BACKGROUND
gene expression profiling, along with other methods to evaluate the global changes in genomes, provides the opportunity to understand whole scale changes present in human biology.
yet the sheer mass of data presented by these techniques often makes subsequent analysis difficult.
techniques such as gene expression profiling may result in the identification of hundreds if not thousands of differentially expressed genes that may be associated with the biological process, but may also represent noise related to the biological and technical variation.
in an economic environment where limited resources are available for the follow-up and validation of potential target genes methods must be provided for the prioritization and sorting of data.
previous methods have relied heavily on the mapping of metabolic pathways or transcription factor binding sites  <cit> .
these processes rely on the premise that the metabolic pathways associated with a given disease are well delineated, or that groups of proteins with very similar structural or functional design are involved in the disease process.
in situations where these assumptions may not be true, alternative methods for the sorting of the data are needed.
here we demonstrate an alternative approach using comparative genomics and animal models of human prostate cancer to sort and identify genes involved in the response of prostate cancer cells to the proposed chemopreventive agent selenium  <cit> .
this process takes advantage of the continued sequencing of multiple animal genomes and the ability to produce gene expression profiles in multiple species.
through the use of these techniques one can leverage established animal models to identify genes associated with human disease processes, as is demonstrated here with the identification of insulin-like growth factor- <dig> binding protein  <dig>  and retinoid-x-receptor alpha .
RESULTS
generation of common genes and homologs
sequence validated gene libraries for both the rat and human dnas were obtained from research genetics , and were supplemented with additional dna samples obtained from the university of iowa rat clone sequencing program  <cit> .
the majority of the rat dnas, and a subset of the human dnas were resequenced by dr. j. quackenbush at tigr through a joint program in genomic applications consortium.
the genebank accession numbers for the  <dig>  individual human or rat clones present in the recent slide printings were used to query the ncbi unigene database to return the associated unigene ids.
unigene ids were returned for virtually all identified clones, and were placed in an oracle database where they were compared with the downloaded ncbi homologene dataset  of rat, mouse, and human homologues.
of the  <dig>  clones,  <dig> genes were identified with homologues present on both the rat and human slides.
this homologue set was used for the subsequent comparisons across species.
similar global and prostate gene expression profiles between rat and human prostate cancer cell lines
we have sought to compare the rat and human prostate cancer transcriptomes in an effort to judge the degree of similarity between the two cell types.
because the use of differentially expressed genes would bias the comparison by eliminating the majority of genes that do not show any difference, we used the absolute level of expression for each gene and compared the rat and human genes for significant differences in absolute expression levels.
in order to derive the absolute level of expression for individual genes in human or rat prostate cancer cells we used expression values derived from the associated self-self hybridizations performed for each cell line.
the experiments were facilitated by the use of slides that have been quality controlled for the quantity of spotted target dna through the use of a fitc label third dye  <cit> .
these slides were subsequently imaged for fitc fluorescence and sorted based on the similar amounts of target dna present on each slide  <cit> .
using the third dye quality control correlation coefficients of greater than  <dig>  are routinely achieved between slide replicates  <cit> .
in this manner comparisons of bound hybridized probe can be made across slides with a degree of confidence.
rna samples from cells were harvested, labeled, and homotypically hybridized to establish the baseline level of consistency within the hybridizations.
performing slice analysis on the normalized homotypic gene expression data across all the self-self hybridization slides within a species and retaining genes that demonstrated consistent expression patterns within two standard deviations of the mean expression value was performed to remove a degree of error from the technical replicates.
using the third dye as a baseline for comparison, these common expressed genes were then broken down into their component cy <dig> or cy <dig> expression vectors and used to build the transcriptomes for each gene using their absolute expression values.
these transcriptomes were then used to compare expression values between the rat and human cell lines.
these genes were annotated and gene homologues identified from the ncbi homologene <cit>  dataset of rat-human homologues.
thus from a dataset of  <dig> homologues,  <dig> genes were found that were present within this experimental dataset and expressed in both the rat and human prostate cancer cell lines, and thus could be used for comparative genomics.
these samples were processed using the multiexperiment viewer mircoarray statistical analysis and visualization program developed by tigr  <cit> .
files were loaded and visualized for comparison across the  <dig> common expressed genes in a self-organizing tree algorhythm  <cit>   and analyzed for similarities in global expression patterns.
the hierarchical clustering in self-organizing trees failed to demonstrate a pattern of clustering between species.
t-test analysis  <cit>  between the human and rat cell lines identified  <dig> genes  which demonstrated significantly different expression patterns between species .
thus in these comparisons,  <dig> genes, or 98% of the genes examined, failed to demonstrate a statistically significant difference in expression between the human and rat prostate cancer cell lines.
using principle components analysis  these studies can be visualized, and demonstrate while there is some clustering of the rat and human prostate cancer cell lines, the differences are not significant.
thus when comparing gene expression patterns in rat and human cell lines one will detect significant species-specific differences in expression in  <dig> out of every  <dig> genes, with the majority of the genes demonstrating similar expression patterns.
the presence of a large quantity of genes whose expression may be related to general cellular functions, as opposed to prostate specific metabolism, could infuse a significant amount of homogeneity to the data.
in the presence of such homogeneity it may be impossible to identify the true differences that are related to prostate cellular function, and thus the perceived similarities may be artifactual.
to address this issue we sought to repeat the analysis using only prostate related genes.
to generate a list of such genes we used cdnas in eight normal human prostate cdna libraries present in the nci cancer genome anatomy project  <cit> .
generation of a list of common genes proved impossible, as the combination of more than four of the cdna lists resulted in the number of common genes being reduced to zero.
a similar result was obtained when one attempted to generate a list of commonly expressed genes across multiple different cancer cdna libraries.
as an alternative approach we developed a list of  <dig>  expressed genes were identified based on their presence in at least one of the eight normal human prostate cdna libraries.
the human unigene ids for each of the expressed genes were then used to identify the associated rat homologues from homologene  <cit>  and yielded  <dig>  homologous rat genes , of which  <dig>   had associated prostate cancer gene expression data.
these  <dig>  prostate expressed genes were then used to repeat the comparative genomics.
similar visual and clustering results were identified for the prostate transcriptomes.
t-test analysis  <cit>  between the human and rat cell lines identified  <dig> prostate expressed genes  which demonstrated significant differential expression between species  failed to demonstrate a significant difference in expression across species.
thus even when only prostate expressed genes are considered, similar results were obtained.
between the rat and human prostate cancer cell lines the patterns of expression are similar for  <dig> of  <dig> genes examined.
comparison of global and prostate specific differential gene expression profiles between rat and human prostate cancer cell lines treated with selenium
while global gene expression profiles appear to be similar between rat and human prostate cancer cell lines one wonders whether the response to specific physiologic stimuli may elicit similar transcriptional changes.
if so, one may be able to infer a degree of homology in their biological response to the stimuli.
this has already been observed on a physiological level for the rat models of prostate cancer.
for example, rat and human prostate cancers respond very similarly to chemotheraputic and environmental agents including hormonal agents , cyclophosphamide , high fat diets , and soy isoflavones   <cit> .
in an effort to evaluate these similar biological responses we have compared the transcriptomes between rat and human prostate cancer cell lines treated with the proposed prostate cancer chemopreventive agent selenium.
samples from the human pc <dig> and rat pa-iii cell lines were treated with selenium and examined for differential gene expression profiling.
these two cell lines were chosen based on their similar biologic characteristics, as both cell lines were derived from androgen independent metastatic tumors, and thus represent tumors with similar biologic potential  <cit> .
the cells were treated with twenty-five micromolar selenium for either  <dig> hours or  <dig> days, to identify both immediate changes in gene transcription or changes related to the long term exposure to selenium.
due to our interest in prostate cancer we have attempted to choose a form and concentration of selenium that would be reflected in the ongoing prevention trials such as the select prostate cancer prevention trial  <cit> .
in this trial patients receive selenium in the form of selenized baker's yeast.
previous hplc and electrospray mass spectroscopy studies have demonstrated that 85% of the selenium in yeast is present as selenomethionine  <cit> .
selenomethionine has previously been used in in-vitro studies of prostate cancer cells <cit> .
these studies demonstrated an inhibition of prostate cancer cell proliferation over a broad range of concentrations, while an ic <dig> and/or decreased expression was seen at concentrations above  <dig> micromolar selenomethionine.
to avoid the general effects of cell inhibition or cell death while focusing on the effect of selenium we chose a lower concentration of  <dig> micromolar selenomethionine.
these changes, while not resulting in increased cell death, did cause decreased cell division and increased doubling time in both species .
common rat and human homologous genes demonstrating differential expression by greater than two standard deviations were identified and included  <dig> genes after  <dig> hours and  <dig> genes after  <dig> days of exposure to selenium.
when the expression patterns of these genes were compared across species by t-test and principle component analysis as outlined above  <dig> genes  were found to have statistically significant differences in expression between species .
thus when comparing rat and human samples, while the majority of the gene expression changes are similar, in at least one in four genes  one can detect significant species specific differences in expression alteration when cells are treated with selenium.
yet similar physiologic changes  were observed in both species.
these changes represent the desired physiologic changes one would expect for the chemopreventive effects of selenium, and could be dissected by examining the common transcriptional changes seen in both species with respect to selenium.
combined differential expression patterns for selenium responsive genes identify common gene pathways
because some of the differences in the rat and human prostate cancer cell line transcriptomes may be related to confounding variables such as culture methods, cell passage number, or time in culture, an effort was made to focus on genes that are common, and as such may define the similar selenium based cell proliferative changes.
the subsets of  <dig> and  <dig> differentially expressed genes  were analyzed for genes that demonstrate similar changes in expression with respect to selenium across species.
of these differentially expressed genes,  <dig> and  <dig> demonstrated up-regulation in rat and human cells at  <dig> hours and  <dig> days respectively.
likewise,  <dig>  and  <dig>  demonstrated down-regulation in the presence of selenium.
when these subsets were further analyzed to identify genes with similar levels of up or down-regulation   <dig> genes were identified at  <dig> hours and  <dig> at  <dig> days .
these genes included  <dig> ests or genes with limited associated data, and  <dig> defined genes with associated gene data.
twenty-four of the genes were common to selenium treatment at both  <dig> hours and  <dig> days.
additional information related to these genes was obtained using the geneinfo data mining tool.
this tool was developed by the authors  to allow for the rapid identification of supplemental data from the biomedical literature related to genes of interest.
in brief, the tool allows one to cut and paste a list of genes based on either unigene or genebank ids and search pubmed for associated references based on annotations of the associated gene names.
additional search terms can be stipulated by the user based on their knowledge of the biological process or in response to results received from the previous search.
results are returned in a table that lists the number of references that met the search criteria and provides a hyperlink to the associated references for either downloading or viewing.
in this way the user is allowed to direct queries in an open manner based on their own experience or unpublished data.
in this manner searches were conducted using the list of genes and the search terms "prostate cancer", "selenium", and "apoptosis" .
igfbp <dig> and rxr-alpha are expressed in the prostate, induced by selenium, and downregulated in prostate cancer
of the  <dig> genes identified with similar cross-species differential expression changes with respect to selenium, two genes were identified that had unique features based on their associated references and interrelated functions.
these genes, igfbp <dig> and rxr-alpha were both up-regulated with respect to selenium and could be used to suggest a model for selenium action in prostate cancer.
pxr-alpha is upregulated in both rat and human prostate cancer cells at  <dig> days in response to selenium.
likewise, igfbp <dig> is upregulated after six hours of selenium treatment in both species.
these two genes both contained medline references with respect to prostate cancer, but had not yet been implicated in selenium action.
western blotting performed on the human prostate cancer cell line pc <dig> with respect to selenium validated the bioinformatically identified expression data .
to confirm the role of these two proteins in the prostate immunohistochemical studies on prostate cancer tissue microarrays were performed to identify igfbp <dig> and rxr-alpha in both normal, nodular hyperplasia , high grade prostatic intraepithelial neoplasia , invasive carcinoma, and metastatic prostatic carcinoma .
these studies demonstrate that both igfbp <dig> and rxr-alpha are expressed in the normal human prostatic epithelium .
igfbp <dig> is also expressed in the prostatic basal cells.
patterns of expression were predominantly nuclear, a finding that has been described for both proteins  <cit> .
in addition, staining for igfbp <dig> was also noted in the prostatic stroma, consistent with igfbp3's associated function as a secreted protein.
decreased levels of igfbp <dig> was noted in prostatic cancers when compared to normal prostate epithelium .
along with this decreased expression there was a distinct shift in the protein localization nuclear to cytoplasmic was observed , and in cases where expression was still present, there were decreased numbers and intensity of cell staining.
igfbp <dig> expression was similar in hgpin, invasive carcinoma, and metastatic carcinoma.
the level and pattern of igfbp <dig> expression in nodular hyperplasia was similar to that seen in normal prostate tissues, and significantly different from the expression seen in cancer samples .
rxr-alpha expression was also significantly downregulated in prostate cancer when compared to normal prostate epithelium or nodular hyperplasia , and was similar to that seen in hgpin and metastatic carcinoma.
rxr-alpha expression was consistently nuclear in the samples studied, and while the intensity of staining was similar, in the remaining positive cancer cases there were decreased numbers of cells staining .
discussion
leveraging cross-species bioinformatics in the prioritization of gene data
through the use of cross-species comparisons of the number of differentially expressed genes to be examined after  <dig> hours and  <dig> days of selenium treatment was dropped from  <dig> and  <dig> to  <dig> and  <dig> respectively, an 87– <dig> percent reduction of the sample size.
even with the use of multiple timepoints, the number of differentially expressed genes was only reduced in a single species study to  <dig>  less than half.
by using comparative genomics the final dataset was reduced to  <dig> genes, providing a greater than  <dig> fold enrichment of the data.
thus by leveraging the additional biological species the ability to reduce the final analysis pool was substantial.
this process only works if the species used have biological relevance to the disease in question.
the choice of rat prostate cancer cell lines was made based on their use as an animal model for the study of prostate cancer  <cit> .
the animal systems have been extensively used in the study of hormonal carcinogenesis, and in particular have been of value as a model of environmental and dietary effects on prostate cancer  <cit> .
previous studies have identified similar effects of rat animal models and prostate cancer cell lines to soy based diets  <cit> , high fat diets  <cit> , hormonal chemotherapeutics  and standard chemotherapy  <cit> .
while comparative gene expression profiling has been performed, this has usually been through cross-species hybridizations to leverage rna studies in species where sufficient expressed transcripts in a given species have not been identified for the production of species-specific gene expression slides, in particular for microbial genomes  <cit> .
thus the approach taken here leverages the production of species-specific gene expression profiles along with the increasing amount of gene homolog data generated by the sequencing of additional animal genomes.
it is expected that with future genome efforts additional cross-species studies will be possible that leverage the knowledge of additional animal models in the study of disease.
similarities in prostate cancer transcriptomes across species
for both overall and prostate expressed genes, we have failed to identify a significant difference in the transcriptomes between rat and human prostate cancer cell lines.
this general similarity in transcriptomes may be due to the inherent biological similarities of the cell lines and/or their underlying biological origin.
while the studies sought to utilize prostate cancer cell lines with similar biological potentials  the degree of diversity present within the samples may account for some of the residual differences still identified.
in addition, the extended period of time that these cell lines have been used has allowed for the continued in-vitro evolution of the cells, and could possibly extend those genomic differences.
yet the common clustering of the rat and human cell lines together suggests there are still significant similarities in their biological potential.
this is also demonstrated by the similar biological potential of the cell lines when treated with a given stimulus, in this example selenium.
this parallels the similar physiological properties observed in the rat models of human prostate cancer.
based on these features we demonstrate that it is possible to identify functionally significant genes related to selenium response by using comparative genomics.
these findings also support the use of animal models in the study of human prostate cancer by suggesting that there is enough inherent genomic similarity that valuable insights may be gained from animal systems.
comparative genomics identifies functionally significant genes with respect to selenium chemoprevention
a true test of the profiling method is the identification of genes that have a functional significance to the experimental system.
in this case we have identified a series of genes, which when examined with additional data mining techniques, identifies genes with associated roles related to apoptosis , antioxidant protection , cell cycle , and protein balance .
in addition, the ability to sort the identified genes by their associated biomedical literature allowed the focus to shift to igfbp <dig> and rxralpha.
retinoids, through the retinoid x receptor, have been shown to induce the expression of igfbp <dig>  <cit> .
in concert these two proteins act to induce apoptosis in cancer cell lines  <cit> .
in particular, recent data has shown that these proteins work in synergy to enhance apoptosis in prostate cancer, and that there is a physical interaction between these two proteins in prostate cancer cells <cit> .
further validation and confirmatory data is presented here that demonstrates the selenium induced expression and interaction between both rxralpha and igfbp <dig> in prostate cancer cells, along with their expression in normal prostate epithelium and subsequent down-regulation in malignant prostatic epithelium.
this allows one to pose a model by which the restoration of igfbp <dig> and rxralpha levels by selenium treatment may lead to the disruption of prostate tumorigenesis.
this model is testable, and if validated, would present not only a mechanism by which selenium may exert its effect, but provide a biomarker for assaying the effect of selenium supplementation in the ongoing prostate cancer prevention clinical trials.
CONCLUSIONS
using gene profiling on highly controlled spotted cdna arrays we have demonstrated that similar baseline and selenium induced gene expression profiles can be identified between rat and human prostate cancer cells.
this has allowed us to filter our gene expression data to identify genes whose transcriptional response to selenium is similar across species, and by so doing focus our discovery process on specific common physiologic pathways.
two such proteins, rxr-alpha and igfbp- <dig>  which may be located in a common pathway, have been identified as dysregulated in human prostate cancers.
this provides further support that the cross-species methods employed here can identify genes with roles in human prostate cancer.

 gene expression profiling, along with other methods to evaluate the global changes in genomes, provides the opportunity to understand whole scale changes present in human biology. yet the sheer mass of data presented by these techniques often makes subsequent analysis difficult. techniques such as gene expression profiling may result in the identification of hundreds if not thousands of differentially expressed genes that may be associated with the biological process, but may also represent noise related to the biological and technical variation. in an economic environment where limited resources are available for the follow-up and validation of potential target genes methods must be provided for the prioritization and sorting of data. previous methods have relied heavily on the mapping of metabolic pathways or transcription factor binding sites. these processes rely on the premise that the metabolic pathways associated with a given disease are well delineated, or that groups of proteins with very similar structural or functional design are involved in the disease process. in situations where these assumptions may not be true, alternative methods for the sorting of the data are needed. here we demonstrate an alternative approach using comparative genomics and animal models of human prostate cancer to sort and identify genes involved in the response of prostate cancer cells to the proposed chemopreventive agent selenium. this process takes advantage of the continued sequencing of multiple animal genomes and the ability to produce gene expression profiles in multiple species. through the use of these techniques one can leverage established animal models to identify genes associated with human disease processes, as is demonstrated here with the identification of insulin-like growth factor- binding protein and retinoid-x-receptor alpha.  generation of common genes and homologs sequence validated gene libraries for both the rat and human dnas were obtained from research genetics, and were supplemented with additional dna samples obtained from the university of iowa rat clone sequencing program. the majority of the rat dnas, and a subset of the human dnas were resequenced by dr. j. quackenbush at tigr through a joint program in genomic applications consortium. the genebank accession numbers for the individual human or rat clones present in the recent slide printings were used to query the ncbi unigene database to return the associated unigene ids. unigene ids were returned for virtually all identified clones, and were placed in an oracle database where they were compared with the downloaded ncbi homologene dataset of rat, mouse, and human homologues. of the clones, genes were identified with homologues present on both the rat and human slides. this homologue set was used for the subsequent comparisons across species. similar global and prostate gene expression profiles between rat and human prostate cancer cell lines we have sought to compare the rat and human prostate cancer transcriptomes in an effort to judge the degree of similarity between the two cell types. because the use of differentially expressed genes would bias the comparison by eliminating the majority of genes that do not show any difference, we used the absolute level of expression for each gene and compared the rat and human genes for significant differences in absolute expression levels. in order to derive the absolute level of expression for individual genes in human or rat prostate cancer cells we used expression values derived from the associated self-self hybridizations performed for each cell line. the experiments were facilitated by the use of slides that have been quality controlled for the quantity of spotted target dna through the use of a fitc label third dye. these slides were subsequently imaged for fitc fluorescence and sorted based on the similar amounts of target dna present on each slide. using the third dye quality control correlation coefficients of greater than are routinely achieved between slide replicates. in this manner comparisons of bound hybridized probe can be made across slides with a degree of confidence. rna samples from cells were harvested, labeled, and homotypically hybridized to establish the baseline level of consistency within the hybridizations. performing slice analysis on the normalized homotypic gene expression data across all the self-self hybridization slides within a species and retaining genes that demonstrated consistent expression patterns within two standard deviations of the mean expression value was performed to remove a degree of error from the technical replicates. using the third dye as a baseline for comparison, these common expressed genes were then broken down into their component cy or cy expression vectors and used to build the transcriptomes for each gene using their absolute expression values. these transcriptomes were then used to compare expression values between the rat and human cell lines. these genes were annotated and gene homologues identified from the ncbi homologene dataset of rat-human homologues. thus from a dataset of homologues, genes were found that were present within this experimental dataset and expressed in both the rat and human prostate cancer cell lines, and thus could be used for comparative genomics. these samples were processed using the multiexperiment viewer mircoarray statistical analysis and visualization program developed by tigr. files were loaded and visualized for comparison across the common expressed genes in a self-organizing tree algorhythm and analyzed for similarities in global expression patterns. the hierarchical clustering in self-organizing trees failed to demonstrate a pattern of clustering between species. t-test analysis between the human and rat cell lines identified genes which demonstrated significantly different expression patterns between species. thus in these comparisons, genes, or 98% of the genes examined, failed to demonstrate a statistically significant difference in expression between the human and rat prostate cancer cell lines. using principle components analysis these studies can be visualized, and demonstrate while there is some clustering of the rat and human prostate cancer cell lines, the differences are not significant. thus when comparing gene expression patterns in rat and human cell lines one will detect significant species-specific differences in expression in out of every genes, with the majority of the genes demonstrating similar expression patterns. the presence of a large quantity of genes whose expression may be related to general cellular functions, as opposed to prostate specific metabolism, could infuse a significant amount of homogeneity to the data. in the presence of such homogeneity it may be impossible to identify the true differences that are related to prostate cellular function, and thus the perceived similarities may be artifactual. to address this issue we sought to repeat the analysis using only prostate related genes. to generate a list of such genes we used cdnas in eight normal human prostate cdna libraries present in the nci cancer genome anatomy project. generation of a list of common genes proved impossible, as the combination of more than four of the cdna lists resulted in the number of common genes being reduced to zero. a similar result was obtained when one attempted to generate a list of commonly expressed genes across multiple different cancer cdna libraries. as an alternative approach we developed a list of expressed genes were identified based on their presence in at least one of the eight normal human prostate cdna libraries. the human unigene ids for each of the expressed genes were then used to identify the associated rat homologues from homologene and yielded homologous rat genes, of which had associated prostate cancer gene expression data. these prostate expressed genes were then used to repeat the comparative genomics. similar visual and clustering results were identified for the prostate transcriptomes. t-test analysis between the human and rat cell lines identified prostate expressed genes which demonstrated significant differential expression between species failed to demonstrate a significant difference in expression across species. thus even when only prostate expressed genes are considered, similar results were obtained. between the rat and human prostate cancer cell lines the patterns of expression are similar for of genes examined. comparison of global and prostate specific differential gene expression profiles between rat and human prostate cancer cell lines treated with selenium while global gene expression profiles appear to be similar between rat and human prostate cancer cell lines one wonders whether the response to specific physiologic stimuli may elicit similar transcriptional changes. if so, one may be able to infer a degree of homology in their biological response to the stimuli. this has already been observed on a physiological level for the rat models of prostate cancer. for example, rat and human prostate cancers respond very similarly to chemotheraputic and environmental agents including hormonal agents, cyclophosphamide, high fat diets, and soy isoflavones. in an effort to evaluate these similar biological responses we have compared the transcriptomes between rat and human prostate cancer cell lines treated with the proposed prostate cancer chemopreventive agent selenium. samples from the human pc and rat pa-iii cell lines were treated with selenium and examined for differential gene expression profiling. these two cell lines were chosen based on their similar biologic characteristics, as both cell lines were derived from androgen independent metastatic tumors, and thus represent tumors with similar biologic potential. the cells were treated with twenty-five micromolar selenium for either hours or days, to identify both immediate changes in gene transcription or changes related to the long term exposure to selenium. due to our interest in prostate cancer we have attempted to choose a form and concentration of selenium that would be reflected in the ongoing prevention trials such as the select prostate cancer prevention trial. in this trial patients receive selenium in the form of selenized baker's yeast. previous hplc and electrospray mass spectroscopy studies have demonstrated that 85% of the selenium in yeast is present as selenomethionine. selenomethionine has previously been used in in-vitro studies of prostate cancer cells. these studies demonstrated an inhibition of prostate cancer cell proliferation over a broad range of concentrations, while an ic and/or decreased expression was seen at concentrations above micromolar selenomethionine. to avoid the general effects of cell inhibition or cell death while focusing on the effect of selenium we chose a lower concentration of micromolar selenomethionine. these changes, while not resulting in increased cell death, did cause decreased cell division and increased doubling time in both species. common rat and human homologous genes demonstrating differential expression by greater than two standard deviations were identified and included genes after hours and genes after days of exposure to selenium. when the expression patterns of these genes were compared across species by t-test and principle component analysis as outlined above genes were found to have statistically significant differences in expression between species. thus when comparing rat and human samples, while the majority of the gene expression changes are similar, in at least one in four genes one can detect significant species specific differences in expression alteration when cells are treated with selenium. yet similar physiologic changes were observed in both species. these changes represent the desired physiologic changes one would expect for the chemopreventive effects of selenium, and could be dissected by examining the common transcriptional changes seen in both species with respect to selenium. combined differential expression patterns for selenium responsive genes identify common gene pathways because some of the differences in the rat and human prostate cancer cell line transcriptomes may be related to confounding variables such as culture methods, cell passage number, or time in culture, an effort was made to focus on genes that are common, and as such may define the similar selenium based cell proliferative changes. the subsets of and differentially expressed genes were analyzed for genes that demonstrate similar changes in expression with respect to selenium across species. of these differentially expressed genes, and demonstrated up-regulation in rat and human cells at hours and days respectively. likewise, and demonstrated down-regulation in the presence of selenium. when these subsets were further analyzed to identify genes with similar levels of up or down-regulation genes were identified at hours and at days. these genes included ests or genes with limited associated data, and defined genes with associated gene data. twenty-four of the genes were common to selenium treatment at both hours and days. additional information related to these genes was obtained using the geneinfo data mining tool. this tool was developed by the authors to allow for the rapid identification of supplemental data from the biomedical literature related to genes of interest. in brief, the tool allows one to cut and paste a list of genes based on either unigene or genebank ids and search pubmed for associated references based on annotations of the associated gene names. additional search terms can be stipulated by the user based on their knowledge of the biological process or in response to results received from the previous search. results are returned in a table that lists the number of references that met the search criteria and provides a hyperlink to the associated references for either downloading or viewing. in this way the user is allowed to direct queries in an open manner based on their own experience or unpublished data. in this manner searches were conducted using the list of genes and the search terms "prostate cancer", "selenium", and "apoptosis". igfbp and rxr-alpha are expressed in the prostate, induced by selenium, and downregulated in prostate cancer of the genes identified with similar cross-species differential expression changes with respect to selenium, two genes were identified that had unique features based on their associated references and interrelated functions. these genes, igfbp and rxr-alpha were both up-regulated with respect to selenium and could be used to suggest a model for selenium action in prostate cancer. pxr-alpha is upregulated in both rat and human prostate cancer cells at days in response to selenium. likewise, igfbp is upregulated after six hours of selenium treatment in both species. these two genes both contained medline references with respect to prostate cancer, but had not yet been implicated in selenium action. western blotting performed on the human prostate cancer cell line pc with respect to selenium validated the bioinformatically identified expression data. to confirm the role of these two proteins in the prostate immunohistochemical studies on prostate cancer tissue microarrays were performed to identify igfbp and rxr-alpha in both normal, nodular hyperplasia, high grade prostatic intraepithelial neoplasia, invasive carcinoma, and metastatic prostatic carcinoma. these studies demonstrate that both igfbp and rxr-alpha are expressed in the normal human prostatic epithelium. igfbp is also expressed in the prostatic basal cells. patterns of expression were predominantly nuclear, a finding that has been described for both proteins. in addition, staining for igfbp was also noted in the prostatic stroma, consistent with igfbp3's associated function as a secreted protein. decreased levels of igfbp was noted in prostatic cancers when compared to normal prostate epithelium. along with this decreased expression there was a distinct shift in the protein localization nuclear to cytoplasmic was observed, and in cases where expression was still present, there were decreased numbers and intensity of cell staining. igfbp expression was similar in hgpin, invasive carcinoma, and metastatic carcinoma. the level and pattern of igfbp expression in nodular hyperplasia was similar to that seen in normal prostate tissues, and significantly different from the expression seen in cancer samples. rxr-alpha expression was also significantly downregulated in prostate cancer when compared to normal prostate epithelium or nodular hyperplasia, and was similar to that seen in hgpin and metastatic carcinoma. rxr-alpha expression was consistently nuclear in the samples studied, and while the intensity of staining was similar, in the remaining positive cancer cases there were decreased numbers of cells staining. discussion leveraging cross-species bioinformatics in the prioritization of gene data through the use of cross-species comparisons of the number of differentially expressed genes to be examined after hours and days of selenium treatment was dropped from and to and respectively, an 87– percent reduction of the sample size. even with the use of multiple timepoints, the number of differentially expressed genes was only reduced in a single species study to less than half. by using comparative genomics the final dataset was reduced to genes, providing a greater than fold enrichment of the data. thus by leveraging the additional biological species the ability to reduce the final analysis pool was substantial. this process only works if the species used have biological relevance to the disease in question. the choice of rat prostate cancer cell lines was made based on their use as an animal model for the study of prostate cancer. the animal systems have been extensively used in the study of hormonal carcinogenesis, and in particular have been of value as a model of environmental and dietary effects on prostate cancer. previous studies have identified similar effects of rat animal models and prostate cancer cell lines to soy based diets, high fat diets, hormonal chemotherapeutics and standard chemotherapy. while comparative gene expression profiling has been performed, this has usually been through cross-species hybridizations to leverage rna studies in species where sufficient expressed transcripts in a given species have not been identified for the production of species-specific gene expression slides, in particular for microbial genomes. thus the approach taken here leverages the production of species-specific gene expression profiles along with the increasing amount of gene homolog data generated by the sequencing of additional animal genomes. it is expected that with future genome efforts additional cross-species studies will be possible that leverage the knowledge of additional animal models in the study of disease. similarities in prostate cancer transcriptomes across species for both overall and prostate expressed genes, we have failed to identify a significant difference in the transcriptomes between rat and human prostate cancer cell lines. this general similarity in transcriptomes may be due to the inherent biological similarities of the cell lines and/or their underlying biological origin. while the studies sought to utilize prostate cancer cell lines with similar biological potentials the degree of diversity present within the samples may account for some of the residual differences still identified. in addition, the extended period of time that these cell lines have been used has allowed for the continued in-vitro evolution of the cells, and could possibly extend those genomic differences. yet the common clustering of the rat and human cell lines together suggests there are still significant similarities in their biological potential. this is also demonstrated by the similar biological potential of the cell lines when treated with a given stimulus, in this example selenium. this parallels the similar physiological properties observed in the rat models of human prostate cancer. based on these features we demonstrate that it is possible to identify functionally significant genes related to selenium response by using comparative genomics. these findings also support the use of animal models in the study of human prostate cancer by suggesting that there is enough inherent genomic similarity that valuable insights may be gained from animal systems. comparative genomics identifies functionally significant genes with respect to selenium chemoprevention a true test of the profiling method is the identification of genes that have a functional significance to the experimental system. in this case we have identified a series of genes, which when examined with additional data mining techniques, identifies genes with associated roles related to apoptosis, antioxidant protection, cell cycle, and protein balance. in addition, the ability to sort the identified genes by their associated biomedical literature allowed the focus to shift to igfbp and rxralpha. retinoids, through the retinoid x receptor, have been shown to induce the expression of igfbp. in concert these two proteins act to induce apoptosis in cancer cell lines. in particular, recent data has shown that these proteins work in synergy to enhance apoptosis in prostate cancer, and that there is a physical interaction between these two proteins in prostate cancer cells. further validation and confirmatory data is presented here that demonstrates the selenium induced expression and interaction between both rxralpha and igfbp in prostate cancer cells, along with their expression in normal prostate epithelium and subsequent down-regulation in malignant prostatic epithelium. this allows one to pose a model by which the restoration of igfbp and rxralpha levels by selenium treatment may lead to the disruption of prostate tumorigenesis. this model is testable, and if validated, would present not only a mechanism by which selenium may exert its effect, but provide a biomarker for assaying the effect of selenium supplementation in the ongoing prostate cancer prevention clinical trials.  using gene profiling on highly controlled spotted cdna arrays we have demonstrated that similar baseline and selenium induced gene expression profiles can be identified between rat and human prostate cancer cells. this has allowed us to filter our gene expression data to identify genes whose transcriptional response to selenium is similar across species, and by so doing focus our discovery process on specific common physiologic pathways. two such proteins, rxr-alpha and igfbp- which may be located in a common pathway, have been identified as dysregulated in human prostate cancers. this provides further support that the cross-species methods employed here can identify genes with roles in human prostate cancer. 
using both the human pc 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. 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. 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. further analysis and data mining for two genes, the insulin like growth factor binding protein 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. 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. gene expression technologies have the ability to generate vast amounts of data, yet there often resides only limited resources for subsequent validation studies.
 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.  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 pc 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 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.  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. 