The effect of preincubation of bone marrow cells on the binding of colony-stimulating factor

In previous studies, the binding of 125I-CSF to fresh murine bone marrow cells was delayed for 6 to 16 hr at 37 degrees C and only minimal binding was observed at 4 degrees C. In the present experiments, marrow cells were preincubated for varying intervals at 37 degrees C in medium devoid of CSF prior to the addition of 125I-CSF. Preincubation for 6 to 24 hr led to a progressive increase in binding sites, so that cellular uptake of radiolabeled CSF was detected within 5 min of exposure to the cells. With preincubated cells, a marked difference was noted in binding kinetics at 4 degrees and 37 degrees C. The increase in cell-bound radioactivity at 4 degrees C with time yielded a curvilinear plot with 31% of maximum uptake at 5 min and 71% at 1 hr. In contrast, maximum cell uptake occurred within 5 min at 37 degrees C, with a plateau in cell bound radioactivity thereafter. This difference appeared to result from continuous internalization of the tracer at 37 degrees C, as judged by the finding of 67% of the radioactivity in the cytosol fraction after 1 hr exposure to tracer. Only 15% was detected in this fraction after 4 degrees incubation. Moreover, a high proportion of cytosol radioactivity appeared to be degraded after binding at 37 degrees C, as judged by a decrease in molecular weight of radioactive material on SDS-acrylamide gels. Degradation of the tracer was further demonstrated by secretion of radioactive peptides into the medium after binding. Only 8% secretion was noted at 4 degrees C; however, this increased to 70% to 80% at 37 degrees C. Development of binding sites in vitro appeared to require new protein synthesis, since addition of 1 microgram/ml cycloheximide during the preincubation prevented the subsequent CSF binding. Thus CSF binding sites are generated by in vitro incubation of marrow cells. Radiolabeled CSF attaches to surface binding sites, is internalized rapidly, and is degraded into lower-molecular-weight peptides. These findings appear to explain the continued requirement of CSF for in vitro colony formation.