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Direct evidence of mitochondrial G-quadruplex DNA by using fluorescent anti-cancer agents.

Huang WC, Tseng TY, Chen YT, Chang CC, Wang ZF, Wang CL, Hsu TN, Li PT, Chen CT, Lin JJ, Lou PJ, Chang TC - Nucleic Acids Res. (2015)

Bottom Line: In this paper, we provide the first evidence supporting the presence of G4 in the mitochondrial DNA (mtDNA) of live cells.In this study, we use fluorescence lifetime imaging microscopy to verify the existence of mtDNA G4s in live cells.Bioactivity studies indicate that interactions between these anti-cancer agents and mtDNA G4 can suppress mitochondrial gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan.

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Related in: MedlinePlus

(A) FLIM images of live cells incubated with 1 μM o-BMVC-12C-P overnight. For clarity, the images were presented in pseudocolors of white (decay time ≥ 2.4 ns) resulting from interaction with G4s and red (decay time < 2.4 ns) due to interaction with others. Several white spots were detected in the time-discrete image of HeLa cancer cells, but not observed in the time-discrete image of MRC-5 normal cells. Scale bar is 10 μm. (B) The histograms of the fluorescence decay time of 0.2 μM o-BMVC-12C-P upon interaction with calf thymus (green) and mt6363 (red) together with o-BMVC-12C-P in isolated mitochondria (black). (C) Reverse transcription RT-PCR was used to evaluate the suppression of ND3 and COX I expression by o-BMVC-12C-P. HeLa cells were incubated with o-BMVC-12C-P at different concentrations for 1–3 d. Here β-actin and a non-G4 forming gene sequence were used as controls. (D) PCR stop assay was used to determine whether the suppression of ND3 and COX I expression is due to the stabilization of the G4s formed by mtDNA of ND3 and COX I by o-BMVC-12C-P. The paused bands indicated the G4 stabilization by the ligand. The mutated sequences were designed specifically for not able to form G4.
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Figure 5: (A) FLIM images of live cells incubated with 1 μM o-BMVC-12C-P overnight. For clarity, the images were presented in pseudocolors of white (decay time ≥ 2.4 ns) resulting from interaction with G4s and red (decay time < 2.4 ns) due to interaction with others. Several white spots were detected in the time-discrete image of HeLa cancer cells, but not observed in the time-discrete image of MRC-5 normal cells. Scale bar is 10 μm. (B) The histograms of the fluorescence decay time of 0.2 μM o-BMVC-12C-P upon interaction with calf thymus (green) and mt6363 (red) together with o-BMVC-12C-P in isolated mitochondria (black). (C) Reverse transcription RT-PCR was used to evaluate the suppression of ND3 and COX I expression by o-BMVC-12C-P. HeLa cells were incubated with o-BMVC-12C-P at different concentrations for 1–3 d. Here β-actin and a non-G4 forming gene sequence were used as controls. (D) PCR stop assay was used to determine whether the suppression of ND3 and COX I expression is due to the stabilization of the G4s formed by mtDNA of ND3 and COX I by o-BMVC-12C-P. The paused bands indicated the G4 stabilization by the ligand. The mutated sequences were designed specifically for not able to form G4.

Mentions: Figure 5A presents a time-discrete FLIM image of o-BMVC-12C-P to reveal the possible existence of G4s in HeLa cancer cells and MRC-5 normal cells under the same experimental conditions. Several white spots characterized by fluorescence decay time (≥2.4 ns) were detected in the study of HeLa cancer cells (24/24), but they were not observed in MRC-5 normal cells (15/15). The absence of white spots in the normal cells can be explained by relatively far lower uptake of o-BMVC-12C-P in the mitochondria of MRC-5 normal cells. This is because the o-BMVC-12C-P localizes primarily in the lysosome of normal cells. Nevertheless, the detection of the white spots in cancer cells supports the existence of G4s in cancer cells, which is very likely contributed by mtDNA G4s.


Direct evidence of mitochondrial G-quadruplex DNA by using fluorescent anti-cancer agents.

Huang WC, Tseng TY, Chen YT, Chang CC, Wang ZF, Wang CL, Hsu TN, Li PT, Chen CT, Lin JJ, Lou PJ, Chang TC - Nucleic Acids Res. (2015)

(A) FLIM images of live cells incubated with 1 μM o-BMVC-12C-P overnight. For clarity, the images were presented in pseudocolors of white (decay time ≥ 2.4 ns) resulting from interaction with G4s and red (decay time < 2.4 ns) due to interaction with others. Several white spots were detected in the time-discrete image of HeLa cancer cells, but not observed in the time-discrete image of MRC-5 normal cells. Scale bar is 10 μm. (B) The histograms of the fluorescence decay time of 0.2 μM o-BMVC-12C-P upon interaction with calf thymus (green) and mt6363 (red) together with o-BMVC-12C-P in isolated mitochondria (black). (C) Reverse transcription RT-PCR was used to evaluate the suppression of ND3 and COX I expression by o-BMVC-12C-P. HeLa cells were incubated with o-BMVC-12C-P at different concentrations for 1–3 d. Here β-actin and a non-G4 forming gene sequence were used as controls. (D) PCR stop assay was used to determine whether the suppression of ND3 and COX I expression is due to the stabilization of the G4s formed by mtDNA of ND3 and COX I by o-BMVC-12C-P. The paused bands indicated the G4 stabilization by the ligand. The mutated sequences were designed specifically for not able to form G4.
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Related In: Results  -  Collection

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Figure 5: (A) FLIM images of live cells incubated with 1 μM o-BMVC-12C-P overnight. For clarity, the images were presented in pseudocolors of white (decay time ≥ 2.4 ns) resulting from interaction with G4s and red (decay time < 2.4 ns) due to interaction with others. Several white spots were detected in the time-discrete image of HeLa cancer cells, but not observed in the time-discrete image of MRC-5 normal cells. Scale bar is 10 μm. (B) The histograms of the fluorescence decay time of 0.2 μM o-BMVC-12C-P upon interaction with calf thymus (green) and mt6363 (red) together with o-BMVC-12C-P in isolated mitochondria (black). (C) Reverse transcription RT-PCR was used to evaluate the suppression of ND3 and COX I expression by o-BMVC-12C-P. HeLa cells were incubated with o-BMVC-12C-P at different concentrations for 1–3 d. Here β-actin and a non-G4 forming gene sequence were used as controls. (D) PCR stop assay was used to determine whether the suppression of ND3 and COX I expression is due to the stabilization of the G4s formed by mtDNA of ND3 and COX I by o-BMVC-12C-P. The paused bands indicated the G4 stabilization by the ligand. The mutated sequences were designed specifically for not able to form G4.
Mentions: Figure 5A presents a time-discrete FLIM image of o-BMVC-12C-P to reveal the possible existence of G4s in HeLa cancer cells and MRC-5 normal cells under the same experimental conditions. Several white spots characterized by fluorescence decay time (≥2.4 ns) were detected in the study of HeLa cancer cells (24/24), but they were not observed in MRC-5 normal cells (15/15). The absence of white spots in the normal cells can be explained by relatively far lower uptake of o-BMVC-12C-P in the mitochondria of MRC-5 normal cells. This is because the o-BMVC-12C-P localizes primarily in the lysosome of normal cells. Nevertheless, the detection of the white spots in cancer cells supports the existence of G4s in cancer cells, which is very likely contributed by mtDNA G4s.

Bottom Line: In this paper, we provide the first evidence supporting the presence of G4 in the mitochondrial DNA (mtDNA) of live cells.In this study, we use fluorescence lifetime imaging microscopy to verify the existence of mtDNA G4s in live cells.Bioactivity studies indicate that interactions between these anti-cancer agents and mtDNA G4 can suppress mitochondrial gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 10617, Taiwan.

Show MeSH
Related in: MedlinePlus