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Cisplatin selects short forms of the mitochondrial DNA OriB variant (16184 – 16193 poly-cytosine tract), which confer resistance to cisplatin

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ABSTRACT

A number of alternations in mitochondrial DNA (mtDNA) have been reported in different types of cancers, and the role of mtDNA in cancer has been attracting increasing interest. In order to investigate the relationship between mtDNA alternations and chemosensitivity, we constructed cybrid (trans-mitochondrial hybrid) cell lines carrying a HeLa nucleus and the mtDNA of healthy individuals because of the presence of somatic alternations in the mtDNA of many cancer cells. After a treatment with 1.0 μg/mL cisplatin for 10 days, we isolated 100 cisplatin-resistant clones, 70 of which carried the shorter mtDNA OriB variant (16184–16193 poly-cytosine tract), which was located in the control region of mtDNA. Whole mtDNA sequencing of 10 clones revealed no additional alternations. Re-construction of the HeLa nucleus and mtDNA from cisplatin-resistant cells showed that cisplatin resistance was only acquired by mtDNA alternations in the control region, and not by possible alternation(s) in the nuclear genome.

No MeSH data available.


Survival assessment of re-constructed cybrids exposed to 1.0 μg/mL of cisplatin for 7 days.Cells were double-stained with Hoechst 33342 and propidium iodide. Hoechst-positive and propidium iodide-negative cells were interpreted as surviving cells. (A) Cells were imaged with a fluoromicroscope and counted using ImageJ. (B) Cells were treated with trypsin and subjected to a flow cytometric analysis. Error bars indicate S.E.M. (n = 3). **P < 0.01; ***P < 0.001.
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f5: Survival assessment of re-constructed cybrids exposed to 1.0 μg/mL of cisplatin for 7 days.Cells were double-stained with Hoechst 33342 and propidium iodide. Hoechst-positive and propidium iodide-negative cells were interpreted as surviving cells. (A) Cells were imaged with a fluoromicroscope and counted using ImageJ. (B) Cells were treated with trypsin and subjected to a flow cytometric analysis. Error bars indicate S.E.M. (n = 3). **P < 0.01; ***P < 0.001.

Mentions: In order to assess cisplatin sensitivity, 9W4c, R13c, and their parental cybrids were cultivated in the presence of 1.0 μg/mL cisplatin for 7 days. Surviving cells were counted under a fluoromicroscope after double-staining with Hoechst 33342 and propidium iodide. Newly constructed R13c cells were more resistant to cisplatin than 9W4c cells, similar to their parental cybrids (Fig. 5A), and this was confirmed by a flow cytometric analysis (Fig. 5B). These results indicate that the differences observed in cisplatin resistance between R13c and 9W4c only arose from mtDNA. Therefore, the length of the mtDNA poly-C tract of the OriB variant affects cisplatin resistance.


Cisplatin selects short forms of the mitochondrial DNA OriB variant (16184 – 16193 poly-cytosine tract), which confer resistance to cisplatin
Survival assessment of re-constructed cybrids exposed to 1.0 μg/mL of cisplatin for 7 days.Cells were double-stained with Hoechst 33342 and propidium iodide. Hoechst-positive and propidium iodide-negative cells were interpreted as surviving cells. (A) Cells were imaged with a fluoromicroscope and counted using ImageJ. (B) Cells were treated with trypsin and subjected to a flow cytometric analysis. Error bars indicate S.E.M. (n = 3). **P < 0.01; ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385546&req=5

f5: Survival assessment of re-constructed cybrids exposed to 1.0 μg/mL of cisplatin for 7 days.Cells were double-stained with Hoechst 33342 and propidium iodide. Hoechst-positive and propidium iodide-negative cells were interpreted as surviving cells. (A) Cells were imaged with a fluoromicroscope and counted using ImageJ. (B) Cells were treated with trypsin and subjected to a flow cytometric analysis. Error bars indicate S.E.M. (n = 3). **P < 0.01; ***P < 0.001.
Mentions: In order to assess cisplatin sensitivity, 9W4c, R13c, and their parental cybrids were cultivated in the presence of 1.0 μg/mL cisplatin for 7 days. Surviving cells were counted under a fluoromicroscope after double-staining with Hoechst 33342 and propidium iodide. Newly constructed R13c cells were more resistant to cisplatin than 9W4c cells, similar to their parental cybrids (Fig. 5A), and this was confirmed by a flow cytometric analysis (Fig. 5B). These results indicate that the differences observed in cisplatin resistance between R13c and 9W4c only arose from mtDNA. Therefore, the length of the mtDNA poly-C tract of the OriB variant affects cisplatin resistance.

View Article: PubMed Central - PubMed

ABSTRACT

A number of alternations in mitochondrial DNA (mtDNA) have been reported in different types of cancers, and the role of mtDNA in cancer has been attracting increasing interest. In order to investigate the relationship between mtDNA alternations and chemosensitivity, we constructed cybrid (trans-mitochondrial hybrid) cell lines carrying a HeLa nucleus and the mtDNA of healthy individuals because of the presence of somatic alternations in the mtDNA of many cancer cells. After a treatment with 1.0&thinsp;&mu;g/mL cisplatin for 10 days, we isolated 100 cisplatin-resistant clones, 70 of which carried the shorter mtDNA OriB variant (16184&ndash;16193 poly-cytosine tract), which was located in the control region of mtDNA. Whole mtDNA sequencing of 10 clones revealed no additional alternations. Re-construction of the HeLa nucleus and mtDNA from cisplatin-resistant cells showed that cisplatin resistance was only acquired by mtDNA alternations in the control region, and not by possible alternation(s) in the nuclear genome.

No MeSH data available.