title: Effects of deregulated Raf activation on integrin, cytokine-receptor expression and the induction of apoptosis in hematopoietic cells.
abstract: The effects of deregulated Raf activation on the growth and differentiation of hematopoietic cells were investigated. The cytokine-dependent murine myeloid FDC-P1 and human erythroleukemic TF-1 cell lines were transformed to grow in response to deregulated Raf expression in the absence of exogenous cytokines. The conditionally active Raf proteins were regulated by beta-estradiol as cDNAs containing the Raf catalytic, but lacking negative-regulatory domains, were ligated to the hormone binding domain of the estrogen receptor (deltaRaf:ER). Continuous deltaRaf expression prevented apoptosis in the absence of exogenous cytokines and altered the morphology of the FD/deltaRaf:ER cells as they grew in large aggregated masses (>100 cells) whereas the parental cytokine-dependent FDC-P1 cells grew in smaller grape-like clusters (< 10 cells). FD/deltaRaf-1:ER cells growing in response to Raf activation displayed decreased levels of the Mac-2 and Mac-3 molecules on their cell surface. In contrast, when these cells were cultured in IL-3, higher levels of these adhesion molecules were detected. Expression of activated Raf oncoproteins also abrogated cytokine dependency and prevented apoptosis of TF-1 cells. Moreover, the differentiation status of these Raf-responsive cells was more immature upon Raf activation as culture with the differentiation-inducing agent phorbol 12 myristate 13-acetate (PMA) and beta-estradiol resulted in decreased levels of the CD11b and CD18 integrin molecules on the cell surface. In contrast when the Raf-responsive cells were induced to differentiate with PMA and GM-CSF, in the absence of deltaRaf:ER activation, increased levels of the CD11b and CD18 molecules were detected. Retinoic acid (RA) inhibited 3H-thymidine incorporation in response to GM-CSF. Interestingly, Raf activation counterbalanced the inhibition of DNA synthesis caused by RA but not PMA. Thus deregulated Raf expression can alter cytokine dependency, integrin expression and the stage of differentiation. These Raf-responsive cell lines will be useful in elucidating the roles of the MAP kinase cascade on hematopoietic cell differentiation and malignant transformation.
authors.affiliation: Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University, Greenville, NC 27858, USA.
authors.forename: C
authors.initials: C
authors.lastname: Weinstein-Oppenheimer
authors.forename: L S
authors.initials: LS
authors.lastname: Steelman
authors.forename: P A
authors.initials: PA
authors.lastname: Algate
authors.forename: W L
authors.initials: WL
authors.lastname: Blalock
authors.forename: C
authors.initials: C
authors.lastname: Burrows
authors.forename: P E
authors.initials: PE
authors.lastname: Hoyle
authors.forename: J T
authors.initials: JT
authors.lastname: Lee
authors.forename: P W
authors.initials: PW
authors.lastname: Moye
authors.forename: J G
authors.initials: JG
authors.lastname: Shelton
authors.forename: R
authors.initials: R
authors.lastname: Franklin
authors.forename: J A
authors.initials: JA
authors.lastname: McCubrey
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D018845:CD11 Antigens / Q000096:biosynthesis / Q000235:genetics; D002449:Cell Aggregation / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D004958:Estradiol / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D016023:Integrins / Q000096:biosynthesis* / Q000235:genetics; D007377:Interleukin-3 / Q000494:pharmacology; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D051379:Mice; D008811:Mice, Inbred DBA; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D023461:Myeloid Progenitor Cells / Q000187:drug effects* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021122:Protein Subunits; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D018121:Receptors, Cytokine / Q000096:biosynthesis* / Q000235:genetics; D011960:Receptors, Estrogen / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
chemical_list: D018845:CD11 Antigens; D018076:DNA, Complementary; D016023:Integrins; D007377:Interleukin-3; D009363:Neoplasm Proteins; D021122:Protein Subunits; D018121:Receptors, Cytokine; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D013629:Tamoxifen; C016601:afimoxifene; D004958:Estradiol; D014212:Tretinoin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019908:Proto-Oncogene Proteins c-raf; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020930:MAP Kinase Kinase Kinases; D013755:Tetradecanoylphorbol Acetate