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.

'I am smart and I am not joking': Aiming high in the middle years of schooling

In this paper, we draw on accounts from students to inform a Middle Schooling movement that has been variously described as "arrested", "unfinished" and "exhausted". We propose that if the Middle Schooling movement is to understand the changing worlds of students and develop new approaches in the middle years of schooling, then it is important to draw on the insights that individual students can provide by conducting research with "students-as-informants". The early adolescent informants to this paper report high hopes for their futures (despite their lower socio-economic surroundings), which reinforces the importance of supporting successful learner identities and highlights the role of schooling in the decline of adolescent student aspirations. However, their insights did not stop at the individual learner, with students also identifying cultural and structural constraints to reform. As such, we argue that students may be both an important resource for inquiry into individual school reform and for the Middle Schooling movement internationally.

Exploring the Pro‐Am interface between production and produsage

This conference celebrates the passing of 40 years since the establishment of the Internet (dating this, presumably, to the first connection between two nodes on ARPANET in October 1969). For a gathering of media scholars such as this, however, it may be just as important not only to mark the first testing of the core technologies upon which much of our present‐day Net continues to build, but also to reflect on another recent milestone: the 20th anniversary of what is today arguably the chief interface through which billions around the world access and experience the Internet – the World Wide Web, launched by Tim Berners‐Lee in 1989.

Stripping Sight Bare : Alison Jones’ ‘Portraits by Proxy’

In this paper I analyse UK artist Alison Jones’ sonic interventions Portrait of the Artist by Proxy (2008), Voyeurism by Proxy (2008) and Art, Lies and Audio Tapes (2009). In Portrait of the Artist by Proxy, Jones – who, due to deteriorating vision, has not seen her reflection in a mirror in years – asks and trusts participants to audio-describe her own image back to her. In Voyeurism by Proxy, Jones asks participants to audio-describe erotic drawings by Gustav Klimt. In Art, Lies and Audio Tapes, Jones asks participants to audio-describe other artworks, such as W.F. Yeames’ And When Did You Last see Your Father?. In these portraits by proxy, Jones opens her image, and other images, to interpretation. In doing so, Jones draws attention to the way sight is privileged as a mode of access to fixed, fundamental truths in Western culture – a mode assumed to be untainted by filters that skew perception of the object. “In a culture where vision is by far the dominant sense,” Jones says, “and as a visual artist with a visual impairment, I am reliant on audio-description …Inevitably, there are limitations imposed by language, time and the interpreter’s background knowledge of the subject viewed, as well as their personal bias of what is deemed important to impart in their description” . In these works, Jones strips these background knowledges, biases and assumptions bare. She reveals different perceptions, as well as tendencies or censor, edit or exaggerate descriptions. In this paper, I investigate how, by revealing unconscious biases, Jones’ works renders herself and her participants vulnerable to a change of perception. I also examine how Jones’ later editing of the audio-descriptions allows her to show the instabilities of sight, and, in Portrait of the Artist by Proxy, to reclaim authorship of her own image.

Gifted adolescents : addressing the 'Who Am I?' question

Despite the complication giftedness can add to the task of developing a personal sense of self during early adolescence little qualitative study is done with this group. This paper reports on a study that invited gifted young adolescents to author about their perceptions of themselves and their lives. The study used digital writing in the form of journal entries delivered by email to generate personal narratives from 12 participants over a 6 month period. With the researcher acting as an empathic listener/responder participants were supported in the expression of their thoughts and observations as authors about themselves and their lives. Findings suggest that the opportunity to self-reflect and to self express in the form of digital writing can offer a positive pathway to growth in self-understanding among gifted young adolescents. Furthermore, the involvement of an adult as an interested and responsive listener in an email relationship appears to facilitate a synergy for healthy self-disclosure.

Exploring the Pro-Am interface between production and produsage

This conference celebrates the passing of 40 years since the establishment of the Internet (dating this, presumably, to the first connection between two nodes on ARPANET in October 1969). For a gathering of media scholars such as this, however, it may be just as important not only to mark the first testing of the core technologies upon which much of our present-day Net continues to build, but also to reflect on another recent milestone: the 20th anniversary of what is today arguably the chief interface through which billions around the world access and experience the Internet – the World Wide Web, launched by Tim Berners-Lee in 1989.

Elucidation of double bond position in unsaturated lipids by ozone electrospray ionization mass spectrometry

The position(s) of carbon-carbon double bonds within lipids can dramatically affect their structure and reactivity and thus has a direct bearing on biological function. Commonly employed mass spectrometric approaches to the characterization of complex lipids, however, fail to localize sites of unsaturation within the molecular structure and thus cannot distinguish naturally occurring regioisomers. In a recent communication \[Thomas, M. C.; Mitchell, T. W.; Blanksby, S. J. J. Am. Chem. Soc. 2006, 128, 58-59], we have presented a new technique for the elucidation of double bond position in glycerophospholipids using ozone-induced fragmentation within the source of a conventional electrospray ionization mass spectrometer. Here we report the on-line analysis, using ozone electrospray mass spectrometry (OzESI-MS), of a broad range of common unsaturated lipids including acidic and neutral glycerophospholipids, sphingomyelins, and triacylglycerols. All lipids analyzed are found to form a pair of chemically induced fragment ions diagnostic of the position of each double bond(s) regardless of the polarity, the number of charges, or the adduction (e.g., \[M - H](-), \[M - 2H](2-), \[M + H](+), \[M + Na](+), \[M + NH4](+)). The ability of OzESI-MS to distinguish lipids that differ only in the position of the double bonds is demonstrated using the glycerophosphocholine standards, GPCho(9Z-18:1/9Z-18:1) and GPCho(6Z-18:1/6Z-18:1). While these regioisomers cannot be differentiated by their conventional tandem mass spectra, the OzESI-MS spectra reveal abundant fragment ions of distinctive mass-to-charge ratio (m/z). The approach is found to be sufficiently robust to be used in conjunction with the m/z 184 precursor ion scans commonly employed for the identification of phosphocholine-containing lipids in shotgun lipidomic analyses. This tandem OzESI-MS approach was used, in conjunction with conventional tandem mass spectral analysis, for the structural characterization of an unknown sphingolipid in a crude lipid extract obtained from a human lens. The OzESI-MS data confirm the presence of two regioisomers, namely, SM(d18:0/15Z-24:1) and SM(d18:0/17Z-24:1), and suggest the possible presence of a third isomer, SM(d18:0/19Z-24:1), in lower abundance. The data presented herein demonstrate that OzESI-MS is a broadly applicable, on-line approach for structure determination and, when used in conjunction with established tandem mass spectrometric methods, can provide near complete structural characterization of a range of important lipid classes. As such, OzESI-MS may provide important new insight into the molecular diversity of naturally occurring lipids.

Olfactomedin-4 regulation by estrogen in the human endometrium requires epidermal growth factor signaling

Olfactomedin-4 (OLFM-4) is an extracellular matrix protein that is highly expressed in human endometrium. We have examined the regulation and function of OLFM-4 in normal endometrium and in cases of endometriosis and endometrial cancer. OLFM-4 expression levels are highest in proliferative-phase endometrium, and 17 beta-estradiol up-regulates OLFM-4 mRNA in endometrial explant cultures. Using the luciferase reporter under control of the OLFM-4 promoter, it was shown that both 17 beta-estradiol and OH-tamoxifen induce luciferase activity, and epidermal growth factor receptor-1 is required for this estrogenic response. In turn, EGF activates the OLFM-4 promoter, and estrogen receptor-alpha is needed for the complete EGF response. The cellular functions of OLFM-4 were examined by its expression in OLFM-4-negative HEK-293 cells, which resulted in decreased vimentin expression and cell adherence as well as increased apoptosis resistance. In cases of endometriosis and endometrial cancer, OLFM-4 expression correlated with the presence of epidermal growth factor receptor-1 and estrogen receptor-alpha (or estrogen signaling). An increase of OLFM-4 mRNA was observed in the endometrium of endometriosis patients. No change in OLFM-4 expression levels were observed in patients with endometrial cancer relative with controts. In conclusion, cross-talk between estrogen and EGF signaling regulates OLFM-4 expression. The role of OLFM-4 in endometrial tissue remodeling before the secretory phase and during the predisposition and early events in endometriosis can be postulated but requires additional investigation. (Am J Pathol 2010, 177:2495-2508: DOI: 10.2353/ajpath.2010.100026

Using ICD-10-AM codes to characterise hospital-acquired complications

This paper describes the limitations of using the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision, Australian Modification (ICD-10-AM) to characterise patient harm in hospitals. Limitations were identified during a project to use diagnoses flagged by Victorian coders as hospital-acquired to devise a classification of 144 categories of hospital acquired diagnoses (the Classification of Hospital Acquired Diagnoses or CHADx). CHADx is a comprehensive data monitoring system designed to allow hospitals to monitor their complication rates month-to-month using a standard method. Difficulties in identifying a single event from linear sequences of codes due to the absence of code linkage were the major obstacles to developing the classification. Obstetric and perinatal episodes also presented challenges in distinguishing condition onset, that is, whether conditions were present on admission or arose after formal admission to hospital. Used in the appropriate way, the CHADx allows hospitals to identify areas for future patient safety and quality initiatives. The value of timing information and code linkage should be recognised in the planning stages of any future electronic systems.

Varying sun protection of young children by migrant and Australian-born mothers

OBJECTIVE: To compare sun protection by Australian-born and migrant mothers of three-year-old children. METHODS: Australian-born and migrant mothers taking part in the Environments for Healthy Living prospective birth-cohort study were asked standard questions about their child's sun protection. Children were given a skin cancer susceptibility score based on grandparents' ethnic origin. Logistic regression was used to estimate odds ratios (ORs) to measure the association of sun protection of children according to mothers' migrant status adjusted for socio-demographic characteristics. RESULTS: A total of 613 Australian-born and 224 migrant mothers of three-year-old children were studied. Mothers who had migrated less than four years ago were more likely to allow their three-year-old to spend more than two hours outdoors between 10 am and 3 pm compared to Australian-born mothers (OR=2.80, 95%CI 1.20-6.57). Mothers from high latitude countries (>45 degrees) were more likely to apply sunscreen to their child than those from lower latitude countries (OR=3.15, 95%CI 1.03-9.61). CONCLUSIONS AND IMPLICATIONS: Strategies should aim to increase general awareness about the need for sun protection of young children, and recent migrants should be alerted to the harms of excessive sun exposure.

“I understand. I am a participant”: Navigating the ‘fuzzy’ boundaries of visual methods in qualitative longitudinal research

In this chapter we discuss how utilising the participatory visual methodology, photovoice, in an aged care context with its unique communal setting raised several ‘fuzzy boundary’ ethical dilemmas. To illustrate these challenges, we draw on immersive field notes from an ongoing qualitative longitudinal research (QLR) exploring the lived experience of aged care from the perspective of older residents, and focus on interactions with one participant, 81 year old Cassie. We explore how the camera, which is integral to the photovoice method, altered the researcher/participant ethical dynamics by becoming a continual ‘connector’ to the researcher. The camera took on a distinct agency, acting as a non-threatening ‘portal’ that lengthened contact, provided informal opportunities to alter the relationship dynamics and enabled unplanned participant revelation.

Now that I have had this experience I am looking at studying…

The Community Aspirations Program in Education (CAP-ED) was started by CQUniversity’s Office of Indigenous Engagement in 2013. CAP-ED’s aim was to focus on building aspirations through small manageable learning projects, and to increase Aboriginal and Torres Strait Islander students’ participation in higher education. Initially the scope of the project was to develop and deliver an accredited certificate-level program to help Indigenous students transition into tertiary education by a) improving pathways and b) addressing their current knowledge gaps. However, the initial investigatory process and community consultation found that a more localised, targeted and intimate approach would work more effectively. In addition, a free or affordable certificate course that would meet community needs was beyond the financial scope of the project. From here, the Office of Indigenous Engagement began to explore other possibilities.

Who am I? Developing pre-service teacher identity in a digital world

The notions of identity and teacher education have attracted considerable research over the years, revealing a strong correlation between teacher beliefs and practices and the resultant impact on pedagogical practices in the classroom. In an era where the use of digital technologies should be synonymous with teacher pedagogical practices and transforming education, there is a growing need for pre-service teachers to develop an identity that resonates with pedagogical practices that engage and connect with students in a positive and productive way. With many educational institutions also mandating that educators use digital technologies as a tool to support and enhance teaching, pre-service teacher education needs to ensure that students understand and develop a positive identity within this digital world. Current literature acknowledges that many educators adopt digital technologies in the classroom without sometimes fully understanding its scope or impact. It is within this context that this paper reports on a three-year study of first year pre-service education students and their understanding of identity in a digital world. More specifically, the study identifies how students currently use social and digital media in their personal and professional lives to identify themselves online in order to promote a positive image. The study also seeks to identify how these technologies and an understanding of identity can be utilised to promote a positive first year experience.