Isolation and characterization of dominant and recessive IL-3-independent hematopoietic transformants

Kiser, Kim; Colombi, Marco; Moroni, Christoph

doi:10.1038/sj.onc.1209673

Cited by 5 publications

(12 citation statements)

References 40 publications

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“…These findings suggest that the Pten mutant 6.5 is sustained by different signalling pathways depending on whether it is growing in the presence or absence of IL-3. With IL-3, growth signals are transmitted via the Jak-Stat pathway as previously shown for the parental cell line (Kiser et al, 2006). In the absence of IL-3, the mTORC2 pathway provides an alternative growth mode that compensates for the absence of Jak-Stat signalling but consequently results in mTOR (mTORC2) pathway addiction.…”

Section: Il-3-sensitive Mtor Addictionmentioning

confidence: 94%

“…Clone 15 is a subclone of PB-3c; 15V4 is a v-H-ras-expressing clone 15 (Nair et al, 1992). In all, 6.5 and 6.8 are IL-3-independent clones that were isolated from 15V4-GFP after frameshift mutagenesis with ICR191 as described and selected for proliferation in absence of IL-3 (Kiser et al, 2006). IL-3-independent SHP cells were generated from IL-3-dependent 15V4 cells following knockdown of Pten via lentiviral shRNA targeting and simultaneous introduction of the blasticidin-resistance marker.…”

Section: Cell Culture and Cell Linesmentioning

confidence: 99%

“…Murine, IL-3-dependent PB-3c mast cells and its derivatives were cultured as described (Kiser et al, 2006). Clone 15 is a subclone of PB-3c; 15V4 is a v-H-ras-expressing clone 15 (Nair et al, 1992).…”

Section: Cell Culture and Cell Linesmentioning

confidence: 99%

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Genome-wide shRNA screen reveals increased mitochondrial dependence upon mTORC2 addiction

et al. 2010

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Section: Il-3-sensitive Mtor Addictionmentioning

confidence: 94%

Section: Cell Culture and Cell Linesmentioning

confidence: 99%

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Genome-wide shRNA screen reveals increased mitochondrial dependence upon mTORC2 addiction

et al. 2010

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“…18 Murine, PB-3c-15v4 mast cells as well as their derivatives were cultured in IMDM (Sigma) supplemented with 10% fetal calf serum, penicillin, streptomycin, 50 µM β-mercaptoethanol, and, if required, 2 ng/mL murine IL-3 (Sigma). 15V4 cells were transformed by a human bcrabl (oncogene gift of Dr. T. Klimkait, University of Basel, Switzerland) using the retroviral packaging system of Morita et al 19 as described.…”

Section: Cellsmentioning

confidence: 99%

“…Interestingly, addition of interleukin (IL)-3 into the cell culture medium efficiently rescued the effect of rapamycin via the activation of the Jak-Stat pathway. 18 The pleiotropic role of PTEN and the convergence of signals from other oncogene and tumor suppressor networks to activate the Akt-mTOR pathway indicate that many points of intervention for pathway inhibition may exist in a cell and that the 6.5 cell line offers an ideal screening tool. Thus, we performed an HTS with clone 6.5 cells in the presence and absence of IL-3.…”

Section: Introductionmentioning

confidence: 99%

An Isogenic Cell Panel Identifies Compounds That Inhibit Proliferation of mTOR-Pathway Addicted Cells by Different Mechanisms

et al. 2014

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The mTOR pathway is a critical integrator of nutrient and growth factor signaling. Once activated, mTOR promotes cell growth and proliferation. Several components of the mTOR pathway are frequently deregulated in tumors, leading to constitutive activation of the pathway and thus contribute to uncontrolled cell growth. We performed a high-throughput screen with an isogenic cell line system to identify compounds specifically inhibiting proliferation of PTEN/mTOR-pathway addicted cells. We show here the characterization and mode of action of two such compound classes. One compound class inhibits components of the PTEN/mTOR signaling pathway, such as S6 ribosomal protein phosphorylation, and leads to cyclin D3 downregulation. These compounds are not adenosine triphosphate competitive inhibitors for kinases in the pathway, nor do they require FKBP12 for activity like rapamycin. The other compound class turned out to be a farnesylation inhibitor, blocking the activity of GTPases, as well as an inducer of oxidative stress. Our results demonstrate that an isogenic cell system with few specific mutations in oncogenes and tumor suppressor genes can identify different classes of compounds selectively inhibiting proliferation of PTEN/mTOR pathway-addicted isogenic clones. The identified mechanisms are in line with the known cellular signaling networks activated by the altered oncogenes and suppressor genes in the isogenic system.

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Anti-apoptotic function of Xbp1 as an IL-3 signaling molecule in hematopoietic cells

et al. 2011

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Cytokine signaling is critical for proliferation, survival and differentiation of hematopoietic cell, and interleukin-3 (IL-3) is required for maintenance of many hematopoietic cell lines, such as BaF3. We have isolated apoptosis-resistant clones of BaF3 using retroviral insertional mutagenesis and the Xbp1 locus was identified as a retroviral integration site. Expression and splicing of the Xbp1 transcript was conserved in the resistant clone but was promptly disappeared on IL-3 withdrawal in parental BaF3. IL-3 stimulation of BaF3 cells enhanced Xbp1 promoter activity and induced phosphorylation of the endoplasmic reticulum stress sensor protein IRE1, resulting in the increase in Xbp1S that activates unfolded protein response. When downstream signaling from IL-3 was blocked by LY294002 and/or dn-Stat5, Xbp1 expression was downregulated and IRE1 phosphorylation was suppressed. Inhibition of IL-3 signaling as well as knockdown of Xbp1-induced apoptosis in BaF3 cells. In contrast, constitutive expression of Xbp1S protected BaF3 from apoptosis during IL-3 depletion. However, cell cycle arrest at the G1 stage was observed in BaF3 and myeloid differentiation was induced in IL-3-dependent 32Dcl3 cells. Expression of apoptosis-, cell cycle- and differentiation-related genes was modulated by Xbp1S expression. These results indicate that the proper transcriptional and splicing regulation of Xbp1 by IL-3 signaling is important in homeostasis of hematopoietic cells.

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Isolation and characterization of dominant and recessive IL-3-independent hematopoietic transformants

Cited by 5 publications

References 40 publications

Genome-wide shRNA screen reveals increased mitochondrial dependence upon mTORC2 addiction

Genome-wide shRNA screen reveals increased mitochondrial dependence upon mTORC2 addiction

An Isogenic Cell Panel Identifies Compounds That Inhibit Proliferation of mTOR-Pathway Addicted Cells by Different Mechanisms

Anti-apoptotic function of Xbp1 as an IL-3 signaling molecule in hematopoietic cells

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