(Caspase-3) and C-Jun NH2-Terminal Kinase 1 by Benzo(A)Pyrene1

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[CANCER RESEARCH 58. 21(12-2106. May 15. I9%| Induction of Apoptosis and Activation of Interleukin 1/8-Converting Enzyme/Ced-3 Protease (Caspase-3) and c-Jun NH2-Terminal Kinase 1 by Benzo(a)pyrene1

Wei Lei, Rong Yu, Sandhya Mandlekar, and A-N. Tony Kong2

Department of Pharmaceutics ami Pharmucotknamics, Center for Pharmaceutical Biotechnology, College of Pharmacy, University of Illinois, Chicago, Illinois 60612

ABSTRACT PAHs including DMBA and TCDD (dioxin). have been shown to affect cellular signal transduction pathways and induce programmed Benzo(a)pyrene (BaP), a prototype of polycyclic aromatic hydrocar cell death or apoptosis. DMBA has been shown to induce apoptosis in bons (PAHs), is a potent procarcinogen generated during the combustion an A20.1 murine B-cell line (4), TCDD induced cytotoxicity in mouse of fossil fuels and cigarette smoke. In addition to the carcinogenic and mutagenic effects, RaP and other PAHs, including 7,12-dimethylben- thy mie epithelial cells (5) and induced apoptosis in mouse thymocytes i[a (anthracene and 2,3,7,8-tetrachlorodibenzo|p]dioxin, have been shown in vivo (6), and BaP has been shown to induce cell death in mouse to induce programmed cell death or apoptosis. However, the molecular epidermis (7) and cell cycle G, arrest in NIH 3T3 fibroblasts (8). mechanisms by which PAHs such as BaP induce apoptosis are not clear. However, the molecular mechanisms by which PAHs induce cell To investigate the molecular events leading to apoptosis induced by BaP, death and apoptosis remain unclear. we studied the involvement of the interleukin 1/J-converting enzyme Apoptosis, frequently referred to as "programmed cell death", is an (ICE)/Ced-3 family of proteases (caspases) and c-Jun NH2-terminal kinase active and physiological mode of cell death, and it was first reported I (JNKI), which have been shown to mediate numerous extracellular by Kerr et al. (9) in 1972. Apoptosis plays important roles in a variety stimuli-induced apoptosis. On treatment of mouse Hepa Iclc? hepatoma of biological processes. Most growth factors prevent apoptosis, cells with BaP, the induction of apoptosis, as determined by genome whereas the inflammatory cytokines, such as TNF-a or Fas (CD95) digestion, was observed at concentrations of 1-30 /LIMafter 24 h of treatments. Importantly, at the apoptosis-inducing concentrations, BaP induce apoptosis. In addition to these physiological regulators of also induced the activation of an ICE/Ced-3 cysteine protease caspase-3 apoptosis. many environmental stresses also induce apoptosis. Recent but not caspasc-l (ICE). The activation of caspase-3 by BaP preceded studies have implicated that oxidative stress, calcium, ATP, and apoptosis. Furthermore, a specific inhibitor of caspase-3-like proteases, mitochondria membrane potential are causal agents of apoptosis (10- acetyl-Asp-Glu-Val-Asp-aldehyde, significantly blocked caspase-3 activity 18). Despite the diversity of apoptosis-inducing agents, numerous and attenuated apoptosis induced by BaP. Treatment with BaP also experiments indicate that signals leading to the activation of a family caused a time- and dose-dependent activation of JNKI activity. Interest of intracellular cysteine proteases, the caspases, may play a pivotal ingly, a much lower concentration (5 n\i), as well as much earlier kinetics, role in the initiation and execution of apoptosis induced by various were observed in JNKI activation as compared with caspase-3 activation stimuli (19-25). To date, at least 10 different members of caspases in or induction of apoptosis by BaP. In summary, our results demonstrate that BaP induced apoptosis in the mouse hepatoma Hepalclc? cell line mammalian cells have been identified (26). Among these caspases, via a caspase-dependcnt pathway, which may be independent of JNK caspase-1/ICE, which was originally identified as a cysteine protease activation. responsible for the processing of interleukin IÃŸ(27), has been well characterized. Crystal structure analyses indicate that a catalytically active form of caspase-1 is a tetramer consisting of (p20)2/(plO)-,, INTRODUCTION which are derived from the cleavage of a Mr 45,000 proenzyme (p45), BaP3 is the prototype of a multitude of PAHs, which is thought to presumably by autocatalysis (28, 29). Overexpression of caspase-1 be a persistent procarcinogen and toxicant generated from the com triggers apoptosis in rodent fibroblasts and strongly potentiates Fas/ APO-1-mediated apoptosis in a different type of cells (19). However, bustion of fossil fuel and cigarette smoke (1). Although BaP itself is caspase-1-knockout mice do not show apparent defects in apoptosis not genotoxic. its biological effects can be mediated by binding to (30). Another emerging apoptotic protease is caspase-3, previously AHR. and BaP can induce the gene expression of more than a dozen genes, including members of the cytochrome P450 (2. 3). Cytochrome called CPP32/Yama/Apopain, which shares even closer homology with Ced-3, the death protease of Caenorhabditis elegans, than does P450 can oxidize BaP to toxic and reactive intermediate species ICE (31-33). Similar to caspase-1, caspase-3 is synthesized as an which, if not detoxified, irreversibly damage nucleic acids and pro teins by covalent binding or oxidation within the target cells (2, 3). In inactive proenzyme that requires proteolytic activation. Unlike caspase-1, however, caspase-3 does not appear to undergo autocata- addition to the carcinogenic and mutagenic effects. BaP and other lytic cleavage. This suggests that activation of caspase-3 may involve another aspartate-specific protease. Once activated, caspase-3 may Received 2/6/98: accepted 3/18/98. The costs of publication of this article were defrayed in part by the payment of page cleave a variety of substrates, including poly(ADP-ribose) polymerase charges. This article must therefore be hereby marked advertisement in accordance with (32), DNA-dependent protein kinase (34), M, 70,000 subunit of the 18 U.S.C. Section 1734 solely to indicate this fact. 'This work was supported by NIH Grants R29-GM49172 and ROI-ES06887 (to Ul small ribonucleoprotein (35), GDP dissociation inhibitor for the A-N.T. K.). Ras-related Rho family GTPases (D4-GDI; Ref. 36), protein kinase 2 To whom requests for reprints should be addressed, at Department of Pharmaceutics Co (37), and DNA fragmentation factor (38). Given the high homol and Pharmacodynamics, Center for Pharmaceutical Biotechnology MC870, College of Pharmacy. University of Illinois. 900 South Ashland Avenue. MBRB Room 3102. ogy with Ced-3 and the diversity of substrates, caspase-3 may serve as Chicago. IL 60607-7173. Phone: (312)413-9646; Fax: (312)413-9303; E-mail: a general mediator of apoptosis in a variety of cells and tissues. [email protected]. 1The abbreviations used are: BaP, ben/.o(i/)pyrene; AHR. aryl hydrocarbon receptor; Recently, another mediator, c-Jun N-terminal kinases (JNKI and PAH. polycyclic aromatic hydrocarbon: DMBA. 7.12-dimethylben/[Â«]anthracene; JNK2), also called stress-activated protein kinases, have been identi TCDD, 2.3.7,8-tctrachlorodiben/.o-/>-dioxin; TNF. tumor necrosis factor; ICE, interleukin l ÃŸ-converting en/.yme; JNK, c-Jun NH2-terminal kinase: Ac-DEVD-CHO, acetyl-Asp- fied to be involved in the regulation of apoptosis induced by a variety Glu-Val-Asp-aldehyde; Ac-DEVD-MCA. acetyl-Asp-Glu-Val-Asp^-methylcoumaryl-?- of stimuli, such as cellular stresses (39, 40), inflammatory cytokine amide: Ac-YVAD-MCA. acetyl-Tyr-Val-Ala-Asp-4-methylcoumaryl-7-amide; DAPI. TNF-a (41), and growth factor withdrawal (42). JNK/stress-activated 4,6-diamidino-2-phenylindole: AMC. amono-4-methylcoumarin; MTS. 3-(4.5-dimethyl- thia/.ol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophcnyl)-2W-tetrazolium; GST, glu- protein kinases are members of the mitogen-activated protein kinase tathione 5-transfera.se. family, which convert various extracellular signals into intracellular 2102

dissolved in 50 /Â¿Iof10 mM Tris (pH 8.0) and 1 mM EDTA buffer. DNA fragments (about 10 /xg) were resolved by electrophoresis in 1.5%agarose gel and stained with ethidium bromide. Nuclear Staining Assay. After treatmentof Hepalclc? cells with BaP. with or without Ac-DEVD-CHO. floating cells were collected by ccntrifuga- tion at 2000 x g for 15 min and attached cells were first trypsinized and then harvested by centrifugation. The cells from the two parts above were com bined, washed once with ice-cold PBS buffer, and fixed in a solution of methanol:acetic acid (3:1) for 30 min. Fixed cells were placed on slides and. after evaporation of fixing solution, were sluined with 1 jig/ml DAPI for 15 min. Apoptotic cells with condensed, or fragmented nuclei were visualized and counted under a fluorescence microscope. Fluorogenic Assay of ICE-like Protease or Caspase Activity. After treatment, cells were washed twice with ice-cold PBS butter and lysed in lysis buffer [50 mMTris (pH 7.4). 50 mM0-glycerophosphate, 15 mM MgCl,. 15 20 mMEDTA. 100/IMphenylmethylsulfonyl fluoride, 1mMDTT, and 150fig/ml digitonin). Total proteins (10 /xg) were incubated with 200 mMAsp-Glu-Val- 15 20 25 30 Asp-4-methylcoumaryl-7-amide or Tyr-Val-Ala-Asp-4-methylcoumaryl-7- BaP Concentration amide in 50^1 of assay buffer [lOOmMHEPES (pH 7.5). 10%sucrose, 10HIM DTT, and 0.1% 3-[(3-cholamidopropyl)dimethylamino]-l-propanesulfonate] Fig. 1. BaP causes a dose-dependent inhibition of cell growth in Hepale Ic7 cells. Cells at 37Â°Cfor2 h. The release of AMC was monitored by a spectrofluorometer were treated with different concentrations of BaP for 24 h. Cell survival was determined by MTS assay. The percentage of survival was calculated as a ratio of A4g0nmof (PerSeptive Biosystems, Inc., Framingham. MA), using an excitation wave BaP-treated cells and control cells related to 100%. Data points are means for nine wells; length of 360 nm and an emission wavelength of 460 nm. bars, SE. /;/ Vitro Immunocomplex Kinase Assay of JNKI Activity. The JNKI activity assay was described previously (45) and briefly summarized here. After treatment, the cells were washed with ice-cold PBS and lysed in 150 fÂ¿l of lysis buffer [10 mM Tris-HCl (pH 7.4), 50 mM sodium chloride. 30 mM responses through serial phosphorylation cascades (43). Once acti sodium PP,, 50 mM sodium fluoride. 100 JUMsodium orthovanadate. 2 mM vated, JNK can phosphorylate various transcription factors, such as iodoacetic acid. 5 /J.MZnCI:. 1 mMphenylmethylsulfonyl fluoride, and 0.5% c-Jun, ATF-2, p62TCF/Elk-1, and ATF-3, which result in immediate- Triton-X-100]. The lysates were homogenized by passing through a 23-gauge early gene induction (44). Although a growing body of evidence needle three times. The homogenate was centrifuged at 12.500rpm for 15 min suggests that both JNK and ICE-Ced-3 proteases are important reg at 4Â°C.Thesupernatant was transferred into a clean tube, and an equal amount ulators of apoptosis, the relationship between these two pathways is of protein was incubated with rabbit anti-JNKl polyclonal antibody (Santa not clear. Cruz Biotechnology. Inc.. Santa Cruz. CA) prebound to protein A-Sepharose 4B conjugate for 90 min at 4Â°C.Theimmunocomplex was washed twice with Here, we report the induction of apoptosis by BaP in mouse Hepalclc? hepatoma cell line. At apoptosis-inducing concentrations, lysis buffer and twice with kinase assay buffer [20 mMHEPES (pH 7.9). 10 BaP activated the ICE/Ced-3 protease, caspase-3, but not caspase-1. A mMMgCU. 2 mMMnCl,. 50 mMÃŸ-glycerolphosphate.10mM(>-nitrophenyl caspase-3 inhibitor inhibited caspase-3 activation and attenuated ap phosphate, and 0.1 mMsodium orthovandate]. Kinase reactions were initiated by the addition of 30 fi\ of kinase assay buffer containing 10fig of GST-c-Jun optosis induced by BaP, suggesting that caspase-3 may play a pivotal (amino acid 1-79) fusion protein (46), 2 /j.Ciof [y-12P]ATP,and 20 /UMATP, role in BaP-induced apoptosis in this cell line. BaP also activated incubated for 30 min at 3()Â°C.and terminated hy adding 10 /xl of 4X JNKI, another potential regulator of apoptosis, in a time- and dose- dependent fashion. BaP(l (5 MATERIALS AND METHODS

Cell Culture, Chemicals, and Treatment. Mouse hepatoma cell line Hepalclc? (American Type Culture Collection, Rockville. MD) was main tained in MEM containing 10% heat-inactivated fetal bovine serum, supple mented with penicillin and streptomycin. BaP (Sigma Chemical Co.. St. Louis. MO) was dissolved in DMSO in 1000X stock. For BaP treatment, cells were 70-80% confluent, and the medium was removed and replaced with phenol red-free medium containing different concentrations of BaP. Fluorogenic peptide substrates Ac-YVAD-MCA and Ac-DEVD-MCA were purchased from Peptide International (Louisville. KY). Caspase-3-specific inhibitor, Ac- DEVD-CHO, was purchased from Calbiochem (San Diego, CA). For the pretreatment with caspase inhibitor, cells were incubated with the inhibitor for 1 h, followed by the BaP treatment. DAPI was purchased from Sigma. MTS Assay for Cell Death Induced by BaP. Cells were seeded in 96-well plates at a density of IO4cells/well. Twenty-four h later, cells were treated with different doses of BaP for 24 h. The MTS assay was performed with CellTiter 96 AQueousnonradioactivecell proliferation assay kit (Promega Corp., Madi son, WI). The absorbance was read at 490 nm on a ELISA reader, and the percentage of cell survival was obtained. DNA Fragmentation Assay. After treatment,cells were lysed in buffer containing 40 mMTris (pH 8.0), 150 mMNaCl, 25 mMEDTA. and 0.5% SDS. Fig. 2. BaP induces DNA fragmentation in Hepalclc7 cells. Cells were treated with I DNA was isolated with an equal volume of neutral phenol:chloroform:isoamyl and 5 fiMBaP for 8-Ã•6h. DNA was isolated and dissolved in 10 /JMTris (pH 8.0) and alcohol mixture, precipitated with 0.1 volume of 3 Msodium acetate (pH 5.2), l Â¿IMEDTA(pH 8.0) buffer. DNA (~IO fig) were resolved by electrophoresis in 1.5% and 2 volumes of 100% ethanol at -20Â°Covernight. Precipitated DNA was agarose gel and stained with ethidium bromide. 2103

shown in Fig. 1 indicated that BaP decreased cell viability in the concentration between 1 and 10 /AM.Based on this result of cell killing concentrations, we next studied whether the cell death induced by BaP was due to apoptosis. Because one of the biochemical hallmarks of apoptosis is the cleavage of chromatin into nucleosomal fragments, a DNA fragmentation assay was performed to detect genome digestion. o Cells were treated with 1 or 5 /AMBaP for 8, 12, 24, and 36 h, and o â€¢¿o DNA was isolated and electrophoresed in 1.5% agarose gel. Fig. 2 e shows that a DNA ladder was observed with 24 and 36 h exposure to BaP. This result indicated that BaP induced Hepalclc? cells to o u, undergo apoptosis. There is growing evidence that caspases, especially caspase-1 and caspase-3, play important roles in apoptotic process. In this study, a fluorogenic assay of protease activity was performed to examine whether these two caspases contribute to the BaP-induced apoptosis. The dose-dependent response was first investigated. As shown in Fig. 3A, 10 JAMof BaP stimulated a substantial induction of caspase-3 BaP Concentration (|Jvi) activity. No caspase-1 activity was observed at any concentration. The kinetics of caspase activation was then examined with 10 /J.Mof BaP. Fig. 3ÃŸshows that substantial activation of caspase-3 activity was

35 -,

a 15 'o 03 â€¢¿Â§ O 12 9 o JO [i. lâ€¢¿a 6 e 2 3 o IL O Time (h): 36 12 24 36 36 12 24 36 BaP (5 uM): + Time (hours) DEVO (50 uM): Fig. 3. BaP activates caspase-3 (CPP32ÃŒbut not cuspase-1 (ICE} protease. A. dose- dependent activation of caspase-3 and caspase-1 activities by BaP. Cells were treated with different doses of BaP for 24 h. Cells were harvested and lysed. 10 /xg of total proteins B were incubated with 2(X) /UMAc-DEVD-MCA or Ac-YVAD-MCA at 37Â°Cfor 2 h. The 50 release of AMC was monitored by a spectrofluoromcter with an excitation wave of 360 nm and an emission wave of 460 nm. Fold of induction of caspase activity was calculated by the ratio of AMC released from treated cells to control cells. Bars, SE. B, kinetics study 40 of caspases activation by BaP. Cells were treated with 10 /AMBaP for different time periods, and protease activity was determined as above. Bar\, SE. o O 30

Laemmli's buffer and heating at 95Â°Cfor 5 min. The products were resolved o. o in 10% SDS-PAGE and stained with Coomassie blue. The phosphorylation of o. 20 GST-c-Jun was visualized by autoradiography and quantitated with a phosphorimager (Ambis, Inc.. San Diego, CA). 10

RESULTS AND DISCUSSION O Previous studies have shown that BaP and other PAHs including Time (h): 36 DMBA and TCDD induce apoptosis in the A20.1 murine B-cell line BaP (5 MM). DEVO (50 MM): (4), mouse thymic epithelial cells (5), and mouse thymocytes in vivo (6). However, the molecular mechanisms by which PAHs induce Fig. 4. Effects of caspase inhibitor on the activation of caspase and apoptosis induced by BaP. A, inhibition of caspase-3 activity by caspase-3 inhibitor. Cells were pretreated apoptosis remain unknown. Here, mouse hepatoma cell line with 50 Â¿AMcaspase-3 inhibitor, Ac-DEVD-CHO, for 1 h, and then treated for an HepalclcV was selected to study the mechanisms of BaP- and other additional 12, 24. and 36 h with 5 /AM BaP. Caspase-3 activity was determined as PAH-induced apoptosis. The effect of BaP on cell viability was tested. described above in Fig. 3. B. inhibition of BaP-induced apoptosis by caspase-3 inhibitor. Cells were treated with different concentrations (0.01-30 JUM)of BaP Pretreatment with Ac-DEVD-CHO and followed by BaP was performed as above. The percentage of apoptotic cells was determined by DAPI staining (with condensed or for 24 h, and cell viability was measured by MTS test. The results fragmented nucleus). Columns, means for three independent experiments; bars, SE. 2104

of JNKI activation by BaP, we exposed the cells to 0. l JU.MofBaP for different time periods. As shown in Fig. 5ÃŸ,JNKI activity rapidly BaP(nM) increased at 15 min, peaked at 60 min, and was sustained through 150 0 .001 .005 0.01 .05 0.1 1.0 min after exposure to BaP. This activation pattern indicated that BaP stimulated rapid activation of JNKI activity at a much lower concen Â«â€¢â€¢Â»W<â€¢Â»4MP â€¢¿â€¢Â» 4- GST-c-Jun tration (as low as 5 nM) than that required for activating caspase-3 or 1 1.2 2.7 8.7 9.1 10.1 9.5 Â«-Fold induction inducing apoptosis, implicating differential activation of JNKI. caspase-3 activities, and apoptosis by BaP. Previous studies have demonstrated that other PAHs, such as TCDD (dioxin), stimulated a rapid and transient increase in intracellular Ca2 +. followed by the induction of immediate early proto- oncogenes c-fos,jun-B, c-jun, andjun-D in hepatoma Hepalclc7 cells B (50). Future investigations would address whether the activation of Time (min) 15 30 60 90 120 150 JNKs by BaP is involved in the induction of these early genes and their physiological consequences. Furthermore, whether the activation â€¢¿GST-c-Jun of caspase-3 and JNKI by BaP will require the AHR remain un known. Recently, Fernandez-Salguero et al. (51) reported that the AHR-deficient mice are relatively resistant to TCDD-induced lesions 2.4 7.4 10.7 10.4 7.8 9.4 4~ Fold induction in the liver, thymus, and other tissues, suggesting that the AHR may Fig. 5. BaP activates JNKI. A, dose-response of JNKI activation by BaP. Hepalclc7 mediate these biological effects. cells were treated with different concentrations of BaP for 90 min. Activation of JNKI was studied using /n vitro immunocomplex kinase assay with 10 /ig of GST-c-Jun as In summary, our results demonstrated that BaP induces apoptosis in substrate and quantified on a phosphorimager. Fold of induction of JNKI activity was the mouse hepatoma Hepalclc7 cell line, which may be involved in calculated as the relative activity to the control cells (0 JIM). B. kinetics of JNKI activation by BaP. Cells were treated with 0.1 (Â¿MBaP for the indicated time periods, and JNKI cytotoxic effects by this agent or other related PAHs. Our results activity was measured as in A. indicate that the activation of caspase-3 may play a critical role in BaP-induced apoptosis, which may be independent of JNKI activa tion. observed between 12 and 24 h of BaP treatments. The activation of caspase-3 activity preceded BaP-induced apoptosis. Again, no ACKNOWLEDGMENTS caspase-1 activation was seen at any time point. To further characterize the relevance of caspase-3 activation in We thank Drs. ArmingLin and Michael Karin (University of California. San BaP-induced apoptosis, we examined the effect of a caspase-3- Diego. CA) for providing the GST-c-Jun cDNA plusmid. Dr. David S. Ucker specific inhibitor, Ac-DEVD-CHO, on apoptosis. Caspase-3 activity for stimulating discussions, and the University of Illinois at Chicago Cancer was measured in the presence of 5 /XMof BaP for 12, 24, and 36 h, Center for use of equipment. with or without Ac-DEVD-CHO. As shown as Fig. 4A, 50 JU.Mof Ac-DEVD-CHO completely inhibited the activation of caspase-3 in REFERENCES duced by BaP. No significant effect was observed of the inhibitor 1. Phillips. D. H. Fifty years of benzo(Â«)pyrene. Nature (Lond.). 303: 468-472. 1983. itself on caspase-3 activity. Because the morphological nuclear 2. Conney, A. H. Induction of microsomal enzymes by foreign chemicals and carcino- genesis by polycyclic aromatic hydrocarbons: G. H. A. Clowes Memorial lecture. changes of apoptotic cells can be visualized and counted with a Cancer Res.. 42: 4875-4917. 1982. nuclear staining assay (DAPI staining) under fluorescence micro 3. Nebert. D. W., and Gon/ale/. F. J. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. Induction of Apoptosis and Activation of Interleukin 1β -Converting Enzyme/Ced-3 Protease (Caspase-3) and c-Jun NH2 -Terminal Kinase 1 by Benzo(a)pyrene

Wei Lei, Rong Yu, Sandhya Mandlekar, et al.

Cancer Res 1998;58:2102-2106.

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