title: Drp1 is dispensable for apoptotic cytochrome c release in primed MCF10A and fibroblast cells but affects Bcl-2 antagonist-induced respiratory changes.
abstract: OBJECTIVE
  Dynamin-related protein 1 (Drp1) mediates mitochondrial fission and is thought to promote Bax/Bak-induced cytochrome c release during apoptosis. Conformationally active Bax, Bak and Bax/Bak-activating BH3-only proteins, such as Bim, are restrained by anti-apoptotic Bcl-2 proteins in cells that are 'primed for death'. Inhibition of Bcl-2/Bcl-xL/Bcl-w by the antagonist ABT-737 causes rapid apoptosis of primed cells. Hence, we determined whether Drp1 is required for cytochrome c release, respiratory alterations and apoptosis of cells that are already primed for death.
  
  
  METHODS
  We tested the Drp1 inhibitor mdivi-1 for inhibition of cytochrome c release in MCF10A cells primed by Bcl-2 overexpression. We measured ATP synthesis-dependent, -independent and cytochrome c-limited maximal oxygen consumption rates (OCRs) and cell death of immortalized wild-type (WT) and Drp1 knockout (KO) mouse embryonic fibroblasts (MEFs) treated with ABT-737.
  
  
  RESULTS
  Mdivi-1 failed to attenuate ABT-737-induced cytochrome c release. ABT-737 decreased maximal OCR measured in the presence of uncoupler in both WT and Drp1 KO MEF, consistent with respiratory impairment due to release of cytochrome c. However, Drp1 KO MEF were slightly less sensitive to this ABT-737-induced respiratory inhibition compared with WT, and were resistant to an initial ABT-737-induced increase in ATP synthesis-independent O2 consumption. Nevertheless, caspase-dependent cell death was not reduced. Pro-apoptotic Bax was unaltered, whereas Bak was up-regulated in Drp1 KO MEF.
  
  
  CONCLUSIONS
  The findings indicate that once fibroblast cells are primed for death, Drp1 is not required for apoptosis. However, Drp1 may contribute to ABT-737-induced respiratory changes and the kinetics of cytochrome c release.
authors.affiliation: Department of Anesthesiology and the Shock, Trauma and Anesthesiology Research (STAR) Center, University of Maryland School of Medicine, Baltimore, MD, USA.
authors.forename: P
authors.initials: P
authors.lastname: Clerc
authors.forename: S X
authors.initials: SX
authors.lastname: Ge
authors.forename: H
authors.initials: H
authors.lastname: Hwang
authors.forename: J
authors.initials: J
authors.lastname: Waddell
authors.forename: B A
authors.initials: BA
authors.lastname: Roelofs
authors.forename: M
authors.initials: M
authors.lastname: Karbowski
authors.forename: H
authors.initials: H
authors.lastname: Sesaki
authors.forename: B M
authors.initials: BM
authors.lastname: Polster
mesh_terms: D000818:Animals; D001713:Biphenyl Compounds / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D045304:Cytochromes c / Q000037:antagonists & inhibitors / Q000378:metabolism*; D034281:Dynamins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D024101:Mitochondrial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D009596:Nitrophenols / Q000494:pharmacology; D010101:Oxygen Consumption / Q000187:drug effects*; D010879:Piperazines / Q000494:pharmacology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D052999:Quinazolinones / Q000494:pharmacology*; D013449:Sulfonamides / Q000494:pharmacology; D015854:Up-Regulation; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: C549041:3-(2,4-dichloro-5-methoxyphenyl)-2-sulfanyl-4(3H)-quinazolinone; C501332:ABT-737; C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; D001713:Biphenyl Compounds; D008869:Microtubule-Associated Proteins; D024101:Mitochondrial Proteins; D009596:Nitrophenols; D010879:Piperazines; D019253:Proto-Oncogene Proteins c-bcl-2; D052999:Quinazolinones; D013449:Sulfonamides; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D020558:GTP Phosphohydrolases; C110768:DNM1L protein, human; C486391:Dnm1l protein, mouse; D034281:Dynamins
keywords: Bak; Bax; Bcl-xL; Bim; apoptosis; caspases; fission; mitochondria; oxygen; primed for death