Photo-oxidative Stress Down-modulates the Activity of Nuclear Factor-κB via Involvement of Caspase-1, Leading to Apoptosis of Photoreceptor Cells
1999; Elsevier BV; Volume: 274; Issue: 6 Linguagem: Inglês
10.1074/jbc.274.6.3734
ISSN1083-351X
AutoresRaghu R. Krishnamoorthy, Matthew J. Crawford, Madan M. Chaturvedi, Sushil K. Jain, Bharat B. Aggarwal, Muayyad R. Al‐Ubaidi, Neeraj Agarwal,
Tópico(s)Bioactive Compounds and Antitumor Agents
ResumoThe mechanisms of photoreceptor cell death via apoptosis, in retinal dystrophies, are largely not understood. In the present report we show that visible light exposure of mouse cultured 661W photoreceptor cells at 4.5 milliwatt/cm2 caused a significant increase in oxidative damage of 661W cells, leading to apoptosis of these cells. These cells show constitutive expression of nuclear factor-κB (NF-κB), and light exposure of photoreceptor cells results in lowering of NF-κB levels in both the nuclear and cytosolic fractions in a time-dependent manner. Immunoblot analysis of IκBα and p50, and p65 (RelA) subunits of NF-κB, suggested that photo-oxidative stress results in their depletion. Immunocytochemical studies using antibody to RelA subunit of NF-κB further revealed the presence of this subunit constitutively both in the nucleus and cytoplasm of the 661W cells. Upon exposure to photo-oxidative stress, a depletion of the cytoplasmic and nuclear RelA subunit was observed. The depletion of NF-κB appears to be mediated through involvement of caspase-1. Furthermore, transfection of these cells with a dominant negative mutant IκBα greatly enhanced the kinetics of down modulation of NF-κB, resulting in a faster photo-oxidative stress-induced apoptosis. Taken together, these studies show that the presence of NF-κB RelA subunit in the nucleus is essential for protection of photoreceptor cells against apoptosis mediated by an oxidative pathway. The mechanisms of photoreceptor cell death via apoptosis, in retinal dystrophies, are largely not understood. In the present report we show that visible light exposure of mouse cultured 661W photoreceptor cells at 4.5 milliwatt/cm2 caused a significant increase in oxidative damage of 661W cells, leading to apoptosis of these cells. These cells show constitutive expression of nuclear factor-κB (NF-κB), and light exposure of photoreceptor cells results in lowering of NF-κB levels in both the nuclear and cytosolic fractions in a time-dependent manner. Immunoblot analysis of IκBα and p50, and p65 (RelA) subunits of NF-κB, suggested that photo-oxidative stress results in their depletion. Immunocytochemical studies using antibody to RelA subunit of NF-κB further revealed the presence of this subunit constitutively both in the nucleus and cytoplasm of the 661W cells. Upon exposure to photo-oxidative stress, a depletion of the cytoplasmic and nuclear RelA subunit was observed. The depletion of NF-κB appears to be mediated through involvement of caspase-1. Furthermore, transfection of these cells with a dominant negative mutant IκBα greatly enhanced the kinetics of down modulation of NF-κB, resulting in a faster photo-oxidative stress-induced apoptosis. Taken together, these studies show that the presence of NF-κB RelA subunit in the nucleus is essential for protection of photoreceptor cells against apoptosis mediated by an oxidative pathway. Nuclear factor-κB (NF-κB) 1The abbreviations used are: NF-κB, nuclear factor-κB; EMSA, electrophoretic mobility shift assay; TUNEL, terminal deoxynucleotidyl transferase mediated fluoresceinated dUTP nick end labeling; GAPDH, glyceraldehyde phosphate dehydrogenase; GSH, glutathione-reduced; NAC, N-acetylcysteine; ALLN, N-acetylleucylleucylnorleucinal; IκBα, inhibitory subunit of NF-κB; IκB αΔN, super-repressor of IκB α; FITC, fluorescein isothiocyanate; TNF-α, tumor necrosis factor-α; ROI, reactive oxygen intermediates; MDCK, Madin-Darby canine kidney.1The abbreviations used are: NF-κB, nuclear factor-κB; EMSA, electrophoretic mobility shift assay; TUNEL, terminal deoxynucleotidyl transferase mediated fluoresceinated dUTP nick end labeling; GAPDH, glyceraldehyde phosphate dehydrogenase; GSH, glutathione-reduced; NAC, N-acetylcysteine; ALLN, N-acetylleucylleucylnorleucinal; IκBα, inhibitory subunit of NF-κB; IκB αΔN, super-repressor of IκB α; FITC, fluorescein isothiocyanate; TNF-α, tumor necrosis factor-α; ROI, reactive oxygen intermediates; MDCK, Madin-Darby canine kidney. is a widely distributed transcription factor that plays a role in the regulation of a number of cellular and viral genes involved in early defense reactions in higher organisms (1Baeuerle P.A. Hankel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4563) Google Scholar). NF-κB exists in an inactive form bound to the inhibitory protein IκBα or IκBβ (2Baeuerle P.A. Baltimore D. Cell. 1988; 53: 211-217Abstract Full Text PDF PubMed Scopus (789) Google Scholar, 3Thompson J. Phillips R. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (690) Google Scholar, 4Zabel U. Baeuerle P.A. Cell. 1990; 61: 255-265Abstract Full Text PDF PubMed Scopus (304) Google Scholar). Treatment of cells with inducers such as lipopolysaccharide, interleukin-1, and tumor necrosis factor-α (TNF-α), generally result in degradation of IκB proteins. This releases NF-κB of its inhibitory constraint, facilitating its translocation to the nucleus, resulting in regulation of expression of genes encoding cytokines, hematopoietic growth factors, and cellular adhesion molecules. NF-κB exhibits its DNA binding activity in its dimeric form, and the most commonly occurring dimer is that of the p50 and the p65 (RelA) subunits. NF-κB has been shown to be constitutively active in several cell types, including B cells (5Grilli M. Jason J.S. Lenardo M. Int. Rev. Cytol. 1993; 143: 1-62Crossref PubMed Scopus (880) Google Scholar), thymocytes (6Sen J. Venkataraman L. Shinkai Y. Pierce J. Alt F. Burakoff S. Sen R. J. Immunol. 1995; 154: 3213-3221PubMed Google Scholar), and neurons (7Kaltschmidt C. Kaltschmidt B. Neumann H. Wekerle H. Baeuerle P.A. Mol. Cell. Biol. 1994; 14: 3981-3992Crossref PubMed Google Scholar). Most of the earlier studies on NF-κB focused on its role in immunological and inflammation responses (1Baeuerle P.A. Hankel T. Annu. Rev. Immunol. 1994; 12: 141-179Crossref PubMed Scopus (4563) Google Scholar, 8Liou H.-C. Baltimore D. Curr. Opin. Cell Biol. 1993; 5: 477-487Crossref PubMed Scopus (516) Google Scholar, 9Siebenlist U. Fanzoso G. Brown K. Annu. 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In the current studies we have used a transformed mouse photoreceptor cell line 661W. 2M. J. Crawford, R. R. Krishnamoorthy, H. J. Sheedlo, D. T. Organisciak, R. S. Roque, N. Agarwal, and M. R. Al-Ubaidi, submitted for publication. 2M. J. Crawford, R. R. Krishnamoorthy, H. J. Sheedlo, D. T. Organisciak, R. S. Roque, N. Agarwal, and M. R. Al-Ubaidi, submitted for publication. In vivo studies have shown that exposure of rats to constant light results in apoptosis of photoreceptor cells (25Abler A.S. Chang C.J. Fu J. Tso M.O.M. Investig. Ophthalmol. Vis. Sci. 1994; 35 (suppl.): 1517Google Scholar, 26Tso M.O.M. Zhang C. Abler A.S. Chang C.J. Wong F. Chang G.Q. Lam T.T. Investig. Ophthalmol. Vis. Sci. 1994; 35: 2693-2699PubMed Google Scholar, 27Li S. Chang C.J. Abler A.S. Tso M.O.M. Anderson R.E. LaVail M.M. Hollyfield J.G. Degenerative Diseases of the Retina. Plenum Press, New York1995: 27-38Crossref Google Scholar, 28Organisciak D.T. Kutty R.K. Leffak M. Wong P. Messing S. Wiggert B. 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Winkler B.S. Osborne N.N. Chader G.J. Progress in Retinal and Eye Research. 13. Elsevier Science Publishing Co., Inc., New York1994: 1-29Google Scholar) and in vitro experimental conditions. In the current study, we assessed the contribution of an oxidative stress paradigm to the propensity of photoreceptor cells to proceed to cell death via apoptosis, using cultured photoreceptor cells. We provide evidence in this paper that visible light exposure to photoreceptor cells results in oxidative damage leading to apoptosis via down-modulation of NF-κB. NF-κB, which was constitutively expressed in the 661W cells, was found to be progressively down-regulated upon exposure of the cells to light. By immunocytochemistry using NF-κB RelA antibody, the NF-κB activity appeared to be localized both in the nucleus and cytoplasm of dark-exposed 661W cells. Upon exposure to light the nuclear and cytoplasmic NF-κB RelA immunolabeling was largely diminished in these cells. Pretreatment of the cells with various antioxidants prevented to a great extent the down-modulation of NF-κB and also protected the cells from apoptosis. Furthermore, transient transfection of the 661W cells with a dominant negative IκBαΔN (super-repressor) caused a rapid decline in NF-κB binding activity in the cells, leading to a faster kinetics of photo-oxidative stress-induced apoptosis. Down-modulation of NF-κB in these cells appears to be mediated by caspase-1. Our results suggest that NF-κB, which is constitutively expressed in 661W photoreceptor cells, undergoes degradation when subjected to oxidative stress leading to apoptosis of the photoreceptor cells. Thus, the presence of NF-κB in the nucleus is essential for photoreceptor cell survival and protection against oxidative stress induced apoptosis. The following materials were purchased from the indicated sources: fetal bovine serum from JRH Biosciences, Lenexa, KS; paraformaldehyde and H2O2 from EM Sciences, Gibbstown, NJ; HEPES, phenylmethylsulfonyl fluoride, ALLN, and dithiothreitol from Sigma; poly(dI-dC)·poly(dI-dC) from Amersham Pharmacia Biotech; and polynucleotide kinase from New England Biolabs, Beverly, MA. p50 subunit of NF-κB, a goat polyclonal IgG; p65, of NF-κB, a rabbit polyclonal IgG; and IκBα rabbit polyclonal IgG were from Santa Cruz Biotechnology, Santa Cruz, CA. GAPDH (chicken anti-rabbit GAPDH immunoaffinity-purified monospecific antibody) was kindly supplied by Drs. Glaser and Cross (34Glaser P.E. Cross R.W. Biochemistry. 1995; 34: 12193-12203Crossref PubMed Scopus (166) Google Scholar). β-Tubulin, a mouse monoclonal antibody, was from Sigma. Peroxidase-labeled secondary antibodies either anti-rabbit IgG or anti-mouse IgG were from Kirkegaard and Perry Laboratories Inc., Gaithersburg, MD. Anti-cyclin D1 antibody was against amino acids 1–295, which represents full-length cyclin D1 of human origin, and was obtained from Santa Cruz Biotechnology. Fluorescein isothiocyanate-labeled anti-rabbit IgG from Vector Laboratories, Burlingame, CA. The 661W cells were originally isolated from a transgenic mouse line expressing the construct HIT1 comprising of SV40 T-antigen driven by the human interphotoreceptor retinol binding protein promoter (35Al-Ubaidi M.R. Font R.L. Quiambao A.B. Keener M.J. Liou G.I. Overbeek P.A. Baehr W. J. Cell Biol. 1992; 119: 1681-1687Crossref PubMed Scopus (182) Google Scholar). The construct HIT1 resulted in SV40 T-antigen expression and retinal and brain tumors. 661W cells are routinely grown in complete medium consisting of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 1% penicillin/streptomycin, at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The 661W cells were seeded either in 35/60/100-mm tissue culture dishes or on round coverslips kept in 35-mm dishes and exposed to fluorescent visible light at 4.5 milliwatt/cm2 for varying durations up to 4 h at 37 °C in tissue culture. The accompanying control cells were shielded from light for similar intervals and left in similar conditions as the cells in light-exposed paradigm. The TUNEL procedure as described by Gavrieli et al. (36Gavrieli Y. Sherman Y. Ben-Sasson S.A. J. Cell Biol. 1992; 119: 490-501Crossref Scopus (9100) Google Scholar) was employed to study apoptosis, using a commercially available apoptosis kit (in situ cell death detection kit, Boehringer Mannheim) as per the supplier's instructions. The membrane lipid peroxidation of light-exposed cultured cells was studied by measuring the malonyldialdehyde levels by a colorimetric method involving thiobarbituric acid adduct formation (37Jain S.K. J. Biol. Chem. 1989; 264: 21340-21345Abstract Full Text PDF PubMed Google Scholar). The GSH levels in light-exposed cells was studied by using the 5,5′-dithiobis(2-nitrobenzoic acid) reagent (38Beutler E. Duronand O. Kelley B.M. J. Lab. Clin. Med. 1963; 61: 882-890PubMed Google Scholar). Protein extracts from 661W cultured cells exposed to light were subjected to immunoblot analysis (39Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44642) Google Scholar, 40Agarwal N. Nir I. Papermaster D.S. J. Neurosci. 1991; 10: 3275-3285Crossref Google Scholar) using specific antibodies for IκBα, p50, and RelA subunit of NF-κB at 1:500 dilution. Cytoplasmic extracts were used for IκBα analysis, whereas nuclear extracts were used to study RelA and p50 subunits of NF-κB. Control blots were run using total cellular extracts and an antibody to GAPDH at 1:1000 dilution. The binding of primary antibodies was detected by using peroxidase labeled appropriate secondary antibodies, which were detected by using diaminobenzidine as substrate. The 661W cells were exposed to light and fixed in 4% paraformaldehyde. The immunofluorescence for p65 subunit of NF-κB was done by using a specific antibody against p65 and a fluorescein isothiocyanate-labeled goat anti-rabbit secondary antibody (20Agarwal N. Jomary C. Jones S.E. O'Rourke K. Chaitin M.H. Wordinger R.J. Murphy B.F. Biochem. Biophys. Res. Commun. 1996; 225: 84-91Crossref PubMed Scopus (25) Google Scholar). The immunofluorescent cells were photographed using a Nikon Microphot-FXA photomicroscope. The 661W cells were exposed to light for the desired amount of time, and the nuclear and cytoplasmic extracts were prepared (41Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2207) Google Scholar). Briefly, the cells were suspended in 100 μl of buffer C (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 10% glycerol, 1 mm dithiothreitol, 0.5 mmphenylmethylsulfonyl fluoride) and incubated on ice for 15 min. 3 μl of 10% Nonidet P-40 was added to the suspension and briefly vortexed. Following this, the nuclei were pelleted by centrifugation at low speed. The supernatant (cytoplasmic extract) was collected and stored at −80 °C. The nuclear pellet was resuspended in 70 μl of buffer D (20 mm HEPES, pH 7.9, 400 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 20% glycerol, 1 mm dithiothreitol, 0.5 mmphenylmethylsulfonyl fluoride). The suspension was incubated for 20 min at 4 °C followed by a centrifugation at 8000 g for 5 min. The supernatant containing the nuclear protein extract was transferred to a fresh microcentrifuge tube and stored at −80 °C. Protein concentrations of the cytoplasmic and the nuclear extracts were measured with a detergent-compatible Protein Assay Kit (Bio-Rad), using bovine serum albumin as a standard. A double-stranded oligonucleotide containing the NF-κB DNA-binding consensus sequence, 5′-AGT TGA GGG GAC TTT CCC AGG C-3′, and a double-stranded mutant oligonucleotide, 5′-AGT TGAGGC GAC TTT CCC AGG C-3′ (Santa Cruz Biotechnology) were used to study the DNA binding activity of NF-κB by EMSA as described (42Shen Y. Rattan V. Sultana C. Kalra V.K. Am. J. Physiol. 1996; 270: H1624-H1633Crossref PubMed Google Scholar). For supershift assay, 4 μg of nuclear extract was incubated with 1 μg of antibodies for 30 min at room temperature and analyzed by EMSA. The dominant negative IκBα (super-repressor) construct IκBαΔN was obtained from Dr. Dean Ballard, Vanderbilt University, Nashville, TN (43Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar). IκBαΔN is a deletion mutant in which the N-terminal 36 amino acids are deleted from the IκBα protein. The 661W cells were transiently transfected with the construct using the LipofectAMINE reagent (Life Technologies, Inc.), as per the manufacturer's instructions using 8 μl of LipofectAMINE and 5 μg of the IκBα super-repressor plasmid DNA for 1 ml of transfection mix. The untransfected control cells were treated in a similar manner except for the exclusion of the plasmid DNA. The transfected cells and their controls were used 48 h post-transfection for making either total cellular extract for immunoblot analysis or cytoplasmic and nuclear extracts for EMSAs and for TUNEL assay as described above. The 661W cells were exposed to light for up to 4 h, and membrane lipid peroxide formation and GSH levels were measured. There was almost a 2-fold increase in malonyldialdehyde formation following light exposure, as compared with controls (TableI) and inclusion of NAC in the medium before light exposure of cultured cells, prevented the increase in MDA levels (Table I). These results indicate that photic injury to photoreceptor cells occurs due to a possible involvement of an oxidative pathway. To explore this possibility further, we used other anti-oxidants such as thiourea and mannitol in our studies. As shown, photo-oxidative stress resulted in significant lowering of GSH levels as compared with control cells maintained in dark. The presence of thiourea (7 mm) in the medium of 661W cells was protective against photo-oxidative damage, as seen by the maintenance of GSH levels close to control values (Table I).Table IEffect of photo-oxidative stress on malonyldialdehyde formation and glutathione levels in cultured photoreceptor cellsTreatmentMDAGSH levelsnmol/mg proteinμmol/mg proteinControls0.306 ± 0.109 (n = 4)0.0258 ± 0.005 (n = 3)Light-exposed0.598 ± 0.040 (n = 4)aSignificant at p < 0.01.0.010 ± 0.003 (n = 3)aSignificant at p < 0.01.N-Acetylcysteine (2 mm) pretreated and light-exposed0.197 ± 0.067 (n = 4)bSignificant at p < 0.05.NDcND = not determined.Thiourea (7 mm) pretreated and light-exposedND0.037 ± NDThe values are expressed as mean ± S.E. of mean. Statistical analysis was done by Student's t test.a Significant at p < 0.01.b Significant at p < 0.05.c ND = not determined. Open table in a new tab The values are expressed as mean ± S.E. of mean. Statistical analysis was done by Student's t test. The photoreceptor cells were exposed to light for various time intervals for 15, 30, and 60 min. There was no change in NF-κB activity up to 30 min of light exposure in both the nucleus (Fig. 1 a, lanes 2and 3 for nucleus) and cytoplasm (Fig. 1 a,lanes 6 and 7) compared with dark-exposed control cells (Fig. 1 a, lanes 1 and 5, for nucleus and cytoplasm, respectively). Upon 60 min of light exposure, there was approximately 80% loss of NF-κB binding activity in both the nucleus and cytoplasm (lanes 4 and 8, respectively). These results indicate that the cultured photoreceptor cells express NF-κB constitutively and that the activity of NF-κB decreases on exposure to photo-oxidative stress.Figure 3Immunoblot analysis of NF-κB p50, RelA, IκB α subunit in 661W cells exposed to photo-oxidative stress. The cells were exposed to light for 2 h in presence or absence of NAC (2 mm) and thiourea (7 mm), and the levels of NF-κB subunits p50, RelA, and IκBα subunit were studied by immunoblot analysis. There was a decrease in the levels of IκBα, p50, and RelA subunit of NF-κB upon light exposure (lane 2), and these were protected to varying extents by pretreatment of the cells with NAC and thiourea (lanes 3 and 4), respectively. The GAPDH levels were not altered between the experimental groups studied. "D" and "L" represent dark- and light-exposed 661W cells, respectively. "Nac" and "Thio" indicate N-acetylcysteine and thiourea, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The specificity of the binding of NF-κB was shown by competition with cold NF-κB consensus oligonucleotide (Fig. 1 b, lanes 2–4). As expected, a lack of competition was observed with cold NF-κB mutant oligonucleotide (Fig. 1 b, lanes 6–8). The DNA protein complex seen in the EMSA appears to be a heterodimer of p50 and p65 subunits of NF-κB, as revealed by a decrease in the binding upon additional incubation with the antibodies to the p65 and p50 (Fig. 1 c) subunits, in a supershift assay. An unrelated antibody, anti-cyclin D1 used as a negative control, did not inhibit the DNA protein complex formation (Fig.1 c). These results confirm the identity of the NF-κB DNA-protein complex seen in the EMSAs. In order to establish the involvement of oxidative damage in the lowering of NF-κB activity during conditions of photo-oxidative stress, we studied the effects of antioxidants, namely NAC, mannitol, and thiourea under these conditions (Fig.2 a). Lanes 1,5, and 9 represent NF-κB binding activity in dark-exposed cells. Lanes 2, 6, and 10represent NF-κB binding activity in dark-exposed cells in the presence of NAC, mannitol, and thiourea, respectively. The presence of these anti-oxidants did not appreciably alter the NF-κB binding activity in dark-exposed control cells. As expected, the activity of NF-κB decreased on exposure to photo-oxidative stress (lanes 3, 7, and 11). The inclusion of NAC, mannitol, and thiourea in the growth medium prior to light exposure partially protected against the down-modulation of NF-κB (Fig.2 a, lanes 4, 8, and 12, respectively) in light-exposed 661W cells. The differences in the extent of protection of NF-κB levels in these groups may be attributed to differences in efficacy of these anti-oxidants in affording protection against photo-oxidative damage. These results indicate that oxidative damage plays a major role in decreasing the NF-κB activity in cultured photoreceptor cells exposed to light. It remains to be seen if these antioxidants offer an additive protection of NF-κB levels, if used simultaneously. Our results so far suggest that light may down-modulate NF-κB through generation of ROIs. To further confirm the role of ROIs in this process we treated the cells with H2O2for various times up to 120 min and measured NF-κB activation and apoptosis. The EMSA revealed no significant change in the NF-κB binding activity in these cells treated with 300 μmH2O2 for 30 and 60 min (Fig. 2 b,lanes 2 and 3 and lanes 6 and 7 for nuclear and cytoplasmic extracts, respectively), compared with untreated control cells (lanes 1 and5 for nucleus and cytoplasm, respectively). However, treatment of H2O2 for 120 min resulted in a modest increase in the NF-κB binding activity both in the nucleus (lane 4) and the cytoplasm (lane 8). The TUNEL assay revealed no significant increase in the number of apoptotic cells on treatment with H2O2 compared with untreated controls for all the durations of H2O2treatment (data not shown). Therefore, this data indicate that ROIs alone are not sufficient for light induced down-regulation of NF-κB and activation of apoptosis in these cells. To assess the specificity of response of 661W cells to photo-oxidative stress, we studied the effect of light exposure on MDCK cells, using them as an unrelated control. The light exposure of MDCK cells did not cause a decrease in NF-κB binding activity, in both the nucleus and cytoplasm (data not shown). These results indicate that the cell-specific response of 661W cells to light is different from that of MDCK cells. To further confirm the down-modulation of NF-κB, the protein levels of IκBα, p50, and RelA subunits of NF-κB were studied in 661W cells exposed to light, with or without pretreatment with NAC and thiourea, by immunoblot analysis using specific antibodies. The light-exposed cells showed lowering of IκBα, p50, and RelA subunit of NF-κB, as compared with dark controls. Pretreatment of the cells with both anti-oxidants protected the levels of NF-κB p50, RelA, and IκBα subunits, albeit partially, upon exposure to light (Fig.3). To ensure that light exposure does not result in a generalized protein degradation, a control protein GAPDH was included, which was not greatly altered in all samples under these experimental conditions. On quantitation, there was a ≅50% decrease of IκBα protein levels on 2-h light exposure. On the other hand, there was ≅90% decrease in p50 and RelA subunit with no change in GAPDH levels under similar conditions. Inclusion of anti-oxidants protected to a large extent against degradation of these proteins. IκBα was protected 100%, whereas p50 and p65 were protected to 40–45% of control values. Based on the results of GAPDH protein levels, these data suggest that down-modulation of IκBα and p50 and p65 subunit of NF-κB is not due to random protein degradation. To establish that oxidative damage along with a down-modulation of NF-κB results in apoptosis of these cells, we studied the effect of photo-oxidative stress on apoptosis of these cells in presence or absence of the anti-oxidant, NAC. We found that exposure of 661W cells to light up to 1 h did not result in any significant apoptosis of cultured photoreceptor cells (Fig.4 A). However, after 2 and 4 h of light exposure, many cells underwent apoptosis (Fig. 4,C and E, respectively), compared with dark-exposed control cells as seen by incorporation of fluoresceinated dUTP in the nuclei of apoptotic cells containing fragmented DNA. Approximately, 80% of the cells were seen to undergo apoptosis in 661W cells exposed to light for 4 h (Fi
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