Activation of Raf-1 and mitogen-activated protein kinases during monocytic differentiation of human myeloid leukemia cells.
Treatment of human myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC), is associated with induction of monocytic differentiation. Since PKC can act immediately upstream to the cytoplasmic Raf-1 serine/threonine protein kinase, we studied activation of Raf-1 during induction of the differentiated monocytic phenotype. The results demonstrate that Raf-1 is activated during TPA-induced monocytic differentiation of HL-60 cells. In contrast, there was little effect of TPA on this kinase in an HL-60 variant, designated HL-525, which is resistant to TPA-induced differentiation. Treatment of both HL-60 and HL-525 cells with okadaic acid, an inhibitor of serine/threonine protein phosphatases 1 and 2A, was associated with Raf-1 activation and induction of the monocytic phenotype. Since Raf-1 can activate the mitogen-activated protein (MAP) kinases, we also studied the relationship between MAP kinase activation and monocytic differentiation. Treatment of HL-60, but not HL-525, cells with TPA was associated with increased MAP kinase activity as determined by phosphorylation of myelin basic protein and the c-Jun Y peptide. Okadaic acid-induced differentiation of both HL-60 and HL-525 cells was similarly accompanied by increases in MAP kinase activity. These findings indicated that activation of Raf-1/MAP kinase signaling is associated with induction of a differentiated monocytic phenotype and that okadaic acid bypasses a defect in this cascade in TPA-treated HL-525 cells. While recent studies have shown that HL-525 cells are deficient in PKC beta, the present results demonstrate that PKC beta expression is up-regulated in the HL-525 variant by treatment with retinoic acid. The results also demonstrate that retinoic acid-treated HL-525 cells respond to TPA with activation of Raf-1 and MAP kinase, as well as induction of monocytic differentiation. Taken together, the results indicate that activation of Raf-1/MAP kinase signaling is associated with monocytic differentiation and that stimulation of serine/threonine protein phosphorylation by TPA or okadaic acid is sufficient for reversal of the leukemic HL-60 phenotype.
Kharbanda
S
S
Laboratory of Clinical Pharmacology, Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Saleem
A
A
Emoto
Y
Y
Stone
R
R
Rapp
U
U
Kufe
D
D
D000595:Amino Acid Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D004988:Ethers, Cyclic / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D016758:Genes, jun; D006801:Humans; D066298:In Vitro Techniques; D007951:Leukemia, Myeloid / Q000473:pathology*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology*; D019319:Okadaic Acid; D011493:Protein Kinase C / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D012333:RNA, Messenger / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004988:Ethers, Cyclic; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D019319:Okadaic Acid; D014212:Tretinoin; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D013755:Tetradecanoylphorbol Acetate