Complex regulation of membrane-type matrix metalloproteinase expression and matrix metalloproteinase-2 activation by concanavalin A in MDA-MB-231 human breast cancer cells

Matrix Metalloproteinase-2 (MMP-2) is secreted as a zymogen, the activation of which has been associated with metastatic progression in human breast cancer (HBC). Concanavalin A (Con A) has been found to induce activation of MMP-2 in invasive HBC cell lines. Con A effects on the expression of mRNA for membrane-type matrix metalloproteinase (MT-MMP), a newly described cell surface-associated MMP, showed a close temporal correlation with induction of MMP-2 activation. It is surprising that MT-MMP mRNA is constitutively present in the uninduced MDA-MB-231 cell, despite a lack of MMP-2 activation. We have used actinomycin D to demonstrate a partial requirement for de novo gene expression in the induction of MMP-2 activation by Con A in MDA-MB-231 HBC cells. Furthermore, this transcriptional response to Con A appeared to require the continued presence of Con A for its manifestation. The nontranscriptional component of the Con A induction manifests rapidly, is quite substantial, and persists strongly despite actinomycin D abrogation of both constitutive and Con A-induced MT-MMP. Cycloheximide analyses suggest that protein synthesis may be involved in this rapid transcription-independent response. These studies suggest that Con A induces MMP-2-activation in part by up-regulation of MT-MMP expression but has a more complicated mode of action, involving additional nontranscriptional effects, which apparently require protein synthesis.

Expression of 67 kDa laminin receptor in human breast cancer cells : regulation by progestins

The level of 67 kDa laminin receptor (67LR) expression on breast and colon tumor cell surfaces was previously shown to be correlated with the capacity of tumor cells to metastasize. In the present work we investigate the effects of progestins and estrogen on the expression of 67LR in two sublines of the T47D human breast cancer cells: weakly tumorigenic, poorly invasive parental T47D cells and a highly tumorigenic, more invasive T47Dco subclone. Inmmunoblotting with an affinity purified antibody directed against a synthetic peptide recognizes the 67LR in these cells. 67LR expression in the T47Dco subclone is 5,5-fold higher than in their parental T47D cells. Treatment of T47D cells with 1 nM of the synthetic progestin R5020 results in a 4-fold increase in 67LR protein expression. Estrogen also induced 67LR expression, but only by 1.5-fold. The progestin-stimulated expression of the 67LR correlates with a 4.3-fold increase in attachment of T47D cells to laminin. A monoclonal antibody, mAb 13, directed against β1 integrin, completely blocks the attachment of T47D cells to fibronectin, only partially inhibits the attachment of T47D cells to laminin, and appears not to affect the progestin-stimulated laminin attachment of T47D cells. A new antiprogestin, ZK 112.993, significantly inhibits both progestin-stimulated 67LR expression and the increased attachment to laminin. These results suggest a possible role for progestin in mediating one of the multiple events thought to be important in metastasis of steroid receptor positive human breast cancer cells.

Towards a global framework for advertising self regulation

One of the forces which has indelibly shaped marketing is the internet. It has not only changed the way we communicate, but our marketing practices and our advertising self-regulation process (Kerr, Mortimer, Dickinson and Waller 2012). This special session seeks to build a new global framework to regulate advertising activity in this uncharted online environment. It looks back to how advertising has been traditionally self-regulated and looks forward to identify the key issues for marketers, consumers, regulators and the media. This special session explores and reinforces the fundamental purpose of the conference, as well as addressing the urgent needs of marketers, consumers and regulators.

MT1-MMP-dependent and -independent regulation of gelatinase A activation in long-term, ascorbate-treated fibroblast cultures: Regulation by fibrillar collagen

Human skin fibroblasts were cultured long-term in the presence of ascorbic acid to allow formation of a three-dimensional collagen matrix, and the effects of this on activation of secreted matrix metalloproteinase-2 (MMP-2) were examined. Accumulation of collagen over time correlated with increased levels of both mature MMP-2 and cell-associated membrane type 1-MMP (MT1-MMP), and subsequently increased mRNA levels for MT1-MMP, providing temporal resolution of the "nontranscriptional" and "transcriptional" effects of collagen on MT-1MMP functionality. MMP-2 activation by these cultures was blocked by inhibitors of prolyl-4-hydroxylase, or when fibroblasts derived from the collagen α1(I) gene-deficient Mov-13 mouse were used. MMP-2 activation by the Mov-13 fibroblasts was rescued by transfection of a full-length α1(I) collagen cDNA, and to our surprise, also by transfection with an α1(I) collagen cDNA carrying a mutation at the C-proteinase cleavage, which almost abrogated fibrillogenesis. Although studies with ascorbate-cultured MT1-MMP-/- fibroblasts showed that MT1-MMP played a significant role in the collagen-induced MMP-2 activation, a residual MT1-MMP-independent activation of MMP-2 was seen which resembled the level of MMP-2 activation persisting when wild-type fibroblasts were cultured in the presence of both ascorbic acid and MMP inhibitors. We were also unable to block this residual activation with inhibitors specific for serinyl, aspartyl, or cysteinyl enzymes.

MTI-MMP expression promotes tumor growth and angiogenesis through an up-regulation of vascular endothelial growth factor expression

Membrane type 1 metalloprotease (MT1-MMP) is a transmembrane metalloprotease that plays a major role in the extracellular matrix remodeling, directly by degrading several of its components and indirectly by activating pro-MMP2. We investigated the effects of MT1-MMP overexpression on in vitro and in vivo properties of human breast adenocarcinoma MCF7 cells, which do not express MT1-MMP or MMP-2. MT1-MMP and MMP-2 cDNAs were either transfected alone or cotransfected. All clones overexpressing MT1-MMP 1) were able to activate endogenous or exogenous pro-MMP-2, 2) displayed an enhanced in vitro invasiveness through matrigel-coated filters independent of MMP-2 transfection, 3) induced the rapid development of highly vascularized tumors when injected subcutanously in nude mice, and 4) promoted blood vessels sprouting in the rat aortic ring assay. These effects were observed in all clones overexpressing MT1-MMP regardless of MMP-2 expression levels, suggesting that the production of MMP-2 by tumor cells themselves does not play a critical role in these events. The angiogenic phenotype of MT1-MMP-producing cells was associated with an up-regulation of VEGF expression. These results emphasize the importance of MT1-MMP during tumor angiogenesis and open new opportunities for the development of antiangiogenic strategies combining inhibitors of MT1-MMP and VEGF antagonists. - Sounni, N. E., Devy, L., Hajitou, A., Frankenne, F., Munaut, C., Gilles, C., Deroanne, C., Thompson, E. W., Foidart, J. M., Noel, A. MT1-MMP expression promotes tumor growth and angiogenesis through an up-regulation of vascular endothelial growth factor expression.

Regulation of breast cancer cells by hormones and growth factors : effects on proliferation and basement membrane invasiveness

The current understanding of the regulation of breast cancer cell proliferation and invasiveness by hormones and growth factors is reviewed. It has been shown that polypeptide growth factors are involved in hormone-independent breast cancer, and are sometimes oestrogen-regulated in hormone-responsive models. Basement-membrane invasiveness, relating to the metastatic potential of these cells, is also stimulated by oestrogen in hormone-dependent models, elevated in hormone-independent models, and is growth factor sensitive. Further understanding of the differential effects of growth factors on breast cancer cell proliferation and invasiveness should facilitate better therapeutic exploitation of regulation at this level.

Reduced excision repair cross-complementing 1 expression associates with enhanced papilloma formation and fibroblast transformation after genetic disruption of secreted protein acidic and rich in cysteine

SPARC (secreted protein acidic and rich in cysteine)/ osteonectin/BM-40 is a matricellular protein implicated in development, cell transformation and tumorigenesis. We have examined the role of SPARC in cell transformation induced chemically with 7,12-dimethylbenz[a]anthracene (DMBA) and 12- tetradecanoylphorbol-13-acetate (TPA) in embryonic fibroblasts and in the skin of mice. Embryonic fibroblasts from SPARCnull mice showed increases in cell proliferation, enhanced sensitivity to DMBA and a higher number of DMBA/TPA-induced transformation foci. The number of DMBA-DNA adducts was 9 times higher in SPARCnull fibroblasts and their stability was lower than wild-type fibroblasts, consistent with a reduction in excision repair cross-complementing 1 the nucleotide excision repair enzyme in these cells. The SPARCnull mice showed an increase in both the speed and number of papillomas forming after topical administration of DMBA/TPA to the skin. These papillomas showed reduced growth and reduced progression to a more malignant phenotype, indicating that the effect of SPARC on tumorigenesis depends upon the transformation stage and/or tissue context. These data reinforce a growing number of observations in which SPARC has shown opposite effects on different tumor types/stages.

Regulation of proliferation, invasion and growth factor synthesis in breast cancer by steroids

Endogenous ovarian estrogens and progestins appear to play a critical role in the development and progression of breast cancer. Local productions of growth factors probably also contribute to malignant proliferation, while production and activation of collagenolytic enzymes may be equally critical for local invasive processes. The current review focusses on characterization of growth factor-receptor systems operant in normal and malignant breast epithelium. In addition, the determinants of local invasion are reviewed: attachment, modality, and proteose secretion. Finally, data are discussed concerning the regulation of both proliferation and invasion by hormones and antihormonal agents in hormone-dependent breast cancer. The results suggest new potential pharmacologic targets to explore to suppress onset and progression of breast cancer.

Mapping eQTLs in the Norfolk Island genetic isolate identifies candidate genes for CVD risk traits

Cardiovascular disease (CVD) affects millions of people worldwide and is influenced by numerous factors, including lifestyle and genetics. Expression quantitative trait loci (eQTLs) influence gene expression and are good candidates for CVD risk. Founder-effect pedigrees can provide additional power to map genes associated with disease risk. Therefore, we identified eQTLs in the genetic isolate of Norfolk Island (NI) and tested for associations between these and CVD risk factors. We measured genome-wide transcript levels of blood lymphocytes in 330 individuals and used pedigree-based heritability analysis to identify heritable transcripts. eQTLs were identified by genome-wide association testing of these transcripts. Testing for association between CVD risk factors (i.e., blood lipids, blood pressure, and body fat indices) and eQTLs revealed 1,712 heritable transcripts (p < 0.05) with heritability values ranging from 0.18 to 0.84. From these, we identified 200 cis-acting and 70 trans-acting eQTLs (p < 1.84 × 10(-7)) An eQTL-centric analysis of CVD risk traits revealed multiple associations, including 12 previously associated with CVD-related traits. Trait versus eQTL regression modeling identified four CVD risk candidates (NAAA, PAPSS1, NME1, and PRDX1), all of which have known biological roles in disease. In addition, we implicated several genes previously associated with CVD risk traits, including MTHFR and FN3KRP. We have successfully identified a panel of eQTLs in the NI pedigree and used this to implicate several genes in CVD risk. Future studies are required for further assessing the functional importance of these eQTLs and whether the findings here also relate to outbred populations.

Irradiation, cisplatin, and 5-azacytidine upregulate cytomegalovirus promoter in tumors and muscles: Implementation of non-invasive fluorescence imaging

Purpose: The cytomegalovirus (CMV) promoter is one of the most commonly used promoters for expression of transgenes in mammalian cells. The aim of our study was to evaluate the role of methylation and upregulation of the CMV promoter by irradiation and the chemotherapeutic agent cisplatin in vivo using non-invasive fluorescence in vivo imaging. Procedures: Murine fibrosarcoma LPB and mammary carcinoma TS/A cells were stably transfected with plasmids encoding CMV and p21 promoter-driven green fluorescent protein (GFP) gene. Solid TS/A tumors were induced by subcutaneous injection of fluorescent tumor cells, while leg muscles were transiently transfected with plasmid encoding GFP under the control of the CMV promoter. Cells, tumors, and legs were treated either by DNA methylation inhibitor 5-azacytidine, irradiation, or cisplatin. GFP expression was determined using a fluorescence microplate reader in vitro and by non-invasive fluorescence imaging in vivo. Results: Treatment of cells, tumors, and legs with 5-azacytidine (re)activated the CMV promoter. Furthermore, treatment with irradiation or cisplatin resulted in significant upregulation of GFP expression both in vitro and in vivo. Conclusions: Observed alterations in the activity of the CMV promoter limit the usefulness of this widely used promoter as a constitutive promoter. On the other hand, inducibility of CMV promoters can be beneficially used in gene therapy when combined with standard cancer treatment, such as radiotherapy and chemotherapy. © 2010 The Author(s).

Effect of atmospheric gas plasmas on cancer cell signaling

Cancer is one of the most life-threatening diseases with many forms still regarded as incurable. The conventional cancer treatments have unwanted side effects such as the death of normal cells. A therapy that can accurately target and effectively kill tumor cells could address the inadequacies of the available therapies. Atmospheric gas plasmas (AGP) that are able to specifically kill cancerous cells offer a promising alternative approach compared to conventional therapies. AGP have been shown to exploit tumor-specific genetic defects and a recent trial in mice has confirmed its antitumor effects. The mechanism by which the AGP act on tumor cells but not normal cells is not fully understood. A review of the current literature suggests that reactive oxygen species (ROS) generated by AGP induce death of cancer cells by impairing the function of intracellular regulatory factors. The majority of cancer cells are defective in tumor suppressors that interfere normal cell growth pathways. It appears that pro-oncogene or tumor suppressor-dependent regulation of antioxidant/or ROS signaling pathways may be involved in AGP-induced cancer cell death. The toxic effects of ROS are mitigated by normal cells by adjustment of their metabolic pathways. On the other hand, tumor cells are mostly defective in several regulatory signaling pathways which lead to the loss of metabolic balance within the cells and consequently, the regulation of cell growth. This review article evaluates the impact of AGP on the activation of cellular signaling and its importance for exploring mechanisms for safe and efficient anticancer therapies.

Genetic and phenotypic evidence supports evolutionary divergence of the American flamingo (Phoenicopterus ruber) population in the Galapagos islands

The Galapagos archipelago is characterized by a high degree of endemism across many taxa, linked to the archpelago's oceanic origin and distance from other colonizing land masses. A population of ~ 500 American Flamingos (Phoenicopterus ruber) resides in Galapagos, which is thought to share an historical origin with the American Flamingo currently found in the Caribbean region. Genetic and phenotypic parameters in American Flamingos from Galapagos and from the Caribbean were investigated. Microsatellite and microchondrial DNA markers data showed that the American Flamingo population in Galapagos differs genetically from that in the Caribbean. American Flamingos in Galapagos form a clade which differs by a single common nucleotide substitution from American Flamingos in the Caribbean. The genetic differentiation is also evident from nuclear DNA in that microsatellite data reveal a number of private alleles for the American Flamingo in Galapagos. Analysis of skeletal measurements showed that American Flamingos in Galapagos are smaller than those in the Caribbean primarily due to shorter tarsus length, and differences in body shape sexual dimorphism. American Flamingo eggs from Galapagos have smaller linear dimensions and volumes than those from the Caribbean. The findings are consistent with reproductive isolation of American Flamingo population in Galapagos.

Regulation of microRNA-1288 in colorectal cancer : altered expression and its clinicopathological significance

We aim to examine the miR-1288 expression in cancer cell lines and a large cohort of patients with colorectal cancer. Two colon cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were recruited. The miRNA expressions of miR-1288 were tested on these cell lines by using quantitative real-time polymerase chain reaction (qRT-PCR). An exogenous miR-1288 (mimic) was used to detect cell proliferation and cell cycle changes in SW480 using MTT calorimetric assay and flow cytometry, respectively. In addition, tissues from 122 patients with surgical resection of colorectum (82 adenocarcinomas, 20 adenomas, and 20 non-neoplastic tissues) were tested for miR-1288 expression by qRT-PCR. The colon cancer cell lines showed reduced expression of miR-1288 compared to normal colonic epithelial cell line. Over expression of miR-1288 in SW480 cell line showed increased cell proliferation and increased G2-M phase cells. In tissues, reduced miR-1288 expression was noted in majority of colorectal adenocarcinoma compared to colorectal adenoma and non-neoplastic tissues. Reduced or absent expression of miR-1288 was noted in 76% (n = 62/82) of the cancers. The expression levels of miR-1288 were higher in distal colorectal adenocarcinomas (P = 0.013) and in cancers of lower T staging (P = 0.033). To conclude, alternation of miR-1288 expression is important in the progression of colorectal cancer. The differential regulation of miR-1288 was found to be related to cancer location and pathological staging in colorectal cancers.