A conditionally-active form of MEK1 results in autocrine tranformation of human and mouse hematopoietic cells.
The Raf/MEK/MAP kinase cascade plays a critical role in transducing growth signals from activated cell surface receptors. Using deltaMEK1:ER, a conditionally-active form of MEK1, we demonstrate the ability of this dual specificity protein kinase to abrogate the cytokine-dependency of the human and murine hematopoietic cells lines TF-1, FDC-P1 and FL5.12. Cytokine-independent cells were obtained from TF-1, FDC-P1 and FL5.12 cells at frequencies of 2.5 x 10(-3), 5 x 10(-5) and 10(-7) respectively, indicating that not all cells expressing deltaMEK1:ER were factor-independent. In general, cells that were converted to a cytokine-independent phenotype displayed a higher level of MAP kinase activity in response to deltaMEK1:ER activation than those that remained cytokine-dependent. deltaME-K1:ER-responsive cells could be maintained long-term in the presence of beta-estradiol as well as the estrogen-receptor antagonist 4-Hydroxy-Tamoxifen and the anti-estrogen ICI 164383. Removal of hormone led to the rapid cessation of cell growth in a manner similar to that observed when cytokine is withdrawn from the parental cells. Treatment of deltaMEKI:ER-responsive cells with a specific and selective inhibitor, PD98059, prevented growth in response to beta-estradiol. GM-CSF mRNA transcripts were detected in the MEK1-responsive cells indicating that the activated deltaMEK1:ER may induce a pathway leading to autocrine proliferation. Treatment of MEK1-responsive cells with an anti-GM-CSF antibody, but not a control antibody, suppressed cell growth. The cell lines described here will be useful for elaborating the ability of the MAP kinase pathway to regulate cell proliferation in hematopoietic cells.
Blalock
W L
WL
Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina 27858, USA.
Pearce
M
M
Steelman
L S
LS
Franklin
R A
RA
McCarthy
S A
SA
Cherwinski
H
H
McMahon
M
M
McCubrey
J A
JA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D019898:Autocrine Communication; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D016207:Cytokines / Q000494:pharmacology; D004789:Enzyme Activation; D004790:Enzyme Induction; D004958:Estradiol / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005813:Genes, Synthetic; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000037:antagonists & inhibitors / Q000276:immunology / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D048369:MAP Kinase Kinase 1; D020935:MAP Kinase Signaling System; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000502:physiology; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D014162:Transfection
D000911:Antibodies, Monoclonal; D016207:Cytokines; D005419:Flavonoids; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D013629:Tamoxifen; C016601:afimoxifene; D004958:Estradiol; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D017346:Protein Serine-Threonine Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; C482127:Map2k1 protein, mouse; D020929:Mitogen-Activated Protein Kinase Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one