Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.
Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.
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DOI：
10.1371/journal.pone.0155391
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Harris RS
中科院分区：
文献类型：
--
作者：
Akre MK;Starrett GJ;Quist JS;Temiz NA;Carpenter MA;Tutt AN;Grigoriadis A;Harris RS
分子、细胞和临床研究相结合，证明了DNA胞嘧啶脱氨酶APOBEC3B(A3B)对乳腺癌、头颈部、肺癌、膀胱癌、宫颈癌、卵巢癌和其他类型癌症的总突变负荷的贡献。然而，可归因于这种酶的突变的完整图景尚未在受控的人类细胞系统中确定。我们报告了一种用于A3B诱导、基因组DNA脱氨基和突变的条件和等基因系统。人293来源的细胞被设计为表达多西环素诱导的A3B-EGFP或EGFP构建物。细胞受到10轮A3B-EGFP暴露，每轮都导致80%-90%的细胞死亡。控制池接受平行的无毒EGFP暴露，每一轮都进行稀释，以模拟A3B-EGFP引起的种群波动。对TP53和MYC部分的定向测序显示，在A3B-EGFP暴露的池中，突变积累更多。产生克隆，并使用微阵列分析来识别那些具有最多SNP改变的克隆，以进行全基因组测序。A3B-EGFP暴露的克隆显示C-T转换突变的全局性增加，A3B偏好的三核苷酸基序内胞嘧啶突变的丰富，以及更多的拷贝数异常。令人惊讶的是，对照和A3B-EGFP克隆也都引发了具有缺陷错配修复特征的强烈突变子表型。尽管有这种额外的突变过程，但这里描述的基于293的系统仍然产生了A3B催化的人类细胞突变的全基因组视图，并产生了一个用于进一步研究同时突变机制在癌细胞中的复合效应的系统。
Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B) to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80–90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.