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An increase in the expression and total activity of endogenous p60c-Src in several factor-independent mutants of a human GM-CSF-dependent leukemia cell line (TF-1)

Oncogene volume 22, pages 7170–7180 (2003)Cite this article

Abstract

Growth factor independence of hematopoietic cells can be induced by ectopic expression of a variety of oncogenes encoding receptor or cytoplasmic tyrosine kinases. To examine whether the activation of tyrosine kinases occurs in factor-independent mutants in vivo, the tyrosine-phosphorylated proteins from 14 factor-independent mutants of a GM-CSF-dependent cell line (TF-1) were analysed. These mutants did not secrete any growth-stimulating activity for TF-1 cells, suggesting that activation of intracellular signaling rather than an autocrine stimulation by secreted growth factors is responsible for their factor-independent growth. In 11 out of 14 GM-CSF-independent mutants analysed, a constitutively tyrosine-phosphorylated protein of 60 kDa was detected, which was subsequently identified as p60c-Src. The kinase activity of p60c-Src was increased up to 12-fold in these mutants, which was at least in part due to overexpression of the c-src gene on the RNA and protein level. The Src substrate Sam68 showed an increased phosphorylation in mutants with high Src activity, suggesting that p60c-Src triggers downstream signaling in these cells. Treatment of the factor-independent mutants with the Src kinase inhibitor PP2 resulted in a reduced proliferation, demonstrating that Src kinases are essential for these cells for maximal proliferation. Further analysis of factor-independent mutants with low or undetectable Src activity revealed a constitutive phosphorylation of the common β chain of the GM-CSF receptor and STAT5. Our data indicate an increase in the expression and total activity of endogenous p60c-Src in several GM-CSF-independent TF-1 mutants, further underlining the role of Src in the process of autonomous growth of hematopoietic cells.

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Abbreviations

SH2:

src homology 2

GM-CSF:

granulocyte–macrophage colony-stimulating factor

IL-3:

interleukin-3

BM:

bone marrow

PBMC:

peripheral blood mononuclear cells

FCS:

fetal calf serum

SDS:

sodium dodecyl sulfate

PAGE:

polyacrylamide gel electrophoresis

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Acknowledgements

We thank W Fiedler and DK Hossfeld from the Department of Hematology/Oncology, University Hospital Eppendorf, Hamburg, for providing recombinant human GM-CSF, JB Bolen and WJM van de Ven for monoclonal antibodies against Src or Fes, respectively, and T Kitamura for providing TF-1 cells. We are grateful to Ulla Bergholz, Wiebke Wegner, Stefanie Ehmer and Eileen Magbanua for excellent technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (JU255/2-3) and Thyssen Stiftung (CS).

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  1. Zentrum für Experimentelle Medizin, Institut für Biochemie und Molekularbiologie I, Zelluläre Signaltransduktion, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, Hamburg, D-20246, Germany

    Stefan Horn & Manfred Jücker

  2. Abteilung für Zell- und Virusgenetik, Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistr. 52, Hamburg, D-20251, Germany

    Johann Meyer, Carol Stocking & Wolfram Ostertag

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Correspondence to Manfred Jücker.

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Horn, S., Meyer, J., Stocking, C. et al. An increase in the expression and total activity of endogenous p60c-Src in several factor-independent mutants of a human GM-CSF-dependent leukemia cell line (TF-1). Oncogene 22, 7170–7180 (2003). https://doi.org/10.1038/sj.onc.1206856

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