Gene Regulation Profiles Research Articles

KLF10 Mediated Epigenetic Dysregulation of Epithelial CD40/CD154 Promotes Endometriosis.

Endometriosis is a highly prevalent, chronic, heterogeneous, fibro-inflammatory disease that remains recalcitrant to conventional therapy. We previously showed that loss of KLF11, a transcription factor implicated in uterine disease, results in progression of endometriosis. Despite extensive homology, co-expression, and human disease association, loss of the paralog Klf10 causes a unique inflammatory, cystic endometriosis phenotype in contrast to fibrotic progression seen with loss of Klf11. We identify here for the first time a novel role for KLF10 in endometriosis. In an animal endometriosis model, unlike wild-type controls, Klf10−/− animals developed cystic lesions with massive immune infiltrate and minimal peri-lesional fibrosis. The Klf10−/− disease progression phenotype also contrasted with prolific fibrosis and minimal immune cell infiltration seen in Klf11−/− animals. We further found that lesion genotype rather than that of the host determined each unique disease progression phenotype. Mechanistically, KLF10 regulated CD40/CD154-mediated immune pathways. Both inflammatory as well as fibrotic phenotypes are the commonest clinical manifestations in chronic fibro-inflammatory diseases such as endometriosis. The complementary, paralogous Klf10 and Klf11 models therefore offer novel insights into the mechanisms of inflammation and fibrosis in a disease-relevant context. Our data suggests that divergence in underlying gene dysregulation critically determines disease-phenotype predominance rather than the conventional paradigm of inflammation being precedent to fibrotic scarring. Heterogeneity in clinical progression and treatment response are thus likely from disparate gene regulation profiles. Characterization of disease phenotype-associated gene dysregulation offers novel approaches for developing targeted, individualized therapy for recurrent and recalcitrant chronic disease.

Gene expression profiles in guided bone regeneration using combinations of different biomaterials: a pilot animal study.

The aim of this study was to investigate the gene expression profile related to guided bone regeneration (GBR) at the early healing stage while using combinations of different biomaterials. Cranial defects in 4 New Zealand rabbits were filled with A) biphasic calcium phosphate/experimental pericardium-derived collagen membrane, B) Bio-Oss® /Bio-Gide® , C) biphasic calcium phosphate/strontium hydroxyapatite-containing collagen membrane and D) Bio-Oss® /strontium hydroxyapatite-containing collagen membrane. Seven days after surgery, one animal was subjected to histological observation and histomorphometric analysis, and three animals to real-time quantitative reverse transcription polymerase chain reaction (PCR). An RT2 Profiler PCR Array (PANZ-026Z, QIAGEN, QIAGEN Sciences, Germantown, MD, USA) was conducted to observe the gene expression profile of groups A, C and D as compared with the control group B. The analysis showed 9 of the 84 genes on the array to be significantly different in the three experimental groups (six genes in group D, four in group C and one in group A). Group D demonstrated the most changes in gene expression profile at day 7. Genes that were significantly down-regulated (AHSG, EGF) or up-regulated (CDH11, MMP13, GLI1 and MCSF) are responsible for early-stage bone formation, bone remodeling and pre-osteoclast development. The gene expression profile of this group correlated with the histological findings, as this group showed the higher formation of osteoid as compared with the other groups. Gene expression patterns at early-stage healing of GBR-treated defects appear to be related to the biomaterial used. The combination of Bio-Oss® and strontium hydroxyapatite-containing collagen membrane showed the most pro-osteogenic gene regulation profile (group D), implying the stimulation of key transcriptional factors, which appeared to translate into the up-regulation of the osteogenic process and earlier bone formation.

Intracerebral interleukin-10 injection modulates post-ischemic neuroinflammation: An experimental microarray study

Stroke induces a profound neuroinflammatory reaction that leads to secondary cerebral tissue injury. Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that is endogenously produced by immune cells and limits this inflammatory reaction. Previously, therapeutic administration of IL-10 has been shown to be neuroprotective in experimental stroke. However, the signaling pathways affected by this approach are largely unknown. The aim of this study was to verify the neuroprotective effects of IL-10 in an experimental mouse stroke model and to analyze the pathways modulated by this approach. Therefore, we performed a whole genome microarray analysis comparing the cerebral gene expression profile at two time points after cortical stroke in IL-10-treated and control C57Bl/6J mice. We administered IL-10 locally by intracerebroventricular injection. We were able to validate a reduction of infarct volume by IL-10 administration and characterized the kinetics of endogenous cerebral IL-10 expression after stroke. The microarray analysis revealed that IL-10 treatment effectively downregulated pro-inflammatory signaling cascades which were upregulated by the ischemic lesion in the acute phase after the stroke. This is the first study characterizing the global gene regulation profile of IL-10 immunotherapy for ischemic stroke. Our results emphasize the key role of IL-10 as a neuroprotective cytokine and suggest several novel downstream pathways for further investigation to better understand the mechanisms of post-stroke neuroinflammation.

Ethylene response factors gene regulation and expression profiles under different stresses in rice

Stresses can cause large yield reductions in cultivated plants. The response to these stresses occurs via a plethora of signalling pathways, where a large number of genes is induced or repressed. Among the environmental stress responsive genes, there are the members of the ethylene response factors (ERF) gene family. The mRNA levels of different ERF are regulated by many hormones and molecules produced under different stress conditions. In this study, with the goal of identifying the response of rice ERF genes to environmental stress, it was analysed the transcriptional expression profile of 114 of these genes under stress by anoxia, salt and Magnaporthe grisea. Also, aiming to characterize how the regulation of ERF genes occurs, the amount of known cis regulatory elements in the promoter region of these genes and their association with the expression profiles under the tested conditions were also assessed. The results indicate that some ERF members present the same specific expression profiles under different environmental stresses, while others do not. Within the ERF family, the regulation of gene expression is complex for some genes which have many cis elements in their promoters, but simple for others, demonstrating high levels of divergence among them. The findings demonstrate the importance of the study of each ERF separately, since it is not possible to establish general rules for regulation and probably for the function of these genes.

Abstract P6-05-10: Progestins exert divergent growth effects and regulate tumor-unique gene cohorts in patient derived breast cancer xenografts

Abstract Background: Progesterone is an important hormone for development and normal function of the breast; however, its role in established breast cancers is less clear. Progestins have been implicated in regulating tumor cell growth, signaling, differentiation state, and stem/progenitor properties in breast cancer cells. High dose progestin treatment can be an effective therapy for some advanced breast tumors, through an unknown mechanism. Progesterone receptors (PRs) are considered positive prognostic indicators, although their levels vary considerably among luminal subtype breast tumors. Progestin/PR regulated genes in tumor cells have only been determined in cultured breast cancer cells and mouse mammary tumor models. Here we define tumor unique progestin-dependent growth effects and gene regulation profiles in patient-derived luminal breast tumor xenografts. Methods: For these studies, three luminal estrogen receptor (ER)+PR+ transplantable xenografts were used that were derived from one pleural effusion and two previously untreated primary tumors. All three tumors were estrogen dependent to various degrees and contained variable levels of ER (5–90%) and PR (5–90%). Tumors were grown in vivo under continuous placebo, estrogen, or estrogen plus progestin conditions for 8–10 weeks and growth parameters monitored. Gene expression profiles were determined from dissected tumors using Affymetrix® GeneChip human gene 1.1 ST microarrays and results analyzed using Partek Genomics Suite software. Specific gene regulation was confirmed by q-RT-PCR and immunohistochemistry. Results: Progestins had an inhibitory, neutral, and stimulatory effect on estrogen dependent growth in each of the three tumor lines. Accordingly, there was relatively little overlap in PR regulated genes between the three tumors. In the tumor in which progestin treatment had a potent anti-tumor effect, progestins were strong repressors of estrogen/ER-regulated genes. Gene expression patterns between the natural hormone progesterone and the synthetic drug MPA were compared in one tumor line and found to be mostly similar. The percent of PR+ tumor cells may have influenced the potency of gene regulation. Unique progestin regulated genes were discovered in this human tumor model system that were involved in immune response, cytokine signaling, and stem/progenitor cell features. Conclusions: Women with breast cancer are exposed to progestins naturally or through hormonal therapies and these may have a profound effect on tumor biology. In some tumors, progestins are potently anti-proliferative, while in others they elicit the opposite effect and potentiate estrogen induced tumor growth. The diversity of progestin-regulated genes in each tumor underscores the hormone's context dependent effects. Determining progestin dependent gene signatures in patient tumors may pinpoint appropriate candidates for progestin therapy in advanced tumors and/or provide a prognostic tool for predicting tumor progression. Funded by: NIH R01 CA140985 (C.A.S.), the Grohne Fund (P.K.), and the University of Colorado Cancer Center (C.A.S., P.K.) Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-05-10.

Transcriptome and Coexpression Network Analysis of the Human Glioma Cell Line Hs683 Exposed to Candoxin

Gliomas are the most common primary tumours of the central nervous system. Snake venom, such as candoxin (CDX) isolated from Bungarus candidus, inhibits glioma cell proliferation. This study explored the gene regulation profile of CDX-treated human glioma Hs683 cells. Using microarray technology and bioinformatics analyses the underlying molecular mechanism of action of CDX was evaluated by constructing gene regulation and protein-protein interaction co expression networks. CDX treatment induced a large number of related genes at the transcriptional level. The MYC gene (v-myc myelocytomatosis viral oncogene homologue [avian]) had a key role in the response of Hs683 cells to CDX treatment. Activation of MYC upregulated NDRG1 (N-myc downstream regulated 1), WNT10B (wingless-type mouse mammary tumour virus integration site family, member 10B), CASP9 (caspase 9, apoptosis-related cysteine peptidase) and CDKN2A (cyclin-dependent kinase inhibitor 2A), and downregulated ID3 (inhibitor of DNA binding 3, dominant negative helix-loop-helix protein) and SLC1A4 (solute carrier family 1 [glutamate/neutral amino acid transporter], member 4). In addition, a subnetwork was constructed among SPP1 (secreted phosphoprotein 1), SDC1 (syndecan 1) and CD44 based on protein-protein interactions, and these genes were predicted to be involved in glioma cell invasion. These findings might provide novel therapeutic targets for glioma chemotherapy.

Abstract SY41-03: Vascular normalization by histidine-rich glycoprotein

Abstract Histidine-rich glycoprotein (HRG) is a 75 KDa heparin-binding plasma protein exclusively synthesized by hepatocytes. Structurally, HRG belongs to the family of type 3 secreted cystatins and is composed of an NH2-terminal cystatin-like region, a central Zn2+-binding domain rich in histidine and proline residues, and a C-terminal tail. HRG is stored in platelet alpha-granules but is also present free in plasma at relatively high concentrations (0.1-0.15 mg/ml in human plasma). Several reports have shown potent inhibition of tumor angiogenesis by HRG and peptides derived from its central His/Pro-rich domain, leading to reduced vascularization and growth of different syngeneic tumor models (1). HRG inhibits adhesion and chemotaxis of primary endothelial cells in vitro, which provides at least a partial mechanism for the effect of HRG in attenuation of tumor angiogenesis (2). However, HRG interacts with a wide range of ligands, at least in vitro, and exerts diverse biological functions (3). Thus, HRG binds to different components of both the coagulation and fibrinolytic systems, components of the immune system such as complement proteins and Fcg receptors, immunoglobulins (IgGs), immunocomplexes and glycosaminoglycans. Through some of these interactions, HRG modulates immune responsiveness. For example, HRG facilitates the clearance of immune complexes and dying/dead cells via an FcgR-I dependent mechanism HRG also enhances the ability of immune complexes to activate complement, allowing faster clearance of necrotic material (4), and it has been implicated in the defense at the local site of bacterial infection (5). We have asked how this wide range of effects could be amalgamated. In a recent study, we reported that HRG inhibits tumor growth and metastasis by polarizing tumor-associated macrophages (TAMs) to the M1 phenotype, leading to anti-tumor immunity and vessel normalization (6). Infiltration of TAMs into the tumor tissue is associated with an unfavorable prognosis. However, not only the number of infiltrating TAMs but also their phenotype regulates tumorigenesis (7, 8). In non-progressing or regressing tumors, TAMs are biased to a classic macrophage activation M1-like program, characterized by pro-inflammatory activity, antigen presentation and tumor lysis. In malignant tumors, TAMs resemble alternatively activated macrophages (M2-type), which increase angiogenesis and tumor cell intra/extra-vasation and growth; they suppress anti-tumor immunity by preventing activation of dendritic cells, cytotoxic T lymphocytes and natural killer cells. Using an hrg gene-inactivated mouse model, we now provide genetic evidence for HRGs effect on macrophage polarization. HRG ablation leads to increased tumor growth rate and metastatic dissemination, enhanced tumor vessel abnormalization, critical changes in the activation state of macrophages that lead to a tumor-promoting immune response, and a constitutive M2-like polarity of peritoneal macrophages. Furthermore, microarray analyses of the HRG-induced gene regulation profile in peritoneal macrophages ex vivo, show a pattern in excellent agreement with the inhibitory immune profile in HRG-deficient tumors. Combined our data show that HRG regulates TAM polarization in tumors, leading to reduced angiogenesis and induction of anti-tumor immunity, moreover, constitutive loss of HRG has a clear impact on the expression profile of circulating macrophages. Macrophage polarization by HRG offers new therapeutic opportunities for anti-cancer and anti-angiogenic treatment.

Comparative transcriptomics of extreme phenotypes of human HIV-1 infection and SIV infection in sooty mangabey and rhesus macaque

High levels of HIV-1 replication during the chronic phase of infection usually correlate with rapid progression to severe immunodeficiency. However, a minority of highly viremic individuals remains asymptomatic and maintains high CD4⁺ T cell counts. This tolerant profile is poorly understood and reminiscent of the widely studied nonprogressive disease model of SIV infection in natural hosts. Here, we identify transcriptome differences between rapid progressors (RPs) and viremic nonprogressors (VNPs) and highlight several genes relevant for the understanding of HIV-1-induced immunosuppression. RPs were characterized by a specific transcriptome profile of CD4⁺ and CD8⁺ T cells similar to that observed in pathogenic SIV-infected rhesus macaques. In contrast, VNPs exhibited lower expression of interferon-stimulated genes and shared a common gene regulation profile with nonpathogenic SIV-infected sooty mangabeys. A short list of genes associated with VNP, including CASP1, CD38, LAG3, TNFSF13B, SOCS1, and EEF1D, showed significant correlation with time to disease progression when evaluated in an independent set of CD4⁺ T cell expression data. This work characterizes 2 minimally studied clinical patterns of progression to AIDS, whose analysis may inform our understanding of HIV pathogenesis.

Global Gene Expression during the Human Organogenesis: From Transcription Profiles to Function Predictions

Human embryogenesis includes an integrated set of complex yet coordinated development of different organs and tissues, which is regulated by the spatiotemporal expression of many genes. Deciphering the gene regulation profile is essential for understanding the molecular basis of human embryo development. While molecular and genetic studies in mouse have served as a valuable tool to understand mammalian development, significant differences exists in human and mouse development at morphological and genomic levels. Thus it is important to carry out research directly on human embryonic development. Here we will review some recent studies on gene regulation during human embryogenesis with particular focus on the period of organogenesis, which had not been well studied previously. We will highlight a gene expression database of human embryos from the 4(th) to the 9(th) week. The analysis of gene regulation during this period reveals that genes functioning in a given developmental process tend to be coordinately regulated during human embryogenesis. This feature allows us to use this database to identify new genes important for a particular developmental process/pathway and deduce the potential function of a novel gene during organogenesis. Such a gene expression atlas should serve as an important resource for molecular study of human development and pathogenesis.

Adhesion formation of primary human osteoblasts and the functional response of mesenchymal stem cells to 330 nm deep microgrooves

The surface microtexture of an orthopaedic device can regulate cellular adhesion, a process fundamental in the initiation of osteoinduction and osteogenesis. Advances in fabrication techniques have evolved to include the field of surface modification; in particular, nanotechnology has allowed for the development of experimental nanoscale substrates for investigation into cell nanofeature interactions. Here primary human osteoblasts (HOBs) were cultured on ordered nanoscale groove/ridge arrays fabricated by photolithography. Grooves were 330nm deep and either 10, 25 or 100microm in width. Adhesion subtypes in HOBs were quantified by immunofluorescent microscopy and cell-substrate interactions were investigated via immunocytochemistry with scanning electron microscopy. To further investigate the effects of these substrates on cellular function, 1.7K gene microarray analysis was used to establish gene regulation profiles of mesenchymal stem cells cultured on these nanotopographies. Nanotopographies significantly affected the formation of focal complexes (FXs), focal adhesions (FAs) and supermature adhesions (SMAs). Planar control substrates induced widespread adhesion formation; 100microm wide groove/ridge arrays did not significantly affect adhesion formation yet induced upregulation of genes involved in skeletal development and increased osteospecific function; 25microm wide groove/ridge arrays were associated with a reduction in SMA and an increase in FX formation; and 10microm wide groove/ridge arrays significantly reduced osteoblast adhesion and induced an interplay of up- and downregulation of gene expression. This study indicates that groove/ridge topographies are important modulators of both cellular adhesion and osteospecific function and, critically, that groove/ridge width is important in determining cellular response.

Alterations in Antral Cytokine Gene Expression in Peptic Ulcer Patients During Ulcer Healing and After Helicobacter pylori Eradication

Earlier studies have shown that the antral immune response in Helicobacter pylori infection has a mixed Th1-Th2-T-regulatory profile. After eradication, a chronic inflammation remains in some patients, but a follow-up study with a comprehensive cytokine profile in has not previously been published. Twelve patients with H. pylori positive peptic ulcer disease (five antral and seven duodenal) were enrolled and cytokine gene expressions in antral biopsies were determined (1) at entry, (2) after resolving the ulcer with proton pump inhibitor (PPI) treatment and (3) after eradication. The second endoscopy was performed 4 weeks after ending the PPI treatment, and the third endoscopy was performed after a mean of 10 months after eradication. Inflammation was graded according to the updated Sydney system. Interleukin (IL)1beta, IL8, IL12A, IL18, TNFalpha, IFNgamma, IL4, IL6 and IL10 expression levels were analysed by real-time RT-PCR. Mixed mononuclear and neutrophil infiltrates were seen at entry and after ulcer healing. After eradication, low-grade mononuclear infiltrates were found. The cytokine expression levels after ulcer healing (H. pylori positive gastritis) were not significantly different from the levels at entry (ulcer). After eradication, attenuation of the Th1 cytokines except for TNFalpha and a persisting increase of IL4 levels were observed, whereas the IL10 expression was markedly reduced. The present data did not indicate a specific ulcer promoting cytokine gene regulation profile. However, after eradication a chronic low-grade inflammation was seen with reduced Th1, prolonged Th2 and disappearance of the T-regulatory response.

Gene regulation profiles by progesterone and dexamethasone in human endometrial cancer Ishikawa H cells

Objective.Progesterone and glucocorticoids such as dexamethasone mediate distinct biological functions, yet they bind to receptors that recognize the same consensus DNA response element. In breast cancer, progestins are associated with the incidence and progression of tumors, whereas glucocorticoids are growth-suppressive in mammary cancer cells; the differential effects of these two steroids are less well understood in the hormone-dependent disease cancer of the uterine endometrium. We set out to identify genes that are regulated by progesterone through progesterone receptors and dexamethasone through glucocorticoid receptors in a well-differentiated human endometrial cancer cell line. Methods.PR- and GR-positive Ishikawa H endometrial cancer cells were treated with vehicle, dexamethasone (100 nM) or progesterone (100 nM) for 2 h, 6 h, 12 h and 24 h, and RNA was isolated. Affymetrix microarrays were performed using the human HG-U133A chip, querying the expression of 22,000 genes. Expression of genes of particular interest was confirmed by real-time RT-PCR. Results.Expression analysis demonstrated that dexamethasone and progesterone regulate overlapping but distinct sets of genes and presumably exert many similar but also unique biological effects. Using real-time RT-PCR, we confirmed three particular genes of interest: the transcript for cysteine 1 (legumain), a gene associated with metastasis, that is strongly downregulated by progesterone, upstream c-fos relating transcription factor-2 (USF-2), an anti-proliferative factor that is induced by both progesterone and dexamethasone and N-cadherin, a cellular adhesion molecule downregulated by dexamethasone. Conclusion.These studies provide new insight into the effects of progesterone and dexamethasone in endometrial cancer cells and provide an extensive list of regulated pathways which can be assessed in the future as biomarkers and molecular targets for new therapies. Taken together, our findings indicate that progesterone and dexamethasone are primarily growth inhibitors in Ishikawa H endometrial cancer cells.

Gene regulation profile reveals consistent anticancer properties of progesterone in hormone-independent breast cancer cells transfected with progesterone receptor

Absence of estrogen receptor (ER) and progesterone receptor (PR) is the hallmark of most hormone-independent breast cancers. Previous studies demonstrated that reactivation of PR expression in hormone-independent MDA-MB-231 breast cancer cells enabled progesterone to suppress cell growth both in vitro and in vivo. We determined the whole genomic effect of progesterone in PR-transfected MDA-MB-231 cells. We identified 151 progesterone-regulated genes with expression changes > 3-fold after 24 hr treatment. Most are novel progesterone target genes. Real-time RT-PCR analysis of 55 genes showed a 100% confirmation rate. Twenty-six genes were regulated at both 3 and 24 hr. Studies using translation inhibitor suggest that most of the 26 genes are primary progesterone target genes. Progesterone consistently suppressed the expression of genes required for cell proliferation and metastasis and increased the expression of many tumor-suppressor genes. Progesterone also consistently decreased the expression of DNA repair and chromosome maintenance genes, which may be part of the mechanism leading to cell cycle arrest. These data suggest potential usefulness of progestin in combating ER-negative but PR-positive breast cancer and indicate that progesterone can exert a strong anticancer effect in hormone-independent breast cancer following PR reactivation. The identification of many novel progesterone target genes open up new avenues for in-depth elucidation of progesterone-mediated molecular networks.

Regulation of gene expression by PRA-910, a novel progesterone receptor modulator, in T47D cells

Progestins play an important role in women’s health and are used in oral contraception, hormone therapy, and treatment of reproductive disorders. The effects of progestins upon gene expression in breast epithelium are poorly understood. In an attempt to characterize the molecular mechanism of progestin action, we used a gene expression profiling approach to examine the action of a novel progestin in the T47D cell model, a human breast cancer cell line. PRA-910 is a novel, nonsteroidal progesterone receptor modulator (PRM) with species-specific activities identified in a screen for selective PRMs. To understand the mechanism of action for PRA-910 in T47D cells, we compared its gene regulation to progesterone (P4) and RU486 through Affymetrix U95A GeneChip ® analysis and TaqMan RT–PCR. PRA-910, P4, and RU486 regulated 50, 108, and 16 genes by threefold or greater versus vehicle, respectively, with 18 genes having similar regulation for P4 and PRA-910. These data confirm and extend previous findings for T47D cells. We also obtained time course, concentration–response, cyclohexamide sensitivity, and PR-specificity data for two progestin-regulated genes, ATP1A1 and CLDN8. Our data demonstrate that PRA-910 has a unique gene regulation profile distinct from both P4 and RU486. Further investigation of the underlying mechanism for these differences is ongoing.

A novel antiinflammatory maintains glucocorticoid efficacy with reduced side effects.

Glucocorticoids (GCs) are commonly used to treat inflammatory disease; unfortunately, the long-term use of these steroids leads to a large number of debilitating side effects. The antiinflammatory effects of GCs are a result of GC receptor (GR)-mediated inhibition of expression of proinflammatory genes as well as GR-mediated activation of antiinflammatory genes. Similarly, side effects are most likely due to both activated and repressed GR target genes in affected tissues. An as yet unachieved pharmaceutical goal is the development of a compound capable of separating detrimental side effects from antiinflammatory activity. We describe the discovery and characterization of AL-438, a GR ligand that exhibits an altered gene regulation profile, able to repress and activate only a subset of the genes normally regulated by GCs. When tested in vivo, AL-438 retains full antiinflammatory efficacy and potency comparable to steroids but its negative effects on bone metabolism and glucose control are reduced at equivalently antiinflammatory doses. The mechanism underlying this selective in vitro and in vivo activity may be the result of differential cofactor recruitment in response to ligand. AL-438 reduces the interaction between GR and peroxisomal proliferator-activated receptor gamma coactivator-1, a cofactor critical for steroid-mediated glucose up-regulation, while maintaining normal interactions with GR-interacting protein 1. This compound serves as a prototype for a unique, nonsteroidal alternative to conventional GCs in treating inflammatory disease.

Overlapping but Distinct Gene Regulation Profiles by Glucocorticoids and Progestins in Human Breast Cancer Cells

Glucocorticoids and progestins bind to receptors that share many structural and functional similarities, including virtually identical DNA recognition specificity. Nonetheless, the two hormones mediate very distinct biological functions. For example, progestins are associated with the incidence and progression of breast cancer, whereas glucocorticoids are growth suppressive in mammary cancer cells. To understand the mechanisms that engender biological specificity, it is necessary to identify genes that are differentially regulated by the two receptors. Here we employ Affymetrix oligonucleotide arrays to compare glucocorticoid- and progestin-regulated gene expression in a human breast cancer cell line. This global analysis reveals that the two hormones regulate overlapping but distinct sets of genes, including 31 genes that are differentially regulated. Surprisingly, the set of differentially regulated genes was almost as large as the set of genes regulated by both hormones. Examination of the set of differentially regulated genes suggests mechanisms behind the distinct growth effects of the two hormones in breast cancer. The differential regulation of four genes representing different regulatory patterns was confirmed by RT-PCR and Northern blot analyses. Treatment with cycloheximide or RU486 indicates that the regulation is a primary, receptor-mediated event. Detailed analyses of genes identified in these studies will furnish a mechanistic understanding of differential regulation by glucocorticoids and progestins.

Overlapping but distinct gene regulation profiles by glucocorticoids and progestins in human breast cancer cells.

Glucocorticoids and progestins bind to receptors that share many structural and functional similarities, including virtually identical DNA recognition specificity. Nonetheless, the two hormones mediate very distinct biological functions. For example, progestins are associated with the incidence and progression of breast cancer, whereas glucocorticoids are growth suppressive in mammary cancer cells. To understand the mechanisms that engender biological specificity, it is necessary to identify genes that are differentially regulated by the two receptors. Here we employ Affymetrix oligonucleotide arrays to compare glucocorticoid- and progestin-regulated gene expression in a human breast cancer cell line. This global analysis reveals that the two hormones regulate overlapping but distinct sets of genes, including 31 genes that are differentially regulated. Surprisingly, the set of differentially regulated genes was almost as large as the set of genes regulated by both hormones. Examination of the set of differentially regulated genes suggests mechanisms behind the distinct growth effects of the two hormones in breast cancer. The differential regulation of four genes representing different regulatory patterns was confirmed by RT-PCR and Northern blot analyses. Treatment with cycloheximide or RU486 indicates that the regulation is a primary, receptor-mediated event. Detailed analyses of genes identified in these studies will furnish a mechanistic understanding of differential regulation by glucocorticoids and progestins.

Identification of genes expressed with temporal-spatial restriction to developing cerebellar neuron precursors by a functional genomic approach.

Hedgehog pathway activation is required for proliferation of cerebellar granule cell neuron precursors during development and is etiologic in certain cerebellar tumors. To identify genes expressed specifically in granule cell neuron precursors, we used oligonucleotide microarrays to analyze regulation of 13,179 genes/expressed sequence tags in heterogeneous primary cultures of neonatal mouse cerebellum that respond to the mitogen Sonic hedgehog. In conjunction, we applied experiment-specific noise models to render a gene-by-gene robust indication of up-regulation in Sonic hedgehog-treated cultures. Twelve genes so identified were tested, and 10 (83%) showed appropriate expression in the external granular layer (EGL) of the postnatal day (PN) 7 cerebellum and down-regulation by PN 15, as verified by in situ hybridization. Whole-organ profiling of the developing cerebellum was carried out from PN 1 to 30 to generate a database of temporal gene regulation profiles (TRPs). From the database an algorithm was developed to capture the TRP typical of EGL-specific genes. The "TRP-EGL" accurately predicted expression in vivo of an additional 18 genes/expressed sequence tags with a sensitivity of 80% and a specificity of 88%. We then compared the positive predictive value of our analytical procedure with other widely used methods, as verified by the TRP-EGL in silico. These findings suggest that replicate experiments and incorporation of noise models increase analytical specificity. They further show that genome-wide methods are an effective means to identify stage-specific gene expression in the developing granule cell lineage.