Gene Expression Changes in a Tumor Xenograft by a Py-Im Polyamide
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ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens
PROVIDER: GSE40218 | GEO | 2012/10/02
SECONDARY ACCESSION(S): PRJNA173215
REPOSITORIES: GEO
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Project description:Gene regulation by DNA binding small molecules could have important therapeutic applications. This study reports the investigation of a DNA-binding pyrrole-imidazole polyamide targeted to bind the DNA sequence 5M-bM-^@M-^Y-WGGWWW-3M-bM-^@M-^Y with reference to its potency in a subcutaneous xenograft tumor model. The molecule is capable of trafficking to the tumor site following subcutaneous injection and modulates transcription of select genes in vivo. A FITC-labeled analogue of this polyamide can be detected in tumor-derived cells by confocal microscopy. RNA deep sequencing (RNA-seq) of tumor tissue allowed the identification of further affected genes, a representative panel of which were interrogated by qRT-PCR and correlated with cell culture expression levels. Xenografts. Grafting with A549-luc-C8. Experiments were performed in female SCID-beige mice (Charles River) between 8 and 12 weeks of age. Cells were injected into the left flank area of the animals as suspensions of 25 x 106 mL-1 in RPMI, 200 M-BM-5L per injection. Treatment and tumor proliferation monitoring. Mice were treated following the schedule delineated in treatment protocol. Tumor proliferation was monitored using the XENOGEN imaging device. The animals were anesthetized with 2 5 % isoflurane and subsequently transferred to the imaging chamber, whereupon the isoflurane levels were reduced to 1-2.5 %. The floor of the imager was heated to +37 M-BM-:C to avoid hypothermia. Breathing frequency was monitored and not allowed to drop below 1 s-1, adjusting the isoflurane levels accordingly at all times. Endpoint criteria and euthanasia. Animal endpoint criteria encompassed weight loss of over 15 %, restriction of motor function by the engrafted tumor, dehydration of over 10 % and moribund behavior. Where appropriate, the animals were euthanized by asphyxiation in a CO2 chamber. Tumor tissue harvest. Animals were resected and tumors excised using standard forceps, scissors and surgical blades. The tumors were combined into one sample per condition and mechanically sheared in TRIZOL, employing a specialized device (tissue tearer, model 985370). Total RNA workup was performed following the standard TRIZOL procedure, followed by a DNAse digest.
2012-10-02 | E-GEOD-40218 | biostudies-arrayexpress
Project description:The crucial role of androgen receptor in prostate cancer development is well documented, and its inhibition is a mainstay of prostate cancer treatment. Here we analyze the perturbations to the androgen receptor cistrome caused by a minor groove binding molecule that is designed to target a sequence found in a subset of androgen response elements. We find treatment with this pyrrole-imidazole polyamide exhibits sequence selectively in its repression of androgen receptor binding in vivo. Differentially changed loci are enriched for sequences resembling ARE half-sites that match the Py-Im polyamide binding preferences determined in vitro. Comparatively, permutations of ARE half-site bearing single or double mismatches to the Py-Im polyamide binding sequence are not enriched. This study represents an indirect determination of Py-Im polyamide binding preference in vivo using an unbiased approach.
2019-02-22 | GSE125552 | GEO
Project description:We report that a DNA minor groove binding hairpin pyrrole-imidazole (Py-Im) polyamide interferes with RNA polymerase II (RNAP2) activity in cell culture. Genome-wide mapping of RNAP2 binding shows reduction of occupancy preferentially at transcription start sites (TSS), while occupancy at enhancer sites are is unchanged. Genome-wide mapping of RNA polymerase II in LNCaP cells treated with DHT and DHT with Py-Im polyamide.
2013-01-31 | E-GEOD-43253 | biostudies-arrayexpress
Project description:We report that a DNA minor groove binding hairpin pyrrole-imidazole (Py-Im) polyamide interferes with RNA polymerase II (RNAP2) activity in cell culture. Genome-wide mapping of RNAP2 binding shows reduction of occupancy preferentially at transcription start sites (TSS), while occupancy at enhancer sites are is unchanged.
2013-01-31 | GSE43253 | GEO
Project description:Pyrrole-imidazole polyamides (PIPs) have been shown to inhibit gene expression by interrupting the DNA-protein interface. Human Ectopic viral integration site 1 (EVI1) is an oncogenic transcription factor which plays a key role in many aggressive forms of cancer. We have developed a novel pyrrole–imidazole polyamide, PIP1 targeting the REL/ELK1 binding site in the EVI1 minimal promoter that can significantly repress the expression of EVI1 in MDA-MB-231 cells. Whole-transcriptome analysis revealed that a fraction of EVI1-driven genes were modulated by PIP1.
2014-07-28 | GSE59502 | GEO
Project description:Pyrrole-imidazole polyamides are versatile DNA minor groove binders and attractive therapeutic options against oncological targets, especially upon functionalization with an alkylating agent such as endo-seco-CBI. These molecules also provide an alternative for oncogenes deemed “undruggable” at the protein level, where the absence of solvent-accessible pockets or structural crevices prevent the formation of protein-inhibitor ligands; nevertheless, the genome-wide effect of pyrrole-imidazole polyamide binding remain largely unclear to-date. Here we propose a next-generation sequencing-based workflow combined with whole genome expression arrays to address such issue using a candidate anti-cancer alkylating agent, KR12, against codon 12 mutant KRAS. Biotinylating KR12 enables the means to identify its genome-wide effects in living cells and possible biological implications via a coupled workflow of enrichment-based sequencing and expression microarrays. The subsequent computational pathway and expression analyses allow the identification of its genomic binding sites, as well as a route to explore a polyamide’s possible genome-wide effects. Among the 3,343 KR12 binding sites identified in the human LS180 colorectal cancer genome, the reduction of KR12-bound gene expressions was also observed. Additionally, the coupled microarray-sequencing analysis also revealed some insights about the effect of local chromatin structure on pyrrole-imidazole polyamide, which had not been fully understood to-date. A comparative analysis with KR12 in a different human colorectal cancer genome SW480 also showed agreeable agreements of KR12 binding affecting gene expressions. Combination of these analyses thus suggested the possibility of applying this approach to other pyrrole-imidazole polyamides to reveal further biological details about the effect of polyamide binding in a genome.
2016-11-01 | GSE86598 | GEO
Project description:Pyrrole-imidazole polyamides are versatile DNA minor groove binders and attractive therapeutic options against oncological targets, especially upon functionalization with an alkylating agent such as endo-seco-CBI. These molecules also provide an alternative for oncogenes deemed “undruggable” at the protein level, where the absence of solvent-accessible pockets or structural crevices prevent the formation of protein-inhibitor ligands; nevertheless, the genome-wide effect of pyrrole-imidazole polyamide binding remain largely unclear to-date. Here we propose a next-generation sequencing-based workflow combined with whole genome expression arrays to address such issue using a candidate anti-cancer alkylating agent, KR12, against codon 12 mutant KRAS. Biotinylating KR12 enables the means to identify its genome-wide effects in living cells and possible biological implications via a coupled workflow of enrichment-based sequencing and expression microarrays. The subsequent computational pathway and expression analyses allow the identification of its genomic binding sites, as well as a route to explore a polyamide’s possible genome-wide effects. Among the 3,343 KR12 binding sites identified in the human LS180 colorectal cancer genome, the reduction of KR12-bound gene expressions was also observed. Additionally, the coupled microarray-sequencing analysis also revealed some insights about the effect of local chromatin structure on pyrrole-imidazole polyamide, which had not been fully understood to-date. A comparative analysis with KR12 in a different human colorectal cancer genome SW480 also showed agreeable agreements of KR12 binding affecting gene expressions. Combination of these analyses thus suggested the possibility of applying this approach to other pyrrole-imidazole polyamides to reveal further biological details about the effect of polyamide binding in a genome.
2016-11-01 | GSE86597 | GEO
Project description:Pyrrole-imidazole polyamides (PIPs) have been shown to inhibit gene expression by interrupting the DNA-protein interface. Human Ectopic viral integration site 1 (EVI1) is an oncogenic transcription factor which plays a key role in many aggressive forms of cancer. We have developed a novel pyrroleM-bM-^@M-^Simidazole polyamide, PIP1 targeting the REL/ELK1 binding site in the EVI1 minimal promoter that can significantly repress the expression of EVI1 in MDA-MB-231 cells. Whole-transcriptome analysis revealed that a fraction of EVI1-driven genes were modulated by PIP1. Global expression changes in MDA-MB-231 cells were evaluated after treating the cells with PIP1 and DMSO for 48 hours. The vehicle DMSO is used as a negative control. Each condition is performed in technical replicates.
2014-07-28 | E-GEOD-59502 | biostudies-arrayexpress
Project description:Cyclic eight-ring pyrrole-imidazole polyamides are sequence-specific DNA-binding small molecules that are cell permeable and can regulate endogenous gene expression. Syntheses of cyclic polyamides have been achieved by solid-phase and solution-phase methods. A rapid solution-phase oligomerization approach to eight-ring symmetrical cyclic polyamides yields 12- and 16-membered macrocycles as well. A preference for DNA binding by the 8- and 16-membered oligomers was observed over the 12-ring macrocycle, which we attributed to a conformational constraint not present in the smaller and larger systems.
| S-EPMC2736337 | biostudies-literature
Project description:Cyclic Py-Im polyamides containing two GABA turn units exhibit enhanced DNA binding affinity, but extensive studies of their biological properties have been hindered due to synthetic inaccessibility. A facile modular approach toward cyclic polyamides has been developed via microwave-assisted solid-phase synthesis of hairpin amino acid oligomer intermediates followed by macrocyclization. A focused library of cyclic polyamides 1-7 targeted to the androgen response element (ARE) and the estrogen response element (ERE) were synthesized in 12-17% overall yield. The Fmoc protection strategy also allows for selective modifications on the GABA turn units that have been shown to improve cellular uptake properties. The DNA binding affinities of a library of cyclic polyamides were measured by DNA thermal denaturation assays and compared to the corresponding hairpin polyamides. Fluorescein-labeled cyclic polyamides have been synthesized and imaged via confocal microscopy in A549 and T47D cell lines. The IC(50) values of compounds 1-7 and 9-11 were determined, revealing remarkably varying levels of cytotoxicity.
| S-EPMC3538502 | biostudies-literature