ABSTRACT
BACKGROUND: Gene expression technologies have the ability to generate vast amounts of data, yet there often resides only limited resources for subsequent validation studies. This necessitates the ability to perform sorting and prioritization of the output data. Previously described methodologies have used functional pathways or transcriptional regulatory grouping to sort genes for further study. In this paper we demonstrate a comparative genomics based method to leverage data from animal models to prioritize genes for validation. This approach allows one to develop a disease-based focus for the prioritization of gene data, a process that is essential for systems that lack significant functional pathway data yet have defined animal models. This method is made possible through the use of highly controlled spotted cDNA slide production and the use of comparative bioinformatics databases without the use of cross-species slide hybridizations. RESULTS: Using gene expression profiling we have demonstrated a similar whole transcriptome gene expression patterns in prostate cancer cells from human and rat prostate cancer cell lines both at baseline expression levels and after treatment with physiologic concentrations of the proposed chemopreventive agent Selenium. Using both the human PC3 and rat PAII prostate cancer cell lines have gone on to identify a subset of one hundred and fifty-four genes that demonstrate a similar level of differential expression to Selenium treatment in both species. Further analysis and data mining for two genes, the Insulin like Growth Factor Binding protein 3, and Retinoic X Receptor alpha, demonstrates an association with prostate cancer, functional pathway links, and protein-protein interactions that make these genes prime candidates for explaining the mechanism of Selenium's chemopreventive effect in prostate cancer. These genes are subsequently validated by western blots showing Selenium based induction and using tissue microarrays to demonstrate a significant association between downregulated protein expression and tumorigenesis, a process that is the reverse of what is seen in the presence of Selenium. CONCLUSIONS: Thus the outlined process demonstrates similar baseline and selenium induced gene expression profiles between rat and human prostate cancers, and provides a method for identifying testable functional pathways for the action of Selenium's chemopreventive properties in prostate cancer.
Subject(s)
Adenocarcinoma/genetics , Anticarcinogenic Agents/therapeutic use , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Neoplasm Proteins/genetics , Prostatic Neoplasms/genetics , Selenium/therapeutic use , Adenocarcinoma/drug therapy , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Adenocarcinoma/prevention & control , Animals , Anticarcinogenic Agents/administration & dosage , Anticarcinogenic Agents/pharmacology , Cell Line, Tumor/drug effects , Cell Line, Tumor/metabolism , DNA, Neoplasm/analysis , DNA, Neoplasm/genetics , Expressed Sequence Tags , Fluorescein-5-isothiocyanate , Fluorescent Dyes , Gene Expression Profiling/instrumentation , Gene Expression Profiling/methods , Humans , Insulin-Like Growth Factor Binding Protein 3/biosynthesis , Insulin-Like Growth Factor Binding Protein 3/genetics , Male , Neoplasm Proteins/biosynthesis , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Prostatic Neoplasms/prevention & control , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Neoplasm/biosynthesis , RNA, Neoplasm/genetics , Rats , Retinoid X Receptor alpha/biosynthesis , Retinoid X Receptor alpha/genetics , Selenium/administration & dosage , Selenium/pharmacology , Selenomethionine/administration & dosage , Selenomethionine/pharmacology , Selenomethionine/therapeutic use , Species Specificity , Subtraction TechniqueABSTRACT
BACKGROUND: Our increasing use of genetic and genomic strategies to understand human prostate cancer means that we need access to simplified and integrated information present in the associated biomedical literature. In particular, microarray gene expression studies and associated genetic mapping studies in prostate cancer would benefit from a generalized understanding of the prior work associated with this disease. This would allow us to focus subsequent laboratory studies to genomic regions already related to prostate cancer by other scientific methods. We have developed a database of prostate cancer related chromosomal information from the existing biomedical literature. The input material was based on a broad literature search with subsequent hand annotation of information relevant to prostate cancer. DESCRIPTION: The database was then analyzed for identifiable trends in the whole scale literature. We have used this database, named ChromSorter PC, to present graphical summaries of chromosomal regions associated with prostate cancer broken down by age, ethnicity and experimental method. In addition we have placed the database information on the human genome using the Generic Genome Browser tool that allows the visualization of the data with respect to user generated datasets. CONCLUSIONS: We have used this database as an additional dataset for the filtering of genes identified through genetics and genomics studies as warranting follow-up validation studies. We would like to make this dataset publicly available for use by other groups. Using the Genome Browser allows for the graphical analysis of the associated data http://www.prostategenomics.org/datamining/chrom-sorter_pc.html. Additional material from the database can be obtained by contacting the authors (mdatta@mcw.edu).
Subject(s)
Chromosomes, Human , Databases, Genetic , Prostatic Neoplasms/genetics , Age Factors , Aged , Computer Graphics , Humans , Male , Middle Aged , Prostatic Neoplasms/ethnologyABSTRACT
Tissue microarrays are a novel technology with the potential to impact cancer research by reducing the time, materials, and costs related to specimen-based marker validation. The process uses small cores of specimen tissue for molecular studies, maximizing the quantity of specimens that can be analyzed on a single slide and the results that can be obtained from a single antibody study. However, this process can be tedious and requires a significant time commitment for array production, particularly for the hand-produced tissue array blocks. In addition, this process has significant repetitive motions, risking repetitive stress injury for technical personnel. For these reasons, we have sought a simple, inexpensive system for automation of the existing microarray technologies. Using this system, slides containing as many as 400 specimens can be constructed in a simple and reproducible manner. Automation of the tissue microarray apparatus is accomplished by attaching two stepper motors to the micrometers of the apparatus that control array movement, and it has the advantages of standardizing the spacing between each specimen and eliminating repetitive motions by the user. A computer program is used to run the motors, allowing the user to input commands based on the desired moving distance. After assimilation of the motors, motor control boards, and corresponding program, the final product was tested and demonstrated to provide consistent, reproducible operation. Tissue microarrays were generated with specimen tissue diameters of 1.5 mm, 1.0 mm, and 0.6 mm with core densities upwards of 300 samples per slide.
