Seuwen K, Steiner U, Adam G
Exp Cell Res. 1984 Sep;154(1):10-24. doi: 10.1016/0014-4827(84)90664-5.
The method for differential fluorescence staining of cellular RNA and DNA by acridine orange (AO) was optimized for 3T3 and SV40-3T3 cells. Cellular contents of DNA and of ribosomal RNA (rRNA) were determined by dual-channel flow cytometry during cell-density-dependent proliferation and after stimulation of quiescent cells. With increasing density of 3T3 cells, cellular content of rRNA decreases by about 60%, whereas SV40-3T3 cells do not exhibit a significant dependence of rRNA content on cell density. 3T3 cells stimulated early after becoming quiescent resume reaccumulation of rRNA after a delay of only 4 h, whereas cells maintained at quiescence for several days exhibit a delay of about 12 h before a significant rise of rRNA is observed. The extent of rise of cellular rRNA content after different regimens of stimulation of quiescent 3T3 cells does not correlate well with the fraction of cells entering the cell cycle. These and other reported instances of discordance between rRNA content and stimulation into the cell cycle are resolved by showing that of the two signals governing entry into the cell cycle only the progression signal, but not the competence signal is associated with reaccumulation of cellular rRNA. The present results are consistent with the progression function being in essence the achievement of a threshold number of ribosomes per cell, which in conjunction with the competence signal is sufficient for initiation of the cell cycle.
针对3T3和SV40 - 3T3细胞，优化了吖啶橙（AO）对细胞RNA和DNA进行差异荧光染色的方法。在细胞密度依赖性增殖过程中以及静止细胞受到刺激后，通过双通道流式细胞术测定DNA和核糖体RNA（rRNA）的细胞含量。随着3T3细胞密度的增加，rRNA的细胞含量下降约60%，而SV40 - 3T3细胞的rRNA含量对细胞密度没有明显依赖性。静止后早期受到刺激的3T3细胞在仅延迟4小时后就恢复了rRNA的重新积累，而保持静止数天的细胞在观察到rRNA显著升高之前有大约12小时的延迟。静止的3T3细胞在不同刺激方案后细胞rRNA含量升高的程度与进入细胞周期的细胞比例相关性不佳。通过表明在控制进入细胞周期的两个信号中，只有进展信号而非感受态信号与细胞rRNA的重新积累相关，解决了这些以及其他报道的rRNA含量与进入细胞周期刺激之间不一致的情况。目前的结果与进展功能本质上是实现每个细胞核糖体数量的阈值一致，该阈值与感受态信号一起足以启动细胞周期。
