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Apoptosis and matrix metalloproteinases remove redundant tissue. They are independently described in similar situations but no causal relationship has been identified. This thesis hypothesizes that the process of apoptosis induces MMP expression. A new cell separation technique was developed allowing rapid separation of apoptotic and non-apoptotic cells with 95% purity, something not previously described. Biotinylated Annexin- V was used to label apoptotic cells and streptavadin-coated dynabeads and a magnetic particle separator used to extract them. Apoptosis was induced in cell lines derived from colon and gastric cancers and the cells separated and analysed using quantitative RT-PCR and real-time PCR. In fibrosarcoma cells apoptosis effects large increases in MMP7 mRNA and decreases for the inhibitor TIMP 4 whereas in malignant epithelial cells apoptosis increases production of MTI-MMP, MMP2 and TIMP2 mRNA. In co-culture, apoptotic epithelial cells induce mRNA production in fibrosarcoma cells for MTI-MMP and TIMP2~ MTI-MMP' combined with TIMP2 is pivotal in the activation of MMP2. These results support the hypothesis that the process of apoptosis leads to increased MMP production. As chemotherapy largely functions by inducing apoptosis, the demonstration that apoptosis may lead to increased matrix destruction and potential tumour shedding could explain why, for many malignancies adjuvant or nee-adjuvant therapy is ineffective and why, for others, efficacy is not as good in-vivo as predicted in-vitro.

The platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) families of receptor tyrosine kinases (RTKs) are evolutionarily related cell-surface receptors which regulate physiological and pathological angiogenesis. Neuropilins (NRPs) are transmembrane glycoproteins which function as co-receptors for VEGFR to mediate vascular development and angiogenesis. RTK signalling has a long established role in tumour-cell biology and downstream cellular effects of RTK activation, such as, sustained cell proliferation and invasion are known hallmarks of cancer. NRPs are also up-regulated in tumour cell lines and clinical specimens, and a major focus of NRP research has been to understand the role of NRPs in cancer, which to date has largely been attributed to NRPs contribution to VEGFR activation. Emerging evidence for NRPs in regulating the activation of adhesion molecules, growth factors and RTKs (other than VEGFR) illustrate that NRP has a much broader role in cancer. In cell types including smooth muscle cells, stem cells and tumour cells, there is now evidence that NRP-1 regulates PDGFR activation and signalling. Identification of the molecules that regulate PDGFR signalling will advance the understanding of tumour cell biology and contribute to the development of targeted therapies.To date, few studies have evaluated the role of NRP-1/PDGFR signalling in cancer. The objective of this study was therefore, to elucidate the cellular mechanisms of NRP-1/PDGFR signalling, and to investigate how this cellular crosstalk modulates PDGFR-stimulated signalling, survival and migration of tumour cells. A subset of mesenchymal tumour cell lines that expressed NRP-1 and PDGFR-α and/or PDGFR-β and were identified to investigate NRP-1/PDGFR crosstalk. In these cell lines, NRP-1 could associate with PDGFR-α and PDGFR-β independent of PDGF growth factor stimulation. NRP-1 did not regulate PDGF-stimulated phosphorylation of PDGFR-α or PDGFR-β yet, in a subset of the cell lines, NRP-1 contributed to the activation of the MAPK-ERK and PI3K pathways. NRP-1 did not regulate PDGF-stimulated cell proliferation, yet NRP-1 knockdown attenuated PDGF-stimulated cell migration in certain cell lines. Together, this study has provided evidence of NRP-1/ PDGFR crosstalk, which affects the migratory potential of a subset of mesenchymal tumour cells. In these cell lines, NRP-1 knockdown does not inhibit the overall phosphorylation of PDGFR, yet does have subtle effects on specific downstream PDGFR pathways.

Most human cancers carry mutations in cell cycle regulators that result in deregulated Cdk activity, which can either be amplification of the cyclins, elimination of the Cdk inhibitors or mutations in the Cdks. These modifications have a high prognostic value. Cdk2 activity has been shown to be upregulated in different kind of tumours (mammary and prostate carcinomas, and lymphomas) due to the mutation of its regulators, p27KiP1 and cyc/in E; and this alteration has a high prognostic value. Moreover, an insensible INK4 point mutation in Cdk4 has been described in human melanomas. To evaluate the importance of Cdks in neoplastic development, the loci encoding Cdk4, Cdk6 and Cdk2 were ablated to study the effect of Cdk deficiency in tumour development. To this end, the corresponding Cdk knock out mice were crossed with the K_Ras+/LSLG12V;RERertert strain that carries an endogenous K-Ras oncogene whose expression is dependent on Cre-mediated recombination. Postnatal expression of this oncogene leads to the development of lung adenomas and adenocarcinomas. Primary MEFs derived from K- Ras+ILSLG12V;RERrrtlert embryos lacking either Cdk4, Cdk6 or Cdk2 displayed decreased proliferation in culture and prevented the growth in low serum condition. However, no obvious differences were detected in immortal MEFs regardless of the missing Cdk.

The majority of cancer related deaths occur due to the invasive growth of metastatic lesions. In the early stages of metastasis, circulating cell interact with the endothelial cells to establish at a distant site. In inflammation endothelial activation results in induction of adhesion molecules on the endothelium that participate in the homing of leukocytes. Because of the interactions of metastatic cells with the endothelium, the question was whether some of the characteristic molecules of endothelial activation were induced during metastasis. In vivo pulmonary metastatic models were used to characterize the expression profile of endothelial activation. Immunohistochemistry identified VCAM-1 to be induced on the pulmonary endothelium following tumour cell arrest. VCAM-1 upregulation was not observed prior to tumour cells arrest or within the first hours. In contrast, tumour cell arrest appeared to be required for endothelial activation, arguing against a mechanism analogous to leukocyte homing. The upregulation of VCAM-1 upon tumour cell arrest corresponded with the initiation of platelet clot formation around the tumour cell and recruitment of leukocytes to the site, both previously shown to be essential for metastasis. Disruption of both phenomena, either through genetic or pharmacological manipulation, demonstrated that in contrast to the recruited leukocytes, platelets were involved in inducing endothelial activation. Another protein investigated was VAP-1. In contrast to VCAM-1, central to VAP-1 adhesive function is its enzymatic activity. Blocking the functions of either molecule highlighted their role in facilitating the recruitment of the leukocyte population to the tumour cell. Disruption of which led to a significant attenuation of metastasis. While VCAM-1 and VAP-1 function appears critical in the early steps of metastasis, their inhibition had no effect at later stages of pulmonary colonization.

Posttranslational modifications are of great importance in the mediation of transcriptional effects, necessary for signalling in cancer. A characteristic example of such modifications is acetylation of the p53 tumour suppressor, a transcription factor involved in several crucial cellular functions including cell-cycle arrest and apoptosis. p53 is stabilised under hypoxic and DNA damaging-conditions. However, only in the latter scenario is p53 fully capable of inducing the expression of its proapoptotic targets through acetylation. The hypoxia inducible factor 1 (HIF-1) transcription factor is stabilised at low oxygen levels to mediate a cellular adaptive response under these conditions, promoting cell survival. As these two opposing transcription factors share a common transcriptional regulator, p300/CBP, this study focused on deciphering the p300/CBP complex components under differential stress to determine its composition required for cellular responses elicited in response to DNA damage or hypoxia, in an effort to investigate a possible link between differential posttranslational modifications and the resulting cell fate. Hence, the aim of this study was to investigate the roles of p300/CBP components in dictating transcriptional regulation of both HIF-1 and p53 in hypoxic conditions. To carry out this study, the proapoptotic BID gene was the system used, as its promoter contains a p53 response element and a HIF-1 response element (HRE). The p300/CBP associated factors PCAF and Strap were appointed as potent candidates for posttranslational modifications under differential conditions, as they are stress-responsive cofactors. Under DNA damage, PCAF acetylates p53 at K320 and Strap augments p300 binding to p53, both of which amplify the p53 response. Evidence from this study demonstrates that under hypoxia-mimicking conditions PCAF-mediated p53 acetylation at K320 is reduced to a greater extent compared to p300/CBP acetylation at K382. The limited amounts of acetylated p53 at K320 are preferentially recruited to the promoter of the cell cycle arrest p21WAF-1/CIP-1 gene that appears to be unaffected by hypoxia, but fail to be recruited to the BID promoter, rendering p53 incapable of upregulating proapoptotic BID in hypoxic conditions. In addition, under the same conditions, PCAF was found to acetylate, and direct HIF-1 to a particular subset of its targets, leading to alterations in the net physiological effect. Moreover, the intrinsic acetyl transferase activity of PCAF was shown to increase the stability of HIF-1. An additional role was attributed to PCAF in relation to apoptosis, albeit from another angle. BID protein translocation to the cytoplasm in hypoxic conditions was facilitated by ectopically expressed PCAF.Strap was found to be preferentially recruited to the HRE of the BID promoter in hypoxic conditions, and to exert a transrepression effect that appeared to be p53-dependent. Strap also interacted with specific PCAF isoforms depending on the type of cellular stress. Contrary to PCAF, ectopically expressed Strap did not have any effect on BID subcellular distribution. This study has provided additional insight in the mechanisms by which cofactors are involved in cell fate, either by affecting activity and stability of HIF-1 and p53, or having a direct effect on Bcl-2 member subcellular distribution.