Dataset Information

Identification of survival genes in human glioblastoma cells by small interfering RNA screening.

ABSTRACT: Target identification and validation remain difficult steps in the drug discovery process, and uncovering the core genes and pathways that are fundamental for cancer cell survival may facilitate this process. Glioblastoma represents a challenging form of cancer for chemotherapy. Therefore, we assayed 16,560 short interfering RNA (siRNA) aimed at identifying which of the 5520 unique therapeutically targetable gene products were important for the survival of human glioblastoma. We analyzed the viability of T98G glioma cells 96 h after siRNA transfection with two orthogonal statistical methods and identified 55 survival genes that encoded proteases, kinases, and transferases. It is noteworthy that 22% (12/55) of the survival genes were constituents of the 20S and 26S proteasome subunits. An expression survey of a panel of glioma cell lines demonstrated expression of the proteasome component PSMB4, and the validity of the proteasome complex as a target for survival inhibition was confirmed in a series of glioma and nonglioma cell lines by pharmacological inhibition and RNA interference. Biological networks were built with the other survival genes using a protein-protein interaction network, which identified clusters of cellular processes, including protein ubiquitination, purine and pyrimidine metabolism, nucleotide excision repair, and NF-kappaB signaling. The results of this study should broaden our understanding of the core genes and pathways that regulate cell survival; through either small molecule inhibition or RNA interference, we highlight the potential significance of proteasome inhibition.

SUBMITTER: Thaker NG

PROVIDER: S-EPMC2784725 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

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Identification of survival genes in human glioblastoma cells by small interfering RNA screening.

Thaker Nikhil G NG Zhang Fang F McDonald Peter R PR Shun Tong Ying TY Lewen Michael D MD Pollack Ian F IF Lazo John S JS

Molecular pharmacology 20090925 6

Target identification and validation remain difficult steps in the drug discovery process, and uncovering the core genes and pathways that are fundamental for cancer cell survival may facilitate this process. Glioblastoma represents a challenging form of cancer for chemotherapy. Therefore, we assayed 16,560 short interfering RNA (siRNA) aimed at identifying which of the 5520 unique therapeutically targetable gene products were important for the survival of human glioblastoma. We analyzed the via ...[more]

PMID: 19783622

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Project description:Currently, there is a serious absence of pharmaceutically attractive small molecules that mitigate the lethal effects of an accidental or intentional public exposure to toxic doses of ionizing radiation. Moreover, cellular systems that emulate the radiobiologically relevant cell populations and that are suitable for high-throughput screening have not been established. Therefore, we examined two human pluripotent embryonal carcinoma cell lines for use in an unbiased phenotypic small interfering RNA (siRNA) assay to identify proteins with the potential of being drug targets for the protection of human cell populations against clinically relevant ionizing radiation doses that cause acute radiation syndrome. Of the two human cell lines tested, NCCIT cells had optimal growth characteristics in a 384 well format, exhibited radiation sensitivity (D(0) = 1.3 ± 0.1 Gy and ñ = 2.0 ± 0.6) comparable to the radiosensitivity of stem cell populations associated with human death within 30 days after total-body irradiation. Moreover, they internalized siRNA after 4 Gy irradiation enabling siRNA library screening. Therefore, we used the human NCCIT cell line for the radiation mitigation study with a siRNA library that silenced 5,520 genes known or hypothesized to be potential therapeutic targets. Exploiting computational methodologies, we identified 113 siRNAs with potential radiomitigative properties, which were further refined to 29 siRNAs with phosphoinositide-3-kinase regulatory subunit 1 (p85?) being among the highest confidence candidate gene products. Colony formation assays revealed radiation mitigation when the phosphoinositide-3-kinase inhibitor LY294002 was given after irradiation of 32D cl 3 cells (D(0) = 1.3 ± 0.1 Gy and ñ = 2.3 ± 0.3 for the vehicle control treated cells compared to D(0) = 1.2 ± 0.1 Gy and ñ = 6.0 ± 0.8 for the LY294002 treated cells, P = 0.0004). LY294002 and two other PI3K inhibitors, PI 828 and GSK 1059615, also mitigated radiation-induced apoptosis in NCCIT cells. Treatment of mice with a single intraperitoneal LY294002 dose of 30 mg/kg at 10 min, 4, or 24 h after LD(50/30) whole-body dose of irradiation (9.25 Gy) enhanced survival. This study documents that an unbiased siRNA assay can identify new genes, signaling pathways, and chemotypes as radiation mitigators and implicate the PI3K pathway in the human radiation response.

Dataset Information

Identification of survival genes in human glioblastoma cells by small interfering RNA screening.

Publications

Identification of survival genes in human glioblastoma cells by small interfering RNA screening.

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