Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

Implication of 3D culture system for study of prostate cancer (CaP) mediated bone metastasis

Introduction : For the past decade, three dimensional (3D) culture has served as a foundation for regenerative medicine study. With an increasing awareness of the importance of cell-cell and cell-extracellular matrix interactions which are lacking in 2D culture system, 3D culture system has been employed for many other applications namely cancer research. Through development of various biomaterials and utilization of tissue engineering technology, many in vivo physiological responses are now better understood. The cellular and molecular communication of cancer cells and their microenvironment, for instance can be studied in vitro in 3D culture system without relying on animal models alone. Predilection of prostate cancer (CaP) to bone remains obscure due to the complexity of the mechanisms and lack of proper model for the studies. In this study, we aim to investigate the interaction between CaP cells and osteoblasts simulating the natural bone metastasis. We also further investigate the invasiveness of CaP cells and response of androgen sensitve CaP cells, LNCaP to synthetic androgen.----- Method : Human osteoblast (hOB) scaffolds were prepared by seeding hOB on medical grade polycaprolactone-tricalcium phosphate (mPLC-TCP) scaffolds and induced to produce bone matrix. CaP cell lines namely wild type PC3 (PC3-N), overexpressed prostate specific antigen PC3 (PC3k3s5) and LNCaP were seeded on hOB scaffolds as co-cultures. Morphology of cells was examined by Phalloidin-DAPI and SEM imaging. Gelatin zymography was performed on the 48 hours conditioned media (CM) from co-cultures to determine matrix metalloproteinase (MMP) activity. Gene expression of hOB/LNCaP co-cultures which were treated for 48 hours with 1nM synthetic androgen R1881 were analysed by quantitative real time PCR (qRT-PCR).----- Results : Co-culture of PCC/hOB revealed that the morphology of PCCs on the tissue engineered bone matrix varied from homogenous to heterogenous clusters. Enzymatically inactive pro-MMP2 was detected in CM from hOBs and PCCs cultured on scaffolds. Elevation in MMP9 activity was found only in hOB/PC3N co-culture. hOB/LNCaP co-culture showed increase in expression of key enzymes associated with steroid production which also corresponded to an increase in prostate specific antigen (PSA) and MMP9.----- Conclusions : Upregulation of MMP9 indicates involvement of ECM degradation during cancer invasion and bone metastases. Expression of enzymes involved in CaP progression, PSA, which is not expressed in osteoblasts, demonstrates that crosstalk between PCCs and osteoblasts may play a part in the aggressiveness of CaP. The presence of steroidogenic enzymes, particularly, RDH5, in osteoblasts and stimulated expression in co-culture, may indicate osteoblast production of potent androgens, fuelling cancer cell proliferation. Based on these results, this practical 3D culture system may provide greater understanding into CaP mediated bone metastasis. This allows the role of the CaP/hOB interaction with regards to invasive property and steroidogenesis to be further explored.

Exercise for health : a breast cancer recovery program - quality of life benefits [Conference Abstract]

Introduction: Five-year survival from breast cancer in Australia is 87%. Hence, ensuring a good quality of life (QOL) has become a focal point of cancer research and clinical interest. Exercise during and after treatment has been identified as a potential strategy to optimise QOL of women diagnosed with breast cancer.----- Methods: Exercise for Health is a randomised controlled trial of an eight-month, exercise intervention delivered by Exercise Physiologists. An objective of this study was to assess the impact of the exercise program during and following treatment on QOL. Queensland women diagnosed with unilateral breast cancer in 2006/07 were eligible to participate. Those living in urban-Brisbane (n=194) were allocated to either the face-to-face exercise group, the telephone exercise group, or the usual-care group, and those living in rural Queensland (n=143) were allocated to the telephone exercise group or the usual-care group. QOL, as assessed by the Functional Assessment of Cancer Therapy-Breast (FACT-B+4) questionnaire, was measured at 4-6 weeks (pre-intervention), 6 months (mid-intervention) and 12 months (three months post-intervention) post-surgery.----- Results: Significant (P<0.01) increases in QOL were observed between pre-intervention and three months post-intervention 12 months post-surgery for all women. Women in the exercise groups experienced greater mean positive changes in QOL during this time (+10 points) compared with the usual-care groups (+5 to +7 points) after adjusting for baseline QOL. Although all groups experienced an overall increase in QOL, approximately 20% of urban and rural women in the usual-care groups reported a decline in QOL, compared with 10% of women in the exercise groups.----- Conclusions: This work highlights the potential importance of participating in physical activity to optimise QOL following a diagnosis of breast cancer. Results suggest that the telephone may be an effective medium for delivering exercise counselling to newly diagnosed breast cancer patients.

Enhancement of DNA repair using topical T4 endonuclease V does not inhibit melanoma formation in Cdk4R24C/R24C/Tyr-NrasQ61K mice following neonatal UVR

To further investigate the use of DNA repair-enhancing agents for skin cancer prevention, we treated Cdk4R24C/R24C/NrasQ61K mice topically with the T4 endonuclease V DNA repair enzyme (known as Dimericine) immediately prior to neonatal ultraviolet radiation (UVR) exposure, which has a powerful effect in exacerbating melanoma development in the mouse model. Dimericine has been shown to reduce the incidence of basal-cell and squamous cell carcinoma. Unexpectedly, we saw no difference in penetrance or age of onset of melanoma after neonatal UVR between Dimericine-treated and control animals, although the drug reduced DNA damage and cellular proliferation in the skin. Interestingly, epidermal melanocytes removed cyclobutane pyrimidine dimers (CPDs) more efficiently than surrounding keratinocytes. Our study indicates that neonatal UVR-initiated melanomas may be driven by mechanisms other than solely that of a large CPD load and/or their inefficient repair. This is further suggestive of different mechanisms by which UVR may enhance the transformation of keratinocytes and melanocytes.

Vitamin D and sun protection : the impact of mixed public health messages in Australia

Exposure of the skin to sunlight can cause skin cancer and is also necessary for cutaneous vitamin D production. Media reports have highlighted the purported health benefits of vitamin D. Our aim was to examine attitudes and behaviours related to sun protection and vitamin D. A cross-sectional study of 2,001 residents in Queensland, Australia aged 20-70 years was undertaken. Information collected included: skin cancer risk factors; perceptions about levels of sun exposure required to maintain vitamin D; belief that sun protection increases risk of vitamin D deficiency; intention, and actual change in sun protection practices for adults and children. Multivariate models examined predictors of attitudinal and behavioural change. One-third (32%) believed a fair-skinned adult, and 31% thought a child required at least 30 minutes per day in summer sun to maintain vitamin D levels. Reductions in sun protection were reported by 21% of adults and 14% of children. Factors associated with belief that sun protection may result in not obtaining enough vitamin D included aged ≥ 60 years (OR=1.35, 95% CI 1.09-1.66) and having skin that tanned easily (OR=1.96, 95% CI 1.38-2.78). Participants from low income households, and those who frequently used sun protective clothing were more likely to have reduced sun protection practices (OR=1.33, 95% CI 1.10-1.73 and OR=1.73, 95% CI 1.36-2.20, respectively). This study provides evidence of reductions in sun protection practices in a population living in a high UV environment. There is an urgent need to re-focus messages regarding sun exposure and for continued sun protection practices.

Androgen levels increase by intratumoral de novo steroidogenesis during progression of castration-resistant prostate cancer

Although systemic androgen deprivation prolongs life in advanced prostate cancer, remissions are temporary because patients almost uniformly progress to a state of a castration-resistant prostate cancer (CRPC) as indicated by recurring PSA. This complex process of progression does not seem to be stochastic as the timing and phenotype are highly predictable, including the observation that most androgen-regulated genes are reactivated despite castrate levels of serum androgens. Recent evidence indicates that intraprostatic levels of androgens remain moderately high following systemic androgen deprivation therapy, whereas the androgen receptor (AR) remains functional, and silencing the AR expression following castration suppresses tumor growth and blocks the expression of genes known to be regulated by androgens. From these observations, we hypothesized that CRPC progression is not independent of androgen-driven activity and that androgens may be synthesized de novo in CRPC tumors leading to AR activation. Using the LNCaP xenograft model, we showed that tumor androgens increase during CRPC progression in correlation to PSA up-regulation. We show here that all enzymes necessary for androgen synthesis are expressed in prostate cancer tumors and some seem to be up-regulated during CRPC progression. Using an ex vivo radiotracing assays coupled to high-performance liquid chromatography-radiometric/mass spectrometry detection, we show that tumor explants isolated from CRPC progression are capable of de novo conversion of [(14)C]acetic acid to dihydrotestosterone and uptake of [(3)H]progesterone allows detection of the production of six other steroids upstream of dihydrotestosterone. This evidence suggests that de novo androgen synthesis may be a driving mechanism leading to CRPC progression following castration.

Can tissue engineering concepts advance tumor biology research?

Advances in tissue engineering have traditionally led to the design of scaffold- or matrix-based culture systems that better reflect the biological, physical and biochemical environment of the natural extracellular matrix. Although their clinical applications in regenerative medicine tend to receive most of the attention, it is obvious that other areas of biomedical research could be well served by the powerful tools that have already been developed in tissue engineering. In this article, we review the recent literature to demonstrate how tissue engineering platforms can enhance in vitro and in vivo models of tumorigenesis and thus hold great promise to contribute to future cancer research.

Public health and clinical dilemmas resulting from imprecise vitamin D tests

Objective: To determine whether there are clinical and public health dilemmas resulting from the reproducibility of routine vitamin D assays. Methods: Blinded agreement studies were conducted in eight clinical laboratories using two commonly used assays to measure serum 25-hydroxyvitamin D (25(OH)D) levels in Australasia and Canada (DiaSorin Radioimmunoassay (RIA) and DiaSorin LIAISON® one). Results: Only one laboratory measured 25(OH)D with excellent precision. Replicate 25(OH)D measurements varied by up to 97% and 15% of paired results differed by more than 50%. Thirteen percent of subjects received one result indicating insufficiency [25-50 nmol/l] and another suggesting adequacy [>50 nmol/l]). Agreement ranged from poor to excellent for laboratories using the manual RIA, while the precision of the semi-automated Liaison® system was consistently poor. Conclusions: Recent interest in the relevance of vitamin D to human health has increased demand for 25(OH)D testing and associated costs. Our results suggest clinicians and public health authorities are making decisions about treatment or changes to public health policy based on imprecise data. Clinicians, researchers and policy makers should be made aware of the imprecision of current 25(OH)D testing so that they exercise caution when using these assays for clinical practice, and when interpreting the findings of epidemiological studies based on vitamin D levels measured using these assays. Development of a rapid, reproducible, accurate and robust assay should be a priority due to interest in populationbased screening programs and research to inform public health policy about the amount of sun exposure required for human health. In the interim, 25(OH)D results should routinely include a statement of measurement uncertainty.

Elucidating the links between UV radiation and vitamin D synthesis : using an in vitro model

Ultraviolet radiation (UV) is the carcinogen that causes the most common malignancy in humans – skin cancer. However, moderate UV exposure is essential for producing vitaminDin our skin. VitaminDincreases the absorption of calcium from the diet, and adequate calcium is necessary for the building and maintenance of bones. Thus, low levels of vitamin D can cause osteomalacia and rickets and contribute to osteoporosis. Emerging evidence also suggests vitamin D may protect against falls, internal cancers, psychiatric conditions, autoimmune diseases and cardiovascular diseases. Since the dominant source of vitamin D is sunlight exposure, there is a need to understand what is a “balanced” level of sun exposure to maintain an adequate level of vitamin D but minimise the risks of eye damage, skin damage and skin cancer resulting from excessive UV exposure. There are many steps in the pathway from incoming solar UV to the eventual vitamin D status of humans (measured as 25-hydroxyvitamin D in the blood), and our knowledge about many of these steps is currently incomplete. This project begins by investigating the levels of UV available for synthesising vitamin D, and how these levels vary across seasons, latitudes and times of the day. The thesis then covers experiments conducted with an in vitro model, which was developed to study several aspects of vitamin D synthesis. Results from the model suggest the relationship between UV dose and vitamin D is not linear. This is an important input into public health messages regarding ‘safe’ UV exposure: larger doses of UV, beyond a certain limit, may not continue to produce vitamin D; however, they will increase the risk of skin cancers and eye damage. The model also showed that, when given identical doses of UV, the amount of vitamin D produced was impacted by temperature. In humans, a temperature-dependent reaction must occur in the top layers of human skin, prior to vitamin D entering the bloodstream. The hypothesis will be raised that cooler temperatures (occurring in winter and at high latitudes) may reduce vitamin D production in humans. Finally, the model has also been used to study the wavelengths of UV thought to be responsible for producing vitamin D. It appears that vitamin D production is limited to a small range of UV wavelengths, which may be narrower than previously thought. Together, these results suggest that further research is needed into the ability of humans to synthesise vitamin D from sunlight. In particular, more information is needed about the dose-response relationship in humans and to investigate the proposed impact of temperature. Having an accurate action spectrum will also be essential for measuring the available levels of vitamin D-effective UV. As this research continues, it will contribute to the scientific evidence-base needed for devising a public health message that will balance the risks of excessive UV exposure with maintaining adequate vitamin D.

The power of a single prototype : sustainable fashion textile design and the prevention of carcinogenic melanoma

Is there a role for prototyping (sketching, pattern making and sampling) in addressing real world problems of sustainability (People, Profit, and Planet), in this case social/healthcare issues, through fashion and textiles research? Skin cancer and related illnesses are a major cause of disfigurement and death in New Zealand and Australia where the rates of Melanoma, a serious form of skin cancer, are four times higher than in the Northern Hemisphere regions of USA, UK and Canada (IARC, 1992). In 2007, AUT University (Auckland University of Technology) Fashion Department and the Health Promotion Department of Cancer Society - Auckland Division (CSA) developed a prototype hat aimed at exploring a barrier type solution to prevent facial and neck skin damage. This is a paradigm shift from the usual medical research model. This paper provides an overview of the project and examines how a fashion prototype has been used to communicate emergent social, environmental, personal, physiological and technological concerns to the trans-disciplinary research team. The authors consider how the design of a product can enhance and support sustainable design practice while contributing a potential solution to an ongoing health issue. Analysis of this case study provides an insight into prototyping in fashion and textiles design, user engagement and the importance of requirements analysis in relation to sustainable development. The analysis and a successful outcome of the final prototype have provided a gateway to future collaborative research and product development.

Seasonal Variation in Measured Solar Ultraviolet Radiation Exposure of Adults in Subtropical Australia

Generating accurate population-specific public health messages regarding sun protection requires knowledge about seasonal variation in sun exposure in different environments. To address this issue for a subtropical area of Australia, we used polysulphone badges to measure UVR for the township of Nambour (26° latitude) and personal UVR exposure among Nambour residents who were taking part in a skin cancer prevention trial. Badges were worn by participants for two winter and two summer days. The ambient UVR was approximately three times as high in summer as in winter. However, participants received more than twice the proportion of available UVR in winter as in summer (6.5%vs 2.7%, P < 0.05), resulting in an average ratio of summer to winter personal UVR exposure of 1.35. The average absolute difference in daily dose between summer and winter was only one-seventh of a minimal erythemal dose. Extrapolating from our data, we estimate that ca. 42% of the total exposure received in the 6 months of winter (June–August) and summer (December–February) is received during the three winter months. Our data show that in Queensland a substantial proportion of people’s annual UVR dose is obtained in winter, underscoring the need for dissemination of sun protection messages throughout the year in subtropical and tropical climates.