Dadke S S, Rao K V
Cellular Carcinogenesis Laboratory, Cancer Research Institute, Tata Memorial Centre, Parel, Bombay, India.
Tumori. 1998 Jan-Feb;84(1):14-20. doi: 10.1177/030089169808400103.
The existence of endogenous growth inhibitors was postulated in 1914 by Boveri. However, most regretfully, progress in the isolation, characterization and mechanisms of actions of endogenous growth-inhibitory proteins is scanty compared to the information available on growth-stimulatory proteins. Accordingly, the major purpose of the present study was to isolate and characterize an endogenous growth-inhibitory protein from normal rat liver so that its role during liver carcinogenesis could be evaluated.
For protein purification, a combination of alcohol precipitation, gel permeation chromatography and ion exchange chromatography techniques was utilized. For characterization and mechanisms, the methods utilized were DNA synthesis, immunoblotting, immunohistochemistry, protein sequencing, DNA-agarose electrophoresis and Hoechst staining.
The purified protein inhibited the growth of several cell lines in culture as measured by the rate of DNA synthesis using 3H-thymidine. In SDS-PAGE stained by the silver staining method, the molecular weight of the polypeptide was found to be 14 kD. Polyclonal antiserum was raised against this 14 kD polypeptide in rabbit. Immunoblotting experiments showed that the antibody recognizes specifically the 14 kD polypeptide and immunolocalization studies showed that the polypeptide is predominantly a cytoplasmic protein. Addition of antibody and inhibitory polypeptide simultaneously to the cultures more or less abolished the inhibitory activity of the polypeptide. Sequencing of the N-terminal 17 amino acids of the growth-inhibitory polypeptide showed Val-Leu-Leu-Ala-Glu-Ala-Glu-Thr-Ala-Ile-Val-Asn-Gly-Leu-Asp-Lys-Ile. Comparing this sequence using a BLAST protein data base indicated that there was no significant homology between the sequence of the growth-inhibitory polypeptide and protein sequences deposited with the data bank, suggesting that this could be a novel growth-inhibitory polypeptide. The mechanisms of growth inhibition appeared to be apoptosis as determined by electrophoretic analysis of DNA fragmentation and staining of the cells with the dye Hoechst 33342.
A growth-inhibitory protein of 14 kD can be isolated from normal rat liver. The physiologic role of the protein in liver appears to be either growth regulatory or apoptotic.
1914年，博韦里提出内源性生长抑制剂的存在。然而，非常遗憾的是，与生长刺激蛋白的现有信息相比，内源性生长抑制蛋白在分离、特性鉴定及作用机制方面的进展甚微。因此，本研究的主要目的是从正常大鼠肝脏中分离并鉴定一种内源性生长抑制蛋白，以便评估其在肝癌发生过程中的作用。
蛋白质纯化采用乙醇沉淀、凝胶过滤色谱和离子交换色谱技术相结合的方法。特性鉴定及作用机制研究采用的方法包括DNA合成、免疫印迹、免疫组织化学、蛋白质测序、DNA-琼脂糖电泳和Hoechst染色。
通过使用³H-胸腺嘧啶核苷测量DNA合成速率，发现纯化后的蛋白质可抑制几种培养细胞系的生长。在银染法染色的SDS-PAGE中，该多肽的分子量为14kD。用此14kD多肽在兔体内制备了多克隆抗血清。免疫印迹实验表明，该抗体能特异性识别14kD多肽，免疫定位研究表明，该多肽主要是一种细胞质蛋白。同时向培养物中添加抗体和抑制性多肽，或多或少会消除该多肽的抑制活性。生长抑制多肽N端17个氨基酸的序列为Val-Leu-Leu-Ala-Glu-Ala-Glu-Thr-Ala-Ile-Val-Asn-Gly-Leu-Asp-Lys-Ile。使用BLAST蛋白质数据库比较该序列表明，生长抑制多肽的序列与数据库中保存的蛋白质序列之间没有显著同源性，这表明这可能是一种新型的生长抑制多肽。通过DNA片段化的电泳分析以及用Hoechst 33342染料对细胞进行染色确定，生长抑制机制似乎是细胞凋亡。
可从正常大鼠肝脏中分离出一种14kD的生长抑制蛋白。该蛋白在肝脏中的生理作用似乎是生长调节或诱导凋亡。
