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Autocrine and possible intracrine regulation of HL-60 cell proliferation by macrophage colony-stimulating factor
1State Key Laboratory for Experimental Hematology, Institute of Hematology, Chinese Academy of Medical Sciences & Peking Union Medical College, 288 Nanjing Road, Tianjin 300020, People's Republic of China.
Abstract:
The abnormal expression of macrophage colony stimulating factor (M-CSF) isoforms, i.e. membrane bound M-CSF (m-M-CSF) and intracellular M-CSF (c-M-CSF), and their receptor were reported in some leukemia and tumor cells. Furthermore, the nuclear localization of them may be related to poor prognosis and metastasis, while the mechanism is uncertain. We previously reported that m-M-CSF and its receptor played auto-juxtacrine and adhesion molecule role in human leukemia cell line J6-1. In this paper, we show that HL-60 cells highly express M-CSF and its receptor. The localization of positive reactions was mainly in cytoplasma and nuclear in HL-60 cells. In cytoplasma and nuclear, three isoforms of M-CSF were found with molecular weight (MW) of 20, 16 and 14 kDa, while one type of m-CSF receptor (M-CSFR) was discovered with MW of 120 kDa. Immunoprecipitation assay showed that these ligands could exist separately or binding with their receptor. Monoclonal antibody (McAb) against M-CSF and anti-sense oligodeoxynucleotides (ASON) blocking M-CSF expression inhibited the proliferation of HL-60 cells. McAb and ASON regulated the expression of cyclin D1/E, CDK2/4 and p16. Simultaneous administration of both McAb and ASON inhibited the proliferation of HL-60 cells and modulate the expression of cyclins at greater degrees. Our results suggested an autocrine and possible an intracrine loop of M-CSF/M-CSFR in HL-60 cells.
Insights
Macrophage colony-stimulating factor (M-CSF) and its receptor are expressed in HL-60 leukemia cells, suggesting an autocrine loop. Blocking M-CSF inhibited cell proliferation and regulated key cell cycle proteins.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Abnormal expression of macrophage colony-stimulating factor (M-CSF) isoforms, including membrane-bound (m-M-CSF) and intracellular (c-M-CSF), and their receptor (M-CSFR) is observed in leukemia and tumor cells.
- Nuclear localization of M-CSF and M-CSFR may correlate with poor prognosis and metastasis, but the underlying mechanisms remain unclear.
- Previous studies indicated a role for m-M-CSF and its receptor in auto-juxtacrine signaling and cell adhesion in human leukemia cell line J6-1.
Purpose of the Study:
- To investigate the expression, localization, and functional role of M-CSF and M-CSFR in the HL-60 human leukemia cell line.
- To explore the potential autocrine and intracrine mechanisms involving M-CSF/M-CSFR signaling in HL-60 cells.
- To assess the effect of M-CSF blockade on HL-60 cell proliferation and cell cycle regulation.
Main Methods:
- Immunohistochemistry and Western blotting to detect M-CSF and M-CSFR expression and localization in HL-60 cells.
- Immunoprecipitation assays to determine ligand-receptor binding status.
- Treatment of HL-60 cells with monoclonal antibody (McAb) against M-CSF and anti-sense oligodeoxynucleotides (ASON) to inhibit M-CSF expression.
- Analysis of cell proliferation and expression of cell cycle regulatory proteins (cyclin D1/E, CDK2/4, p16).
Main Results:
- HL-60 cells exhibited high expression of M-CSF and M-CSFR, localized primarily in the cytoplasm and nucleus.
- Three M-CSF isoforms (20, 16, 14 kDa) and one M-CSFR (120 kDa) were identified.
- M-CSF ligands were found to exist independently or bound to their receptor.
- Inhibition of M-CSF using McAb or ASON significantly reduced HL-60 cell proliferation.
- M-CSF blockade modulated the expression of cyclin D1/E, CDK2/4, and p16.
- Combined administration of McAb and ASON resulted in greater inhibition of proliferation and modulation of cyclins.
Conclusions:
- HL-60 cells demonstrate an autocrine and potentially intracrine loop involving M-CSF/M-CSFR signaling.
- M-CSF/M-CSFR signaling plays a crucial role in HL-60 cell proliferation.
- Targeting M-CSF represents a potential therapeutic strategy for certain types of leukemia.