Munshi N C, Gabig T G
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
J Clin Invest. 1990 Jan;85(1):300-4. doi: 10.1172/JCI114428.
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.
为了在分子水平上研究造血细胞对生长因子的增殖反应，我们开发了一种无细胞系统，用于生长因子依赖性启动基因组DNA复制。在剥夺白细胞介素-3（IL-3）10小时后，从依赖IL-3的细胞系NFS/N1-H7中分离出细胞核。从增殖的NFS/N1-H7细胞中制备含胞质和膜的亚细胞组分。在与未标记的鸟苷三磷酸（GTP）、三磷酸腺苷（ATP）、尿苷三磷酸（UTP）、胞苷三磷酸（CTP）、脱氧鸟苷三磷酸（dGTP）、脱氧腺苷三磷酸（dATP）、脱氧胞苷三磷酸（dCTP）和[3H]胸苷三磷酸（[3H]dTTP）的混合物孵育16小时期间，来自非增殖细胞（±IL-3）的细胞核基本上没有掺入[3H]胸腺嘧啶核苷。当将IL-3、胞质组分和来自增殖细胞的含膜组分的组合添加到来自非增殖细胞的细胞核中时，在12小时的延迟期后，[3H]胸腺嘧啶核苷掺入DNA的量开始激增，在16小时达到最大速率，并在24小时达到860皮摩尔胸腺嘧啶核苷/10（6）个细胞核的水平（相当于约56%的小鼠基因组总DNA的复制）。这种DNA合成被特异性DNA聚合酶α抑制剂阿非迪霉素抑制约90%。从系统中删除单个细胞组分或IL-3会导致DNA复制显著减少（-膜，80±4%；-胞质溶胶，90%±4%；-IL-3，74±7%抑制）。该模型需要一种生长因子（IL-3）、一个含有其受体以及可能还有其他膜相关组分的可沉降细胞组分和一个胞质组分。它似乎概括了IL-3与其受体结合诱导细胞周期从早期G1期进入S期所需的分子事件。
