Harrison L, Skorvaga M, Cunningham R P, Hendry J H, Margison G P
Cancer Research Campaign Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, United Kingdom.
Radiat Res. 1992 Oct;132(1):30-9.
The Escherichia coli nth gene encodes endonuclease III, which catalyses the glycolytic removal of various oxidized thymine residues from DNA. A truncated version of nth, with the prokaryotic regulatory sequences removed, was ligated into the retrovirus-based vector pZipneoSV(X)1 and transfected into the radiosensitive Chinese hamster ovary cell line, xrs7. Following selection with G418, two clones (x7nth1 and x7nth6) were shown by Southern analysis to contain the nth gene. No substantial difference in gamma-ray sensitivity was detected between xrs7, clones x7nth1 and x7nth6, and the parent vector transfected clone (x7neo1). However, clones containing the nth gene were more resistant to hydrogen peroxide cytotoxicity [D0's for x7nth1 and x7nth6 were 0.072 microgram/ml (4 microM) and 0.046 microgram/ml, respectively, compared with D0's of 0.034 and 0.027 microgram/ml for xrs7 and x7neo1, respectively] but markedly more sensitive to bleomycin sulfate cytotoxicity than xrs7 and x7neo1 (e.g., 1D0's for x7nth6 and xrs7 were 0.05 and 0.12 microgram/ml, while 2D0's for x7nth1 and xrs7 were 0.35 and 0.48 microgram/ml, respectively). Alterations in sensitivity to hydrogen peroxide and bleomycin sulfate could not be explained by differences in the distribution of the cell-cycle phases and growth rate of nth-containing clones and control cell lines. These results are consistent with the hypothesis that modified thymine lesions are potentially cytotoxic. Hence, when cells incur a high level of endonuclease III-repairable damage relative to strand breakage, such as after treatment with hydrogen peroxide, increased repair capacity increases survival. Gamma radiation produces a lower level of endonuclease III-repairable damage relative to all the other types of lesions produced; hence increased repair capacity has no measurable effect on cell survival. The increased sensitivity of x7nth1 and x7nth6 to bleomycin sulfate toxicity may indicate that, when thymine damage and single-strand breaks are in close proximity on opposite strands of the DNA, endonuclease III, which incises DNA at the site of damaged residues, can increase the number of double-strand breaks and hence decrease the level of cell survival.
大肠杆菌nth基因编码内切核酸酶III，该酶催化从DNA中糖基化去除各种氧化的胸腺嘧啶残基。将去除了原核调控序列的nth截短版本连接到基于逆转录病毒的载体pZipneoSV(X)1中，并转染到对辐射敏感的中国仓鼠卵巢细胞系xrs7中。用G418筛选后，通过Southern分析表明两个克隆（x7nth1和x7nth6）含有nth基因。在xrs7、克隆x7nth1和x7nth6以及转染亲本载体的克隆（x7neo1）之间未检测到γ射线敏感性有实质性差异。然而，含有nth基因的克隆对过氧化氢细胞毒性更具抗性[x7nth1和x7nth6的D0分别为0.072微克/毫升（4微摩尔）和0.046微克/毫升，而xrs7和x7neo1的D0分别为0.034和0.027微克/毫升]，但对博来霉素硫酸盐细胞毒性的敏感性明显高于xrs7和x7neo1（例如，x7nth6和xrs7的1D0分别为0.05和0.12微克/毫升，而x7nth1和xrs7的2D0分别为0.35和0.48微克/毫升）。对过氧化氢和博来霉素硫酸盐敏感性的改变无法用含nth克隆和对照细胞系的细胞周期阶段分布及生长速率的差异来解释。这些结果与修饰的胸腺嘧啶损伤具有潜在细胞毒性这一假设一致。因此，当细胞相对于链断裂产生高水平的可被内切核酸酶III修复的损伤时，如用过氧化氢处理后，增加的修复能力会提高存活率。γ辐射产生的相对于所有其他类型损伤而言较低水平的可被内切核酸酶III修复的损伤；因此增加的修复能力对细胞存活没有可测量的影响。x7nth1和x7nth6对博来霉素硫酸盐毒性的敏感性增加可能表明，当胸腺嘧啶损伤和单链断裂在DNA的相反链上紧邻时，在受损残基位点切割DNA的内切核酸酶III可增加双链断裂的数量，从而降低细胞存活水平。
