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In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice
Published on: November 22, 2017
Quantitative assessment of mitophagy in irradiated cancer cells
Emma Guilbaud1, Sheila Spada1, Norma Bloy1
1Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, United States.
Abstract:
Mitophagy is a finely regulated mechanism through which eukaryotic cells selectively dispose of supernumerary, permeabilized or otherwise damaged mitochondria through lysosomal degradation. Dysfunctional mitochondria are prone to release potentially cytotoxic factors including reactive oxygen species (ROS) and caspase activators, such as cytochrome c, somatic (CYCS). Thus, proficient mitophagic responses mediate prominent cytoprotective functions. Moreover, the rapid degradation of permeabilized mitochondria limits the release of mitochondrial components that may drive inflammatory reactions, such as mitochondrial DNA (mtDNA) and transcription factor A, mitochondrial (TFAM), implying that mitophagy also mediates potent anti-inflammatory effects. Here, we detail a simple, flow cytometry-assisted protocol for the specific measurement of mitophagic responses as driven by radiation therapy (RT) in mouse hormone receptor (HR)+ mammary carcinoma TS/A cells. With some variations, this method - which relies on the mitochondria-restricted expression of a fluorescent reporter that is sensitive to pH and hence changes excitation wavelength within lysosomes (mt-mKeima) - can be adapted to a variety of human and mouse cancer cell lines and/or straightforwardly implemented on fluorescence microscopy platforms.
Insights
This study presents a new flow cytometry method to measure mitophagy, a cellular process for clearing damaged mitochondria. This technique helps understand how radiation therapy affects mitophagy in cancer cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Mitophagy is crucial for cellular health, removing damaged mitochondria to prevent the release of cytotoxic factors like reactive oxygen species (ROS).
- Dysfunctional mitochondria can release inflammatory molecules such as mitochondrial DNA (mtDNA), highlighting mitophagy's anti-inflammatory role.
- Efficient mitophagy is vital for cytoprotection and limiting inflammation.
Purpose of the Study:
- To develop and present a simple, flow cytometry-assisted protocol for measuring radiation therapy-induced mitophagy.
- To specifically quantify mitophagic responses in mouse hormone receptor-positive mammary carcinoma cells.
Main Methods:
- Utilized a mitochondria-restricted fluorescent reporter (mt-mKeima) that changes excitation wavelength based on lysosomal pH.
- Employed flow cytometry for specific measurement of mitophagic activity.
- Applied the protocol to mouse hormone receptor-positive mammary carcinoma TS/A cells treated with radiation therapy.
Main Results:
- Successfully detailed a flow cytometry-assisted protocol for measuring mitophagy.
- Demonstrated the protocol's applicability in assessing radiation therapy-induced mitophagy in cancer cells.
- The method relies on the pH-sensitive fluorescent properties of mt-mKeima within lysosomes.
Conclusions:
- The presented method offers a straightforward approach to quantify mitophagy.
- This protocol can be adapted for various cancer cell lines and fluorescence microscopy platforms.
- It provides a valuable tool for studying the role of mitophagy in cancer therapy response.
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