Little J B, Nagasawa H, Pfenning T, Vetrovs H
Department of Cancer Biology, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Radiat Res. 1997 Oct;148(4):299-307.
The frequency of mutations at the Hprt locus was measured in clonal populations of Chinese hamster ovary cells derived from single cells surviving exposure to 0-12 Gy of X rays or 2 Gy of alpha particles. Approximately 8-9% of 446 clonal populations examined 23 population doublings after irradiation showed high frequencies of late-arising mutations as indicated by mutant fractions 10(2)-10(4)-fold above background. The frequency with which such clones occurred was similar for alpha-particle irradiation and X irradiation, with no apparent dose dependence for X irradiation over the range of 4-12 Gy. The molecular structure of Hprt mutations was determined by analysis by multiplex polymerase chain reaction of all nine exons. Of mutations induced directly after exposure to X rays, 75% involved partial or total gene deletions. Only 19-23% of late-arising (delayed) mutations were associated with deletions, the preponderance of these being partial deletions involving one or two exons. This spectrum was very similar to that for spontaneously arising mutations. To determine whether delayed mutations were non-clonal, the spectrum of exons deleted was examined among 29 mutants with partial deletions derived from a single clonal population. The results indicated that at least 15 of these mutants arose independently. To examine the relationship between the occurrence of delayed mutations and chromosomal instability, 60 Hprt mutant subclones isolated from a clonal population showing a high frequency of delayed mutations were serially cultivated in vitro. Of these, 14 showed a slow-growth phenotype with a high frequency of polyploid cells (10-38%) and a markedly enhanced frequency of non-clonal chromosomal rearrangements including both chromosome-type and chromatid-type aberrations. These clones also showed a 3- to 30-fold increase in the frequency of ouabain-resistant mutations; no ouabain-resistant mutants were induced directly by X irradiation. These results suggest that among clones showing a high frequency of delayed mutations there may be a subpopulation of cells that are particularly unstable; selection for the slow-growth phenotype has the effect of selecting for this chromosomally unstable subpopulation.
在源自经0 - 12 Gy X射线或2 Gy α粒子照射后存活的单细胞的中国仓鼠卵巢细胞克隆群体中，测量了次黄嘌呤 - 鸟嘌呤磷酸核糖转移酶（Hprt）位点的突变频率。在照射后23次群体倍增时检测的446个克隆群体中，约8 - 9%表现出高频率的晚期出现突变，突变率比背景高10² - 10⁴倍。α粒子照射和X射线照射产生此类克隆的频率相似，在4 - 12 Gy范围内，X射线照射没有明显的剂量依赖性。通过对所有九个外显子进行多重聚合酶链反应分析，确定了Hprt突变的分子结构。在暴露于X射线后直接诱导的突变中，75%涉及部分或全部基因缺失。晚期出现（延迟）的突变中只有19 - 23%与缺失有关，其中大多数是涉及一个或两个外显子的部分缺失。这个谱与自发产生的突变非常相似。为了确定延迟突变是否是非克隆性的，在来自单个克隆群体的29个有部分缺失的突变体中检查了缺失的外显子谱。结果表明，这些突变体中至少有15个是独立产生的。为了研究延迟突变的发生与染色体不稳定性之间的关系，从一个显示高频率延迟突变的克隆群体中分离出60个Hprt突变亚克隆，在体外进行连续培养。其中，14个表现出生长缓慢的表型，多倍体细胞频率高（10 - 38%），非克隆性染色体重排频率明显增加，包括染色体型和染色单体型畸变。这些克隆的哇巴因抗性突变频率也增加了3至30倍；X射线照射未直接诱导出哇巴因抗性突变体。这些结果表明，在显示高频率延迟突变的克隆中，可能存在一个特别不稳定的细胞亚群；选择生长缓慢的表型具有选择这个染色体不稳定亚群的作用。
