Abstract
BackgroundGenomic mutations caused by cytotoxic agents used in cancer chemotherapy may cause secondary malignancies as well as contribute to the evolution of treatment-resistant tumour cells. The stable diploid genome of the chicken DT40 lymphoblast cell line, an established DNA repair model system, is well suited to accurately assay genomic mutations.ResultsWe use whole genome sequencing of multiple DT40 clones to determine the mutagenic effect of eight common cytotoxics used for the treatment of millions of patients worldwide. We determine the spontaneous mutagenesis rate at 2.3 × 10–10 per base per cell division and find that cisplatin, cyclophosphamide and etoposide induce extra base substitutions with distinct spectra. After four cycles of exposure, cisplatin induces 0.8 mutations per Mb, equivalent to the median mutational burden in common leukaemias. Cisplatin-induced mutations, including short insertions and deletions, are mainly located at sites of putative intrastrand crosslinks. We find two of the newly defined cisplatin-specific mutation types as causes of the reversion of BRCA2 mutations in emerging cisplatin-resistant tumours or cell clones. Gemcitabine, 5-fluorouracil, hydroxyurea, doxorubicin and paclitaxel have no measurable mutagenic effect. The cisplatin-induced mutation spectrum shows good correlation with cancer mutation signatures attributed to smoking and other sources of guanine-directed base damage.ConclusionThis study provides support for the use of cell line mutagenesis assays to validate or predict the mutagenic effect of environmental and iatrogenic exposures. Our results suggest genetic reversion due to cisplatin-induced mutations as a distinct mechanism for developing resistance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0963-7) contains supplementary material, which is available to authorized users.
Highlights
Genomic mutations caused by cytotoxic agents used in cancer chemotherapy may cause secondary malignancies as well as contribute to the evolution of treatment-resistant tumour cells
In vitro use of eight chemotherapeutic agents Isogenic wild-type DT40 cells derived from a single cell clone were treated with eight different commonly used cytotoxic agents representing each of the main classes of cancer chemotherapeutics
Cyclophosphamide-induced mutations show only weak correlation with the rare signature 25 of unknown aetiology (Fig. 6a). These results demonstrate that while mutagenesis analysis in cell lines can model the mutational processes observed in cancer, as evidenced by exome sequencing of mutagen-treated mouse and human cells [26,27,28] and whole genome sequencing of individual mouse embryonic fibroblast clones [25], it is unlikely that mutations induced by cisplatin or cyclophosphamide treatment significantly contributed to COSMIC signatures
Summary
Genomic mutations caused by cytotoxic agents used in cancer chemotherapy may cause secondary malignancies as well as contribute to the evolution of treatment-resistant tumour cells. Cytotoxic drugs have been in use for cancer therapy since the 1950s, and remain the first line treatment for most cancers today These drugs inhibit cell proliferation through a range of different mechanisms, including directly damaging DNA, interfering with DNA metabolism and interfering with the mitotic machinery. Szikriszt et al Genome Biology (2016) 17:99 important currently approved tests are the comet assay for detecting DNA breaks, the chromosome aberration assay and the micronucleus formation test [2]. These assays give indirect and imprecise predictions of carcinogenic potential [3], as a finding of genotoxicity only reveals that a compound has potential to cause genomic mutations, without measuring the outcome in a surviving cell. The comprehensive detection of all genomic changes of all types only became available with affordable whole genome sequencing
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