Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer.

BACKGROUND: Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting. RESULTS: In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells. CONCLUSION: A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton.

Ubiquitous expression of the rtTA2S-M2 inducible system in transgenic mice driven by the human hnRNPA2B1/CBX3 CpG island.

BACKGROUND: A sensitive, ubiquitously expressed tetracycline inducible system would be a valuable tool in mouse transgenesis. However, this has been difficult to obtain due to position effects observed at different chromosomal sites of transgene integration, which negatively affect expression in many tissues. The aim of this study was to test the utility of a mammalian methylation-free CpG island to drive ubiquitous expression of the sensitive doxycycline (Dox) inducible rtTA2S-M2 Tet-transactivator in transgenic mice. RESULTS: An 8 kb genomic fragment from the methylation-free CpG island of the human hnRNPA2B1-CBX3 housekeeping gene locus was tested. In a number of transgenic mouse lines obtained, rtTA2S-M2 expression was detected in many tissues examined. Characterisation of the highest expressing rtTA2S-M2 transgenic mouse line demonstrated Dox-inducible GFP transgene expression in many tissues. Using this line we also show highly sensitive quantitative induction with low doses of Dox of an assayable plasma protein transgene under the control of a Tet Responsive Element (TRE). The utility of this rtTA2S-M2 line for inducible expression in mouse embryos was also demonstrated using a GATA-6 Tet-inducible transgene to show specific phenotypes in the embryonic lung, as well as broader effects resulting from the inducible widespread overexpression of the transgene. CONCLUSION: The ubiquitously expressing rtTA2S-M2 transgenic mouse line described here provides a very useful tool for studying the effects of the widespread, inducible overexpression of genes during embryonic development and in adult mice.

TF Target Mapper: a BLAST search tool for the identification of Transcription Factor target genes.

BACKGROUND: In the current era of high throughput genomics a major challenge is the genome-wide identification of target genes for specific transcription factors. Chromatin immunoprecipitation (ChIP) allows the isolation of in vivo binding sites of transcription factors and provides a powerful tool for examining gene regulation. Crosslinked chromatin is immunoprecipitated with antibodies against specific transcription factors, thus enriching for sequences bound in vivo by these factors in the immunoprecipitated DNA. Cloning and sequencing the immunoprecipitated sequences allows identification of transcription factor target genes. Routinely, thousands of such sequenced clones are used in BLAST searches to map their exact location in the genome and the genes located in the vicinity. These genes represent potential targets of the transcription factor of interest. Such bioinformatics analysis is very laborious if performed manually and for this reason there is a need for developing bioinformatic tools to automate and facilitate it. RESULTS: In order to facilitate this analysis we generated TF Target Mapper (Transcription Factor Target Mapper). TF Target Mapper is a BLAST search tool allowing rapid extraction of annotated information on genes around each hit. It combines sequence cleaning/filtering, pattern searching and BLAST searches with extraction of information on genes located around each BLAST hit and comparisons of the output list of genes or gene ontology IDs with user-implemented lists. We successfully applied and tested TF Target Mapper to analyse sequences bound in vivo by the transcription factor GATA-1. We show that TF Target Mapper efficiently extracted information on genes around ChIPed sequences, thus identifying known (e.g. alpha-globin and zeta-globin) and potentially novel GATA-1 gene targets. CONCLUSION: TF Target Mapper is a very efficient BLAST search tool that allows the rapid extraction of annotated information on the genes around each hit. It can contribute to the comprehensive bioinformatic transcriptome/regulome analysis, by providing insight into the mechanisms of action of specific transcription factors, thus helping to elucidate the pathways these factors regulate.

An embryonic-specific repressor element located 3' to the Agamma-globin gene influences transcription of the human beta-globin locus in transgenic mice.

OBJECTIVE: Persistent expression of the human fetal gamma-globin genes in the adult stage is often associated with naturally occurring deletions in the human beta-globin locus. The mapping of the 5' breakpoints of these deletions within the Agamma- to delta-globin intergenic region has suggested that regulatory elements involved in the silencing of the gamma-globin genes in the adult may be present. We previously identified two elements in this region, termed Enh and F, located 3' to the Agamma-globin gene acting as silencers in transient transfection assays. Here, we tested directly the in vivo significance of these elements in the developmental regulation of the human beta-like globin genes. MATERIALS AND METHODS. We selectively deleted both Enh and F elements in the context of a 185-kb human beta-globin locus PAC (P1 phage artificial chromosome) and tested the effects of this deletion on the expression of the human P-like globin genes in transgenic mice. RESULTS: The Enh/F deletion resulted in an increase in epsilon- and gamma-globin mRNA levels in the embryonic yolk sac stage of erythropoiesis, which appears to be due to an increase in the rate of transcription rather than to an increase in the number of cells transcribing the human globin locus. However, the human developmental switching from fetal gamma-globin to adult beta-globin gene expression in transgenic mice was not affected by this deletion. CONCLUSION: These results identify Enh and F as locus-wide regulatory elements capable of down-regulating transcription of the human beta-globin locus in an embryonic-specific manner.

Persistent gamma-globin expression in adult transgenic mice is mediated by HPFH-2, HPFH-3, and HPFH-6 breakpoint sequences.

Deletions at the 3' end of the human beta-globin locus are associated with the hereditary persistence of fetal hemoglobin (HPFH) in adults, potentially through the juxtaposition of enhancer elements in the vicinity of the fetal gamma-globin genes. We have tested how sequences at the HPFH-2, HPFH-3, and HPFH-6 breakpoints, which act as enhancers in vitro, affect the silencing of a locus control region A gamma (LCRA gamma) transgene in the adult stage of mice. We found persistent A gamma expression in the adult blood of most of the multicopy HPFH-2, HPFH-3, or HPFH-6 lines, in contrast to the control LCRA gamma lines which were silenced. Cre-mediated generation of single copy lines showed persistent gamma gene expression maintained in some of the HPFH-2 and HPFH-6 lines, but not in any of the HPFH-3 or LCRA gamma lines. In the HPFH-2 and HPFH-6 lines, persistent gamma gene expression correlated with euchromatic transgene integrations. Thus, our observations provide support for a model whereby HPFH conditions arise from the juxtaposition of enhancers as well as permissive chromatin subdomains in the vicinity of the gamma-globin genes.