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First published online 26 March 2003
doi: 10.1242/jcs.00390

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Oxidant-induced cell death in retinal pigment epithelium cells mediated through the release of apoptosis-inducing factor

¹ National Eye Institute, Building 10 – Room 10N119, 9000 Rockville Pike, Bethesda, Maryland 20892-1857, USA
² Department of Ophthalmology, University of Miami, Miami, Florida

View larger version (95K): [in a new window]   Fig. 1. Cytoxocity and morphology of cells exposed to menadione. (A) Dependence of U937 () and ARPE-19 () cell survival on menadione concentration. 1x104 cells were exposed to menadione (1 µM to 250 µM) for 4 hours and allowed to recover for 24 hours. Cell viability was determined by XTT assay as described in Materials and Methods. Data are represented as mean±s.d. (n=3). (B) Both U937 cells and ARPE-19 cells were exposed to 50 µM menadione for 4 hours and were observed immediately under inverted microscopy. (Inserts) Control ARPE-GFP cells in which GFP is localized to the membrane (left, arrowheads) under similar conditions were observed under confocal fluorescent microscopy and showed the presence of membrane blebs (right, arrows). Images represent three independent experiments. Bar, top, 50 µm; bottom and inserts, 30 µm.

View larger version (49K): [in a new window]   Fig. 2. Detection of caspase cleavage in whole cells and cell-free extracts. (A) 50-100 µg of whole lysates of U937 and ARPE-19 cells were analyzed by western blotting using antibodies against both pro- and cleaved forms of caspase-9 (top), -3 (middle) and PARP (bottom) in control cells and 4 hours after exposure to 50 µM menadione. (B) Cytosolic extracts of 5x104 cells were prepared as described in Materials and Methods, incubated with 10 µM of cytochrome c and 1 mM ATP for 37°C for 2 hours, separated on a reducing gel and analyzed by western blotting using antibodies against caspase-9 (top) and -3 (bottom). (C) ARPE-19 cells were preincubated with Z-VAD.fmk (100 µM) for 1 hour and exposed to various concentrations of menadione (1 µM to 100 µM) for 2 hours. Cell viability was determined using the XTT assay as indicated in Fig.1A. (), menadione alone; (), menadione with Z-VAD.fmk.

View larger version (35K): [in a new window]   Fig. 3. Nuclear changes in apoptotic cells. (A) Following exposure to 50 µM menadione for 6 hours, cells were fixed in 4% paraformaldehyde for 5 minutes and put on a coverslip with DAPI-containing mounting media and observed under epi-flurorescent microscopy. Bar, 10 µm. (B) 10 µg genomic DNA from U937 and ARPE-19 cells exposed to 50 µM menadione for 6 hours was separated either by 1.0% agarose gel electrophoresis (left) or pulse field gel electrophoresis (right) and visualized under UV illuminescence.

View larger version (48K): [in a new window]   Fig. 4. Mitochondrial permeability changes during apoptotic cell death in ARPE-19 cells. (A) ARPE-19 cells, treated with 50 µM menadione for 4 hours were incubated with CMXRos, fixed, immunolabeled with anti-AIF antibodies and put on a coverslip with DAPI-containing mounting media. In control cells (top), immunostaining of AIF colocalized with CMXRos and was excluded from the DAPI-labeled nuclei. In menadione-treated cells (bottom), AIF is colocalized with DAPI-labeled nuclei, and CMXRos staining is diminished. Bar, 10 µm. (B) Redistribution of AIF and cytochrome c during ARPE-19 cell death. Whole cells or subcellular extracts of control or ARPE-19 cells treated with 50 µM menadione for 4 hours were analyzed for AIF and cytochrome c translocation using western blot analysis. Actin was used to normalize whole cell and cytosolic loading. Cytochrome c oxidase II and Oct-1 were used for normalization of mitochondrial and nuclear fraction loading, respectively. (Data represent two independent experiments). (C) The values are the percentage of ARPE-19 cells treated as in A displaying nuclear AIF and loss of CMXRos staining obtained by averaging three fields per slide in which approximately 150 cells per slide were counted. The results shown are mean±s.d., which are representative of two independent experiments. (*, P<0.001, ANOVA/t-test, unpaired).

View larger version (37K): [in a new window]   Fig. 5. HGF/SF treatment of apoptotic ARPE-19 cells. (A) ARPE-19 cells were incubated with HGF/SF (50 ng/ml for 48 hours) and subsequently exposed to menadione (50 µM for 2 hours). After 24 hours recovery, cell viability was determined using a XTT assay as described in Fig. 1A. (B) Cells either left untreated or treated with HGF, menadione alone or HGF/SF + menadione as described in (A). AIF immunohistochemistry was performed as in Materials and Methods. The value is the percentage of cells displaying nuclear AIF obtained by an average of three fields per slide in which approximately 150 cells per slide were counted. The results shown are mean +/– std, representative of two independent experiments. (*, P<0.001, ANOVA/t-test, unpaired). (C) Appearance of ARPE-19 cells treated with menadione and HGF/SF as in (A). Immunohistochemistry for AIF and counter-staining with CMXRos or DAPI was performed as in Fig. 4A.

View larger version (29K): [in a new window]   Fig. 6. HGF/SF effects on AIF nuclear distribution and large scale DNA fragmentation. Nuclear extracts were analyzed by western blotting for AIF (top) obtained 6 hours following either no treatment or treatment with HGF/SF (50 ng/ml for 48 hours) and menadione (50 µM for 2 hours). The blot was stripped and re-blotted with anti-Oct-1 antibody (middle) to normalize for protein loading. Pulse field gel electrophoresis was performed (as described in Materials and Methods) on 10 µg of genomic DNA from ARPE-19 cells exposed to similar conditions (bottom).