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/10(6) nuclei at 24 h (corresponding to replication of approximately 56% total mouse genomic DNA). This DNA synthesis was inhibited approximately 90% by the specific DNA polymerase alpha 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 recapitulate the molecular events required for progression from early G1 to S phase of the cell cycle induced by IL-3 binding to its receptor.
Munshi
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Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Gabig
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D000818:Animals; D016590:Aphidicolin; D001973:Bromodeoxyuridine / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002474:Cell-Free System; D004261:DNA Replication / Q000187:drug effects*; D004224:Diterpenes / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism; D007377:Interleukin-3 / Q000494:pharmacology*; D007700:Kinetics; D051379:Mice; D013698:Templates, Genetic
D004224:Diterpenes; D007377:Interleukin-3; D016590:Aphidicolin; D001973:Bromodeoxyuridine