Ball K L, Lain S, Fâhraeus R, Smythe C, Lane D P
CRC Cell Transformation Research Group, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, UK.
Curr Biol. 1997 Jan 1;7(1):71-80. doi: 10.1016/s0960-9822(06)00029-7.
A common event in the development of human neoplasia is the inactivation of a damage-inducible cell-cycle checkpoint pathway regulated by p53. One approach to the restoration of this pathway is to mimic the activity of key downstream effectors. The cyclin-dependent kinase (Cdk) inhibitor p21(WAF1) is one such molecule, as it is a major mediator of the p53-dependent growth-arrest pathway, and can, by itself, mediate growth suppression. The primary function of the p21(WAF1) protein appears to be the inhibition of G1 cyclin-Cdk complexes. Thus, if we can identify the region(s) of p21(WAF1) that contain its inhibitor activity they may provide a template from which to develop novel anti-proliferative drugs for use in tumours with a defective p53 pathway.
We report on the discovery of small synthetic peptides based on the sequence of p21(WAF1) that bind to and inhibit cyclin D1-Cdk4. The peptides and the full-length protein are inhibitory at similar concentrations. A 20 amino-acid peptide based on the carboxy-terminal domain of p21(WAF1) inhibits Cdk4 activity with a concentration for half-maximal inhibition (l0.5) of 46 nM, and it is only four-fold less active than the full-length protein. The length of the peptide has been minimized and key hydrophobic residues forming the inhibitory domain have been defined. When introduced into cells, both a 20 amino-acid and truncated eight amino-acid peptide blocked phosphorylation of the retinoblastoma protein (pRb) and induced a potent G1/S growth arrest. These data support a physiological role for the carboxyl terminus of p21(WAF1) in the inhibition of Cdk4 activity.
We have discovered that a small peptide is sufficient to mimic p21(WAF1) function and inhibit the activity of a critical G1 cyclin-Cdk complex, preventing pRb phosphorylation and producing a G1 cell-cycle arrest in tissue culture cell systems. This makes cyclin D1-Cdk4 a realistic and exciting target for the design of novel synthetic compounds that can act as anti-proliferative agents in human cells.
人类肿瘤发生过程中的一个常见事件是由p53调控的损伤诱导细胞周期检查点通路失活。恢复该通路的一种方法是模拟关键下游效应器的活性。细胞周期蛋白依赖性激酶（Cdk）抑制剂p21（WAF1）就是这样一种分子，因为它是p53依赖性生长停滞通路的主要介导因子，并且自身就能介导生长抑制。p21（WAF1）蛋白的主要功能似乎是抑制G1期细胞周期蛋白 - Cdk复合物。因此，如果我们能够确定p21（WAF1）中包含其抑制活性的区域，它们可能会提供一个模板，用于开发新型抗增殖药物，用于p53通路缺陷的肿瘤。
我们报告了基于p21（WAF1）序列发现能结合并抑制细胞周期蛋白D1 - Cdk4的小合成肽。这些肽和全长蛋白在相似浓度下具有抑制作用。基于p21（WAF1）羧基末端结构域的一个20个氨基酸的肽抑制Cdk4活性时，半数最大抑制浓度（IC0.5）为46 nM，其活性仅比全长蛋白低四倍。肽的长度已被最小化，并且已确定形成抑制结构域的关键疏水残基。当导入细胞时，一个20个氨基酸的肽和截短的8个氨基酸的肽都能阻断视网膜母细胞瘤蛋白（pRb）的磷酸化，并诱导有效的G1/S期生长停滞。这些数据支持p21（WAF1）羧基末端在抑制Cdk4活性方面具有生理作用。
我们发现一个小肽足以模拟p21（WAF1）的功能并抑制关键的G1期细胞周期蛋白 - Cdk复合物的活性，阻止pRb磷酸化并在组织培养细胞系统中产生G1期细胞周期停滞。这使得细胞周期蛋白D1 - Cdk4成为设计新型合成化合物的一个现实且令人兴奋的靶点，这些化合物可在人类细胞中作为抗增殖剂发挥作用。
