Change in expression of genes after retinoic acid treatment of stellate cells: time course
ABSTRACT: We evaluated the change in expression of genes after treatment of stellate cells with retinoic acid to understand how the stellate cells can de-differentiate and effect their physiological behaviour in pathological conditions. We then tested the changes in the gene expression in 2D and 3D culture conditions for pancreatic stellate cells and in a pancreatic cancer model. Keywords: gene expression change, time course Overall design: We treated a pancreatic stellate cell line on plastic with 1 micromolar all-trans retinoic acid (ATRA) for timepoints of 30 mins, 4 hours, 12 hours, 24 hours and 168 hours. RNA was extracted and hybridized on Illumina Human microarrays. We looked for target genes regulated by ATRA and evaluated for time course change.
Project description:We evaluated the change in expression of genes after treatment of stellate cells with retinoic acid to understand how the stellate cells can de-differentiate and effect their physiological behaviour in pathological conditions. We then tested the changes in the gene expression in 2D and 3D culture conditions for pancreatic stellate cells and in a pancreatic cancer model. Keywords: gene expression change, time course We treated a pancreatic stellate cell line on plastic with 1 micromolar all-trans retinoic acid (ATRA) for timepoints of 30 mins, 4 hours, 12 hours, 24 hours and 168 hours. RNA was extracted and hybridized on Illumina Human microarrays. We looked for target genes regulated by ATRA and evaluated for time course change.
Project description:Haffez2017 - RAR interaction with synthetic
This model is described in the article:
The molecular basis of the
interactions between synthetic retinoic acid analogues and the
retinoic acid receptors
Hesham Haffez, David R. Chisholm,
Roy Valentine, Ehmke Pohl, Christopher Redfern and Andrew
All-trans-retinoic acid (ATRA) and its synthetic analogues
EC23 and EC19 direct cellular differentiation by interacting as
ligands for the retinoic acid receptor (RARÎ±,
Î² and Î³) family of nuclear receptor
proteins. To date, a number of crystal structures of natural
and synthetic ligands complexed to their target proteins have
been solved, providing molecular level snap-shots of ligand
binding. However, a deeper understanding of receptor and ligand
flexibility and conformational freedom is required to develop
stable and effective ATRA analogues for clinical use.
Therefore, we have used molecular modelling techniques to
define RAR interactions with ATRA and two synthetic analogues,
EC19 and EC23, and compared their predicted biochemical
activities to experimental measurements of relative ligand
affinity and recruitment of coactivator proteins. A
comprehensive molecular docking approach that explored the
conformational space of the ligands indicated that ATRA is able
to bind the three RAR proteins in a number of conformations
with one extended structure being favoured. In contrast the
biologically-distinct isomer, 9-cis-retinoic acid (9CRA),
showed significantly less conformational flexibility in the RAR
binding pockets. These findings were used to inform docking
studies of the synthetic retinoids EC23 and EC19, and their
respective methyl esters. EC23 was found to be an excellent
mimic for ATRA, and occupied similar binding modes to ATRA in
all three target RAR proteins. In comparison, EC19 exhibited an
alternative binding mode which reduces the strength of key
polar interactions in RARÎ±/Î³ but is
well-suited to the larger RARÎ² binding pocket. In
contrast, docking of the corresponding esters revealed the loss
of key polar interactions which may explain the much reduced
biological activity. Our computational results were
complemented using an in vitro binding assay based on FRET
measurements, which showed that EC23 was a strongly binding,
pan-agonist of the RARs, while EC19 exhibited specificity for
RARÎ², as predicted by the docking studies. These
findings can account for the distinct behaviour of EC23 and
EC19 in cellular differentiation assays, and additionally, the
methods described herein can be further applied to the
understanding of the molecular basis for the selectivity of
different retinoids to RARÎ±, Î² and
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Project description:tumor-stroma crosstalk drives pancreatic carcinogenesis we used time-resolved genome-wide transcriptional profiling to analyse changes caused by co-exposure of pancreatic tumor and stellate cells Primary pancreatic Stellate cells (PSC) were treated with a cumulative supernatant of pancreatic tumor cell lines (n=8) and harvested at 1-7, and 24 hours post exposure for RNA extraction and hybridization on Affymetrix microarrays. The 8 tumor cell lines are pancreatic ductal adenocarcinoma lines: AsPC1, BxPC3, Capan1, Colo357, MiaPaca2, Panc1, Su8686, and T3M4
Project description:All-trans retinoic acid (ATRA) has been shown to have anti-proliferative effects, particularly in the context of cancer. However, the effects of ATRA on gene and microRNA expression in solid tumors have not been investigated. In this study, we performed gene expression and microRNA analysis of the squamous cell carcinoma cell line, ME180, following treatment with 10 micromolar all-trans retinoic acid (ATRA) for 1, 3, and 6 hours. Results provide insight into the temporal regulation of genes and microRNAs by retinoids. Overall design: 16 samples (4 DMSO-Treated, 4-1 hour 10μM ATRA, 4-3 hours 10μM ATRA, 4-6 hours 10μM ATRA)
Project description:This study used Illumina single-end RNA-sequencing to examine gene expression differences between 2 mouse-derived pancreatic stellate cell lines (PSC4, PSC5) grown either in 2D monolayers, as 3D quiescent cultures, or as 3D activated transwell cocultures with 2 mouse tumor-derived pancreatic ductal organoid lines (T4, T5). Mouse pancreatic stellate cell (PSC) lines were derived from the pancreata of wild-type C57Bl/6J mice. Mouse tumor organoid lines were derived from mouse pancreata containing pancreatic ductal adenocarcinoma (PDAC) from the KrasLSL-G12D; Trp53LSL-R162H; Pdx1-Cre mouse model. We measured genes differentially expressed among 2D, quiescent, and 3D activated PSCs that may reflect the expression changes of heterogeneous CAF population in pancreatic tumors. Overall design: Gene expression comparison of 2 murine pancreatic stellate cell lines grown either in 2D monolayers, 3D quiescent cultures, or 3D activated transwell cocultures with 2 murine pancreatic ductal adenocarcinoma-derived pancreatic ductal organoid lines.
Project description:In SKBR3 cells, simultaneous targeting of RARë± with all-trans retinoic acid (ATRA) and HER2 with lapatinib results in synergistic anti-tumor responses. SKBR3 cells were treated with vehicle (DMSO), lapatinib (100 nM), ATRA (100 nM) or lapatinib+ATRA for 36 hours and miRNA expression profile was determined by one-color Agilent microarray experiments.
Project description:There is emerging evidence that, beyond their cholesterol lowering properties, statins exhibit important antileukemic effects in vitro and in vivo, but the precise mechanisms by which they generate such responses remain to be determined. We have previously shown that statins promote differentiation of acute promyelocytic leukemia (APL) cells and enhance generation of all-trans-retinoic acid (ATRA)-dependent antileukemic responses. We now provide evidence that statin-dependent leukemic cell differentiation requires engagement and activation of the JNK kinase pathway. In addition, in experiments to define the molecular targets and mediators of statin-induced differentiation we found a remarkable effect of statins on ATRA-dependent gene transcription, evidenced by the selective induction of over 400 genes by the combination of atorvastatin and ATRA. Altogether, our studies identify novel statin molecular targets linked to differentiation, establish that statins modulate ATRA-dependent transcription, and suggest that combined use of statins with retinoids may provide a novel approach to enhance antileukemic responses in APL and possibly other leukemias. Overall design: To determine whether the effects of statin-treatment on ATRA-induced leukemic cell differentiation reflect induction of specific genes, the patterns of gene expression induced by ATRA, atorvastatin, or by the combination of atorvastatin + ATRA were subsequently examined using DNA microarrays. We analyzed three prototypic situations (ATRA, atorvastatin, ATRA + atorvastatin) using Illumina Sentrix® Human-6 Expression BeadChips over three points time course (8, 24, 48 hours), and time course points were replicated in three independent experiments performed at different days. A total of 36 arrays were hybridized. After average probe intensity 9 calculation, log2 transformation and normalization, probes characterized by low quality signal or invariant expression within the experimental conditions were discarded. A total of 11287 out of the 47293 RefSeq BeadChip probes were used to identify significantly differentially expressed genes. Statistical analysis was performed using a two step regression strategy. 1901 RefSeqs were found significantly differentially expressed in at least one condition over the time course treatments. After calculating the log2 (fold change) variation for all treatments with respect to the corresponding control time point, 782 out of 1901 RefSeq probes were characterized by a |log2(fold change)| ≥ 1 for at least one of the time points.
Project description:Differences in the expression profile of hepatic and pancreatic stellate cells are investigated. Aim is to identify organ and disease specific transcriptome signatures of stellate cells, comparing hepatic and pancreatic stellate cells obtained from tissues of chronic inflammation, and primary or metastatic cancers of the pancreas. Tissues of chronic pancreatitis (n=6), pancreatic ductal adenocarcinoma (n=5), liver cirrhosis (n=5) and liver metastasis of pancreatic ductal adenocarcinoma (n=6) were collected and stellate cells were isolated by the outgrowth method. Using cDNA microarrays, differentially expressed genes are identified.
Project description:Gene expression data from BE(2)-C cells treated in triplicate with either vehicle (DMSO), 5 μM all-trans retinoic acid (ATRA), 1 mM valproic acid (VPA), or 5 μM ATRA + 1 mM VPA for 6, 24, or 72 hours. Genome-wide expression profiling was performed using Affymetrix U133A microarrays. While cytotoxic chemotherapy remains the hallmark of cancer treatment, intensive regimens fall short in many malignancies, including high-risk neuroblastoma. One alternative strategy is to therapeutically promote tumor differentiation. We created a gene expression signature to measure neuroblast maturation, adapted it to a high-throughput platform, and screened a diversity oriented synthesis-generated small-molecule library for differentiation inducers. We identified BRD8430, containing a nine-membered lactam, an ortho-amino anilide functionality, and three chiral centers, as a selective Class I histone deacetylase (HDAC) inhibitor (HDAC1 > 2 > 3). Further investigation demonstrated that selective HDAC1/HDAC2 inhibition using compounds or RNA interference induced differentiation and decreased viability in neuroblastoma cell lines. Combined treatment with 13-cis retinoic acid augmented these effects and enhanced activation of retinoic acid signaling. Therefore, by applying a chemical genomic screening approach we identified selective HDAC1/HDAC2 inhibition as a strategy to induce neuroblastoma differentiation. BE(2)-C cells were treated in triplicate with either vehicle (DMSO), 5 μM all-trans retinoic acid (ATRA), 1 mM valproic acid (VPA), or 5 μM ATRA + 1 mM VPA for 6, 24, or 72 hours. Genome-wide expression profiling was performed using Affymetrix U133A microarrays [HT-HG_U133A Early Access].