Nuclear poly(ADP-ribose) polymerase-1 rapidly triggers mitochondrial dysfunction.
To obtain further information on time course and mechanisms of cell death after poly(ADP-ribose) polymerase-1 (PARP-1) hyperactivation, we used HeLa cells exposed for 1 h to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. This treatment activated PARP-1 and caused a rapid drop of cellular NAD(H) and ATP contents, culminating 8-12 h later in cell death. PARP-1 antagonists fully prevented nucleotide depletion and death. Interestingly, in the early 60 min after challenge with N-methyl-N'-nitro-N-nitrosoguanidine, mitochondrial membrane potential and superoxide production significantly increased, whereas cellular ADP contents decreased. Again, these events were prevented by PARP-1 inhibitors, suggesting that PARP-1 hyperactivity leads to mitochondrial state 4 respiration. Mitochondrial membrane potential collapsed at later time points (3 h), when mitochondria released apoptosis-inducing factor and cytochrome c. Using immunocytochemistry and targeted luciferase transfection, we found that, despite an exclusive localization of PARP-1 and poly(ADP-ribose) in the nucleus, ATP levels first decreased in mitochondria and then in the cytoplasm of cells undergoing PARP-1 activation. PARP-1 inhibitors rescued ATP (but not NAD(H) levels) in cells undergoing hyper-poly(ADP-ribosyl)ation. Glycolysis played a central role in the energy recovery, whereas mitochondria consumed ATP in the early recovery phase and produced ATP in the late phase after PARP-1 inhibition, further indicating that nuclear poly(ADP-ribosyl)ation rapidly modulates mitochondrial functioning. Together, our data provide evidence for rapid nucleus-mitochondria cross-talk during hyper-poly(ADP-ribosyl)ation-dependent cell death.
Cipriani
Giulia
G
Department of Pharmacology, University of Florence, 50139 Florence, Italy.
Rapizzi
Elena
E
Vannacci
Alfredo
A
Rizzuto
Rosario
R
Moroni
Flavio
F
Chiarugi
Alberto
A
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000477:Alkylating Agents / Q000494:pharmacology; D015153:Blotting, Western; D016923:Cell Death; D002467:Cell Nucleus / Q000201:enzymology* / Q000378:metabolism; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D006019:Glycolysis; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology; D015530:Nuclear Matrix / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry; D010100:Oxygen / Q000378:metabolism; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors
D000477:Alkylating Agents; D009711:Nucleotides; D013481:Superoxides; D008769:Methylnitronitrosoguanidine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D010100:Oxygen