Plagemenn P G, Richey D P, Zylka J M, Erbe J
J Cell Biol. 1975 Jan;64(1):29-41. doi: 10.1083/jcb.64.1.29.
Populations of Novikoff rat hepatoma cells (subline N1S1-67) were monitored for the rates of transport of various substrates and for their incorporation into acid-insoluble material as a function of the age of cultures of randomly growing cells in suspension as well as during traverse of the cells through the cell cycle. Populations of cells were synchronized by a double hydroxyurea block or by successive treatment with hydroxyurea and Colcemid. Kinetic analyses showed that changes in transport rates related to the age of cultures or the cell cycle stage reflecte alterations in the V max of the transport processes, whereas the Km remained constant, indicating that changes in transport rates reflect alterations in the number of functional transport sites. The transport sites for uridine and 2-deoxy-D-glucose increased continuously during traverse of the cells through the cell cycle, whereas those for choline and hypoxanthine were formed early in the cell cycle. Increases in thymidine transport sites were confined to the S phase. Synchronized cells deprived of serum failed to exhibit normal increases in transport sites, although the cells divided normally at the end of the cell cycle. Arrest of the cells in mitosis by treatment with Colcemid prevented any further increases in transport rates. The formation of functional transport sites was also dependent on de novo synthesis of RNA and protein. Inhibition of DNA synthesis in early S phase inhibited the increase in thymidine transport rates which normally occurs during the S phase, but had no effect on the formation of the other transport systems. Transport rates also fluctuated markedly with the age of the cultures of randomly growing cells, reaching maximum levels in the mid-exponential phase of growth. The transport systems for thymidine and uridine were rapidly lost upon inhibition of protein and RNA synthesis, and thus seem to be metabolically unstable, whereas the transport systems for choline and 2-deoxy-D-glucose were stable under the same conditions.
监测诺维科夫大鼠肝癌细胞（亚系N1S1-67）群体，观察其对各种底物的转运速率以及它们掺入酸不溶性物质的情况，这些指标是随机生长的悬浮培养细胞的培养年龄以及细胞穿越细胞周期的函数。通过双羟基脲阻断或依次用羟基脲和秋水仙酰胺处理使细胞群体同步化。动力学分析表明，与培养年龄或细胞周期阶段相关的转运速率变化反映了转运过程中Vmax的改变，而Km保持恒定，这表明转运速率的变化反映了功能性转运位点数量的改变。在细胞穿越细胞周期的过程中，尿苷和2-脱氧-D-葡萄糖的转运位点持续增加，而胆碱和次黄嘌呤的转运位点在细胞周期早期形成。胸苷转运位点的增加仅限于S期。剥夺血清的同步化细胞未能表现出转运位点的正常增加，尽管细胞在细胞周期结束时正常分裂。用秋水仙酰胺处理使细胞停滞在有丝分裂期可阻止转运速率的进一步增加。功能性转运位点的形成也依赖于RNA和蛋白质的从头合成。在S期早期抑制DNA合成可抑制通常在S期发生的胸苷转运速率的增加，但对其他转运系统的形成没有影响。转运速率也随随机生长细胞培养物的年龄而显著波动，在生长的指数中期达到最高水平。胸苷和尿苷的转运系统在蛋白质和RNA合成受到抑制后迅速丧失，因此似乎在代谢上不稳定，而胆碱和2-脱氧-D-葡萄糖的转运系统在相同条件下是稳定的。
