Parfett Craig L, Healy Caroline
Mutagenesis Section, Healthy Environments and Consumer Safety Branch, Health Canada, Environmental Health Centre 0803A, Tunney's Pasture, Ottawa, Ont., Canada K1A 0L2.
Mutat Res. 2006 Apr 30;604(1-2):42-52. doi: 10.1016/j.mrgentox.2005.12.008. Epub 2006 Feb 3.
The ability of tumour promoters to alter DNA stability within regions that contain tandemly repeated sequences (TRSs), was studied in a cell culture model of multi-stage carcinogenesis. Non-cytotoxic concentrations of TPA (12-O-tetradecanoyl-phorbol-13-acetate) and xanthine oxidase with xanthine substrate, sufficient to promote morphological transformation in C3H/10T1/2 cultures, were tested for their effects on mutation frequencies in TRSs by a DNA fingerprinting approach. Specifically, restriction digests of genomic DNA samples from randomly selected, non-transformed clones, isolated from cultures after several days exposure to promoters, were visualized by Southern hybridizations with the multi-locus pentamer repeat sequence probe, Ms6-Hm (Pc-1). Basal and promoter-induced frequencies of sub-clone TRS fingerprint polymorphisms were estimated in five cell populations: an uncloned stock culture, three populations established from normal-appearing sub-clones, and one clonal population established from a 3-methylcholanthrene (MCA)-transformed focus. Basal variant fingerprint frequencies spanned a range from 0.0 to 0.43% mutants/band among cells from the four untransformed populations. Both TPA and xanthine oxidase treatments significantly increased recorded mutation frequencies, 2.3- and 2.7-fold, respectively, using combined data from the progenitor population and three untransformed clones. The untreated MCA-transformed clonal population appeared to contain a single, pre-existing mutant restriction fragment, but additional mutations were induced thereafter, in response to the promoting treatments. Taken together, the measured increases in mutations were highly significant and suggest that promoters of cell transformation in the C3H/10T1/2 cell line might induce a genome-wide instability targeted to regions containing Ms6-Hm sequence motifs.
在多阶段致癌作用的细胞培养模型中，研究了肿瘤促进剂改变含有串联重复序列（TRS）区域内DNA稳定性的能力。用DNA指纹图谱方法测试了非细胞毒性浓度的佛波酯（12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯）和黄嘌呤氧化酶与黄嘌呤底物，它们足以促进C3H/10T1/2培养物中的形态转化，测试其对TRS中突变频率的影响。具体而言，从暴露于促进剂数天后的培养物中分离出的随机选择的未转化克隆的基因组DNA样品的限制性消化物，通过与多位点五聚体重复序列探针Ms6 - Hm（Pc - 1）的Southern杂交进行可视化。在五个细胞群体中估计了亚克隆TRS指纹多态性的基础频率和促进剂诱导频率：一个未克隆的原代培养物、三个由外观正常的亚克隆建立的群体以及一个由3 - 甲基胆蒽（MCA）转化灶建立的克隆群体。在四个未转化群体的细胞中，基础变异指纹频率范围为0.0至0.43%突变体/条带。使用来自祖代群体和三个未转化克隆的合并数据，佛波酯和黄嘌呤氧化酶处理均显著增加了记录的突变频率，分别增加了2.3倍和2.7倍。未经处理的MCA转化克隆群体似乎含有一个预先存在的单一突变限制性片段，但此后响应促进处理诱导了额外的突变。总体而言，测得的突变增加非常显著，表明C3H/10T1/2细胞系中的细胞转化促进剂可能诱导针对含有Ms6 - Hm序列基序区域的全基因组不稳定性。
