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1.  Caveolin-1 Regulates Genomic Action of the Glucocorticoid Receptor in Neural Stem Cells 
Molecular and Cellular Biology  2014;34(14):2611-2623.
While glucocorticoids (GCs) are used clinically to treat many conditions, their neonatal and prenatal usage is increasingly controversial due to reports of delayed adverse outcomes, especially their effects on brain development. Such alterations may reflect the impact of GCs on neural progenitor/stem cell (NPSC) function. We previously demonstrated that the lipid raft protein caveolin-1 (Cav-1) was required for rapid GC signaling in embryonic mouse NPSCs operating through plasma membrane-bound glucocorticoid receptors (GRs). We show here that genomic GR signaling in NPSCs requires Cav-1. Loss of Cav-1 impacts the transcriptional response of many GR target genes (e.g., the serum- and glucocorticoid-regulated kinase 1 gene) that are likely to mediate the antiproliferative effects of GCs. Microarray analysis of wild-type C57 or Cav-1-deficient NPSCs identified approximately 100 genes that are differentially regulated by GC treatment. These changes in hormone responsiveness in Cav-1 knockout NPSCs are associated with the loss of GC-regulated phosphorylation of GR at serine 211 but not at serine 226. Chromatin recruitment of total GR to regulatory regions of target genes such as Fkbp-5, RhoJ, and Sgk-1, as well as p211-GR recruitment to Sgk-1, are compromised in Cav-1 knockout NPSCs. Cav-1 is therefore a multifunctional regulator of GR in NPSCs influencing both rapid and genomic action of the receptor to impact cell proliferation.
PMCID: PMC4097667  PMID: 24777604
2.  Serum protein profiles predict coronary artery disease in symptomatic patients referred for coronary angiography 
BMC Medicine  2012;10:157.
More than a million diagnostic cardiac catheterizations are performed annually in the US for evaluation of coronary artery anatomy and the presence of atherosclerosis. Nearly half of these patients have no significant coronary lesions or do not require mechanical or surgical revascularization. Consequently, the ability to rule out clinically significant coronary artery disease (CAD) using low cost, low risk tests of serum biomarkers in even a small percentage of patients with normal coronary arteries could be highly beneficial.
Serum from 359 symptomatic subjects referred for catheterization was interrogated for proteins involved in atherogenesis, atherosclerosis, and plaque vulnerability. Coronary angiography classified 150 patients without flow-limiting CAD who did not require percutaneous intervention (PCI) while 209 required coronary revascularization (stents, angioplasty, or coronary artery bypass graft surgery). Continuous variables were compared across the two patient groups for each analyte including calculation of false discovery rate (FDR ≤ 1%) and Q value (P value for statistical significance adjusted to ≤ 0.01).
Significant differences were detected in circulating proteins from patients requiring revascularization including increased apolipoprotein B100 (APO-B100), C-reactive protein (CRP), fibrinogen, vascular cell adhesion molecule 1 (VCAM-1), myeloperoxidase (MPO), resistin, osteopontin, interleukin (IL)-1β, IL-6, IL-10 and N-terminal fragment protein precursor brain natriuretic peptide (NT-pBNP) and decreased apolipoprotein A1 (APO-A1). Biomarker classification signatures comprising up to 5 analytes were identified using a tunable scoring function trained against 239 samples and validated with 120 additional samples. A total of 14 overlapping signatures classified patients without significant coronary disease (38% to 59% specificity) while maintaining 95% sensitivity for patients requiring revascularization. Osteopontin (14 times) and resistin (10 times) were most frequently represented among these diagnostic signatures. The most efficacious protein signature in validation studies comprised osteopontin (OPN), resistin, matrix metalloproteinase 7 (MMP7) and interferon γ (IFNγ) as a four-marker panel while the addition of either CRP or adiponectin (ACRP-30) yielded comparable results in five protein signatures.
Proteins in the serum of CAD patients predominantly reflected (1) a positive acute phase, inflammatory response and (2) alterations in lipid metabolism, transport, peroxidation and accumulation. There were surprisingly few indicators of growth factor activation or extracellular matrix remodeling in the serum of CAD patients except for elevated OPN. These data suggest that many symptomatic patients without significant CAD could be identified by a targeted multiplex serum protein test without cardiac catheterization thereby eliminating exposure to ionizing radiation and decreasing the economic burden of angiographic testing for these patients.
PMCID: PMC3566965  PMID: 23216991
atherosclerosis; biomarkers; cardiac catheterization; coronary angiography; coronary stenosis; multiplex proteomics
3.  Transcriptional profiling reveals elevated Sox2 in DNA polymerase ß null mouse embryonic fibroblasts 
There are over 150 human proteins that have been categorized as bona fide DNA repair proteins. These DNA repair proteins maintain the integrity of the genome, reducing the onset of cancer, disease and aging phenotypes. Variations in expression and/or function would therefore impact genome integrity as well as the cellular response to genotoxins. Global gene expression analysis is an effective approach to uncover defects in DNA repair gene expression and to discover cellular and/or organismal effects brought about by external stimuli such as environmental genotoxicants, chemotherapeutic regimens, viral infections as well as developmental and age-related stimuli. Given the significance of genome stability in cell survival and response to stimuli, we have hypothesized that cells may undergo transcriptional re-programming to accommodate defects in basal DNA repair capacity to promote survival. As a test of this hypothesis, we have compared the transcriptome in three DNA polymerase ß knockout (Polß-KO) mouse embryonic fibroblasts (MEFs) and the corresponding wild-type (WT) littermate control cell lines. Each Polß-KO cell line was found to have a range of genes up-regulated, when compared to its WT littermate control cell line. Interestingly, six (6) genes were commonly up regulated in all three Polß-KO cell lines, including Sox2, one of several genes associated with the induction of pluripotent stem cells. Herein, we present these findings and suggest that loss of DNA repair and the induction of cellular transcriptional re-programming may, in part, contribute to tumor formation and the cellular response to external stimuli.
PMCID: PMC3512183  PMID: 23226616
DNA polymerase ß; mouse embryonic fibroblast; Sox2; gene expression profiling; transcriptional reprogramming
4.  Identification and Expansion of a Unique Stem Cell Population from Adult Mouse Gallbladder 
Hepatology (Baltimore, Md.)  2011;54(5):1830-1841.
The identification of resident stem cells in the mouse gallbladder is to date, unexplored. In addition, the relationship between adult gallbladder stem cells and intrahepatic bile duct (IHBD) cells is not well understood. The goal of this study was to isolate stem cells from an adult mouse gallbladder and determine if they were unique compared to IHBD cells. By limiting dilution analyses and index sorts, we found that an EpCAM+CD49fhi sub-population from primary gallbladder is enriched in colony forming cells compared to EpCAM+CD49flo cells. EpCAM+CD49fhi cells expressed CD29, CD133 and Sca1 but were negative for lineage markers CD31, CD45 and F4/80. Using a novel feeder cell culture system, we observed long-term (>passage 20) and clonal expansion of the EpCAM+CD49fhi cells in vitro. In a matrigel differentiation assay, EpCAM+CD49f+ cells expanding in vitro underwent organotypic morphogenesis forming ductular structures and cysts. These structures are similar to, and recapitulate a transport function of primary gallbladder. EpCAM+CD49f+ cells also engraft into the subcutaneous space of recipient mice. We compared primary gallbladder and IHBD cells by flow cytometry and found phenotypic differences in expression of CD49f, CD49e, CD81, CD26, CD54 and CD166. In addition, oligonucleotide microarrays showed that the expanded EpCAM+CD49f+ gallbladder cells and IHBD cells exhibit differences related to lipid and drug metabolism. Notable genes that were different are cytochrome P450, glutathione-S-transferase, Indian hedgehog and solute carrier family genes.
we have isolated an epithelial cell population from primary mouse gallbladder with stem cell characteristics and found it to be unique compared to IHBD cells.
PMCID: PMC3205206  PMID: 21793026
Intrahepatic bile duct cells; EpCAM; CD49f; biliary cells; gallbladder cells
5.  Immunohistochemical Staining of Slit2 in Primary and Metastatic Prostatic Adenocarcinoma1 
Translational Oncology  2011;4(5):314-320.
BACKGROUND: Conflicting roles for Slit2, a protein involved in mediating the processes of cell migration and chemotactic response, have been previously described in prostate cancer. Here we use immunohistochemistry to evaluate the expression of Slit2 in normal donor prostate (NDP), benign prostatic hyperplasia (BPH), high-grade prostatic intraepithelial neoplasia (HGPIN), normal tissue adjacent to prostatic adenocarcinoma (NAC), primary prostatic adenocarcinoma (PCa), and metastatic prostatic adenocarcinoma (Mets). METHODS: Tissue microarrays were immunostained for Slit2. The staining intensities were quantified using automated image analysis software. The data was statistically analyzed using one-way analysis of variance with subsequent Tukey tests for multiple comparisons or a nonparametric equivalent. Eleven cases of NDP, 35 cases of NAC, 15 cases of BPH, 35 cases of HGPIN, 106 cases of PCa, and 37 cases of Mets were analyzed. RESULTS: Specimens of PCa and HGPIN had the highest absolute staining for Slit2. Significant differences were seen between PCa and NDP (P < .05), PCa and NAC (P < .05), HGPIN and NDP (P < .05), and HGPIN and NAC (P < .05). Whereas the average Mets staining was not significantly different from NDP or NAC, several individual Mets cases featured intense staining. CONCLUSIONS: To our knowledge, this represents the first study comparing the immunohistochemical profiles of Slit2 in PCa and Mets to specimens of HGPIN, BPH, NDP, and NAC. These findings suggest that Slit2 expression can be increased in HGPIN, PCa, and Mets, making it a potentially important biomarker for prostate cancer.
PMCID: PMC3162306  PMID: 21966548
6.  Immunohistochemical analysis of ezrin-radixin-moesin-binding phosphoprotein 50 in prostatic adenocarcinoma 
BMC Urology  2011;11:12.
Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is an adapter protein which has been shown to play an active role in a wide variety of cellular processes, including interactions with proteins related to both tumor suppression and oncogenesis. Here we use immunohistochemistry to evaluate EBP50's expression in normal donor prostate (NDP), benign prostatic hyperplasia (BPH), high grade prostatic intraepithelial neoplasia (HGPIN), normal tissue adjacent to prostatic adenocarcinoma (NAC), primary prostatic adenocarcinoma (PCa), and metastatic prostatic adenocarcinoma (Mets).
Tissue microarrays were immunohistochemically stained for EBP50, with the staining intensities quantified using automated image analysis software. The data were statistically analyzed using one-way ANOVA with subsequent Tukey tests for multiple comparisons. Eleven cases of NDP, 37 cases of NAC, 15 cases of BPH, 35 cases of HGPIN, 103 cases of PCa, and 36 cases of Mets were analyzed in the microarrays.
Specimens of PCa and Mets had the lowest absolute staining for EBP50. Mets staining was significantly lower than NDP (p = 0.027), BPH (p = 0.012), NAC (p < 0.001), HGPIN (p < 0.001), and PCa (p = 0.006). Additionally, HGPIN staining was significantly higher than NAC (p < 0.009) and PCa (p < 0.001).
To our knowledge, this represents the first study comparing the immunohistochemical profiles of EBP50 in PCa and Mets to specimens of HGPIN, BPH, NDP, and NAC and suggests that EBP50 expression is decreased in Mets. Given that PCa also had significantly higher expression than Mets, future studies are warranted to assess EBP50's potential as a prognostic biomarker for prostate cancer.
PMCID: PMC3132203  PMID: 21672215
7.  Identifier mapping performance for integrating transcriptomics and proteomics experimental results 
BMC Bioinformatics  2011;12:213.
Studies integrating transcriptomic data with proteomic data can illuminate the proteome more clearly than either separately. Integromic studies can deepen understanding of the dynamic complex regulatory relationship between the transcriptome and the proteome. Integrating these data dictates a reliable mapping between the identifier nomenclature resultant from the two high-throughput platforms. However, this kind of analysis is well known to be hampered by lack of standardization of identifier nomenclature among proteins, genes, and microarray probe sets. Therefore data integration may also play a role in critiquing the fallible gene identifications that both platforms emit.
We compared three freely available internet-based identifier mapping resources for mapping UniProt accessions (ACCs) to Affymetrix probesets identifications (IDs): DAVID, EnVision, and NetAffx. Liquid chromatography-tandem mass spectrometry analyses of 91 endometrial cancer and 7 noncancer samples generated 11,879 distinct ACCs. For each ACC, we compared the retrieval sets of probeset IDs from each mapping resource. We confirmed a high level of discrepancy among the mapping resources. On the same samples, mRNA expression was available. Therefore, to evaluate the quality of each ACC-to-probeset match, we calculated proteome-transcriptome correlations, and compared the resources presuming that better mapping of identifiers should generate a higher proportion of mapped pairs with strong inter-platform correlations. A mixture model for the correlations fitted well and supported regression analysis, providing a window into the performance of the mapping resources. The resources have added and dropped matches over two years, but their overall performance has not changed.
The methods presented here serve to achieve concrete context-specific insight, to support well-informed decisions in choosing an ID mapping strategy for "omic" data merging.
PMCID: PMC3124437  PMID: 21619611
8.  A novel SNP analysis method to detect copy number alterations with an unbiased reference signal directly from tumor samples 
BMC Medical Genomics  2011;4:14.
Genomic instability in cancer leads to abnormal genome copy number alterations (CNA) as a mechanism underlying tumorigenesis. Using microarrays and other technologies, tumor CNA are detected by comparing tumor sample CN to normal reference sample CN. While advances in microarray technology have improved detection of copy number alterations, the increase in the number of measured signals, noise from array probes, variations in signal-to-noise ratio across batches and disparity across laboratories leads to significant limitations for the accurate identification of CNA regions when comparing tumor and normal samples.
To address these limitations, we designed a novel "Virtual Normal" algorithm (VN), which allowed for construction of an unbiased reference signal directly from test samples within an experiment using any publicly available normal reference set as a baseline thus eliminating the need for an in-lab normal reference set.
The algorithm was tested using an optimal, paired tumor/normal data set as well as previously uncharacterized pediatric malignant gliomas for which a normal reference set was not available. Using Affymetrix 250K Sty microarrays, we demonstrated improved signal-to-noise ratio and detected significant copy number alterations using the VN algorithm that were validated by independent PCR analysis of the target CNA regions.
We developed and validated an algorithm to provide a virtual normal reference signal directly from tumor samples and minimize noise in the derivation of the raw CN signal. The algorithm reduces the variability of assays performed across different reagent and array batches, methods of sample preservation, multiple personnel, and among different laboratories. This approach may be valuable when matched normal samples are unavailable or the paired normal specimens have been subjected to variations in methods of preservation.
PMCID: PMC3041647  PMID: 21269491
9.  Immunohistochemical profiles of claudin-3 in primary and metastatic prostatic adenocarcinoma 
Diagnostic Pathology  2011;6:12.
Claudins are integral membrane proteins that are involved in forming cellular tight junctions. One member of the claudin family, claudin-3, has been shown to be overexpressed in breast, ovarian, and pancreatic cancer. Here we use immunohistochemistry to evaluate its expression in benign prostatic hyperplasia (BPH), prostatic intraepithelial neoplasia (PIN), normal tissue adjacent to prostatic adenocarcinoma (NAC), primary prostatic adenocarcinoma (PCa), and metastatic prostatic adenocarcinoma (Mets).
Tissue microarrays were immunohistochemically stained for claudin-3, with the staining intensities subsequently quantified and statistically analyzed using a one-way ANOVA with subsequent Tukey tests for multiple comparisons or a nonparametric equivalent. Fifty-three cases of NAC, 17 cases of BPH, 35 cases of PIN, 107 cases of PCa, and 55 cases of Mets were analyzed in the microarrays.
PCa and Mets had the highest absolute staining for claudin-3. Both had significantly higher staining than BPH (p < 0.05 in both cases) and NAC (p < 0.05 in both cases). PIN had a lower, but non-significant, staining score than PCa and Mets, but a statistically higher score than both BPH and NAC (p < 0.05 for both cases). No significant differences were observed between PCa, Mets, and PIN.
To our knowledge, this represents one of the first studies comparing the immunohistochemical profiles of claudin-3 in PCa and NAC to specimens of PIN, BPH, and Mets. These findings provide further evidence that claudin-3 may serve as an important biomarker for prostate cancer, both primary and metastatic, but does not provide evidence that claudin-3 can be used to predict risk of metastasis.
PMCID: PMC3033791  PMID: 21255442
10.  Immunohistochemical staining of radixin and moesin in prostatic adenocarcinoma 
Some members of the Protein 4.1 superfamily are believed to be involved in cell proliferation and growth, or in the regulation of these processes. While the expression levels of two members of this family, radixin and moesin, have been studied in many tumor types, to our knowledge they have not been investigated in prostate cancer.
Tissue microarrays were immunohistochemically stained for either radixin or moesin, with the staining intensities subsequently quantified and statistically analyzed using One-Way ANOVA or nonparametric equivalent with subsequent Student-Newman-Keuls tests for multiple comparisons. There were 11 cases of normal donor prostates (NDP), 14 cases of benign prostatic hyperplasia (BPH), 23 cases of high-grade prostatic intraepithelial neoplasia (HGPIN), 88 cases of prostatic adenocarcinoma (PCa), and 25 cases of normal tissue adjacent to adenocarcinoma (NAC) analyzed in the microarrays.
NDP, BPH, and HGPIN had higher absolute staining scores for radixin than PCa and NAC, but with a significant difference observed between only HGPIN and PCa (p = < 0.001) and HGPIN and NAC (p = 0.001). In the moesin-stained specimens, PCa, NAC, HGPIN, and BPH all received absolute higher staining scores than NDP, but the differences were not significant. Stage 4 moesin-stained PCa had a significantly reduced staining intensity compared to Stage 2 (p = 0.003).
To our knowledge, these studies represent the first reports on the expression profiles of radixin and moesin in prostatic adenocarcinoma. The current study has shown that there were statistically significant differences observed between HGPIN and PCa and HGPIN and NAC in terms of radixin expression. The differences in the moesin profiles by tissue type were not statistically significant. Additional larger studies with these markers may further elucidate their potential roles in prostatic neoplasia progression.
PMCID: PMC3029218  PMID: 21235778
11.  Gene expression profiles of prostate cancer reveal involvement of multiple molecular pathways in the metastatic process 
BMC Cancer  2007;7:64.
Prostate cancer is characterized by heterogeneity in the clinical course that often does not correlate with morphologic features of the tumor. Metastasis reflects the most adverse outcome of prostate cancer, and to date there are no reliable morphologic features or serum biomarkers that can reliably predict which patients are at higher risk of developing metastatic disease. Understanding the differences in the biology of metastatic and organ confined primary tumors is essential for developing new prognostic markers and therapeutic targets.
Using Affymetrix oligonucleotide arrays, we analyzed gene expression profiles of 24 androgen-ablation resistant metastatic samples obtained from 4 patients and a previously published dataset of 64 primary prostate tumor samples. Differential gene expression was analyzed after removing potentially uninformative stromal genes, addressing the differences in cellular content between primary and metastatic tumors.
The metastatic samples are highly heterogenous in expression; however, differential expression analysis shows that 415 genes are upregulated and 364 genes are downregulated at least 2 fold in every patient with metastasis. The expression profile of metastatic samples reveals changes in expression of a unique set of genes representing both the androgen ablation related pathways and other metastasis related gene networks such as cell adhesion, bone remodelling and cell cycle. The differentially expressed genes include metabolic enzymes, transcription factors such as Forkhead Box M1 (FoxM1) and cell adhesion molecules such as Osteopontin (SPP1).
We hypothesize that these genes have a role in the biology of metastatic disease and that they represent potential therapeutic targets for prostate cancer.
PMCID: PMC1865555  PMID: 17430594
12.  Differences in gene expression in prostate cancer, normal appearing prostate tissue adjacent to cancer and prostate tissue from cancer free organ donors 
BMC Cancer  2005;5:45.
Typical high throughput microarrays experiments compare gene expression across two specimen classes – an experimental class and baseline (or comparison) class. The choice of specimen classes is a major factor in the differential gene expression patterns revealed by these experiments. In most studies of prostate cancer, histologically malignant tissue is chosen as the experimental class while normal appearing prostate tissue adjacent to the tumor (adjacent normal) is chosen as the baseline against which comparison is made. However, normal appearing prostate tissue from tumor free organ donors represents an alterative source of baseline tissue for differential expression studies.
To examine the effect of using donor normal tissue as opposed to adjacent normal tissue as a baseline for prostate cancer expression studies, we compared, using oligonucleotide microarrays, the expression profiles of primary prostate cancer (tumor), adjacent normal tissue and normal tissue from tumor free donors.
Statistical analysis using Significance Analysis of Microarrays (SAM) demonstrates the presence of unique gene expression profiles for each of these specimen classes. The tumor v donor expression profile was more extensive that the tumor v adjacent normal profile. The differentially expressed gene lists from tumor v donor, tumor v adjacent normal and adjacent normal v donor comparisons were examined to identify regulated genes. When donors were used as the baseline, similar genes are highly regulated in both tumor and adjacent normal tissue. Significantly, both tumor and adjacent normal tissue exhibit significant up regulation of proliferation related genes including transcription factors, signal transducers and growth regulators compared to donor tissue. These genes were not picked up in a direct comparison of tumor and adjacent normal tissues.
The up-regulation of these gene types in both tissue types is an unexpected finding and suggests that normal appearing prostate tissue can undergo genetic changes in response to or in expectation of morphologic cancer. A possible field effect surrounding prostate cancers and the implications of these findings for characterizing gene expression changes in prostate tumors are discussed.
PMCID: PMC1173092  PMID: 15892885

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