Some evidences for an intracrine loop in the macrophage colony-stimulating factor (M-csf) System

The mechanism of action of the pleiotropic cytokine was incompletely understood. Previously, it was demonstrated that M-CSF and its receptor are co-expressed in the cytoplasm and nucleus of some human leukemic cell lines such as HL-60, suggesting the possibility of intracrine regulation mechanism for autonomous growth. To verify this hypothesis, we used an anti-M-CSF monoclonal antibody (mAb), B5, and antisense oligomer, which inhibited M-CSF transcripts, to block either interaction between exogenous M-CSF and its receptor, or extracellular interaction between membrane-bound M-CSF and its receptor, or possible intracellular interaction between M-CSF and its receptor. The results indicated that the in vitro inhibition rates of cell growth were 40-50% by both treatments separately, whereas it reached 80% by the treatment of the combination of antisense oligomer of M-CSF and mAb. This inhibition of cell growth could be reversed by removal of M-CSF antisense oligomer and anti-M-CSF mAb. In addition, we found that cells treated with antisense oligomer greatly decreased the expression of M-CSF protein and that both B5 and antisense oligomer down-regulated the expression of cyclin D1, up-regulated the expression of cyclin-dependent kinase (cdk) inhibitor p16 by means of immunofluorescence and flow cytometry. The sense oligomer control did not inhibit cell growth. Furthermore, we demonstrated that intracellular M-CSF was co-precipitated with its receptor by immunoprecipitation and Western blot, suggesting that intracellular M-CSF was present in receptor-bound state. Taken together, these results indicated that there should be an intracrine loop in these cells. To further study the roles of the intracrine loop, we constructed an expression plasmid of antisense gene fragment of M-CSF receptor, which was conducted into HL-60 by lipofectin method. The results showed that this it could cause G1 arrest of HL-60 cells and induce expression of cdk inhibitor p16 and inhibit expression of cyclin D1, but had little effect on expressions of cyclin E, cdk2 and cdk4, which suggested antisense gene-mediated G1 arrest correlated with the up-regulation of p16 and down-regulation of cyclin D1. Mounting evidences suggest that intracrine regulation may be crucial for cell transformation and tumorigenesis, so our findings are helpful to explain how autonomous growth factors produced by malignant cells might play an important role in diseases.