The presence or absence of IL-3 during long-term culture of Flt3-ITD and c-Kit-D816V expressing Ba/F3 cells influences signaling outcome

Kazi, Julhash U.; Sun, Jianmin; Rönnstrand, Lars

doi:10.1016/j.exphem.2013.03.005

Cited by 16 publications

(14 citation statements)

References 10 publications

(5 reference statements)

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“…Therefore, we generated the Y823F mutant of c-Kit and stably transfected it into Ba/F3 cells. Ba/F3 is an immortalized murine bone marrow-derived pro-B cell line that is dependent on IL-3 for its growth (20). We have used Ba/F3 cells in this study because this cell line does not express c-Kit endogenously and, therefore, can be used to overexpress both the wild-type and mutant forms of human c-Kit.…”

Section: The Activation Loop Y823f Mutation Accelerates Receptormentioning

confidence: 99%

Phosphorylation of the Activation Loop Tyrosine 823 in c-Kit Is Crucial for Cell Survival and Proliferation

Agarwal

Kazi

2013

Journal of Biological Chemistry

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Section: The Activation Loop Y823f Mutation Accelerates Receptormentioning

confidence: 99%

Phosphorylation of the Activation Loop Tyrosine 823 in c-Kit Is Crucial for Cell Survival and Proliferation

Agarwal

Kazi

2013

Journal of Biological Chemistry

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“…Selection of factor independent Ba/F3 cells in the absence of IL-3 has been previously proposed, possibly as the result of enrichment for cells with stronger activation of downstream signaling [40]. Retroviral infection and reverse transcription is an inherently mutagenic process [41], making it an inevitability that an infected cell line has a library of clonal mutations in the infected transgene.…”

Section: Discussionmentioning

confidence: 99%

Analysis of acquired mutations in transgenes arising in Ba/F3 transformation assays: findings and recommendations

et al. 2017

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The identification and functional validation of potentially oncogenic mutations in leukemia is an essential step toward a future of personalized targeted therapy. To assess the oncogenic capacity of individual mutations, reliable and scalable in vitro experimental approaches are required. Since 1988, researchers have used the IL-3 dependent Ba/F3 transformation assay to validate the oncogenic potential of mutations to drive factor-independent growth. Here we report a previously unrecognized phenomenon whereby Ba/F3 cells, engineered to express weakly transforming mutations, present with additional acquired mutations in the expressed transgene following factor withdrawal. Using four mutations with known transformative capacity in three cytokine receptors (CSF2RB, CSF3R and IL7R), we demonstrate that the mutated receptors are highly susceptible to acquiring additional mutations. These acquired mutations of unknown functional significance are selected by factor withdrawal but appear to exist prior to the removal of growth factor. This anomaly has the potential to confound efforts to both validate and characterize oncogenic mutations in leukemia, particularly when it is not standard practice to sequence validate cDNAs from transformed Ba/F3 lines. We present specific recommendations to detect and mitigate this phenomenon in future research using Ba/F3 transformation assays, along with methods to make the Ba/F3 assay more quantitative.

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“…We tested cell proliferation and survival using constitutively active mutants of FLT3 and cKit which are known to induce IL-3-independent Ba/F3 cell survival [16]. We observed that CSK depletion resulted in an increased proliferation of Ba/F3-FLT3-ITD (Fig.…”

Section: Csk Depletion Increases Oncogenic Flt3-and C-kit-mediated Cementioning

confidence: 99%

“…Since IL3-withdrawal interferes functional redundancy of oncogenic FLT3 and c-Kit signaling [16], we used IL-3 containing medium for the culture of all transfected Ba/F3 cells.…”

Section: Cell Culturementioning

confidence: 99%

The tyrosine kinase CSK associates with FLT3 and c-Kit receptors and regulates downstream signaling

Kazi

Vaapil

Agarwal

et al. 2013

Cellular Signalling

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The tyrosine kinase CSK associates with FLT3 and c-Kit receptors and regulates downstream signaling.Uddin, Kazi; Vaapil, Marica; Agarwal, Shruti; Bracco, Enrico; Påhlman, Sven; Rönnstrand, Lars Link to publication Citation for published version (APA): Kazi, J. U., Vaapil, M., Agarwal, S., Bracco, E., Påhlman, S., & Rönnstrand, L. (2013). The tyrosine kinase CSK associates with FLT3 and c-Kit receptors and regulates downstream signaling. Cellular Signalling, 25(9), 1852 -1860 . DOI: 10.1016 /j.cellsig.2013 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.• Users may download and print one copy of any publication from the public portal for the purpose of private study or research.• You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal

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The presence or absence of IL-3 during long-term culture of Flt3-ITD and c-Kit-D816V expressing Ba/F3 cells influences signaling outcome

Cited by 16 publications

References 10 publications

Phosphorylation of the Activation Loop Tyrosine 823 in c-Kit Is Crucial for Cell Survival and Proliferation

Phosphorylation of the Activation Loop Tyrosine 823 in c-Kit Is Crucial for Cell Survival and Proliferation

Analysis of acquired mutations in transgenes arising in Ba/F3 transformation assays: findings and recommendations

The tyrosine kinase CSK associates with FLT3 and c-Kit receptors and regulates downstream signaling

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