Related Experiment Videos
Hck expression correlates with granulocyte-macrophage colony-stimulating factor-induced proliferation in HL-60 cells
D Linnekin1, O M Howard, L Park
1Laboratory of Leukocyte Biology, National Cancer Institute, Frederick Cancer Research and Development Center, MD.
Insights
Granulocyte-macrophage colony-stimulating factor receptors (GM-CSFRs) on HL-60 cells are uncoupled. The src-like tyrosine kinase hck is critical for functional coupling of GM-CSFR to cellular responses.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- HL-60 cells express GM-CSFR but do not respond to GM-CSF.
- Pretreatment with DMSO, retinoic acid, or vitamin D3 enables GM-CSF response.
- These treatments increase hck tyrosine kinase expression.
Purpose of the Study:
- To investigate the role of hck in GM-CSF signal transduction.
- To determine if hck mediates the recoupling of GM-CSFR in HL-60 cells.
Main Methods:
- Overexpression of hck in HL-60 cells to create HL-60/hck cell line.
- Stimulation with GM-CSF.
- Analysis of GM-CSF receptor (GM-CSFR) coupling, hck activation, protein tyrosine phosphorylation, and cell proliferation.
Main Results:
- GM-CSF stimulation increased hck autophosphorylation in DMSO-treated cells.
- Overexpression of hck in HL-60/hck cells restored GM-CSF responsiveness.
- HL-60/hck cells showed GM-CSF-induced hck activation, tyrosine phosphorylation, and proliferation.
Conclusions:
- Cytokine receptors can exist in an uncoupled state.
- Appropriate levels of hck are essential for the functional coupling of GM-CSFR to biological responses in HL-60 cells.
Abstract:
The human myeloid cell line HL-60 expresses approximately 300 high-affinity granulocyte-macrophage colony-stimulating factor receptors (GM-CSFRs), yet treatment of these cells with GM-CSF does not result in enhanced cellular proliferation or increases in protein tyrosine phosphorylation. In contrast, GM-CSF induces rapid increases in protein tyrosine phosphorylation and proliferative responses in HL-60 cells pretreated for 3 days in dimethyl sulfoxide (DMSO). Similarly, HL-60 cells pretreated with retinoic acid or 1,25 dihydroxyvitamin D3 were also capable of responding to GM-CSF. Interestingly, each of these treatments resulted in increased expression of the src-like tyrosine kinase hck. Stimulation with GM-CSF increased hck autophosphorylation in DMSO-treated HL-60 cells, suggesting that hck is a component of the GM-CSF signal transduction pathway. To determine if hck has a role in the DMSO-induced recoupling of the GM-CSFR, we overexpressed hck in HL-60 cells. The resulting cell line (HL-60/hck) expresses hck mRNA and protein at levels comparable with DMSO-treated HL-60 cells. Stimulation of HL-60/hck cells with GM-CSF results in activation of hck, increases in protein tyrosine phosphorylation, and increased proliferation. These results show that cytokine receptors can exist in an uncoupled form and suggest that in HL-60 cells, appropriate levels of the src-like tyrosine kinase hck are critical for functional coupling of the GM-CSFR to biologic responses.