In vitro and in vivo examination of the cell surface glycoprotein CDCP1

A number of reports have demonstrated the importance of the CUB domaincontaining protein 1 (CDCP1) in facilitating cancer progression in animal models and the potential of this protein as a prognostic marker in several malignancies. CDCP1 facilitates metastasis formation in animal models by negatively regulating anoikis, a type of apoptosis triggered by the loss of attachment signalling from cell-cell contacts or cell-extra cellular matrix (ECM) contacts. Due to the important role CDCP1 plays in cancer progression in model systems, it is considered a potential drug target to prevent the metastatic spread of cancers. CDCP1 is a highly glycosylated 836 amino acid cell surface protein. It has structural features potentially facilitating protein-protein interactions including 14 N-glycosylation sites, three CUB-like domains, 20 cysteine residues likely to be involved in disulfide bond formation and five intracellular tyrosine residues. CDCP1 interacts with a variety of proteins including Src family kinases (SFKs) and protein kinase C ä (PKCä). Efforts to understand the mechanisms regulating these interactions have largely focussed on three CDCP1 tyrosine residues Y734, Y743 and Y762. CDCP1-Y734 is the site where SFKs phosphorylate and bind to CDCP1 and mediate subsequent phosphorylation of CDCP1-Y743 and -Y762 which leads to binding of PKCä at CDCP1-Y762. The resulting trimeric protein complex of SFK•CDCP1•PKCä has been proposed to mediate an anti-apoptotic cell phenotype in vitro, and to promote metastasis in vivo. The effect of mutation of the three tyrosines on interactions of CDCP1 with SFKs and PKCä and the consequences on cell phenotype in vitro and in vivo have not been examined. CDCP1 has a predicted molecular weight of ~90 kDa but is usually detected as a protein which migrates at ~135 kDa by Western blot analysis due to its high degree of glycosylation. A low molecular weight form of CDCP1 (LMWCDCP1) of ~70 kDa has been found in a variety of cancer cell lines. The mechanisms leading to the generation of LMW-CDCP1 in vivo are not well understood but an involvement of proteases in this process has been proposed. Serine proteases including plasmin and trypsin are able to proteolytically process CDCP1. In addition, the recombinant protease domain of the serine protease matriptase is also able to cleave the recombinant extracellular portion of CDCP1. Whether matriptase is able to proteolytically process CDCP1 on the cell surface has not been examined. Importantly, proteolytic processing of CDCP1 by trypsin leads to phosphorylation of its cell surface-retained portion which suggests that this event leads to initiation of an intracellular signalling cascade. This project aimed to further examine the biology of CDCP1 with a main of focus on exploring the roles played by CDCP1 tyrosine residues. To achieve this HeLa cells stably expressing CDCP1 or the CDCP1 tyrosine mutants Y734F, Y743F and Y762F were generated. These cell lines were used to examine: • The roles of the tyrosine residues Y734, Y743 and Y762 in mediating interactions of CDCP1 with binding proteins and to examine the effect of the stable expression on HeLa cell morphology. • The ability of the serine protease matriptase to proteolytically process cell surface CDCP1 and to examine the consequences of this event on HeLa cell phenotype and cell signalling in vitro. • The importance of these residues in processes associated with cancer progression in vitro including adhesion, proliferation and migration. • The role of these residues on metastatic phenotype in vivo and the ability of a function-blocking anti-CDCP1 antibody to inhibit metastasis in the chicken embryo chorioallantoic membrane (CAM) assay. Interestingly, biochemical experiments carried out in this study revealed that mutation of certain CDCP1 tyrosine residues impacts on interactions of this protein with binding proteins. For example, binding of SFKs as well as PKCä to CDCP1 was markedly decreased in HeLa-CDCP1-Y734F cells, and binding of PKCä was also reduced in HeLa-CDCP1-Y762F cells. In contrast, HeLa-CDCP1-Y743F cells did not display altered interactions with CDCP1 binding proteins. Importantly, observed differences in interactions of CDCP1 with binding partners impacted on basal phosphorylation of CDCP1. It was found that HeLa-CDCP1, HeLa-CDCP1-Y743F and -Y762F displayed strong basal levels of CDCP1 phosphorylation. In contrast, HeLa-CDCP1-Y734F cells did not display CDCP1 phosphorylation but exhibited constitutive phosphorylation of focal adhesion kinase (FAK) at tyrosine 861. Significantly, subsequent investigations to examine this observation suggested that CDCP1-Y734 and FAK-Y861 are competitive substrates for SFK-mediated phosphorylation. It appeared that SFK-mediated phosphorylation of CDCP1- Y734 and FAK-Y861 is an equilibrium which shifts depending on the level of CDCP1 expression in HeLa cells. This suggests that the level of CDCP1 expression may act as a regulatory mechanism allowing cells to switch from a FAK-Y861 mediated pathway to a CDCP1-Y734 mediated pathway. This is the first time that a link between SFKs, CDCP1 and FAK has been demonstrated. One of the most interesting observations from this work was that CDCP1 altered HeLa cell morphology causing an elongated and fibroblastic-like appearance. Importantly, this morphological change depended on CDCP1- Y734. In addition, it was observed that this change in cell morphology was accompanied by increased phosphorylation of SFK-Y416. This suggests that interactions of SFKs with CDCP1-Y734 increases SFK activity since SFKY416 is critical in regulating kinase activity of these proteins. The essential role of SFKs in mediating CDCP1-induced HeLa cell morphological changes was demonstrated using the SFK-selective inhibitor SU6656. This inhibitor caused reversion of HeLa-CDCP1 cell morphology to an epithelial appearance characteristic of HeLa-vector cells. Significantly, in vitro studies revealed that certain CDCP1-mediated cell phenotypes are mediated by cellular pathways dependent on CDCP1 tyrosine residues whereas others are independent of these sites. For example, CDCP1 expression caused a marked increase in HeLa cell motility that was independent of CDCP1 tyrosine residues. In contrast, CDCP1- induced decrease in HeLa cell proliferation was most prominent in HeLa- CDCP1-Y762F cells, potentially indicating a role for this site in regulating proliferation in HeLa cells. Another cellular event which was identified to require phosphorylation of a particular CDCP1 tyrosine residue is adhesion to fibronectin. It was observed that the CDCP1-mediated strong decrease in adhesion to fibronectin is mostly restored in HeLa-CDCP1-Y743F cells. This suggests a possible role for CDCP1-Y743 in causing a CDCP1-mediated decrease in adhesion. Data from in vivo experiments indicated that HeLa-CDCP1-Y734F cells are more metastic than HeLa-CDCP1 cells in vivo. This indicates that interaction of CDCP1 with SFKs and PKCä may not be required for CDCP1-mediated metastasis formation of HeLa cells in vivo. The metastatic phenotype of these cells may be caused by signalling involving FAK since HeLa-CDCP1- Y734F cells are the only CDCP1 expressing cells displaying constitutive phosphorylation of FAK-Y861. HeLa-CDCP1-Y762F cells displayed a very low metastatic ability which suggests that this CDCP1 tyrosine residue is important in mediating a pro-metastatic phenotype in HeLa cells. More detailed exploration of cellular events occurring downstream of CDCP1-Y734 and -Y762 may provide important insights into the mechanisms altering the metastatic ability of CDCP1 expressing HeLa cells. Complementing the in vivo studies, anti-CDCP1 antibodies were employed to assess whether these antibodies are able to inhibit metastasis of CDCP1 and CDCP1 tyrosine mutants expressing HeLa cells. It was found that HeLa- CDCP1-Y734F cells were the only cell line which was markedly reduced in the ability to metastasise. In contrast, the ability of HeLa-CDCP1, HeLa- CDCP1-Y743F and -Y762F cells to metastasise in vivo was not inhibited. These data suggest a possible role of interactions of CDCP1 with SFKs, occurring at CDCP1-Y734, in preventing an anti-metastatic effect of anti- CDCP1 antibodies in vivo. The proposal that SFKs may play a role in regulating anti-metastatic effects of anti-CDCP1 antibodies was supported by another experiment where differences between HeLa-CDCP1 cells and CDCP1 expressing HeLa cells (HeLa-CDCP1-S) from collaborators at the Scripps Research Institute were examined. It was found that HeLa-CDCP1-S cells express different SFKs than CDCP1 expressing HeLa cells generated for this study. This is important since HeLa-CDCP1-S cells can be inhibited in their metastatic ability using anti-CDCP1 antibodies in vivo. Importantly, these data suggest that further examinations of the roles of SFKs in facilitating anti-metastatic effects of anti-CDCP1 antibodies may give insights into how CDCP1 can be blocked to prevent metastasis in vivo. This project also explored the ability of the serine protease matriptase to proteolytically process cell surface localised CDCP1 because it is unknown whether matriptase can cleave cell surface CDCP1 as it has been reported for other proteases such as trypsin and plasmin. Furthermore, the consequences of matriptase-mediated proteolysis on cell phenotype in vitro and cell signalling were examined since recent reports suggested that proteolysis of CDCP1 leads to its phosphorylation and may initiate cell signalling and consequently alter cell phenotype. It was found that matriptase is able to proteolytically process cell surface CDCP1 at low nanomolar concentrations which suggests that cleavage of CDCP1 by matriptase may facilitate the generation of LWM-CDCP1 in vivo. To examine whether matriptase-mediated proteolysis induced cell signalling anti-phospho Erk 1/2 Western blot analysis was performed as this pathway has previously been examined to study signalling in response to proteolytic processing of cell surface proteins. It was found that matriptase-mediated proteolysis in CDCP1 expressing HeLa cells initiated intracellular signalling via Erk 1/2. Interestingly, this increase in phosphorylation of Erk 1/2 was also observed in HeLa-vector cells. This suggested that initiation of cell signalling via Erk 1/2 phosphorylation as a result of matriptase-mediated proteolysis occurs by pathways independent of CDCP1. Subsequent investigations measuring the flux of free calcium ions and by using a protease-activated receptor 2 (PAR2) agonist peptide confirmed this hypothesis. These data suggested that matriptase-mediated proteolysis results in cell signalling via a pathway induced by the activation of PAR2 rather than by CDCP1. This indicates that induction of cell signalling in HeLa cells as a consequence of matriptase-mediated proteolysis occurs via signalling pathways which do not involve phosphorylation of Erk 1/2. Consequently, it appears that future attempts should focus on the examination of cellular pathways other than Erk 1/2 to elucidate cell signalling initiated by matriptase-mediated proteolytic processing of CDCP1. The data presented in this thesis has explored in vitro and in vivo aspects of the biology of CDCP1. The observations summarised above will permit the design of future studies to more precisely determine the role of CDCP1 and its binding partners in processes relevant to cancer progression. This may contribute to further defining CDCP1 as a target for cancer treatment.

Children re-read and re-write their neighborhoods : critical literacies and identity work

This chapter considers the complex literate repertoires of 21st century children in multicultural primary classrooms in Adelaide South Australia. It draws on the curricular and pedagogical work of two experienced primary school teachers who explore culture, race and class, by positioning children as textual producers across a variety of media. In particular we discuss two child-authored texts – A is for Arndale – a local alphabet book co-authored by children aged between eight and ten, and – Cooking Afghani Style - a magazine style film produced by a multi-aged class of children (aged eight to thirteen) recently arrived in Australia. In the process of making these texts, primary children engaged in reading as a cultural practice – re-reading and re-writing their neighbourhoods and identities (both individual and collective). This involved frequent excursions to local key sites, both familiar and unfamiliar to the children. They investigated how diverse children experienced and lived their lives in particular places within changing communities.

Critical literacies in place : teachers who work for just and sustainable communities

This chapter draws upon theories of social justice, critical literacy and place-based pedagogies and two research projects to discuss how teachers are working ethically and creatively towards a sustainable and just society in their place(s).

Critical reading comprehension in an era of accountability

This paper argues for the need for critical reading comprehension in an era of accountability that often promotes reading comprehension as readily assessable through students answering multiple choice questions of unseen texts. Based upon a 1 year study investigating literacy in Years 4–9 the ways strong-performing primary schools develop serious and in-depth reading for learning are explored. School and teacher features which allow for the development of sophisticated pedagogical repertoires and space for critical reading comprehension, without losing the complexity of curriculum offerings, are outlined. How one experienced middle primary teacher operates strategically, ethically and critically in supporting her ESL students to learn to read is illustrated. The teacher’s work is situated within the complex accountability demands faced by classroom teachers. This was accomplished by a teacher whose pedagogical repertoire has been assembled across a career teaching in low-SES high ESL communities in a school with a balanced literacy program and high level of collegial support. Risks for schools and teachers whose circumstances work against their capacities for prioritisation and strategic decision-making are identified and discussed.

Integrated literacies : every online player wins a prize

QUT Library’s model of learning support brings together academic literacy (study skills) and information literacy (research skills). The blended portfolio enables holistic planning and development, seamless services, connected learning resources and more authentic curriculum-embedded education. The model reinforces the Library’s strategic focus on learning service innovation and active engagement in teaching and learning. ----- ----- ----- The online learning strategy is a critical component of the broader literacies framework. This strategy unifies new and existing online resources (e.g.: Pilot, QUT cite|write and IFN001|AIRS Online) to augment learner capability. Across the suite, prudent application of emerging technologies with visual communications and learning design delivers a wide range of adaptive study tools. Separately and together, these resources meet the learning needs and styles of a diverse cohort providing positive and individual learning opportunities. Deliberate articulation with strategic directions regarding First Year Experience, assessment, retention and curriculum alignment assures that the Library’s initiatives move in step with institutional objectives relating to enhancing the student experience and flexible blended learning. ----- ----- ----- The release of Studywell in 2010 emphasises the continuing commitment to blended literacy education. Targeting undergraduate learners (particularly 1st year/transition), this online environment provides 24/7 access to practical study and research tools. Studywell’s design and application of technology creates a “discovery infrastructure” [1] which facilitates greater self-directed learning and interaction with content. ----- ----- ----- This paper presents QUT Library’s online learning strategy within the context of the parent “integrated literacies” framework. Highlighting the key online learning resources, the paper describes the inter-relationships between those resources to develop complementary literacies. The paper details broad aspects of the overarching learning and study support framework as well as the online strategy, including strategic positioning, quality and evaluation processes, maintenance, development, implementation, and client engagement and satisfaction with the learning resources.

Strategy in developing cell based therapy for large bone

Regeneration of osseous defects by tissue-engineering approach provides a novel means of treatment utilizing cell biology, materials science, and molecular biology. The concept of in vitro cultured osteoblasts having an ability to induce new bone formation has been demonstrated in the critical size defects using small animal models. The bone derived cells can be incorporated into bioengineered scaffolds and synthesize bone matrix, which on implantation can induce new bone formation. In search of optimal cell delivery materials, the extracellular matrix as cell carriers for the repair and regeneration of tissues is receiving increased attention. We have investigated extracellular matrix formed by osteoblasts in vitro as a scaffold for osteoblasts transplantation and found a mineralized matrix, formed by human osteoblasts in vitro, can initiate bone formation by activating endogenous mesenchymal cells. To repair the large bone defects, osteogenic or stem cells need to be prefabricated in a large three dimensional scaffold usually made of synthetic biomaterials, which have inadequate interaction with cells and lead to in vivo foreign body reactions. The interstitial extracellular matrix has been applied to modify biomaterials surface and identified vitronectin, which binds the heparin domain and RGD (Arg-Gly-Asp) sequence can modulate cell spreading, migration and matrix formation on biomaterials. We also synthesized a tri-block copolymer, methoxy-terminated poly(ethylene glycol)(MPEG)-polyL-lactide(PLLA)-polylysine(PLL) for human osteoblasts delivery. We identified osteogenic activity can be regulated by the molecular weight and composition of the triblock copolymers. Due to the sequential loss of lineage differentiation potential during the culture of bone marrow stromal cells that hinderers their potential clinical application, we have developed a clonal culture system and established several stem cell clones with fast growing and multi-differentiation properties. Using proteomics and subtractive immunization, several differential proteins have been identified and verified their potential application in stem cell characterization and tissue regeneration

Altered cell interactions of subchondral bone osteoblasts and articular chondrocytes in osteoarthritis is through the mediation of ERK1/2 phosphorylation

Introduction: Osteoarthritis (OA) is the most common musculoskeletal disorder and represents a major health burden to society. In the course of the pathological development of OA, articular cartilage chondrocytes (ACCs) undergo a typical phenotype changes characterized by the expression of hypertrophic differentiation markers. Also, the adjacent subchondral bone shows signs of abnormal mineral density and enhanced production of bone turnover markers, indicative of osteoblast dysfunction. However, the mechanism(s) by which these changes occur during the OA development are not completely understood. Materials and Methods: ACCs and subchondral bone osteoblasts (SBOs) were harvested from OA and healthy patients for the cross-talk studies between normal and OA ACCs and SBOs. The involvement of mitogen activated protein kinase (MAPK) signalling pathway during the cell-cell interactions was analysed by zymography, ELISA and western blotting methods. Results: The direct and in-direct co-culture studies showed that OA (ACCs and SBOs) cells induced osteoarthritic changes of normal (ACC and SBOs) cells. This altered cell interaction induced by OA cells significantly aggravated the proteolytic activity, which resulted cartilage degeneration. The altered cell interaction appeared to significantly activate ERK 1/2 phosphorylation and inhibition of MAPK-ERK 1/2 pathway reversed the osteoarthrtitic phenotypic changes. Discussion and Conclusion: Our study has demonstrated that the altered bi-directional communication of SBOs and ACCs are critical for initiation and progression of OA related changes and that this process is mediated by MAPK signalling pathways. Targeting these altered interactions by the use of MAPK inhibitors may provide the scientific rationale for the development of novel therapeutic strategies in the treatment and management of OA related disorders.

Critical literacy : foundational notes

This article traces the lineage of critical literacy from Freire through critical pedagogies and discourse analysis. It discusses the need for a contingent definition of critical literacy, given the increasingly sophisticated nature of texts and discourses.

Critical literacy finds a "Place": Writing and social action in a low-income Australian grade 2/3 classroom

In a study of socioeconomically disadvantaged children's acquisition of school literacies, a university research team investigated how a group of teachers negotiated critical literacies and explored notions of social power with elementary children in a suburban school located in an area of high poverty. Here we focus on a grade 2/3 classroom where the teacher and children became involved in a local urban renewal project and on how in the process the children wrote about place and power. Using the students' concerns about their neighborhood, the teacher engaged her class in a critical literacy project that not only involved a complex set of literate practices but also taught the children about power and the possibilities for local civic action. In particular, we discuss examples of children's drawing and writing about their neighborhoods and their lives. We explore how children's writing and drawing might be key elements in developing "critical literacies" in elementary school settings. We consider how such classroom writing can be a mediator of emotions, intellectual and academic learning, social practice, and political activism.

Innovation, interaction, and inclusion : heritage luxury brands in collusion with the consumer

Many luxury heritage brands operate on the misconception that heritage is interchangeable with history rather than representative of the emotional response they originally developed in their customer. This idea of heritage as static history inhibits innovation, prevents dynamic renewal and impedes their ability to redefine, strengthen and position their brand in current and emerging marketplaces. This paper examines a number of heritage luxury brands that have successfully identified the original emotional responses they developed in their customers and, through innovative approaches in design, marketing, branding and distribution evoke these responses in contemporary consumers. Using heritage and innovation hand-in-hand, these brands have continued to grow and develop a vision of heritage that incorporates both historical and contemporary ideas to meet emerging customer needs. While what constitutes a ‘luxury’ item is constantly challenged in this era of accessible luxury products, up-scaling and aspirational spending, this paper sees consumers’ emotional needs as the key element in defining the concept of luxury. These emotional qualities consistently remain relevant due to their ability to enhance a positive sense of identity for the brand user. Luxury is about the ‘experience’ not just the product providing the consumer with a sense of enhanced status or identity through invoked feelings of exclusivity, authenticity, quality, uniqueness and culture. This paper will analyse luxury heritage brands that have successfully combined these emotional values with those of their ‘heritage’ to create an aura of authenticity and nostalgia that appeals to contemporary consumers. Like luxury, the line where clothing becomes fashion is blurred in the contemporary fashion industry; however, consumer emotion again plays an important role. For example, clothing becomes ‘fashion’ for consumers when it affects their self perception rather than fulfilling basic functions of shelter and protection. Successful luxury heritage brands can enhance consumers’ sense of self by involving them in the ‘experience’ and ‘personality’ of the brand so they see it as a reflection of their own exclusiveness, authentic uniqueness, belonging and cultural value. Innovation is a valuable tool for heritage luxury brands to successfully generate these desired emotional responses and meet the evolving needs of contemporary consumers. While traditionally fashion has been a monologue from brand to consumer, new technology has given consumers a voice to engage brands in a conversation to express their evolving needs, ideas and feedback. As a result, in this consumer-empowered era of information sharing, this paper defines innovation as the ability of heritage luxury brands to develop new design and branding strategies in response to this consumer feedback while retaining the emotional core values of their heritage. This paper analyses how luxury heritage brands can effectively position themselves in the contemporary marketplace by separating heritage from history to incorporate innovative strategies that will appeal to consumer needs of today and tomorrow.

Improving team dynamics and innovation : the “Aspect Design” approach applied to interaction design

Interaction Design is a fast developing branch of Industrial Design. The availability of cheap microprocessors and sensor electronics allow interactions between people and products that were until recently impossible. This has added additional layers of complexity to the design process. Novice designers find it difficult to effectively juggle these complexities and typically tend to focus on one aspect at a time. They also tend to take a linear, step-by-step approach to the design process in contrast to expert designers who pursue “parallel lines of thought” whilst simultaneously co-evolving both problem and solution. (Lawson, 1993) This paper explores an approach that encourages designers (in this case novice designers) to take a parallel rather than linear approach to the design process. It also addresses the problem of social loafing that tends to occur in team activities.

Study of the interaction between 10-hydroxycamptothecine and DNA with the use of ethidium bromide dye as a fluorescence probe

The interaction of 10-hydroxycamptothecine (HCPT) with DNA under pseudo-physiological conditions (Tris-HCl buffer of pH 7.4), using ethidium bromide (EB) dye as a probe, was investigated with the use of spectrofluorimetry, UV-vis spectrometry and viscosity measurement. The binding constant and binding number for HCPT with DNA were evaluated as (7.1 ± 0.5) × 104 M-1 and 1.1, respectively, by multivariate curve resolution-alternating least squares (MCR-ALS). Moreover, parallel factor analysis (PARAFAC) was applied to resolve the three-way fluorescence data obtained from the interaction system, and the concentration information for the three components of the system at equilibrium was simultaneously obtained. It was found that there was a cooperative interaction between the HCPT-DNA complex and EB, which produced a ternary complex of HCPT-DNA-EB. © 2011 Elsevier B.V.

Intimate transactions : art, exhibition and interaction within distributed network environments

The broad research questions of the book are: How can successful, interdisciplinary collaboration contribute to research innovation through Practice-led research? What contributes to the design, production and curation of successful new media art? What are the implications of exhibiting it across dual sites for artists, curators and participant audiences? Is it possible to create an 'intimate transaction' between people who are separated by vast distances but joined by interfaces and distributed networks? Centred on a new media work of the same name by the Transmute Collective (led by Keith Armstrong), this book provides insights from multidisciplinary perspectives. Visual, sound and performance artists, furniture designers, spatial architects, technology systems designers, and curators who collaborated in the production of Intimate Transactions discuss their design philosophies, working processes and resolution of this major new media work. Analytical and philosophical essays by international writers complement these writings on production. They consider how new media art, like Intimate Transactions, challenges traditional understandings of art, curatorial installation and exhibition experience because of the need to take into account interaction, the reconfiguration of space, co-presence, performativity and inter-site collaboration.