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Normal germ cell development in the human testis is crucial for subsequent fertility and reproductive health. Disruption of testis development in fetal life can result in deleterious health consequences such as testicular dysgenesis syndrome (TDS), which includes disorders, such as cryptorchidism, hypospadias, infertility and testicular germ cell tumours (TGCT). A rat model of TDS in which rats are exposed to phthalates in utero has been validated, but does result in the development of TGCT. In humans, TGCTs result from transformation of pre-neoplastic carcinoma in-situ (CIS) cells and these CIS cells are believed to arise from human fetal germ cells during their transition from gonocyte to spermatogonia, based on their morphology and protein expression profile. It has been proposed asynchronous differentiation of germ cells in the human fetal testis may predispose fetal germ cells to become CIS cells. Studying the development of these tumours in humans is difficult because of their fetal origins and prolonged duration from initiation of impaired development to invasive disease. For this reason the use of relevant animal models that can mimic normal and abnormal germ cell development may provide new insight into how TGCT develop. The Common Marmoset monkey, a New World primate exhibits many similarities to the human in terms of reproductive biology and could represent such a model. This thesis aimed to further characterise the origins of CIS cells in the human testis by investigating the protein expression profile of CIS cells in patients with TGCT and comparing them to established markers of human fetal germ cell types using immunohistochemistry and immunofluorescence. Quantification of the various subpopulations of CIS and proliferation within these populations was performed. The thesis also investigated the Common Marmoset monkey as a potential model of normal testis and germ cell development by comparing the differentiation and proliferation profile of germ cells with those of the human during fetal and early postnatal life. During the present studies methods were successfully developed that enabled us to use testicular xenografts to recapitulate normal development of immature testes from marmoset and human. This involved grafting pieces of testis tissue subcutaneously under the dorsal skin of immunodeficient mice and retrieving them several weeks later to investigate their development during the grafting period. Xenografts using tissue from fetal, neonatal and juvenile marmosets were performed in addition to testes from first and second trimester human fetuses. Finally the present studies aimed to use the marmoset and the xenografting approach as systems in which to examine the effects of gonadotrophin suppression and phthalate treatment on germ cell differentiation and proliferation, with particular attention to the potential for development of CIS and TGCT. Heterogeneous phenotypes of CIS cells were identified, mostly consistent with those seen in the normal human fetal testis, however some of these CIS cells did not exhibit the same phenotype as germ cells identified in normal fetal testes. In addition it was shown that some of the proteins considered to be ‘classical’ markers of CIS cells, such as the pluripotent transcription factor OCT4, were not expressed in a proportion of the CIS cells. The proliferation index of CIS cells is also significantly higher in those subpopulations with the most ‘undifferentiated’ phenotype (i.e. OCT4+/VASA-). The present studies have generated novel data showing that the marmoset is a good model of fetal and neonatal germ cell development, with similarities to the human in terms of an asynchronous and prolonged period of differentiation and proliferation of germ cells from gonocyte to spermatogonia. This feature is also common to the human, but not a characteristic of the rodent. Fetal, neonatal and pre-pubertal germ cell development can be re-capitulated by xenografting tissue from marmoset and human testes into nude mouse hosts. Human fetal testis grafts produced testosterone and were responsive to hCG stimulation. First trimester human testis xenografts that have not developed fully formed seminiferous cords prior to grafting can complete the process of cord formation whilst grafted in host mice. In addition, germ cells in fetal human and marmoset xenografts can differentiate and proliferate in a similar manner to that seen in the intact non-grafted testis. In the intact neonatal marmoset, suppression of gonadotrophins resulted in a 30% decrease in proliferation, however differentiation of gonocytes is not affected. In-utero treatment of neonatal marmosets with mono-n-butyl phthalate was associated with unusual ‘gonocyte’ clusters, however, di-n-butyl phthalate treatment of mice carrying fetal marmoset xenografts resulted in no visible effects on germ cell differentiation or proliferation and did not result in the development of CIS or TGCT. In conclusion, this thesis has shown that there are many subpopulations of CIS cells of which many have not been previously described. These subpopulations have different characteristics, such as variable proliferation rates and this may indicate the potential for progression or invasiveness. These subpopulations have similar protein expression phenotypes to normal human fetal germ cells although the present studies have identified some CIS cells with phenotypes that are not found in the normal human testis. This thesis has demonstrated that the marmoset is a comparable model to the human in terms of asynchronous fetal germ cell development, which may predispose this species to the development of CIS/TGCT. In addition to the use of intact marmosets, these studies have also demonstrated for the first time that testis xenografting provides a comparable system for testis cord formation, germ cell differentiation and proliferation in fetal/postnatal marmosets and fetal human testis. In addition the marmoset and xenografting models have indicated that phthalates may have minor effects on testis development in the human and marmoset but do not result in CIS or TGCT. These model systems are suitable for further investigation of normal and disrupted testis development.

Osteosarcoma is the most common primary malignancy of bone. 5-year survival has remained stable at around 60-70% for 40 years. However, a number of patients will suffer from recurrent and/or metastatic disease representing a large unmet clinical need. CSPG4 is a transmembrane protein which is expressed on a number of progenitor cells and tumour types. Preliminary work had found CSPG4 present in osteosarcoma tumour samples. In this study, CSPG4 mRNA and protein expression was demonstrated in clinical samples and model cell lines. CSPG4 mRNA is overexpressed in osteosarcoma samples compared to mature osteoblast cells, the putative cell of origin for osteosarcoma. In a cohort of patients, CSPG4 protein expression was found on 86% of samples. Furthermore, CSPG4 expression was demonstrated in U2OS, MG63, HOS, HOS-MNNG and 143B osteosarcoma cell lines. CSPG4 protein expression was successfully deleted in 143B cells using CRISPR/Cas9 technology. Two stable CSPG4-negative cell lines were produced. CSPG4 expression was then reintroduced into negative cell lines, as well as the parental 143B cell line. This created a panel of 6 cell lines with differing CSPG4 expression. Furthermore, siRNA treatment of U2OS, MG63, 143B and U87MG cell lines reduced CSPG4 expression. These cells provided another panel with varying CSPG4 expression for in vitro investigation. In vitro experiments failed to demonstrate a role for CSPG4 in osteosarcoma tumorigenesis. The CRISPR/Cas9 cell panel found that CSPG4 expression did not influence cell proliferation, adhesion and spreading on fibronectin or collagen-I, cell migration, chemosensitivity or anchorage-independent growth. Similarly, the siRNA cell panel found that CSPG4 expression did not influence cell proliferation or anchorage-independent growth. In vivo experimentation did not demonstrate a role for CSPG4 in mediating osteosarcoma tumour growth or metastatic spread. Treatment with a sc-Fv antibody fragment failed to demonstrate specific toxicity of CSPG4-positive cell lines. These results indicate that CSPG4 plays no role in osteosarcoma tumour cell behaviour. However, due to its wide expression pattern it represents a viable therapeutic option for drug targeting.

Tumour cell dissemination is a major issue with the treatment of cancer, thus new therapeutic strategies which can control this process are needed. Antagonism of integrins highly expressed in tumours is one potential strategy. The integrins are transmembrane glycoprotein adhesive receptors. Two of the integrins, αVβ3 and αIIbβ3, are highly expressed in a number of tumours and induce bi-directional signalling through their interaction with extracellular matrix proteins, and growth factor receptors. Through this signalling they play an important role in a number of cellular processes that are involved in tumour dissemination such as tumour growth, migration, invasion, metastasis and angiogenesis. Dual αIIbβ3 and αVβ3 integrin antagonism will have a direct effect on β3-expressing tumour cells that leads to the inhibition of cell migration and dissemination. Furthermore, through targeting tumour cell interaction with endothelial cells and platelets, this will also lead to inhibition of angiogenesis and metastasis. The aim of this project was to characterise the expression of αVβ3 and αIIbβ3 integrin in a panel of tumour cell lines and in human tumour xenograft samples, and to develop and utilise cell-based models to investigate potential novel β3 antagonists. The expression of αV and β3 subunits was detected in xenograft tissue using immunoblotting techniques. A panel of cell lines of different tumour types including melanoma, prostate, breast, colon and non small cell lung carcinoma was then characterised for αVβ3 and αIIbβ3 integrin expression using immunoblotting and immunocytochemistry. Melanoma cell lines demonstrated the strongest αVβ3 expression. No αIIbβ3 integrin expression was seen in any of the cell lines evaluated. A selection of cell lines with varying αVβ3 expression were then used to develop a functional test for cell migration, the scratch wound healing assay. Migration of tumour cells that expressed αVβ3 integrin was inhibited by the known β3 antagonists, cRGDfV peptide and LM609 antibody. A panel of 12 potential novel β3 integrin antagonists was screened for cytotoxicity and activity in the validated scratch assay. ICT9055 was the most effective antagonist in inhibition of M14 cell migration as determined by the scratch assay, with an IC₅₀ of < 0.1 μM. Therefore the work presented in this thesis has established models and tools for evaluating potential novel β3 integrin antagonists, and identified a promising molecule to progress for further preclinical evaluation.

>Magister Scientiae - MSc / The plant Hypoxis hemerocallidea, also known as the African potato, is commonly used as a traditional medicine to treat diabetes in South Africa. The mechanism by which it lowers blood glucose levels is not known. The main aim of this research was to study the possible hypoglycemic effect of HH using RIN-5 F pancreatic tumor cells. To accomplish this, the study was divided into three parts: (1) to test whether exposure of RIN-5F cells to glucose and HH extract affect the cell proliferation and cell viability, (2) to test whether the HH extract have an effect on insulin secretion, and (3) to test whether the HH extract has an effect on alpha amylase and alpha glucosiadase enzyme activity. The RIN-5F cells were exposed to different concentrations of glucose (5, 10, 20, 37.5, 50, 55, 74, and 92.3 mM) for different times (1, 3, 6 and 24 hours). The RIN-5F cells were also exposed to different concentrations of HH (50, 100, 150, 200 and 500 μ/ml) for different times (1, 3, 6 and 24 hours). Cell proliferation was evaluated using crystal violet staining and cell viability was evaluated using the XTT assay. To evaluate the effect of glucose and HH on RIN-5 F cell insulin secretion the cells were exposed to HH (100 μg/ml or 500 μg/ml) and / or glucose (2 mM or 50 mM) for 30 or 90 minutes. Insulin, α-amylase activity and α-glucosidase activity were evaluated by using commercially available colorimetric assays. Enzymatic activity in the presence of HH was compared with positive controls for α-amylase activity or α-glucosidase activity. Results are expressed as means ± SEM or median. Statistical differences among groups were analyzed by analyses of variance. P < 0.05 was considered as significant. An increase in the cell viability and cell proliferation was found when RIN-5 F cells were exposed to high glucose concentrations and a high dose of HH extract for a short time period (1, 3 and 6 hours). When the cells were exposed to the HH extract over 24 hours, HH did not affect cell viability significantly. Insulin secretion from RIN-5 F cells was increased when exposed to low glucose (2 mM) or high glucose (50 mM) for 30 minutes. Insulin secretion was increased from RIN5F cells after exposure to low HH (100 μg/ml) or high HH (500 μg/ml) for 30 minutes. Exposure of RIN5-F cells to HH for 90 minutes caused a further increase in insulin secretion from (4.3±0.17 mIU/mg protein; P ≤ 0.01) in 100 μg/ml, to (7.87±0.17 mIU/mg protein; P ≤ 0.001) in 500 μg/ml. At both 30 minutes and 90 minutes, insulin secretion was significantly higher when cells where exposed to 500 μg/ml HH compared to 100 μg/ml HH. Insulin secretion by cells exposed to 2 mM glucose + 100 μg/ml HH (4.69±0.16 mIU/mg protein; P ≤ 0.001) was significantly higher than when exposed to 2 mM glucose only (2.27±0.17 mIU/mg protein), while the insulin secretion in 2 mM glucose + 500 μg/ml HH (2.56±0.17 mIU/mg protein; P > 0.05) was not significantly different from that in 2 mM glucose treated cells (2.27±0.17 mIU/mg protein). Similar results are obtained after 90 minutes. In the presence of high-glucose (50 mM), at both 30 minutes and 90 minutes, insulin secretion was significantly decreased when cells where exposed to low concentration of HH (100 μg/ml) and high concentration of HH (500 μg/ml). The HH extract produced α-amylase enzyme inhibition. The maximum inhibition was at a concentration of 10μg/ml (922±117U/ml; P ≤ 0.01). The 5 μg/ml concentrations failed to produce significant inhibition. The HH extract had significant α- glucosidase inhibitory activity at a concentration of 5μg/ml (0.12±0.3U/ml; P ≤ 0.001) or 10μg/ml (0.13±0.3U/ml; P ≤ 0.001). In conclusion, based on its ability to inhibit α-amylase and α- glucosidase activity HH has the potential to be used in control of blood glucose levels. The HH aqueous extract increased insulin secretion under our basic experimental conditions and in the presence of low glucose levels, but not at high (50 mM) glucose concentrations. Insulin secretion in the presence of different glucose concentrations, in the presence of HH, needs further investigation. It is recommended that the ability of HH to stimulate insulin secretion be evaluated at 15-20 mM glucose.

Tumour cell dissemination is a major issue with the treatment of cancer, thus new therapeutic strategies which can control this process are needed. Antagonism of integrins highly expressed in tumours is one potential strategy. The integrins are transmembrane glycoprotein adhesive receptors. Two of the integrins, αVβ3 and αIIbβ3, are highly expressed in a number of tumours and induce bi-directional signalling through their interaction with extracellular matrix proteins, and growth factor receptors. Through this signalling they play an important role in a number of cellular processes that are involved in tumour dissemination such as tumour growth, migration, invasion, metastasis and angiogenesis. Dual αIIbβ3 and αVβ3 integrin antagonism will have a direct effect on β3-expressing tumour cells that leads to the inhibition of cell migration and dissemination. Furthermore, through targeting tumour cell interaction with endothelial cells and platelets, this will also lead to inhibition of angiogenesis and metastasis. The aim of this project was to characterise the expression of αVβ3 and αIIbβ3 integrin in a panel of tumour cell lines and in human tumour xenograft samples, and to develop and utilise cell-based models to investigate potential novel β3 antagonists. The expression of αV and β3 subunits was detected in xenograft tissue using immunoblotting techniques. A panel of cell lines of different tumour types including melanoma, prostate, breast, colon and non small cell lung carcinoma was then characterised for αVβ3 and αIIbβ3 integrin expression using immunoblotting and immunocytochemistry. Melanoma cell lines demonstrated the strongest αVβ3 expression. No αIIbβ3 integrin expression was seen in any of the cell lines evaluated. A selection of cell lines with varying αVβ3 expression were then used to develop a functional test for cell migration, the scratch wound healing assay. Migration of tumour cells that expressed αVβ3 integrin was inhibited by the known β3 antagonists, cRGDfV peptide and LM609 antibody. A panel of 12 potential novel β3 integrin antagonists was screened for cytotoxicity and activity in the validated scratch assay. ICT9055 was the most effective antagonist in inhibition of M14 cell migration as determined by the scratch assay, with an IC50 of < 0.1 µM. Therefore the work presented in this thesis has established models and tools for evaluating potential novel β3 integrin antagonists, and identified a promising molecule to progress for further preclinical evaluation. / Public Authority for Applied Education and Training (PAAET)

This study aimed to investigate the pharmacology of selected pyrrolobenzodiazepine (PBD) compounds shown to have cytotoxic activity with predicted DNA sequence selectivity. Research focused upon the PBD dimer, SJG-136, selected for clinical trials, and the novel PBD monomer compounds D709119, MMY-SJG and SJG-303. SJG-136, a novel sequence-selective DNA minor groove cross-linking agent, was shown to have potent tumour cell type selective cytotoxicity in in vitro assays. Pharmacokinetic studies in mice via both the i.p. and i.v. route (dosed at the maximum tolerated dose (MTD)) showed that SJG-136 reaches concentrations in plasma well in excess of the in vitro IC50 values for 1 h exposure, and was detected in tumour and brain samples also above the in vitro IC50 values. Furthermore, SJG-136 showed linear pharmacokinetics over a 3-fold drug dose range. Metabolism studies showed SJG-136 is readily metabolised in vitro by hepatic microsomes, predominantly to a monodemethylated metabolite; this metabolite could be detected in vivo. Analytical method development work was also conducted for the imminent Phase I clinical trial of SJG-136 resulting in a sensitive and selective bio-analytical detection protocol. Comet analysis showed that SJG-136 dosed at the MTD and ⅓MTD causes significant interstrand DNA cross-linking in lymphocytes in vivo. In vitro studies demonstrated that SJG-136 localises within the cell nucleus, and acts to disrupt cell division via a G2/M block in the cell cycle at realistic concentrations and exposure times that are achievable in vivo. In vivo pharmacokinetic studies of D709119 showed the compound is easily detectable in mouse plasma following i.p. dosing at the MTD, but could not be detected in either tumour or brain samples. In vitro cytotoxicity studies revealed D709119 to have potent activity across a selection of tumour cell lines. SJG-136, D709119, MMY-SJG, SJG-303 and DC-81 demonstrated a non-enzyme-catalysed reactivity with the biologically relevant thiol, reduced glutathione (GSH). Studies demonstrated that reactivity of the PBD compounds toward GSH was dependent on GSH concentrations. At levels of GSH found in plasma, the PBD compounds showed considerably lower reactivity with GSH than at intracellular GSH levels. SJG-136 and D709119 also showed favourable pharmacokinetic profiles in mice, and warrant further study for anti-tumour activity in vivo and progression to use in patients.