Regulation of myeloid cell growth by distinct effectors of Ras.
To examine the biochemical pathways by which activated Ha-Ras(G12V) (Ha-RasV12) induces factor-independent growth of myeloid cells, Ha-Ras effector loop mutations, including Y40C, T35S, and E37G, were analysed in a mouse factor-dependent myeloid cell line, WT19. Expression of a single effector loop mutant, Ha-Ras(G12V, Y40C) (Ha-RasV12C40), inhibited factor-withdrawal apoptosis, suggesting that activation of the phosphatidylinositol 3'-kinase (Pl3K) pathway is essential to prevent cell death. Neither Ha-Ras (G12V, T35S) (Ha-RasV12S35), which activates the Rafl signaling pathway, nor Ha-Ras(G12V, E37G) (Ha-RasV12G37), which stimulates the RalGDS pathway, did not have significant effects on factor-withdrawal apoptosis of myeloid cells. Although Ha-RasV12C40 inhibited apoptosis, it did not stimulate entry into the cell cycle. Cell lines containing the combination of Ha-RasV12G37 and Ha-RasV12C40 were capable of factor-independent cell growth, while expression of the other combinations of the Ha-Ras effector mutants were not. The combined expression of Bcl-2 and Ha-RasV12G37 was not sufficient to stimulate factor independent growth, suggesting that Ha-RasV12C40 activates additional signals, besides blocking apoptosis, which are critical for factor-independent growth of myeloid cells. In factor-starved myeloid cells, inducible expression of Ha-RasV12G37 results in decreased level of p27Kip1 protein, a cyclin-dependent kinase inhibitor (CKI). These data suggest that the factor-independent growth of myeloid cells requires the activation of at least two pathways, one inhibiting factor-withdrawal apoptosis, and another causing cell cycle progression.
Matsuguchi
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Division of Medical Oncology, University of Colorado Health Science Center, Denver 80262, USA.
Kraft
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D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000502:physiology*; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D050760:Cyclin-Dependent Kinase Inhibitor p27; D005786:Gene Expression Regulation; D011905:Genes, ras; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D013329:Structure-Activity Relationship; D014162:Transfection; D025521:Tumor Suppressor Proteins
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016283:Proto-Oncogene Proteins p21(ras)