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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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Mitochondrially targeted mt125Top1-103 leads to mtDNA loss and the formation of rho° petite. (A) Time course of mt125Top1-103 dependent petite formation. Cells were grown in galactose-containing liquid medium (expression). At the indicated times cells were retrieved, grown on glucose containing plates (no expression) and replica-plated onto glycerol. Petite cells were determined by color (white; open diamonds) and their inability to grow on glycerol (black diamonds). Average values of two independent experiments are shown. (B) CHEF analysis of the mtDNA copy number. In-plug isolated, total cellular DNA was ApaI digested, gel electrophoresed and analyzed by Southern blot. Shown are the agarose gel before Southern blotting (rDNA fragments, open triangle) and the signals obtained using a specific probe against mitochondrial (right top, black triangle) and nuclear DNA (right bottom) after Southern blotting. (C) Determination of the relative mtDNA copy number. The mtCOX2 hybridization signal obtained at time point 0 was normalized to the nuclear probe and set as 100%. Average values of two independent experiments are shown. (D) Wide-field fluorescence microscopy analysis of petite cells after 48 h of mt125Top1-103 expression. DAPI (white) and Mitotracker (red) staining of rho+ (w/o induction) and rho° (48 h of induction) is shown. White bar represents 5 µm.
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Figure 4: Mitochondrially targeted mt125Top1-103 leads to mtDNA loss and the formation of rho° petite. (A) Time course of mt125Top1-103 dependent petite formation. Cells were grown in galactose-containing liquid medium (expression). At the indicated times cells were retrieved, grown on glucose containing plates (no expression) and replica-plated onto glycerol. Petite cells were determined by color (white; open diamonds) and their inability to grow on glycerol (black diamonds). Average values of two independent experiments are shown. (B) CHEF analysis of the mtDNA copy number. In-plug isolated, total cellular DNA was ApaI digested, gel electrophoresed and analyzed by Southern blot. Shown are the agarose gel before Southern blotting (rDNA fragments, open triangle) and the signals obtained using a specific probe against mitochondrial (right top, black triangle) and nuclear DNA (right bottom) after Southern blotting. (C) Determination of the relative mtDNA copy number. The mtCOX2 hybridization signal obtained at time point 0 was normalized to the nuclear probe and set as 100%. Average values of two independent experiments are shown. (D) Wide-field fluorescence microscopy analysis of petite cells after 48 h of mt125Top1-103 expression. DAPI (white) and Mitotracker (red) staining of rho+ (w/o induction) and rho° (48 h of induction) is shown. White bar represents 5 µm.

Mentions: Respiration-deficient petite cells result from the loss of nuclear encoded functions, which are essential for the mitochondrial respiration capacity, or from mtDNA rearrangement, mutation or loss. Targeting of the toxic mt125Top1-103 to mitochondria results in mtDNA damage, which is predicted to impede mtDNA replication and to promote mtDNA loss. In order to quantify the extent of respiration-deficient cells, the mt125TOP1-103 construct placed under the control of the GAL1 promoter was expressed up to 48 h, and petite formation was assayed by visual inspection of white-versus red-pigmented colonies as well as growth inhibition in nonfermentable medium at the indicated time-point (Figure 4A). Within 48 h of mt125Top1-103 expression, conversion of wild-type into petite cells was nearly complete. About 97% of colonies expressing mt125Top1-103 turned white (7% in the control) and only 15% of the colonies were proficient to form colonies in nonfermentable medium (99% in the control). We wondered if the observed time-dependent increase in petite cells was due to the formation of respiration-deficient but mtDNA-containing rho– or due to the appearance of mtDNA-less rho° cells, respectively. Therefore, DNA was extracted in agarose plugs, in-gel digested with ApaI and analyzed by Southern blot (Figure 4B). After hybridization with a probe specific for the mitochondrial COX2 gene, a clear drop in mtDNA was seen after 48 h expression of the mt125Top1-103 construct (compare signals obtained for nuclear and mtDNA after 48 h of cellular growth in galactose or glucose). Quantification of the mtDNA content in respect to nuclear DNA (Figure 4C) showed that the overall mtDNA content was reduced to about 25% within 48 h of growth in galactose. To confirm that prolonged mt125Top1-103 expression induces the formation of rho° cells, cells expressing mt125Top1-103 for 48 h were stained with the DNA-intercalating agent DAPI and analyzed by fluorescence microscopy. In vivo staining of mtDNA by DAPI was practically absent in a high percentage of cells (see example in Figure 4D and Figure S2) confirming that mt125Top1-103 expression leads to mtDNA less rho° cells.Figure 4.


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)

Mitochondrially targeted mt125Top1-103 leads to mtDNA loss and the formation of rho° petite. (A) Time course of mt125Top1-103 dependent petite formation. Cells were grown in galactose-containing liquid medium (expression). At the indicated times cells were retrieved, grown on glucose containing plates (no expression) and replica-plated onto glycerol. Petite cells were determined by color (white; open diamonds) and their inability to grow on glycerol (black diamonds). Average values of two independent experiments are shown. (B) CHEF analysis of the mtDNA copy number. In-plug isolated, total cellular DNA was ApaI digested, gel electrophoresed and analyzed by Southern blot. Shown are the agarose gel before Southern blotting (rDNA fragments, open triangle) and the signals obtained using a specific probe against mitochondrial (right top, black triangle) and nuclear DNA (right bottom) after Southern blotting. (C) Determination of the relative mtDNA copy number. The mtCOX2 hybridization signal obtained at time point 0 was normalized to the nuclear probe and set as 100%. Average values of two independent experiments are shown. (D) Wide-field fluorescence microscopy analysis of petite cells after 48 h of mt125Top1-103 expression. DAPI (white) and Mitotracker (red) staining of rho+ (w/o induction) and rho° (48 h of induction) is shown. White bar represents 5 µm.
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Figure 4: Mitochondrially targeted mt125Top1-103 leads to mtDNA loss and the formation of rho° petite. (A) Time course of mt125Top1-103 dependent petite formation. Cells were grown in galactose-containing liquid medium (expression). At the indicated times cells were retrieved, grown on glucose containing plates (no expression) and replica-plated onto glycerol. Petite cells were determined by color (white; open diamonds) and their inability to grow on glycerol (black diamonds). Average values of two independent experiments are shown. (B) CHEF analysis of the mtDNA copy number. In-plug isolated, total cellular DNA was ApaI digested, gel electrophoresed and analyzed by Southern blot. Shown are the agarose gel before Southern blotting (rDNA fragments, open triangle) and the signals obtained using a specific probe against mitochondrial (right top, black triangle) and nuclear DNA (right bottom) after Southern blotting. (C) Determination of the relative mtDNA copy number. The mtCOX2 hybridization signal obtained at time point 0 was normalized to the nuclear probe and set as 100%. Average values of two independent experiments are shown. (D) Wide-field fluorescence microscopy analysis of petite cells after 48 h of mt125Top1-103 expression. DAPI (white) and Mitotracker (red) staining of rho+ (w/o induction) and rho° (48 h of induction) is shown. White bar represents 5 µm.
Mentions: Respiration-deficient petite cells result from the loss of nuclear encoded functions, which are essential for the mitochondrial respiration capacity, or from mtDNA rearrangement, mutation or loss. Targeting of the toxic mt125Top1-103 to mitochondria results in mtDNA damage, which is predicted to impede mtDNA replication and to promote mtDNA loss. In order to quantify the extent of respiration-deficient cells, the mt125TOP1-103 construct placed under the control of the GAL1 promoter was expressed up to 48 h, and petite formation was assayed by visual inspection of white-versus red-pigmented colonies as well as growth inhibition in nonfermentable medium at the indicated time-point (Figure 4A). Within 48 h of mt125Top1-103 expression, conversion of wild-type into petite cells was nearly complete. About 97% of colonies expressing mt125Top1-103 turned white (7% in the control) and only 15% of the colonies were proficient to form colonies in nonfermentable medium (99% in the control). We wondered if the observed time-dependent increase in petite cells was due to the formation of respiration-deficient but mtDNA-containing rho– or due to the appearance of mtDNA-less rho° cells, respectively. Therefore, DNA was extracted in agarose plugs, in-gel digested with ApaI and analyzed by Southern blot (Figure 4B). After hybridization with a probe specific for the mitochondrial COX2 gene, a clear drop in mtDNA was seen after 48 h expression of the mt125Top1-103 construct (compare signals obtained for nuclear and mtDNA after 48 h of cellular growth in galactose or glucose). Quantification of the mtDNA content in respect to nuclear DNA (Figure 4C) showed that the overall mtDNA content was reduced to about 25% within 48 h of growth in galactose. To confirm that prolonged mt125Top1-103 expression induces the formation of rho° cells, cells expressing mt125Top1-103 for 48 h were stained with the DNA-intercalating agent DAPI and analyzed by fluorescence microscopy. In vivo staining of mtDNA by DAPI was practically absent in a high percentage of cells (see example in Figure 4D and Figure S2) confirming that mt125Top1-103 expression leads to mtDNA less rho° cells.Figure 4.

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