[An in vitro nuclear run-on assay for nuclear function of angiogenin].
A sensitive and quantitative in vitro analysis method was established to detect the nascent RNAs stimulated by angiogenin using the nuclei isolated from human umbilical vein endothelial cells (HUVE). Angiogenin was mixed with nuclei in the reaction buffer, then transcription was initiated by adding the NTPs mixture. The RNA products were measured quantitatively by [alpha-(32)P]CTP incorporation with a liquid scintillation counter, either after removing free isotope by using spin column, or cutting the electrophoresis lane and counting after autoradiography. It was found that the optimum reacting temperature was 30 degrees, the most suitable reaction time was 30 min for this system, and the transcription enhancement activity of angiogenin was dose-dependent with the feasible concentration being 1 mg/L. Higher concentration of angiogenin degraded the RNA products in the system, suggesting that there is a mechanism to control the entry and accumulation of angiogenin in the target cells, which ensured angiogenin to play its role properly in the cells. Based on the evidence that angiogenin bound to DNA in nucleolus and enhanced RNA transcription, it was proposed that angiogenin might act as a trans-acting factor in nucleus to regulate RNA transcription, and inhibition of angiogenin-stimulated RNA transcription might be a promising target for screening anti-angiogenesis inhibitor.
Li
Ling-Yun
LY
Department of Public Health, Zhejiang University School of Medicine, Hangzhou 310031, China. zpxu@zju.edu.cn
Zhang
Yao-Zhou
YZ
Zheng
Yi-Fan
YF
Hu
Guo-Fu
GF
Xu
Zheng-Ping
ZP
D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D012313:RNA / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000494:pharmacology*; D013696:Temperature; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects
D012313:RNA; C046366:angiogenin; D012259:Ribonuclease, Pancreatic