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Introduction: Over the past decades, considerable advances in understanding of cell biology at genetic, epigenetic and proteomic levels led to development of new strategies for better outcome of cancer therapy. One of these new strategies is targeting the class I PI3K/Akt/mTOR signaling pathway, in that this pathway plays a key role in regulation of many cellular functions, including proliferation, survival, metabolism, autophagy and motility. Dysregulation of the class I PI3K/Akt/mTOR pathway has been documented in a variety of human tumours and inhibition of this pathway has been observed to hamper tumour proliferation in vitro and prevent tumour progression in vivo and in clinic. More recently, emerging evidence suggests that the class I PI3K/Akt/mTOR pathway is associated with Cancer Stem Cell (CSC) biology, in light of maintenance, viability and conventional therapy resistance of CSCs. The CSC theory conceptualizes that a subset of tumour cells with Stem Cell-like properties, including self-renewal, multipotency, differentiation, and resistance to chemotherapy and radiotherapy, can recapitulate new tumours and resistance to cancer therapy. Materials and Methods: To explore class I PI3K/Akt/mTOR signaling pathway and CSCs as therapeutic targets in canine oncology, in one series of experiments, smallmolecular inhibitors Wortmannin, ZSTK474, KP372-1 and Rapamycin, which selectively target pan-class I PI3K, pan-class I PI3K, Akt and mTOR, respectively, were utilized to treat canine cancer cell lines using inhibitors alone or in combination with conventional therapeutic drugs. The human acute lymphoblastic leukaemia of T-cell origin cell line (Jurkat T cell line) was used as a comparative control. In another, a stem cell culture system was performed to isolate CSCs from canine glioma J3T cell line. Subsequently, microarray analysis of transcriptional expression profiles of J3T spheres (the putative CSCs) versus J3T parental cells was performed. Results: In this study, small molecules ZSTK474 and KP372-1 were found to significantly decrease cell viability at lower micromolar and nanomolar ranges, respectively. Rapamycin decreased cell viability at lower micromolar concentrations. However, the efficacy of Wortmannin varied from one cell line to another. Dissection of the mechanism of these inhibitors using Western Blot analysis and annexin V staining showed that all inhibitors functioned by decreasing phosphorylation of class I PI3K pathway members. Notably, the efficacy of Wortmannin for this pathway inhibition is confined to certain cell lines. In addition, Wortmannin had shorter drug duration than the other three inhibitors. Annexin V staining showed that KP372-1 was a potent inducer of apoptosis, with decreasing potency in hierarchy order, Rapamycin, Wortmannin and ZSTK474. The data obtained from the combination of pan-class I PI3K inhibitor (Wortmannin or ZSTK474) and mTOR inhibitor (Rapamycin) suggested that additive/synergistic effects were, in part, due to inactivation of Akt. The class I PI3K pathway inhibitors enhanced the efficacy of Doxorubicin in SB cells but not in canine REM, 3132 and J3T cells. The CSC colonies of canine glioma J3T cells were successfully isolated and expanded in the neurosphere formation assay. By microarray analysis, several class I PI3K signaling network-associated genes, particularly IGFBP2 (27-fold), FYN (9.3- fold), and DDIT4 (8.5-fold), were found to be highly up-regulated in the J3T CSCs. However, the genes encoding components, such as Akt1 and eIF4E, of class I PI3K/Akt/mTOR axis signaling were either unchanged or down-regulated in the CSCs. The majority of the genes encoding translation initiation factors were also downregulated in the CSCs. Conclusions: This study demonstrates that class I PI3K/Akt/mTOR signaling pathway is critical for proliferation and survival of cell lines derived from human acute lymphoblastic leukemia of T cell origin (Jurkat T cell line) and a variety of canine tumours. However, it appears that this pathway is dispensible for maintainence and viability of the CSCs isolated from canine gloma J3T cell line. This study suggests that the strategy of dual inhibition of class I PI3K and mTOR kinases may have better outcomes than the combination inhibitors of this pathway (such as ZSTK474 and KP372-1) with Doxorubicin in canine oncology.

The well conserved Notch signalling pathway plays a crucial role in vascular development and physiology. Delta-like 4 (DLL4) and Jagged1 (JAG1) are two key notch ligands implicated in angiogenesis. Both ligands were shown to have opposite effects on vasculature. DLL4-Notch signalling inhibits sprouting resulting in fewer but better perfused blood vessels, promoting tumour growth. In contrast to DLL4, very little is known about JAG1- Notch signalling in tumour angiogenesis and its influence on tumour growth and progression. The overall aim of this work is to study the functional difference between DLL4 and JAG1-Notch signalling. The effects of murine DLL4 and murine JAG1 over-expression on tumour growth and angiogenesis was also investigated in a mouse U87 xenograft model. Firstly, the downstream target genes of DLL4 and JAG1-Notch signalling were established through microarray and QPCR. Angiogenic assays such as sprouting, network formation and migration assays were employed to study the functional effects of these two ligands in endothelial cells. The thesis firstly demonstrates that JAG1 has opposing effects on endothelial cells compared to DLL4 by increasing sprout coverage and network formation. JAG1 is less potent than DLL4 in stimulation of Notch target genes in primary endothelial cell (HUVEC) but both displayed equal potency in HMEC-1, an immortalised endothelial cell line. The growth of U87 cell lines which over-expressed murine DLL4 or murine JAG1 was slower compared to wild-type U87 cell line in vitro. JAG1- and DLL4- Notch signaling have different effects on vessel formation, which impacted on the tumour growth in vivo. Interestingly, tumours over-expressing mDLL4 had less but larger vessels compared to control, whereas mJAG1 produced more yet functional vessels; both tumours had significantly reduced pericyte coverage. Both U87 mDLL4 and mJAG1 over-expressing tumours showed increased resistance towards anti-VEGF therapy, compared to control tumours. Sensitivity to therapy was restored in combinational treatment with DBZ and bevacizumab. The mechanism behind the differential responsiveness of the Notch receptors to DLL4 or JAG1 ligands could either reflect modulation by fringes, a family of glycosyltransferases that regulate Notch signalling or by a positive feedback loop present for DLL4-Notch signalling only. Fringe was found to be abundantly expressed in endothelial cells and highly vascularised tumours. This work has highlighted some key novel differences between the two Notch Ligands.

Cytochrome P450 enzymes are a superfamily of constitutive and inducible haemoproteins, with a central role in the oxidative metabolism of a wide range of compounds. These enzymes have been shown to play important roles both in tumour development and mediation of chemotherapy. Although cytoclirome P450 was first identified in the liver, evidence has accumulated for the presence of cytochrome P450 in extrahepatic tissues including the brain. The presence of the xenobiotic metabolising cytochrome P450 enzymes in the brain is intriguing because of the possible ramifications on function from small changes in P450 levels. o This project has highlighted the presence of several cytochrome P450 enzymes belonging to families 1, 2 and 3 in normal and neoplastic human brain. o In addition, it is the first study to look at cytochrome P450 expression in a number of different human brains using a variety of molecular biology and biochemical techniques. This methodology has provided some indication as to the inter-individual differences in the levels and types of cytochrome P450 enzymes in human brain tissue. o All the P450s examined were identified in specific regions of brain with CYPlAl and CYP2C8 mRNA being the most fi'equently expressed forms. CYP2D6 mRNA was localised primarily to the substantia nigra of the mid brain. The distribution of individual P450s in brain is important in determining the response of the brain to xenobiotics. o This project has also established the presence of several cytochrome P450 enzymes in neoplastic human brain by immunohistochemistry. CYPlAl and CYPIBI were over-expressed in the majority of tumours at high intensity, and almost 50% of tumours exhibited strong CYP3A expression. P450s are important in the aetiology and treatment of various cancers, the over-expression of CYPIBI was demonstrated in the majority of astrocytomas investigated, with no concomitant expression observed in the adjacent normal tissue. Data are emerging that CYPIBI has the capacity to metabolise a variety of putative human carcinogens, including polycyclic aromatic hydrocarbons and heterocyclic amines. In addition, preliminary findings from our group suggest CYPIBI may metabolise key chemotherapeutic drugs. This observations highlights CYPIBI as a possible target for gene therapy and CYPIB1 activated anti-cancer drugs. The major mechanism of induction of members of the CYPl gene family is via the Ah receptor (AhR) complex. To obtain a clearer picture of the mechanisms involved in the regulation of the CYPIBI gene in the brain, five glioma cell lines were investigated, only one of the five (MOG-G-CCM) exhibited constitutive AhR mRNA expression. The lack of the AhR mRNA expression which is the main mechanism of induction of members of the CYPl gene family paralleled the lack of cytochrome P450 gene expression in four out of the five cell lines. A key finding of this study suggests that, although the glioma cell line (MOG-G- CCM) may constitutively express CYPIBI mRNA, the presence of an inducing agent is required for subsequent protein expression. This finding requires further examination to investigate the factors involved in the regulation of the CYPIBI gene. It may be possibly that CYPIBI mRNA is present in normal tissue, and that translation/production of the accompanying protein is usually repressed suggesting that the expression of CYPIBI protein observed in tumour tissue may be due to derepression.

Faculty of Health Sciences School of Oral Health Sciences 9401428a vcvibha / There is considerable confusion regarding the origin and classification of the AOT with the most recent WHO classification including the AOT as a non-inductive tumour or hamartoma showing metaplastic mineralisation. This study reviewed the clinical and epidemiological features of 51 AOTs retrieved from the archives of the Division of Oral Pathology, University of the Witwatersrand. In addition a detailed histological analysis, including histochemical and immunohistochemical investigations, was undertaken with a view to provide evidence for induction in AOTs. 4μ haematoxylin and eosin sections were examined. Selected cases were stained with PAS, alcian blue at pH 2.5, Congo red, reticulin, mucicarmine, von Gieson, Masson’s trichrome and Prussian blue. Melanin bleach was performed on certain sections. Immunohistochemistry was performed in the presence of adequate preparations and controls with MNF 116 and Vimentin antisera. Analysis of the clinical and epidemiologic data revealed that the AOT in our series had the same clinicopathological features as those reported from other parts of the world. This data will be included in a review article being prepared to commemorate the 100th anniversary of the description of this lesion. AOTs occur in both follicular (64%) and extrafollicular forms (21%) most commonly in the anterior maxilla (62.7%) in females (63.6%) in the second decade (66.6%) where they are frequently associated with unerupted canines (42%). There is some evidence ii suggesting that extrafollicular AOTs may originate in other odontogenic cysts and that this might explain why some AOTs grow to a large size and behave aggressively causing root resorption and expansion. Histologically the unique and important presence of tall columnar cells resembling ameloblasts or odontoblasts were identified in 5 cases of AOT. These cells were arranged in a circular configuration and were actively secreting PAS positive material, which we have interpreted as dental matrix material. We have called these ‘circular secretory units’. The tall columnar cells did not always surround the entire secretory unit suggesting either that there was a variable rate of differentiation or that having completed their function these cells change shape and become unrecognisable. We regard these circular secretory units as providing definite evidence of induction. Further evidence of induction is provided by the presence of clusters or strands of odontogenic epithelium intimately associated with a lace-like pattern of dental matrix material. No evidence of residual ectomesenchyme was found, but this does not rule out the possibility that induction has indeed occurred. We can also find no evidence linking the circular secretory units with the pseudo-ductular spaces, which characterise the AOT. In conclusion, based on our observations, we recommend that the AOT be classified as a benign tumour with inductive capacity.