Induction of growth alterations in factor-dependent hematopoietic progenitor cell lines by cocultivation with irradiated bone marrow stromal cell lines

We studied the production of hemopoietins by x-irradiated plateau-phase cultures of cloned marrow stromal cell lines derived from C3H/HeJ marrow, termed D2XRII and clone 11. The production of CSF in agar overlay of control or 10,000 rad irradiated stromal cultures was quantitated by induction of colonies in: overlaid fresh marrow, IL-3- dependent cell line 32D cl 3, or GM-CSF/IL-3-dependent cell lines FDCP- 1 or bg/bg cl 1. Conditioned media were tested for CSF by bioassay using fresh marrow cells, for M-CSF (CSF-1) by RIA, and for IL-3 and GM- CSF by microwell proliferation assay with 32D cl 3 and FDCP-1 cells, respectively. X-irradiation to doses that decreased CSF-1 to 40% of control levels (greater than 5,000 rad) resulted in a 30-fold increase in growth of FDCP-1 or bg/bg cl 1 cells in liquid co-culture or agar culture overlay with no detectable growth of 32D cl 3. The frequency of subculture of nonautocrine, factor independent (FI) variant clonal lines of FDCP-1 or bg/bg cl 1 cells was increased over 1000-fold by 15 weeks cocultivation with irradiated stromal cell cultures. FI subclonal lines formed tumors in syngeneic mice and contained no detectable poly A messenger RNA for GM-CSF or IL-3, and no elevation in c-myc, c-abl, c- src, or erb-B onc gene-specific messenger RNA compared to parent factor- dependent lines. These data indicate that x-irradiated plateau phase marrow stromal cells produce increased levels of cell contact-mediated biologically active hemopoietin(s) other than M-CSF, GM-CSF, or IL-3 and induce nonautocrine factor-independent malignant cell lines in vitro.