Induction of growth factor-independence and GM-CSF secretion by the v-src oncogene in murine myeloid cells does not require membrane association of pp60v-src.
Introduction of v-src or c-src527F, a transforming mutant of the c-src proto-oncogene, into the growth factor-dependent cell line FDCP-1 resulted in growth factor independence. Temperature-shift studies with cells carrying the tsLA29 mutant of v-src demonstrated that growth factor independence was oncogene-dependent; that is, the cells were growth factor-independent at the permissive temperature but became growth factor-dependent at the nonpermissive temperature. Introduction of the c-src proto-oncogene did not result in growth factor independence. The c-src2A,527F mutant, which encodes an activated tyrosine kinase but does not transform fibroblasts due to a mutation in the membrane localization sequence, induced growth factor independence. This suggests that the presence of an activated tyrosine kinase is necessary for this process but that membrane localization is not. Bioassays indicated that conditioned medium from growth factor-independent cells contained a growth factor identified by antibody neutralization studies as granulocyte-macrophage colony-stimulating factor (GM-CSF). Secretion of GM-CSF was confirmed by a quantitative enzyme-linked immunosorbent assay (ELISA) specific for GM-CSF. The presence of GM-CSF mRNA in src-infected FDCP-1 cells was demonstrated by PCR amplification of cDNAs with primers specific for GM-CSF. While GM-CSF mRNA was detected in FDCP/ts29 cells grown at 34 degrees C, it was not observed in cells infected with the tsLA29 mutant grown at the nonpermissive temperature of 39 degrees C. Transfection of v-src-infected FDCP-1 cells with a GM-CSF promoter reporter plasmid revealed src-dependent expression of luciferase; that is, while expression was observed at the permissive temperature, no expression was detected in FDCP/ts29 clone 6 cells grown at the nonpermissive temperature. No expression of the GM-CSF promoter reporter plasmid was observed in uninfected FDCP-1 cells.
Ostermeyer
A G
AG
Department of Pathology, SUNY at Stony Brook, NY, USA.
Anderson
S M
SM
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000502:physiology*; D002472:Cell Transformation, Viral / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D016391:Genes, src; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000378:metabolism / Q000502:physiology*; D007377:Interleukin-3 / Q000494:pharmacology*; D051379:Mice; D016685:Mitomycin / Q000494:pharmacology; D009154:Mutation; D015688:Oncogene Protein pp60(v-src) / Q000235:genetics / Q000502:physiology*; D011401:Promoter Regions, Genetic; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014162:Transfection
D004274:DNA, Recombinant; D007377:Interleukin-3; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016685:Mitomycin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D015688:Oncogene Protein pp60(v-src); D016392:Proto-Oncogene Proteins pp60(c-src)