Gene expression profiling in human breast cancer – toward personalised therapeutics?

The most integrated approach toward understanding the multiple molecular events and mechanisms by which cancer may develop is the application of gene expression profiling using microarray technologies. As molecular alterations in breast cancer are complex and involve cross-talk between multiple cellular signalling pathways, microarray technology provides a means of capturing and comparing the expression patterns of the entire genome across multiple samples in a high throughput manner. Since the development of microarray technologies, together with the advances in RNA extraction methodologies, gene expression studies have revolutionised the means by which genes suitable as targets for drug development and individualised cancer treatment can be identified. As of the mid-1990s, expression microarrays have been extensively applied to the study of cancer and no cancer type has seen as much genomic attention as breast cancer. The most abundant area of breast cancer genomics has been the clarification and interpretation of gene expression patterns that unite both biological and clinical aspects of tumours. It is hoped that one day molecular profiling will transform diagnosis and therapeutic selection in human breast cancer toward more individualised regimes. Here, we review a number of prominent microarray profiling studies focussed on human breast cancer and examine their strengths, their limitations, clinical implications including prognostic relevance and gene signature significance along with potential improvements for the next generation of microarray studies.

Mathematical modelling of soft callus formation in early murine bone repair

Fracture healing is a complicated coupling of many processes. Yet despite the apparent complexity, fracture repair is usually effective. There is, however, no comprehensive mathematical model addressing the multiple interactions of cells, cytokines and oxygen that includes extra-cellular matrix production and that results in the formation of the early stage soft callus. This thesis develops a one dimensional continuum transport model in the context of early fracture healing. Although fracture healing is a complex interplay of many local factors, critical components are identified and used to construct an hypothesis about regulation of the evolution of early callus formation. Multiple cell lines, cellular differentiation, oxygen levels and cytokine concentrations are examined as factors affecting this model of early bone repair. The model presumes diffusive and chemotactic cell migration mechanisms. It is proposed that the initial signalling regime and oxygen availability arising as consequences of bone fracture, are sufficient to determine the quantity and quality of early soft callus formation. Readily available software and purpose written algorithms have been used to obtain numerical solutions representative of various initial conditions. These numerical distributions of cellular populations reflect available histology obtained from murine osteotomies. The behaviour of the numerical system in response to differing initial conditions can be described by alternative in vivo healing pathways. An experimental basis, as illustrated in murine fracture histology, has been utilised to validate the mathematical model outcomes. The model developed in this thesis has potential for future extension, to incorporate processes leading to woven bone deposition, while maintaining the characteristics that regulate early callus formation.

Timing and distribution of protein ingestion during prolonged recovery from resistance exercise alters myofibrillar protein synthesis

Quantity and timing of protein ingestion are major factors regulating myofibrillar protein synthesis (MPS). However, the effect of specific ingestion patterns on MPS throughout a 12 h period is unknown. We determined how different distributions of protein feeding during 12 h recovery after resistance exercise affects anabolic responses in skeletal muscle. Twenty-four healthy trained males were assigned to three groups (n = 8/group) and undertook a bout of resistance exercise followed by ingestion of 80 g of whey protein throughout 12 h recovery in one of the following protocols: 8 × 10 g every 1.5 h (PULSE); 4 × 20 g every 3 h (intermediate: INT); or 2 × 40 g every 6 h (BOLUS). Muscle biopsies were obtained at rest and after 1, 4, 6, 7 and 12 h post exercise. Resting and post-exercise MPS (l-[ring-(13)C6] phenylalanine), and muscle mRNA abundance and cell signalling were assessed. All ingestion protocols increased MPS above rest throughout 1-12 h recovery (88-148%, P < 0.02), but INT elicited greater MPS than PULSE and BOLUS (31-48%, P < 0.02). In general signalling showed a BOLUS>INT>PULSE hierarchy in magnitude of phosphorylation. MuRF-1 and SLC38A2 mRNA were differentially expressed with BOLUS. In conclusion, 20 g of whey protein consumed every 3 h was superior to either PULSE or BOLUS feeding patterns for stimulating MPS throughout the day. This study provides novel information on the effect of modulating the distribution of protein intake on anabolic responses in skeletal muscle and has the potential to maximize outcomes of resistance training for attaining peak muscle mass.

mTOR function in skeletal muscle : a focal point for overnutrition and exercise

The mammalian target of rapamycin (mTOR) is a highly conserved atypical serine-threonine kinase that controls numerous functions essential for cell homeostasis and adaptation in mammalian cells via 2 distinct protein complex formations. Moreover, mTOR is a key regulatory protein in the insulin signalling cascade and has also been characterized as an insulin-independent nutrient sensor that may represent a critical mediator in obesity-related impairments of insulin action in skeletal muscle. Exercise characterizes a remedial modality that enhances mTOR activity and subsequently promotes beneficial metabolic adaptation in skeletal muscle. Thus, the metabolic effects of nutrients and exercise have the capacity to converge at the mTOR protein complexes and subsequently modify mTOR function. Accordingly, the aim of the present review is to highlight the role of mTOR in the regulation of insulin action in response to overnutrition and the capacity for exercise to enhance mTOR activity in skeletal muscle.

The molecular bases of training adaptation

Skeletal muscle is a malleable tissue capable of altering the type and amount of protein in response to disruptions to cellular homeostasis. The process of exercise-induced adaptation in skeletal muscle involves a multitude of signalling mechanisms initiating replication of specific DNA genetic sequences, enabling subsequent translation of the genetic message and ultimately generating a series of amino acids that form new proteins. The functional consequences of these adaptations are determined by training volume, intensity and frequency, and the half-life of the protein. Moreover, many features of the training adaptation are specific to the type of stimulus, such as the mode of exercise. Prolonged endurance training elicits a variety of metabolic and morphological changes, including mitochondrial biogenesis, fast-to-slow fibre-type transformation and substrate metabolism. In contrast, heavy resistance exercise stimulates synthesis of contractile proteins responsible for muscle hypertrophy and increases in maximal contractile force output. Concomitant with the vastly different functional outcomes induced by these diverse exercise modes, the genetic and molecular mechanisms of adaptation are distinct. With recent advances in technology, it is now possible to study the effects of various training interventions on a variety of signalling proteins and early-response genes in skeletal muscle. Although it cannot presently be claimed that such scientific endeavours have influenced the training practices of elite athletes, these new and exciting technologies have provided insight into how current training techniques result in specific muscular adaptations, and may ultimately provide clues for future and novel training methodologies. Greater knowledge of the mechanisms and interaction of exercise-induced adaptive pathways in skeletal muscle is important for our understanding of the aetiology of disease, maintenance of metabolic and functional capacity with aging, and training for athletic performance. This article highlights the effects of exercise on molecular and genetic mechanisms of training adaptation in skeletal muscle.

Ratchetting of railhead in the vicinity of the gap of the insulated rail joints

Insulated Rail Joints (IRJs) are designed to electrically isolate two rails in rail tracks to control the signalling system for safer train operations. Unfortunately the gapped section of the IRJs is structurally weak and often fails prematurely especially in heavy haul tracks, which adversely affects service reliability and efficiency. The IRJs suffer from a number of failure modes; the railhead ratchetting at the gap is, however, regarded as the root cause and attended to in this thesis. Ratchetting increases with the increase in wheel loads; in the absence of a life prediction model, effective management of the IRJs for increased wagon wheel loads has become very challenging. Therefore, the main aim of this thesis is to determine method to predict IRJs' service life. The distinct discontinuity of the railhead at the gap makes the Hertzian theory and the rolling contact shakedown map, commonly used in the continuously welded rails, not applicable to examine the metal ratchetting of the IRJs. Finite Element (FE) technique is, therefore, used to explore the railhead metal ratchetting characteristics in this thesis, the boundary conditions of which has been determined from a full scale study of the IRJ specimens under rolling contact of the loaded wheels. A special purpose test set up containing full-scale wagon wheel was used to apply rolling wheel loads on the railhead edges of the test specimens. The state of the rail end face strains was determined using a non-contact digital imaging technique and used for calibrating the FE model. The basic material parameters for this FE model were obtained through independent uniaxial, monotonic tensile tests on specimens cut from the head hardened virgin rails. The monotonic tensile test data have been used to establish a cyclic load simulation model of the railhead steel specimen; the simulated cyclic load test has provided the necessary data for the three decomposed kinematic hardening plastic strain accumulation model of Chaboche. A performance based service life prediction algorithm for the IRJs was established using the plastic strain accumulation obtained from the Chaboche model. The predicted service lives of IRJs using this algorithm have agreed well with the published data. The finite element model has been used to carry out a sensitivity study on the effects of wheel diameter to the railhead metal plasticity. This study revealed that the depth of the plastic zone at the railhead edges is independent of the wheel diameter; however, large wheel diameter is shown to increase the IRJs' service life.

Ginger - mechanism of action in chemotherapy-induced nausea and vomiting: A review

Despite advances in anti-emetic therapy, chemotherapy-induced nausea and vomiting (CINV) still poses a significant burden to patients undergoing chemotherapy. Nausea, in particular, is still highly prevalent in this population. Ginger has been traditionally used as a folk remedy for gastrointestinal complaints and has been suggested as a viable adjuvant treatment for nausea and vomiting in the cancer context. Substantial research has revealed ginger to possess properties that could exert multiple beneficial effects on chemotherapy patients who experience nausea and vomiting. Bioactive compounds within the rhizome of ginger, particularly the gingerol and shogaol class of compounds, interact with several pathways that are directly implicated in CINV in addition to pathways that could play secondary roles by exacerbating symptoms. These properties include 5-HT3, substance P and acetylcholine receptor antagonism; anti-inflammatory properties; and modulation of cellular redox signalling, vasopressin release, gastrointestinal motility, and gastric emptying rate. This review outlines these proposed mechanisms by discussing the results of clinical, in vitro and animal studies both within the chemotherapy context and in other relevant fields. The evidence presented in this review indicates that ginger possesses multiple properties that could be beneficial in reducing chemotherapy-induced nausea and vomiting.

MMP-9 and the epidermal growth factor signal pathway in non-small cell lung cancer

Background Matrix metalloproteinase (MMP)-9 is an endopeptidase that digests basement membrane type-IV collagen. Enhanced expression has been related to tumour progression in a number of systems. The control of MMP expression is complex, but recently epidermal growth actor receptor (EGFR) activity has been implicated in up-regulation of MMP-9 in tumour cells in vitro. Aims To evaluate interrelations between MMP-9 and EGFR expression in non-small cell lung cancer (NSCLC) and to assess the impact of expression on survival. Methods This is a retrospective study of 152 patients who underwent resection for stage I-IIIa NSCLC with a post-operative survival >60 days. Minimum follow-up was 2 years. Standard ABC immunohistochemistry was performed on 4μm paraffin-embedded sections from the tumour periphery using monoclonal antibodies to MMP-9 and EGFR. Results: MMP-9 was expressed in the tumour cells of 79/152 (52%) cases. EGFR expression was found in 86/152 (57%) cases [membranous 51/152 (34%), cytoplasmic 35/152 (23%)]. MMP-9 expression was associated with poor outcome (p=0.04). Membranous, cytoplasmic and overall EGFR expression were not associated with outcome (p=0.29, p=0.85 and p=0.41 respectively). There was a strong correlation between MMP-9 expression and EGFR expression (p=0.001) and EGFR membranous expression (p=0.01) but not with cytoplasmic EGFR expression (p=0.28). Co-expression of MMP-9 and EGFR (36%) conferred a worse prognosis (p=0.003). Subset analysis revealed only MMP-9 and membranous EGFR co-expression (22%) was associated with poor outcome (p=0.008). Conclusions Our results show that MMP-9 and EGFR are co-expressed in NSCLC. This finding suggests the EGFR signalling pathway may play an important role in the invasive behaviour of NSCLC via specific upregulation of MMP-9. The co-expression of these markers also confers a poor prognosis.

Receptor tyrosine kinases and their activation in melanoma

Receptor tyrosine kinases (RTKs) and their downstream signalling pathways have long been hypothesized to play key roles in melanoma development. A decade ago, evidence was derived largely from animal models, RTK expression studies and detection of activated RAS isoforms in a small fraction of melanomas. Predictions that overexpression of specific RTKs implied increased kinase activity and that some RTKs would show activating mutations in melanoma were largely untested. However, technological advances including rapid gene sequencing, siRNA methods and phospho-RTK arrays now give a more complete picture. Mutated forms of RTK genes including KIT, ERBB4, the EPH and FGFR families and others are known in melanoma. Additional over- or underexpressed RTKs and also protein tyrosine phosphatases (PTPs) have been reported, and activities measured. Complex interactions between RTKs and PTPs are implicated in the abnormal signalling driving aberrant growth and survival in malignant melanocytes, and indeed in normal melanocytic signalling including the response to ultraviolet radiation. Kinases are considered druggable targets, so characterization of global RTK activity in melanoma should assist the rational development of tyrosine kinase inhibitors for clinical use. © 2011 John Wiley & Sons A/S.

Regulation of EP receptors in non-small cell lung cancer by epigenetic modifications

Background: Cyclooxygenase (COX)-2 is frequently overexpressed in non-small cell lung cancer (NSCLC) and results in increased levels of prostaglandin E2 (PGE 2), an important signalling molecule implicated in tumourigenesis. PGE 2 exerts its effects through the E prostanoid (EP) receptors (EPs1-4). Methods: The expression and epigenetic regulation of the EPs were evaluated in a series of resected fresh frozen NSCLC tumours and cell lines. Results: EP expression was dysregulated in NSCLC being up and downregulated compared to matched control samples. For EPs1, 3 and 4 no discernible pattern emerged. EP2 mRNA however was frequently downregulated, with low levels being observed in 13/20 samples as compared to upregulation in 5/20 samples examined. In NSCLC cell lines DNA CpG methylation was found to be important for the regulation of EP3 expression, the demethylating agent decitabine upregulating expression. Histone acetylation was also found to be a critical regulator of EP expression, with the histone deacteylase inhibitors trichostatin A, phenylbutyrate and suberoylanilide hydroxamic acid inducing increased expression of EPs2-4. Direct chromatin remodelling was demonstrated at the promoters for EPs2-4. Conclusions: These results indicate that EP expression is variably altered from tumour to tumour in NSCLC. EP2 expression appears to be predominantly downregulated and may have an important role in the pathogenesis of the disease. Epigenetic regulation of the EPs may be central to the precise role COX-2 may play in the evolution of individual tumours. © 2009 Elsevier Ltd. All rights reserved.

Vorinostat/SAHA-induced apoptosis in malignant mesothelioma is FLIP/caspase 8-dependent and HR23B-independent

Introduction: Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence. The caspase 8 inhibitor FLIP is an anti-apoptotic protein over-expressed in several cancer types including MPM. The histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) is currently being evaluated in relapsed mesothelioma. We examined the roles of FLIP and caspase 8 in regulating SAHA-induced apoptosis in MPM. Methods: The mechanism of SAHA-induced apoptosis was assessed in 7 MPM cell lines and in a multicellular spheroid model. SiRNA and overexpression approaches were used, and cell death was assessed by flow cytometry, Western blotting and clonogenic assays. Results: RNAi-mediated FLIP silencing resulted in caspase 8-dependent apoptosis in MPM cell line models. SAHA potently down-regulated FLIP protein expression in all 7 MPM cell lines and in a multicellular spheroid model of MPM. In 6/7 MPM cell lines, SAHA treatment resulted in significant levels of apoptosis induction. Moreover, this apoptosis was caspase 8-dependent in all six sensitive cell lines. SAHA-induced apoptosis was also inhibited by stable FLIP overexpression. In contrast, down-regulation of HR23B, a candidate predictive biomarker for HDAC inhibitors, significantly inhibited SAHA-induced apoptosis in only 1/6 SAHA-sensitive MPM cell lines. Analysis of MPM patient samples demonstrated significant inter-patient variations in FLIP and caspase 8 expressions. In addition, SAHA enhanced cisplatin-induced apoptosis in a FLIP-dependent manner. Conclusions: These results indicate that FLIP is a major target for SAHA in MPM and identifies FLIP, caspase 8 and associated signalling molecules as candidate biomarkers for SAHA in this disease. © 2011 Elsevier Ltd. All rights reserved.

Chronic immune activation and inflammation as the cause of malignancy

Several chronic infections known to be associated with malignancy have established oncogenic properties. However the existence of chronic inflammatory conditions that do not have an established infective cause and are associated with the development of tumours strongly suggests that the inflammatory process itself provides the prerequisite environment for the development of malignancy. This environment includes upregulation of mediators of the inflammatory response such as cyclo-oxygenase (COX)-2 leading to the production of inflammatory cytokines and prostaglandins which themselves may suppress cell mediated immune responses and promote angiogenesis. These factors may also impact on cell growth and survival signalling pathways resulting in induction of cell proliferation and inhibition of apoptosis. Furthermore, chronic inflammation may lead to the production of reactive oxygen species and metabolites such as malondialdehyde within the affected cells that may in turn induce DNA damage and mutations and, as a result, be carcinogenic. Here it is proposed that the conditions provided by a chronic inflammatory environment are so essential for the progression of the neoplastic process that therapeutic intervention aimed at inhibiting inflammation, reducing angiogenesis and stimulating cell mediated immune responses may have a major role in reducing the incidence of common cancers. © 2001 Cancer Research Campaign http://www.bjcancer.com.

On the allocation of a takeover purchase price under AASB1013

The purpose of this paper is to document and explain the allocation of takeover purchase price to identifiable intangible assets (IIAs), purchased goodwill, and/or target net tangible assets in an accounting environment unconstrained with respect to IIA accounting policy choice. Using a sample of Australian acquisitions during the unconstrained accounting environment from 1988 to 2004, we find the percentage allocation of purchase price to IIAs averaged 19.09%. The percentage allocation to IIAs is significantly positively related to return on assets and insignificantly related to leverage, contrary to opportunism. Efficiency suggests an explanation: profitable firms acquire and capitalise a higher percentage of IIAs in acquisitions. The target's investment opportunity set is significantly positively related to the percentage allocation to IIAs, consistent with information-signalling. The paper contributes to the accounting policy choice literature by showing how Australian firms make the one-off accounting policy choice in regards allocation of takeover purchase price (which is often a substantial dollar amount to) in an environment where accounting for IIAs was unconstrained.

A stimulatory effect of Ca3ZrSi2O9 bioceramics on cementogenic/osteogenic differentiation of periodontal ligament cells

The regeneration of periodontal tissues to cure periodontitis remains a medical challenge. Therefore, it is of great importance to develop a novel biomaterial that could induce cementogenesis and osteogenesis in periodontal tissue engineering. Calcium silicate (Ca–Si) based ceramics have been found to be potential bioactive materials due to their osteostimulatory effect. Recently, it is reported that zirconium modified calcium-silicate-based (Ca3ZrSi2O9) ceramics stimulate cell proliferation and osteogenic differentiation of osteoblasts. However, it is unknown whether Ca3ZrSi2O9 ceramics possess specific cementogenic stimulation for human periodontal ligament cells (hPDLCs) in periodontal tissue regeneration in vitro. The purpose of this study was to investigate whether Ca3ZrSi2O9 ceramic disks and their ionic extracts could stimulate cell growth and cementogenic/osteogenic differentiation of hPDLCs; the possible molecular mechanism involved in this process was also explored by investigating the Wnt/β-catenin signalling pathway of hPDLCs. Our results showed that Ca3ZrSi2O9 ceramic disks supported cell adhesion, proliferation and significantly up-regulated relative alkaline phosphatase (ALP) activity, cementogenic/osteogenic gene expression (CEMP1, CAP, ALP and OPN) and Wnt/β-catenin signalling pathway-related genes (AXIN2 and CTNNB) for hPDLCs, compared to that of β-tricalcium phosphate (β-TCP) bioceramic disks and blank controls. The ionic extracts from Ca3ZrSi2O9 powders also significantly enhanced relative ALP activity, cementogenic/osteogenic and Wnt/β-catenin-related gene expression of hPDLCs. The present results demonstrate that Ca3ZrSi2O9 ceramics are capable of stimulating cementogenic/osteogenic differentiation of hPDLCs possibly via activation of the Wnt/β-catenin signalling pathway, suggesting that Ca3ZrSi2O9 ceramics have the potential to be used for periodontal tissue regeneration.

Role of Human Topoisomerase I in DNA Repair and Apoptosis

Human topoisomerase I (htopoI) is an enzyme that up to now was believed to function mainly in the removal of torsional stress generated during transcription and replication. In 1998, it was found that htopoI might play another important role in the cellular response to DNA damage -- the so-called htopoI damage response. Since this initial discovery, many studies have suggested that the htopoI damage response is involved in DNA repair as well as in apoptosis. Here we discuss the earliest as well as the latest results in this field. Combining all of the published and as yet unpublished results, we suggest and discuss a model of how htopoI may function during DNA repair and apoptosis. Furthermore, numerous results show that the htopoI damage response is not a spontaneous event, but is strictly regulated by cellular signalling pathways. We discuss which pathways may be involved and how the htopoI damage response is activated. Although the htopoI damage response was discovered several years ago, research in this area is just beginning. The future will surely bring more clarity regarding the precise mechanism behind the involvement of htopoI in DNA repair and apoptosis, as well as its implications for a broader understanding of how the human organism ensures genomic stability.