Cells Preconditioned with Mild, Transient UVA Irradiation Acquire Resistance to Oxidative Stress and UVA-induced Apoptosis
2003; Elsevier BV; Volume: 278; Issue: 42 Linguagem: Inglês
10.1074/jbc.m305766200
ISSN1083-351X
AutoresYusong Yang, Abha Sharma, Rajendra Sharma, Brad Patrick, Sharad S. Singhal, Piotr Zimniak, Sanjay Awasthi, Sanjay Awasthi,
Tópico(s)Vitamin C and Antioxidants Research
ResumoBecause 4-hydroxynonenal (4-HNE) has been suggested to be involved in oxidative stress-mediated apoptosis (Cheng, J. Z., Sharma, R., Yang, Y., Singhal, S. S., Sharma, A., Saini, M. K., Singh, S. V., Zimniak, P., Awasthi, S., and Awasthi, Y. C. (2001) J. Biol. Chem. 276, 41213–41223) and UVA irradiation also causes lipid peroxidation, we have examined the role of 4-HNE in UVA-mediated apoptosis. K562 cells irradiated with UVA (3.0 milliwatts/cm2) for 5, 15, and 30 min showed a time dependent increase in 4-HNE levels. As judged by the activation of caspases, apoptosis was observed only in cells irradiated for 30 min. Within 2 h of recovery in normal medium, 4-HNE levels in 5 and 15 min UVA, irradiated cells returned to the basal or even lower levels but in cells irradiated for 30 min, 4-HNE levels remained consistently higher. The cells irradiated with UVA for 5 min and allowed to recover for 2 h in normal medium (UVA-preconditioned cells) showed a remarkable induction of hGST5.8, which catalyzes conjugation of 4-HNE to glutathione (GSH), and RLIP76 (Ral BP-1), which mediates the transport of the conjugate, GS-HNE. In cells irradiated with UVA for 30 min the induction of RLIP76 or hGST5.8 was not observed. The preconditioned cells transported GS-HNE into the medium at a rate about 2-fold higher than the controls and the transport was inhibited (65%) by coating the cells with anti-RLIP76 IgG. Upon treatment with xanthine/xanthine oxidase (XA/XO), 4-HNE, or prolonged UVA exposure, the control cells showed a sustained activation of c-Jun N-terminal kinase (JNK) and apoptosis. However, in the UVA-preconditioned cells, apoptosis was not observed, and JNK activation was inhibited. This resistance of preconditioned cells to XA/XO-, 4-HNE-, or UVA-induced apoptosis could be abrogated when these cells were coated with anti-RLIP76 IgG to block the efflux of GS-HNE. These studies strongly suggest a role of 4-HNE in UVA-mediated apoptosis. Because 4-hydroxynonenal (4-HNE) has been suggested to be involved in oxidative stress-mediated apoptosis (Cheng, J. Z., Sharma, R., Yang, Y., Singhal, S. S., Sharma, A., Saini, M. K., Singh, S. V., Zimniak, P., Awasthi, S., and Awasthi, Y. C. (2001) J. Biol. Chem. 276, 41213–41223) and UVA irradiation also causes lipid peroxidation, we have examined the role of 4-HNE in UVA-mediated apoptosis. K562 cells irradiated with UVA (3.0 milliwatts/cm2) for 5, 15, and 30 min showed a time dependent increase in 4-HNE levels. As judged by the activation of caspases, apoptosis was observed only in cells irradiated for 30 min. Within 2 h of recovery in normal medium, 4-HNE levels in 5 and 15 min UVA, irradiated cells returned to the basal or even lower levels but in cells irradiated for 30 min, 4-HNE levels remained consistently higher. The cells irradiated with UVA for 5 min and allowed to recover for 2 h in normal medium (UVA-preconditioned cells) showed a remarkable induction of hGST5.8, which catalyzes conjugation of 4-HNE to glutathione (GSH), and RLIP76 (Ral BP-1), which mediates the transport of the conjugate, GS-HNE. In cells irradiated with UVA for 30 min the induction of RLIP76 or hGST5.8 was not observed. The preconditioned cells transported GS-HNE into the medium at a rate about 2-fold higher than the controls and the transport was inhibited (65%) by coating the cells with anti-RLIP76 IgG. Upon treatment with xanthine/xanthine oxidase (XA/XO), 4-HNE, or prolonged UVA exposure, the control cells showed a sustained activation of c-Jun N-terminal kinase (JNK) and apoptosis. However, in the UVA-preconditioned cells, apoptosis was not observed, and JNK activation was inhibited. This resistance of preconditioned cells to XA/XO-, 4-HNE-, or UVA-induced apoptosis could be abrogated when these cells were coated with anti-RLIP76 IgG to block the efflux of GS-HNE. These studies strongly suggest a role of 4-HNE in UVA-mediated apoptosis. UVA irradiation affects cellular signaling mechanisms and is known to cause the activation of transcription factors such as NF-κB and the stress-related kinases including extracellularly regulated kinase (ERK), c-Jun N-terminal kinase (JNK), 1The abbreviations used are: JNK, c-Jun N-terminal kinase; ROS, reactive oxygen species; 4-HNE, 4-hydroxy-2-nonenal; GST, glutathione S-transferase; RLIP76, 76-kDa Ral-binding GTPase-activating protein (RalBP1); GPx, glutathione peroxidase; SOD, superoxide dismutase; CAT, catalase; XA/XO, xanthine/xanthine oxidase; GS-HNE, glutathione conjugate of 4-HNE; K562 cells, human erythroleukemia cells; CDNB, 1-chloro-2,4-dinitrobenzene; DAPI, 4′,6-diamidino-2-phenylindole; SAPK, stress-activated protein kinase; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline. and p38 in different cell lines (1Vile G.F. Tanew-Ilitschew A. Tyrrell R.M. Photochem. Photobiol. 1995; 62: 463-468Crossref PubMed Scopus (133) Google Scholar, 2Zhang Y. Zhong S. Dong Z. Chen N. Bode A.M. Ma W. Dong Z. J. Biol. Chem. 2001; 276: 14572-14580Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). UVA causes oxidative stress in cells via the formation of reactive oxygen species (ROS) including singlet oxygen and hydrogen peroxide, which besides damaging other cellular constituents including DNA, can initiate lipid peroxidation in membranes (3Morliere P. Moysan A. Santus R. Huppe G. Maziere J.C. Dubertret L. Biochim. Biophys. Acta. 1991; 1084: 261-268Crossref PubMed Scopus (144) Google Scholar, 4Girotti A.W. J. Photochem. Photobiol. 2001; B 63: 103-113Crossref Scopus (479) Google Scholar, 5De Gruijl F.R. Skin Pharmacol. Appl. Skin. Physiol. 2002; 15: 316-320Crossref PubMed Scopus (245) Google Scholar). UVA irradiation causes the release of iron through degradation of ferritin, which can potentiate ROS-induced oxidative damage and lipid peroxidation (6Pourzand C. Watkin R.D. Brown J.E. Tyrrell R.M. Proc. Natl. Acad. Sci. U. S. 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Exposure of cells to 4-HNE causes a wide range of biological outcomes such as necrosis, apoptosis, differentiation, and proliferation in a concentration-dependent manner (9Cheng J.Z. Singhal S.S. Saini M. Singhal J. Piper J.T. Van Kuijk F.J. Zimniak P. Awasthi Y.C. Awasthi S. Arch Biochem. Biophys. 1999; 372: 29-36Crossref PubMed Scopus (111) Google Scholar, 10Kruman I. Bruce-Keller A.J. Bredesen D. Waeg G. Mattson M.P. J. Neurosci. 1999; 17: 5089-5100Crossref Google Scholar, 11Ruef J. Rao G.N. Li F. Bode C. Patterson C. Bhatnagar A. Runge M.S. Circulation. 1998; 97: 1071-1078Crossref PubMed Scopus (135) Google Scholar). The intracellular concentrations of 4-HNE are regulated by a coordinated action of specific glutathione S-transferase isozymes that catalyze the Michael addition of 4-HNE to GSH to form the conjugate GS-HNE, and transporters, including RLIP76 (RalBP1), which catalyze the ATP-dependent efflux of GS-HNE from cells (12Cheng J.Z. Sharma R. Yang Y. Singhal S.S. Sharma A. 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Awasthi Y.C. Biochim. Biophys. Acta. 1994; 1204: 279-286Crossref PubMed Scopus (63) Google Scholar, 18Hubatsch I. Ridderstrom M. Mannervik B. Biochem. J. 1998; 330: 175-179Crossref PubMed Scopus (314) Google Scholar). Transfection of HL-60 and K562 cells with murine enzyme mGSTA4-4 with substrate preference to 4-HNE has been shown to protect cells from apoptosis caused by oxidative stress as well as 4-HNE (9Cheng J.Z. Singhal S.S. Saini M. Singhal J. Piper J.T. Van Kuijk F.J. Zimniak P. Awasthi Y.C. Awasthi S. Arch Biochem. Biophys. 1999; 372: 29-36Crossref PubMed Scopus (111) Google Scholar, 19Cheng J.Z. Singhal S.S. Sharma A. Saini M. Yang Y. Awasthi S. Zimniak P. Awasthi Y.C. Arch. Biochem. Biophys. 2001; 392: 197-207Crossref PubMed Scopus (101) Google Scholar). Also, a concomitant induction of hGST5.8 and RLIP76 has been shown to provide protection to several human cell lines in culture against oxidative stress-mediated apoptosis (12Cheng J.Z. Sharma R. Yang Y. Singhal S.S. Sharma A. Saini M.K. Singh S.V. Zimniak P. Awasthi S. Awasthi Y.C. J. Biol. Chem. 2001; 276: 41213-41223Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Previous studies with a number of cell lines of human origin have demonstrated that cells preconditioned with mild, transient oxidative stress or heat shock acquire resistance to apoptosis caused by oxidative stress through an accelerated metabolism and exclusion of 4-HNE due to the induction of hGST5.8 and RLIP76. A coordinated action of these proteins causes an increased efflux of the GS-HNE across the cell membranes, lowers the intracellular concentration of 4-HNE, and protects against oxidative stress-induced apoptosis (12Cheng J.Z. Sharma R. Yang Y. Singhal S.S. Sharma A. Saini M.K. Singh S.V. Zimniak P. Awasthi S. Awasthi Y.C. J. Biol. Chem. 2001; 276: 41213-41223Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). These studies strongly indicate a role of 4-HNE in oxidative stress-mediated apoptosis. Although UVA exposure causes lipid peroxidation (3Morliere P. Moysan A. Santus R. Huppe G. Maziere J.C. Dubertret L. Biochim. Biophys. Acta. 1991; 1084: 261-268Crossref PubMed Scopus (144) Google Scholar, 4Girotti A.W. J. Photochem. Photobiol. 2001; B 63: 103-113Crossref Scopus (479) Google Scholar, 5De Gruijl F.R. Skin Pharmacol. Appl. Skin. Physiol. 2002; 15: 316-320Crossref PubMed Scopus (245) Google Scholar) and also leads to apoptosis (20Pourzand C. Rossier G. Reelfs O. Borner C. Tyrrell R.M. Cancer Res. 1997; 57: 1405-1411PubMed Google Scholar, 21He Y.Y. Huang J.L. Ramirez D.C. Chignell C.F. J. Biol. Chem. 2003; 278: 8058-8064Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), it is not known if lipid peroxidation products, particularly 4-HNE, are involved in UVA-mediated signaling for apoptosis. Therefore, present studies were designed to examine a possible role of 4-HNE in UVA-induced apoptosis. K562 cells were selected for these studies because we have previously characterized the enzyme systems, which regulate the intracellular concentrations of 4-HNE (12Cheng J.Z. Sharma R. Yang Y. Singhal S.S. Sharma A. Saini M.K. Singh S.V. Zimniak P. Awasthi S. Awasthi Y.C. J. Biol. Chem. 2001; 276: 41213-41223Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 22Singhal S.S. Piper J.T. Saini M.K. Cheng J.Z. Awasthi Y.C. Awasthi S. Biochem. Arch. 1999; 15: 163-176Google Scholar) in these cells. It was hypothesized that if 4-HNE was involved in UVA-mediated apoptosis, then upon UVA exposure, 4-HNE levels should rise. This should be accompanied by a defense response of the cells against 4-HNE toxicity resulting in the induction of hGST5.8 and RLIP76. If this response occurs at non-toxic/non-lethal doses of UVA irradiation, cells preconditioned with mild and transient UVA exposure should become more resistant to apoptosis caused by prolonged UV exposures. Furthermore, if UVA preconditioned cells also acquire resistance to oxidative stress-induced apoptosis, 4-HNE could be considered to be a common mediator of apoptosis in a variety of stress situations. To address the above questions, we have studied the effect of UVA exposure on the formation of 4-HNE, induction of hGST5.8 and RLIP76, and the efflux of GS-HNE from K562 cells. The effect of mild, transient UVA exposure on major antioxidant enzymes, which metabolize ROS has also been studied. Furthermore, we have examined whether cells exposed to mild UVA exposure acquire resistance to apoptosis caused by xanthine/xanthine oxidase (oxidative stress) and prolonged UVA exposure and if so, whether this protection can be abrogated by inhibiting the efflux of GS-HNE. Results of these studies show that preconditioning with mild UVA exposure imparts to cells a partial resistance against oxidative stress- and UVA-induced apoptosis by preventing the activation of JNK and caspase, and that this resistance can be abolished by inhibiting the efflux of GS-HNE. These results strongly suggest an involvement of 4-HNE in UVA-induced signaling for apoptosis, and point out similarities in the mechanisms of apoptosis induced by oxidative stress and UVA. Materials—1-chloro-2,4-dinitrobenzene (CDNB), glutathione (GSH), xanthine (2,6-dihydroxypurine) (XA), and xanthine oxidase (XO) were from Sigma. RPMI 1640 medium, fetal bovine serum, phosphate-buffered saline (PBS), and penicillin/streptomycin were purchased from Invitrogen. 4-HNE was purchased from Cayman Chemical Co. (Ann Arbor, MI) and hydrogen peroxide was obtained from Fisher Scientific (Fair Lawn, NJ). 4-[3H]HNE was synthesized by us as described previously (12Cheng J.Z. Sharma R. Yang Y. Singhal S.S. Sharma A. Saini M.K. Singh S.V. Zimniak P. Awasthi S. Awasthi Y.C. J. Biol. Chem. 2001; 276: 41213-41223Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). All reagents for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western transfer were purchased from Invitrogen. Antibodies—Polyclonal antibodies raised in rabbits against the Alpha, Mu, and Pi classes of human GSTs were the same as those used in our previous studies (23Yang Y. Cheng J.Z. Singhal S.S. Saini M. Pandya U. Awasthi S. Awasthi Y.C. J. Biol. Chem. 2001; 276: 19220-19230Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Polyclonal antibodies against recombinant mGSTA4-4, the mouse ortholog of hGST5.8, were raised in rabbits. In earlier studies, these antibodies have been shown to be specific to hGST5.8 among the human GSTs (24Cheng J.Z. Yang Y. Singh S.P. Singhal S.S. Awasthi S. Pan S.S. Singh S.V. Zimniak P. Awasthi Y.C. Biochem. Biophys. Res. Commun. 2001; 282: 1268-1274Crossref PubMed Scopus (52) Google Scholar). Polyclonal antibodies raised in rabbit against the bacterially expressed recombinant RLIP76 were the same as those used in our previous studies (13Awasthi S. Cheng J.Z. Singhal S.S. Saini M.K. Pandya U. Pikula S. Bandorowicz-Pikula J. Singh S.V. Zimniak P. Awasthi Y.C. Biochemistry. 2000; 39: 9327-9334Crossref PubMed Scopus (132) Google Scholar). Purified IgG obtained by sequentially passing these antibodies over DEAE-52 and protein A-Sepharose columns were used in all the experiments. Polyclonal antibodies against recombinant hGSTA4-4 were raised in chicken as described by us previously (24Cheng J.Z. Yang Y. Singh S.P. Singhal S.S. Awasthi S. Pan S.S. Singh S.V. Zimniak P. Awasthi Y.C. Biochem. Biophys. Res. Commun. 2001; 282: 1268-1274Crossref PubMed Scopus (52) Google Scholar). Monoclonal antibody against phosphorylated JNK (G-7) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against β-actin was acquired from Sigma. Cell Lines and Cultures—The human erythroleukemia K562 cells obtained from the American Type Culture Collection were grown as suspension cultures in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum and 1% penicillin/streptomycin, and maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. UV Irradiation—K562 (5 × 106) cells were collected by centrifugation, washed with PBS, and resuspended in 5 ml of PBS. The cells were then transferred into a Petri dish (60 mm diameter) and irradiated under a 365-nm UV lamp (Model UVL-56, UVP Inc., San Gabriel, CA) in dark at a rate of 3 mW/cm2 determined by the chemical actinometry method of Hatchard and Parker (25Hatchard C.G. Parker C.A. Proc. R. Soc. London, Ser. A. 1956; 235: 518Crossref Google Scholar). After the UVA exposure for fixed time periods, the cells were pelleted, and PBS was replaced by normal culture medium. The cells were allowed a 2 h recovery time at 37 °C. In parallel, sham irradiated cells were subjected to an identical experimental protocol except that UV irradiation was omitted. Preparation of Cell Extracts and Western Blot Analyses—The treated cells were pelleted, washed, and resuspended in RIPA buffer containing 1× PBS, pH 7.4, 1% Nonidet P-40 or Igepal CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride, and 2 μg/ml pepstatin. The extract was passed through a 23-gauge needle to shear DNA, and was kept on ice for 30–60 min. The cell lysate was then centrifuged at 15,000 × g for 20 min at 4 °C, and the resulting supernatant was used for Western blot analysis. For detection of phosphorylated JNK, cell extracts were suspended in 20 mm Tris-HCl, pH 7.4 containing 150 mm sodium chloride, 1 mm EDTA, 1 mm EGTA, 1 mm NaF, 1 mm sodium vanadate, 2 mm phenylmethylsulfonyl fluoride, 1% Nonidet P-40, and freshly added protease and phosphatase inhibitor cocktails (Sigma), and were lysed by sonication (3 times, 5 s each at 40 watts). Cell lysates containing 25–100 μg of protein were subjected to SDS-PAGE according to the method of Laemmli (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). For the detection of the expression of hGST5.8 in K562 cells, 250 μg of protein was loaded. For detection of phosphorylated JNK, 50 μg of protein was loaded. Western blot analysis was performed essentially according to the method of Towbin et al. (27Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). The chemiluminiscent reagents from Pierce were used to develop the immunoblots by following the manufacturer's instructions. Determination of Intracellular 4-HNE Levels—Biotech LPO-586 228 kit (Oxis International, Portland, OR) was used to measure concentrations of MDA and MDA plus 4-HNE in K562 cells before recovery and after 2 h of recovery. For each determination, 1 × 107 cells were collected by centrifugation at 500 × g for 10 min and washed twice with PBS. The pellet was resuspended in 0.2 ml of 20 mm Tris-HCl, pH 7.4, containing 5 mm BHT and frozen at –70 °C until assayed. To each sample, 650 μl of N-methyl-2-phenylindole and 150 μl of either 12 n HCl (for MDA determination) or 15.4 m methanesulfonic acid (for 4-HNE plus MDA determination) were added. The reaction mixture was mixed by vortexing and incubated at 45 °C for 60 min. After centrifugation at 15000 × g for 10 min, the absorbance of the supernatant was determined at 586 nm. Standards of MDA and 4-HNE were prepared by the hydrolysis of 1,1,3,3-tetramethoxypropane in HCl and 4-HNE diethylacetal in methanesulfonic acid, respectively. Extinction coefficients for MDA and 4-HNE (1.1 × 105m–1 cm–1 and 1.3 × 105m–1 cm–1, respectively) determined from the standard curves were used, and the values were expressed as pmol of 4-HNE/mg of protein. Enzyme Assays—Control or UVA-irradiated K562 cells were harvested by centrifugation and washed with PBS. The cells were resuspended in 10 mm potassium phosphate buffer, pH 7.0, containing 1.4 mm 2-mercaptoethanol (buffer A). After lysis by sonication, the lysates were centrifuged for 45 min at 28,000 × g at 4 °C. The supernatants were then assayed for their activities against different substrates. GST activity toward CDNB was determined by the method of Habig et al. (28Habig W.H. Pabst M.J. Jakoby W.B. J. Biol. Chem. 1974; 249: 7130-7139Abstract Full Text PDF PubMed Google Scholar) and that toward 4-HNE was determined according to the procedure described by Alin et al. (29Alin P. Danielson U.H. Mannervik B. FEBS Lett. 1985; 179: 267-270Crossref PubMed Scopus (369) Google Scholar). Catalase and superoxide dismutase (SOD) activities were determined by the methods described by Beers and Sizer (30Beers Jr., R.F. Sizer I.W. J. Biol. Chem. 1952; 195: 133-140Abstract Full Text PDF PubMed Google Scholar), and Paoletti and Mocali (31Paoletti F. Mocali A. Methods Enzymol. 1990; 186: 209-220Crossref PubMed Scopus (428) Google Scholar), respectively. 2-Mercaptoethanol was excluded from the buffer A in preparation of the supernatants used for the determination of SOD activity. Measurement of GS-HNE Efflux from the Cells—In order to investigate the effect of GS-HNE efflux on the mechanism of UVA, oxidative stress, and 4-HNE-induced apoptosis, control or UVA irradiated K562 cells were allowed to recover for an hour, treated with preimmune IgG or anti-RLIP76 IgG at a final concentration of 20 μg/ml, and incubated for an additional 1 h. In some experiments, as noted in the text, the antibody coating step was omitted. Instead, the cells were allowed to rest for 2 h, so that the total recovery period was 2 h in each treatment. After washing with PBS to remove IgG. The cells were then loaded with 4-[3H]HNE by incubating with 20 μm 4-[3H]HNE (specific activity 3,800 cpm/nmol) in culture medium for 30 min. The cells loaded with 4-[3H]HNE were harvested by centrifugation at 500 × g, washed with PBS (2 × 2 ml), resuspended in 2 ml of PBS, and incubated for 10 min at 37 °C for measuring the efflux of GS-[3H]HNE from the cells. After the incubation period, cells were harvested, the medium was quantitatively separated, and radioactivity was measured in the medium and in cells. For the identification of GS-HNE conjugate, the medium was lyophilized, extracted with 200 μl of 70% ethanol, and the conjugate was isolated and characterized by HPLC analysis and mass spectrometry as described previously (12Cheng J.Z. Sharma R. Yang Y. Singhal S.S. Sharma A. Saini M.K. Singh S.V. Zimniak P. Awasthi S. Awasthi Y.C. J. Biol. Chem. 2001; 276: 41213-41223Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Determinations were made in triplicate in parallel experiments using equal number of cells under identical conditions. Caspase Assay for in Situ Apoptosis—Control or 5 min of UVA-irradiated cells were allowed to recover for 2 h in complete medium. Apoptosis in separate experiments was induced by one of the following treatments (i) 20 μm 4-HNE for 2 h at 37 °C, (ii) UVA irradiation (3.0 mW/cm2) for 30 min, and (iii) 30 μm xanthine (XA) for 1 h prior to the addition of xanthine oxidase (XO, 20 milliunits) and incubation for additional 15 h. Where mentioned, after 5 min of UVA irradiation, cells were incubated for 1 h in complete medium followed by preincubation with preimmune IgG or anti RLIP76 IgG at a final concentration of 20 μg/ml for an additional 1 h and, after washing with PBS, were treated with 4-HNE, XA/XO, or UVA irradiation as described above. Apoptotic cells were detected by staining with 10 μm CaspACE FITC-VAD-FMK (Promega) in situ marker for 30 min in the dark. The slides were rinsed with PBS twice, fixed with 4% paraformaldehyde for 1 h, mounted in a medium containing DAPI (1.5 μg/ml), and observed under fluorescent microscopy (Olympus, Japan). Effects of Short Time UVA Irradiation on 4-HNE Levels and Apoptosis—In order to investigate a possible role of 4-HNE in UVA-mediated signaling for apoptosis, K562 human erythroleukemia cells were subjected to UVA (365 nm) irradiation (3.0 mW/cm2) up to a period of 30 min, and the intracellular levels of 4-HNE were examined at different time intervals. The results of these experiments presented in Fig. 1 showed that 5, 15, and 30 min of UVA irradiation caused ∼1.4-fold, 2-fold, and 2.6-fold increase, respectively, in the 4-HNE levels over that observed in the control cells. After UVA exposure, the cells were allowed to recover in complete medium at 37 °C for 2 h, and 4-HNE concentrations were re-examined after the recovery period. Results of these experiments (Fig. 1) showed that after 2 h of recovery, 4-HNE levels in the cells irradiated for relatively short time were remarkably reduced. For example, in cells irradiated for 5 min and rested for 2 h, the 4-HNE level was only about 40% of that observed immediately after UVA exposure. In fact, the 4-HNE level in these rested cells was even lower than the basal level observed without UVA irradiation. Likewise, in cells subjected to 15 min of UVA irradiation, the 4-HNE level was remarkably reduced after recovery period and was comparable to the basal 4-HNE level before irradiation. On the contrary, the 4-HNE level in cells irradiated for 30 min remained persistently high even after the recovery period (Fig. 1). These results indicated that UVA exposure caused an enhanced formation of 4-HNE and suggested that cells exposed to UVA for a short time acquired the capability to dispose of 4-HNE at an accelerated rate but lose this capability if irradiated for a prolonged period with UVA. Effect of UVA on Apoptosis—UVA-irradiated cells after 2 h of resting were examined for apoptosis using the CaspACE FITC-VAD-FMK marker which specifically detects caspase activation in situ (Fig. 2). Results of these studies showed that after 2 h recovery, cells exposed to 5 and 15 min of UVA showed no significant activation of caspases indicating lack of significant apoptosis. However, a major fraction of the cells exposed to 30 min of UVA showed caspase activation, suggesting onset of apoptosis, which was also indicated by the characteristic nuclear condensation observed in DAPI-stained cells. These results are consistent with earlier reports that UVA causes apoptosis (20Pourzand C. Rossier G. Reelfs O. Borner C. Tyrrell R.M. Cancer Res. 1997; 57: 1405-1411PubMed Google Scholar, 21He Y.Y. Huang J.L. Ramirez D.C. Chignell C.F. J. Biol. Chem. 2003; 278: 8058-8064Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and suggest that the intracellular 4-HNE levels may correlate with the extent of UVA-mediated apoptosis. Effects of UVA on Expression of Enzymes Regulating Intracellular Concentrations of 4-HNE—Since our results indicated that 4-HNE concentrations in cells exposed to 5 and 15 min of UVA were decreased after a 2-h recovery, we examined the expression of 4-HNE-metabolizing enzymes in cells rested after UVA exposure. Previous studies have shown that the majority of cellular 4-HNE is metabolized through its conjugation to GSH, catalyzed by GSTs (32Srivastava S. Chandra A. Wang L.F. Seifert Jr., W.E. DaGue B.B. Ansari N.H. Srivastava S.K. Bhatnagar A. J. Biol. Chem. 1998; 273: 10893-10900Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). In humans, there are two GST isozymes, hGSTA4-4 (18Hubatsch I. Ridderstrom M. Mannervik B. Biochem. J. 1998; 330: 175-179Crossref PubMed Scopus (314) Google Scholar), and hGST5.8 (17Singhal S.S. Zimniak P. Sharma R. Srivastava S.K. Awasthi S. Awasthi Y.C. Biochim. Biophys. Acta. 1994; 1204: 279-286Crossref PubMed Scopus (63) Google Scholar), which display high catalytic efficiency toward 4-HNE (33Yang Y. Sharma R. Zimniak P. Awasthi Y.C. Toxicol. Appl. Pharmacol. 2002; 182: 105-115Crossref PubMed Scopus (67) Google Scholar). Results presented in Fig. 3, A and B showed that hGST5.8 expression was strongly induced upon 5 min (20-fold) and 15 min (7-fold) of UVA irradiation but returned to barely detectable basal level in cells subjected to 30 min of UVA irradiation. These results are consistent with the results on 4-HNE levels presented in Fig. 1 and show that the 4-HNE-metabolizing GST isozyme hGST5.8 is remarkably induced upon initial exposure to UVA but this transient induction is abolished upon a prolonged exposure to UVA. The other 4-HNE-metabolizing GST isozyme, hGSTA4-4, was undetectable in K562 cells under physiological conditions as well as after UVA irradiation (data not presented). UV exposure also did not affect the Pi and Mu class GSTs (data not presented), which comprise the bulk of constitutive GST protein in K562 cells (22Singhal S.S. Piper J.T. Saini M.K. Cheng J.Z. Awasthi Y.C. Awasthi S. Biochem. Arch. 1999; 15: 163-176Google Scholar). Accumulation of GS-HNE generated by GST-catalyzed conjugation of 4-HNE to GSH is inhibitory to GSTs. Therefore, GS-HNE should be pumped out of the cells to sustain the continuing conjugation of 4-HNE. Previous studies have shown that RLIP76 mediates the ATP-dependent transport of GSH conjugates, and that it accounts for ∼70% of the total transport activity toward GS-HNE in various human cells (14Sharma R. Singhal S.S. Cheng J.Z. Yang Y. Sharma A. Zimniak P. Awasthi S. Awasthi Y.C. Arch. Biochem. Biophys. 2001; 391: 71-79Crossref Scopus (53) Google Scholar, 15Awasthi S. Sharma R. Singhal S.S. Zimniak P. Awasthi Y.C. Drug Metab. Dispos. 2002; 30: 1300-1310Crossref PubMed Scopus (70) Google Scholar). We there
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