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PLoS One. 2016 May 10;11(5):e0155391. doi: 10.1371/journal.pone.0155391. eCollection 2016.

Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

Author information

1
Department of Biochemistry, Molecular Biology, and Biophysics, Institute for Molecular Virology, Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States of America.
2
Breast Cancer Now Research Unit, Research Oncology, Guy's Hospital, King's College London, London, United Kingdom.
3
Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN, United States of America.

Abstract

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.

PMID:
27163364
PMCID:
PMC4862684
DOI:
10.1371/journal.pone.0155391
[Indexed for MEDLINE]
Free PMC Article

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