Chu E, Copur S M, Ju J, Chen T M, Khleif S, Voeller D M, Mizunuma N, Patel M, Maley G F, Maley F, Allegra C J
Department of Medicine and Pharmacology, Yale Cancer Center and VA CT Cancer Center, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Mol Cell Biol. 1999 Feb;19(2):1582-94. doi: 10.1128/MCB.19.2.1582.
A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.
从培养的人结肠癌细胞中分离出一种由胸苷酸合成酶（TS）和肿瘤抑制基因p53的mRNA组成的胸苷酸合成酶-核糖核蛋白（RNP）复合物。RNA凝胶迁移实验证实了TS蛋白与p53 mRNA的蛋白质编码区之间存在特异性相互作用，体外翻译研究表明这种相互作用导致p53 mRNA翻译的特异性抑制。为了证明TS蛋白-p53 mRNA相互作用的潜在生物学作用，蛋白质免疫印迹分析显示，与各自的亲本H630和H35细胞系相比，TS过表达的人结肠癌H630-R10和大鼠肝癌H35(F/F)细胞系中p53蛋白水平几乎检测不到。多核糖体分析显示，与H35(F/F)细胞相比，H35细胞中的p53 mRNA与高分子量多核糖体相关。虽然亲本细胞系和TS过表达细胞系中p53 mRNA的表达水平相同，但以RNP复合物形式与TS结合的p53 RNA水平在TS过表达细胞中显著更高。还利用四环素诱导系统在人结肠癌RKO细胞中研究了TS对p53表达的影响。用四环素衍生物强力霉素处理RKO细胞，导致TS蛋白表达增加15倍，p53蛋白表达几乎完全受到抑制。然而，在有无强力霉素的情况下，转染的RKO细胞中p53 mRNA水平相同。综上所述，这些发现表明TS在翻译水平上调节p53的表达。本研究确定了一种调节p53基因表达的新途径，并扩展了目前对TS作为细胞基因表达调节因子潜在作用的理解。
