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.

Family-focused weight management program for five- to nine-year-olds incorporating parenting skills training with healthy lifestyle information to support behaviour modification

his case study aims to describe how general parenting principles can be used as part of parent-led, family-focused child weight management that is in line with current Australian Clinical Practice Guidelines. A parent-led, family-focused child weight management program was designed for use by dietitians with parents of young children (five- to nine-year-olds). The program utilises the cornerstones of overweight treatment: diet, activity, behaviour modification and family support delivered in an age-appropriate, family-focused manner. Parents participate in 16 sessions (4 parenting-focused, 8 lifestyle-focused and 4 individual telephone support calls) conducted weekly, fortnightly then monthly over six months. This case study illustrates how a family used the program, resulting in reduced degree of overweight and stabilised waist circumference in the child over 12 months. In conclusion, linking parenting skills to healthy family lifestyle education provides an innovative approach to family-focused child weight management. It addresses key Australian Clinical Practice Guidelines, works at the family level, and provides a means for dietitians to easily adopt age-appropriate behaviour modification as part of their practice.

Civics and citizenship education in the national history curriculum : conducting the same music or rehearsing an incomplete tune?

This paper raises some questions about teaching and teacher education in the social sciences in response to the decision to implement a national curriculum in Australia. In particular, it contends that the decision to focus on discipline-specific knowledge in the social sciences will not necessarily meet the hopes of the Melbourne Declaration and deliver a 21st century curriculum that prepares students for the future. In doing so, it suggests that social educators need to engage with the broader discourse and political context shaping the push for curriculum reform in Australia and makes reference to the marginalisation of civics and citizenship education in the latest draft of the Australian curriculum: History.

Sex, drugs, and deterrence : applying Stafford and Warr’s reconceptualization of deterrence theory to drug driving across the genders

A consistent finding in the literature is that males report greater usage of drugs and subsequently greater amounts of drug driving. Research also suggests that vicarious influences may be more pertinent to males than to females. Utilising Stafford and Warr’s (1993) reconceptualization of deterrence theory, this study sought to determine if the relative deterrent impact of zero-tolerance drug driving laws is disparate between genders. A sample of motorists’ (N = 899) completed a self-report questionnaire assessing participants frequency of drug driving and personal and vicarious experiences with punishment and punishment avoidance. Results show that males were significantly more likely to report future intentions of drug driving. Additionally, vicarious experiences of punishment avoidance was a more influential predictor of future drug driving instances for males with personal experiences of punishment avoidance a more influential predictor for females. These findings can inform gender sensitive media campaigns and interventions for convicted drug drivers.

Age and gender differences in ambulance utilisation in Queensland

Introduction Queensland has the highest ambulance utilisation (150 per 1000 population) in Australia and growing 4.4% annually. However, the impact of gender and age on utilisation is unknown. Methods & Materials Data on ambulance utilisation from Queensland Ambulance Service for the period 2002-2009 were analysed. Results Between 2002 and 2009, the number of ambulance patients per 1000 population increased overall by 17% (females) and 18% (males). The utilisation rate remained highest among the elderly but grew differently across age groups. For females, the rates were 55% (0-14yo), 73% (15-29yo), 38% (30-44yo), 22% (45-59yo), -9% (60-74yo) and -6% (75,+ yo); for males they were 48%, 59%, 38%, 17%, -13% and -2% respectively. Within the same age groups and period, the population adjusted number of males per 100 females (M:F ratio) changed from 134 to 128 (-5% growth), 98 to 91 (-8%), 101 to 100 (-0.4%), 115 to 111 (-3%), 114 to 108 (-5%) and 106 to 111 (4%). Conclusion Understanding the impact of patients’ demographic profiles on service utilisation and broader effects on the emergency health system is imperative for policy-making, demand management, designing public health campaigns and health promotions. Gender and age characteristics of ambulance users in Queensland appear to be changing most noticeably in the youngest and oldest groups. Physical and mental health, attitudinal, lifestyle, parenting, financial and socio-cultural reasons may account for these trends, but little evidence exists. A theoretical framework will be discussed to contextualise the findings.

Out of time: The moral temporality of sex, crime and taboo

This paper discusses the relationship between law and morality. Morality does not necessarily coincide with the law, but it contributes to it. An act may be legal but nevertheless considered to be immoral in a particular society. For example, the use of pornography may be considered by many to be immoral. Nevertheless, the sale and distribution of non-violent, non-child related, sexually explicit material is legal (or regulated) in many jurisdictions. Many laws are informed by, and even created by, morality. This paper examines the historical influence of morality on the law and on society in general. It aims to develop a theoretical framework for examining legal moralism and the social construction of morality and crime as well as the relationship between sex, desire and taboo. Here, we refer to the moral temporality of sex and taboo, which examines the way in which moral judgments about sex and what is considered taboo change over time, and the kinds of justifications that are employed in support of changing moralities. It unpacks the way in which abstract and highly tenuous concepts such as ‘‘desire’’, ‘‘art’’ and ‘‘entertainment’’ may be ‘‘out of time’’ with morality, and how morality shapes laws over time, fabricating justifications from within socially constructed communities of practice. This theoretical framework maps the way in which these concepts have become temporally dominated by heteronormative structures such as the family, marriage, reproduction, and longevity. It is argued that the logic of these structures is inexorably tied to the heterosexual life-path, charting individual lives and relationships through explicit phases of childhood, adolescence and adulthood that, in the twenty-first century, delimit the boundaries of taboo surrounding sex more than any other time in history.

Sex, crime and morality

Over the last few decades, there has been a marked increase in media and debate surrounding a specific group of offences in modern Democratic nations which bear the brunt of the label ‘crimes against morality’. Included within this group are offences related to prostitution and pornography, homosexuality and incest and child sexual abuse. This book examines the nexus between sex, crime and morality from a theoretical perspective. This is the first academic text to offer an examination and analysis of the philosophical underpinnings of sex-related crimes and social attitudes towards them and the historical, anthropological and moral reasons for differentiating these crimes in contemporary western culture. The book is divided into three sections corresponding to three theoretical frameworks: Part 1 examines the moral temporality of sex and taboo as a foundation for legislation governing sex crimes Part 2 focuses on the geography of sex and deviance, specifically notions of public morality and the public private divide Part 3 examines the moral economy of sex and harm, including the social construction of harm. Sex, Crime and Morality will be key reading for students of criminology, criminal justice, gender studies and ethics, and will also be of interest to justice professionals.

Absence of malice: Constructing the female sex offender

It could be argued that all crimes have a general moral basis, condemned as ‘wrong’ or ‘bad’ in the society in which they are proscribed, however, there are a specific group of offences in modern democratic nations which bear the brunt of the label, crimes against morality. Included within this group are offences related to prostitution and pornography, homosexuality and incest, as well as child sexual abuse. While the places where sex and morality meet have shifted over time, these two concepts continue to form the basis of much criminal legislation and associated criminal justice responses. Offenders of sexual mores are positioned as the reviled corruptors of innocent children, the purveyors of disease, an indictment on the breakdown of the family and/or the secularisation of society, and a corruptive force (Davidson 2008, Kincaid 1998). Other types of offending may divide public and political opinion, but the consensus on sex crimes appears constant.

The Cannabis Expectancy Questionnaire for Men who have Sex with Men (CEQ-MSM): A measure of substance-related beliefs

Objective Substance-related expectancies are associated with substance use and post-substance use thoughts, feelings and behaviours. The expectancies held by specific cultural or sub-cultural groups have rarely been investigated. This research maps expectancies specific to gay and other men who have sex with men (MSM) and their relationship with substance patterns and behaviours following use, including sexual practices (e.g., unprotected anal intercourse). This study describes the development of a measure of such beliefs for cannabis, the Cannabis Expectancy Questionnaire for Men who have Sex with Men (CEQ-MSM). Method Items selected through a focus group and interviews were piloted on 180 self-identified gay or other MSM via an online questionnaire. Results Factor analysis revealed six distinct substance reinforcement domains (“Enhanced sexual experience”, “Sexual negotiation”, “Cognitive impairment”, “Social and emotional facilitation”, “Enhanced sexual desire”, and “Sexual inhibition”). The scale was associated with consumption patterns of cannabis, and in a crucial test of discriminant validity not with the consumption of alcohol or stimulants. Conclusions The CEQ-MSM represents a reliable and valid measure of outcome expectancies, related to cannabis among MSM. Future applications of the CEQ-MSM in health promotion, clinical settings and research may contribute to reducing harm associated with substance use among MSM, including HIV transmission.

The Drinking Expectancy Questionnaire for Men who have Sex with Men (DEQ-MSM): A measure of substance-related beliefs

Introduction and Aims. Alcohol expectancies are associated with drinking behaviour and post-drinking use thoughts, feelings and behaviours. The expectancies held by specific cultural or sub-cultural groups have rarely been investigated. This research maps expectancies specific to gay and other men who have sex with men (MSM) and their relationship with substance use. This study describes the specific development of a measure of such beliefs for alcohol, the Drinking Expectancy Questionnaire for Men who have Sex with Men (DEQ-MSM). Design and Methods. Items selected through a focus group and interviews were piloted on 220 self-identified gay or other MSM via an online questionnaire. Results. Factor analysis revealed three distinct substance reinforcement domains ('Cognitive impairment', 'Sexual activity' and 'Social and emotional facilitation'). These factors were associated with consumption patterns of alcohol, and in a crucial test of discriminant validity were not associated with the consumption of cannabis or stimulants. Similarities and differences with existing measures will also be discussed. Discussion and Conclusions. The DEQ-MSM represents a reliable and valid measure of outcome expectancies, related to alcohol use among MSM, and represents an important advance as no known existing alcohol expectancy measure, to date, has been developed and/or normed for use among this group. Future applications of the DEQ-MSM in health promotion, clinical settings and research may contribute to reducing harm associated with alcohol use among MSM, including the development of alcohol use among young gay men.