The identification of tumour antigens (TAs) represents an ongoing challenge to the development of novel cancer diagnostic, prognostic and therapeutic strategies. A group of proteins, the cancer testis (CT) antigens are promising targets for such clinical applications. Their encoding genes show expression restricted to the immunologically privileged testes but their expression is also found in cells with a cancerous phenotype. To facilitate and automate the identification of novel CT genes, bioinformatic analytical pipelines based on publicly available microarray and expressed sequence tag (EST) data were developed and implemented as web tools to support wider application. Human germline-associated datasets were generated and the developed screening pipelines were subsequently used to analyse these datasets, leading to the identification of a. novel cohort of meiosis-speci fic genes, the meiCT genes that exhibit t he characteristics of CT genes and may have oncogenic features. In general, frequent germline gene expression found in cancer could reflect a soma-to-germline transformation occurring in human cells in the course of the development of cancer. The expression of germline-specific genes, in particular of meiotic genes, could lead to the production of proteins that cause oncogenic events and thus contribute to tumorigenesis and to the acquisition of tumour characteristics.

Cancer is of paramount medical concern as an increasingly major contributor of disease-related fatalities of significant prevalence - particularly in the context of current statisticaLlstochastical epidemiological studies which predict that one in three people will contract cancer at some stage of their lives, whilst one in four of these patients will die as a consequence of their particular neoplastic-associated condition. The human Rad9 protein exists in two full-length isoforms (termed Rad9A and Rad9B) whose respective differentially-elevated levels and related expression profiles are distinctive for specific tumour cell tissue types. Most known functions of the DNA damage response protein Rad9 are executed via the well- characterised Rad9-Rad l-Hus 1 ("9-1-1") protein complex, which is loaded onto chromatin in close vicinity to DNA lesion sites. The chromatin-loaded "9-1-1" complex functions as both a DNA damage "sliding-clamp" sensor and a recruitment platform which modulates and co-ordinates the activities of a wide variety of different proteins implicated in cell cycle checkpoint signalling, steroidal nuclear receptor signalling, protein chaperoning and DNA repair - via associative protein-protein interactions with the C-terminal tail domain of the Rad9 sub-unit. This toroidal, heterotrimeric "9-1-1" DNA sliding-clamp complex is highly conserved and its recently resolved crystal structure shows a functional similarity to the homotrimeric PCNA DNA sliding-clamp complex.

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Hereditary leiomyomatosis and renal cell cancer (HLRCC) and hereditary paragangliomatosis (HPGL) are familial cancer syndromes, caused by germline mutations in genes encoding the Tricarboxylic Acid Cycle (TCAC) enzymes fumarate hydratase (FH) and succinate dehydrogenase (SDH) respectively. Both FH and SDH function as tumour-suppressor genes and the conditions are inherited as autosomal dominant traits. Germline FH mutations predispose individuals to develop benign smooth muscle tumours of the skin and uterus (leiomyomas) and renal carcinoma, whilst individuals with mutations in SDHB, -C, and -D develop paragangliomas and phaeochromocytomas. In order to study the genetic and functional consequences of FH and SDH mutations, and to elucidate mechanisms of tumourigenesis, which are poorly understood, I have undertaken a comprehensive analysis of tumours from both HPGL and HLRCC patients, using gene and protein expression analysis, metabolomic profiling and cytogenetic analysis of HPGL tumours. Tumours from both syndromes over- express hypoxia-inducible factor-alpha (HIFla), the central signalling protein in hypoxia, and HIFIa-target genes including vascular endothelial growth factor (VEGF) and Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3). HLRCC and HPGL tumours accumulate the TCAC intermediates succinate and fumarate which have been shown to up-regulate HIFla in vitro by inhibiting the prolyl hydroxylases (PHD) that target HIFlct for proteosomal degradation. Therefore, 'pseudo-hypoxia' - the constitutive expression of HIFla in normoxic conditions - is likely to contribute largely to the tumourigenesis of HLRCC and HPGL and is most likely to occur as a direct result of accumulation of TCAC intermediates and PHD inhibition. To further investigate the tumourigenesis of HLRCC, I have successfully created a conditional Fhl (the mouse homologue of human FH) mouse knockout which causes hypertrophy when targeted to smooth muscle. I aim to create further temporal and tissue specific knockouts of Fhl, and if these mice develop tumours provide a model of HLRCC for the testing of anti-cancer drugs and therapies.

The c-myc oncogene is over-expressed or deregulated in many human cancers. c-myc encodes a transcription factor, the oncoprotein c-Myc (Myc), which acts as a master regulator of genes involved in such diverse cellular processes as replication and growth, loss of differentiation, invasion, and angiogenesis. Myc can also act as its own tumour suppressor by promoting cell death in the form of apoptosis. Thus, for putative cancer cells to arise, apoptosis must be blocked. Conditional MycERTAM transgenic mice allow regulated activation of Myc in distinct cell populations (skin suprabasal keratinocytes and pancreatic islet β-cells) and have highlighted contrasting behaviour between these two adult tissues in vivo: proliferation in the skin, and apoptosis in the pancreas. Given the crucial dependence on tissue location in vivo, we still do not know enough about the key divergence in Myc-regulated genes and proteins under conditions favouring opposing outcomes. To address this, we performed high-throughput transcriptome analysis using oligonucleotide microarrays. The in vivo transcriptional response to deregulated Myc was analysed for skin keratinocytes and laser-captured pancreatic islets following a time-course of MycERTAM activation. Due to the multi-factorial nature of the experimental design, novel statistical tools were developed allowing the use of linear models for inference of changes in gene-expression based on multiple experimental variables. Comparison of the transcriptional response between the two tissues identified potential signalling pathways which may promote apoptosis of β-cells or survival of skin keratinocytes: the DNA damage response pathway, and the Insulin-like growth factor 1 (Igf1) signalling pathway respectively. In addition, a marked change in expression was detected in members of the steroid hormone-regulated Kallikrein serine protease family in suprabasal keratinocytes but not for β-cells. These have been found to play an important role in regulating Igf1/Igf1-receptor ligation through proteolysis of the Igf1 binding proteins, are previously categorised markers for several human cancers, and may indicate a tissue-specific regulatory mechanism for determining ultimate Myc function in vivo.

p53 is a tumour suppressor protein that is stabilised and activated by DNA damage. DNA damage-induced p53 is able to bring about either cell cycle arrest or apoptosis by the induction of p53-responsive genes such as mdm2 and p21 waf-I. Mdm2 regulates p53 function by blocking the transcriptional transactivation domain of p53 and also by targeting p53 for degradation via an ubiquitin-mediated pathway. Increases in the levels and activity of p53 are brought about by post-translational modifications. The most widely studied modification of p53 is phosphorylation, mediated by several DNA damageactivated kinases. Poly(ADP-Ribose) Polymerase-l (PARP-l) is also a DNA damageactivated enzyme which covalently modifies several target proteins by poly(ADPribosylation). It is well established that PARP-1 plays a key role in DNA base excision repair. More recently, several studies have implicated PARP-1 in the regulation of p53 function in response to DNA damage, although the nature of this relationship has been controversial. This study aimed to clarify and investigate further the role of PARP-1 in p53 regulation using PARP-1 proficient and PARP-1 deficient mouse embryonic fibroblasts (MEFs) as well as a novel potent PARP-1 inhibitor (AGI4361; Ki < 6nM). In this study, both primary and immortalised PARP-l MEFs were used. Initial experiments revealed a tendency for PARP-l +/+ MEFs to develop p53 mutations during immortalisation. Interestingly. PARP-1 -/- MEFs retained wild-type p53, suggesting that the absence of PARP-l bypasses the requirement for p53 to be mutated during the immortalisation of MEFs. As these cells could not be used to analyse p53 responses, experiments were perfonned on primary PARP-l MEFs. However. the primary PARP-l- - MEFs were found to grow very slowly compared to their PARP-1 proficient counterparts. Interestingly. treatment of primary PARP-1+1+ MEFs with AG14361 had a similar effect on cellular growth. This growth inhibition in the absence of PARP-1 was only evident in primary and not immortalised cells. It was therefore decided to stably transfect immortalised PARP-l-- MEFs, expressing wild-type p53, with a plasmid construct containing PARP-l to produce an isogenic cell line pair. These cells have been used, together with a human colorectal carcinoma cell line (HCT-116) and the potent PARP-1 inhibitor AG14361 to analyse the p53 response to different DNA damaging agents. In response to ionising radiation and ultra violet radiation, the absence of PARP-1 did not alter the induction or activity of p53. In response to the alkylating agent temozolomide, treatment of PARP-l proficient MEFs with AG14361 potentiated the increase in p53 protein levels without affecting the transcriptional transactivation activity of p53, possibly due to an impaired repair of the DNA damage and hence increased signalling to p53 due to the persistence of DNA strand breaks. However, similar results were not obtained in the absence of PARP-1 protein (P ARP-1-/- MEFs) or in HCT -116 cells treated with AG 14361 The data presented do not support the hypothesis that PARP-1 is directly involved in the DNA damage induced regulation of p53. There may, however, be an altered p53 response in the absence of PARP-l when cells are treated with particular DNA damaging agents, due to an impaired DNA repair pathway.

The susceptibility of human lungs to carcinogens depends on the metabolic balance between activation and detoxification pathways, though for the tumour to develop depcnds on activation of cell immortalisation pathway. The correlation of these pathways has not been reported. The present study described the correlation in transcription of genes of two phases of drug metabolism pathways and immortalisation pathway in four lung cell lines namely; a normal lung cell line (CDD32Lu), alveolar adenocarcinoma (A549), pleural adenocarcinoma (1-1460), and a drug resistance large cell carcinoma (COR-L23/5010). The levels of transcripts of Phase I (CYPL4I, CYPIA2 and CYP2EI), phase II (GSTMJ) and immortalisation (hTERT) genes were investigated. There was evidence suggesting that the transcription of CYPJAI, and CYP/A2 is of cell specific since CYPIAJ transcribed in the A549 eell line only, while CYPIA2 was transcribed in the H460 cell line. The present study validates the ability of CYPIA2 to be expressed in cell line. Moreover, the present study showed abundant expression of CYP2E/ and GSTMJ mRNA in normal and lung cancer cell lines suggesting that these genes may play no active role in lung carcinogenesis. In addition, the transcription level of immortalisation gene (hTERT) and telomerase activity was determined and observed in the A549 cell line only. The novel finding of this research is the cb-transcription of CYPJAJ and JITERT in the A549 cell line. The co-transcription was further analysed by induction of CYPJAJ in all cell lines with the AhR ligands TCDD and 3-MC. The finding reveals the existence of CYPIAI and hTERT co-transcription. Despite the fact that transcription of CYPJAI was observed CYP1A1 activity was not detected even after cell treatment with CYP1A1 inducers. This was possibly due to scarcity of CYP1A! and limited level of haem in the extrahepatic tissue. This study demonstrated a novel basal and induced co-transcription of CYP1AJ and hTERT. The regulation of co transcription was analysed by silencing CYPIAJ using siRNA technology and observing hTERT knockdown. Silencing of CYP1AI was subsequently downregulate hTERT transcription and reduces cells viability. The mechanism of co-transcription was investigated to rule out the involvement of suggested AhR signalling pathway. This was carried out by determining the level of mRNA expression of those genes, which their proteins were involved in the AhR signalling pathway. The results obtained suggest the role of the AhR signalling pathway in the co-transcription of CYPJAJ and hTERT. The data obtained from gene knockdown experiments revealed silencing of CYPJAJ alters hTERT expression and cell proliferation. The present study suggests that siRNA technology can be used as a reliable tool for the validation of co-transcription. Moreover, the concomitant silencing of CYPJAJ and hTERT and inhibition of cells proliferation not only validate the co-transcription but also a valuable finding to be considered as a novel therapeutic target, which may contribute to management of lung cancer. The transcription of CYPIAJ and hTERT has been identified as a cancer risk marker in a diverse range of cancers, the data of the present study suggests the use of CYPJA] 5iRNA as optional gene therapy in cancer management, where CYPJAJ plays a major risk.

In response to DNA damage, the ATM and ATR kinases proliferate a signal that is transduced, either directly or via Chk2 and Chk1, to effector proteins, forming the DNA damage response (DDR). The effector proteins delay cell cycle progression, through checkpoints, and activate specific DNA repair mechanisms essential for preserving genome integrity and preventing cancer formation. Bloom's syndrome (BS) patients, which lack the BLM protein show genome instability and have a predisposition to cancer. BLM is phosphorylated by the DDR kinases ATM, ATR and Chk1. These phosphorylation events are essential for BLM to maintain replication fork integrity, preserve the S phase checkpoint and activate BLM to interact with other DDR proteins. In this study I have shown that BLM, isolated from mitotic cells, is phosphorylated on amino acid residue serine 26 (S26). BS cells lacking native BLM, but expressing a variant of BLM protein that cannot be phosphorylated at S26, fail to fully activate the G2/M checkpoint following UV irradiation or treatment with inhibitors of DNA topoisomerase H. Consequently, these cells are more sensitive to killing by these agents than are BS cells expressing wildtype BLM. The Chk1 and Aurora B kinases are able to phosphorylate BLM on S26 in vitro. Moreover, loss of Aurora B kinase activity leads to reduction of S26 phosphorylation in mitotic cells. Cells treated with inhibitors of Aurora B fail to fully active the G2/M checkpoint after UV DNA damage. Taken together, these data suggest, that Aurora B kinase phosphorylates BLM on S26 and that this is required to fully activate the G2/M checkpoint.