GROWTH RELATED SIGNAL TRANSDUCTION PATHWAYS IN CARCINOGENESIS

这个项目的主要目标是分析对增长的刺激和 抑制因子诱导的磷酸化蛋白表达的调节 大鼠肝细胞培养及其受体介导性的研究 信号通路。低传代非同步化大鼠肝上皮 用32-P-原磷酸盐预先标记RLE细胞，并用 转化生长因子-β1(5 ng/ml)作用15分钟和60分钟。十五分钟后 转化生长因子-β1对磷酸化异构体表达的影响 在两种多肽中，2种(等电点5.00/85 kDa)和3种(4.90/84 kDa)明显 下降，但在60分钟后恢复到本构水平。按顺序 要解决这样一个问题，即观察到的 磷酸蛋白2和3的表达是特异性的结果 磷酸化途径的调节或是瞬时信号的结果 减少合成2或3，一种“三重标记”技术 是发展起来的。多肽2和3的分析，利用这个 技术表明多肽的表达显著减少。 3由于合成减少或降解增加 多肽3为转化生长因子-β1治疗的结果。减少的 另一方面，多肽2的磷酸化可能是由于 一种特定的转化生长因子-β1磷酸酶的激活(S)。以前的工作有 结果表明，转化生长因子-β1介导了其在体外的生长抑制作用。 细胞周期的G1晚期；因此，RLE细胞是同步的 洛伐他汀/甲伐他汀，在G1期的不同时间，32- 将P-正磷酸盐预标细胞分别处理15分钟和60分钟 转化生长因子-β1(5 ng/ml)。在7小时和8小时时， 两种多肽(等电点7.00/22 kDa和7.10/20 kDa)的磷酸化 在用转化生长因子-β1治疗15分钟后，两者都恢复为结构性 治疗60分钟后的血药浓度。RLE细胞的类似处理 转化生长因子-β1在较早(6小时)或较晚(9、10或11小时) 细胞周期对这些多肽的表达没有影响。 到目前为止的结果表明，转化生长因子-β1诱导了快速和短暂的 对多肽的特定亚群的调制，其中一些可能是 参与了转化生长因子-β1的生长抑制作用。

The main objective of this project is to analyze growth stimulatory and inhibitory factor induced modulation of phosphoprotein expression in cultured rat liver cells in an attempt to delineate receptor mediated signaling pathways. Low passage unsynchronized rat liver epithelial (RLE) cells were prelabeled with 32-P-ortho- phosphate and treated with TGF-beta1 (5 ng/ml) for 15 and 60 minutes. Fifteen minutes after treatment with TGF-beta1, the expression of the phosphorylated isoforms of two polypeptides, 2 (pI 5.00/85 kDa) and 3 (4.90/84 kDa) were markedly decreased but returned to constitutive levels after 60 minutes. In order to address the question of whether the observed decrease in the expression of phosphoproteins 2 and 3 was the result of specific modulation of phosphorylation pathways or was the result of a transient decrease in the synthesis of either 2 or 3, a "triple labeling" technique was developed. Analysis of polypeptides 2 and 3, utilizing this technique, indicated a marked decrease in the expression of poly-peptide 3 as a result of either a decreased synthesis or increased degradation of polypeptide 3 as a result of TGF-beta1 treatment. The decreased phosphorylation of polypeptide 2, on the other hand, may be the result of an activation of a specific TGF-beta1 phosphatase(s). Previous work has demonstrated that TGF-beta1 mediates its growth inhibitory action in the late G1 stages of the cell cycle; therefore, RLE cells were synchronized with lovastatin/mevalonate and, at various times during the G1 phase, 32- P-orthophosphate prelabeled cells were treated for 15 and 60 minutes with TGF-beta1 (5 ng/ml). At 7 and 8 hours there was a marked increase in the phosphorylation of two polypeptides (pI 7.00/22 kDa and 7.10/20 kDa) after 15 minutes treatment with TGF-beta1; both returned to constitutive levels after 60 minutes treatment. Similar treatment of RLE cells with TGF-beta1 either earlier (6 hours) or later (9, 10 or 11 hours) in the cell cycle had no effect on the expression of these polypeptides. Results to date indicate that TGF-beta1 induces rapid and transient modulation of specific subsets of polypeptides some of which may be involved in the growth inhibitory effects of TGF-beta1.