Photoreceptor cell apoptosis induced by the 2-nitroimidazole radiosensitizer, CI-1010, is mediated by p53-linked activation of caspase-3.
The nitroimidazole radiosensitizer CI-1010 ((R)-alpha-[[(2-bromoethyl)-amino]methyl]-2-nitro-1H-imidazole-1-ethanol monohydrobromide) causes selective, irreversible, retinal photoreceptor apoptosis in vivo. The mouse 661 W photoreceptor cell line was used as a neuronotypic model of CI-1010-mediated retinal degeneration. Exposure to CI-1010 for 24 h induced apoptosis in 661 W cells, as determined by ultrastructural analysis, agarose electrophoresis and analysis of TUNEL-positive nuclei. CI-1010 caused a loss of viability in 661 W cells, as measured by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). A clear link was established between the onset of apoptosis and activity of caspase-3 and caspase-8, prior to poly[ADP-ribose]polymerase (PARP) cleavage. Pretreatment with caspase inhibitors, ZVAD.fmk or DEVD-CHO, prevented morphological changes in most CI-1010-treated cells. Evaluation of mitochondrial inner membrane potential (Deltapsi(m)) in live 661 W cells using the fluorescent dye, tetramethylrhodamine methyl ester revealed retention of (Deltapsi(m)) until after caspase activation. Absence of cytochrome c in the cytoplasm in treated cells further supports the hypothesis of a mitochondrial-independent mechanism of cell death. Significant increase in DNA crosslinks observed in 661 W cells correlates with induction and phosphorylation of p53 at multiple serine sites. Cell cycle analysis of 661 W cells reveals a G(2) arrest in response to CI-1010-induced DNA damage and neuronal cell death. Increased protein expression of Bax, Fas, and FasL, concomitant to the loss of Bcl-xL in treated 661 W cells may be modulated by p53. This study evaluates in vitro mechanisms of CI-1010-induced cell death in photoreceptors and provides evidence in support of a p53-linked activation of caspase-3 in response to DNA damage caused by CI-1010.
Miller
Terry J
TJ
Toxicology Program, Department of Environmental Health Sciences, University of Michigan, 1420 Washington Heights, SPH II, Ann Arbor, MI 48109-2029, USA.
Schneider
Randal J
RJ
Miller
James A
JA
Martin
Brad P
BP
Al-Ubaidi
Muayyad R
MR
Agarwal
Neeraj
N
Dethloff
Lloyd A
LA
Philbert
Martin A
MA
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D002461:Cell Line, Transformed; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D053222:Fas Ligand Protein / Q000096:biosynthesis / Q000378:metabolism; D005434:Flow Cytometry; D051379:Mice; D009593:Nitroimidazoles; D010786:Photoreceptor Cells / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011838:Radiation-Sensitizing Agents; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000096:biosynthesis / Q000378:metabolism; D051020:bcl-X Protein / Q000096:biosynthesis / Q000378:metabolism
C494827:Bax protein, mouse; C494812:Bcl2l1 protein, mouse; D053222:Fas Ligand Protein; D009593:Nitroimidazoles; D011838:Radiation-Sensitizing Agents; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; C067834:RB 6145; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D053148:Caspase 3