Growth factor-dependent initiation of DNA replication in nuclei isolated from an interleukin 3-dependent murine myeloid cell line

To study the proliferative response of hematopoietic cells to growth factors at the molecular level, we developed a cell-free system for growth factor-dependent initiation of genomic DNA replication. Nuclei were isolated from the IL-3-dependent cell line NFS/N1-H7 after a 10-h period of IL-3 deprivation. Cytosolic and membrane-containing subcellular fractions were prepared from proliferating NFS/N1-H7 cells. Nuclei from the nonproliferating cells (±IL-3) showed essentially no incorporation of [³H]thymidine during a 16-h incubation with a mixture of unlabeled GTP, ATP, UTP, CTP, dGTP, dATP, dCTP, and [³H]dTTP. When the combination of IL-3, a cytosolic fraction, and a membrane-containing fraction from proliferating cells was added to nuclei from nonproliferating cells, a burst of [³H]thymidine incorporation into DNA began after a 12-h lag period, attained a maximal rate at 16 h, and reached a level of 860 pmol thymidine/10⁶ nuclei at 24 h (corresponding to replication of ~56% total mouse genomic DNA). This DNA synthesis was inhibited ~90% by the specific DNA polymerase α inhibitor aphidicolin. Deletion of a single cellular component or IL-3 from the system resulted in a marked reduction of DNA replication (^-membrane, 80 ± 4%; ^-cytosol, 90% ± 4%; ^-IL-3, 74 ± 7% inhibition). This model requires a growth factor (IL-3), a sedimentable cell fraction containing its receptor and possibly additional membrane-associated components, and a cytosolic fraction. It appears to recaptulate the molecular events required for progression from early G₁ to S phase of the cell cycle induced by IL-3 binding to its receptor.