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A comprehensive survey of the mutagenic impact of common cancer cytotoxics.

Szikriszt B, Póti Á, Pipek O, Krzystanek M, Kanu N, Molnár J, Ribli D, Szeltner Z, Tusnády GE, Csabai I, Szallasi Z, Swanton C, Szüts D - Genome Biol. (2016)

Bottom Line: Gemcitabine, 5-fluorouracil, hydroxyurea, doxorubicin and paclitaxel have no measurable mutagenic effect.This 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 1117, Budapest, Hungary.

ABSTRACT

Background: 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. 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.

Results: We 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.

Conclusion: This 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.

No MeSH data available.


Related in: MedlinePlus

Number and spectrum of treatment-induced SNVs. a The mean number of observed SNVs per genome following the described treatment regimen with the indicated drugs. Error bars indicate SEM. b Base substitution spectrum of mutations that arose from the mock treatment, as well as cisplatin and cyclophosphamide treatments. c The mean number of mutations per sample and base substitution spectrum of the indicated treatments. Significant differences from the mock treatment (p <0.05, Student’s t-test) are indicated with an asterisk. d Triplet mutation spectra of the mock, cisplatin and cyclophosphamide treatments. The middle base of each triplet, listed at the bottom, mutated as indicated at the top of the panel. The number of mutations of each type was normalised to the frequency of occurrence of that base triplet in the chicken genome, and the resulting mutation rates are shown
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Fig2: Number and spectrum of treatment-induced SNVs. a The mean number of observed SNVs per genome following the described treatment regimen with the indicated drugs. Error bars indicate SEM. b Base substitution spectrum of mutations that arose from the mock treatment, as well as cisplatin and cyclophosphamide treatments. c The mean number of mutations per sample and base substitution spectrum of the indicated treatments. Significant differences from the mock treatment (p <0.05, Student’s t-test) are indicated with an asterisk. d Triplet mutation spectra of the mock, cisplatin and cyclophosphamide treatments. The middle base of each triplet, listed at the bottom, mutated as indicated at the top of the panel. The number of mutations of each type was normalised to the frequency of occurrence of that base triplet in the chicken genome, and the resulting mutation rates are shown

Mentions: Following a mock treatment regimen spanning approximately 100 cell generations, we detected 47 ± 20 (SD) novel SNVs in three post-treatment clones (Table 2, Additional file 1: Table S1). It is likely that almost all the identified mutations truly arose during the mock treatment, as these were identified as unique mutations among all the whole genome sequences obtained for this study, and the same mutation detection method found no unique SNVs – which would be false positives – in the pre-treatment starting clone (Table 2). Of the six possible base substitutions (C > A, C > G, C > T, T > A, T > C, T > G), C > T transitions and C > A transversions were the most common among the spontaneous mutations (Fig. 2c, d). The observed mutation number, projected to the 2.06 × 109 base pair diploid genome is equivalent to about 2.3 × 10–10 mutations per base per cell division. When mutations are viewed in the context of the neighbouring bases, and the spontaneous ‘triplet mutation spectrum’ is normalised to the frequency of genomic occurrence of each triplet, it becomes apparent that NCG > NTG mutations are most common, presumably due to C > T base substitutions at methylated CpG sequences [35]. We calculated that NCG > NTG mutations were 15× more common than the mean mutation rate. Non-normalised triplet spectra are shown in Additional file 2: Figure S1.Table 2


A comprehensive survey of the mutagenic impact of common cancer cytotoxics.

Szikriszt B, Póti Á, Pipek O, Krzystanek M, Kanu N, Molnár J, Ribli D, Szeltner Z, Tusnády GE, Csabai I, Szallasi Z, Swanton C, Szüts D - Genome Biol. (2016)

Number and spectrum of treatment-induced SNVs. a The mean number of observed SNVs per genome following the described treatment regimen with the indicated drugs. Error bars indicate SEM. b Base substitution spectrum of mutations that arose from the mock treatment, as well as cisplatin and cyclophosphamide treatments. c The mean number of mutations per sample and base substitution spectrum of the indicated treatments. Significant differences from the mock treatment (p <0.05, Student’s t-test) are indicated with an asterisk. d Triplet mutation spectra of the mock, cisplatin and cyclophosphamide treatments. The middle base of each triplet, listed at the bottom, mutated as indicated at the top of the panel. The number of mutations of each type was normalised to the frequency of occurrence of that base triplet in the chicken genome, and the resulting mutation rates are shown
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4862131&req=5

Fig2: Number and spectrum of treatment-induced SNVs. a The mean number of observed SNVs per genome following the described treatment regimen with the indicated drugs. Error bars indicate SEM. b Base substitution spectrum of mutations that arose from the mock treatment, as well as cisplatin and cyclophosphamide treatments. c The mean number of mutations per sample and base substitution spectrum of the indicated treatments. Significant differences from the mock treatment (p <0.05, Student’s t-test) are indicated with an asterisk. d Triplet mutation spectra of the mock, cisplatin and cyclophosphamide treatments. The middle base of each triplet, listed at the bottom, mutated as indicated at the top of the panel. The number of mutations of each type was normalised to the frequency of occurrence of that base triplet in the chicken genome, and the resulting mutation rates are shown
Mentions: Following a mock treatment regimen spanning approximately 100 cell generations, we detected 47 ± 20 (SD) novel SNVs in three post-treatment clones (Table 2, Additional file 1: Table S1). It is likely that almost all the identified mutations truly arose during the mock treatment, as these were identified as unique mutations among all the whole genome sequences obtained for this study, and the same mutation detection method found no unique SNVs – which would be false positives – in the pre-treatment starting clone (Table 2). Of the six possible base substitutions (C > A, C > G, C > T, T > A, T > C, T > G), C > T transitions and C > A transversions were the most common among the spontaneous mutations (Fig. 2c, d). The observed mutation number, projected to the 2.06 × 109 base pair diploid genome is equivalent to about 2.3 × 10–10 mutations per base per cell division. When mutations are viewed in the context of the neighbouring bases, and the spontaneous ‘triplet mutation spectrum’ is normalised to the frequency of genomic occurrence of each triplet, it becomes apparent that NCG > NTG mutations are most common, presumably due to C > T base substitutions at methylated CpG sequences [35]. We calculated that NCG > NTG mutations were 15× more common than the mean mutation rate. Non-normalised triplet spectra are shown in Additional file 2: Figure S1.Table 2

Bottom Line: Gemcitabine, 5-fluorouracil, hydroxyurea, doxorubicin and paclitaxel have no measurable mutagenic effect.This 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, 1117, Budapest, Hungary.

ABSTRACT

Background: 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. 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.

Results: We 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.

Conclusion: This 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.

No MeSH data available.


Related in: MedlinePlus