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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

SNV mutation spacing, dinucleotide mutations and proposed mechanisms of mono- and dinucleotide mutations. a The distance of each SNV mutation from the previous SNV on the same chromosome is plotted against the genomic position of the mutation. Thin dashed lines indicate chromosome boundaries. Chromosomes are shown in numerical order; chromosome Z is shown last on the right. The colour of each dot illustrates the type of mutation according to the key at the bottom of the panel. Mutations with an intermutation distance of one are part of dinucleotide mutations. One sequenced clone of each is shown. bSequence analysis of the 183 dinucleotide mutations detected following cisplatin treatment. The change in the 5’ base is shown in the rows, while the 3’ base in the columns. The equivalent mutations on the two strands are added together, e.g. GG > TT is shown as CC > AA. The most common mutation types are grouped together below the table and their sequences are indicated using the purine-rich strand to aid interpretation. cSchematic models for the replicative process that may generate each of the most common classes of cisplatin-induced mononucleotide (c) and dinucleotide (d) mutations. Putative intrastrand crosslinks are marked, the uncertain lesion at mutated GA sequences is indicated with a question mark. Non-canonical base pairing is shown with a zig-zag symbol. The contribution of each mutation class to the total number of observed SNVs is shown
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Fig3: SNV mutation spacing, dinucleotide mutations and proposed mechanisms of mono- and dinucleotide mutations. a The distance of each SNV mutation from the previous SNV on the same chromosome is plotted against the genomic position of the mutation. Thin dashed lines indicate chromosome boundaries. Chromosomes are shown in numerical order; chromosome Z is shown last on the right. The colour of each dot illustrates the type of mutation according to the key at the bottom of the panel. Mutations with an intermutation distance of one are part of dinucleotide mutations. One sequenced clone of each is shown. bSequence analysis of the 183 dinucleotide mutations detected following cisplatin treatment. The change in the 5’ base is shown in the rows, while the 3’ base in the columns. The equivalent mutations on the two strands are added together, e.g. GG > TT is shown as CC > AA. The most common mutation types are grouped together below the table and their sequences are indicated using the purine-rich strand to aid interpretation. cSchematic models for the replicative process that may generate each of the most common classes of cisplatin-induced mononucleotide (c) and dinucleotide (d) mutations. Putative intrastrand crosslinks are marked, the uncertain lesion at mutated GA sequences is indicated with a question mark. Non-canonical base pairing is shown with a zig-zag symbol. The contribution of each mutation class to the total number of observed SNVs is shown

Mentions: Cisplatin induced the greatest number of SNVs among the eight tested drugs (Fig. 2a). We performed a detailed analysis of cisplatin-induced mutations to better understand the mutagenic mechanisms. We detected 812 ± 193 SNVs per sequenced post-treatment clone. C/G > A/T transversions were most common, accounting for 57 % of all SNVs, but all six classes of base substitutions increased at least fourfold (Fig. 2b). Looking at cisplatin-induced SNVs in the context of the neighbouring bases, it is apparent that NCC > NAC mutations are most common, accounting for 40 % of all SNV cases. Further common changes are NCT > NAT and NTC > NAC, arising in 12 % and 9 % of the SNV cases (Fig. 2d, Additional file 2: Figure S1 and Figure S3 and Additional file 1: Table S2). As the overwhelming majority of cisplatin-induced DNA lesions are intrastrand crosslinks between neighbouring purines [36, 37], these three SNV types could represent mutations opposite the 3’ base of crosslinked GG, AG and GA dinucleotides, respectively. In case of GG and AG intrastrand crosslinks, these mutations arise through the incorrect incorporation of an adenosine opposite the 3’ G of the lesion (Fig. 3c). However, GA crosslinks have not been observed in the above reports. Therefore, we catalogued the bases surrounding the 211 observed TC > AC (GA > GT) mutations, and found that 159 incidences happened at TCC > ACC or TCT > ACT sequences, suggesting that the adjacent base pair 3’ to a GG or AG intrastrand crosslink can also mutate. Of the remaining 52 mutations, ten happened at the 5’ base of potential AG crosslinks at CTC > CAC sequences, but in the remaining cases the only potential site for a bipurine crosslink is at GA (Additional file 2: Figure S2). We conclude that cisplatin induces mutagenic lesions at GA dinucleotides, where the lesions may be hitherto unobserved intrastrand GA crosslinks or monoadducts. To complete the analysis of cisplatin-induced single nucleotide mutations, we note an enrichment of CCA > CAA and CTN > CAN base changes, suggesting adenosine mis-incorporation opposite the 5’ base of crosslinked GG or AG dinucleotides.Fig. 3


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)

SNV mutation spacing, dinucleotide mutations and proposed mechanisms of mono- and dinucleotide mutations. a The distance of each SNV mutation from the previous SNV on the same chromosome is plotted against the genomic position of the mutation. Thin dashed lines indicate chromosome boundaries. Chromosomes are shown in numerical order; chromosome Z is shown last on the right. The colour of each dot illustrates the type of mutation according to the key at the bottom of the panel. Mutations with an intermutation distance of one are part of dinucleotide mutations. One sequenced clone of each is shown. bSequence analysis of the 183 dinucleotide mutations detected following cisplatin treatment. The change in the 5’ base is shown in the rows, while the 3’ base in the columns. The equivalent mutations on the two strands are added together, e.g. GG > TT is shown as CC > AA. The most common mutation types are grouped together below the table and their sequences are indicated using the purine-rich strand to aid interpretation. cSchematic models for the replicative process that may generate each of the most common classes of cisplatin-induced mononucleotide (c) and dinucleotide (d) mutations. Putative intrastrand crosslinks are marked, the uncertain lesion at mutated GA sequences is indicated with a question mark. Non-canonical base pairing is shown with a zig-zag symbol. The contribution of each mutation class to the total number of observed SNVs is shown
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: SNV mutation spacing, dinucleotide mutations and proposed mechanisms of mono- and dinucleotide mutations. a The distance of each SNV mutation from the previous SNV on the same chromosome is plotted against the genomic position of the mutation. Thin dashed lines indicate chromosome boundaries. Chromosomes are shown in numerical order; chromosome Z is shown last on the right. The colour of each dot illustrates the type of mutation according to the key at the bottom of the panel. Mutations with an intermutation distance of one are part of dinucleotide mutations. One sequenced clone of each is shown. bSequence analysis of the 183 dinucleotide mutations detected following cisplatin treatment. The change in the 5’ base is shown in the rows, while the 3’ base in the columns. The equivalent mutations on the two strands are added together, e.g. GG > TT is shown as CC > AA. The most common mutation types are grouped together below the table and their sequences are indicated using the purine-rich strand to aid interpretation. cSchematic models for the replicative process that may generate each of the most common classes of cisplatin-induced mononucleotide (c) and dinucleotide (d) mutations. Putative intrastrand crosslinks are marked, the uncertain lesion at mutated GA sequences is indicated with a question mark. Non-canonical base pairing is shown with a zig-zag symbol. The contribution of each mutation class to the total number of observed SNVs is shown
Mentions: Cisplatin induced the greatest number of SNVs among the eight tested drugs (Fig. 2a). We performed a detailed analysis of cisplatin-induced mutations to better understand the mutagenic mechanisms. We detected 812 ± 193 SNVs per sequenced post-treatment clone. C/G > A/T transversions were most common, accounting for 57 % of all SNVs, but all six classes of base substitutions increased at least fourfold (Fig. 2b). Looking at cisplatin-induced SNVs in the context of the neighbouring bases, it is apparent that NCC > NAC mutations are most common, accounting for 40 % of all SNV cases. Further common changes are NCT > NAT and NTC > NAC, arising in 12 % and 9 % of the SNV cases (Fig. 2d, Additional file 2: Figure S1 and Figure S3 and Additional file 1: Table S2). As the overwhelming majority of cisplatin-induced DNA lesions are intrastrand crosslinks between neighbouring purines [36, 37], these three SNV types could represent mutations opposite the 3’ base of crosslinked GG, AG and GA dinucleotides, respectively. In case of GG and AG intrastrand crosslinks, these mutations arise through the incorrect incorporation of an adenosine opposite the 3’ G of the lesion (Fig. 3c). However, GA crosslinks have not been observed in the above reports. Therefore, we catalogued the bases surrounding the 211 observed TC > AC (GA > GT) mutations, and found that 159 incidences happened at TCC > ACC or TCT > ACT sequences, suggesting that the adjacent base pair 3’ to a GG or AG intrastrand crosslink can also mutate. Of the remaining 52 mutations, ten happened at the 5’ base of potential AG crosslinks at CTC > CAC sequences, but in the remaining cases the only potential site for a bipurine crosslink is at GA (Additional file 2: Figure S2). We conclude that cisplatin induces mutagenic lesions at GA dinucleotides, where the lesions may be hitherto unobserved intrastrand GA crosslinks or monoadducts. To complete the analysis of cisplatin-induced single nucleotide mutations, we note an enrichment of CCA > CAA and CTN > CAN base changes, suggesting adenosine mis-incorporation opposite the 5’ base of crosslinked GG or AG dinucleotides.Fig. 3

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