Bladder cancer, one of the most frequently diagnosed cancers, is a significant source of morbidity and mortality throughout the world. According to the American Cancer Society (2005), approximately 63,210 new cases will be diagnosed in the United States and bladder cancer will account for nearly 13,180 deaths. The current standard for detection of bladder cancer relies on cystoscopy, an invasive procedure, and cytology. Cytology has a high specificity, but lacks sensitivity in detection of low-grade tumors, as well as requires a trained pathologist for review. Because current diagnostic tools are less than optimal and because bladder cancer has a high rate of recurrence and long term monitoring is a necessity, a better diagnostic tool is needed. There is now a great interest in researching urine markers for bladder cancer. Our lab previously identified six nuclear structural proteins (BLCA 1-6) that are specifically expressed in bladder cancer tissue. The nuclear matrix is the support scaffold of the cell nucleus. This structure has a variety of functions, many of which have implications in cancer progression. The purpose of this dissertation is to examine changes in nuclear structural proteins. The hypothesis we propose is that changes in structural elements of the nucleus are involved in the progression of bladder cancer and can be developed into markers of this disease. Specifically this study had three goals. 1) to determine if BLCA-1 could be developed into a biomarker of bladder cancer, 2) to clone the gene encoding BLCA-1, and 3) to examine functional aspects of BLCA-4. A urine-based immunoassay was developed that can detect BLCA-1 in patients with bladder cancer with a specificity of 87% and sensitivity of 80%. Furthermore, this protein can be detected in serum of individuals with bladder cancer and may associate with the stage of disease. We also demonstrated that BLCA-4 can confer a growth advantage to cells over-expressing this protein. Over-expression of BLCA-4 led to many gene expression changes. BLCA-4 may play a role in bladder cancer pathobiology by altering genes that enhance proliferation and invasion, maintain blood flow for tumor cell survival, or enhance angiogenesis. Finally, we have been successful in cloning part of the cDNA that encodes for BLCA-1 and it appears to have a close homology to a novel metastasis related gene. In summary, this project has demonstrated that bladder cancer specific nuclear matrix proteins can be developed into markers of the disease and may play a functional role in bladder cancer pathobiology.

The utilization of high-throughput -omics strategies, such as proteomics, in the analysis of breast cancer will function to define central molecular characteristics across a disease that is associated with a high degree of molecular heterogeneity. Data reported herein details the investigation of key subjects in breast cancer biology focused on the characterization of endogenous and experimentally-induced disease biology characteristics utilizing the application of LC-MS based proteomic analyses of both in vitro models of breast cancer as well as primary clinical samples. Results include a combined global and functional proteomic strategy to identify governing functional roles for mutually, differentially abundant proteins observed across three divergent cell line models of breast cancer. Further, evidence is presented which provides insights into the regulatory activity of the breast cancer-associated microRNA (miR-145) in several cell line models of breast cancer in which expression of this microRNA has been restored. Lastly, robust analyses are detailed focused on the identification of differential protein characteristics indicative of disease stage as well as of recurrent disease in breast cancer derived from proteomic analysis of formalin-fixed, paraffin embedded (FFPE) clinical samples. These studies contribute to the field of proteomics in the form of 1) providing robust experimental workflows directed towards investigation of functional themes and associated functional targets in large protein data sets 2) detailing strategies for navigating the application of proteomic analysis to microRNA target discovery and 3) further development and utilization of methodologies towards the proteomic analysis of clinical, FFPE tissue samples. Furthermore, these studies benefit the breast cancer community on several fronts including 1) the elucidation of provocative protein candidates which warrant further investigation for their role in regulating disease mechanisms underlying v breast cancer biology and 2) through the discovery of diagnostic markers indicative of discrete subtypes and stages of disease progression in breast cancer. The results reported herein detail disease-specific protein abundance characteristics associated with neoplastic progression in breast cancer that will benefit further expansion of the basic biological understanding of this disease and describes novel proteins for further evaluation as biomarker candidates for the diagnosis of breast cancer.

The Phosphatase of Regenerating Liver (PRL) family, consisting of PRL-1, PRL-2, and PRL-3, is a group of prenylated phosphatases that are candidate cancer biomarkers and therapeutic targets. Individual PRLs are over-expressed in a variety of cancer cell lines and tissues, and elevated PRL expression has been associated with tumorigenesis and metastasis. Although several studies have documented that altered expression of PRL-1 or PRL-3 can influence cell proliferation, migration and invasion, there is an absence of knowledge about the biological functions of PRL-2. Thus, the current study was designed to evaluate the role of PRL-2 in cell migration and invasion in human cancer cells. I found that four lung cancer cells, including A549, over-expressed PRL-2 when compared with normal lung cells. PRL-2 suppression by siRNA or shRNA markedly inhibited cell migration and invasion. PRL-2 suppression by siRNA decreased p130Cas and vinculin expression, increased phosphorylation of Ezrin on tyrosine 146, and decreased ERK phosphorylation upon serum stimulation. There were no significant changes in total p53, Akt and c-Src expression levels or their phosphorylation status, suggesting PRL-2 suppression could inhibit tumor cell migration and invasion through a Src-independent p130Cas signaling pathway. Ectopic expression of wild type PRL-2, a catalytic inactive C101S mutant, and a C-terminal CAAX deletion revealed a requirement for both the PRL-2 catalytic functionality and prenylation site. Expression of wild type but not the mutant forms of PRL-2 caused ERK phosphorylation and nuclear translocation, and promoted tumor cell migration and invasion. These results support a model in which PRL-2 promotes cell migration and invasion through an ERK-dependent signaling pathway. In addition, thienopyridone, a previously reported PRL inhibitor, showed antiproliferative activity in a concentration-dependent manner, and decreased cell migration and invasion. In summary, these studies demonstrate for the first time that PRL-2 regulates cell migration and invasion in non-small cell lung cancer, and I propose that PRL-2 stimulates cell migration and invasion through an ERK signaling pathway.

The majority of current pharmacological treatments for cancer target rapidly dividing cells, a characteristic of most cancer cells. Unfortunately, these treatments also affect cells that normally divide at a rapid rate, such as cells of the digestive tract, hair follicles, and bone marrow, which limits the efficacy of chemotherapy due to toxic side effects. Reducing the drug dose to evade these side effects, however, often impairs efficacy and encourages drug resistance. Therefore, new unbiased approaches are required to identify new drug combinations with existing effective cancer chemotherapeutics. I therefore exploited data from a short interfering RNA (siRNA) high throughput screen targeting 5,520 unique druggable genes, which comprises gene products that are theoretically good targets for drug development. I used the siRNA screening methodology to identify novel combination chemotherapies for the treatment of glioblastoma multiforme (GBM), the most common and aggressive form of human primary brain tumors. My hypothesis is that unrecognized chemosensitivity nodes exist for the microtubule destabilizing agent vinblastine. GBM cells were treated with a sub-lethal concentration of vinblastine and identified gene products that sensitized cells to vinblastine. Using a series of statistical methods, followed by target identification assays, I found gene products that sensitized GBM cells to vinblastine, implicating siRNA screening technology as an efficient, unbiased method for identifying potentially novel anticancer treatments.

Ataxia telangiectasia-mutated (ATM) is a serine/threonine protein kinase that has critical functions in the cellular responses to DNA damage, including cell cycle checkpoint activation and DNA repair. Since ataxia telangiectasia individuals, who have homozygous mutations in ATM, are exquisitely radiosensitive there is considerable interest in inhibiting the kinase activity of ATM to increase the efficacy of targeted radiotherapy. In this dissertation work, I sought to understand the cellular responses to radiation when ATM kinase activity is transiently inhibited using the small molecule ATM kinase inhibitor KU55933. During my PhD, our laboratory has shown that transient ATM kinase inhibition one hour post-irradiation results in radiosensitization, increased chromosome aberrations and abrogation of sister chromatid exchange. I contributed to these findings by showing that the cellular radiosensitization seen in H460 cells with kinase-inhibited ATM was identical to that seen when ATM protein was disrupted using siRNA prior to the insult. In addition, I demonstrated that 15 minutes of ATM kinase activity post-irradiation is sufficient to trigger the G2/M cell cycle checkpoint, and that subsequent transient inhibition of ATM with KU55933 does not affect recovery from this checkpoint. To gain a more global view of the functional consequences of kinase-inhibited ATM following irradiation, I utilized a SILAC-based tandem mass spectrometry approach, combined with a subcellular fractionation protocol, to determine ATM kinase-dependent spatial proteome dynamics in response to radiation-induced DNA damage. Analysis of the chromatin-associated proteome revealed that the retention of 53BP1 at chromatin is decreased when the kinase activity of ATM is inhibited following ionizing radiation (IR). Using fluorescence recovery after photobleaching in live cells, I determined that the stability of IR-induced GFP-53BP1 foci is decreased when the kinase activity of ATM is inhibited following IR. These results provide a roadmap for understanding ATM kinase-dependent spatial protein dynamics in response to DNA damage.

Thesis (Ph. D.)--University of Queensland, 2003. / Includes bibliographical references.

Thesis--Ph. D., University of Hong Kong.

When aptamers emerged almost two decades ago, “selection-ologists” quickly realized the aptamer’s clinical potential - both as a diagnostic tool and as a therapeutic. Since that time, nearly hundreds of medically relevant targets have been successfully selected against. Moreover, many have proven efficacy in tissue culture and animal models. However, only one has successfully advanced to clinical use. Several key limitations in aptamer-based drugs may explain the dearth of aptamers in the pharmacy. Issues of expression level, delivery, and targeting will need to be addressed before aptamers can reach their full clinical potential. This work broaches on aspects of these limitations and leads into ways of transitioning the aptamer into clinical use. / text

The introduction of antiretroviral drugs has dramatically increased the lifespan of human immunodeficiency virus (HIV)-infected patients, shifting the major concern towards long-term morbidity and mortality, particularly an increased risk of cardiovascular complications. Antiretroviral therapy has been proposed to be one of the contributing factors. However, existing evidence for the role of antiretroviral therapy in the development of cardiovascular diseases is controversial. Therefore, in the present thesis, the effects of several antiretroviral drugs on the vascular system were investigated. In view of the contribution of vascular inflammation in the development of cardiovascular diseases, the first study examined the effects of acute treatment of efavirenz, indinavir, saquinavir, lopinavir and ritonavir on the release of major inflammatory markers, interleukin (IL)-8, soluble intercellular adhesion molecule 1 (ICAM-1) and monocyte adhesion molecule 1 (MCP-1) in human umbilical vein endothelial cells in the absence or presence of lipopolysaccharide, a pro-inflammatory stimulus. The results demonstrated that efavirenz and the combination of lopinavir and ritonavir, at concentrations present in human plasma, reduced the IL-8 release, but not that of soluble ICAM-1 and MCP-1 in endothelial cells exposed to lipopolysaccharide. The data, therefore, suggest that efavirenz and lopinavir plus ritonavir may possibly have anti-inflammatory effects. Since these findings seems to contradict with the increased incidence of cardiovascular diseases associated with antiretroviral therapy, in vivo experiments were performed to further characterized the effects of antiretroviral drugs on the cardiovascular system. In the second study, the atherogenic effects of long-term treatment (eight weeks) with efavirenz, abacavir and lamivudine, alone or in combination (as used clinically), were investigated in apolipoprotein E deficient (Apo-E-/-) mice (hyperlipidemic/atherosclerotic model) and the corresponding wild-type mice of both genders. All drug treatments had no effects on the lipid profile, nitrotyrosine expression in the liver (an indication of oxidative stress) and the degree of atherosclerotic lesions in all mice. Efavirenz and lamivudine did not have any significant effects on acetylcholine- and sodium nitroprusside-induced relaxations in all mice. Abacavir and the combination of the three drugs did not have any effects on acetylcholine-induced relaxation in aortae of wild-type mice, but impaired acetylcholine-induced relaxation in those of male Apo-E-/- mice without affecting sodium nitroprusside-induced relaxation. The reduction in relaxation was likely mediated by the cyclooxygenase pathway since indomethacin restored the reduction in relaxation. In male Apo-E-/- mice, IL-6 levels were increased by abacavir and the combined treatment, whereas serum amyloid P component (SAP) levels remained unchanged. Although no differences in the development of atherosclerotic lesions were observed, female Apo-E-/- mice receiving abacavir had better lipid profiles, no impairment in acetylcholine-induced relaxation and decreased serum IL-6 and SAP levels compared to male Apo-E-/- mice, revealing a vasculoprotective role of the female gender. In conclusion, the data suggest that certain, but not all, antiretroviral drugs may increase the risk of cardiovascular diseases, and that this risk may be exacerbated in hyperlipidemia but reduced in females. Antiretroviral drugs should be cautiously prescribed to HIV-infected patients to minimize cardiovascular adverse effects. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy

(Uncorrected OCR) Abstract Abstract of thesis entitled "A comparative study of some possible mechanisms governing the intraluminal and intravascular release of 5-HT from the rabbit jejunum" submitted by WONG Joseph Chi-yan for the degree of Doctor of Philosophy (Pharmacology) at the University of Hong Kong in April 1973. Experiments were carried out on rabbit jejunal segments in. vivo and in vitro under conditions of intraluminal and intravascular perfusion. 5-HT was released into the effluent of both intraluminal- and intravascular perfusate during perfusion with Krebs solution under quiescent conditions. Enhanced 5-HT release was observed during active peristalsis elicited by increasing the intraluminalpressure during intraluminal perfusion. The addition of hydrochloric acid in physiological amounts to give pH values of 7.2, 6, 5 and 4 in Krebs solution revealed a progressive increase in 5-HT release as the pH fell, during intraluminal perfusion. Enhanced 5-HT release was also observed during intraluminal perfusions with 0.3 M and 0.6 M sucrose solutions; 0.6 M glucose solution and 7.5% egg albumin in Krebs solution. During intraluminal perfusion, Sennosides A & BlO ~g/ml., Nicotine 1 ~g/ml. and Morphine 10 ~g/ml. in Krebs solution, all enhanced 5-HT release; but not 1% Monosodium L-glutamate. Monoamine oxidase inhibitor, Nialamide, in a concentration of 100 ~g/ml. prevented . the destruction of both endogenous and exogenous 5-HT. Nicotine 1 ~g/ml. and Morphine 10 ~g/ml. did not enhance 5-HT release during intravascular perfusion. From the above observations, it is concluded that, in rabbits, 5-HT is involved in the normal physiological functions of digestion, absorption and motility. It possibly functions as a local hormone in regulating these processes. The intravascular release of 5-HT demonstrated in these experiments may not have any direct physiologic~l significance. It is not appropriate to compare the two perfusion methods in terms of the net amount of released 5-HT. From histological observations, it is suspected that the results of intraluminal perfusion are complicated by the shedding off of intestinal epithelial cells, into the perfusing fluid. Further investigations are needed to evaluate the reliability of this method in the study of the phenomenon of 115-HT release II under such experimental conditions, and the interpretation which can be put upon the results obtained. ii / abstract / toc / Pharmacology / Doctoral / Doctor of Philosophy