Depression is highly prevalent in coronary artery disease (CAD) and confers an increased risk of morbidity and mortality, yet mechanisms are unknown. Platelet activating factor (PAF) lipids are associated not only with CAD but also with inflammation, oxidative/nitrosative stress, vascular endothelial dysfunction and platelet reactivity which are proposed etiopathological mechanisms for depression. This study investigated the relationship between PAF species and depressive symptoms in 20 CAD patients. Plasma analyses were performed using electrospray ionization mass spectrometry (precursor ion scan). Primary analysis revealed no association between the potent pro-inflammatory PAF PC(O-16:0/2:0) and depressive symptoms measured by the Hamilton Depression Rating Scale [HAM-D] (F=0.405, p=0.533) or Beck Depression Inventory [BDI]-II (F=0.120, p=0.733) in a linear regression. Exploratory analyses revealed potential associations between greater PC(O-18:1/0:0) and greater HAM-D score and greater PC(O-22:6/2:0) concentrations with a greater BDI-II score. This study suggests that specific PAFs might be biomarkers for depressive symptoms in CAD patients.

Depression is highly prevalent in coronary artery disease (CAD) and confers an increased risk of morbidity and mortality, yet mechanisms are unknown. Platelet activating factor (PAF) lipids are associated not only with CAD but also with inflammation, oxidative/nitrosative stress, vascular endothelial dysfunction and platelet reactivity which are proposed etiopathological mechanisms for depression. This study investigated the relationship between PAF species and depressive symptoms in 20 CAD patients. Plasma analyses were performed using electrospray ionization mass spectrometry (precursor ion scan). Primary analysis revealed no association between the potent pro-inflammatory PAF PC(O-16:0/2:0) and depressive symptoms measured by the Hamilton Depression Rating Scale [HAM-D] (F=0.405, p=0.533) or Beck Depression Inventory [BDI]-II (F=0.120, p=0.733) in a linear regression. Exploratory analyses revealed potential associations between greater PC(O-18:1/0:0) and greater HAM-D score and greater PC(O-22:6/2:0) concentrations with a greater BDI-II score. This study suggests that specific PAFs might be biomarkers for depressive symptoms in CAD patients.

Rad/Rem/Rem2/Gem/Kir (RGK) proteins are Ras-like GTPases with diverse (and expanding) functions including: regulating cytoskeleton dynamics, cell proliferation, synaptogenesis, and inhibition of high voltage-dependent calcium (CaV) channels. Furthermore, they have tissue-specific distribution with Rem and Rad most highly expressed in the heart. Indeed, the importance of Rem and Rad in the cardiovascular system is underscored by a number of studies linking them to disease states including cardiac hypertrophy, cardiac fibrosis, and inflammation. A hallmark feature of RGK proteins is their ability to inhibit current through CaV channels (ICa) and in fact, they are recognized as the most potent endogenous inhibitors of ICa. However, how RGK proteins are regulated and what their physiological role is are unknown. Understanding these points is critical for defining the patho-physiological roles of RGK proteins. My thesis work contributes towards the RGK field on two fronts: First, we demonstrate that RGK proteins are non-canonical G-proteins in the context of their ability to undergo nucleotide regulation and second, we reveal a novel paradigm of RGK-mediated inhibition on CaV channels. In Chapters 2 and 3, we show that Rem and Rad are are non-canonical G-proteins with respect to the regulatory role of their guanine nucleotide binding pocket (GNBP). Canonical Raslike G-proteins contain a conserved G-domain that encompass a GNBP and is important for guanine nucleotide binding and hydrolysis. Since RGK proteins also possess a G-domain and GNBP as well as demonstrate bona fide nucleotide binding, it was initially thought that they were regulated in a manner similar to other Ras proteins. However, subsequent studies suggested that RGK proteins may not obey such a classical model and as a result, the regulatory role of their GNBP in the G-domain was unclear. By using a wide range of functional measurements (CaV1.2 currents, Ca2+ transients, β-subunit binding), we demonstrate that RGK proteins Rem and Rad are non-canonical G-proteins. Utilizing point mutants that abolish GTPbinding and prevent GTPase activity (RemT94N and RadS105N), we show that only some cellular functions are dependent on an operational nucleotide binding pocket while others are unperturbed. Specifically, Rem- and Rad-mediated inhibition of ICa is independent of guanine nucleotide regulation whereas protein interactions with the b-subunit of CaV channels (CaVβ) and protein stability are sensitive to nucleotide regulation. We also discover skeletal and cardiac actin to be novel binding partners of Rem. And lastly, we observe differences between the effects of Rem and Rad on their degree of ICa inhibition in cardiac myocytes. Thus, Rem and Rad are non-canonical G-proteins with respect to the regulatory role of their GNBP. In collaboration with a close colleague, Akil Puckerin, Chapter 4 reveals a novel mechanism behind RGK-mediated inhibition of ICa. Together, we show RGK proteins display different modes of inhibition against specific CaV channels and that we can utilize this property to design calcium channel blockers which inhibit CaV channels in an isoform specific manner. We demonstrate this by designing Rem and Rad mutants which have diminished CaVβ capacity, termed Rem-βNULL and Rad-βNULL, respectively. Characterization of these mutants using wholecell patch clamp experiments revealed that Rem-βNULL inhibits only CaV1.2 whereas Rad-βNULL inhibits only CaV1.2 and CaV2.2. Thus, our results describe the first genetically encoded calcium channel blocker that can selectively distinguish amongst L-type channels. Altogether, this thesis work contributes towards our understanding of RGK protein regulation function and the underlying mechanisms by which they inhibit ICa. These findings advance the field both from a mechanistic and physiological standpoint, and will be of great importance towards investigating the patho-physiological role of RGK proteins.

Copies of author's previously published articles inserted. / Includes bibliographical references. / xiii, 231, [145] leaves, [2] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to analyse the interactions and to identify synergism occurring between the combinations of adrenaline with ADP and 5-HT with ADP ; and to identify the intraplatelet mechanism involved in signalling the initiation of the enhanced aggregation response. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 1995?

Understanding the specific mechanisms regulating chemotherapeutic drug anti-cancer activities will uncover novel strategies to enhance the efficacy of these drugs in clinical settings. Activating Transcription Factor 3 (ATF3) is a stress inducible gene whose expression has been associated with survival outcomes in cancer models. This study characterizes the chemotherapeutic drugs, cisplatin and Histone Deacetylase Inhibitor (HDACi), M344 as novel inducers of ATF3 expression. Cisplatin is a DNA damaging agent widely used in various tumour types including lung, head and neck, and ovarian carcinomas. The HDAC inhibitor, SAHA, has recently been approved as a single agent in the treatment of subcutaneous T-cell lymphoma and HDACis themselves show potential for synergistic anti-cancer effects when used in combination with established chemotherapeutic drugs, including cisplatin. This study evaluates the mechanisms by which cisplatin and HDACi induce ATF3, as well as the role ATF3 plays as a mediator of cisplatin-induced cytotoxicity and the enhanced cytotoxicity between HDACi and cisplatin in combination. In this study, we demonstrate that cytotoxic doses of cisplatin and carboplatin consistently induced ATF3 expression in a panel of human tumour derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response (ISR) independent mechanism, all previously implicated in stress mediated ATF3 induction. Analysis of MAPKinase pathway involvement in ATF3 induction by cisplatin revealed a MAPKinase dependent mechanism. Cisplatin treatment, in combination with specific inhibitors to each MAPKinase pathway (JNK, ERK and p38) resulted in decreased ATF3 induction at the protein level. MAPKinase pathway inhibition led to decreased ATF3 mRNA expression and a reduction in the cytotoxic effects of cisplatin as measured by MTT cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific shRNAs also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3 -/- MEFs were shown to be less sensitive to cisplatin induced cytotoxicity as compared with ATF3+/+ MEFs. Taken together, we identified cisplatin as a MAPKinase pathway dependent inducer of ATF3 whose expression regulates in part cisplatin’s cytotoxic effects. Furthermore, we demonstrated that the HDAC inhibitor M344 was also an inducer of ATF3 expression at the protein and mRNA level in the same human derived cancer cell lines. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatment was also shown to enhance the cytotoxic effects of cisplatin in these cancer cell lines. Unlike cisplatin, the mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways. Utilizing ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) M334 induction of ATF3 was shown to depend on the presence of ATF4, a known regulator of ATF3 expression as part of the ISR pathway. HDACi treatment did not affect the level of histone acetylation associated with the ATF3 promoter as determined through Chromatin immunoprecipitation (ChIP) analysis, suggesting that ATF3 induction was not a direct effect of HDACi mediated histone acetylation. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.

Platinum-based chemotherapies are effective agents in the treatment of a wide variety of human cancers. However, patients with recurrent disease can become resistant to platinum-based chemotherapy, leading to low overall survival rates. Activating transcription factor 3 (ATF3) is a stress-inducible gene that is a regulator of cisplatin-induced cytotoxicity. ATF3 protein expression was upregulated after cytotoxic doses of cisplatin treatment in a panel of cell lines. A chromatin immunoprecipitation assay showed that upon treatment with cisplatin, ATF3 directly bound to the CHOP gene promoter and this correlated with an increase in CHOP protein expression. In a 1200 compound library screen performed on cancer cell lines, disulfiram, a dithiocarbamate drug, was identified as an enhancer of the cytotoxic effects of cisplatin. This increased cytotoxic action was likely due to disulfiram and cisplatin’s ability to induce ATF3 independently through two separate mechanisms, namely the MAPK and integrated stress pathways. Furthermore, ATF3 protein and mRNA levels were variable amongst human ovarian and lung cancer tissues, suggesting the potential for basal expression of ATF3 to be predictive of cisplatin treatment response. Thus, understanding ATF3’s role in cisplatin-induced cytotoxicity will lead to novel therapeutic approaches that could improve this drug’s efficacy.

Baz/PAR3 is a key regulator of epithelial cell polarity (ECP). To identify proteins functioning with Baz, I completed a baz genetic interaction screen by localizing 15 GFP-tagged candidates. Then I tested the role of a top candidate, dASAP (Drosophila Arf GTPase-activating protein with SH3 domain, Ankyrin repeat and PH domain), in Drosophila ECP. To determine whether dASAP might interact with polarity players, I defined the localization of dASAP throughout embryogenesis with GFP-tagged proteins and an anti-dASAP antibody. To study how loss of dASAP function affects ECP, I generated a deletion allele by imprecise P-element excision. To evaluate how each of the six domains of dASAP contributes to its localization and functions, I generated constructs deleting each domain. I found associations between dASAP, actin and the apical domain. The six domains may act redundantly to localize dASAP, although interactions between domains may affect the degree of membrane association.

Baz/PAR3 is a key regulator of epithelial cell polarity (ECP). To identify proteins functioning with Baz, I completed a baz genetic interaction screen by localizing 15 GFP-tagged candidates. Then I tested the role of a top candidate, dASAP (Drosophila Arf GTPase-activating protein with SH3 domain, Ankyrin repeat and PH domain), in Drosophila ECP. To determine whether dASAP might interact with polarity players, I defined the localization of dASAP throughout embryogenesis with GFP-tagged proteins and an anti-dASAP antibody. To study how loss of dASAP function affects ECP, I generated a deletion allele by imprecise P-element excision. To evaluate how each of the six domains of dASAP contributes to its localization and functions, I generated constructs deleting each domain. I found associations between dASAP, actin and the apical domain. The six domains may act redundantly to localize dASAP, although interactions between domains may affect the degree of membrane association.

The adaptor subunit of phosphatidylinositol 3'-kinases (PI3K), p85, is involved in many different biological processes. Recent studies have shown that one of these functions is to serve as a GTPase activating protein (GAP) towards Rab5, a small monomeric G-protein. Rab5, like other G-proteins, can bind to either GDP or GTP in vivo, assuming its inactive and active form, respectively. The p85 protein has been shown to associate with both the nucleotide-bound and nucleotide-free states of Rab5. It has also been shown that p85 associates with activated, phosphorylated platelet-derived growth factor receptors (PDGFRs) via its two SH2 domains, and that upon binding there is a conformational change in the p85 protein which leads to a derepression of p110 kinase activity. The purpose of this study was to analyze if binding of the activated PDGFR peptides to p85 affects its Rab5GAP activity, as well as to measure the binding affinity of p85 towards Rab5 in each of its nucleotide-bound states. GAP assays were performed to measure the effect that peptide analogs of both the activated and inactivated PDGFR had on p85 Rab5GAP activity, while the binding affinity of p85 towards Rab5 was measured using surface plasmon resonance. The results of this study suggest that PDGFR peptides have no significant effect on p85 Rab5GAP activity. Furthermore, p85 appears to have a higher magnitude of binding to nucleotide-associated Rab5 proteins, than nucleotide-free Rab5 proteins. It also appears that p85 forms more stable complexes with Rab5-GTP than with Rab5-GDP. These results further support previous studies that show p85 to be an important regulator of Rab5-mediated endosomal fusion and show that this activity is not regulated by binding to the activated PDGFR itself.

Understanding the specific mechanisms regulating chemotherapeutic drug anti-cancer activities will uncover novel strategies to enhance the efficacy of these drugs in clinical settings. Activating Transcription Factor 3 (ATF3) is a stress inducible gene whose expression has been associated with survival outcomes in cancer models. This study characterizes the chemotherapeutic drugs, cisplatin and Histone Deacetylase Inhibitor (HDACi), M344 as novel inducers of ATF3 expression. Cisplatin is a DNA damaging agent widely used in various tumour types including lung, head and neck, and ovarian carcinomas. The HDAC inhibitor, SAHA, has recently been approved as a single agent in the treatment of subcutaneous T-cell lymphoma and HDACis themselves show potential for synergistic anti-cancer effects when used in combination with established chemotherapeutic drugs, including cisplatin. This study evaluates the mechanisms by which cisplatin and HDACi induce ATF3, as well as the role ATF3 plays as a mediator of cisplatin-induced cytotoxicity and the enhanced cytotoxicity between HDACi and cisplatin in combination. In this study, we demonstrate that cytotoxic doses of cisplatin and carboplatin consistently induced ATF3 expression in a panel of human tumour derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response (ISR) independent mechanism, all previously implicated in stress mediated ATF3 induction. Analysis of MAPKinase pathway involvement in ATF3 induction by cisplatin revealed a MAPKinase dependent mechanism. Cisplatin treatment, in combination with specific inhibitors to each MAPKinase pathway (JNK, ERK and p38) resulted in decreased ATF3 induction at the protein level. MAPKinase pathway inhibition led to decreased ATF3 mRNA expression and a reduction in the cytotoxic effects of cisplatin as measured by MTT cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific shRNAs also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3 -/- MEFs were shown to be less sensitive to cisplatin induced cytotoxicity as compared with ATF3+/+ MEFs. Taken together, we identified cisplatin as a MAPKinase pathway dependent inducer of ATF3 whose expression regulates in part cisplatin’s cytotoxic effects. Furthermore, we demonstrated that the HDAC inhibitor M344 was also an inducer of ATF3 expression at the protein and mRNA level in the same human derived cancer cell lines. Combination treatment with M344 and cisplatin lead to increased induction of ATF3 compared with cisplatin alone. Utilizing the MTT cell viability assay, M344 treatment was also shown to enhance the cytotoxic effects of cisplatin in these cancer cell lines. Unlike cisplatin, the mechanism of ATF3 induction by M344 was found to be independent of MAPKinase pathways. Utilizing ATF4 heterozygote (+/-) and knock out (-/-) mouse embryonic fibroblast (MEF) M334 induction of ATF3 was shown to depend on the presence of ATF4, a known regulator of ATF3 expression as part of the ISR pathway. HDACi treatment did not affect the level of histone acetylation associated with the ATF3 promoter as determined through Chromatin immunoprecipitation (ChIP) analysis, suggesting that ATF3 induction was not a direct effect of HDACi mediated histone acetylation. We also demonstrated that ATF3 regulates the enhanced cytotoxicity of M344 in combination with cisplatin as evidenced by attenuation of cytotoxicity in shRNAs targeting ATF3 expressing cells. This study identifies the pro-apoptotic factor, ATF3 as a novel target of M344, as well as a mediator of the co-operative effects of cisplatin and M344 induced tumour cell cytotoxicity.