Expression of M-CSF and its receptor (C-FMS) during factor-independent cell line evolution from hematopoietic progenitor cells cocultivated with gamma irradiated marrow stromal cell lines.
Gamma irradiation of plateau-phase clonal bone marrow stromal cell lines produces factor-independent growth of cocultivated clonal interleukin-3/granulocyte-macrophage colony-stimulating factor-dependent hematopoietic progenitor cell lines. The process is associated with three biologic changes including: (i) adherence of hematopoietic cells to stromal cells forming 'cobblestone islands'; (ii) an intermediate stage [during which the cells show proliferation in suspension in the presence in leukemogenic stromal factor (LSF), a factor similar to macrophage colony-stimulating factor (M-CSF) released by irradiated stromal cells, and transient hematopoietic cell surface expression of MAC-1, and c-fms (M-CSF receptor)]; and (iii) a third stage of factor-independence. A monoclonal antibody to M-CSF receptor inhibited proliferation of intermediate stage but not all factor-independent cell subclones. In the present studies, a subclonal factor-independent malignant subline of FDC-P1JL26 derived by cocultivation with gamma-irradiated stromal cells as well as the parent clone and intermediate stage cells were shown to express significant levels of M-CSF polyA+ mRNA and M-CSF of at least two sizes (23 and 15 kDa) as detected by 35S-methionine labelling and immunoprecipitation with polyclonal anti-M-CSF antiserum. There was no significant difference in intracellular M-CSF protein size between cells at each of the three stages of biologic change. This M-CSF was not detected on the cell surface by fluorescence-activated cell sorting (FACS). In contrast, c-fms expression at the cell surface was detected by FACS analysis and c-fms polyA+ mRNA was only detected during the intermediate stage of induction of factor-independence. FDC-P1JL26 parent cells, the subclone stimulated by LSF, and the factor-independent subclone, showed little or no detectable autophosphorylation of the c-fms receptor at tyrosine. There was no detectable rearrangement of the M-CSF or c-fms genes by Southern analysis between clonal lines during the three stages. While we cannot rule out an autocrine mechanism or mutated c-fms receptor mechanism, the data also suggest that evolution of hemopoietic cell factor-independence during cocultivation with irradiated stromal cells may involve a mechanism distal to the c-fms receptor/M-CSF interaction.
Greenberger
J S
JS
Department of Radiation Oncology, University of Massachusetts Medical Center, Worcester 01655.
Sakakeeny
M A
MA
Leif
J
J
Anklesaria
P
P
Pierce
J H
JH
Shadduck
R K
RK
D000818:Animals; D015139:Blotting, Southern; D001854:Bone Marrow Cells; D002460:Cell Line; D015870:Gene Expression; D015321:Gene Rearrangement; D005796:Genes; D016236:Genes, fms; D006133:Growth Substances / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D006801:Humans; D016173:Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009374:Neoplasms, Experimental / Q000235:genetics; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D015398:Signal Transduction
D006133:Growth Substances; D012333:RNA, Messenger; D016173:Macrophage Colony-Stimulating Factor; D016186:Receptor, Macrophage Colony-Stimulating Factor