Metastasis-inducing DNA Regulates the Expression of the Osteopontin Gene by Binding the Transcription Factor Tcf-4

Small 1,000-bp fragments of genomic DNA obtained from human malignant breast cancer cell lines when transfected into a benign rat mammary cell line enhance transcription of the osteopontin gene and thereby cause the cells to metastasize in syngeneic rats. To identify the molecular events underlying this process, transient cotransfections of an osteopontin promoter-reporter construct and fragments of one metastasis-inducing DNA (Met-DNA) have identified the active components in the Met-DNA as the binding sites for the T-cell factor (Tcf) family of transcription factors. Incubation of cell extracts with active DNA fragments containing the sequence CAAAG caused retardation of their mobilities on polyacrylamide gels, and Western blotting identified Tcf-4, beta-catenin, and E-cadherin in the relevant DNA complexes in vitro. Transfection of an expression vector for Tcf-4 inhibited the stimulated activity of the osteopontin promoter-reporter construct caused by transiently transfected active fragments of Met-DNA or permanently transfected Met-DNA. This stimulated activity of the osteopontin promoter-reporter construct is accompanied by an increase in endogenous osteopontin mRNA but not in fos or actin mRNAs in the transfected cells. Permanent transfection of the benign rat mammary cell line with a 20-bp fragment from the Met-DNA containing the Tcf recognition sequence CAAAG caused an enhanced permanent production of endogenous osteopontin protein in vitro and induced the cells to metastasize in syngeneic rats in vivo. The corresponding fragment without the CAAAG sequence was without either effect. Therefore, the regulatory effect of the C9-Met-DNA is exerted, at least in part, by a CAAAG sequence that can sequester the endogenous inhibitory Tcf-4 and thereby promote transcription of osteopontin, the direct effector of metastasis in this system.

Association of Functional Heme Oxygenase-1 Gene Promoter Polymorphism with Renal Transplantation Outcomes

Heme oxygenase-1 (HO-1) is a cytoprotective molecule and increased expression in experimental transplant models correlates with reduced graft injury. A functional dinucleotide repeat (GT)n polymorphism, within the HO-1 promoter, regulates gene expression; a short number of repeats (S-allele

The tissue plasminogen activator gene promoter: a novel tool for radiogenic gene therapy of the prostate?

Radiation therapy is a treatment modality routinely used in cancer management so it is not unexpected that radiation-inducible promoters have emerged as an attractive tool for controlled gene therapy. The human tissue plasminogen activator gene promoter (t-PA) has been proposed as a candidate for radiogenic gene therapy, but has not been exploited to date. The purpose of this study was to evaluate the potential of this promoter to drive the expression of a reporter gene, the green fluorescent protein (GFP), in response to radiation exposure. METHODS: To investigate whether the promoter could be used for prostate cancer gene therapy, we initially transfected normal and malignant prostate cells. We then transfected HMEC-1 endothelial cells and ex vivo rat tail artery and monitored GFP levels using Western blotting following the delivery of single doses of ionizing radiation (2, 4, 6 Gy) to test whether the promoter could be used for vascular targeted gene therapy. RESULTS: The t-PA promoter induced GFP expression up to 6-fold in all cell types tested in response to radiation doses within the clinical range. CONCLUSIONS: These results suggest that the t-PA promoter may be incorporated into gene therapy strategies driving therapeutic transgenes in conjunction with radiation therapy.

Association of functional haem oxygenase-1 gene promoter polymorphism with polycystic kidney disease and IgA nephropathy

Background: Haem oxygenase-1 (HO-1) is a cytoprotective molecule that is reported to have a protective role in a variety of experimental models of renal injury. A functional dinucleotide repeat (GT)n polymorphism, within the HO-1 promoter, regulates HO-1 gene expression; a short number of repeats (S-allele &lt;25) increases transcription. We report the first assessment of the role of this HO-1 gene promoter polymorphism in chronic kidney disease due to autosomal dominant polycystic kidney disease (ADPKD) and IgA nephropathy (IgAN).<br/><br/>Methods: The DNA from 160 patients (99% Caucasian) on renal replacement therapy (RRT) was genotyped. The primary renal disease was ADPKD in 100 patients and biopsy-proven IgAN in 60 patients.<br/><br/>Results: Overall, the mean age at commencement of RRT was not significantly different between patients with and without an S-allele (44.1 years versus 45.0 years, P = 0.64). In patients with ADPKD, the age at commencement of RRT was comparable regardless of the HO-1 genotype (47.7 years versus 46.7 years, P = 0.59). The same was true in patients with IgAN (38.3 years versus 42.2 years, P = 0.28).<br/><br/>Conclusion: This suggests that the functional HO-1 promoter polymorphism does not influence renal survival in CKD due to ADPKD or IgAN.

sscMap: An extensible Java application for connecting small-molecule drugs using gene-expression signatures

Background<br/>Connectivity mapping is a process to recognize novel pharmacological and toxicological properties in small molecules by comparing their gene expression signatures with others in a database. A simple and robust method for connectivity mapping with increased specificity and sensitivity was recently developed, and its utility demonstrated using experimentally derived gene signatures.<br/><br/>Results<br/>This paper introduces sscMap (statistically significant connections' map), a Java application designed to undertake connectivity mapping tasks using the recently published method. The software is bundled with a default collection of reference gene-expression profiles based on the publicly available dataset from the Broad Institute Connectivity Map 02, which includes data from over 7000 Affymetrix microarrays, for over 1000 small-molecule compounds, and 6100 treatment instances in 5 human cell lines. In addition, the application allows users to add their custom collections of reference profiles and is applicable to a wide range of other 'omics technologies.<br/><br/>Conclusion<br/>The utility of sscMap is two fold. First, it serves to make statistically significant connections between a user-supplied gene signature and the 6100 core reference profiles based on the Broad Institute expanded dataset. Second, it allows users to apply the same improved method to custom-built reference profiles which can be added to the database for future referencing. The software can be freely downloaded from http://purl.oclc.org/NET/sscMap

A simple and robust method for connecting small-molecule drugs using gene-expression signatures

Background<br/><br/>Interaction of a drug or chemical with a biological system can result in a gene-expression profile or signature characteristic of the event. Using a suitably robust algorithm these signatures can potentially be used to connect molecules with similar pharmacological or toxicological properties by gene expression profile. Lamb et al first proposed the Connectivity Map [Lamb et al (2006), Science 313, 1929–1935] to make successful connections among small molecules, genes, and diseases using genomic signatures.<br/><br/>Results<br/><br/>Here we have built on the principles of the Connectivity Map to present a simpler and more robust method for the construction of reference gene-expression profiles and for the connection scoring scheme, which importantly allows the valuation of statistical significance of all the connections observed. We tested the new method with two randomly generated gene signatures and three experimentally derived gene signatures (for HDAC inhibitors, estrogens, and immunosuppressive drugs, respectively). Our testing with this method indicates that it achieves a higher level of specificity and sensitivity and so advances the original method.<br/><br/>Conclusion<br/><br/>The method presented here not only offers more principled statistical procedures for testing connections, but more importantly it provides effective safeguard against false connections at the same time achieving increased sensitivity. With its robust performance, the method has potential use in the drug development pipeline for the early recognition of pharmacological and toxicological properties in chemicals and new drug candidates, and also more broadly in other 'omics sciences.

Frequency of cytokine gene promoter polymorphisms in the Northern Ireland Cystic Fibrosis population

It has been postulated that cytokine allele frequencies are gender and perhaps geographically-specific. Cytokine release is crucial in the regulation of the type and magnitude of the immune response. This study observed no differences in the frequency of cytokine promoter polymorphisms associated with variant levels of expression in patients with CIF and a non-CF population of Northern Ireland. (c) 2007 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription

Background: Glycogen synthase kinase-3 (GSK-8) is a serine/threonine protein kinase, the activity of which is inhibited by a variety of extracellular stimuli including insulin, growth factors, cell specification factors and cell adhesion. Consequently, inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3.