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Osteosarcoma (OS) is the most common primary malignant bone tumour in humans and dogs. Although medicine has made dramatic progress in treating osteosarcoma by surgery, with chemotherapy given before and after surgery, drug resistance and highly metastatic spread are often responsible for the failure of current therapies. Thus, more effective therapeutic approaches for treating osteosarcoma are needed. Previous results from our laboratory and others had shown that the basic-leucine zipper (bLZip) containing transcription factor, Zhangfei/CREBZF is a potent inhibitor of a variety of other transcription factors and has a dramatic effect on the growth of several cancer cell lines, including dog OS and human medulloblastoma cells. The objective of the studies described in this thesis was to determine the molecular mechanisms by which Zhangfei exerts its effect on dog and human OS cells. Several stressors in the microenvironment of cancer cells directly or indirectly perturb the endoplasmic reticulum (ER), which then activates the Unfolded Protein Response (UPR). The UPR modulates the effects of stress and allows tumours to survive, develop, metastasize and escape therapy. The UPR is regulated by three bLZip transcription factors—ATF6, ATF4 and Xbp1s. Since Zhangfei inhibits Luman/CREB3, a bLZip structurally similar to and closely related to ATF6 and ATF4, I initially focused my efforts on this pathway. I hypothesized that Zhangfei interacts with UPR-related bLZip transcription factors and inhibits their ability to activate the UPR signaling pathways, thereby suppressing the growth of cancer cells and increasing their susceptibility to ER stressors. To test this hypothesis, we monitored cell growth as well as levels of UPR gene transcripts and proteins in several dog and human osteosarcoma cell lines infected with adenovirus vectors expressing Zhangfei, and studied the interactions between Zhangfei and the UPR-mediator, Xbp1s. The results showed that the ectopic expression of Zhangfei in cell lines derived from dog osteosarcomas potently suppressed cell growth and inhibited their ability to activate the UPR. Further studies demonstrated that Zhangfei inhibited the UPR, at least partially, by binding to Xbp1s and suppressing its ability to activate transcription from a promoter containing unfolded protein response elements (UPRE). The leucine zipper of Zhangfei was required for this interaction, which led to the subsequent proteasomal degradation of Xbp1s. However, we also found that the effects of Zhangfei were not universal. While Zhangfei had a profound effect on the growth and UPR in some OS cell lines, it either had only a partial effect, or no effect on others. This suggested that susceptibility (or resistance) to Zhangfei may be an inherent property of OS cell lines. Since the suppressive effects of Zhangfei were not universal, and it had no obvious effects on untransformed cells and some cancer cell lines, I proposed that Zhangfei mediates its effect on cell growth and the UPR through an intermediary that is either not induced or is defective in cells that are unaffected by Zhangfei. I found that this intermediary was the tumour suppressor protein p53. The inhibitory effects of Zhangfei were only observed in the wild-type p53 expressing OS cell line U2OS while Zhangfei had no effect on the p53-null OS cell line MG63. In cells with functional p53, the ectopic expression of Zhangfei caused it to displace the ubiquitin ligase mdm2 and stabilize p53. Suppression of p53 by siRNA partially inhibited the effects of Zhangfei on the UPR and cell growth. In contrast, OS cells lacking functional p53 could be made to respond to Zhangfei if they were transfected to express wild-type p53. These results explain why Zhangfei has a profound effect on some cancer cells while having no obvious effect on others. I also characterized the interaction of Zhangfei and p53 by mapping the interacting domains on both proteins, showing that the bLZip domain of Zhangfei and the N-terminal transactivation domain (NTD) of p53 were required for their interactions. My findings reveal the profoundly inhibitory effects of Zhangfei on OS growth and the UPR, a stress-response known to promote tumour survival. I also show how Zhangfei may exert its effects. My work suggests an alternative modality for the therapy of certain types of OS, and perhaps other tumours with functional p53.

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst stage embryo. They exhibit unlimited proliferation in culture and have the ability to differentiate into all three germ layers of the developing organism, a property defined as pluripotency. Previously it was reported that growth factor-bound protein 2 (Grb2) is required for differentiation of the epiblast, the embryonic tissue that harbours the pluripotent founder cells of the foetus. GRB2 is an adapter protein involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in response to extracellular signals. It has also been implicated in the activation of the phosphoinositol-3-kinase (PI3K) pathway in response to fibroblast growth factor (FGF) signaling. The work presented in this thesis examines the role of Grb2 in ES cells and describes previously unreported contributions of this adaptor protein in regulating ES cell growth and differentiation. It has been previously been shown by others that Grb2 deficient (Grb2-/-) cells grow relatively normally in ES growth medium containing serum. However, in serum free conditions (N2B27 medium) in this project, proliferation of Grb2-/- cells is reduced compared with wild type and “restored” Grb2-/- cells stably expressing a Grb2 cDNA mini gene. Under serum free conditions, Grb2-/- cells grow in tight, refractive colonies. Nanog expression was uniformly upregulated, in contrast to the heterogeneous pattern reported in serum-based medium. Colony expansion on the substratum appears to be compromised, although there is no apparent defect in the initial attachment of Grb2-/- cells. Cell cycle analysis indicates that the slower growth of Grb2-/- cells in serum free medium could be due to lengthening of the G1 phase of the ES cell cycle. In an attempt to identify the signalling deficiency responsible for the growth defect of Grb2-/- cells, MAPK activation was restored by two methods, PMA a ligand that bypasses the requirement for Grb2, and Raf-ER, a conditionally regulated component of the MAPK pathway that acts downstream of Grb2 in the MAPK pathway. Although both approaches increased MAPK signalling they were unable to rescue the growth defect. This suggests that MAPK is not required or alone is not sufficient. Inhibition of Glycogen synthase kinase 3 β (GSK3 β ) is known to augment growth of ES cells under MAPK inhibition. Surprisingly, GSK3 β inhibition did not enhance Grb2-/- cell growth. Under GSK3 β inhibition, Grb2-/- ES cells fail to thrive. It is hypothesised that under these conditions cells undergo hyper-self-renewal at the cost of growth. Grb2-/- ES cells are reported to exhibit limited differentiation potential. To examine the potency of Grb2-/- cells, these cells were subjected to embryoid body (EB) and monolayer differentiation. Analysis of EBs showed a loss of Gata4, Gata6 and endoderm marker gene expression. However, markers of ectoderm (Sox1, Pax6, MAP2), the late epiblast/nascent mesoderm (Brachyury) and markers associated with gastrulation (Twist and Snail) were expressed. Outgrowths of morphologically and immunohistochemically identifiable neuronal cells confirmed differentiation of ectodermal cell types, indicating Grb2 is not required for neuronal differentiation. However, beating cardiomyocytes could not be identified in Grb2-/- EBs, though readily found in restored Grb2-/- cells expressing the Grb2 cDNA. This suggests that there is an essential role for Grb2 in the mesoderm/cardiomyocyte differentiation pathway. This may be due to a defect in GATA factor expression since these factors are essential for cardiogenesis. In serum-free monolayer differentiation, Grb2-/- cells formed neuronal cells. Additional inhibition of the MAPK pathway using a small chemical inhibitor failed to prevent this differentiation. However, biochemical analysis of the cells indicates that this occurs when ERK activation is very low, indicating differentiation was not MAPK-independent. Grb2 mediates FGF-MAPK induced exit from the naïve ground state. These data suggest a Grb2-independent pathway can also facilitate this transition. Grb2 is dispensable for differentiation in to some lineages. However as differentiation of Grb2-/- ES cells is restricted, this indicates Grb2 is required for true pluripotency.

The renin-angiotensin system (RAS) is a major physiological regulator of body fluid volume, electrolyte balance and blood pressure. The principal effector peptides for this system are the angiotensins, particularly angiotensin II (Ang II), whose biological cations are mediated by specific angiotensin receptors. The use of highly specific nonpeptide angiotensin antagonists has allowed identification and characterisation of two major Ang II receptor subtypes, designated AT1 and AT2. The aims of this thesis were to investigate AT1 and AT2 receptor expression at both mRNA and protein level in freshly isolated and primary cultures of rat proximal tubular (PT) cells, and to determine the effect of culture upon Ang II receptor subtype expression. The possible role of Ang II upon receptor expression was also investigated. This study demonstrated that only the AT1 receptor is expressed in freshly isolated rat PT cells, with no evidence for AT2 receptor expression. Although continuously present throughout the culture period, a significant decrease in AT1 receptor expression was observed with time. Conversely AT2 receptor expression was absent in freshly isolated cells but was observed after 24 hours in culture with expression then remaining stable throughout culture. Clearly Ang II receptor expression is not stable during culture. Primary cultures of rat PT cells exhibit a change in receptor expression similar to those observed in vivo following tissue damage and repair, with an increase in AT2 receptor expression possibly mediated by locally released Ang II. Initial studies however, involving incubation of rat PT cells with exogenous Ang II, have demonstrated no effect upon AT1 receptor mRNA expression.

Aryl hydrocarbon receptor nuclear translocator (ARNT) is an important player in xenobiotic and hypoxic responses. In addition to this, my mentor has shown that ARNT is an integral cofactor of NF-kB signaling. However, these initial observations of ARNT-mediated NF-kB modulation were based on simultaneous suppression of the two ARNT isoforms, isoform 1 and 3, and therefore precluded the isolated examination of each isoform’s function. We show here that lymphoid malignancies exhibit higher levels of ARNT isoform 1 compared to ARNT isoform 3. However, normal T and B lymphocytes are seen to harbor equal levels of ARNT isoform 1 and 3. We hypothesize that the increase in ARNT isoform 1 is necessary for the growth of these cancer cells as suppression of isoform 1 resulted in S-phase cell cycle arrest. These findings reveal that ARNT isoform 1 potentiates cell growth by antagonizing a p53 cell cycle inhibitory mechanism and this further suggests that ARNT targeted therapies would benefit chemotherapy regimens. / text