The role of c-Myc on granulocyte colony-stimulating factor-dependent neutrophilic proliferation and differentiation of HL-60 cells.
We have previously suggested that phosphatidylinositol 3-kinase (PI3K)/p70 S6 kinase (p70 S6K) plays an important role in the regulation of neutrophilic differentiation of HL-60 cells on the basis of analysis of transferrin receptor (Trf-R)-positive (Trf-R(+)) and -negative (Trf-R(-)) cells that appear after treatment with dimethyl sulfoxide (DMSO). In the present study, we analyzed the downstream events of p70 S6K in differentiation and proliferation of both cell types, with a particular focus on c-Myc. Similar to p70 S6K, we found that the expression of c-Myc in Trf-R(+) cells is also higher than that in Trf-R(-) cells. Wortmannin, a specific inhibitor of PI3K, partially inhibited G-CSF-induced p70 S6K activity, c-Myc expression, and G-CSF-dependent proliferation, whereas rapamycin, an inhibitor of p70 S6K, completely inhibited p70 S6K activity, c-Myc expression, and G-CSF-dependent proliferation, indicating that the extent of c-Myc inhibition by these inhibitors correlates with a reduction in proliferation, and that c-Myc is downstream from PI3K/p70 S6K. We also determined phosphorylation of the 4E-binding protein 1 (4E-BP1), which is regulated downstream of the mammalian target of rapamycin. The addition of G-CSF failed to enhance the phosphorylation state of 4E-BP1 of HL-60 cells 2 days after DMSO differentiation. An antisense oligonucleotide for c-myc inhibited both G-CSF-dependent enhancement of c-Myc expression and proliferation in Trf-R(+) cells, but did not enhance the differentiation in terms of O(2)(-)-generating ability or fMLP-R expression. In contrast, antisense oligonucleotide for c-myc promoted fMLP-R on non-treated HL-60 cells. We therefore conclude that the PI3K/p70 S6K/c-Myc cascade plays an important role in neutrophilic proliferation in HL-60 cells. Unlike that of rapamycin, however, the antisense oligonucleotide for c-myc could not promote differentiation of Trf-R(+) cells cultured with G-CSF, indicating that another target downstream of p70 S6K may control the differentiation of HL-60 cells in terms of the signal transduction of G-CSF.
Kanayasu-Toyoda
Toshie
T
Division of Cellular and Gene Therapy Products, National Institute of Health, 1-18-1, Kamiyoga, Setagaya-Ku, 158-8501 Tokyo, Japan.
Yamaguchi
Teruhide
T
Oshizawa
Tadashi
T
Uchida
Eriko
E
Hayakawa
Takao
T
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002455:Cell Division / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016179:Granulocyte Colony-Stimulating Factor / Q000502:physiology*; D018922:HL-60 Cells; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D016271:Proto-Oncogene Proteins c-myc / Q000502:physiology*; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000235:genetics / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D000077191:Wortmannin
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D016179:Granulocyte Colony-Stimulating Factor; D007166:Immunosuppressive Agents; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D016271:Proto-Oncogene Proteins c-myc; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D020123:Sirolimus; D000077191:Wortmannin; C087000:EIF4EBP1 protein, human