Loss of epithelial markers and acquisition of vimentin expression in adriamycin- and vinblastine-resistant human breast cancer cell lines

We have previously observed that breast cancer cell lines could exhibit either epithelial or fibroblastic phenotypes as reflected by their morphologies and intermediate filament protein expression (C. L. Sommers, D. Walker-Jones, S. E. Heckford, P. Worland, E. Valverius, R. Clark, M. Stampfer, and E. P. Gelmann, Cancer Res., 49: 4258-4263, 1989). Fibroblastoid, vimentin-expressing breast cancer cell lines are more invasive in vitro and in vivo (E. W. Thompson, S. Paik, N. Brunner, C. L. Sommers, G. Zugmaier, R. Clarke, T. B. Shima, J. Torri, S. Donahue, M. E. Lippman, G. R. Martin, and R. B. Dickson, J. Cell. Physiol., 150: 534-544, 1992). We hypothesized that a breast cancer cell with an epithelial phenotype could undergo a transition to a fibroblastic phenotype, possibly resulting in more invasive capacity. We now show that two Adriamycin-resistant MCF-7 cell lines and a vinblastine-resistant ZR-75-B cell line have undergone such a transition. Adriamycin-resistant MCF-7 cells express vimentin, have diminished keratin 19 expression, have lost cell adhesion molecule uvomorulin expression, and have reduced formation of desmosomes and tight junctions as determined by reduced immunodetection of their components desmoplakins I and II and zonula occludens (ZO)-1. Other MCF-7 cell lines selected for resistance to vinblastine and to Adriamycin and verapamil did not have these characteristics, indicating that drug selection does not invariably cause these phenotypic changes. In addition, to determine if vimentin expression in MCF-7 cells alone could manifest a fibroblastic phenotype, we transfected the full-length human vimentin complementary DNA into MCF-7 cells. Although vimentin expression was achieved in MCF-7 cells, it did not affect the phenotype of the cells in terms of the distribution of keratins, desmoplakins I and II, ZO-1, or uvomorulin or in terms of in vitro invasiveness. We conclude that vimentin expression is a marker for a fibroblastic and invasive phenotype in breast cancer cells but does not by itself give rise to this phenotype.

Assessing the property market impact of stigma removal : high voltage overhead transmission lines removal in Wellington, NZ

This thesis advances the understanding of the impact of stigma on property values. A case study in Wellington, New Zealand, enabled hedonic modelling and an empirical analysis to determine the impact of the stigma from the high voltage transmission line structure and how long the stigma remained after removal. The results reveal a substantial difference between the discount applied to individual properties while the structure is in place, as compared to the overall increase in neighbourhood value once the structure, which created the stigma, is removed.

Epithelial mesenchymal transition traits in human breast cancer cell lines

Epithelial mesenchymal transition (EMT) has long been associated with breast cancer cell invasiveness and evidence of EMT processes in clinical samples is growing rapidly. Genome-wide transcriptional profiling of increasingly larger numbers of human breast cancer (HBC) cell lines have confirmed the existence of a subgroup of cell lines (termed Basal B/Mesenchymal) with enhanced invasive properties and a predominantly mesenchymal gene expression signature, distinct from subgroups with predominantly luminal (termed Luminal) or mixed basal/luminal (termed Basal A) features (Neve et al Cancer Cell 2006). Studies providing molecular and cellular analyses of EMT features in these cell lines are summarised, and the expression levels of EMT-associated factors in these cell lines are analysed. Recent clinical studies supporting the presence of EMT-like changes in vivo are summarised. Human breast cancer cell lines with mesenchymal properties continue to hold out the promise of directing us towards key mechanisms at play in the metastatic dissemination of breast cancer.

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.

LCC15-MB cells are MDA-MB-435 : a review of misidentified breast and prostate cell lines

Current stocks of the LCC15-MB cell line, which we originally isolated from a human breast-bone metastasis, were found to be genetically matched to the MDA-MB-435 cell line from the Lombardi Cancer Center (MDA-MB-435-LCC) using comparative genomic hybridisation, DNA microsatellite analysis and chromosomal number. LCC15-MB stocks used for our previously published studies as well as the earliest available LCC15-MB cells also showed identity to MDA-MB-435-LCC cells. The original karyotype reported for LCC15-MB cells was considerably different to that of MDA-MB-435 cells, indicating that the original LCC15-MB cells were lost to contamination by MDA-MB-435-LCC cells. Chromosome number is the simplest test to distinguish original LCC 15-MB cells (n ∼ 75) from MDA-MB-435 (n ∼ 52). Collectively, our results prove that LCC15-MB cells currently available are MDA-MB-435 cells and we suggest their re-designation as MDA-MB-435-LCC15 cells. We also review the known misclassification of breast and prostate cancer cell lines to date and have initiated a register maintained at http://www.svi.edu.au/cell_lines_registry.doc.

Partitioning of dioxins (PCDDs/Fs) in ambient air at urban residential locations

In this work, 17-polychlorinated dibenzo-pdioxin/furan (PCDD/Fs) isomers were measured in ambient air at four urban sites in Seoul, Korea (from February to June 2009). The concentrations of their summed values RPCDD/Fs) across all four sites ranged from 1,947 (271 WHO05 TEQ) (Jong Ro) to 2,600 (349 WHO05 TEQ) fg/m3 (Yang Jae) with a mean of 2,125 ± 317) fg/m3 (292 WHO05 TEQ fg/m3). The sum values for the two isomer groups of RPCDD and RPCDF were 527 (30 WHO05 TEQ) and 1,598 (263 WHO05 TEQ) fg/m3, respectively. The concentration profile of individual species was dominated by the 2,3,4,7,8-PeCDF isomer, which contributed approximately 36 % of the RPCDD/Fs value. The observed temporal trends in PCDD/F concentrations were characterized by relative enhancement in the winter and spring. The relative contribution of different sources, when assessed by principal component analysis, is explained by the dominance of vehicular emissions along with coal (or gas) burning as the key source of ambient PCDD/Fs in the residential areas studied.

Cell adhesion molecule uvomorulin expression in human breast cancer cell lines : relationship to morphology and invasive capacities

Loss of cell-cell adhesion in carcinoma cells may be an important step in the acquisition of an invasive, metastatic phenotype. We have examined the expression of the epithelial-specific cell adhesion molecule uvomorulin (E-cadherin, cell-CAM 120/80, L-CAM) in human breast cancer cell lines. We find that fibroblastoid, highly invasive, vimentin-expressing breast cancer cell lines do not express uvomorulin. Of the more epithelial-appearing, less invasive, keratin-expressing breast cancer cell lines, some express uvomorulin, and some do not. We examined the morphologies of the cell lines in the reconstituted basement membrane matrix Matrigel and measured the ability of the cells to traverse a Matrigel-coated filter as in vitro models for detachment of carcinoma cells from neighboring cells and invasion through basement membrane into surrounding tissue. Colonies of uvomorulin-positive cells have a characteristic fused appearance in Matrigel, whereas uvomorulin-negative cells appear detached. Cells which are uvomorulin negative and vimentin positive have a stellate morphology in Matrigel. We show that uvomorulin is responsible for the fused colony morphology in Matrigel since treatment of uvomorulin-positive MCF-7 cells with an antibody to uvomorulin caused the cells to detach from one another but did not induce invasiveness in these cells, as measured by their ability to cross a Matrigel-coated polycarbonate filter in a modified Boyden chamber assay. Two uvomorulin-negative, vimentin-negative cell lines are also not highly invasive as measured by this assay. We suggest that loss of uvomorulin-mediated cell-cell adhesion may be one of many changes involved in the progression of a carcinoma cell to an invasive phenotype.

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.

Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44 : correlation with invasive potential

In the present study, we examined a panel of human breast cancer cell lines with regard to their expression of CD44 and ability to bind and degrade hyaluronan. The cell lines expressed varying amounts of different molecular weight forms of CD44 (85-200 kDa) and, in general, those that expressed the greatest amounts of CD44 were the most invasive as judged by in vitro assays. In addition, the ability to bind and degrade hyaluronan was restricted to the cell lines expressing high levels of CD44, and both these functions were blocked by an antibody to CD44 (Hermes-1). Moreover, the rate of [3H]hyaluronan degradation was highly correlated with the amount of CD44 (r = 0.951, P < 0.0001), as well as with the invasive potential of the cells. Scatchard analysis of the [3H]hyaluronan binding of these cells revealed the existence of significant differences in both their binding capacity and their dissociation constant. To determine the source of this deviation, the different molecular weight forms of CD44 were partially separated by gel filtration chromatography. In all cell lines, the 85 kDa form was able to bind hyaluronan, although with different affinities. In contrast, not all of the high molecular weight forms of CD44 had this ability. These results illustrate the diversity of CD44 molecules in invasive tumor cells, and suggest that one of their major functions is to degrade hyaluronan.

Agent-based modelling simulation case study : assessment of airport check-in and evacuation process by considering group travel behaviour of air passengers

Passenger experience has become a major factor that influences the success of an airport. In this context, passenger flow simulation has been used in designing and managing airports. However, most passenger flow simulations failed to consider the group dynamics when developing passenger flow models. In this paper, an agent-based model is presented to simulate passenger behaviour at the airport check-in and evacuation process. The simulation results show that the passenger behaviour can have significant influences on the performance and utilisation of services in airport terminals. The model was created using AnyLogic software and its parameters were initialised using recent research data published in the literature.

A transcriptomic analysis of striped catfish (Pangasianodon hypophthalmus) in response to salinity adaptation: De novo assembly, gene annotation and marker discovery

The striped catfish (Pangasianodon hypophthalmus) culture industry in the Mekong Delta in Vietnam has developed rapidly over the past decade. The culture industry now however, faces some significant challenges, especially related to climate change impacts notably from predicted extensive saltwater intrusion into many low topographical coastal provinces across the Mekong Delta. This problem highlights a need for development of culture stocks that can tolerate more saline culture environments as a response to expansion of saline water-intruded land. While a traditional artificial selection program can potentially address this need, understanding the genomic basis of salinity tolerance can assist development of more productive culture lines. The current study applied a transcriptomic approach using Ion PGM technology to generate expressed sequence tag (EST) resources from the intestine and swim bladder from striped catfish reared at a salinity level of 9 ppt which showed best growth performance. Total sequence data generated was 467.8 Mbp, consisting of 4,116,424 reads with an average length of 112 bp. De novo assembly was employed that generated 51,188 contigs, and allowed identification of 16,116 putative genes based on the GenBank non-redundant database. GO annotation, KEGG pathway mapping, and functional annotation of the EST sequences recovered with a wide diversity of biological functions and processes. In addition, more than 11,600 simple sequence repeats were also detected. This is the first comprehensive analysis of a striped catfish transcriptome, and provides a valuable genomic resource for future selective breeding programs and functional or evolutionary studies of genes that influence salinity tolerance in this important culture species.

Invasive and metastatic properties of MCF-7 cells and ras(H)-transfected MCF-7 cell lines

In vitro invasion and in vivo metastasis assays were performed with a panel of MCF-7 cells transfected with isogenic constructs of mutated ras(H) genes. Both increased levels of ras(H) expression and ras(H) oncogene activation increased activity of derivative cell lines in in vitro invasion assays. In vivo formation of spontaneous metastases was assessed after intradermal inoculation of MCF-7 cells in the vicinity of the mammary fat pads of ovariectomized nude mice. No metastases were seen in the absence of estradiol treatment of the mice. With estradiol supplementation of the mice both the ras(H)-transfected and control transfected cell lines gave a higher incidence of metastases than parental MCF-7 cells. Prolonged treatment of mice with exogenous estradiol (60 days vs. 21 days) resulted in more frequent metastases to liver and lung at the end of the 90-day observation period. In contrast to activated ras(H)-gene enhancement of metastatic capacity of rodent fibroblast and epithelial cell lines, there was no correlation of ras(H) expression with in vivo metastatic capacity of a human mammary carcinoma cell line.

Heregulin regulates the actin cytoskeleton and promotes invasive properties in breast cancer cell lines

The metastatic process requires changes in tumor cell adhesion properties, cell motility and remodeling of the extracellular matrix. The erbB2 proto-oncogene is overexpressed in approximately 30% of breast cancers and is a major prognostic parameter when present in invasive disease. A ligand for the erbB2 receptor has not yet been identified but it can be activated by heterodimerization with heregulin (HRG)-stimulated erbB3 and erbB4 receptors. The HRGs are a family of polypeptide growth factors that have been shown to play a role in embryogenesis, tumor formation, growth and differentiation of breast cancer cells. The erbB3 and erbB4 receptors are involved in transregulation of erbB2 signaling. The work presented here suggests biological roles for HRG including regulation of the actin cytoskeleton and induction of motility and invasion in breast cancer cells. HRG-expressing breast cancer cell lines are characterized by low erbB receptor levels and a high invasive and metastatic index, while those which overexpress erbB2 demonstrate minimal invasive potential in vitro and are non-tumorigenic in vivo. Treatment of the highly tumorigenic and metastatic HRG-expressing breast cancer cell line MDA-MB-231 with an HRG-neutralizing antibody significantly inhibited proliferation in culture and motility in the Boyden chamber assay. Addition of exogenous HRG to non-invasive erbB2 overexpressing cells (SKBr-3) at low concentrations induced formation of pseudopodia, enhanced phagocytic activity and increased chemomigration and invasion in the Boyden chamber assay. The specificity of the chemomigration response to HRG is demonstrated by inhibition with the anti-HRG neutralizing antibody. These results suggest that either HRG can act as an autocrine or paracrine ligand to promote the invasive behavior of breast cancer cells in vitro or thus may enhance the metastatic process in vivo.

Expression of β1 integrin in laminin-adhesion-selected human colon cancer cell lines of varying tumorigenicity

Laminin has been shown to promote the malignant phenotype and the expression of certain laminin receptors has been correlated with the malignant character of the tumors. Here new cell lines were isolated from a human colon cancer cell line (LCC-C1) based on their adhesiveness to laminin. The laminin-adherent subclone formed large tumors in nude mice, whereas the laminin-nonadherent subclone failed to form sizable tumors. Only the laminin-adherent subclone adhered to laminin and invaded through Matrigel-coated filters. The adhesive and invasive ability of the cells was almost completely blocked by low concentrations (1.0 μg/ml) of anti-β1 integrin antibody. The amounts of total cellular β1 integrin protein were similar in the two subclones when compared by Western blot, and the mRNA levels also did not differ. The localization of β1 integrin laminin receptor varied in the two subclones; the laminin-adherent subclone showed a linear distribution along the cell-cell junctions, while the laminin-nonadherent subclone did not stain between the cells. Using laminin-Sepharose affinity chromatography, more β1 integrin was obtained from the laminin-adherent subclone. These findings suggest that alterations in the affinity of β1 integrin for laminin and in its membrane distribution might be involved in the increased tumorigenicity observed in colon cancer cells.