Miller A M, Allen B S, Ziboh V
Tulane Cancer Center, Tulane University, New Orleans, Louisiana 70112, USA.
J Cell Physiol. 1997 Mar;170(3):309-15. doi: 10.1002/(SICI)1097-4652(199703)170:3<309::AID-JCP12>3.0.CO;2-9.
The cell line M-07e requires either Interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF) for proliferation in vitro. Cells deprived of growth factor for up to 48 hours remain viable but no longer divide. The growth-factor-deprived M-07e cells begin to divide within 48 hours of reexposure to IL-3. Flow cytometric analysis of M-07e cells labeled with hypotonic propidium iodide demonstrates that the percentage of cells undergoing DNA synthesis decreases from 24%, in a log phase population of IL-3 stimulated cells, to 1% when cells are deprived of IL-3 for 24 hours. IL-3-deprived cells accumulate predominantly in a flow cytometry peak representative of G0/G1. DNA synthetic activity, as determined by tritiated thymidine uptake and flow cytometry, resumes between 12 and 18 hours after reexposure to IL-3, reaching a peak of up to 40% by 24 hours and returning to log phase levels by 72 hours. Prior to initiation of DNA synthesis, increases are seen in mRNA levels for five-lipoxygenase-activating protein (FLAP). Following reexposure to IL-3, a rapid time-dependent biosynthesis of leukotriene D4 (LTD4) is induced by M-07e cells. When IL-3 is added in the presence of any of three lipoxygenase inhibitors tested (Piriprost, caffeic acid, nordihydroguiaretic acid) or FLAP inhibitor, MK-886, there is dose-dependent inhibition of the resumption of proliferation and of DNA synthesis. Flow cytometric cell cycle analysis demonstrates that the inhibited cells remain in the G0/G1 population and do not progress through the cell cycle. These results are consistent with our previous observation that an intact lipoxygenase pathway is necessary for hematopoietic growth-factor-stimulated colony formation of normal bone marrow myeloid progenitors and suggest that the induction of a lipoxygenase metabolite or metabolites is necessary for myeloid cells to progress through the cell cycle when stimulated by a hematopoietic growth factor.
细胞系M-07e在体外增殖需要白细胞介素-3（IL-3）或粒细胞-巨噬细胞集落刺激因子（GM-CSF）。生长因子剥夺长达48小时的细胞仍可存活但不再分裂。重新暴露于IL-3后48小时内，生长因子剥夺的M-07e细胞开始分裂。用低渗碘化丙啶标记的M-07e细胞的流式细胞术分析表明，进行DNA合成的细胞百分比从IL-3刺激细胞的对数期群体中的24%降至细胞被剥夺IL-3 24小时时的1%。剥夺IL-3的细胞主要积聚在代表G0/G1的流式细胞术峰中。通过氚标记胸腺嘧啶核苷摄取和流式细胞术确定的DNA合成活性在重新暴露于IL-3后12至18小时恢复，到24小时达到高达40%的峰值，并在72小时恢复到对数期水平。在DNA合成开始之前，5-脂氧合酶激活蛋白（FLAP）的mRNA水平升高。重新暴露于IL-3后，M-07e细胞诱导白三烯D4（LTD4）的快速时间依赖性生物合成。当在测试的三种脂氧合酶抑制剂（吡嘧司特、咖啡酸、去甲二氢愈创木酸）或FLAP抑制剂MK-886存在下添加IL-3时，增殖恢复和DNA合成受到剂量依赖性抑制。流式细胞术细胞周期分析表明，受抑制的细胞停留在G0/G1群体中，不通过细胞周期进展。这些结果与我们之前的观察一致，即完整的脂氧合酶途径对于造血生长因子刺激的正常骨髓髓系祖细胞集落形成是必需的，并表明当受到造血生长因子刺激时，一种或多种脂氧合酶代谢产物的诱导对于髓系细胞通过细胞周期进展是必需的。
