Juan G, Li X, Darzynkiewicz Z
Brander Cancer Research Institute, New York Medical College, Valhalla, New York, 10595, USA.
Exp Cell Res. 1998 Oct 10;244(1):83-92. doi: 10.1006/excr.1998.4165.
Expression of pRb and its state of phosphorylation were immunocytochemically assayed in individual HL-60 cells during their proliferation and after induction of differentiation, using mAb which detects hypophosphorylated pRb (pRbP-) combined with mAb which reacts with pRb regardless of its phosphorylation (total pRb; pRbT). Correlated measurements of pRbP-, pRbT, a ratio of pRbP-/pRbT, and cellular DNA content by flow cytometry revealed expression of total pRb and its phosphorylation state vis-à-vis the cell cycle position. Following mitosis (during the exponential phase of cell growth) a mixture of hypo- and hyperphosphorylated pRb was present within the cell for less than 2 h, i.e., early in G1; no hypophosphorylated pRb was detected throughout remainder of the cycle. Cellular pRb content was increasing primarily during G1 and the cell entrance to S was correlated with attainment of a distinct threshold level of pRb. No correlation was seen between the content of pRb per cell and its state of phosphorylation during G1. Cell differentiation whether induced by 1,25-dihydroxyvitamin D3, retinoic acid, or phorbol myristate acetate led to cell arrest primarily in G0/1. The G0/1 cells in these cultures, compared to G1 cells from the untreated cultures, had increased level of both pRbT and pRbP-. However, because the relative increase of pRbP- was disproportionally greater than of pRbT, the pRbP-/pRbT ratio of the differentiating cells was markedly elevated. The cells that still were in S and G2/M in the differentiating cultures also showed the presence of hypophosphorylated pRb. Our data suggest that the mechanism of irreversible cell cycle arrest during terminal differentiation involves both the increase in content of pRb and dephosphorylation of pRb already present within the cell. This provides a large pool of hypophosphorylated pRb that can effectively remove all free E2F, thereby precluding activation of the genes whose transcription is needed to pass the G1 restriction point. In contrast to terminal differentiation the transient quiescence (G0 state) manifests only by dephosphorylation of pRb, without a change in its cellular level.
在HL-60细胞增殖过程中以及诱导分化后，使用检测低磷酸化pRb（pRbP-）的单克隆抗体与能与pRb发生反应而不论其磷酸化状态如何的单克隆抗体（总pRb；pRbT），通过免疫细胞化学方法检测单个HL-60细胞中pRb的表达及其磷酸化状态。通过流式细胞术对pRbP-、pRbT、pRbP-/pRbT比值和细胞DNA含量进行相关测量，揭示了总pRb的表达及其相对于细胞周期位置的磷酸化状态。有丝分裂后（在细胞生长的指数期），低磷酸化和高磷酸化pRb的混合物在细胞内存在不到2小时，即在G1早期；在细胞周期的其余阶段未检测到低磷酸化pRb。细胞pRb含量主要在G1期增加，细胞进入S期与达到pRb的特定阈值水平相关。在G1期，每个细胞的pRb含量与其磷酸化状态之间未发现相关性。无论是由1,25-二羟基维生素D3、视黄酸还是佛波酯肉豆蔻酸酯诱导的细胞分化，主要导致细胞停滞在G0/1期。与未处理培养物中的G1细胞相比，这些培养物中的G0/1细胞中pRbT和pRbP-的水平均有所增加。然而，由于pRbP-的相对增加远大于pRbT，分化细胞的pRbP-/pRbT比值明显升高。在分化培养物中仍处于S期和G2/M期的细胞也显示存在低磷酸化pRb。我们的数据表明，终末分化过程中不可逆细胞周期停滞的机制涉及pRb含量的增加以及细胞内已存在的pRb的去磷酸化。这提供了大量的低磷酸化pRb，其可以有效地去除所有游离的E2F，从而排除了通过G1限制点所需转录的基因的激活。与终末分化相反，短暂静止（G0期）仅表现为pRb的去磷酸化，而其细胞水平没有变化。
