Whitson R H, Itakura K
Department of Molecular Genetics, Beckman Research Institute of the City of Hope, Duarte, California 91010.
J Cell Biochem. 1992 Mar;48(3):305-15. doi: 10.1002/jcb.240480311.
In the rat liver epithelial cell line, WB, the ability of TGF-beta 1 to inhibit DNA synthesis was shown to correlate with its ability to inhibit phosphorylation of the protein product of the retinoblastoma susceptibility gene, pRb. When WB cells were serum-starved, then refed with serum-containing medium, a peak of DNA synthesis occurred at about 18 h. Autoradiographs showed that 43.6% of cell nuclei could be labeled with 3H-thymidine at this time. When TGF-beta 1 was added simultaneously with serum, it blocked DNA synthesis and reduced the number of labeled nucleii to 6.3%. Cells treated with serum alone for 18 h also showed a pronounced increase in the highly phosphorylated form of pRb, as shown by mobility shifts in immunoblots, and in active phosphorylation of pRb, as shown by 32P incorporation. Simultaneous addition of TGF-beta 1 with serum abolished both 32P incorporation into pRb and its mobility shift on immunoblots. The effect of TGF-beta 1 on DNA synthesis measured at 18 h was sharply reduced if the cells were incubated with serum for 8 h (and thus allowed to enter S) before the addition of TGF-beta 1. If TGF-beta 1 was added after 8 h of serum treatment, its ability to inhibit pRb phosphorylation at 18 h was unchanged. If TGF-beta 1 was added after 13 h of serum treatment, its effects on pRb phosphorylation were reduced. Thus, as the cell population moved into S, the ability of TGF-beta 1 to inhibit both pRb phosphorylation and DNA synthesis was lost. In higher passages of WB cells the dose-response for inhibition of DNA synthesis by TGF-beta 1 was shifted to the right. Inhibition of pRb phosphorylation by TGF-beta 1 was also lost in higher passage WB cells. Thus, the passage-dependent loss of sensitivity to inhibition of DNA synthesis accompanied the loss of sensitivity to inhibition of pRb phosphorylation. Since the phosphorylation of pRb is believed to be required for the progression of cells from G1 to S, inhibition of pRb phosphorylation may be either a cause or a consequence of the G1 arrest of WB cells by TGF-beta 1.
在大鼠肝上皮细胞系WB中，已表明转化生长因子β1（TGF-β1）抑制DNA合成的能力与其抑制视网膜母细胞瘤易感基因的蛋白产物pRb磷酸化的能力相关。当WB细胞进行血清饥饿处理，然后再用含血清培养基重新培养时，DNA合成在约18小时出现峰值。放射自显影片显示此时43.6%的细胞核可用3H-胸腺嘧啶核苷标记。当TGF-β1与血清同时添加时，它会阻断DNA合成，并将标记细胞核的数量减少到6.3%。单独用血清处理18小时的细胞也显示出pRb的高度磷酸化形式显著增加，这通过免疫印迹中的迁移率变化得以体现，并且pRb的活性磷酸化也增加，这通过32P掺入得以显示。TGF-β1与血清同时添加会消除32P掺入pRb以及其在免疫印迹上的迁移率变化。如果在添加TGF-β1之前将细胞与血清孵育8小时（从而使其进入S期），那么在18小时测量时TGF-β1对DNA合成的影响会急剧降低。如果在血清处理8小时后添加TGF-β1，其在18小时抑制pRb磷酸化的能力不变。如果在血清处理13小时后添加TGF-β1，其对pRb磷酸化的影响会降低。因此，随着细胞群体进入S期，TGF-β1抑制pRb磷酸化和DNA合成的能力丧失。在WB细胞的传代次数增加时，TGF-β1抑制DNA合成的剂量反应向右移动。在传代次数增加的WB细胞中，TGF-β1对pRb磷酸化的抑制作用也丧失。因此，对DNA合成抑制敏感性的传代依赖性丧失伴随着对pRb磷酸化抑制敏感性的丧失。由于pRb的磷酸化被认为是细胞从G1期进入S期所必需的，pRb磷酸化的抑制可能是TGF-β1使WB细胞G1期停滞的原因或结果。
