Establishment and Characterization of a Novel Human Desmoplastic Small Round Cell Tumor Cell Line, JN-DSRCT-1

Nishio, Jun; Iwasaki, Hiroshi; Ishiguro, Masako; Ohjimi, Yuko; Fujita, Chikako; Yanai, Fumio; Nibu, Keiko; Mitsudome, Akihisa; Kaneko, Yasuhiko; Kikuchi, Masahiro

doi:10.1097/01.lab.0000028059.92642.03

Cited by 41 publications

(43 citation statements)

References 21 publications

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“…These cells were grown in DMEM with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin (Invitrogen). JN-DSRCT-1 cells (24), authenticated by the presence of EWS-WT1 translocation and free of mycoplasma, were grown in DMEM/F-12 media with 10% FBS. Tamoxifen, 4-hydroxytamoxifen (4-HT) and G418 (Sigma), and N2 supplement (Invitrogen) were purchased.…”

Section: Methodsmentioning

confidence: 99%

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EWS–WT1 Oncoprotein Activates Neuronal Reprogramming Factor ASCL1 and Promotes Neural Differentiation

et al. 2014

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The oncogenic fusion gene EWS-WT1 is the defining chromosomal translocation in desmoplastic small roundcell tumors (DSRCT), a rare but aggressive soft tissue sarcoma with a high rate of mortality. EWS-WT1 functions as an aberrant transcription factor that drives tumorigenesis, but the mechanistic basis for its pathogenic activity is not well understood. To address this question, we created a transgenic mouse strain that permits physiologic expression of EWS-WT1 under the native murine Ews promoter. EWS-WT1 expression led to a dramatic induction of many neuronal genes in embryonic fibroblasts and primary DSRCT, most notably the neural reprogramming factor ASCL1. Mechanistic analyses demonstrated that EWS-WT1 directly bound the proximal promoter of ASCL1, activating its transcription through multiple WT1-responsive elements. Conversely, EWS-WT1 silencing in DSRCT cells reduced ASCL1 expression and cell viability. Notably, exposure of DSRCT cells to neuronal induction media increased neural gene expression and induced neurite-like projections, both of which were abrogated by silencing EWS-WT1. Taken together, our findings reveal that EWS-WT1 can activate neural gene expression and direct partial neural differentiation via ASCL1, suggesting agents that promote neural differentiation might offer a novel therapeutic approach to treat DSRCT. Cancer Res; 74(16); 4526-35. Ó2014 AACR.

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Section: Methodsmentioning

confidence: 99%

“…Note that EWS-WT1 migrates higher (62 kDa) in JN-DSRCT-1 cells due to a different EWS translocation breakpoint (24). D, JN-DSRCT-1 cells were transduced with lentivirus carrying three independent shWT1 or control and analyzed by immunoblotting with anti-EWS, anti-ASCL1, or antiactin.…”

Section: Partial Reprogramming Of Fibroblasts To Neuron-like Cells Bymentioning

confidence: 99%

EWS–WT1 Oncoprotein Activates Neuronal Reprogramming Factor ASCL1 and Promotes Neural Differentiation

et al. 2014

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“…PCR primers for Bax, Bcl-xL, and actin were designed using the Oligo 6.0 software from National Biosciences (Plymouth, MN, USA), with GenBank published sequences as templates. The primer set for EWS/WT1 has been previously published (Nishio et al, 2002). A 279-bp Bax fragment was amplified with primers GAGGATGATTGCC GCCGTGGAC (upper) and CGGTGGTGGGGGTGAGGA GG (lower).…”

Section: Rt-pcr Assaysmentioning

confidence: 99%

“…This cell line exhibits the unique morphologic and genetic characteristics of DRSCT and, by RT-PCR, has been demonstrated to express a chimeric transcript in which exon 10 of the EWS gene is fused to exon 8 of the WT1 gene, which generates a WT1 (ÀKTS) isoform (Nishio et al, 2002). Therefore, the JN-DRSCT-1 cell line provides a useful in vitro model to understand the molecular pathology of DSRCT and to study potential new therapies.…”

Section: Introductionmentioning

confidence: 99%

Rapamycin induces apoptosis of JN-DSRCT-1 cells by increasing the Bax : Bcl-xL ratio through concurrent mechanisms dependent and independent of its mTOR inhibitory activity

2005

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Rapamycin, a complex macrolide and potent fungicide, immunosuppressant and anticancer agent, is a highly specific inhibitor of mammalian target of rapamycin (mTOR). Rapamycin has been shown to induce G1-phase cell cycle arrest in diverse tumor cell types, and its derivatives RAD001 and CCI-779 are currently in phase I and phase II clinical trials, respectively, as anticancer agents. In this study, we show that rapamycin induced the apoptotic death of JN-DSRCT-1 cells, the only available in vitro model for Desmoplastic Small Round Cell Tumors (DSRCT), while having only minor effects on their cell cycle. Rapamycin induced apoptosis by increasing the Bax : Bcl-xL ratio as a consequence of the concomitant downregulation of Bcl-xL and upregulation of Bax, both at the post-transcriptional level. Rapamycin also downregulated the levels of EWS/WT1, the fusion protein characteristic of DSRCT. Transient transfection studies using kinase-dead and rapamycin-resistant forms of mTOR demonstrated that only the downregulation of Bcl-xL was caused by the mTOR inhibitory action of rapamycin, which prevented cap-dependent translation initiation, whereas Bax upregulation was induced by rapamycin through a mechanism independent of its mTOR inhibitory activity. Moreover, rapamycin treatment downregulated the mRNA and protein levels of the 26S p44.5 proteasome subunit, suggesting the involvement of the proteasome complex in the mechanisms of rapamycin-induced apoptosis. Treatment of JN-DSRCT-1 cells with MG-132, a proteasome specific inhibitor, also resulted in the induction of apoptosis through a similar increase in the Bax : Bcl-xL ratio specifically caused by inhibiting Bax degradation and turnover. These results suggested that rapamycin induces apoptosis by preventing the degradation of the Bax protein by the proteasome, and that this process is independent of mTOR inhibition. Furthermore, these results strongly support the introduction of the use of rapamycin as a cytotoxic agent for the treatment of DSRCT.

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“…So far, cell lines have been obtained from sarcomas mainly by two methods; seeding material from pleural effusions [5][6][7][8][9][10][11][12] or seeding of cells after enzymatic digestion of tumor material. [13][14][15][16][17][18][19] As we assumed it to be difficult to optimize enzymatic digestion protocols for each single tissue type affected by sarcoma and because pleural effusion is mainly performed in patients with end-stage lung metastases only, we explored here the potential to obtain patient-derived cell lines from sarcoma patients using the explant technique.…”

mentioning

confidence: 99%

Explant culture of sarcoma patients' tissue

Muff

Botter

Husmann

et al. 2016

Laboratory Investigation

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Establishment and Characterization of a Novel Human Desmoplastic Small Round Cell Tumor Cell Line, JN-DSRCT-1

Cited by 41 publications

References 21 publications

EWS–WT1 Oncoprotein Activates Neuronal Reprogramming Factor ASCL1 and Promotes Neural Differentiation

EWS–WT1 Oncoprotein Activates Neuronal Reprogramming Factor ASCL1 and Promotes Neural Differentiation

Rapamycin induces apoptosis of JN-DSRCT-1 cells by increasing the Bax : Bcl-xL ratio through concurrent mechanisms dependent and independent of its mTOR inhibitory activity

Explant culture of sarcoma patients' tissue

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