Project description:BACKGROUND & AIMS- More frequent interaction of bacteria with the colonic epithelium is associated with ulcerative colitis (UC). The identities of all proteins which promote bacterial clearance in colonic epithelial cells are unknown. Previously, we discovered that dCAP-D3 (Chromosome Associated Protein-D3), regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS- Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing CAP-D3 shRNA. CAP-D3 levels in colonic epithelial cells from healthy and UC patient tissues were analyzed by immunoblot. RNA-sequencing identified bacterially-induced CAP-D3 target genes. The role of CAP-D3 target genes in bacterial clearance was analyzed by gentamycin protection assays, immunofluorescent staining, and by using pharmacologic inhibitors. RESULTS- CAP-D3 expression was reduced in colonic epithelial cells from UC patients with active disease. Reduction of CAP-D3 expression inhibited autophagy and decreased intracellular bacterial clearance. The components of the heterodimeric SLC7A5/SLC3A2 amino acid transporter were identified as CAP-D3 target genes; their levels increased in infected, CAP-D3 deficient cell lines and in cells from UC patients. In HT-29 cells, this resulted in earlier SLC7A5 recruitment to Salmonella-containing vacuoles, increased mTOR activity, and enhanced bacterial survival. Inhibition of SLC7A5/SLC3A2 or mTOR activity rescued the bacterial clearance defect in CAP-D3 deficient cells. CONCLUSIONS- CAP-D3 attenuates amino acid transporter transcription to promote bacterial autophagy in colon epithelial cells. CAP-D3 protein levels are decreased in patients with active UC, suggesting that CAP-D3 is a potential therapeutic target to restore mucosal homeostasis in UC patients.
Project description:Signaling through the vitamin D receptor (VDR) has been proposed to suppress the development of epithelial cancers, including prostate cancer. We conducted ChIP-seq to identify the VDR binding sites in the genome of the prostate epithelial cell line, RWPE1. This analysis reveals a large number of VDR binding sites in both control cells and in cells treated with 10 nM 1,25 dihydroxyvitamin D3 for 3 hours. These peaks are associated with genes controlling a wide variety of cellular functions.
Project description:Immuno-stained (keratin 14+ basal marker) frozen prostate sections were subjected to laser-guided microdissection to isolate basal and luminal epithelial prostate cells for expression profiling. RNA was amplified using the AmpTec TRinucleotide kit (AmpTec GmBH). Expression profiling performed using the Invitrogen post-labelling kit and the CRUK whole genome array (WGA) gene set. Keywords: repeat sample
Project description:A major goal in prostate stem cell biology is to identify genes, pathways, or networks that control self-renewal and multilineage differentiation. We hypothesize that 1,25 dihydroxyvitamin D3 can induce differentiation of prostatic progenitor/stem cells, thus serving as an in vitro model with which to study the molecular mechanisms of stem cell differentiation by 1,25 dihydroxyvitamin D3. 1,25 dihydroxyvitamin D3 elicits its effects primarily through transcriptional regulation of genes, so microarray studies were used to gain insight into the cellular response to 1,25 dihydroxyvitamin D3. We used microarrays to detail the global gene expression changes that occur upon 1,25 dihydroxyvitamin D3 treatment of prostatic progenitor/stem cells.
Project description:To identify which lncRNAs are differentially expressed in prostate cancer, the total RNA from prostate cancer cell lines (PC3, LNCaP), normal epithelial prostatic cells and the pool of 10 prostate tumor tissues and 10 adjacent normal prostate tissues were screened using SurePrint G3 human lncRNA microarrays (Agilent). The SurePrint G3 Human Gene array contains 16,472 lincRNAs and 34,127 mRNA genes.
Project description:Effects of sulforaphane and 3,3’-diindolylmethane on genome-wide promoter methylation in normal prostate epithelial cells and prostate cancer cells This study was undertaken to determine the genome-wide effects of sulforaphane (SFN) and 3,3’-diindolylmethane (DIM) on promoter methylation in normal prostate epithelial cells and prostate cancer cells. Nimblegen Human DNA Methylation 3x720K CpG Island Plus RefSeq Promoter Array was used in this study. We hypothesize that both SFN and DIM are effective dietary modulators of DNA methylation due to their inhibitory effects on DNMT expression, and that SFN and DIM can differentially affect the promoter methylation profiles in normal and cancerous prostate epithelial cells.
Project description:Prostate cancer is the second most occurring cancer in men worldwide, and with the advances made with screening for prostate-specific antigen, it has been prone to early diagnosis and over-treatment. To better understand the mechanisms of tumorigenesis and possible treatment responses, we developed a mathematical model of prostate cancer which considers the major signalling pathways known to be deregulated. The model includes pathways such as androgen receptor, MAPK, Wnt, NFkB, PI3K/AKT, MAPK, mTOR, SHH, the cell cycle, the epithelial-mesenchymal transition (EMT), apoptosis and DNA damage pathways. The final model accounts for 133 nodes and 449 edges. We applied a methodology to personalise this Boolean model to molecular data to reflect the heterogeneity and specific response to perturbations of cancer patients, using TCGA and GDSC datasets.
Project description:A major goal in prostate stem cell biology is to identify genes, pathways, or networks that control self-renewal and multilineage differentiation. We hypothesize that 1,25 dihydroxyvitamin D3 can induce differentiation of prostatic progenitor/stem cells, thus serving as an in vitro model with which to study the molecular mechanisms of stem cell differentiation by 1,25 dihydroxyvitamin D3. 1,25 dihydroxyvitamin D3 elicits its effects primarily through transcriptional regulation of genes, so microarray studies were used to gain insight into the cellular response to 1,25 dihydroxyvitamin D3. We used microarrays to detail the global gene expression changes that occur upon 1,25 dihydroxyvitamin D3 treatment of prostatic progenitor/stem cells. Adult mouse prostate progenitor/stem cells were plated at 1 x 10^5 cells per 10 cm culture dish and grown to 70% confluency before treatment with vehicle (0.1% ethanol) or 100 nM 1,25(OH)2D3 in cell culture media (n = 3 or 4). Cells were treated with control or experimental media for 6 hrs or 48 hrs before RNA isolation. The RNA from 6 hrs and 48 hrs was used to probe Affymetrix 430A oligonucleotide arrays (GPL339).
Project description:Effects of sulforaphane and 3,3’-diindolylmethane on genome-wide promoter methylation in normal prostate epithelial cells and prostate cancer cells This study was undertaken to determine the genome-wide effects of sulforaphane (SFN) and 3,3’-diindolylmethane (DIM) on promoter methylation in normal prostate epithelial cells and prostate cancer cells. Nimblegen Human DNA Methylation 3x720K CpG Island Plus RefSeq Promoter Array was used in this study. We hypothesize that both SFN and DIM are effective dietary modulators of DNA methylation due to their inhibitory effects on DNMT expression, and that SFN and DIM can differentially affect the promoter methylation profiles in normal and cancerous prostate epithelial cells. Normal prostate epithelial cells (PrEC), androgen-dependent prostate cancer epithelial cells (LnCAP) and androgen-independent prostate cancer epithelial cells (PC3) were treated with vehicle control, 15uM SFN, or 15uM DIM for 48h in triplicates
Project description:BACKGROUND & AIMS- More frequent interaction of bacteria with the colonic epithelium is associated with ulcerative colitis (UC). The identities of all proteins which promote bacterial clearance in colonic epithelial cells are unknown. Previously, we discovered that dCAP-D3 (Chromosome Associated Protein-D3), regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS- Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing CAP-D3 shRNA. CAP-D3 levels in colonic epithelial cells from healthy and UC patient tissues were analyzed by immunoblot. RNA-sequencing identified bacterially-induced CAP-D3 target genes. The role of CAP-D3 target genes in bacterial clearance was analyzed by gentamycin protection assays, immunofluorescent staining, and by using pharmacologic inhibitors. RESULTS- CAP-D3 expression was reduced in colonic epithelial cells from UC patients with active disease. Reduction of CAP-D3 expression inhibited autophagy and decreased intracellular bacterial clearance. The components of the heterodimeric SLC7A5/SLC3A2 amino acid transporter were identified as CAP-D3 target genes; their levels increased in infected, CAP-D3 deficient cell lines and in cells from UC patients. In HT-29 cells, this resulted in earlier SLC7A5 recruitment to Salmonella-containing vacuoles, increased mTOR activity, and enhanced bacterial survival. Inhibition of SLC7A5/SLC3A2 or mTOR activity rescued the bacterial clearance defect in CAP-D3 deficient cells. CONCLUSIONS- CAP-D3 attenuates amino acid transporter transcription to promote bacterial autophagy in colon epithelial cells. CAP-D3 protein levels are decreased in patients with active UC, suggesting that CAP-D3 is a potential therapeutic target to restore mucosal homeostasis in UC patients. Three RNA samples from 3 independent experiments including timepoints taken at 0, 0.5 and 7 hours post-infection were analyzed on a bioanalyzer for quality; one of the 0.5 hour post-infection samples was excluded at this time due to poor RNA purity. Directional, cDNA libraries made from cellular mRNAs were generated from the other 8 samples and sequenced (paired-end sequencing of 100 bp reads) in the Genomics Core at the University of Chicago on an Illumina HiSeq2000.