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Mitochondrial localization of APE/Ref-1 in thyroid cells
G Tell1, E Crivellato, A Pines
1Dipartimento di Biochimica Biofisica e Chimica delle Macromolecole, Università degli Studi di Trieste, Trieste, Italy. tell@bbcm.univ.trieste.it
Abstract:
Mutations of mitochondrial DNA (mtDNA) are associated with different human diseases, including cancer and aging. Reactive oxygen species produced during oxidative phosphorylation are a major source of mtDNA damage. It is not clear, however, whether DNA repair mechanisms, able to abolish effects due to oxidative damage, are present in mitochondria. APE/Ref-1 is a nuclear protein possessing both redox activity (by which activates, "in vitro", the DNA-binding functions of several transcription factors) and DNA repair activity over apurinic/apyrimidinic sites. Immunohistochemical evidences indicate that in follicular thyroid cells, APE/Ref-1 is located in both nucleus and cytoplasm. Electronmicroscopy immunocytochemistry performed in the rat thyroid FRTL-5 cell line, indicates that part of the cytoplasmatic APE/Ref-1 is located in mitochondria. The presence of APE/Ref-1 inside mitochondria is further demonstrated by western blot analysis after cell fractionation. In the Kimol cell line (which is derived from FRTL-5, transformed by the Ki-ras oncogene) the amount of mitochondrial APE/Ref-1 is reduced by three to fourfold with respect to the normal FRTL-5 cells. These results suggest that: (i) a machinery capable of repairing DNA damaged by oxidative stress is present in mitochondria and (ii) mtDNA repair mechanisms may be impaired during cell transformation.
Insights
Mitochondria possess DNA repair capabilities, with the APE/Ref-1 protein found within them. This mitochondrial DNA repair capacity appears diminished in cancer cells, suggesting a link between impaired repair and cell transformation.
Area of Science:
- Mitochondrial biology
- DNA repair mechanisms
- Cellular transformation
Background:
- Mitochondrial DNA (mtDNA) mutations are linked to human diseases like cancer and aging.
- Oxidative phosphorylation generates reactive oxygen species, a primary cause of mtDNA damage.
- The existence and function of DNA repair mechanisms within mitochondria remain largely uncharacterized.
Purpose of the Study:
- To investigate the presence and localization of DNA repair proteins within mitochondria.
- To explore the role of APE/Ref-1 in mitochondrial DNA repair.
- To assess alterations in mitochondrial DNA repair capacity during cellular transformation.
Main Methods:
- Immunohistochemistry to detect APE/Ref-1 localization in follicular thyroid cells.
- Electron microscopy immunocytochemistry in FRTL-5 cell lines to confirm mitochondrial localization.
- Western blot analysis of cell fractions to quantify mitochondrial APE/Ref-1 levels.
Main Results:
- APE/Ref-1, a nuclear protein with DNA repair functions, was detected in both the nucleus and cytoplasm of follicular thyroid cells.
- Part of the cytoplasmic APE/Ref-1 was localized to mitochondria in rat thyroid FRTL-5 cells.
- Mitochondrial APE/Ref-1 levels were significantly reduced (three to fourfold) in the oncogene-transformed Kimol cell line compared to normal FRTL-5 cells.
Conclusions:
- Mitochondria possess a DNA repair machinery capable of addressing oxidative damage.
- Mitochondrial DNA repair mechanisms may be impaired during the process of cell transformation, potentially contributing to disease development.