Limits...
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.

Show MeSH

Related in: MedlinePlus

Topotecan is capable of inducing petite and accumulates in mitochondria. (A) Drop test assay to test the effect of TPT treatment on yeast growth. The indicated constructs were expressed under control of the GAL1 promoter and cells were grown in plates without or with 20 µg/ml TPT. Note that petite induced in galactose by expression of mt125Top1-103 under control of the GAL1p grew slower than petite induced in galactose by expression of mt125Top1-103 under control of the MET25p (Figure 3). (B) Intracellular localization of CPT and TPT. Using the settings for the detection of DAPI, the intracellular TPT and CPT localization was determined by their auto-fluorescence. Construct pRWE129 was expressed for GFP-localization of the nucleus. CPT is mainly enriched in vacuoles while TPT fluorescence and Mitotracker staining co-localized (white arrow). Cells without Mitotracker straining were used as control to discard the possibility that we detect TPT auto-fluorescence applying the Mitotracker settings. White bar represents 5 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2651790&req=5

Figure 6: Topotecan is capable of inducing petite and accumulates in mitochondria. (A) Drop test assay to test the effect of TPT treatment on yeast growth. The indicated constructs were expressed under control of the GAL1 promoter and cells were grown in plates without or with 20 µg/ml TPT. Note that petite induced in galactose by expression of mt125Top1-103 under control of the GAL1p grew slower than petite induced in galactose by expression of mt125Top1-103 under control of the MET25p (Figure 3). (B) Intracellular localization of CPT and TPT. Using the settings for the detection of DAPI, the intracellular TPT and CPT localization was determined by their auto-fluorescence. Construct pRWE129 was expressed for GFP-localization of the nucleus. CPT is mainly enriched in vacuoles while TPT fluorescence and Mitotracker staining co-localized (white arrow). Cells without Mitotracker straining were used as control to discard the possibility that we detect TPT auto-fluorescence applying the Mitotracker settings. White bar represents 5 µm.

Mentions: While yeast cells are sensitive to CPT treatment, it has been shown that CPT analogs which are soluble in aqueous solutions are much less toxic to yeast cells (37). One such analog is the anticancer drug TPT that induces apoptosis of mammalian tumor cells (38). From microscopic studies it has been inferred that TPT is enriched in mitochondria of mammalian cells (39). We reasoned that the water solubility and positive charge of the TPT molecule (15) could also allow the drug to penetrate mitochondria of S. cerevisiae. Indeed, expression of mt125Top1 in the presence of TPT led to the formation of petite cells (Figure 6A) indicating that TPT reaches the mitochondria in an active form. We determined petite formation by replica-plating of single colonies grown on galactose onto glycerol-containing medium. By counting the colonies growing on glycerol, we found that expression of mt125Top1 in the presence of TPT led to induction of more than 95% petite thus being as potent in generating petite as expression of mt125Top1-103.Figure 6.


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)

Topotecan is capable of inducing petite and accumulates in mitochondria. (A) Drop test assay to test the effect of TPT treatment on yeast growth. The indicated constructs were expressed under control of the GAL1 promoter and cells were grown in plates without or with 20 µg/ml TPT. Note that petite induced in galactose by expression of mt125Top1-103 under control of the GAL1p grew slower than petite induced in galactose by expression of mt125Top1-103 under control of the MET25p (Figure 3). (B) Intracellular localization of CPT and TPT. Using the settings for the detection of DAPI, the intracellular TPT and CPT localization was determined by their auto-fluorescence. Construct pRWE129 was expressed for GFP-localization of the nucleus. CPT is mainly enriched in vacuoles while TPT fluorescence and Mitotracker staining co-localized (white arrow). Cells without Mitotracker straining were used as control to discard the possibility that we detect TPT auto-fluorescence applying the Mitotracker settings. White bar represents 5 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651790&req=5

Figure 6: Topotecan is capable of inducing petite and accumulates in mitochondria. (A) Drop test assay to test the effect of TPT treatment on yeast growth. The indicated constructs were expressed under control of the GAL1 promoter and cells were grown in plates without or with 20 µg/ml TPT. Note that petite induced in galactose by expression of mt125Top1-103 under control of the GAL1p grew slower than petite induced in galactose by expression of mt125Top1-103 under control of the MET25p (Figure 3). (B) Intracellular localization of CPT and TPT. Using the settings for the detection of DAPI, the intracellular TPT and CPT localization was determined by their auto-fluorescence. Construct pRWE129 was expressed for GFP-localization of the nucleus. CPT is mainly enriched in vacuoles while TPT fluorescence and Mitotracker staining co-localized (white arrow). Cells without Mitotracker straining were used as control to discard the possibility that we detect TPT auto-fluorescence applying the Mitotracker settings. White bar represents 5 µm.
Mentions: While yeast cells are sensitive to CPT treatment, it has been shown that CPT analogs which are soluble in aqueous solutions are much less toxic to yeast cells (37). One such analog is the anticancer drug TPT that induces apoptosis of mammalian tumor cells (38). From microscopic studies it has been inferred that TPT is enriched in mitochondria of mammalian cells (39). We reasoned that the water solubility and positive charge of the TPT molecule (15) could also allow the drug to penetrate mitochondria of S. cerevisiae. Indeed, expression of mt125Top1 in the presence of TPT led to the formation of petite cells (Figure 6A) indicating that TPT reaches the mitochondria in an active form. We determined petite formation by replica-plating of single colonies grown on galactose onto glycerol-containing medium. By counting the colonies growing on glycerol, we found that expression of mt125Top1 in the presence of TPT led to induction of more than 95% petite thus being as potent in generating petite as expression of mt125Top1-103.Figure 6.

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