research highlights the potential significance of de novo and somatic mosaic variants in WES variant discovery and prioritisation.Medical Research Counci