Slebos Robbert J C, Oh Daniel S, Umbach David M, Taylor Jack A
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709, USA.
Cancer Res. 2002 Nov 1;62(21):6052-60.
Sporadic microsatellite mutations are frequently observed in lung, bladder, and head and neck tumors with intact DNA mismatch repair. AAAG tetranucleotide repeats appear to be especially prone to the accumulation of these mutations. We hypothesized that occurrences of microsatellite mutations in these cancers may be linked to DNA damage caused by exposure to carcinogens in tobacco smoke. To test this hypothesis, we developed a model system based on reactivation of green fluorescent protein (GFP) in which a plasmid vector carries a microsatellite repeat that places the GFP sequence out of frame for protein translation. In this reporter system, DNA slippage mutations can restore the GFP reading frame and become detectable by flow cytometry as GFP-positive cells. Pools of stably transfected RKO cells were treated at four dose levels each of gamma-irradiation, benzo(a)pyrene diol epoxide, N-methyl-N-nitro-N-nitrosoguanidine (MNNG), t-butyl hydrogen peroxide, and UV irradiation and assayed for GFP-positive cells 48 h later. We studied the microsatellite repeats AAAG, ATAG, CAGT, and CA, as well as a control sequence lacking any repetitive elements. A log-linear regression approach was used to discriminate between the effects of repeat unit and dose for each agent. A statistically significant increase in GFP-positive cells was found with increasing dose with all agents, although repeat unit-specific response patterns were only observed with MNNG, t-butyl hydrogen peroxide, and UV irradiation. With MNNG, significant differences in response were observed between dinucleotide and tetranucleotide repeat units. The effects of UV irradiation were consistent with the predicted number of pyrimidine dimers/repeat unit, with higher GFP activation in repeats that had large numbers of adjacent pyrimidines. We found no evidence to indicate that the AAAG repeat responded to any of the DNA-damaging agents with higher levels of GFP activation than other repeat units. These results provide evidence that DNA damage can induce slippage mutations and increase mutation rates in repeated sequences and that there are sequence-specific responses to different types of DNA damage. Our results are compatible with the hypothesis that sporadic microsatellite mutations in human cancer may reflect DNA damage caused by carcinogen exposure.
在DNA错配修复功能完整的肺癌、膀胱癌和头颈癌中，经常观察到散发性微卫星突变。AAAG四核苷酸重复序列似乎特别容易积累这些突变。我们推测，这些癌症中微卫星突变的发生可能与接触烟草烟雾中的致癌物所导致的DNA损伤有关。为了验证这一假设，我们开发了一种基于绿色荧光蛋白（GFP）再激活的模型系统，其中一个质粒载体携带一个微卫星重复序列，该序列使GFP序列在蛋白质翻译时移码。在这个报告系统中，DNA滑动突变可以恢复GFP阅读框，并通过流式细胞术检测为GFP阳性细胞。将稳定转染的RKO细胞池分别用γ射线、苯并[a]芘二醇环氧化物、N-甲基-N-硝基-N-亚硝基胍（MNNG）、叔丁基过氧化氢和紫外线照射的四个剂量水平进行处理，并在48小时后检测GFP阳性细胞。我们研究了微卫星重复序列AAAG、ATAG、CAGT和CA，以及一个没有任何重复元件的对照序列。采用对数线性回归方法来区分每种试剂的重复单元和剂量的影响。随着所有试剂剂量的增加，GFP阳性细胞数量有统计学意义的增加，尽管仅在MNNG、叔丁基过氧化氢和紫外线照射下观察到重复单元特异性的反应模式。对于MNNG，二核苷酸和四核苷酸重复单元之间观察到显著的反应差异。紫外线照射的影响与预测的每个重复单元嘧啶二聚体数量一致，在具有大量相邻嘧啶的重复序列中GFP激活程度更高。我们没有发现证据表明AAAG重复序列对任何一种DNA损伤剂的GFP激活水平高于其他重复单元。这些结果证明DNA损伤可以诱导滑动突变并增加重复序列中的突变率，并且对不同类型的DNA损伤存在序列特异性反应。我们的结果与人类癌症中散发性微卫星突变可能反映致癌物暴露导致的DNA损伤这一假设相符。
