Broadening of transgenic adenocarcinoma of the mouse prostate (TRAMP) model to represent late stage androgen depletion independent cancer

BACKGROUND The transgenic adenocarcinoma of the mouse prostate (TRAMP) model closely mimics PC-progression as it occurs in humans. However, the timing of disease incidence and progression (especially late stage) makes it logistically difficult to conduct experiments synchronously and economically. The development and characterization of androgen depletion independent (ADI) TRAMP sublines are reported. METHODS Sublines were derived from androgen-sensitive TRAMP-C1 and TRAMP-C2 cell lines by androgen deprivation in vitro and in vivo. Epithelial origin (cytokeratin) and expression of late stage biomarkers (E-cadherin and KAI-1) were evaluated using immunohistochemistry. Androgen receptor (AR) status was assessed through quantitative real time PCR, Western blotting, and immunohistochemistry. Coexpression of AR and E-cadherin was also evaluated. Clonogenicity and invasive potential were measured by soft agar and matrigel invasion assays. Proliferation/survival of sublines in response to androgen was assessed by WST-1 assay. In vivo growth of subcutaneous tumors was assessed in castrated and sham-castrated C57BL/6 mice. RESULTS The sublines were epithelial and displayed ADI in vitro and in vivo. Compared to the parental lines, these showed (1) significantly faster growth rates in vitro and in vivo independent of androgen depletion, (2) greater tumorigenic, and invasive potential in vitro. All showed substantial downregulation in expression levels of tumor suppressor, E-cadherin, and metastatis suppressor, KAI-1. Interestingly, the percentage of cells expressing AR with downregulated E-cadherin was higher in ADI cells, suggesting a possible interaction between the two pathways. CONCLUSIONS The TRAMP model now encompasses ADI sublines potentially representing different phenotypes with increased tumorigenicity and invasiveness.

Identifying optimal lipid raft characteristics required to promote nanoscale protein-protein interactions on the plasma membrane

The dynamic lateral segregation of signaling proteins into microdomains is proposed to facilitate signal transduction, but the constraints on microdomain size, mobility, and diffusion that might realize this function are undefined. Here we interrogate a stochastic spatial model of the plasma membrane to determine how microdomains affect protein dynamics. Taking lipid rafts as representative microdomains, we show that reduced protein mobility in rafts segregates dynamically partitioning proteins, but the equilibrium concentration is largely independent of raft size and mobility. Rafts weakly impede small-scale protein diffusion but more strongly impede long-range protein mobility. The long-range mobility of raft-partitioning and raft-excluded proteins, however, is reduced to a similar extent. Dynamic partitioning into rafts increases specific interprotein collision rates, but to maximize this critical, biologically relevant function, rafts must be small (diameter, 6 to 14 nm) and mobile. Intermolecular collisions can also be favored by the selective capture and exclusion of proteins by rafts, although this mechanism is generally less efficient than simple dynamic partitioning. Generalizing these results, we conclude that microdomains can readily operate as protein concentrators or isolators but there appear to be significant constraints on size and mobility if microdomains are also required to function as reaction chambers that facilitate nanoscale protein-protein interactions. These results may have significant implications for the many signaling cascades that are scaffolded or assembled in plasma membrane microdomains.

Estrogen receptor-Beta activated apoptosis in benign hyperplasia and cancer of the prostate is androgen independent and TNF-alpha mediated

Prostate cancer (PCa) and benign prostatic hyperplasia (BPH) are androgen-dependent diseases commonly treated by inhibiting androgen action. However, androgen ablation or castration fail to target androgen-independent cells implicated in disease etiology and recurrence. Mechanistically different to castration, this study shows beneficial proapoptotic actions of estrogen receptor–β (ERβ) in BPH and PCa. ERβ agonist induces apoptosis in prostatic stromal, luminal and castrate-resistant basal epithelial cells of estrogen-deficient aromatase knock-out mice. This occurs via extrinsic (caspase-8) pathways, without reducing serum hormones, and perturbs the regenerative capacity of the epithelium. TNFα knock-out mice fail to respond to ERβ agonist, demonstrating the requirement for TNFα signaling. In human tissues, ERβ agonist induces apoptosis in stroma and epithelium of xenografted BPH specimens, including in the CD133+ enriched putative stem/progenitor cells isolated from BPH-1 cells in vitro. In PCa, ERβ causes apoptosis in Gleason Grade 7 xenografted tissues and androgen-independent cells lines (PC3 and DU145) via caspase-8. These data provide evidence of the beneficial effects of ERβ agonist on epithelium and stroma of BPH, as well as androgen-independent tumor cells implicated in recurrent disease. Our data are indicative of the therapeutic potential of ERβ agonist for treatment of PCa and/or BPH with or without androgen withdrawal.

An examination of monotony and hypovigilance, independent of fatigue : relevance to road safety

Driving is a vigilance task, requiring sustained attention to maintain performance and avoid crashes. Hypovigilance (i.e., marked reduction in vigilance) while driving manifests as poor driving performance and is commonly attributed to fatigue (Dinges, 1995). However, poor driving performance has been found to be more frequent when driving in monotonous road environments, suggesting that monotony plays a role in generating hypovigilance (Thiffault & Bergeron, 2003b). Research to date has tended to conceptualise monotony as a uni-dimensional task characteristic, typically used over a prolonged period of time to facilitate other factors under investigation, most notably fatigue. However, more often than not, more than one exogenous factor relating to the task or operating environment is manipulated to vary or generate monotony (Mascord & Heath, 1992). Here we aimed to explore whether monotony is a multi-dimensional construct that is determined by characteristics of both the task proper and the task environment. The general assumption that monotony is a task characteristic used solely to elicit hypovigilance or poor performance related to fatigue appears to have led to there being little rigorous investigation into the exact nature of the relationship. While the two concepts are undoubtedly linked, the independent effect of monotony on hypovigilance remains largely ignored. Notwithstanding, there is evidence that monotony effects can emerge very early in vigilance tasks and are not necessarily accompanied by fatigue (see Meuter, Rakotonirainy, Johns, & Wagner, 2005). This phenomenon raises a largely untested, empirical question explored in two studies: Can hypovigilance emerge as a consequence of task and/or environmental monotony, independent of time on task and fatigue? In Study 1, using a short computerised vigilance task requiring responses to be withheld to infrequent targets, we explored the differential impacts of stimuli and task demand manipulations on the development of a monotonous context and the associated effects on vigilance performance (as indexed by respone errors and response times), independent of fatigue and time on task. The role of individual differences (sensation seeking, extroversion and cognitive failures) in moderating monotony effects was also considered. The results indicate that monotony affects sustained attention, with hypovigilance and associated performance worse in monotonous than in non-monotonous contexts. Critically, performance decrements emerged early in the task (within 4.3 minutes) and remained consistent over the course of the experiment (21.5 minutes), suggesting that monotony effects can operate independent of time on task and fatigue. A combination of low task demands and low stimulus variability form a monotonous context characterised by hypovigilance and poor task performance. Variations to task demand and stimulus variability were also found to independently affect performance, suggesting that monotony is a multi-dimensional construct relating to both task monotony (associated with the task itself) and environmental monotony (related to characteristics of the stimulus). Consequently, it can be concluded that monotony is multi-dimensional and is characterised by low variability in stimuli and/or task demands. The proposition that individual differences emerge under conditions of varying monotony with high sensation seekers and/or extroverts performing worse in monotonous contexts was only partially supported. Using a driving simulator, the findings of Study 1 were extended to a driving context to identify the behavioural and psychophysiological indices of monotony-related hypovigilance associated with variations to road design and road side scenery (Study 2). Supporting the proposition that monotony is a multi-dimensional construct, road design variability emerged as a key moderating characteristic of environmental monotony, resulting in poor driving performance indexed by decrements in steering wheel measures (mean lateral position). Sensation seeking also emerged as a moderating factor, where participants high in sensation seeking tendencies displayed worse driving behaviour in monotonous conditions. Importantly, impaired driving performance was observed within 8 minutes of commencing the driving task characterised by environmental monotony (low variability in road design) and was not accompanied by a decline in psychophysiological arousal. In addition, no subjective declines in alertness were reported. With fatigue effects associated with prolonged driving (van der Hulst, Meijman, & Rothengatter, 2001) and indexed by drowsiness, this pattern of results indicates that monotony can affect driver vigilance, independent of time on task and fatigue. Perceptual load theory (Lavie, 1995, 2005) and mindlessness theory (Robertson, Manly, Andrade, Baddley, & Yiend, 1997) provide useful theoretical frameworks for explaining and predicting monotony effects by positing that the low load (of task and/or stimuli) associated with a monotonous task results in spare attentional capacity which spills over involuntarily, resulting in the processing of task-irrelevant stimuli or task unrelated thoughts. That is, individuals – even when not fatigued - become easily distracted when performing a highly monotonous task, resulting in hypovigilance and impaired performance. The implications for road safety, including the likely effectiveness of fatigue countermeasures to mitigate monotony-related driver hypovigilance are discussed.

Haplotype analysis of two recurrent CDKN2A mutations in 10 melanoma families : evidence for common founders and independent mutations

Germ-line mutations in CDKN2A have been shown to predispose to cutaneous malignant melanoma. We have identified 2 new melanoma kindreds which carry a duplication of a 24bp repeat present in the 5' region of CDKN2A previously identified in melanoma families from Australia and the United States. This mutation has now been reported in 5 melanoma families from 3 continents: Europe, North America, and Australasia. The M53I mutation in exon 2 of CDKN2A has also been documented in 5 melanoma families from Australia and North America. The aim of this study was to determine whether the occurrence of the mutations in these families from geographically diverse populations represented mutation hotspots within CDKN2A or were due to common ancestors. Haplotypes of 11 microsatellite markers flanking CDKN2A were constructed in 5 families carrying the M53I mutation and 5 families carrying the 24bp duplication. There were some differences in the segregating haplotypes due primarily to recombinations and mutations within the short tandem-repeat markers; however, the data provide evidence to indicate that there were at least 3 independent 24bp duplication events and possibly only 1 original M53I mutation. This is the first study to date which indicates common founders in melanoma families from different continents.

p53-independent NOXA induction overcomes apoptotic resistance of malignant melanomas

Once melanoma metastasizes, no effective treatment modalities prolong survival in most patients. This notorious refractoriness to therapy challenges investigators to identify agents that overcome melanoma resistance to apoptosis. Whereas many survival pathways contribute to the death-defying phenotype in melanoma, a defect in apoptotic machinery previously highlighted inactivation of Apaf-1, an apoptosome component engaged after mitochondrial damage. During studies involving Notch signaling in melanoma, we observed a gamma-secretase tripeptide inhibitor (GSI; z-Leu-Leu-Nle-CHO), selected from a group of compounds originally used in Alzheimer's disease, induced apoptosis in nine of nine melanoma lines. GSI only induced G2-M growth arrest (but not killing) in five of five normal melanocyte cultures tested. Effective killing of melanoma cells by GSI involved new protein synthesis and a mitochondrial-based pathway mediated by up-regulation of BH3-only members (Bim and NOXA). p53 activation was not necessary for up-regulation of NOXA in melanoma cells. Blocking GSI-induced NOXA using an antisense (but not control) oligonucleotide significantly reduced the apoptotic response. GSI also killed melanoma cell lines with low Apaf-1 levels. We conclude that GSI is highly effective in killing melanoma cells while sparing normal melanocytes. Direct enhancement of BH3-only proteins executes an apoptotic program overcoming resistance of this lethal tumor. Identification of a p53-independent apoptotic pathway in melanoma cells, including cells with low Apaf-1, bypasses an impediment to current cytotoxic therapy and provides new targets for future therapeutic trials involving chemoresistant tumors.

Scaling digital radiographs for templating in total hip arthroplasty using conventional acetate templates independent of calibration markers

We describe a scaling method for templating digital radiographs using conventional acetate templates independent of template magnification without the need for a calibration marker. The mean magnification factor for the radiology department was determined (119.8%, range117%-123.4%). This fixed magnification factor was used to scale the radiographs by the method described. 32 femoral heads on postoperative THR radiographs were then measured and compared to the actual size. The mean absolute accuracy was within 0.5% of actual head size (range 0 to 3%) with a mean absolute difference of 0.16mm (range 0-1mm, SD 0.26mm). Intraclass Correlation Coefficient (ICC) showed excellent reliability for both inter and intraobserver measurements with ICC scores of 0.993 (95% CI 0.988-0.996) for interobserver measurements and intraobserver measurements ranging between 0.990-0.993 (95% CI 0.980-0.997).