title: Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.
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.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, Institute for Molecular Virology, Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States of America.
authors.forename: Monica K
authors.identifier: 0000-0002-1050-7417
authors.initials: MK
authors.lastname: Akre
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, Institute for Molecular Virology, Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States of America.
authors.forename: Gabriel J
authors.identifier: 0000-0001-5871-5306
authors.initials: GJ
authors.lastname: Starrett
authors.affiliation: Breast Cancer Now Research Unit, Research Oncology, Guy's Hospital, King's College London, London, United Kingdom.
authors.forename: Jelmar S
authors.initials: JS
authors.lastname: Quist
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, Institute for Molecular Virology, Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States of America.
authors.forename: Nuri A
authors.initials: NA
authors.lastname: Temiz
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, Institute for Molecular Virology, Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States of America.
authors.forename: Michael A
authors.identifier: 0000-0001-9524-5323
authors.initials: MA
authors.lastname: Carpenter
authors.affiliation: Breast Cancer Now Research Unit, Research Oncology, Guy's Hospital, King's College London, London, United Kingdom.
authors.forename: Andrew N J
authors.initials: AN
authors.lastname: Tutt
authors.affiliation: Breast Cancer Now Research Unit, Research Oncology, Guy's Hospital, King's College London, London, United Kingdom.
authors.forename: Anita
authors.initials: A
authors.lastname: Grigoriadis
authors.affiliation: Department of Biochemistry, Molecular Biology, and Biophysics, Institute for Molecular Virology, Masonic Cancer Center, University of Minnesota, Minneapolis, MN, United States of America.|Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN, United States of America.
authors.forename: Reuben S
authors.initials: RS
authors.lastname: Harris
mesh_terms: D001483:Base Sequence; D016923:Cell Death / Q000235:genetics; D060846:Cell Engineering / Q000379:methods*; D002460:Cell Line; D002999:Clone Cells; D003564:Cytidine Deaminase / Q000235:genetics* / Q000378:metabolism; D003596:Cytosine / Q000378:metabolism; D053843:DNA Mismatch Repair; D004318:Doxycycline / Q000494:pharmacology; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015778:Minor Histocompatibility Antigens / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D013941:Thymine / Q000378:metabolism; D046888:Tissue Array Analysis; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: C489427:MYC protein, human; D015778:Minor Histocompatibility Antigens; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003596:Cytosine; C495794:APOBEC3B protein, human; D003564:Cytidine Deaminase; D004318:Doxycycline; D013941:Thymine