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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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Related in: MedlinePlus

Drop test assay to assess the effect of nuclear and mitochondrial expression of MET25p-driven constructs. Targeting of the toxic n125Top1-103 to the nucleus leads to growth inhibition in fermentable (glucose) and nonfermentable (glycerol) growth medium. Targeting of the toxic mt125Top1-103 to mitochondria leads to growth of white colonies in glucose and growth inhibition in glycerol, both of which indicate the formation of respiration deficient ‘petite’ cells.
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Figure 3: Drop test assay to assess the effect of nuclear and mitochondrial expression of MET25p-driven constructs. Targeting of the toxic n125Top1-103 to the nucleus leads to growth inhibition in fermentable (glucose) and nonfermentable (glycerol) growth medium. Targeting of the toxic mt125Top1-103 to mitochondria leads to growth of white colonies in glucose and growth inhibition in glycerol, both of which indicate the formation of respiration deficient ‘petite’ cells.

Mentions: Growth inhibition on nonfermentable carbon sources such as glycerol is a hallmark of respiration deficient rho−, so-called ‘petite’ cells (35). In order to test the effect of nuclear or mitochondrial targeting of the Top1-103 protein on cell viability and respiration capacity we placed expression of the bio-fluorescent Top1-103 chimera under control of the MET25 promoter. This promoter has the advantage that cells can be grown in medium with different carbons sources because its expression is induced in the absence of methionine. As shown in the drop test in Figure 3, targeting of n125Top1-103 to the nucleus greatly reduced growth and viability of cells. Targeting of mt125Top1-103 to the mitochondria did not affect cell viability in fermentable medium (glucose). However, the accumulation of a red pigmentation characteristic of ade2− cells was suppressed and instead, white-colored colonies were obtained. The appearance of white colonies is an indication of respiration-deficient petite cells where mitochondria are not functional. Accordingly, the formation of petite cells was confirmed by growth inhibition in nonfermentable medium (glycerol). Thus, targeting of Top1-103-mediated SSBs to the nucleus affects cells viability, while mitochondrial targeting induces petite cells.Figure 3.


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)

Drop test assay to assess the effect of nuclear and mitochondrial expression of MET25p-driven constructs. Targeting of the toxic n125Top1-103 to the nucleus leads to growth inhibition in fermentable (glucose) and nonfermentable (glycerol) growth medium. Targeting of the toxic mt125Top1-103 to mitochondria leads to growth of white colonies in glucose and growth inhibition in glycerol, both of which indicate the formation of respiration deficient ‘petite’ cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Drop test assay to assess the effect of nuclear and mitochondrial expression of MET25p-driven constructs. Targeting of the toxic n125Top1-103 to the nucleus leads to growth inhibition in fermentable (glucose) and nonfermentable (glycerol) growth medium. Targeting of the toxic mt125Top1-103 to mitochondria leads to growth of white colonies in glucose and growth inhibition in glycerol, both of which indicate the formation of respiration deficient ‘petite’ cells.
Mentions: Growth inhibition on nonfermentable carbon sources such as glycerol is a hallmark of respiration deficient rho−, so-called ‘petite’ cells (35). In order to test the effect of nuclear or mitochondrial targeting of the Top1-103 protein on cell viability and respiration capacity we placed expression of the bio-fluorescent Top1-103 chimera under control of the MET25 promoter. This promoter has the advantage that cells can be grown in medium with different carbons sources because its expression is induced in the absence of methionine. As shown in the drop test in Figure 3, targeting of n125Top1-103 to the nucleus greatly reduced growth and viability of cells. Targeting of mt125Top1-103 to the mitochondria did not affect cell viability in fermentable medium (glucose). However, the accumulation of a red pigmentation characteristic of ade2− cells was suppressed and instead, white-colored colonies were obtained. The appearance of white colonies is an indication of respiration-deficient petite cells where mitochondria are not functional. Accordingly, the formation of petite cells was confirmed by growth inhibition in nonfermentable medium (glycerol). Thus, targeting of Top1-103-mediated SSBs to the nucleus affects cells viability, while mitochondrial targeting induces petite cells.Figure 3.

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