* Department of Cell Biology and Anatomy and
College of Pharmacy, The University of Arizona, Tucson, Arizona; and
Department of Medical Pharmacology and Toxicology, Texas A&M University System Health Sciences Center, College Station, Texas
Received September 28, 2000; accepted November 27, 2000
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ABSTRACT |
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Key Words: sodium arsenite; in vitro; precision-cut lung slices; NFB; AP-1; stress proteins.
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INTRODUCTION |
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The mechanism(s) responsible for causing cancer by arsenic in humans is not fully known, because it is not mutagenic in bacteria or mammalian cells (Lofroth and Ames, 1978; Rossman et al., 1980
). Furthermore, there are no suitable animal models for investigating carcinogenicity of arsenic compounds. Several mechanisms by which arsenic may cause genetic damage have been proposed, including the generation of reactive oxygen species (Applegate et al., 1991
; Wang et al., 1996
; Yamanaka et al., 1991
). Arsenic has been classified as an atypical carcinogen, as it does not fall into the category of initiator or promoter (Barrett et al., 1989
; Brown and Kitchin, 1996
). However, recent investigations reveal that arsenite activates NADH oxidase to produce superoxide, resulting in DNA strand breaks and large deletion mutations in mammalian cells (Hei et al., 1998
; Lynn et al., 2000
). It can also cause inhibition of DNA repair by inhibiting DNA ligase (Lynn et al., 1997
).
Since no animal models are available for the investigation of mechanisms by which arsenic causes cancer, in vitro systems become important in studying mechanisms of toxicity and elucidating early changes in gene expression after arsenic exposure. Precision-cut lung slices are a versatile in vitro system for toxicity studies, since they preserve the structural architecture of the lung as well as the cellular heterogeneity. They also preserve cell-cell interactions and cell-matrix interactions. Lung slices from one animal can be used for one experiment as well as control and treatment group, thus reducing variability. Precision-cut lung slices have been used previously in various toxicological studies (Fisher et al., 1994; Jones et al., 1992
; Morin et al., 1999
; Price et al., 1995
).
Exposure to arsenic results in the activation of several signaling pathways: the mitogen-activated protein kinase (MAPK), and the NFB signaling pathways. The activation of these pathways, which leads to the expression of stress proteins, is considered to be an important stress response that enables cells to survive, to undergo proliferation, or to experience apoptosis. There are 4 major MAPK pathways: extracellular signal-regulated kinase or ERK (Boulton et al., 1991
), c-jun N-terminal kinase or stress-activated protein kinase, abbreviated JNK/SAPK (Davis, 1994
), p38 MAP kinase (Stein et al., 1997
), and big MAP kinase or BMK/ERK5 (Lee et al., 1995
). MAPK pathways regulate the expression of transcription factors such as AP-1, ATF-2, ELK-1 (Karin, 1995
), and HSF (Lee and Corry, 1998
). AP-1 and ATF-2 are responsible for the expression of protooncogenes c-jun (Karin et al., 1997
), and ELK-1 for the expression of fos (Cavigelli et al., 1995
). AP-1 complex can consist of multiple protein complexes (Cohen et al., 1989
). It can be a heterodimer of the jun family of transcription factors: c-jun, jun-B, jun-D, or the fos-family of transcription factors: c-fos, fos-B, Fra1 and Fra2 (Angel and Karin, 1991
). AP-1 can also consist of jun-jun homodimers (Karin, 1995
).
NFB is activated due to oxidative stress and is involved in the expression of antioxidant enzymes such as superoxide dismutase (Iwanaga et al., 1988) and
-glutamylcysteine synthetase, which is the rate-limiting enzyme of glutathione synthesis (Meyrick and Magnuson, 1994
). In unstimulated cells, NF
B is localized in the cytoplasm, bound to its inhibitory subunit I
B (Beg et al., 1992
). Phosphorylation of I
B by I
B kinase (Brown et al., 1995
) results in the dissociation of I
B from NF
B and ubiquitination of I
B (Scherer et al., 1995
). As a result, the nuclear localization signal on NF
B becomes unmasked and it migrates into the nucleus and binds to DNA (Jung et al., 1995
). The exact mechanisms by which reactive oxygen species activate NF
B are not fully known. However, there is evidence to believe that the redox-sensitive enzyme, thioredoxin peroxidase, regulates the activity of NF
B by modulating the phosphorylation of I
B (Jin et al., 1997
).
The purpose of the present study was to examine the acute toxicity of low concentrations of sodium arsenite in lung slices, and to determine whether arsenite exposure results in the expression and activation of transcription factors c-jun/AP-1 and NFB. These transcription factors are involved in the regulation of genes induced under stressful conditions, such as stress proteins HSP-32 and HSP 72.
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MATERIALS AND METHODS |
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Animals.
Male Sprague-Dawley rats (275300 g) were purchased from Harlen (Indianapolis, IN). The animals were allowed to acclimatize for 1 week before use. Food (standard laboratory chow) and water were freely available to the rats.
Preparation and incubation of lung slices.
Rats were sacrificed by carbon dioxide asphyxiation. The skin was dissected from the abdomen to the neck and the pleural cavity and trachea were exposed. The trachea was cannulated, and warm 5% agarose solution (40°C) in Waymouth MB751/2 was instilled as described earlier (Stefaniak et al., 1992). The trachea and lungs were immediately removed en bloc and placed in ice-cold Krebs-Henseleit buffer (pH 7.4) for the agarose to gel. Tissue cores of 10-mm diameter were prepared using a sharpened stainless steel tube. Precision-cut lung slices were prepared using a Krumdieck tissue slicer (Alabama Research and Development Corporation, Munsford, AL) and ice-cold Krebs-Hensleit buffer (pH 7.4) gassed with 95% O2:5% CO2. Slice thickness was 500600 µ. Three lung slices were loaded onto a semicircular screen with 2 steel rings at each end, and the screen was placed in a glass scintillation vial containing 1.7 ml Waymouth MB751/2 to which 0.5% gentamycin and Fungibact solution (10 ml/l) has been added. The vials were incubated at 37°C for 124 h on a dynamic roller incubator, with continuous passage of 95% O2:5% CO2 (1 L/min). Sodium arsenite was added after a 1-h preincubation period. Different concentrations of sodium arsenite (0.1, 1.0, 10, and 100 µM) were obtained by diluting a stock solution (10 mM, pH 7.4) in Waymouth medium. Control slices were incubated in the medium only.
Viability.
Analyzing the intracellular K+ content assessed viability of lung slices. Three slices (from the same vial and from the same animal) were pooled into 1 ml of deionized water and homogenized by sonication. Proteins were precipitated by adding 40 µl of perchloroacetic acid to 800 µl of homogenate and the samples were centrifuged at 14,000 rpm for 15 min. The supernatant fraction was used for K+ assay by flame photometry, as described earlier (Azri et al., 1990). The pellet was dried overnight and dissolved in 1 ml of 1 M sodium hydroxide. The protein content was analyzed by the Bradford Method (Bradford, 1976
). The results were expressed as µmol K+/mg protein.
Histopathology.
Lung slices were incubated for 4 h with 10-µM arsenite. Control slices were incubated without arsenite. The slices were fixed in 10% neutral buffered formalin, embedded in paraffin, and processed for light microscopy. Sections were stained with hematoxylin and eosin.
Western blot analysis of stress proteins.
After incubation for 4 h with 0100 µM arsenite, 3 lung slices from the same vial were homogenized in 300 µl lysis buffer (1% SDS, 1.0 mM sodium orthovanadate, 10 mM TrisHCl pH 7.4) containing 1 µl/ml Protease Inhibitor Cocktail (Sigma Chemical Co.). Sliced homogenate was centrifuged at 14,000 rpm for 15 min. and the supernatant fraction was used for the analysis. Protein content was determined by Bradford Assay (Bradford, 1976). Protein (40 µg) was mixed with an equal volume of sample buffer (BIO RAD, Hercules, CA), containing 10% ß-mercaptoethanol and boiled for 5 min. Samples were then loaded onto a 10% gel, and the proteins were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDSPAGE, 130 V) until the dye front reached the bottom of the gel. The proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (400 mÅ for 30 min), and the membranes were blocked overnight with 5% nonfat milk in TBST (0.1 M Tris base pH 7.5, 0.1 M NaCl, 0.1% Tween-20). Immunoperoxidase staining for HSP 72, HSP 60, HSP 32, and HSP 90 were performed using specific monoclonal antibodies for each stress protein (StressGen, Victoria, BC, Canada), and horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using a DAB Substrate Kit for Peroxidase (Vector Laboratories, Burlingame, CA), which utilizes the enzymatic conversion of 3,3'-diaminobenzidine (DAB) to a chromogen by horseradish peroxidase. Densitometric analysis of the bands was performed using Scion Image (NIH).
Preparation of nuclear proteins.
Nuclear protein was isolated, as previously described (Parrish et al., 1999), from sodium arsenite-treated or control lung slices incubated for 4 h. Six slices from the same condition were pooled and homogenized in 1 ml of HEGD buffer (25 mM HEPES, 1.5 mM EDTA, 10% glycerol, 0.15 mg/ml dithiothreitol, 0.1 mg/ml phenylmethylsulfonyl fluoride, and 1 µl/ml Protease Inhibitor Cocktail (Sigma Chemical Co.) using a glass homogenizer, and was centrifuged at 14,000 rpm for 15 min. The supernatant was aspirated and the pellet resuspended in 80 µl of ice-cold HEGDK buffer (HEGD buffer containing 0.5-M KCl) and incubated on ice for 1 h. The samples were microfuged at 14,000 rpm for 15 min, and the nuclear protein extract in the supernatant was transferred to a fresh tube. The nuclear protein extracts were stored at 20°C and used within a few days.
Gel-shift assay for AP-1 and NFB.
Gel-shift assay was performed using a Digoxigenin Kit # 1635 (Boehringer Mannheim) according to the manufacturer's directions. NFB (5'-AGT TGA GGG GAC TTT CCC AGG C-3') and AP-1 (5'd[CGC TTG ATG AGT CAG CCG GAA]-3') double-stranded consensus oligonucleotides (Promega, Madison, WI) were 3' end-labeled with Dig-11-ddUTP using terminal transferase. Nuclear protein (10 µg) from control or 10 µM arsenite-treated lung slices was incubated for 15 min. at room temperature with 4 µl binding buffer, 1 µl poly [D(I-C)], 1 µl poly L-lysine, 1 µl digoxigenin labeled AP-1 or NF
B (400 pg/µl), and nuclease-free water to make up to 20 µl. Two hundred-µM H2O2-treated lung slices were used as a positive control. The specificity of the reaction was determined by the addition of excess unlabeled oligonucleotide for competition. The samples were loaded onto a 5% nondenaturing polyacrylamide gel for electrophoresis, and after electroblotting onto a nylon membrane, oligonucleotides were cross-linked to nylon membrane using a UV crosslinker. The digoxigenin-labeled oligonucleotides on the membrane were bound by anti-digoxigenin antibody linked to alkaline phosphatase. Chemiluminescence is produced when the substrate CSPD (disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5chloro)tricyclo[3.3.1.137]decan}-4-yl)phenyl phosphate) is dephosphorylated by alkaline phosphatase and is recorded on x-ray film (Kodak Biomax) for 1530 s.
Western blot analysis of nuclear c-jun/AP-1 and NFB proteins.
Nuclear proteins from lung slices exposed to 0, 0.1, 1.0, 10, or 100 µM of sodium arsenite for 4 h were prepared as mentioned above. Western blot analysis was performed as described for the stress proteins, with a few modifications. Ten µg protein was loaded onto a 10% gel and SDS/PAGE was performed. Proteins were transferred onto PVDF membranes, and the membranes were incubated with primary antibodies to NFB (Chemicon, Temecula, CA) or c-jun/AP-1 (Oncogene, Boston, MA). NF
B is a mouse monoclonal antibody to the p65 subunit, which recognizes an epitope overlapping the nuclear localization signal. Thus it recognizes the activated form of NF
B. C-jun/AP-1 is a rabbit polyclonal antibody raised against an amino acid sequence in the DNA binding domain of jun protein, and it recognizes the activated form of AP-1 as well as c-jun protein. For the detection of NF
B, the membranes were incubated with biotinylated goat anti-mouse antibody for 1 h. The membranes were then incubated with streptavidin-HRP complex (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h. For the detection of c-jun/AP-1, membranes were incubated with HRP-labeled goat anti-rabbit antibody for 1 h. Protein bands were detected by using enhanced chemiluminescence reagents (ECL Plus Kit, Amersham Pharmacia Biotech) and exposure to x-ray films (Kodak Biomax). The density of the bands was analyzed using Scion Image (NIH).
Localization of c-jun/AP-1 and NFB by confocal microscopic immunofluorescence detection.
Lung slices were incubated for 4 h with 10 µM arsenite. Control slices were incubated without arsenite. The slices were fixed in 10% neutral buffered formalin, embedded in paraffin, and 5-µ sections were cut. Lung sections were deparaffinized and antigen retrieval was performed by placing slides in citrate buffer (pH. 6.1) and heating in a microwave oven on high power for 35 min or until boiling point was reached. The slides were microwaved for another 5 min on Defrost setting and allowed to cool to room temperature (2530 min). The tissues were permeabilized by incubation with 0.1% NP-40 (Sigma Chemical Co.) in phosphate-buffered saline. The sections were incubated with anti-NFB against the p65 sub unit (which detects activated form of NF
B), or anti-c-jun/AP-1 (which detects activated AP-1 or c-jun protein) at 37°C for 1 h. This was followed by incubation with the appropriate biotinylated secondary antibody for 1 h at 37°C. After the washing step, sections were incubated with Cy-5 conjugated to Streptavidin, and RNA was digested using DNase-free RNase. The nuclei were stained with YOYO iodide. The sections were mounted in DAKO mounting medium and stored at 4°C. Samples were viewed using a Leica TCS confocal microscope with a Kr/Ar laser.
Statistics.
Values represent mean ± standard error of 34 animals. Three slices from the same vial were pooled for each condition unless mentioned otherwise. Data was analyzed by ANOVA, followed by Dunnett's multiple comparison test. P < 0.05 was considered significant.
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RESULTS |
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Localization of c-jun/AP-1 and NFB by Confocal Microscopy
Nuclear localization of c-jun/AP-1 was observed mainly in the nuclei of alveolar type-II cells and macrophages, indicating activation and nuclear translocation of this transcription factor. Cytoplasm of the above cells had very little staining for c-jun/AP-1 (Figs. 8B and 8D). Bronchial epithelial cells too exhibited positive staining for AP-1. Only minimal staining was seen in control tissue (Figs. 8A and 8C
). NF
B was observed in the nuclei and cytoplasm of type II epithelial cells and macrophages (Figs. 9B and 9D
). However, the intensity of staining for NF
B was less as compared to that of c-Jun/AP-1. Cytoplasm of type-II epithelial cells and macrophages seemed to accept more staining for NF
B than did the nuclei. In control tissue, only minimal staining was observed (Figs. 9A and 9C
).
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DISCUSSION |
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Stress proteins are constitutively expressed at low levels in eukaryotic as well as prokaryotic cells and are well conserved during evolution. Their expression is increased due to a variety of cellular stresses such as heat shock, xenobiotics, radiation, metals, ischemia, cytokines, and oxygen-free radicals. Stress proteins play a role in protecting cells from irreversible damage and help recovery of cells after a toxic insult. Expression of HSP 32 is particularly increased as a response to hyperoxic lung injury in vivo (Lee et al., 1996). Exposure of rats to arsenite for 12 weeks resulted in a depletion of glutathione (GSH), and an increase in oxidized glutathione (GSSG) and malondialdehyde in liver and brain (Flora, 1999
), indicating oxidative injury. In other studies, arsenite has been shown to cause damage to cellular macromolecules by generating reactive oxygen species (Chen et al., 1998
; Liu and Jan, 2000
; Rhee et al., 2000
). Reactive oxygen species include, H2O2, superoxide anion (O-2), hydroxyl radical (OH), as well as organic peroxides and radicals. These can damage cells by lipid peroxidation and DNA and protein oxidation. In bovine aortic cells, arsenite caused DNA strand breaks. These could be decreased by nitric oxide synthase inhibitors, superoxide scavengers, and peroxynitrite scavengers, indicating that arsenite increases superoxide and nitric oxide formation, and nitric oxide and superoxide probably react to produce peroxynitrite (Liu and Jan, 2000
). In the present study, concentrations as low as 1 µM arsenite, which did not cause K+ leakage, induced HSP 32. Since both, HSP 32 (Lee et al., 1996
) and HSP 72 (Gomer et al., 1996
) are increased after oxidative stress, arsenic may also induce these genes by inducing oxidative stress. However, other than induction of HSP-32, we have not examined indices of oxidative stress in this study.
HSP 32 plays a role in protecting cells against the damaging effects of oxygen-free radicals. It is the rate-limiting enzyme in heme degradation. Heme, a prooxidant, is converted by HSP 32 into biliverdin and bilirubin, which are potent physiological antioxidants (Stocker et al., 1987, 1990
). Iron, which is released from heme during its degradation, may generate oxygen-free radicals by Fenton-type reactions (Halliwell and Gutteridge, 1984
). However, iron induces ferritin production (Munro, 1993
), and ferritin binds free iron and further protects cells from oxidant-mediated cellular damage (Balla et al., 1992
; Cermak et al., 1993
).
In the present study, an increase in HSP 72 was evident only after 100 µM arsenite, unlike HSP 32, which was increased by 10 µM arsenite. One hundred µM arsenite resulted in a decrease of HSP 32 to control levels. Similar observations were found in the chicken hepatoma cell line, LMH, where dose-dependent induction in HSP 32 occurred as observed by luciferase reporter gene expression. In the above studies, the most effective dose for HSP 32 induction was 75 µM, and higher doses resulted in a decrease in HSP 32 (Elbirt et al., 1998). Other studies have investigated the expression of stress proteins in liver slices and renal slices. In liver slices, 10 µM arsenite increased HSP 72 and HSP 90 (Wijeweera et al., 1995
). Treatment with 0.110 µM arsenite induced HSP 32, but not HSP 72, HSP 90, or HSP 60, in renal slices (Parrish et al., 1999
). In our studies, no increase in either HSP 60 or HSP 90 contents were seen after arsenite exposure. This indicates that there are tissue-specific differences in the induction of stress proteins by arsenite. The fact that HSP 32 was induced by lower concentrations of arsenite, compared to HSP 72 in our studies, suggests that HSP 32 is much more sensitive to macromolecular damage caused by arsenite.
It has been suggested that HSP-72 expression increases after a certain threshold of cellular damage (Tacchini et al., 1993). Only 100-µM arsenite increased HSP 72 in our studies, indicating this concentration of arsenite may have caused subtle changes in the structure of cellular proteins. Damaged or abnormal proteins serve as a signal for the induction of HSP 72 (Ananthan et al., 1986
). The function of HSP 72 is to bring about the proper folding of proteins and to prevent the formation of protein aggregates that result when hydrophobic regions of damaged proteins are exposed (Aufricht et al., 1998
). Arsenite can certainly cause damage to cellular proteins by its reactivity towards thiol groups, as well as due to the production of reactive oxygen species that are generated during its cellular metabolism. From our studies, it is apparent that HSP 32 has a lower threshold for increased expression and is much more sensitive to damage to cellular macromolecules than HSP 72.
Western blot analysis of nuclear proteins from lung slices indicated that arsenite increased c-jun/AP-1 contents in a concentration-dependent manner. Confocal microscopic analysis indicated nuclear localization of c-jun/AP-1 in type-II epithelial cells and macrophages. Gel-shift assays indicated an increased nuclear presence of c-jun/AP-1 and NFB following arsenic exposure. However, an arsenite-induced increase in nuclear NF
B, as indicated by Western blot analysis, was not detected. Confocal microscopy indicated that activated NF
B was localized mainly to the cytoplasm of type-II epithelial cells and macrophages. Very little nuclear staining was seen. This indicates that NF
B is activated in the cytoplasm. However, the levels of nuclear NF
B were too low to detect with Western blot analysis. Confocal microscopy provided information on site-specific localization of AP-1 and NF
B in lung slices after arsenite treatment.
Normal tissue homeostasis requires a balance between cell proliferation and apoptosis, and an imbalance between these two processes may lead to cancer (Manning and Patierno, 1996; Thompson, 1995
). Apoptosis is important in removing genetically damaged cells. However, after a toxic insult, the surrounding surviving cells that contain damaged DNA may be stimulated to proliferate, resulting in neoplastic growth (Manning and Patierno, 1996
). Both AP-1 and NF
B appear to be critically involved in genes that regulate cell proliferation and apoptosis (Baeuerle, 1991
; Karin, 1995
; Lenardo and Baltimore, 1989
). Many of the environmental carcinogens such as arsenite can stimulate apoptosis (Hossain et al., 2000
) as well as proliferation (Simeonova et al., 2000
). Whether proliferation or apoptosis occurs is determined by the balance of several cell-signaling pathways leading to the activation of c-fos and c-jun (Xia et al., 1995
) as well as activation of NF
B (Baldwin, 1996
; Ghosh et al., 1998
). Furthermore, subunits of AP-1 and NF
B can cross talk and both of these factors may be involved in cell transformation (Denhardt, 1996; Stein et al., 1993).
HSP 32 expression is under the regulation of the transcription factors AP-1 and NFB, and both AP-1 and NF
B are known to be expressed under conditions of oxidative stress (Meyer et al., 1993
; Pinkus et al., 1996
; Schulze-Osthoff et al., 1995
). Both human (Lavrovsky et al., 1994
, 1996
; Shibahara et al., 1989
) and rodent (Alam, 1994
; Alam and Den, 1992
, 1995
; Bergeron et al., 1998
; Kurata et al., 1996
; Lavrovsky et al., 2000
) HSP 32 gene promoters have NF
B binding sites. Oxidant-induced lung injury in rat increased HSP 32 and AP-1 binding activity to the promoter region of HSP 32 gene (Lee et al., 1996
).
Both HSP 32 and HSP 72 expression is under the control of another transcription factor, HSF (Stuhlmeier, 2000). HSF binds to heat shock element (HSE) in the upstream promoter region of stress proteins and increase their transcription (Sorger et al., 1987
; Wu, 1984
). Several stressors including arsenite are known to activate HSF (Kato and Okamoto, 1997
; Larson et al., 1988
). Although we did not study expression and activation of HSF, probably arsenite may have activated it in lung slices. Activation of HSF by phosphorylation can be mediated by several members of the MAP kinase family, including ERK, JNK, and P38 (Kim et al., 1997
; Lee and Corry, 1988). Thus, stress protein expression is under a complex regulatory mechanism, which requires integration of signals from different signal transduction pathways that converge upon AP-1, NF
B, and HSF. This may explain why HSP 32 decreased, although AP-1 contents were increased by 100-µM arsenite in our study.
Recent investigations indicate that HSP 72 protein can protect cells from damage caused by various stresses by multiple mechanisms. HSP 72 can function as a chaperonin by refolding denatured proteins (Gething and Sambrook, 1992). In addition, HSP 72 can regulate signal transduction pathways: it can inhibit activation of NF
B by blocking I
B dissociation and degradation (Yoo et al., 2000
). Dissociation of I
B from p65/p50 NF
B complex is needed for the activation and nuclear translocation of this transcription factor (Siebenlist et al., 1994
). Thus, HSP 72 can prevent the expression of NF
B-dependent genes. Furthermore, HSP 72 plays a role in the activation as well as in the deactivation of JNK, which is responsible for the phosphorylation and activation of c-jun/AP-1. This activation of JNK is due to the inhibition of JNK phosphatase by HSP 72. When JNK phosphatase is inhibited, the background activity of JNK activating kinase, SEK1, is sufficient to account for JNK activation (Meriin et al., 1999
).
Increased expression of HSP 72 may protect cells against apoptotic or necrotic cell death. Recent studies indicate that the overexpression of HSP 72 forms a negative-feedback loop involved in the inhibition of apoptosis. This inhibitory effect on apoptosis is due to the prevention of JNK activation by high levels of HSP 72 protein (Gabai et al., 1997). Necrotic cell death is associated with ATP loss, aggregation of cytoskeletal proteins, and bleb formation (Gabai et al., 1993). Protein aggregation due to ATP depletion was inhibited (Kabakov and Gabai, 1995
) in tumor cells that accumulated HSP 72 after mild heat shock.
Arsenite did not increase HSP 60 in lung slices. Although HSP 90 was found to be much more abundant in control slices than the other stress proteins, its expression was not changed by arsenite treatment. HSP 60 is a mitochondrial stress protein which functions as a chaperonin to assist the correct folding of cytoplasmic polypeptides that are targeted for import into the mitochondria (Hartl and Martin, 1995). Exposure of human proximal tubular cells to 100 µM arsenite for 4 h resulted in an increase in HSP-60 content after the cells were allowed to recover for 12 h, and increased levels of this protein were observed for up to 48 h (Somji et al., 2000
). It appears that HSP 60 only increases after the stress factor is removed and the cells are allowed to recover. Similar to HSP 72, HSP 90 protein functions as a chaperonin to bring about proper folding of proteins and prevent aggregation of unfolded proteins (Freeman and Morimoto, 1996
; Young et al., 1997
). While arsenite induces changes in the expression of HSP 90 in the liver (Wijeweera et al., 1995
), such changes were not observed in the lung.
Thus, the present study indicates that precision-cut lung slices are a suitable in vitro system for studying perturbations in gene expression after a toxic insult. Low levels of arsenite resulted in the activation and expression of transcription factors and expression of stress proteins in lung slices. Confocal microscopic analysis provided information on the site-specific localization of transcription factors AP-1 and NFB.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at the Department of Cell Biology and Anatomy, College of Medicine, The University of Arizona, P.O. Box 245044, Tucson, AZ 85724-5044. Fax: (520) 626-2097. E-mail: lantz{at}u.arizona.edu.
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