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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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Localization sequences target Top1p either to the nucleus or mitochondria. (A) Northern blot analysis of TOP1 mRNA upon galactose-induced gene expression. (B) Relative quantification of TOP1 mRNA levels. Relative TOP1 mRNA levels were normalized to 25S mRNA. The value obtained at time point 0 was set as 1. Average values of two independent experiments are shown. (C) Projection of a series of focal plane images derived from confocal fluorescence microscopy. GFP-Top1 expression was induced for 30 min in galactose (green). The nucleus and mitochondria were localized with Hoechst33342 (blue) and Mitotracker (red) dyes, respectively. White bar represents 5 µm. Control cells grown in glucose did not exhibit green fluorescence (data not shown).
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Figure 2: Localization sequences target Top1p either to the nucleus or mitochondria. (A) Northern blot analysis of TOP1 mRNA upon galactose-induced gene expression. (B) Relative quantification of TOP1 mRNA levels. Relative TOP1 mRNA levels were normalized to 25S mRNA. The value obtained at time point 0 was set as 1. Average values of two independent experiments are shown. (C) Projection of a series of focal plane images derived from confocal fluorescence microscopy. GFP-Top1 expression was induced for 30 min in galactose (green). The nucleus and mitochondria were localized with Hoechst33342 (blue) and Mitotracker (red) dyes, respectively. White bar represents 5 µm. Control cells grown in glucose did not exhibit green fluorescence (data not shown).

Mentions: It is possible that accumulation of persistent nuclear DNA SSBs mediated by a toxic Top1-103 protein might affect mRNA transcription levels. Thus, we first analyzed mRNA expression levels of GAL1 promoter driven constructs by northern analysis (Figure 2A). TOP1-103, n125TOP1-103 and mt125TOP-103 mRNAs were not detectable in cells grown in glycerol (no induction) and glucose (repression), while shifting cells to galactose (induction) led to the rapid appearance of mRNAs. In all constructs, expression levels reached a maximum within 60 min (Figure 2B) and mRNA expression levels remained high within 180 min. No significant difference in mRNA levels was obtained comparing expression of n125TOP1-103 with mt125TOP-103, or expression of the TOP-103 constructs with a TOP1 expressing control vector (data not shown). From these results we conclude that nuclear targeting of the Top1-103 protein does not affect TOP1-103 transcription levels within 180 min of induction.Figure 2.


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)

Localization sequences target Top1p either to the nucleus or mitochondria. (A) Northern blot analysis of TOP1 mRNA upon galactose-induced gene expression. (B) Relative quantification of TOP1 mRNA levels. Relative TOP1 mRNA levels were normalized to 25S mRNA. The value obtained at time point 0 was set as 1. Average values of two independent experiments are shown. (C) Projection of a series of focal plane images derived from confocal fluorescence microscopy. GFP-Top1 expression was induced for 30 min in galactose (green). The nucleus and mitochondria were localized with Hoechst33342 (blue) and Mitotracker (red) dyes, respectively. White bar represents 5 µm. Control cells grown in glucose did not exhibit green fluorescence (data not shown).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Localization sequences target Top1p either to the nucleus or mitochondria. (A) Northern blot analysis of TOP1 mRNA upon galactose-induced gene expression. (B) Relative quantification of TOP1 mRNA levels. Relative TOP1 mRNA levels were normalized to 25S mRNA. The value obtained at time point 0 was set as 1. Average values of two independent experiments are shown. (C) Projection of a series of focal plane images derived from confocal fluorescence microscopy. GFP-Top1 expression was induced for 30 min in galactose (green). The nucleus and mitochondria were localized with Hoechst33342 (blue) and Mitotracker (red) dyes, respectively. White bar represents 5 µm. Control cells grown in glucose did not exhibit green fluorescence (data not shown).
Mentions: It is possible that accumulation of persistent nuclear DNA SSBs mediated by a toxic Top1-103 protein might affect mRNA transcription levels. Thus, we first analyzed mRNA expression levels of GAL1 promoter driven constructs by northern analysis (Figure 2A). TOP1-103, n125TOP1-103 and mt125TOP-103 mRNAs were not detectable in cells grown in glycerol (no induction) and glucose (repression), while shifting cells to galactose (induction) led to the rapid appearance of mRNAs. In all constructs, expression levels reached a maximum within 60 min (Figure 2B) and mRNA expression levels remained high within 180 min. No significant difference in mRNA levels was obtained comparing expression of n125TOP1-103 with mt125TOP-103, or expression of the TOP-103 constructs with a TOP1 expressing control vector (data not shown). From these results we conclude that nuclear targeting of the Top1-103 protein does not affect TOP1-103 transcription levels within 180 min of induction.Figure 2.

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