A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen

Moffat, Jason; Grueneberg, Dorre A.; Yang, Xiaoping; Kim, So Young; Kloepfer, Angela M.; Hinkle, Gregory; Piqani, Bruno; Eisenhaure, Thomas; Luo, Biao; Grenier, Jennifer K.; Carpenter, Anne E.; Foo, Shi Yin; Stewart, Sheila A.; Stockwell, Brent R.; Hacohen, Nir; Hahn, William C.; Lander, Eric S.; Sabatini, David M.; Root, David E.

doi:10.1016/j.cell.2006.01.040

Cited by 1,619 publications

(1,389 citation statements)

References 43 publications

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“…The non-target shRNA control vector with the sequence CAACAAGATGAAGAG CACCAA was used as a negative control. According to the manufacturer, a scoring algorithm, consisting of sequence, specificity and position is utilized to generate optimal shRNA sequences (Moffat et al, 2006).…”

Section: Flow Cytometrymentioning

confidence: 99%

Picropodophyllin induces downregulation of the insulin-like growth factor 1 receptor: potential mechanistic involvement of Mdm2 and β-arrestin1

Vasilcanu

Rosengren

et al. 2007

Oncogene

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The insulin-like growth factor 1 receptor (IGF-1R) is crucial for growth and survival of malignant cells. Experience in targeting IGF-1R in cancer models has shown that strategies promoting downregulation of the receptor are much more efficient in inducing apoptosis than those inhibiting the IGF-1R activity. Recently, we found that the cyclolignan picropodophyllin (PPP) inhibits phosphorylation of IGF-1R and activation of downstream signaling without interfering with the highly homologous insulin receptor (IR). Furthermore, PPP treatment caused strong regression of tumor grafts and prolonged survival of animals with systemic tumor disease. Here we demonstrate that PPP also downregulates the IGF-1R, whereas the IR and several other receptors were not affected. PPP-induced IGF-1R downregulation required expression of the MDM2 E3 ligase, which recently was found to ubiquitinate and cause degradation of the IGF-1R. In addition knockdown of barrestin1, the adaptor molecule known to bridges MDM2 and IGF-1R, prevented downregulation of the receptor and significantly decreased PPP-induced cell death. All together these data suggest that PPP downregulates IGF-1R by interfering with the action of b-arrestin1/MDM2 as well as the achieved receptor downregulation contributes to the apoptotic effect of PPP.

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Section: Flow Cytometrymentioning

confidence: 99%

Picropodophyllin induces downregulation of the insulin-like growth factor 1 receptor: potential mechanistic involvement of Mdm2 and β-arrestin1

Vasilcanu

Rosengren

et al. 2007

Oncogene

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“…293T cells were seeded (3 Â 10 6 cells per ml) in 60-mm dish, and viral particles were generated by transfection as described previously. 60,61 To standardize lentiviral transduction assays, viral titers were measured in a benchmark cell line, A549. For growth assays, titers corresponding to multiplicities of infection of 5 and 1 in A549 cells were employed.…”

Section: Lentiviral Shrna Experimentsmentioning

confidence: 99%

c-Jun N-terminal kinase has a pivotal role in the maintenance of self-renewal and tumorigenicity in glioma stem-like cells

Yoon

Kim

et al. 2012

Oncogene

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Uncovering the mechanisms that govern the maintenance of stem-like cancer cells is critical for developing therapeutic strategies for targeting these cells. Constitutive activation of c-Jun N-terminal kinase (JNK) has been reported in gliomas and correlates with histological grade. Here, we found that JNK signaling is crucial for the maintenance of 'stemness' in glioma cells. Sphere-cultured glioma cells showed more phosphorylation of JNK compared with serum-containing monolayer cultures. Importantly, blockade of JNK signaling with SP600125 or small interfering RNAs targeting JNK1 or JNK2 significantly reduced the CD133 þ /Nestin þ population and suppressed sphere formation, colony formation in soft agar, and expression of stem cell markers in sphere-cultured glioma cells. Intriguingly, sphere-cultured glioma cells exhibited enhanced expression of Notch-2, but not Notch-1, -3 or -4, and JNK inhibition almost completely abrogated this increase. Blocking the phosphoinoside 3-kinase (PI3K)/Akt pathway with LY294002 or si-Akt also suppressed the self-renewal of sphere-cultured glioma cells. PI3K, but not Akt, had a role as an upstream kinase in JNK1/2 activation. In addition, treatment with si-JNK greatly increased etoposideand ionizing radiation (IR)-induced cell death in glioma spheres. Consistent with glioma cell lines, glioma stem-like cells isolated from primary patient glioma cells also had a higher activity of JNK and Notch-2 expression. Importantly, inhibition of JNK2 led to a decrease of Notch-2 expression and suppressed the CD133 þ /Nestin þ cell population in patient-derived primary glioma cells. Finally, downregulation of JNK2 almost completely suppressed intracranial tumor formation by glioma cells in nude mice. Taken together, these data demonstrate that JNK signaling is crucial for the maintenance of self-renewal and tumorigenicity of glioma stem-like cells and drug/IR resistance, and can be considered a promising target for eliminating stem-like cancer cells in gliomas.

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“…Silencing of PRDX1 by lentiviral transduction of shRNA constructs Lentiviral particles were produced by co-transfecting pMD2.G, psPAX2 and pLKO.1 constructs (MISSION TRCHs 1.0; Sigma-Aldrich Corporation, St Louis, MO) in HEK293T cells (Moffat et al, 2006) using FuGENE6 (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instruction. Viruses were harvested by ultracentrifugation 48 h after transfection.…”

Section: Patients' Samplesmentioning

confidence: 99%

Downregulation of PRDX1 by promoter hypermethylation is frequent in 1p/19q-deleted oligodendroglial tumours and increases radio- and chemosensitivity of Hs683 glioma cells in vitro

et al. 2011

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Deletions of chromosomal arms 1p and 19q are frequent in oligodendroglial tumours and linked to radio-and chemotherapy response as well as longer survival. The molecular mechanisms underlying this clinically important association are as yet unknown. Here, we studied the peroxiredoxin 1 (PRDX1) gene at 1p34.1 for promoter methylation and expression in primary gliomas and investigated its role in radio-and chemosensitivity of glioma cells in vitro. In total, we screened primary glioma tissues from 93 patients for methylation of the 5 0 -CpG island of PRDX1 by sodium bisulfite sequencing. PRDX1 mRNA and protein expression levels were determined in subsets of the tumours by quantitative PCR and western blot analysis, respectively. PRDX1 hypermethylation and reduced expression were frequently detected in oligodendroglial tumours and secondary glioblastomas, but not in primary glioblastomas. In oligodendroglial tumours, both PRDX1 hypermethylation and reduced mRNA expression were significantly associated with 1p/19q-deletion. Stable knockdown of PRDX1 by lentiviral transduction of shorthairpin (sh)RNA constructs significantly increased apoptosis and reduced cell viability of Hs683 glioma cells exposed to ionizing irradiation or temozolomide in vitro. Taken together, our findings indicate that epigenetic silencing of PRDX1 is frequent in 1p/19q-deleted oligodendroglial tumours and likely contributes to radioand chemosensitivity of these tumours.

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A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen

Cited by 1,619 publications

References 43 publications

Picropodophyllin induces downregulation of the insulin-like growth factor 1 receptor: potential mechanistic involvement of Mdm2 and β-arrestin1

Picropodophyllin induces downregulation of the insulin-like growth factor 1 receptor: potential mechanistic involvement of Mdm2 and β-arrestin1

c-Jun N-terminal kinase has a pivotal role in the maintenance of self-renewal and tumorigenicity in glioma stem-like cells

Downregulation of PRDX1 by promoter hypermethylation is frequent in 1p/19q-deleted oligodendroglial tumours and increases radio- and chemosensitivity of Hs683 glioma cells in vitro

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