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RNA-Immunoprecipitation of PIWIL1/piRNA complexes in COLO 205 cells and deep-sequencing of associated small RNAs with NGS technologies

ABSTRACT: RNA-Immunoprecipitation of PIWIL1/piRNA complexes in COLO 205 cells and deep-sequencing of associated small RNAs with NGS technologies

PROVIDER: PRJEB33352 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR3415730.fastq.gz	Fastqsanger.gz
	ERR3415740.fastq.gz	Fastqsanger.gz
	ERR3415731.fastq.gz	Fastqsanger.gz
	ERR3415732.fastq.gz	Fastqsanger.gz
	ERR3415733.fastq.gz	Fastqsanger.gz

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Project description:We previously identified the induction of growth arrest with phenotypic characteristics of senescence in melanoma cell lines sensitive to diterpene esters, indicating a therapeutic potential. Here we compared the cytostatic effects of two diterpene esters namely TPA (12-O-tetradecanoylphorbol-13-acetate) and PEP008 (20-O-acetyl-ingenol-3-angelate) in sensitive and resistant cell lines derived from melanoma, breast cancer and colon cancer. We showed the diterpene esters to induce senescence-like growth arrest in the sensitive cells at 100-1000 ng/ml. Use of the pan-PKC inhibitor bisindolylmaleimide-l demonstrated that activation of PKC was required for growth arrest. Full genome expression profiling revealed that pivotal genes involved in DNA synthesis and cell cycle control were down-regulated by treatment in all three sensitive solid tumor models. At the protein level, prolonged down-regulation of E2F-1 and proliferating cell nuclear antigen (PCNA), sustained expression of p21WAF1/CIP1 and dephosphorylation of retinoblastoma (Rb) occurred in the sensitive cells. Although activation of extracellular signal-related kinase (ERK) 1/2 by the diterpene esters occurred in both sensitive and resistant cell lines, the HRASLS3 type II tumor suppressor, which appears to have a role in MAPK pathway suppression, was constitutively elevated in the resistant cell lines compared to their sensitive counterparts. Together, these results demonstrate the ability of the PKC activating drugs TPA and PEP008 to induce growth arrest with characteristics of senescence in solid tumor cell lines derived from a variety of tissue types through a similar mechanism. PKC-activating diterpene esters may therefore have therapeutic potential in a range of solid tumors. Experiment Overall Design: We analyzed the transcriptional profiles of the diterpene ester sensitive cell lines MCF7, COLO-205 and SK-MEL-5 following treatment with PEP008 using full genome expression profiling (Affymetrix, U133 Plus 2.0). Cells were treated for 24 h and 24 h plus 72 h recovery with 1000 ng/ml of the drug, before harvesting RNA for analysis. From the cell growth assays, all three cell lines demonstrated permanent growth arrest with diterpene ester treatments at the 1000 ng/ml dose. Mock controls were treated with solvent alone for 24 h. SK-MEL-5 cells were also treated with 1000 ng/ml TPA for 24 h.

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RNA-Immunoprecipitation of PIWIL1/piRNA complexes in COLO 205 cells and deep-sequencing of associated small RNAs with NGS technologies

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