title: Inhibition of p21ras activation blocks proliferation but not differentiation of interleukin-3-dependent myeloid cells.
abstract: Interleukin-3 (IL-3) induces proliferation of immature myeloid cells and mast cells and prevents programmed cell death (apoptosis) in vitro. These activities are exerted through binding of IL-3 to specific, high affinity receptors that then initiate a series of intracellular signaling events. Among the earliest of these signaling events in an IL-3-dependent cell line such as 32Dcl3 are activation of one or more receptor-associated tyrosine kinases followed by activation of p21ras. In an effort to define the functional role of p21ras activation in mediating the effects of IL-3, we constructed a series of sublines of 32Dcl3 in which a dominant inhibitory mutant of Ha-ras (c-Ha-ras(Asn-17)) was expressed under the control of a steroid-inducible promoter. Steroid treatment (dexamethasone, 1 microM) specifically induced c-Ha-ras(Asn-17) protein and mRNA and blocked IL-3-induced accumulation of p21ras-GTP in 32Dcl3/p21rasN17 cell lines, but not in control cells. Dexamethasone slightly inhibited IL-3-dependent proliferation of control 32Dcl3 cell lines (to 80% of maximum), but it completely blocked proliferation of 32Dcl3/p21rasN17 cell lines and induced cell cycle arrest in G0/G1. This proliferative block could be overcome by cotransfection with v-ras, and was reversible if dexamethasone was washed out. Cells arrested by c-Ha-ras(Asn-17) were viable in culture for > 2 weeks, despite their inability to proliferate. Notably, however, these cells remained dependent on IL-3 for viability and initiated apoptosis within 18 h of IL-3 deprivation. Finally, granulocyte colony-stimulating factor-induced differentiation of 32Dcl3/p21rasN17 cells to neutrophils was not affected by steroid-induced expression of c-Ha-ras(Asn-17) and did not require removal of IL-3. These results suggest that IL-3-induced proliferation and maintenance of cell viability are either initiated through separate signal transduction pathways or require different degrees of p21ras activation. Similarly, granulocyte colony-stimulating factor-induced neutrophil differentiation is not blocked by expression of c-Ha-ras(Asn-17).
authors.affiliation: Division of Hematologic Malignancies, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
authors.forename: K
authors.initials: K
authors.lastname: Okuda
authors.forename: T J
authors.initials: TJ
authors.lastname: Ernst
authors.forename: J D
authors.initials: JD
authors.lastname: Griffin
mesh_terms: D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003907:Dexamethasone / Q000494:pharmacology; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology*; D009154:Mutation; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology*
chemical_list: D007377:Interleukin-3; D016179:Granulocyte Colony-Stimulating Factor; D003907:Dexamethasone; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)