Jones S, Willmore E, Durkacz B W
Cancer Research Unit, Medical School, University of Newcastle upon Tyne, UK.
Carcinogenesis. 1994 Nov;15(11):2435-8. doi: 10.1093/carcin/15.11.2435.
Nascent DNA (nDNA) replication intermediates can be isolated and quantified by pH-step alkaline elution (L.C. Erikson et al., Chromosoma, 74, 125-139, 1979). The effects of 5-fluorouracil (FU) and 5-fluoro-2'-deoxyuridine (FdU) on the formation and persistence of nascent DNA structures were studied in Chinese hamster ovary K1 cells. Exogenous thymidine (dT) rescued FdU-induced, but had little effect on FU-induced, cytotoxicity, indicating DNA- and RNA-directed cytotoxic mechanisms respectively. Drug-treated cells were pulse labelled with [3H]dT and the percentage of total counts eluting from and retained by filters estimated at successive pH increments (11.0, 11.3, 11.5 and 12.1). A 1 min pulse of control cells resulted in > 75% of the DNA eluting at pH 12.1 or being retained by the filter. By 1 h, > 90% of the DNA was retained by the filter. FdU treatment resulted in the persistence of DNA in all four pH bands. More than 95% of total DNA synthesized during a 5 min pulse eluted as discrete peaks at each pH. This DNA was processed to higher M(r) species during a 1 h chase, but following a 24 h chase, 68% of the DNA still eluted at pH 12.1. In contrast, FU treatment caused only a transient accumulation (< or = 10 min) of the DNA (e.g. approximately 26% at 5 min) in the pH 11.0 and 11.3 bands only, indicating a selective effect of FU on the maturation of very short size classes of DNA. Neutral elution was used to assess the effect of FdU on double-strand break (dsb) formation in nDNA. Whereas no dsbs were evident in untreated cells, dsbs appeared in FdU-treated cells even following the shortest [3H]dT pulse times (1-10 min). Although these were processed into higher M(r) species with longer pulse times, dsbs were still evident at 2 h.
新生DNA（nDNA）复制中间体可通过pH梯度碱性洗脱法进行分离和定量（L.C.埃里克森等人，《染色体》，第74卷，第125 - 139页，1979年）。研究了5 - 氟尿嘧啶（FU）和5 - 氟 - 2'-脱氧尿苷（FdU）对中国仓鼠卵巢K1细胞中新生DNA结构形成和持久性的影响。外源性胸苷（dT）可挽救FdU诱导的细胞毒性，但对FU诱导的细胞毒性影响很小，分别表明了DNA和RNA导向的细胞毒性机制。用[³H]dT对药物处理的细胞进行脉冲标记，并在连续的pH值增量（11.0、11.3、11.5和12.1）下估计从滤膜上洗脱和保留的总计数百分比。对照细胞1分钟的脉冲导致>75%的DNA在pH 12.1时洗脱或被滤膜保留。到1小时时，>90%的DNA被滤膜保留。FdU处理导致DNA在所有四个pH带中持续存在。在5分钟脉冲期间合成的总DNA中，超过95%在每个pH值下以离散峰的形式洗脱。在1小时的追踪过程中，这种DNA被加工成更高分子量的物种，但在24小时的追踪后，68%的DNA仍在pH 12.1时洗脱。相比之下，FU处理仅在pH 11.0和11.3带中导致DNA短暂积累（≤10分钟）（例如5分钟时约为26%），表明FU对极短尺寸类别的DNA成熟具有选择性作用。使用中性洗脱来评估FdU对nDNA中双链断裂（dsb）形成的影响。在未处理的细胞中未发现明显的dsb，而即使在最短的[³H]dT脉冲时间（1 - 10分钟）后，FdU处理的细胞中也出现了dsb。尽管随着脉冲时间延长这些dsb被加工成更高分子量的物种，但在2小时时dsb仍然明显。
