Bao Hong-Bo, Zhang Chuan-Bao, Wang Jin-Fang, Zhou Chuan-Nong, Liu Fang, Zhao Xiao-Hang, Qian Shi-Jun
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Sep;19(5):561-5.
The uracil in DNA comes from either the misincorporation of dUTP in place of dTTP or deamination of cytosine. In the latter case, it can result in a GC to AT transition mutation if the uracil is not removed before DNA replication. Base excision repair (BER) is a major pathway for removing DNA lesions arising from endogenous processes as well as those induced by exposure to exogenous chemicals or irradiation. BER is initiated by DNA glycosylases that excise aberrant bases from DNA by cleavage of the N-glycosidic bond linking to the base of its deoxyribose sugar. Uracil N-glycosylase (UNG) is the enzyme responsible for the first step in the BER pathway that specifically removes uracil from DNA. The UNG gene undergoes both temporal and spatial regulation mainly at the level of transcription. Normally cancer cells undergo over-proliferation and up-regulate their UNG during tumorigenesis. In this study we examine the correlation between UNG level and carcinogenesis, and explore the possibility of using UNG as a marker for cancer diagnosis. Human UNG gene was amplified from the total RNA of the human choriocarcinoma cell line, JEG-3, by RT-PCR. After purification, the 942bp full-length UNG cDNA coding sequence was digested with EcoR I and Sal I, and cloned into the digested pET-21 to construct a recombinant vector, pUNG. The UNG protein was expressed under the control of T7 promoter in E. coli BL21 (DE3) cells induced with IPTG. After ultrasonic treatment, the cell lysate and precipitate were analyzed by SDS-PAGE and a 39kD band was detected. The plasmid was serially diluted at appropriate concentrations and employed as standards in the subsequent quantification. Total RNAs were extracted from 18 pairs of clinical samples, each pair contains a sample of esophageal squamous cell carcinoma (ESCC) tissue and its surrounding normal esophageal epithelia. The copy numbers of UNG mRNA in these RNA samples were determined by real-time quantitative RT-PCR using a Lightcycler (Roche). UNG was present in 13 cases of ESCC (13/18, n = 18) but absent in all of the normal tissues. The results indicated that there was a correlation between high level of UNG expression and the carcinogenesis of ESCC.
DNA中的尿嘧啶要么来自dUTP错掺入取代dTTP，要么来自胞嘧啶的脱氨基作用。在后一种情况下，如果在DNA复制前尿嘧啶未被去除，可能会导致GC到AT的转换突变。碱基切除修复（BER）是去除内源性过程以及暴露于外源性化学物质或辐射诱导产生的DNA损伤的主要途径。BER由DNA糖基化酶启动，这些酶通过切割与脱氧核糖糖基上的碱基相连的N-糖苷键从DNA中切除异常碱基。尿嘧啶N-糖基化酶（UNG）是负责BER途径第一步的酶，它特异性地从DNA中去除尿嘧啶。UNG基因主要在转录水平上受到时间和空间调控。正常情况下，癌细胞在肿瘤发生过程中会过度增殖并上调其UNG。在本研究中，我们检测UNG水平与致癌作用之间的相关性，并探索将UNG用作癌症诊断标志物的可能性。通过RT-PCR从人绒毛膜癌细胞系JEG-3的总RNA中扩增人UNG基因。纯化后，将942bp的全长UNG cDNA编码序列用EcoR I和Sal I消化，并克隆到经消化的pET-21中构建重组载体pUNG。UNG蛋白在IPTG诱导的大肠杆菌BL21（DE3）细胞中在T7启动子的控制下表达。超声处理后，通过SDS-PAGE分析细胞裂解物和沉淀物，检测到一条39kD的条带。将质粒以适当浓度进行系列稀释，并在随后的定量中用作标准品。从18对临床样本中提取总RNA，每对样本包含一份食管鳞状细胞癌（ESCC）组织及其周围正常食管上皮组织的样本。使用Lightcycler（罗氏）通过实时定量RT-PCR测定这些RNA样本中UNG mRNA的拷贝数。UNG在13例ESCC中存在（13/18，n = 18），但在所有正常组织中均不存在。结果表明UNG高表达水平与ESCC的致癌作用之间存在相关性。
