Isolation of revertants from a factor-independent myeloid cell line.

Mouse myeloid stem cell line D35 was obtained from a primary cell culture of C3H bone marrow cells. This cell grows completely dependent on the multi-lineage colony-stimulating factor (multiCSF; IL-3) or the granulocyte macrophage colony-stimulating factor (GMCSF). By serial dilution of the medium every 3 days, autonomously growing mutants were obtained spontaneously at the rate of 108 -107 . Among 18 mutants, 11 produce GM-CSF by themselves, three produce multi-CSF, and two do not secrete any growth factors that stimulate the parental cell line. All of those mutants that were checked were tumorigenic in nude mice. Four of the GM-CSF-producing mutants turned out to be the result of the insertion of an intracisternal A particle (Dind 1), Rauscher murine leukemia virus (Dind 4), or Friend spleen focus-forming virus (F-SFFV; Dind 5, Dind 9) at either 3' or 5' of GM-CSF locus. To clarify whether aberrant expression of a CSF gene is sufficient for autonomous growth and tumorigenicity, and to investigate possible mechanisms of gene activation, as well as insertion and excision of these retroviruses, we have attempted to obtain revertants that again have a growth dependency on factor. After cultivation of these autonomous mutants with multi-CSF, 106 cells were washed and incubated for 24 h without any growth factor. A final concentration