INDUCTION OF GROWTH FACTOR-INDEPENDENCE AND GM-CSF SECRETION BY THE V-SRC ONCOGENE IN MURINE MYELOID CELLS DOES NOT REQUIRE MEMBRANE ASSOCIATION OF PP60(V-SRC)

Introduction of v-src or C-src(527F), a transforming mutant of the c-s rc proto-oncogene, into the growth factor-dependent cell line FDCP-1 r esulted in growth factor independence. Temperature-shift studies with cells carrying the tsLA29 mutant of v-src demonstrated that growth fac tor independence was oncogene-dependent; that is, the cells were growt h factor-independent at the permissive temperature but became growth f actor-dependent at the nonpermissive temperature. Introduction of the c-src proto-oncogene did not result in growth factor independence. The C-src(2A,527F) mutant, which encodes an activated tyrosine kinase but does not transform fibroblasts due to a mutation in the membrane loca lization sequence, induced growth factor independence. This suggests t hat the presence of an activated tyrosine kinase is necessary for this process but that membrane localization is not. Bioassays indicated th at conditioned medium from growth factor-independent cells contained a growth factor identified by antibody neutralization studies as granul ocyte-macrophage colony-stimulating factor (GM-CSF). Secretion of GM-C SF was confirmed by a quantitative enzyme-linked immunosorbent assay ( ELISA) specific for GM-CSF. The presence of GM-CSF mRNA in src-infecte d FDCP-1 cells was demonstrated by PCR amplification of cDNAs with pri mers 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 wit h the tsLA29 mutant grown at the nonpermissive temperature of 39 degre es C; Transfection of v-src-infected FDCP-1 cells with a GM-CSF promot er reporter plasmid revealed src-dependent expression of luciferase; t hat is, while expression was observed at the permissive temperature, n o expression was detected in FDCP/ts29 clone 6 cells grown at the nonp ermissive temperature. No expression of the GM-CSF promoter reporter p lasmid was observed in uninfected FDCP-1 cells.