Annals of Surgical Oncology

, Volume 5, Issue 3, pp 287–295 | Cite as

Flow cytometric measurement of mitochondrial mass and function: A novel method for assessing chemoresistance

  • Mariangela Mancini
  • Monireh Sedghinasab
  • Katya Knowlton
  • Amy Tam
  • David Hockenberg
  • Benjamin O. Anderson
Best Basic Science Research Paper

Abstract

Background: Chemotherapeutic agents induce apoptosis in cancer cells. Drugs failing to induce apoptosis are likely to have decreased clinical efficacy. We hypothesize that (1) chemotherapeutic agents induce mitochondrial changes and apoptosis through mechanisms associated with reactive oxidant species production; (2) the anti-apoptotic protein Bcl-2 prevents drug-induced mitochondrial changes, reactive oxygen species (ROS) production, and apoptosis; and (3) the assay of drug-induced mitochondrial changes can reflect drug-specific chemoresistance in a given cancer cell line.

Methods: A stable Bcl-2 transfectant of the Bcl-2 negative breast cancer cell line SKBr3 was created (SKBr3/Bcl2-2). Both SKBr3 and SKBr3/Bcl2-2 cells were treated with Herbimycin A (300 ng/mL) or vehicle (1% DMSO). Cell cycle changes were assessed by BRDU staining. Apoptosis was determined by electron microscopy, TUNEL (TdT-mediated dUTP-biotin nick end labeling) staining, and diphenylamine assay of DNA fragmentation. Changes in mitochondrial mass and transmembrane potential (ΔΨm) were assessed by flow cytometric assessment of JC-1 fluorescence. Reactive oxygen species production was measured by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH) fluorescence.

Results: Both SKBr3 and SKBr3/Bcl2-2 cells show cell cycle arrest after Herbimycin treatment. However, SKBr3 cells, but not SKBr3/Bcl2-2 cells, undergo apoptosis. Herbimycin-treated SKBr3 cells show increased mitochondrial mass (JC-1 green fluorescence), with no corresponding increase in ΔΨm (JC-1 red fluorescence). By contrast, Herbimycin-treated SKBr3/Bcl2-2 cells show no change in mitochondrial mass or ΔΨm. Similarly, drug-treated SKBr3 cells, but not SKBr3/Bcl2-2 cells, demonstrate increased reactive oxygen species (ROS) production concomitant with the development of apoptosis.

Conclusion: SKBr3 cells undergoing apoptosis demonstrate mitochondrial changes associated with ROS production. Bcl-2 transfection prevents these changes because it prevents apoptosis and induces chemoresistance to Herbimycin in SKBr3. Flow cytometric measurement of drug induced mitochondrial changes and ROS production may facilitate in vitro assessment of chemosensitivity or chemoresistance in breast cancer.

Key Words

Chemoresistance Apoptosis Breast cancer Mitochondria Bcl-2 gene Reactive oxygen species 

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Copyright information

© The Society of Surgical Oncology, Inc. 1998

Authors and Affiliations

  • Mariangela Mancini
    • 2
    • 1
  • Monireh Sedghinasab
    • 2
    • 1
  • Katya Knowlton
    • 2
    • 1
  • Amy Tam
    • 1
  • David Hockenberg
    • 1
  • Benjamin O. Anderson
    • 2
    • 1
  1. 1.the Divisions of Clinical Research and Molecular MedicineFred Hutchinson Cancer Research CenterSeattle
  2. 2.Dept. of SurgeryUniversity of WashingtonSeattle

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