Expression of c-ets-1 mRNA is associated with an invasive, EMT-derived phenotype in breast carcinoma cell lines

We have previously observed in vitro that some stromal proteinases (MMP- 2, MT1-MMP) were expressed or activated by invasive carcinoma cell lines exhibiting mesenchymal features, presumably acquired through an epithelial to mesenchymal transition (EMT). To examine the potential contribution of c- ets-1 to this phenotype, we have compared here the expression of c-ets-1 with invasiveness in vitro and expression of vimentin, E-cadherin, uPA, MMP-1 and MMP-3 in a panel of human breast cancer cell lines. Our results clearly demonstrate an association between c-ets-1 expression and the invasive, EMT- derived phenotype, which is typified by the expression of vimentin and the lack of E-cadherin. While absent from the two non-invasive, vimentin-negative cell lines, c-ets-1 was abundantly expressed in all the four vimentin- positive lines. However, we could not find a clear quantitative or qualitative relationship between the expression of c-ets-1 and the three proteinases known to he regulated by c-ets-1, except that when they were expressed, it was only in the invasive c-ets-1-positive lines. UPA mRNAs were found in three of the four vimentin-positive lines, MMP-1 in two of the four, and MMP-3 could not be detected in any of the cell lines. Intriguingly, MDA- MB-435 cells, which exhibit the highest metastatic potential of these cell lines in nude mice, expressed vimentin and c-ets-1, but lacked expression of these three proteinases, at least under the culture conditions employed. Taken together, our results show that c-ets-1 expression is associated with an invasive, EMT-derived phenotype in breast cancer cells, although it is apparently not sufficient to ensure the expression of uPA, MMP-1 or MMP-3, in the vimentin-positive cells. Such proteases regulation is undoubtedly qualified by the cellular context. This study therefore advances our understanding of the molecular regulation of invasiveness in EMT-associated carcinoma progression, and suggests that c-ets-1 may contribute to the invasive phenotype in carcinoma cells.

MT1-MMP correlates with MMP-2 activation potential seen after epithelial to mesenchymal transition in human breast carcinoma cells

We have previously reported that human breast carcinoma (HBC) cell lines expressing the mesenchymal intermediate filament protein vimentin (VIM+) are highly invasive in vitro, and highly metastatic in nude mice when compared to their VIM- counterparts. Since only VIM+ cell lines can be induced to activate matrix metalloproteinase-2 (MMP-2) upon stimulation with Concanavalin A (Con A), we have examined here membrane type 1 MMP (MT1-MMP), a cell surface activator of MMP-2. Northern analysis reveals baseline expression of MT1-MMP in five of the six VIM+ cell lines studied (MDA-MB-231, MDA-MB-435, BT-549, Hs578T, MCF-7(ADR)), each of which showed variable activation of exogenous MMP-2 after treatment with Con A. In contrast, the four VIM-, poorly invasive HBC cell lines studied (MCF-7, T47D, MDA-MB 468, ZR-75-1) lacked baseline MT1-MMP mRNA expression, and showed no induction of either MT1-MMP expression or MMP-2-activation with Con A. Such differential MT1-MMP expression was confirmed in vivo using in situ hybridization analysis of nude mouse tumor xenografts of representative cell lines. Western analysis of the MDA-MB-231 cells revealed baseline membrane expression of a 60 kDa species, which was strongly induced by Con A treatment along with a weaker band co-migrating with that from MT1-MMP-transfected COS-1 cells (63 kDa), presumably representing latent MT1-MMP. MT1-MMP immunofluorescence strongly decorated Con A-stimulated MDA-MB-231 cells in a manner consistent with membranous staining, but did not decorate the unstimulated MDA-MB-231 cells or MCF-7 cells under either condition. Collectively, the results suggest the constitutive production of active MT1-MMP which is unavailable for either MMP-2 activation or immuno-decoration until Con A treatment. Since VIM expression arises by virtue of the so-called epithelial to mesenchymal transition (EMT) in invasive embryonic epithelia, we propose that this represents a major metastasis mechanism in breast carcinomas. MT1-MMP on the surface of such 'fibroblastoid' carcinoma cells may mediate a paracrine loop for the utilization of stromally produced MMP-2, and contribute to the poorer survival associated with VIM+ breast carcinomas.

The LCC15-MB human breast cancer cell line expresses osteopontin and exhibits an invasive and metastatic phenotype

We have characterized the LCC15-MB cell line which was recently derived from a breast carcinoma metastasis resected from the femur of a 29-year-old woman. LCC15-MB cells are vimentin (VIM) positive, exhibit a stellate morphology in routine cell culture, and form penetrating colonies when embedded in three-dimensional gels of Matrigel or fibrillar collagen. They show high levels of activity in the Boyden chamber chemomigration and chemoinvasion assays, and like other invasive human breast cancer (HBC) cell lines, LCC15-MB cells activate matrix-metalloproteinase-2 in response to treatment with concanavalin A. In addition, these cells are tumorigenic when implanted subcutaneously in nude mice and recolonize bone after arterial injection. Interestingly, both the primary lesion and the bone metastasis from which LCC15-MB were derived, as well as the resultant cell line, abundantly express the bone matrix protein osteopontin (OPN). OPN is also expressed by the highly metastatic MDA-MB-435 cells, but not other invasive or noninvasive HBC cell lines. Expression of OPN is retained in the subcutaneous xenograft and intraosseous metastases of LCC15-MB as detected by immunohistochemistry. Both VIM and OPN expression have been associated with breast cancer invasion and metastasis, and their expression by the LCC15-MB cell line is consistent with its derivation from a highly aggressive breast cancer. These cells provide a useful model for studying molecular mechanisms important for breast cancer metastasis to bone and, in particular, the implication(s) of OPN and VIM expression in this process.

Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines

Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM- (MCF-7, T47D, ZR-75-1), and ER-/VIM- (MDA-MB-468, SK-Br-3) cell lines were uninvasive, while ER-/VIM+ (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER-/VIM+ cell lines were significantly more invasive than either the ER+/VIM- or ER-/VIM- cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER-/ VIM+ cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-436. Hs578T cells (ER-/VIM+) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER-/VIM+ MCF-7ADR subline was significantly more active than the MCF-7 cells in vitro, but resembled the wild-type MCF-7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF-7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.

Association of MMP-2 activation potential with metastatic progression in human breast cancer cell lines independent of MMP-2 production

Background: Expression of matrix metalloproteinase-2 (MMP-2), the 72-kd type IV collagenase/gelatinase, by cancer cells has been implicated in metastasis through cancer cell invasion of basement membranes mediated by degradation of collagen IV. However, the abundance of this latent proenzyme in normal tissues and fluids suggests that MMP-2 proenzyme utilization is limited by its physiological activation rather than expression alone. We previously reported activation of this proenzyme by normal and malignant fibroblastoid cells cultured on collagen I (vitrogen) gels. Purpose: Our purposes in this study were 1) to determine whether MMP-2 activation is restricted to the more invasive human breast cancer cell lines and 2) to localize the activating mechanism. Methods: Zymography was used to monitor MMP-2 activation through detection of latent MMP-2 (72 kd) and mature species of smaller molecular weight (59 or 62 kd). Human breast cancer cell lines cultured on plastic, vitrogen, and other matrices were thus screened for MMP- 2 activation. Collagen I-cultured cells were exposed to cycloheximide, a protein synthesis inhibitor, or to protease inhibitors to determine the nature of the MMP-2-activating mechanism. Triton X-114 (TX-114) detergent extracts from cells cultured on collagen I or plastic were incubated with latent MMP-2 and analyzed by zymography to localize the MMP-2 activator. Results: MMP-2 activation was only induced by collagen I culture in the more aggressive, highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines (Hs578T, MDA-MB-436, BT549, MDA-MB-231, MDA- MB-435, MCF-7(ADR)) and was independent of MMP-2 production. MMP-2 activation was detected in cells cultured on collagen I gels but not in those cultured on gelatin gels, Matrigel, or thin layers of collagen I or IV, gelatin, or fibronectin. Collagen-induced activation was specific for the enzyme species MMP-2, since MMP-9, the 92-kd type IV collagenase/gelatinase, was not activatable under similar conditions. MMP-2 activation was inhibited by cycloheximide and was sensitive to a metalloproteinase inhibitor but not to aspartyl, serine, or cysteinyl protease inhibitors. MMP-2 activation was detected in the hydrophobic, plasma membrane-enriched, TX-114 extracts from invasive collagen I-cultured cells. Conclusion: Collagen I-induced MMP-2 activation is restricted to highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines, is independent of MMP-2 production, and is associated with metastatic potential. Our findings are consistent with plasma membrane localization of the activator. Implications: The MMP-2 activation mechanism may represent a new target for diagnosis, prognosis, and treatment of human breast cancer.

Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and β-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, β-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses.

Tumor cells are the source of osteopontin and bone sialoprotein expression in human breast cancer

Bone sialoprotein (BSP) and osteopontin (OPN) are secreted glycoproteins with a conserved Arg-Gly-Asp (RGD) integrin-binding motif and are expressed predominantly in bone. The RGD tripeptide is commonly present in extracellular attachment proteins and has been shown to mediate the attachment of osteosarcoma cells and osteoclasts. To determine the origin and incidence of BSP and OPN mRNA expression in primary tumor, a cohort of archival, primary invasive breast carcinoma specimens was analyzed. BSP transcripts were detected in 65% and OPN transcripts in 77% of breast cancers examined. In general, BSP and OPN transcripts were detected in both invasive and in situ carcinoma components. The transcripts were not detected in surrounding stromal cells or in peritumoral macrophages. Despite its abundance in carcinomas, BSP expression was not detected in a panel of 11 human breast cancer cell lines (MCF-7, T47D, SK-Br-3, MDA-MB-453, MDA-MB- 231, MDA-MB-436, BT549, MCF-7(AOR), Hs578T, MDA-MB-435, and LCC15-MB) and OPN expression was detected only in two of these (MDA-MB-435 and LCC15-MB). To examine the possibility that expression of these genes was down-regulated in cell culture, several cell lines were grown as nude mouse xenografts in vivo; however, these tumors also failed to express BSP. OPN expression was identified in all cell lines grown as nude mouse xenografts. Our data suggest that in human primary breast tumors, the origin of BSP and OPN mRNA is predominantly the breast cancer cells and that expression of these transcripts is influenced by the tumor environment.

The impact of nurse practitioner services on cost, quality of care, satisfaction and wait times in the emergency department: A systematic review

Aims To provide the best available evidence to determine the impact of nurse practitioner services on cost, quality of care, satisfaction and waiting times in the emergency department for adult patients. Background The delivery of quality care in the emergency department is one of the most important service indicators in health delivery. Increasing service pressures in the emergency department have resulted in the adoption of service innovation models: the most common and rapidly expanding of these is emergency nurse practitioner services. The rapid uptake of emergency nurse practitioner service in Australia has outpaced the capacity to evaluate this service model in terms of outcomes related to safety and quality of patient care. Previous research is now outdated and not commensurate with the changing domain of delivering emergency care with nurse practitioner services. Data A comprehensive search of four electronic databases from 2006-­‐2013 was conducted to identify research evaluating nurse practitioner service impact in the emergency department. English language articles were sought using MEDLINE, CINAHL, Embase and Cochrane and included two previous systematic reviews completed five and seven years ago. Methods A three step approach was used. Following a comprehensive search, two reviewers assessed identified studies against the inclusion criteria. From the original 1013 studies, 14 papers were retained for critical appraisal on methodological quality by two independent reviewers and data extracted using standardised tools. Results Narrative synthesis was conducted to summarise and report the findings as insufficient data was available for meta-­‐analysis of results. This systematic review has shown that emergency nurse practitioner service has a positive impact on quality of care, patient satisfaction and waiting times. There was insufficient evidence to draw conclusions regarding impact on costs. Conclusion Synthesis of the available research attempts to provide an evidence base for emergency nurse practitioner service to guide healthcare leaders, policy makers and clinicians in reforming emergency department service provision. The findings suggest that further quality research is required for comparative measures of clinical and service effectiveness of emergency nurse practitioner service. In the context of increased health service demand and the need to provide timely and effective care to patients, such measures will assist in delivering quality patient care.

Two-channel waveguide modulator based on surface wave propagating in a semiconductor-metal thin-film structure

The results of theoretical investigations of two-channel waveguide modulator based on Surface Wave (SW) propagation are presented. The structure studied consists of two n-type semiconductor waveguide channels separated from each other by a dielectric gap and coated by a metal. The SW propagates at the semiconductor-metal interface across an external magnetic field which is parallel to the interface. An external dc voltage is applied to the metal surface of one channel to provide a small phase shift between two propagating modes. In a coupled mode approximation, two possible regimes of operation of the structure, namely as a directional coupler and as an electro-optical modulator, are considered. Our results suggest new applications in millimeter and submillimeter wave solid-state electronics and integrated optics.

Multimedia copyright protection

Internet and its widespread usage for multimedia document distribution put the copyright issue in a complete new setting. Multimedia documents, specifically those installed on a web page, are no longer passive as they typically include active applets. Copyright protection safeguards the intellectual property (IP) of multimedia documents, which are either sold or distributed free of charge. In this Chapter, the basic tools for copyright protection are discussed. First, general concepts and the vocabulary used in copyright protection of multimedia documents are discussed. Later, taxonomy of watermarking and fingerprinting techniques are studied. This part is concluded by a review of the literature dealing with IP security. The main part of the chapter discusses the generic watermarking scheme and illustrates it on three specific examples: collusion-free watermarking, spread spectrum watermarking, and software fingerprinting. Future trends and conclusions close the chapter.

Pulmonary innate immunity in children with protracted bacterial bronchitis

Objective To determine bronchoalveolar lavage (BAL) levels of 3 innate immunity components (human alpha-defensin-2 [hBD2], mannose-binding lectin [MBL], and surfactant protein-A [SP-A], the relationship with airway neutrophilia and infection, and cytokine production of stimulated BAL cells in children with current protracted bacterial bronchitis (PBB), children with resolved PBB (PBB well), and controls. Study design BAL of 102 children (mean age 2.8 years) fulfilling predefined criteria of current PBB (n=61), PBB well (n=20), and controls (n=21) was cultured (quantitative bacteriology) and viruses examined by polymerase chain reaction. hBD2, MBL, and SP-A were measured, and cytokine production of lipopolysaccharide-stimulated BAL cells were determined. Results Median hBD2 and MBL levels were significantly higher in the current PBB group (hBD2 = 164.4, IQR 0-435.5pg/mL; MBL = 1.7, 0.4-4ng/mL) than in the PBB well group (hBD2 = 0, IQR 0-85.2; MBL = 0.6, IQR 0.03-2.9) and controls (hBD2 = 3.6, IQR 0-126; MBL = 0.4, IQR 0.02-79). hBD2 was significantly higher in children with airway infection (n = 54; median 76.9, IQR 0-397.3) compared with those without (n = 48; 0, IQR 0-236.3), P=0.04. SP-A levels and cytokine production of stimulated BAL cells were similar between groups. Conclusion In children's airways, hBD2, but not MBL and SP-A, relates to inflammation and infection. In children with PBB, mechanisms involving airway hBD2 and MBL are augmented. These pulmonary innate immunity components and the ability of BAL cells to respond to stimuli are unlikely to be deficient.

Socio-demographic vulnerability to heatwave impacts in Brisbane, Australia : a time series analysis

Objective: Examining the association between socioeconomic disadvantage and heat-related emergency department (ED) visits during heatwave periods in Brisbane, 2000–2008. Methods: Data from 10 public EDs were analysed using a generalised additive model for disease categories, age groups and gender. Results: Cumulative relative risks (RR) for non-external causes other than cardiovascular and respiratory diseases were 1.11 and 1.05 in most and least disadvantaged areas, respectively. The pattern persisted on lags 0–2. Elevated risks were observed for all age groups above 15 years in all areas. However, with RRs of 1.19–1.28, the 65–74 years age group in more disadvantaged areas stood out, compared with RR=1.08 in less disadvantaged areas. This pattern was observed on lag 0 but did not persist. The RRs for male presentations were 1.10 and 1.04 in most and less disadvantaged areas; for females, RR was 1.04 in less disadvantaged areas. This pattern persisted across lags 0–2. Conclusions: Heat-related ED visits increased during heatwaves. However, due to overlapping confidence intervals, variations across socioeconomic areas should be interpreted cautiously. Implications: ED data may be utilised for monitoring heat-related health impacts, particularly on the first day of heatwaves, to facilitate prompt interventions and targeted resource allocation.

Book Review : "The Media Gaze: Representations of Diversities in Canada by Augie Fleras". Vancouver and Toronto: UBC Press, 2011.

The Media Gaze effectively shatters the assumption that Canada, in all its political correctness, is a cultural mosaic free of discrimination and prejudice. While great strides have been made to reduce blatant racism and sexism in Canadian media, Fleras illustrates how discriminatory and oppressive discourses are still very present in news, television, and film.He brings to light the structural, institutional, and practice-oriented means by which the media is systemically biased toward privileging mainstream audiences while misrepresenting minority groups in the public eye...

Unswitch-ABL drugs overcome resistance in chronic myeloid leukemia

ABL inhibitors have revolutionized the clinical management of chronic myeloid leukemia, but the BCR-ABLT315I mutation confers resistance to currently approved drugs. Chan et al. show, in this issue of Cancer Cell, that " switch-control" inhibitors block BCR-ABLT315I activity by preventing ABL from switching from the inactive to active conformation.