The Expression of the Nectin Complex in Human Breast Cancer and the Role of Nectin-3 in the Control of Tight Junctions during Metastasis

Introduction

Nectins are a family of integral protein molecules involved in the formation of functioning Adherens and Tight Junctions (TJ). Aberrant expression is associated with cancer progression but little is known how this effects changes in cell behaviour. This study aimed to ascertain the distribution of Nectins-1 to -4 in human breast cancer and the effect on junctional integrity of Nectin-3 modulation in human endothelial and breast cancer cells.

Methods

A human breast tissue cohort was processed for Q-PCR and immunohistochemistry for analysis of Nectin-1/-2/-3/-4. Nectin-3 over-expression was induced in the human breast cancer cell line MDA-MB-231 and the human endothelial cell line HECV. Functional testing was carried out to ascertain changes in cell behaviour.

Results

Q-PCR revealed a distinct reduction in node positive tumours and in patients with poor outcome. There was increased expression of Nectin-1/-2 in patients with metastatic disease, Nectin-3/-4 was reduced. IHC revealed that Nectin-3 expression showed clear changes in distribution between normal and cancerous cells. Nectin-3 over-expression in MDA-MB-231 cells showed reduced invasion and migration even when treated with HGF. Changes in barrier function resulted in MDAN3 cells showing less change in resistance after 2h treatment with HGF (p<0.001). Nectin-3 transformed endothelial cells were significantly more adhesive, irrespective of treatment with HGF (p<0.05) and had reduced growth. Barrier function revealed that transformed HECV cells had significantly tighter junctions that wildtype cells when treated with HGF (p<0.0001). HGF-induced changes in permeability were also reduced. Overexpression of Nectin-3 produced endothelial cells with significantly reduced ability to form tubules (p<0.0001). Immunoprecipitation studies discovered hitherto novel associations for Nectin-3. Moreover, HGF appeared to exert an effect on Nectin-3 via tyrosine and threonine phosphorylation.

Conclusions

Nectin-3 may be a key component in the formation of cell junctions and be a putative suppressor molecule to the invasion of breast cancer cells.     

Isolation and characterization of polymorphic microsatellite loci in nurse shark (Ginglymostoma cirratum)

We report on the development of nine polymorphic microsatellite loci in nurse shark (Ginglymostoma cirratum) using a combination of enriched and unenriched subgenomic libraries. Based on the small percentage of positive clones in the unenriched library (0.4%) it appears that microsatellites are very scarce in nurse shark genomes. Numbers of alleles at polymorphic loci ranged from two to 15; observed heterozygosity ranged from 0.17 to 0.90. We expect these loci to be useful for studies of breeding structure and paternity.     

The neural gland in tunicates: fine structure and intracellular distribution of phosphatases

Summary The cells comprising the neural gland in the ascidians Ciona, Styela, and Botryllus have been examined for their fine structural features and enzyme cytochemistry. The gland cells are either cuboidal or irregular in outline. They are full of small vesicles, of which some are pinocytotic, as well as larger vacuoles; they become increasingly vacuolated as their shape decreases in regularity. At the same time, glycogen deposits accumulate and the cisternae of the endoplasmic reticulum become distended. Some of the vacuoles contain an electron dense material or a fibrillar substance, but the cells contain no obvious electron opaque secretory granules associated with an extensive Golgi complex such as occur in the vertebrate adenohypophysis.Acid phosphatase is localized in some of the vesicles and vacuoles, indicating that they are a kind of lysosome, the latter possibly representing autophagic vacuoles. Thiamine pyrophosphatase is also found in many vacuoles as well as in the saccules of the Golgi apparatus which in these cells is in the form of dictyosomes.The results suggest a developmental cycle of increasing cytoplasmic vacuolation, ultimately leading to a breakdown and release of the vacuolar products. The significance of these observations is considered, particularly with respect to the hypothesis that the gland represents the ascidian equivalent of the vertebrate pituitary.I am grateful to Miss Yvonne R. Carter for technical assistance with the photography and to Mr. John Rodford for producing the diagram.     

Production of extrachromosomal r-determinant circles from integrated R100.1: Involvement of the E. coli recombination system

Summary The drug resistance plasmid R100.1 can integrate into the E. coli chromosome at several sites on the plasmid. Many of the resulting Hfr strains continuously produce extrachromosomal circular forms of the r-determinant. These r-det plasmids seem incapable of stable autonomous replication. We show that their presence in the cell requires the continuous activity of functional recA and recC genes but does not require the lexA function.The production of r-det circular forms is correlated with an increased copy number of r-det sequences, relative to RTF sequences. This copy number increase is, however, also found in a recA ^- backgound where no circular forms of r-det are found. These results show that a specific replication of r-det sequences, not present in the wild-type R100.1 plasmid, occurs in these R-Hfr strains. They suggest that a rec promoted recombination, posterior to the specific replication event, is needed for the production of circular r-det forms.     

Expression of SPARC during development of the chicken chorioallantoic membrane: evidence for regulated proteolysis in vivo.

SPARC is a secreted glycoprotein that has been shown to disrupt focal adhesions and to regulate the proliferation of endothelial cells in vitro. Moreover, peptides resulting from the proteolysis of SPARC exhibit angiogenic activity. Here we describe the temporal synthesis, turnover, and angiogenic potential of SPARC in the chicken chorioallantoic membrane. Confocal immunofluorescence microscopy revealed specific expression of SPARC protein in endothelial cells, and significantly higher levels of SPARC were observed in smaller newly formed blood vessels in comparison to larger, developmentally older vessels. SPARC mRNA was detected at the earliest stages of chorioallantoic membrane morphogenesis and reached maximal levels at day 13 of embryonic development. Interestingly, steady-state levels of SPARC mRNA did not correlate directly with protein accumulation; moreover, the protein appeared to undergo limited degradation during days 10-15. Incubation of [125I]-SPARC with chorioallantoic membranes of different developmental ages confirmed that extracellular proteolysis occurred during days 9-15, but not at later stages (e.g., days 17-21). Comparison of peptides produced by incubation with chorioallantoic membranes with those generated by plasmin showed an identical pattern of proteolysis. Plasmin activity was present throughout development, and in situ zymography identified sites of plasminogen activator activity that corresponded to areas exhibiting high levels of SPARC expression. Synthetic peptides from a plasmin-sensitive region of SPARC, between amino acids 113-130, stimulated angiogenesis in the chorioallantoic membrane in a dose-dependent manner; in contrast, intact SPARC was inactive in similar assays. We have shown that SPARC is expressed in endothelial cells of newly formed blood vessels in a manner that is both temporally and spatially restricted. Between days 9 and 15 of chorioallantoic membrane development, the protein undergoes proteolytic cleavage that is mediated, in part, by plasmin. SPARC peptides released specifically by plasmin induce angiogenesis in vivo. We therefore propose that SPARC acts as an intrinsic regulator of angiogenesis in vivo.     

Proposed minimum reporting standards for chemical analysis

There is a general consensus that supports the need for standardized reporting of metadata or information describing large-scale metabolomics and other functional genomics data sets. Reporting of standard metadata provides a biological and empirical context for the data, facilitates experimental replication, and enables the re-interrogation and comparison of data by others. Accordingly, the Metabolomics Standards Initiative is building a general consensus concerning the minimum reporting standards for metabolomics experiments of which the Chemical Analysis Working Group (CAWG) is a member of this community effort. This article proposes the minimum reporting standards related to the chemical analysis aspects of metabolomics experiments including: sample preparation, experimental analysis, quality control, metabolite identification, and data pre-processing. These minimum standards currently focus mostly upon mass spectrometry and nuclear magnetic resonance spectroscopy due to the popularity of these techniques in metabolomics. However, additional input concerning other techniques is welcomed and can be provided via the CAWG on-line discussion forum at or . Further, community input related to this document can also be provided via this electronic forum. The contents of this paper do not necessarily reflect any position of the Government or the opinion of the Food and Drug Administration Sponsor: Metabolomics Society http://www.metabolomicssociety.org/ Reference: http://msi-workgroups.sourceforge.net/bio-metadata/reporting/pbc/ http://msi-workgroups.sourceforge.net/chemical-analysis/ Version: Revision: 5.1 Date: 09 January, 2007     

Tissue specific expression of p422 protein, a putative lipid carrier, in mouse adipocytes

The differentiation of 3T3-L1 preadipocytes leads to the expression of a new protein, p422, and its mRNA. This protein has 70% and 20-30% amino acid sequence homology to myelin P2 and the fatty acid binding proteins of liver and intestine, respectively. Investigation of the distribution in mouse tissues of p422 protein by immunoblotting and of p422 mRNA by cDNA hybridization indicates that they are expressed only in adipose tissue. Liver and intestinal fatty acid binding protein mRNA's were not detectable in mouse adipose tissue or in 3T3-L1 adipocytes. It is suggested that p422 functions as an adipocyte fatty acid binding protein.