Pro-matrix metalloproteinase-2 transfection increases orthotopic primary growth and experimental metastasis of MDA-MB-231 human breast cancer cells in nude mice

The ability to activate pro-matrix metalloproteinase (pro-MMP)-2 via membrane type-MMP is a hallmark of human breast cancer cell lines that show increased invasiveness, suggesting that MMP-2 contributes to human breast cancer progression. To investigate this, we have stably transfected pro-MMP-2 into the human breast cancer cell line MDA-MB-231, which lacks MMP-2 expression but does express its cell surface activator, membrane type 1-MMP. Multiple clones were derived and shown to produce pro-MMP-2 and to activate it in response to concanavalin A. In vitro analysis showed that the pro-MMP-2-transfected clones exhibited an increased invasive potential in Boyden chamber and Matrigel outgrowth assays, compared with the parental cells or those transfected with vector only. When inoculated into the mammary fat pad of nude mice, each of the MMP-2-tranfected clones grew faster than each of the vector controls tested. After intracardiac inoculation into nude mice, pro-MMP-2-transfected clones showed a significant increase in the incidence of metastasis to brain, liver, bone, and kidney compared with the vector control clones but not lung. Increased tumor burden was seen in the primary site and in lung metastases, and a trend toward increased burden was seen in bone, however, no change was seen in brain, liver, or kidney. This data supports a role for MMP-2 in breast cancer progression, both in the growth of primary tumors and in their spread to distant organs. MMP-2 may be a useful target for breast cancer therapy when refinement of MMP inhibitors provides for MMP-specific agents.

Transfection of MDA-MB-231 human breast carcinoma cells with bone sialoprotein (BSP) stimulates migration and invasion in vitro and growth of primary and secondary tumors in nude mice

We have investigated the role of bone sialoprotein (BSP), a secreted glycoprotein normally found in bone, in breast cancer progression. To explore functions for BSP in human breast cancer invasion and metastasis, the full-length BSP cDNA was transfected into the MDA-MB-231-BAG human breast cancer cell line under the control of the CMV promoter. Clones expressing BSP and vector control clones were isolated. BSP producing clones showed increased monolayer wound healing, a faster rate of stellate outgrowth in Matrigel and increased rate of invasion into a collagen matrix when compared to control clones. Clones were also examined in models of breast cancer growth and metastasis in vivo. BSP transfected clones showed an increased rate of primary tumor growth following mammary fat pad injection of nude mice. BSP transfected clones and vector control clones metastasized to soft organs and bone at a similar rate after intra-cardiac injection as determined by real-time PCR and X-ray analysis. Although these organs were targets for both BSP transfected and non-transfected cells, the size of the metastatic lesion was shown to be significantly larger for BSP expressing clones. This was determined by real-time PCR analysis for soft organs and by X-ray analysis of bone lesions. For bone this was confirmed by intra-tibial injections of cells in nude mice. We conclude that BSP acts to drive primary and secondary tumor growth of breast cancers in vivo.

An MMP13-selective inhibitor delays primary tumor growth and the onset of tumor-associated osteolytic lesions in experimental models of breast cancer

We investigated the effects of the matrix metalloproteinase 13 (MMP13)-selective inhibitor, 5-(4-{4-[4-(4-fluorophenyl)-1,3-oxazol-2-yl]phenoxy}phenoxy)-5-(2-methoxyethyl) pyrimidine-2,4,6(1H,3H,5H)-trione (Cmpd-1), on the primary tumor growth and breast cancer-associated bone remodeling using xenograft and syngeneic mouse models. We used human breast cancer MDA-MB-231 cells inoculated into the mammary fat pad and left ventricle of BALB/c Nu/Nu mice, respectively, and spontaneously metastasizing 4T1.2-Luc mouse mammary cells inoculated into mammary fat pad of BALB/c mice. In a prevention setting, treatment with Cmpd-1 markedly delayed the growth of primary tumors in both models, and reduced the onset and severity of osteolytic lesions in the MDA-MB-231 intracardiac model. Intervention treatment with Cmpd-1 on established MDA-MB-231 primary tumors also significantly inhibited subsequent growth. In contrast, no effects of Cmpd-1 were observed on soft organ metastatic burden following intracardiac or mammary fat pad inoculations of MDA-MB-231 and 4T1.2-Luc cells respectively. MMP13 immunostaining of clinical primary breast tumors and experimental mice tumors revealed intra-tumoral and stromal expression in most tumors, and vasculature expression in all. MMP13 was also detected in osteoblasts in clinical samples of breast-to-bone metastases. The data suggest that MMP13-selective inhibitors, which lack musculoskeletal side effects, may have therapeutic potential both in primary breast cancer and cancer-induced bone osteolysis.

Cisplatin treatment of primary and metastatic epithelial ovarian carcinomas generates residual cells with mesenchymal stem cell-like profile

Epithelial mesenchymal transition (EMT) and cancer stem cells (CSC) have been associated with resistance to chemotherapy. Eighty percent of ovarian cancer patients initially respond to platinum-based combination therapy but most return with recurrence and ultimate demise. To better understand such chemoresistance we have assessed the potential role of EMT in tumor cells collected from advanced-stage ovarian cancer patients and the ovarian cancer cell line OVCA 433 in response to cisplatin in vitro. We demonstrate that cisplatin-induced transition from epithelial to mesenchymal morphology in residual cancer cells correlated with reduced E-cadherin, and increased N-cadherin and vimentin expression. The mRNA expression of Snail, Slug, Twist, and MMP-2 were significantly enhanced in response to cisplatin and correlated with increased migration. This coincided with increased cell surface expression of CSC-like markers such as CD44, α2 integrin subunit, CD117, CD133, EpCAM, and the expression of stem cell factors Nanog and Oct-4. EMT and CSC-like changes in response to cisplatin correlated with enhanced activation of extracellular signal-regulated kinase (ERK)1/2. The selective MEK inhibitor U0126 inhibited ERK2 activation and partially suppressed cisplatin-induced EMT and CSC markers. In vivo xenotransplantation of cisplatin-treated OVCA 433 cells in zebrafish embryos demonstrated significantly enhanced migration of cells compared to control untreated cells. U0126 inhibited cisplatin-induced migration of cells in vivo, suggesting that ERK2 signaling is critical to cisplatin-induced EMT and CSC phenotypes, and that targeting ERK2 in the presence of cisplatin may reduce the burden of residual tumor, the ultimate cause of recurrence in ovarian cancer patients.

The experiences of teachers teaching children with Down syndrome in the early years of schooling

This innovative collective case study research documented teachers' experiences of teaching children with Down syndrome in the early years of schooling in Australia. Results indicated differences in teachers' conceptualisation of children with Down syndrome as learners and how these variations impacted the way the child was included within the class. Unique to this research was the utilisation of a mind-mapping technique of data collection which effectively captured the individual nature of teachers' experiences, making implicit knowledge explicit through description and interpretation of these experiences. Overall findings indicated that teachers were more likely to include children with Down syndrome into general education classrooms if they operated within a contemporary understanding of disability, had positive support from key stakeholders such as school principals and parents/caregivers, and had access to current information on Down syndrome from professional bodies.

Laminin adhesion-selected primary human colon-cancer cells are more tumorigenic than the parenteral and nonadherent cells

Laminin has been shown to promote the malignant phenotype and the level of the 32/67 Kd laminin receptor has been found to correlate with Dukes' staging of colon cancer. A biopsy of a Dukes' stage B2 human colon carcinoma formed a tumor in a nude mouse after coinjection with Matrigel. The parental tumor and the murine tumor appeared identical at the histological level. A cell line LCC-C1 was established from the murine tumor. The cell line appeared moderately differentiated although it did not produce mucin in vitro; however, the xenograft in vivo did produce low levels of mucin. Laminin adherent and non-adherent cell lines were selected. The parental and the laminin-selected cell subclones adhered equally well to plastic and to fibronectin and showed similar growth rates on plastic. When injected subcutaneously into nude mice, the laminin-adherent cells formed relatively undifferentiated tumors that were twice as large as the parental cell tumors whereas the laminin non adherent cells formed very small, but highly differentiated tumors. These data demonstrate that subpopulations of tumor cells which differ in their tumorigenic properties can be selected based on their adhesion to laminin and thus provide models for studying the mechanisms of tumor growth.

Literacy assessment that counts : mediating, interpreting and contesting translocal policy in a primary school

In Australia, as in many western education systems over the last two decades, discourses of accountability and performativity have reshaped education policy that has in turn reorganised the work of school leaders and teachers. One of the effects of this reorganisation is increased attention to the production, analysis and display of student achievement data. In this paper we examine in detail a sequence of the production and reading of literacy assessment data in a small Catholic school. Our analysis uses institutional ethnography’s concept of the ‘active text’, the text as occurring in a specific place and time even as it is articulated to social relations beyond its immediate context. Through this process we learn from those involved how their everyday work brings into being formalised, textually authorised processes in a local site that ensure the school meets accountability requirements while enabling teachers to resist standardisation of literacy teaching and assessment.

Time course analysis of hypoxia, granulation tissue and blood vessel growth, and remodeling in healing rat cutaneous incisional primary intention wounds

Hypoxia and the development and remodeling of blood vessels and connective tissue in granulation tissue that forms in a wound gap following full-thickness skin incision in the rat were examined as a function of time. A 1.5 cm-long incisional wound was created in rat groin skin and the opposed edges sutured together. Wounds were harvested between 3 days and 16 weeks and hypoxia, percent vascular volume, cell proliferation and apoptosis, α-smooth muscle actin, vascular endothelial growth factor-A, vascular endothelial growth factor receptor-2, and transforming growth factor-β 1 expression in granulation tissue were then assessed. Hypoxia was evident between 3 and 7 days while maximal cell proliferation at 3 days (123.6 ± 22.2 cells/mm 2, p < 0.001 when compared with normal skin) preceded the peak percent vascular volume that occurred at 7 days (15.83 ± 1.10%, p < 0.001 when compared with normal skin). The peak in cell apoptosis occurred at 3 weeks (12.1 ± 1.3 cells/mm 2, p < 0.001 when compared with normal skin). Intense α-smooth muscle actin labeling in myofibroblasts was evident at 7 and 10 days. Vascular endothelial growth factor receptor-2 and vascular endothelial growth factor-A were detectable until 2 and 3 weeks, respectively, while transforming growth factor-β 1 protein was detectable in endothelial cells and myofibroblasts until 3-4 weeks and in the extracellular matrix for 16 weeks. Incisional wound granulation tissue largely developed within 3-7 days in the presence of hypoxia. Remodeling, marked by a decline in the percent vascular volume and increased cellular apoptosis, occurred largely in the absence of detectable hypoxia. The expression of vascular endothelial growth factor-A, vascular endothelial growth factor receptor-2, and transforming growth factor-β 1 is evident prior, during, and after the peak of vascular volume reflecting multiple roles for these factors during wound healing.

Asthma interventions in primary schools : a review

Objective To explore, in depth, the literature for evidence supporting asthma interventions delivered within primary schools and to identify any “gaps” in this research area. Methods A literature search using electronic search engines (i.e. Medline, PubMed, Education Resources Information Center (ERIC), International Pharmaceutical Abstracts (IPA), Cumulative Index to Nursing and Allied Health Literature (CINAHL), Embase and Informit) and the search terms “asthma”, “asthma intervention” and “school-based asthma education program” (and derivatives of these keywords) was conducted. Results Twenty-three articles met the inclusion criteria; of these eight were Randomised Controlled Trials. There was much variety in the type, content, delivery and outcome measures in these 23 studies. The most common intervention type was asthma education delivery. Most studies demonstrated improvement in clinical and humanistic markers, for example, asthma symptoms medication use (decrease in reliever medication use or decrease in the need for rescue oral steroid), inhaler use technique and spacer use competency, lung function and quality of life. Relatively few studies explored the effect of the intervention on academic outcomes. Most studies did not report on the sustainability or cost effectiveness of the intervention tested. Another drawback in the literature was the lack of details about the intervention and inconsistency in instruments selected for measuring outcomes. Conclusion School-based asthma interventions regardless of their heterogeneity have positive clinical, humanistic, health economical and academic outcomes.

Development and pilot study of the Primary Care Practice Improvement Tool (PC-PIT) : an innovative approach

Objective To assess the usability and validity of the Primary Care Practice Improvement Tool (PC-PIT), a practice performance improvement tool based on 13 key elements identified by a systematic review. It was co-created with a range of partners and designed specifically for primary health care. Design This pilot study examined the PC-PIT using a formative assessment framework and mixed-methods research design. Setting and participants Six high-functioning general practices in Queensland, Australia, between February and July 2013. A total of 28 staff participated — 10 general practitioners, six practice or community nurses, 12 administrators (four practice managers; one business manager and eight reception or general administrative staff). Main outcome measures Readability, content validity and staff perceptions of the PC-PIT. Results The PC-PIT offers an appropriate and acceptable approach to internal quality improvement in general practice. Quantitative assessment scores and qualitative data from all staff identified two areas in which the PC-PIT required modification: a reduction in the indicative reading age, and simplification of governance-related terms and concepts. Conclusion The PC-PIT provides an innovative approach to address the complexity of organisational improvement in general practice and primary health care. This initial validation will be used to develop a suite of supporting, high-quality and free-to-access resources to enhance the use of the PC-PIT in general practice. Based on these findings, a national trial is now underway.

Consumers persepctive on pharmacists integration into private primary healthcare clinics in Malaysia

Background: Pharmacists are considered medication experts but are underutilised mainly at the periphery of the primary healthcare team. General medical practitioners (GPs) in Malaysian private healthcare clinics are granted rights to prescribe and dispense medications, thus furhter limiting pharmacists involvement in ensuring safe use of medicines. The integration of pharmacist into private primary healthcare clinics has the potential to reduce medication-relation problems. Objective: To explore the views of consumers on the integration of pharmacists within private primary healthcare clinics in Malaysia. Method: A purposive sample of healthcare consumers in Selangor and Kuala Lumpur, Malaysia were invited to participate in focus groups and semi-structured interviews. Sessions were audio recorded and transcribed verbatim and thematically analysed using NVivo 10. Results: A total of 24 healthcare consumers particpated in two focus groups and six semi-structured interviews. Four major themes were identified: (1) Pharmacists role viewed mainly as supplying medications, (2) Readiness to accept pharmacists in private healthcare clinics, (3) Willingness to pay for pharmacy services, and (4) Concerns about GPs resistance to pharmacist integration. Consumers felt that a pharmacist integrated into private prumary healthcare clinics could offer potential benefits such as counter-checking prescriptions to ensure correct medication is supplied and counselling consumers on their medications and the potential side effects. The potential to increase in costs to consumers and GPs reluctance were perceived as barriers to integration. Conclusion: This study provides insights into consumers perspectives on the roles of pharmacists within private primary healthcare clinics in Malaysia. Consumers generally supported pharmacist integration into private primary healthcare clinics. However, for pharmacists to expand their capacity in providing integrated and collaborative primary care services to consumers, barriers to pharmacist integration need to be addressed.

The expression profiles of the galectin gene family in primary and metastatic papillary thyroid carcinoma with particular emphasis on galectin-1 and galectin-3 expression

INTRODUCTION: Galectin family members have been demonstrated to be abnormally expressed in cancer at the protein and mRNA level. This study investigated the levels of galectin proteins and mRNA expression in a large cohort of patients with papillary thyroid carcinoma and matched lymph node metastases with particular emphasis on galectin-1 and galectin-3. METHODS: mRNA expression of galectin family members (1, 2, 3, 4, 7, 8, 9, 10 and 12) were analysed by real-time polymerase chain reaction in 65 papillary thyroid carcinomas, 30 matched lymph nodes with metastatic papillary thyroid carcinoma and 5 non-cancer thyroid tissues. Galectin-1 and 3 protein expression was determined by immunohistochemistry in these samples. RESULTS: Significant expression differences in all tested galectin family members (1, 2, 3, 4, 7, 8, 9, 10 and 12) were noted for mRNA in papillary thyroid carcinomas, with and without lymph node metastasis. Galectin-1 protein was more strongly expressed than galectin-3 protein in papillary thyroid carcinoma. Galectin-1 protein was found to be overexpressed in 32% of primary papillary thyroid carcinomas. A majority of lymph nodes with metastatic papillary thyroid carcinoma (53%) had significantly increased expression of galectin-1 protein, as did 47% of primaries with metastases. Galectin-1 mRNA levels were decreased in the vast majority (94%) of primary thyroid carcinomas that did not have metastases present. Galectin-3 protein levels were noted to be overexpressed in 15% of primary papillary thyroid carcinomas. In primary papillary thyroid carcinoma with lymph node metastases, 32% had over expression of galectin-3 protein. Overexpression of galectin-3 mRNA was noted in 58% of papillary thyroid carcinomas and 64% of lymph nodes bearing metastatic papillary thyroid carcinoma. Also, primary papillary thyroid carcinoma with lymph node metastases had significantly higher expression of galectin-3 mRNA compared to those without lymph node metastases. CONCLUSION: Galectin family members show altered expression at the mRNA level in papillary thyroid cancers. Overexpression of galectin-1 and 3 proteins were noted in papillary thyroid carcinoma with lymph node metastases. The results presented here demonstrated that galectin-1 and galectin-3 expression have important roles in clinical progression of papillary thyroid carcinoma.

Detection of dynamic primary user with cooperative spectrum sensing

In this paper we demonstrate that existing cooperative spectrum sensing formulated for static primary users cannot accurately detect dynamic primary users regardless of the information fusion method. Performance error occurs as the sensing parameters calculated by the conventional detector result in sensing performance that violates the sensing requirements. Furthermore, the error is accumulated and compounded by the number of cooperating nodes. To address this limitation, we design and implement the duty cycle detection model for the context of cooperative spectrum sensing to accurately calculate the sensing parameters that satisfy the sensing requirements. We show that longer sensing duration is required to compensate for dynamic primary user traffic.

Literacy, poverty and schooling : what matters in young people's education?

This paper draws upon several decades of literacy research in schools in high poverty environments to explore what matters in young people’s education. In dialogue with themes from Kevin Marjoribanks’ work, such as student aspirations, family environments, and teacher expectations, key insights are summarised. Referring to longitudinal case studies and a current ethnographic project, the interplay between literacy, poverty and schooling, and, young people’s aspirations and education outcomes is explored. While the work of educators in high poverty communities continues to be highly demanding, there are some schools and teachers making a durable positive difference to learner dispositions and literate repertoires. Teacher expectations and discursive practices are crucial in this process.

Genome-wide analysis in a murine Dnmt1 knockdown model identifies epigenetically silenced genes in primary human pituitary tumors

DNA methylation at promoter CpG islands (CGI) is an epigenetic modification associated with inappropriate gene silencing in multiple tumor types. In the absence of a human pituitary tumor cell line, small interfering RNA-mediated knockdown of the maintenance methyltransferase DNA methyltransferase (cytosine 5)-1 (Dnmt1) was used in the murine pituitary adenoma cell line AtT-20. Sustained knockdown induced reexpression of the fully methylated and normally imprinted gene neuronatin (Nnat) in a time-dependent manner. Combined bisulfite restriction analysis (COBRA) revealed that reexpression of Nnat was associated with partial CGI demethylation, which was also observed at the H19 differentially methylated region. Subsequent genome-wide microarray analysis identified 91 genes that were significantly differentially expressed in Dnmt1 knockdown cells (10% false discovery rate). The analysis showed that genes associated with the induction of apoptosis, signal transduction, and developmental processes were significantly overrepresented in this list (P < 0.05). Following validation by reverse transcription-PCR and detection of inappropriate CGI methylation by COBRA, four genes (ICAM1, NNAT, RUNX1, and S100A10) were analyzed in primary human pituitary tumors, each displaying significantly reduced mRNA levels relative to normal pituitary (P < 0.05). For two of these genes, NNAT and S100A10, decreased expression was associated with increased promoter CGI methylation. Induced expression of Nnat in stable transfected AtT-20 cells inhibited cell proliferation. To our knowledge, this is the first report of array-based "epigenetic unmasking" in combination with Dnmt1 knockdown and reveals the potential of this strategy toward identifying genes silenced by epigenetic mechanisms across species boundaries.