published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Prostate cancer is the second leading cancer in men. A large amount of polyamines are synthesised in the human prostate and are involved in prostate cell growth and its physiological functions. The content of intracellular polyamines is closely related to cell growth. An increase in cell growth is accompanied by a rise of intracellular polyamine content, and a depletion of intracellular polyamine pools can cause growth arrest or cell death. Therefore, maintaining polyamine concentrations is critical to the cell. Spermidine/spermine N1-acetyltransferase (SSAT) is the first and rate-limiting enzyme in the polyamine catabolic pathway. SSAT gene is highly inducible, with many stimuli including polyamine analogues and some anticancer drugs producing dramatic increases in activity. Many studies have focussed on polyamine analogues as inducers of SSAT activity as increases in SSAT are associated with a growth inhibition in many tumour cells. However, the mechanisms of this inhibition are not fully understood with respect to polyamine content. Additionally, in vivo results in SSAT transgenic mice studies are contradictory. For example, prostate carcinogenesis is reduced in TRAMP mice but Apcmin/+ mice show a promoted intestinal tumorigenesis. It is thus necessary to characterise the regulation of polyamine content and metabolism by SSAT in prostate cancer cells. The aim of the present study was to characterise the role of SSAT in both the growth of LNCaP prostate carcinoma cells and the response of these cells to anticancer drugs. Our hypothesis is that increased SSAT activity will inhibit cell growth and that this is associated with a decrease of intracellular polyamine pools. Furthermore, if SSAT induction is an essential part of the response of cancer cells to anticancer drugs, then altered SSAT activity should affect sensitivity of the cells to the drugs. The present study used a cell culture model of human prostate cancer: LNCaP wild type (WT) and SSAT cDNA transfected prostate carcinoma cell lines. The expression of SSAT in the transfected cell line (SSAT- & SSAT+) was controlled through the “Tet-off” system. This model system provided a background for comparison of effects under basal (WT), low (SSAT-), and high (SSAT+) SSAT activity. Due to our interest in acetylpolyamine derivatives and their low concentrations in cells, a new method for quantifying polyamine concentrations was developed using liquid chromatography-mass spectrometry (LC-MS). This method was highly sensitive and can detect polyamines about 250 fold lower than HPLC, as well as N-acetylpolyamines and N1,N12-diacetylspermine. In addition, a variety of methods were utilised to measure cell growth, enzyme activity, protein expression, polyamine efflux and apoptosis, which includes enzyme assays, western blot, radiochemical labelled assays, flow cytometry, spectrophotometry and fluorescent microscopy. A stable increase in SSAT activity was inhibitory to the cell growth. This inhibition was associated with significant changes in the activity of the polyamine pathway. The alterations included an increase in ODC, APAO, and SMO activity; an accumulation of intracellular N1-acetylspermidine and putrescine; a decrease in intracellular spermidine and spermine; an increased polyamine flux and efflux; and an increase in apoptosis. Combination treatment to the cells with DFMO and MDL72527 partially restored the growth of SSAT+ cells. The original contribution of this study to the field is that the cells with a higher SSAT activity are less sensitive to aspirin and 5-FU, and the sensitivity increased while the overexpressed SSAT activity decreased. The growth inhibition was associated with a depletion of total intracellular polyamine pools by the drug treatments. Moreover, to our knowledge, it is first time that the extracellular polyamine concentrations were quantified by LC-MS in human tumour cells. Overall, an increase in SSAT activity led to an inhibition of prostate cancer cell growth, and vice versa. Thereby, this study suggests that SSAT is a potential target for novel drug discovery for cancer chemotherapy or chemoprevention. For example, a combination treatment could be designed that acts as an inducer of SSAT activity in tumour cells, leading to an inhibition of the cell growth in the first place and increased sensitivity to cytotoxic agents. This would then be followed by an agent to decrease SSAT activity when the sensitivity of cancer cells to the cytotoxic treatment was optimal.

The proliferation and differentiation of normal prostate epithelial cells depend upon the action of the androgens, testosterone and dihydrotestosterone. Prostate cancer cells retain the ability to respond to androgens in the initial stages of cancer development but progressively become independent of exogenous androgens in advanced stages of the disease while maintaining the expression of functional androgen receptor (AR). We hypothesized that prostate cancer cells in the advanced stages of the disease acquire capability to synthesize androgens which activate AR in an intracrine manner. To test this hypothesis, we determined the expression of proteins and enzymes involved in cholesterol uptake, transport and its conversion into testosterone in androgen-dependent and androgen-independent prostate cancer cell lines. Established androgen independent prostate cancer cell lines, PC3 and DU145 cells, expressed mRNA and proteins for scavenger receptor type B 1 (SRB 1), steroidogenic acute regulatory (StAR) protein, metastatic lymph node 64 (MLN64), cytochrome P450 cholesterol side chain cleavage (CYP11), and other enzymes involved in androgen biosynthesis. Expression of all these proteins and enzymes was significantly higher in the androgen-independent derivative of LNCaP prostate cancer cells (C81) than in the androgen-dependent cell line (C33). In serum free cultures, the androgen independent cell line C81 secreted ~5 fold higher testosterone than C33 as determined in the conditioned media by specific immunoassays. These cells also converted radioactive cholesterol into testosterone which was identified by thin layer chromatography. To evaluate the effects of endogenous production of testosterone on the survival and growth of prostate cancer cells, C81 cells were treated with either aminoglutethimide to prevent conversion of cholesterol to pregnenolone or the AR antagonist, bicalutamide. Our results demonstrated growth inhibition ofC8l cells and a reduction in expression of the AR regulated genes, PSA and TMPRSS2 in aminoglutethimide and bicalutaniide treated cells which were reversed by exogenous androgens. These results indicate that prostate cancer cells in advanced stages of the disease synthesize androgens from cholesterol. These androgens activate AR in an intracrine maimer to regulate cell survival and proliferation. The ability of the prostate to synthesize androgens may be associated with progression to castration resistant prostate cancer and possibly represents a therapeutic target for advanced disease.

Cancer metastasis is a multi–step process that initiates with a tumor cell obtaining the ability to migrate. A multitude of changes occur in such a cell including changes to cell adhesion molecules such as integrins. In cancer cells, integrins are known to be involved in migration, invasion and metastasis. Investigation by our group of the α6 integrin led to the discovery of a cleaved form of the integrin lacking the ligand binding domain, called α6p. While it is known that the integrin is cleaved by urokinase plasminogen activator (uPA) little is known about how this process is regulated. There is a need to better understand the players involved in regulation of α6 cleavage as inhibiting this event from occurring may contribute to prolonged or increased patient survival or ultimately a cure.The existence of the integrin–actin complex has been known for many years. In this study actin was identified as a potential regulator of α6 cleavage. Using a diverse set of tumor cell lines (DU145, PC3 and MDA–MB–231) and a number of actin modifing compounds (latrunculin A, jasplakinolide and siRNA) it is reported here that disassembling actin filaments leads to an increase in α6p production. Although the increase in cleavage product did not always correlate with an increase in uPA receptor, an increase in uPAR was observed when actin was complexed by small molecule inhibitors. Taken together the results demonstrate a potential role for actin filaments to protect α6 integrin from uPA–uPAR induced cleavage via a multi–protein complex.

Ethonafide is an anthracene-containing derivative of amonafide that belongs to the azonafide series of anticancer agents. The lack of cross-resistance in MDR-expressing cancer cell lines and the absence of a quinone and hydroquinone moiety make ethonafide a possible less-cardiotoxic replacement for existing anthracene-containing anticancer agents, such as mitoxantrone and doxorubicin. For this study, we investigated the anticancer activity and mechanism of action of ethonafide in human prostate cancer cell lines. Ethonafide was cytotoxic against three human prostate cancer cell lines at nanomolar concentrations. Ethonafide was also found to be better tolerated and more effective at inhibiting tumor growth compared to mitoxantrone in DU 145 prostate cancer-bearing mice. Mechanistically, we found that ethonafide inhibits topoisomerase II activity in human prostate cancer cell lines and equally inhibits purified topoisomerase IIα and recombinant topoisomerase IIβ. The inhibition of topoisomerase II activity was due to stabilization of the cleavable complex, involving both topoisomerase IIα and β. By creating stable DU 145 cell lines with decreased expression of either topoisomerase IIα or β, we found that topoisomerase IIα is necessary for ethonafide-induced cytotoxicity. The decrease in sensitivity to ethonafide was due to a decrease in DNA damage and an increase in DNA repair as measured by the neutral comet assay. Additionally, ethonafide induces potent G₂ cell cycle arrest in the DU 145 human prostate cancer cell line. Ethonafide also induces apoptosis as measured by procaspase and PARP cleavage. In conclusion, we have identified ethonafide as a topoisomerase II poison and determined that it is topoisomerase IIα-specific in the DU 145 human prostate cancer cell line. Due to ethonafide’s activity in vitro and in vivo, decreased toxicity in mice compared to mitoxantrone, and its activity in multi-drug resistant cancer cell lines, ethonafide may be a suitable replacement to mitoxantrone for the treatment of hormone-refractory prostate cancer.

Maintaining reproductive health and diagnosing and treating conditions of infertility in stud dogs is important in canine theriogenology. However, there is still a great deal to be learned about reproductive physiology and factors that affect reproductive organs and semen quality in dogs. This thesis is an investigation of two factors in the male dog; serum 25-hydroxy Vitamin D (25OHVD) and prolactin (PRL) concentrations, and their possible associations with benign prostatic hyperplasia (BPH), prostate volume and/or sperm morphology and motility characteristics. 28 (Vitamin D Study) and 29 (28 plus one for the Prolactin study) client dogs of various breeds from the Ontario Veterinary College and Graham Animal Hospital in Southwestern Ontario, Canada were enrolled in the study from March to December 2009. Of these dogs 22 were successfully collected for semen. BPH was diagnosed using prostate volume measured by ultrasound, as well as clinical signs including blood in the ejaculate. Semen analysis was performed using manual microscopic techniques for morphology and computer assisted sperm analysis equipment for motility. In the vitamin D study, no associations were found between BPH and serum 25OHVD concentrations. In contrast, several sperm motility (motility, progressive motility, beat cross frequency (BCF), distance average path (DAP), curvilinear distance (DCL), linear distance (DSL), average path velocity (VAP), curvilinear velocity (VCL) and straight line velocity (VSL), amplitude lateral head displacement (ALH) and average orientation change (AOC)) and morphology characteristics (percentage normal sperm, head defects and detached heads) had desirable outcomes with 25OHVD concentrations between 120-180 nmol/l. Using bivariable analysis, positive associations were observed with 25OHVD and some semen quality characteristics from 4 to 8 years of age (motility, progressive motility, BCF, DCL, VCL, ALH, AOC) and at transformed prostate volumes smaller than or equal to 4.5 (motility, progressive motility, DCL, VCL, and normal morphology) while negative associations of these semen parameters were found at ages greater than 8 years and transformed prostate volumes greater than or equal to 5.5. Head defects were negatively associated with 25OHVD. Vitamin D may have an impact on spermatogenesis and normal sperm physiology that warrants further research. The prolactin study showed no statistically significant associations between serum PRL and BPH and serum PRL and sperm motility characteristics. However, two sperm morphology characteristics (percentage proximal droplets and percentage midpiece defects) had significant negative associations with PRL concentrations. Age interaction with PRL was also a factor in the percentage of midpiece defects with desirable outcomes associated at 4 years of age compared with older ages. Overall, undesirable outcomes occurred at PRL concentrations less than 2.5 ng/ml. In conclusion, both 25OHVD and PRL may have important roles in spermatogenesis and normal sperm physiology in the dog. / Ontario Veterinary College Pet Trust

Prostate cancer is the most common malignancy among men. The gold standard clinical diagnosis method for prostate cancer is histopathologic analysis of biopsy samples acquired under ultrasound guidance. However, most prostate tumors lack visually distinct appearances on medical images. Therefore, pathologically significant cases of cancer can be missed during biopsy, resulting in false negative or repeated trials. The goal of our research is to augment ultrasound-guided prostate biopsy by adding tissue typing information that can be used for targeted biopsies. As a new paradigm in tissue typing, we hypothesize and demonstrate that if a specific location in tissue undergoes sequential interactions with ultrasound, the time series of echoes, which we call radiofrequency (RF) time series, would carry ``tissue typing'' information. We provide a potential physical explanation for this phenomenon and justify it based on computer simulations of the ultrasound probe and scattering media. We also report laboratory and animal studies that illustrate the utility of the method. We rely on a set of seven spectral and fractal features extracted from RF time series for tissue typing. To show the clinical value of the proposed approach, we report an ex-vivo study involving 35 patients in which the utility of RF time series features for detection of prostate tumors is confirmed. The outcomes are validated using histopathologic disease distribution maps provided for the studied specimen. We show that the RF time series features are powerful tissue typing parameters that result in an area under receiver operating characteristic (ROC) curve of 0.87 in 10-fold cross validation for diagnosis of prostate cancer. They are significantly more accurate and sensitive than spectral features extracted from single RF frames, and also B-scan texture features (area under ROC curve of 0.78 and 0.72, respectively). A combination of these three categories of features results in a feature vector that provides an area under ROC curve of 0.95 in 10-fold cross-validation and 0.82 in leave-one-patient-out cross validation for diagnosis of prostate cancer. Using this hybrid feature vector and support vector machines, we create cancer distribution probability maps that highlight areas of tissue with high risk of cancer. / Thesis (Ph.D, Computing) -- Queen's University, 2008-09-27 07:51:11.45

Background: Quality of patient care includes both technical and non-technical elements of care, referred to as personal care. Previous work has focused on assessing the quality of technical care in prostate cancer radiotherapy, but little work has been done to assess the quality of personal care. Purpose: The purpose of this project was to create a self-administered questionnaire to measure the quality of personal care for patients undergoing radiotherapy for early-stage prostate cancer. Methods: Dimensions and candidate indicators of the quality of personal care were identified through a comprehensive literature search. The indicators were assigned to dimensions and then arranged into steps in the radiotherapy care continuum. A questionnaire was constructed using the indicators to assess patients’ views about the quality of their care as well as the importance of each indicator. Cognitive interviews were conducted with four health care professionals and eight patients to determine the clarity, comprehensiveness and appropriateness of the questionnaire. The questionnaire was then pilot tested on patients undergoing radiotherapy for early-stage prostate cancer. Results: A total of 176 indicators of the quality of personal care were initially identified representing 10 dimensions of care. Cognitive interviews identified problems with the questionnaire primarily related to the clarity and redundancy of the indicators and the appropriateness of the response categories. To reduce burden, the questionnaire was divided into three modules, corresponding to appropriate steps in the continuum of care. Each module was pilot tested on at least 10 patients with an overall response rate of 84%. Most patients responded to all indicators on the questionnaire without difficulty and without distress; however patterns of missing responses indicate a few particular indicators need revision. The results suggest that the design of the questionnaire is appropriate since patients seem to be using the range of response options that are offered. Conclusions: The results suggest that this questionnaire is feasible to administer in a clinic setting and that it does not place a large burden on patients. / Thesis (Master, Community Health & Epidemiology) -- Queen's University, 2010-12-17 07:41:09.823

Ultrasound elevation beamwidth is the out of plane thickness causing image artefacts normally appearing around anechoic areas in the medium. These artefacts could also cause uncertainties in localizing objects (such as a surgical needle) in the ultrasound image slices. This thesis studies the clinical significance of elevation beamwidth artefacts in needle insertion procedures. A new measurement device was constructed to measure the transrectal ultrasound elevation beamwidth. The beam profiles of various lateral and axial distances to the transducer were generated. It is shown that the ultrasound elevation beamwidth converges to a point within its focal zone close to the transducer. This means that the transrectal ultrasound images have the best resolution within the focal zone of the ultrasound close to the transducer. It is also shown that the ultrasound device settings have a considerable impact on the amount of beamwidth artefacts. Needle tip localization error was examined for a curvilinear transrectal ultrasound transducer. Beveled prostate brachytherapy needles were inserted through all holes of a grid template orthogonal to the axial beam axis. The effects of device imaging parameters were also investigated on the amount of localization error. Based on the developed results, it was found that the imaging parameters of an ultrasound device have direct impact on the amount of object localization error from 0.5 mm to 4 mm. The smallest localization error occurs laterally close to the center of the grid template, and axially within the beam’s focal zone. Similarly, the largest localization error occurs laterally around both sides of the grid template, and axially within the beam’s far field. Using the ultrasound device with appropriate imaging settings could minimize the effects of these artefacts. We suggest to reduce the gain setting of the ultrasound device. This will reduce the energies assigned to the off-axis beams and as a result, the elevation beamwidth artefacts are minimized. / Thesis (Master, Computing) -- Queen's University, 2011-09-01 15:27:43.098

"The central focus of this study is an investigation into how the implementation of a clinical pathway for the surgical procedure of transurethral resection of the prostate (TURP) impacted on clinical quality, cost, and patient and staff satisfaction at the Aga Khan University Hospital (AKUH) in Pakistan" / Doctor of Philosophy