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A novel approach for organelle-specific DNA damage targeting reveals different susceptibility of mitochondrial DNA to the anticancer drugs camptothecin and topotecan.

de la Loza MC, Wellinger RE - Nucleic Acids Res. (2009)

Bottom Line: In wild-type cells, toxic topoisomerase I-DNA intermediates are formed as a consequence of topoisomerase I interaction with camptothecin-based anticancer drugs.We reasoned that targeting of topoisomerase I to the mitochondria of top1 Delta cells should lead to petite formation in the presence of camptothecin.Interestingly, camptothecin failed to generate petite; however, its derivative topotecan accumulates in mitochondria and induces petite formation.

View Article: PubMed Central - PubMed

Affiliation: Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla - CSIC, Avda, Américo Vespucio s/n, 41092, Sevilla, Spain.

ABSTRACT
DNA is susceptible of being damaged by chemicals, UV light or gamma irradiation. Nuclear DNA damage invokes both a checkpoint and a repair response. By contrast, little is known about the cellular response to mitochondrial DNA damage. We designed an experimental system that allows organelle-specific DNA damage targeting in Saccharomyces cerevisiae. DNA damage is mediated by a toxic topoisomerase I allele which leads to the formation of persistent DNA single-strand breaks. We show that organelle-specific targeting of a toxic topoisomerase I to either the nucleus or mitochondria leads to nuclear DNA damage and cell death or to loss of mitochondrial DNA and formation of respiration-deficient 'petite' cells, respectively. In wild-type cells, toxic topoisomerase I-DNA intermediates are formed as a consequence of topoisomerase I interaction with camptothecin-based anticancer drugs. We reasoned that targeting of topoisomerase I to the mitochondria of top1 Delta cells should lead to petite formation in the presence of camptothecin. Interestingly, camptothecin failed to generate petite; however, its derivative topotecan accumulates in mitochondria and induces petite formation. Our findings demonstrate that drug modifications can lead to organelle-specific DNA damage and thus opens new perspectives on the role of mitochondrial DNA-damage in cancer treatment.

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CPT has no effect on mitochondrially targeted mt125Top1 protein. (A) Drop test analysis of cell viability in cells expressing 125TOP1 constructs in the presence of CPT. (B) Western analysis of n125Top1 and mt125Top1 purified from yeast. Protein size was determined using an anti-GFP antibody and HRP substrate (top left) and inferred from a coloration of the membrane upon incubation with the antibody (top right, arrows). Coomassie blue staining shows a significant enrichment for 125Top1 proteins (bottom, arrow). (C) Biochemical characterization of 125Top1p activity by in vitro DNA relaxation assays. Arrows indicate supercoiled (SC), relaxed (RL), linear (L) and open circular (OC) forms of plasmid analyzed in the presence (lanes 1 to 3) or absence (lanes 4 to 10) of EtBr. Both n125Top1 and mt125Top1 led to the formation of topoisomers (lanes 2 and 3) and to open circular DNA in the presence of CPT (lanes 7 and 9).
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Figure 5: CPT has no effect on mitochondrially targeted mt125Top1 protein. (A) Drop test analysis of cell viability in cells expressing 125TOP1 constructs in the presence of CPT. (B) Western analysis of n125Top1 and mt125Top1 purified from yeast. Protein size was determined using an anti-GFP antibody and HRP substrate (top left) and inferred from a coloration of the membrane upon incubation with the antibody (top right, arrows). Coomassie blue staining shows a significant enrichment for 125Top1 proteins (bottom, arrow). (C) Biochemical characterization of 125Top1p activity by in vitro DNA relaxation assays. Arrows indicate supercoiled (SC), relaxed (RL), linear (L) and open circular (OC) forms of plasmid analyzed in the presence (lanes 1 to 3) or absence (lanes 4 to 10) of EtBr. Both n125Top1 and mt125Top1 led to the formation of topoisomers (lanes 2 and 3) and to open circular DNA in the presence of CPT (lanes 7 and 9).

Mentions: As outlined before, another approach to induce persistent SSBs takes advantage of the anticancer drug CPT. To test the effect of CPT on organelle-specific Top1 targeting, MET25-promoter-driven constructs were used. Drop test assays revealed that upon targeting of n125Top1 to the nucleus, cellular growth was completely inhibited in the presence of CPT (Figure 5A). Interestingly, targeting of mt125Top1 to the mitochondria neither led to the appearance of white colonies nor impeded cellular growth in nonfermentable medium. This result can be interpreted in two ways: the mitochondrial mt125Top1 is not functional, or that CPT cannot interact with the mt125Top1 protein. In order to test for these possibilities, the mt125Top1 and n125Top1 proteins were purified from top1Δ yeast cells. The proteins were enriched in a two-step protocol by selective ammonium-sulfate precipitation and by FPLC using a Mono S column as described previously [(27), for detailed description see ‘Materials and Methods’ section)]. Western blot analysis using an anti-GFP antibody led to a strong fluorescent signal accompanied by a coloration of the membrane close to 114.4 kDa (Figure 5B top, arrows), which approximately corresponds to the expected molecular weight of the protein chimeras (expected molecular weight: n125Top1 = 104.98 kDa, mt125Top1 =103.66 kDa). Coomassie-staining of a protein gel run in parallel revealed enrichment of a specific band (bottom, arrow) migrating at about the same size, as well as the presence of a few contaminant proteins. From these results we conclude that the two-step purification protocol led to highly enriched Top1 proteins. In order to test for CPT sensitivity of the nuclear and mitochondrial Top1 proteins, a plasmid relaxation assay was carried out and the products were analyzed in the presence and absence of EtBr (36) (Figure 5C). Incubation of supercoiled plasmid DNA with nuclear and mitochondrial Top1 led to the formation of relaxed topoisomers as visualized in an agarose gel without EtBr (lanes 2 and 3). Importantly, electrophoresis of DNA treated with nuclear and mitochondrial Top1 proteins in the presence of EtBr shows an increase of relaxed plasmid (RL, lanes 6 and 8). In the presence of CPT in the reaction, a fraction of the relaxed plasmid was converted into nicked, open circular plasmid DNA (OC, lanes 7 and 9). Thus, the nuclear n125Top1 protein is sensitive to CPT in vivo and in vitro, but the mitochondrial mt125Top1 protein is only sensitive to CPT in vitro.Figure 5.


A novel approach for organelle-specific DNA damage targeting reveals different susceptibility of mitochondrial DNA to the anticancer drugs camptothecin and topotecan.

de la Loza MC, Wellinger RE - Nucleic Acids Res. (2009)

CPT has no effect on mitochondrially targeted mt125Top1 protein. (A) Drop test analysis of cell viability in cells expressing 125TOP1 constructs in the presence of CPT. (B) Western analysis of n125Top1 and mt125Top1 purified from yeast. Protein size was determined using an anti-GFP antibody and HRP substrate (top left) and inferred from a coloration of the membrane upon incubation with the antibody (top right, arrows). Coomassie blue staining shows a significant enrichment for 125Top1 proteins (bottom, arrow). (C) Biochemical characterization of 125Top1p activity by in vitro DNA relaxation assays. Arrows indicate supercoiled (SC), relaxed (RL), linear (L) and open circular (OC) forms of plasmid analyzed in the presence (lanes 1 to 3) or absence (lanes 4 to 10) of EtBr. Both n125Top1 and mt125Top1 led to the formation of topoisomers (lanes 2 and 3) and to open circular DNA in the presence of CPT (lanes 7 and 9).
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Figure 5: CPT has no effect on mitochondrially targeted mt125Top1 protein. (A) Drop test analysis of cell viability in cells expressing 125TOP1 constructs in the presence of CPT. (B) Western analysis of n125Top1 and mt125Top1 purified from yeast. Protein size was determined using an anti-GFP antibody and HRP substrate (top left) and inferred from a coloration of the membrane upon incubation with the antibody (top right, arrows). Coomassie blue staining shows a significant enrichment for 125Top1 proteins (bottom, arrow). (C) Biochemical characterization of 125Top1p activity by in vitro DNA relaxation assays. Arrows indicate supercoiled (SC), relaxed (RL), linear (L) and open circular (OC) forms of plasmid analyzed in the presence (lanes 1 to 3) or absence (lanes 4 to 10) of EtBr. Both n125Top1 and mt125Top1 led to the formation of topoisomers (lanes 2 and 3) and to open circular DNA in the presence of CPT (lanes 7 and 9).
Mentions: As outlined before, another approach to induce persistent SSBs takes advantage of the anticancer drug CPT. To test the effect of CPT on organelle-specific Top1 targeting, MET25-promoter-driven constructs were used. Drop test assays revealed that upon targeting of n125Top1 to the nucleus, cellular growth was completely inhibited in the presence of CPT (Figure 5A). Interestingly, targeting of mt125Top1 to the mitochondria neither led to the appearance of white colonies nor impeded cellular growth in nonfermentable medium. This result can be interpreted in two ways: the mitochondrial mt125Top1 is not functional, or that CPT cannot interact with the mt125Top1 protein. In order to test for these possibilities, the mt125Top1 and n125Top1 proteins were purified from top1Δ yeast cells. The proteins were enriched in a two-step protocol by selective ammonium-sulfate precipitation and by FPLC using a Mono S column as described previously [(27), for detailed description see ‘Materials and Methods’ section)]. Western blot analysis using an anti-GFP antibody led to a strong fluorescent signal accompanied by a coloration of the membrane close to 114.4 kDa (Figure 5B top, arrows), which approximately corresponds to the expected molecular weight of the protein chimeras (expected molecular weight: n125Top1 = 104.98 kDa, mt125Top1 =103.66 kDa). Coomassie-staining of a protein gel run in parallel revealed enrichment of a specific band (bottom, arrow) migrating at about the same size, as well as the presence of a few contaminant proteins. From these results we conclude that the two-step purification protocol led to highly enriched Top1 proteins. In order to test for CPT sensitivity of the nuclear and mitochondrial Top1 proteins, a plasmid relaxation assay was carried out and the products were analyzed in the presence and absence of EtBr (36) (Figure 5C). Incubation of supercoiled plasmid DNA with nuclear and mitochondrial Top1 led to the formation of relaxed topoisomers as visualized in an agarose gel without EtBr (lanes 2 and 3). Importantly, electrophoresis of DNA treated with nuclear and mitochondrial Top1 proteins in the presence of EtBr shows an increase of relaxed plasmid (RL, lanes 6 and 8). In the presence of CPT in the reaction, a fraction of the relaxed plasmid was converted into nicked, open circular plasmid DNA (OC, lanes 7 and 9). Thus, the nuclear n125Top1 protein is sensitive to CPT in vivo and in vitro, but the mitochondrial mt125Top1 protein is only sensitive to CPT in vitro.Figure 5.

Bottom Line: In wild-type cells, toxic topoisomerase I-DNA intermediates are formed as a consequence of topoisomerase I interaction with camptothecin-based anticancer drugs.We reasoned that targeting of topoisomerase I to the mitochondria of top1 Delta cells should lead to petite formation in the presence of camptothecin.Interestingly, camptothecin failed to generate petite; however, its derivative topotecan accumulates in mitochondria and induces petite formation.

View Article: PubMed Central - PubMed

Affiliation: Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla - CSIC, Avda, Américo Vespucio s/n, 41092, Sevilla, Spain.

ABSTRACT
DNA is susceptible of being damaged by chemicals, UV light or gamma irradiation. Nuclear DNA damage invokes both a checkpoint and a repair response. By contrast, little is known about the cellular response to mitochondrial DNA damage. We designed an experimental system that allows organelle-specific DNA damage targeting in Saccharomyces cerevisiae. DNA damage is mediated by a toxic topoisomerase I allele which leads to the formation of persistent DNA single-strand breaks. We show that organelle-specific targeting of a toxic topoisomerase I to either the nucleus or mitochondria leads to nuclear DNA damage and cell death or to loss of mitochondrial DNA and formation of respiration-deficient 'petite' cells, respectively. In wild-type cells, toxic topoisomerase I-DNA intermediates are formed as a consequence of topoisomerase I interaction with camptothecin-based anticancer drugs. We reasoned that targeting of topoisomerase I to the mitochondria of top1 Delta cells should lead to petite formation in the presence of camptothecin. Interestingly, camptothecin failed to generate petite; however, its derivative topotecan accumulates in mitochondria and induces petite formation. Our findings demonstrate that drug modifications can lead to organelle-specific DNA damage and thus opens new perspectives on the role of mitochondrial DNA-damage in cancer treatment.

Show MeSH
Related in: MedlinePlus