The expression of ADAM-9 and -10 in prostate cancer and their regulation by dihydrotestosterone, insulin-like growth Factor-1 and epidermal growth factor

Prostrate Cancer(PCa)is the most common cause of cancer death amongst Western males. PCa occurs in two distinct stages. In its early stage, growth and development is dependent primarily on male sex hormones (androgens) such as testosterone, although other growth factors have roles maintaining PCa cell survival in this stage. In the later stage of PCa development, growth and.maintenance is independent of androgen stimulation and growth factors including Insulin-like Growth Factor -1 (IGf.:·l) and Epidermal Growth Factor (EGF) are thought to have more crucial roles in cell survival and PCa progression. PCa, in its late stages, is highly aggressive and metastatic, that is, tumorigenic cells migrate from the primary site of the body (prostate) and travel via the systemic and lymphatic circulation, residing and colonising in the bone, lymph node, lung, and in more rare cases, the brain. Metastasis involves both cell migration and tissue degradation activities. The degradation of the extracellular matrix (ECM), the tissue surrounding the organ, is mediated in part by members of a family of 26 proteins called the Matrix Metalloproteases (MMPs), whilst ceil adhesion molecules, of which proteins known as Integrins are included, mediate ce11 migration. A family of proteins known as the ADAMs (A Disintegrin . And Metalloprotease domain) were a recently characterised family at the commencement of this study and now comprise 34 members. Because of their dual nature, possessing an active metaiioprotease domain, homologous to that of the MMPs, and an integrin-binding domain capable of regulating cell-cell and cell-ECM contacts, it was thought likely that members of the ADAMs family may have implications for the progression of aggressive cancers such as those ofthe prostate. This study focussed on two particular ADAMs -9 and -10. ADAM-9 has an active metalloprotease domain, which has been shown to degrade constituents of the ECM, including fibronectin, in vitro. It also has an integrin-binding capacity through association with key integrins involved in PCa progression, such as a6~1. ADAM-10 has no such integrin binding activities, but its bovine orthologue, MADM, is able to degrade coHagen type IV, a major component of basement membranes. It is likely human ADAM-10 has the same activity. It is also known to cleave Ll -a protein involved in cell anchorage activities - and collagen type XVII - which is a principal component of the hemidesmosomes of cellular tight junctions. The cleavage of these proteins enables the cell to be released from the surrounding environment and commence migratory activities, as required in metastasis. Previous studies in this laboratory showed the mRNA expression of the five ADAMs -9,- 10, -11, -15 and -17 in PCa cell lines, characteristic of androgen-dependent and androgen independent disease. These studies were furthered by the characterisation of AD AM-9, -10 and -17 mRNA regulation by Dihydrotestosterone (DHT) in the androgen-responsive cell line (LNCaP). ADAM-9 and -10 mRNA levels were elevated in response to DHT stimulation. Further to these observations, the expression of ADAM-9 and -10 was shown in primary prostate biopsies from patients with PCa. ADAM-1 0 was expressed in the cytoplasm and on the ceH membrane in epithelial and basal cells ofbenign prostate glands, but in high-grade PCa glands, ADAM-I 0 expression was localised to the nucleus and its expression levels appeared to be elevated when compared to low-grade PCa glands. These studies provided a strong background for the hypothesis that ADAM-9 and -10 have key roles in the development ofPCa and provided a basis for further studies.The aims of this study were to: 1) characterise the expression, localisation and levels, of ADAM-9 and -10 mRNA and protein in cell models representing characteristics of normal through androgen-dependent to androgen-independent PCa, as well as to expand the primary PCa biopsy data for ADAM-9 and ADAM-10 to encompass PCa bone metastases 2) establish an in vitro cell system, which could express elevated levels of ADAM-1 0 so that functional cell-based assays such as cell migration, invasion and attachment could be carried out, and 3) to extend the previous hormonal regulation data, to fully characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in the hormonal/growth factor responsive cell line LNCaP. For aim 1 (expression of ADAM-9 and -10 mRNA and protein), ADAM-9 and -10 mRNA were characterised by R T -PCR, while their protein products were analysed by Western blot. Both ADAM-9 and -10 mRNA and protein were expressed at readily detectable levels across progressively metastatic PCa cell lines model that represent characteristics of low-grade,. androgen-dependent (LNCaP and C4) to high-grade, androgen-independent (C4-2 and C4-2B) PCa. When the non-tumorigenic prostate cell line RWPE-1 was compared with the metastatic PCa cell line PC-3, differential expression patterns were seen by Western blot analysis. For ADAM-9, the active form was expressed at higher levels in RWPE-1, whilst subcellular fractionation showed that the active form of ADAM-9 was predominantly located in the cell nucleus. For ADAM-I 0, in both of the cell Jines, a nuclear specific isoform of the mature, catalytically active ADAM-I 0 was found. This isoforrn differed by -2 kDa in Mr (smaller) than the cytoplasmic specific isoform. Unprocessed ADAM-I 0 was readily detected in R WPE-1 cell lines but only occasionally detected in PC-3 cell lines. Immunocytochemistry using ADAM-9 and -10 specific antibodies confirmed nuclear, cytoplasmic and membrane expression of both ADAMs in these two cell lines. To examine the possibility of ADAM-9 and -10 being shed into the extracellular environment, membrane vesicles that are constitutively shed from the cell surface and contain membrane-associated proteins were collected from the media of the prostate cell lines RWPE-1, LNCaP and PC-3. ADAM-9 was readily detectable in RWPE- 1 and LNCaP cell membrane vesicles by Western blot analysis, but not in PC-3 cells, whilst the expression of ADAM-I 0 was detected in shed vesicles from each of these prostate cell lines. By Laser Capture Microdissection (LCM), secretory epithelial cells of primary prostate gland biopsies were isolated from benign and malignant glands. These secretory cells, by Western blot analysis, expressed similar Mr bands for ADAM-9 and -10 that were found in PCa cell lines in vitro, indicating that the nuclear specific isoforrn of ADAM-I 0 was present in PCa primary tumours and may represent the predominantly nuclear form of ADAM-I 0 expression, previously shown in high-grade PCa by immunohistochemistry (IHC). ADAM-9 and -10 were also examined by IHC in bone metastases taken from PCa patients at biopsy. Both ADAMs could be detected at levels similar to those shown for Prostate Specific Antigen (PSA) in these biopsies. Furthermore, both ADAM-9 and -10 were predominantly membrane- bound with occasional nuclear expression. For aim 2, to establish a cell system that over-expressed levels of ADAM-10, two fulllength ADAM-I 0 mammalian expression vectors were constructed; ADAM-I 0 was cloned into pcDNA3.1, which contains a CMV promoter, and into pMEP4, containing an inducible metallothionine promoter, whose activity is stimulated by the addition of CdC}z. The efficiency of these two constructs was tested by way of transient transfection in the PCa cell line PC-3, whilst the pcDNA3.1 construct was also tested in the RWPE-1 prostate cell line. Resultant Western blot analysis for all transient transfection assays showed that levels of ADAM-I 0 were not significantly elevated in any case, when compared to levels of the housekeeping gene ~-Tubulin, despite testing various levels of vector DNA, and, for pMEP4, the induction of the transfected cell system with different degrees of stimulation with CdCh to activate the metallothionine promoter post-transfection. Another study in this laboratory found similar results when the same full length ADAM-10 sequence was cloned into a Green Fluorescent Protein (GFP) expressing vector, as no fluorescence was observed by means of transient tran sfection in the same, and other, PCa cell lines. It was hypothesised that the Kozak sequence included in the full-length construct (human ADAMI 0 naturally occurring sequence) is not strong enough to initiate translation in an artificial system, in cells, which, as described in Aim 1, are already expressing readily detectable levels of endogenous ADAM-10. As a result, time constraints prevented any further progress with Aim 2 and functional studies including cell attachment, invasion and migration were unable to be explored. For Aim 3, to characterise the response of ADAM-9 and -10 mRNA and protein levels to DHT, IGF-1, DHT plus IGF-1 and EGF in LNCaP cells, the levels of ADAM-9 and -10 mRNA were not stimulated by DHT or IGF-I alone, despite our previous observations that initially characterised ADAM-9 and -10 mRNA as being responsive to DHT. However, IGF-1 in synergy with DHT did significantly elevate mRNA levels ofboth ADAMs. In the case of ADAM-9 and -10 protein, the same trends of stimulation as found at the rnRNA level were shown by Western blot analysis when ADAM-9 and -10 signal intensity was normalised with the housekeeping protein ~-Tubulin. For EGF treatment, both ADAM-9 and -10 mRNA and protein levels were significantly elevated, and further investigation vm found this to be the case for each of these ADAMs proteins in the nuclear fractions of LNCaP cells. These studies are the first to describe extensively, the expression and hormonal/growth factor regulation of two members of the ADAMs family ( -9 and -1 0) in PCa. These observations imply that the expression of ADAM-9 and -10 have varied roles in PCa whilst it develops from androgen-sensitive (early stage disease), through to an androgeninsensitive (late-stage), metastatic disease. Further studies are now required to investigate the several key areas of focus that this research has revealed, including: • Investigation of the cellular mechanisms that are involved in actively transporting the ADAMs to the cell's nuclear compartment and the ADAMs functional roles in the cell nucleus. • The construction of a full-length human ADAM-10 mammalian expression construct with the introduction of a new Kozak sequence, that elevates ADAM-I 0 expression in an in vitro cell system are required, so that functional assays such as cell invasion, migration and attachment may be carried out to fmd the functional consequences of ADAM expression on cellular behaviour. • The regulation studies also need to be extended by confirming the preliminary observations that the nuclear levels of ADAMs may also be elevated by hormones and growth factors such as DHT, IGF-1 and EGF, as well as the regulation of levels of plasma membrany vesicle associated ADAM expression. Given the data presented in this study, it is likely the ADAMs have differential roles throughout the development of PCa due to their differential cellular localisation and synergistic growth-factor regulation. These observations, along with those further studies outlined above, are necessary in identifying these specific components ofPCa metastasis to which the ADAMs may contribute.

Disruption of androgen regulation in the prostate by the environmental contaminant hexachlorobenzene

Hexachlorobenzene (HCB) is a persistent environmental contaminant that has the potential to interfere with steroid hormone regulation. The prostate requires precise control by androgens to regulate its growth and function. To determine if HCB impacts androgen action in the prostate, we used a number of methods. Our in vitro cell-culture-based assay used a firefly luciferase reporter gene driven by an androgen-responsive promoter. In the presence of dihydrotestosterone, low concentrations (0.5-5 nM) of HCB increased the androgen-responsive production of firefly luciferase and high concentrations of HCB (> 10 microM) suppressed this transcriptional activity. Results from a binding assay showed no evidence of affinity between HCB and the androgen receptor. We also tested HCB for in vivo effects using transgenic mice in which the transgene was a prostate-specific, androgen-responsive promoter upstream of a chloramphenicol acetyl transferase (CAT) reporter gene. In 4-week-old mice, the proportion of dilated prostate acini, a marker of sexual maturity, increased in the low HCB dose group and decreased in the high HCB dose mice. In the 8-week-old mice, there was a significant decrease in both CAT activity and prostate weight upon exposure to 20 mg/kg/day HCB. Therefore, in vitro and in vivo data suggest that HCB weakly agonizes androgen action, and consequently, low levels of HCB enhanced androgen action but high levels of HCB interfered. Environmental contaminants have been implicated in the rising incidence of prostate cancer, and insight into the mechanisms of endocrine disruption will help to clarify their role.

The first Australian nurse practitioner census : a protocol to guide standardized collection of information about an emergent professional group

Internationally, collection of reliable data on new and evolving health-care roles is crucial. We describe a protocol for design and administration of a national census of an emergent health-care role, namely nurse practitioners in Australia using databases held by regulatory authorities. A questionnaire was developed to obtain data on the role and scope of practice of Australian nurse practitioners. Our tool comprised five sections and included a total of 56 questions, using 28 existing items from the National Nursing and Midwifery Labour Force Census and nine items recommended in the Nurse Practitioner Workforce Planning Minimum Data Set. Australian Nurse Registering Authorities (n = 6) distributed the survey on our behalf. This paper outlines our instrument and methods. The survey was administered to 238 authorized Australian nurse practitioners (85% response rate). Rigorous collection of standardized items will ensure health policy is informed by reliable and valid data. We will re-administer the survey 2 years following the first survey to measure change over time.

Inquiry-based science in a primary classroom : professional development impacting practice

The critical factor in determining students' interest and motivation to learn science is the quality of the teaching. However, science typically receives very little time in primary classrooms, with teachers often lacking the confidence to engage in inquiry-based learning because they do not have a sound understanding of science or its associated pedagogical approaches. Developing teacher knowledge in this area is a major challenge. Addressing these concerns with didactic "stand and deliver" modes of Professional Development (PD) has been shown to have little relevance or effectiveness, yet is still the predominant approach used by schools and education authorities. In response to that issue, the constructivist-inspired Primary Connections professional learning program applies contemporary theory relating to the characteristics of effective primary science teaching, the changes required for teachers to use those pedagogies, and professional learning strategies that facilitate such change. This study investigated the nature of teachers' engagement with the various elements of the program. Summative assessments of such PD programs have been undertaken previously, however there was an identified need for a detailed view of the changes in teachers' beliefs and practices during the intervention. This research was a case study of a Primary Connections implementation. PD workshops were presented to a primary school staff, then two teachers were observed as they worked in tandem to implement related curriculum units with their Year 4/5 classes over a six-month period. Data including interviews, classroom observations and written artefacts were analysed to identify common themes and develop a set of assertions related to how teachers changed their beliefs and practices for teaching science. When teachers implement Primary Connections, their students "are more frequently curious in science and more frequently learn interesting things in science" (Hackling & Prain, 2008). This study has found that teachers who observe such changes in their students consequently change their beliefs and practices about teaching science. They enhance science learning by promoting student autonomy through open-ended inquiries, and they and their students enhance their scientific literacy by jointly constructing investigations and explaining their findings. The findings have implications for teachers and for designers of PD programs. Assertions related to teaching science within a pedagogical framework consistent with the Primary Connections model are that: (1) promoting student autonomy enhances science learning; (2) student autonomy presents perceived threats to teachers but these are counteracted by enhanced student engagement and learning; (3) the structured constructivism of Primary Connections resources provides appropriate scaffolding for teachers and students to transition from didactic to inquiry-based learning modes; and (4) authentic science investigations promote understanding of scientific literacy and the "nature of science". The key messages for designers of PD programs are that: (1) effective programs model the pedagogies being promoted; (2) teachers benefit from taking the role of student and engaging in the proposed learning experiences; (3) related curriculum resources foster long-term engagement with new concepts and strategies; (4) change in beliefs and practices occurs after teachers implement the program or strategy and see positive outcomes in their students; and (5) implementing this study's PD model is efficient in terms of resources. Identified topics for further investigation relate to the role of assessment in providing evidence to support change in teachers' beliefs and practices, and of teacher reflection in making such change more sustainable.

Collaborative research to develop a specialist, post-graduate competency-based education curriculum and continuing professional development structure for health librarians in Australia

Through a grant received from the Australian Library and Information Association (ALIA), members of Health Libraries Australia (HLA) are collaborating with a researcher/educator to conduct a twelve month research project with the goal of developing an educational framework for the Australian health librarianship workforce of the future. The collaboration comprises the principal researcher and a representative group of practitioners from different sectors of the health industry who are affiliated with ALIA in various committees, advisory groups and roles. The research has two main aims: to determine the future skills requirements for the health librarian workforce in Australia; and to develop a structured, modular education framework for specialist post-graduate qualifications together with a structure for ongoing continuing professional development. The paper highlights some of the major trends in the health sector and some of the main environmental influences that may act as drivers for change for health librarianship as a profession, and particularly for educating the future workforce. The research methodology is outlined and the main results are described; the findings are discussed with regard to their implications for the development of a structured, competency-based education framework.

On the endogeneity of the pay-performance relationship in professional soccer

In this reply we show that the Nüesch (2009) comment paper to our initial contribution (Torgler and Schmidt 2007) has several shortcomings. He suggests that professional soccer wages seem to buy talent rather than motivation. We therefore provide a larger set of talent proxies and estimations to check whether this assertion is correct. Our results indicate that his conclusion is problematic. We still observe a strong motivational effect, and in some cases the effect is even larger than the talent effect. A further key problem in Nüesch’s contribution is the fact that he neglects to consider the relevance of the relative salary situation.

Adherence to medicines in the older-aged with chronic conditions : does an intervention concerning adherence by an allied health professional help?

Adherence to medicines is a major determinant of the effectiveness of medicines. However, estimates of non-adherence in the older-aged with chronic conditions vary from 40 to 75%. The problems caused by non-adherence in the older-aged include residential care and hospital admissions, progression of the disease, and increased costs to society. The reasons for non-adherence in the older-aged include items related to the medicine (e.g. cost, number of medicines, adverse effects) and those related to person (e.g. cognition, vision, depression). It is also known that there are many ways adherence can be increased (e.g. use of blister packs, cues). It is assumed that interventions by allied health professions, including a discussion of adherence, will improve adherence to medicines in the older aged but the evidence for this has not been reviewed. There is some evidence that telephone counselling about adherence by a nurse or pharmacist does improve adherence, short- and long-term. However, face-to-face intervention counselling at the pharmacy, or during a home visit by a pharmacist, has shown variable results with some studies showing improved adherence and some not. Education programs during hospital stays have not been shown to improve adherence on discharge, but education programs for subjects with hypertension have been shown to improve adherence. In combination with an education program, both counselling and a medicine review program have been shown to improve adherence short-term in the older-aged. Thus, there are many unanswered questions about the most effective interventions to promote adherence. More studies are needed to determine the most appropriate interventions by allied health professions, and these need to consider the disease state, demographics, and socio-economic status of the older-aged subject, and the intensity and duration of intervention needed.

Renewable energy sources and frequency regulation : survey and new perspectives

As the use of renewable energy sources (RESs) increases worldwide, there is a rising interest on their impacts on power system operation and control. An overview of the key issues and new challenges on frequency regulation concerning the integration of renewable energy units into the power systems is presented. Following a brief survey on the existing challenges and recent developments, the impact of power fluctuation produced by variable renewable sources (such as wind and solar units) on sysstem frequency performance is also presented. An updated LFC model is introduced, and power system frequency response in the presence of RESs and associated issues is analysed. The need for the revising of frequency performance standards is emphasised. Finally, non-linear time-domain simulations on the standard 39-bus and 24-bus test systems show that the simulated results agree with those predicted analytically.