Aflatoxin B1 Alters the Expression of p53 in Cytochrome P450-Expressin
http://www.100md.com
《毒物学科学杂志》
Graduate Program in Toxicology
Department of Veterinary Sciences, Utah State University, Logan, UT 84322–4620
3 To whom correspondence should be addressed at Graduate Program in Toxicology, Utah State University, 4620 Old Main Hill, Logan, UT 84322. Fax: (435) 797-1598. E-mail: rogerc@cc.usu.edu.
ABSTRACT
Aflatoxin B1 (AFB1) is a potent dietary hepatocarcinogen in animals and probably in humans. Mutations (and altered expression) of the tumor suppresser gene p53 have been observed in liver tumors from patients exposed to high dietary AFB1. Inhalation of AFB1-laden grain dusts has been associated with an increased incidence of lung cancer in humans as well. We examined the effects of low concentrations of AFB1 on the expression of p53 and MDM2 in human bronchial epithelial cells (BEAS-2B) transfected with cDNA for either cytochrome P450 (CYP) 1A2 (B-CMV1A2) or CYP 3A4 (B3A4), two isozymes that are responsible for AFB1 activation in human liver and possibly the lung. Untreated B-CMV1A2 and B3A4 cells constitutively expressed p53. Exposure to a range (0.015–15 μM for 30 min) of AFB1 concentrations caused a concentration-dependent decline in p53 expression in B-CMV1A2 cells, and to a lesser extent, in B3A4 cells. The AFB1-mediated decrease in p53 continued for at least 12 h after 30-min exposures to 1.5 μM AFB1. Mirroring the decrease in p53 expression was a concentration-dependent increase in the expression of the 76-kDa MDM2 isoform in B-CMV1A2 and B-3A4 cells. Interestingly, AFB1 did not induce DNA laddering, an indicator of apoptotic cell death, but proteolytic activation of caspase-3 was detected in AFB1-treated B-CVM1A2 cells. In total, these data show that low, environmentally-relevant concentrations of AFB1 alter the expression of p53 and MDM2 in these human lung cells, and that cells that stably express CYP 1A2 were more susceptible to this effect than nontransfected, or 3A4-expressing cells.
INTRODUCTION
Aflatoxin B1 (AFB1) is a potent immunotoxicant and hepatocarcinogen in animals and probably in humans (Bondy and Pestka, 2000; Klein et al., 2000). The liver is the primary target organ, because AFB1 requires metabolic activation to form the reputed carcinogenic species AFB1-8,9-epoxide (AFBO) (Mace et al., 1994, 1997), but other organs are also affected by AFB1 exposures (Ball and Coulombe, 1991; Ball et al., 1995; Coulombe et al., 1991; Imaoka et al., 1992; Kato et al., 1994; Kelly et al., 1997; Liu et al., 1990, 1993; Liu and Massey, 1992). In human liver, CYPs 1A2 and 3A4 have been shown to be the principle enzymes responsible for AFB1 activation (Ramsdell et al., 1991; Shimada and Guengerich, 1989) and have also been detected in human lung tissues and cultured lung cells (Mace et al., 1998; Van Vleet et al., 2001; Wei et al., 2001).
Inhalation of respirable AFB1-contaminated grain dusts may pose a cancer hazard to susceptible individuals in certain agricultural occupations (Hayes et al., 1984). AFB1 is activated to AFBO in animal (Ball et al., 1995; Daniels et al., 1990; Daniels and Massey, 1992; Imaoka et al., 1992; Liu et al., 1990; Liu and Massey, 1992) and in human pulmonary tissues (Autrup et al., 1979; Donnelly et al., 1996; Kelly et al., 1997).
The tumor suppressor gene p53 is the most commonly mutated gene in human cancers, with mutations in approximately 50% of all human cancers (Chang et al., 1993; Kew, 1992; May and May, 1995; Nigro et al., 1989). Dietary AFB1 exposure has been linked to GT transversions at codon 249 of p53 in human primary hepatocellular carcinomas (PHC) (Cerutti et al., 1994; Hainaut and Vahakangas, 1997; Soini et al., 1996) and in cultured human hepatocytes exposed to AFB1 (Aguilar et al., 1993; Mace et al., 1997). Therefore, this mutation may inactivate p53, leading to AFB1-induced liver cancers (Hollstein et al., 1993; Lasky and Magder, 1997). It seems reasonable that a similar mutation may be produced by AFB1 in the lung.
After cellular DNA damage, p53 protein is phosphorylated by DNA-dependent protein kinase, which arrests the cell cycle in the G1 phase, thus preventing mitosis and allowing the repair of damaged sequences (Kastan et al., 1992). Thus, by preventing the proliferation of damaged cells, p53 acts to protect the integrity of the genome (Lane, 1992). Induction of p53 leads to increased MDM2 expression, which eventually inhibits p53 expression via a negative-feedback mechanism (Pochampally et al., 1998). To induce MDM2 expression, p53 acts as a transcriptional element on the MDM2-P2 promoter (Hesketh, 1997; Ralhan et al., 2000), but MDM2 induction is at least partly also due to mRNA stabilization (Hsing et al., 2000). To date, several MDM2 proteins have been identified (90, 76, 60, 46, and 35 kDa) (Maxwell, 1994; Mendrysa et al., 2001; Ralhan et al., 2000), which are produced by differential splicing of the mRNA transcript and caspase-3-mediated cleavage of the 90-kDa isoform (Chen et al., 1997; Maxwell, 1994; Mendrysa et al., 2001) and by internal initiation at codon 50 of the mdm2 mRNA (Saucedo et al., 1999).
The proteolytic activation of caspase-3 and DNA ladder formation are key steps in the apoptotic cascade (Maruyama et al., 2001) with caspase-3 activation regarded as a primary mechanism of apoptosis (He et al., 2003). Caspase-3-like activity has been implicated in the processing of MDM2 to a form that stabilizes p53 (Pochampally et al., 1999). Caspase-3 activation can be detected using Western immunoblotting to demonstrate proteolytic cleavage of the procaspase-3 protein (35 kDa) to the largest (17 kDa) proteolytic fragment (Erhardt et al., 2001).
The BEAS-2B cell line, a simian virus 40 (SV-40) large T antigen immortalized version of normal human bronchial epithelial (NHBE) cells, is an in vitro model for the study of the human lung toxicity (Reddel et al., 1988). Infection of normal cells with SV-40 interferes with p53 function, leading to immortalization (Carnero et al., 2000; Hsieh et al., 2000; Peterson et al., 1995; Porras et al., 1999). The SV-40 viral genome codes for the large T antigen, which binds p53, inhibiting its normal growth arrest and cell cycling functions (Levresse et al., 1998; Peterson et al., 1995; Porras et al., 1999).
We recently demonstrated that BEAS-2B cells transfected to stably express CYP 1A2 (BCMV-1A2) and 3A4 (B3A4) activate AFB1 to cytotoxic and DNA-alkylating species (Van Vleet et al., 2002a,b). Because AFB1 affects p53 expression in human liver cancer (Barton et al., 1991), we wished to determine if AFB1 would affect this tumor suppressor system in these immortalized lung cells. Our data indicate that AFB1 perturbs the expression of p53 and related proteins in these cells when critical CYPs are expressed. At the lowest concentrations studied, CYP 1A2-expressing cells were affected to a greater extent than those expressing 3A4, in support of previous results showing that expression of the former isoform may be more relevant to AFB1 toxicity. Despite compromised p53 activity, exposure to AFB1 activates caspase-3 in BCMV1A2 cells.
MATERIALS AND METHODS
Chemicals and reagents.
LHC-8, LHC-9, LHC Basal, epinephrine, retinoic acid, and bovine serum albumin (BSA) stock were obtained from BioWhittaker (Rockville, MD). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). Bovine fibronectin, 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), glycerol, AFB1, trypsin inhibitor, aminosalicylate, -mercaptoethanol, pyronin-Y, staurosporine, caffeine, and Coomassie blue were purchased from Sigma (St. Louis, MO). Collagen was a product of Collagen Corp. (Fremont, CA). CHAPS Cell Extraction buffer, primary polyclonal rabbit anti-human caspase-3 antibody, primary polyclonal rabbit p53 antibody used with the BEAS-2B cells, and secondary antirabbit IgG were purchased from Cell Signaling, (Beverly, MA). Primary monoclonal mouse p53 and MDM2 used with the BCMV-1A2, B3A4, and NHBE cells were from Calbiochem (San Diego, CA). Secondary (goat-anti-mouse) antibody was from Bio-Rad (Hercules, CA). ECL chemiluminescent reagent was from Amersham (Piscataway, NJ). Supersignal West Femto chemiluminescent substrate was from Fisher Scientific, (Pittsburg, PA). BEAS-2B, B-CMV1A2, and B3A4 cells were a generous gift from Dr. Katherine Mace (Nestle Research Centre; Lausanne, Switzerland). Normal human bronchial epithelial (NHBE) cells were purchased from BioWhittaker (San Diego, CA).
Cell culture.
BEAS-2B, B-CMV1A2, and B3A4 cells were cultured as previously described (Van Vleet et al., 2002a) for all experiments excluding caspase-3 activation and basal p53 expression in BEAS-2B wherein flasks were not coated with a plate coat consisting of 5 mg bovine fibronectin, 5 ml collagen, 50 ml BSA stock, and 500 ml LHC Basal. NHBE cells were cultured as previously described (Van Vleet et al., 2001).
Preparation of cell lysates.
Cells were seeded at a density of 9.5 x 105 cells/T75 flask and cultured for 48 h. For time-course studies, cultures were then exposed to 1.5 μM AFB1 for 30 min, after which flasks were washed with PBS, and then fresh media was added to cultures. Cells were harvested via trypsinization, at various time intervals thereafter (1, 2, 4, 6, 9, and 12 h). To determine the effects of a range of AFB1 concentrations on p53 expression, cells were exposed to AFB1 (0.015–15 μM) for 30 min, and flasks were then washed with PBS. The cells were cultured for 6 h in fresh media before they were harvested. Next, cells from each T75 flask were resuspended in 1 ml of LHC-9. The cell density was determined (Counter Model FN; Beckman-Coulter Fullerton, CA), and 0.5 ml of the cell suspension was centrifuged to collect cells. After removing the supernatant, cell lysing buffer (2% sodium dodecyl sulfate (SDS), 12% aminosalicylate, 2% NaCl, and 12% 2-butanol) was added to the pellet at a concentration of 100,000 cells/20 μl. Samples were stored at –80°C until separated by SDS–PAGE. Control groups were also run at each time point (time-course study), or at 6 h after exposure to AFB1 (concentration range study) for 30 min.
Measurement of p53 and MDM2 expression.
Cell lysates (20 μl) were heated in sample buffer (10% SDS, 0.5M Tris-HCl, 20% glycerol, 10% -mercaptoethanol, 0.1% pyronin-Y; 30 μl; total vol = 50 μl) to 70°C for 5 min and loaded into 10–15% SDS-PAGE gels (14 x 11 x 0.1 cm), with duplicate lanes, then electrophoresed for 8 h at 125 V. One-half of each gel was transferred to a Nitrobind nitrocellulose transfer membrane (Micron Separations, Inc.) using a semi-dry blotter (Buchler, Kansas City, MO). The other half of the gel was stained with Coomassie blue for molecular weight analysis. Molecular weight markers served as negative controls for nonspecific binding of antibodies to protein in the immunostained gel portions, and for molecular weight approximations of the Coomassie-stained gel halves. Nitrocellulose membranes were immunostained using the primary antibody (1:5000) in High Salt Tween (HST) blocking buffer (10 mM Tris, 1 M NaCl 0.5% Tween 20, pH 7.4). Membranes were washed with HST, Tris-buffered saline (TBS) (10 mM Tris and 140 mM NaCl, pH 7.4), and TBS-Tween (TBS with 0.1% Tween 20) as described previously (Klein et al., 2000). Secondary antibodies were also diluted in HST (1:2000). Proteins were detected by chemiluminescence generated by horseradish peroxidase-conjugated secondary antibody, using ECL reagent as a substrate, and quantified using a Nucleovision 920 chemiluminescence imaging workstation (Nucleotech Corp., Hayward, CA).
The following method was employed exclusively for examining p53 expression in BEAS-2B cells. Cells were grown to approximately 80% confluence and harvested via trypsinization. Cells were pelleted and resuspended in 100 μl of CHAPS (50 mM PIPES/NaOH (pH 6.5), 2 mM EDTA, 0.1% Chaps, 5 mM DTT, 20 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, and 1 mM PMSF) cell extract buffer and subjected to five freeze–thaw cycles. The cell lysate was then centrifuged at 13,000 rpm (16,060 x g) for 5 min, and the supernatant was retained and frozen at –80°C. Sample protein was measured using the Quick Start Bradford Protein assay kit (Bio-Rad, Hercules CA). Aliquots of the supernatant were diluted 1:1 with SDS sample buffer (2% SDS, 50 mM dithiothreitol (DTT), 0.01% bromophenyl blue, 10% glycerol), boiled for 5 min, and loaded onto 4–15% SDS/Tris–HCl mini acrylamide gradient gels (Bio-Rad Labs, Hercules, CA) at 15 μg protein per well. P53 standard (Oncogene, Boston, MA) was loaded at 10 μl per well. Samples were electrophoresed for 45 min at 200 V on a Bio-Rad Mini-Protean 3 Cell electrophoresis unit (Bio-Rad Labs, Hercules, CA). Gels were transferred at 100 V for 1 h to nitrocellulose transfer membranes (GE Osmonics, Minnetonka, MN) using the Bio-Rad Mini-Protean 3 Cell electrophoresis unit. Nitrocellulose membranes were washed in TBS for 5 min and then incubated in blocking buffer (TBS with 0.1% Tween 20 and 5% nonfat dry milk). Membranes were again washed in TBS-Tween (TBS with 0.1% Tween 20) three times for 5 min each, and immuno-stained overnight (at 4°C) with the primary antibody (1:1000) in 5% BSA, 1x TBS, 0.1% Tween-20 at 4°C with gentle shaking, overnight. Membranes were then washed with TBS-Tween three times. Secondary antibodies were diluted in blocking buffer (1:2000 and 1:1000) and incubated with the membrane at room temperature for 1 h. Proteins were detected by chemiluminescence generated by horseradish peroxidase-conjugated secondary antibody, using Supersignal West Femto chemiluminescent substrate, and captured using a Nucleovision 920 chemiluminescence imaging workstation (Nucleotech Corp., Hayward, CA).
Ladder assay for apoptosis.
B-CMV1A2 cells were seeded at a density of 9.5 x 105 cells/T75 flask, and cultured for 48 h. Actively dividing cells (approx. 50% confluency) were then dosed with AFB1 at various concentrations (0–15 μM) for 30 min and harvested after 6 h of culture in fresh media via trypsinization. To study the time course of apoptosis, actively dividing cells were dosed with 1.5 μM AFB1 for 30 min, washed with PBS, and harvested at various time intervals (1, 2, 4, 8, 12, 16, 20, 24 h) after PBS was replaced with fresh LHC-9. Cells were then harvested, and pellets were resuspended at a concentration of 2 x 106 cells/200 μl in PBS for use in assay. DNA samples (6 μg) were added to 10x loading buffer (1% SDS, 2.5 mg/ml bromophenyl blue, 30% glycerol) and loaded into 7 x 7.5 x 1 cm 1% agarose gels in TBE (0.04 M Tris, 0.04 M boric acid, 0.01 M EDTA). Samples were electrophoresed at 4°C for 35 min at 200 V. Laddering was also examined using the Qiagen DNeasy tissue Kit (Qiagen, Valencia, CA) and the proteinase K method described by Thorburn et al. (2003).
Detection of caspase-3 activation.
B-CMV1A2 and B3A4 cells were seeded at a density of 6.4 x 104 cells/T-75 flask and cultured for 48 h. Actively dividing cells (approx. 70% confluency) were then dosed with either AFB1 (1 μM), Staurosporine (1 μM; positive control), or DMSO (20 μl; negative control) for 4 and 9 h, and harvested in ice cold PBS, via scraping. Caffeine-dosed cells were exposed to 150, 250, 350, and 450 μM concentrations for 24 h. Cells were pelleted and resuspended in 100 μl of CHAPS (50 mM PIPES/NaOH (pH 6.5), 2 mM EDTA, 0.1% Chaps, 5 mM DTT, 20 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, and 1 mM PMSF) cell extract buffer and subjected to five freeze–thaw cycles. The cell lysate was then centrifuged at 16,000 rpm for 5 min, and the supernatant was retained and frozen at –80°C. Sample protein was measured using the Quick Start Bradford Protein Plate reader assay kit (Bio-Rad, Hercules CA) on a Labsystems Multiskan MCC/340 (Fisher Scientific, Pittsburg, PA). Aliquots of the supernatant (25 μl) were diluted 1:1 with SDS sample buffer (2% SDS, 50 mM DTT, 0.01% bromophenyl blue, 10% glycerol), boiled for 5 min, and loaded at 0.5 μg protein per well for AFB1 and 24 μg per well caffeine onto 15% SDS/Tris–HCl mini acrylamide gels (Bio-Rad Labs, Hercules, CA). Samples were electrophoresed for 45 min at 200 V on a Bio-Rad Mini-Protean 3 Cell electrophoresis unit (Bio-Rad Labs, Hercules, CA). Gels were transferred at 100 V for 1 h to nitrocellulose transfer membranes (GE Osmonics, Minnetonka, MN). Nitrocellulose membranes were washed in TBS for 5 min and then incubated in blocking buffer (TBS with 0.1% Tween 20 and 5% nonfat dry milk). Membranes were again washed in TBS-Tween (TBS with 0.1% Tween 20) three times for 5 min each, and immuno-stained overnight (at 4°C) with the primary antibody (1:1000) in blocking buffer. Membranes were then washed with TBS-Tween three times for 5 min. Secondary antibodies were also diluted in blocking buffer (1:2000 anti biotin and 1:1000 anti rabbit) and incubated with the membrane at room temperature for 1 h. Proteins were detected by chemiluminescence generated by horseradish peroxidase-conjugated secondary antibody, using Supersignal West Femto chemiluminescent substrate, and images were captured and archived.
Determination of cell viability.
The IC50 value for caffeine toxicity in BCMV-1A2 cells was determined via MTT in a 96-well format using a Labsystems Multiskan MCC/340 (Fisher scientific, Pittsburgh PA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Aldrich, St. Louis, MO) as previously described (Mosmann, 1983).
Statistical analysis and curve fitting.
Groups were compared for differences using one-way ANOVA (Sigma Stat Software). A p < 0.05 was judged significant. Curves generated from digital densitometry analysis were fit using Sigma Plot logistics curve fitting program (SPSS, Chicago, IL).
RESULTS
All SV-40 transformed cells, BEAS-2B, B-CMV1A2, and B3A4 expressed p53 constitutively (Figs. 1A and 1B). The doublets match those found by other researchers (Matlashewski et al., 1986) and those shown on the manufacturer's instructions. This is in contrast to normal human bronchial epithelial (NHBE) cells, where no constitutive p53 expression was observed (Fig. 1A).
When B-CMV1A2 and B3A4 cells were exposed to 1.5 μM AFB1 for 30 min, p53 expression decreased over time compared to their respective unexposed controls (Figs. 2A and 2B). This decrease in expression continued at least 12 h, with the effect being significantly greater in B-CMV1A2 cells than in B3A4 cells at all time points (Figs. 2A, 2B, and 2C). We then determined whether the inhibitory effect on p53 expression was dependent on AFB1 concentration. When exposed to a range of AFB1 concentrations (0–15 μM) for 30 min, a similar decrease in p53 expression was observed in both B-CMV1A2 and B3A4 cell types in a concentration-dependent manner 6 h post-exposure (Figs. 3A and 3B). Thus, the inhibitory effect of AFB1 on p53 expression was dependent on AFB1 concentration and duration of exposure. In cultures exposed to the highest AFB1 concentration (i.e., 15 μM), the decrease in p53 expression was greater in B3A4 than the B-CMV1A2 cells (Fig. 3C).
Because p53 expression was altered to a greater extent in B-CMV1A2 cells, we then examined the effect of AFB1 on the expression of MDM2 in these cells. As can be seen in Figure 4, AFB1 elicited an increase in MDM2 (76 kDa) expression in B-CMV1A2 cells in both a concentration- (0.15–15 μM; 6 h after 30-min exposures) and time-dependent (0–12 h after 30-min exposures to 1.5 μM AFB1) fashion (Figs. 4A and 4B). When data from Figures 3 and 4 are plotted together, the combined effect of AFB1 on both p53 and MDM2 can clearly be seen in Figure 5. The AFB1 concentration and time-dependent decrease in p53 expression were mirrored by a concomitant increase in MDM2 (76 kDa) expression (Figs. 5A and 5B) at each point. However, only expression of the 76-kDa isoform was consistently affected by AFB1. Other MDM2 proteins—90, 60, and 35 kDa—were detected, but their levels were not affected by AFB1 treatment (data not shown).
DNA ladder formation, an indicator of the onset of an irreversible stage in apoptosis, was examined in B-CMV1A2 cells. B-CMV1A2 cells were exposed to 1.5 μM at a range of post-AFB1 exposure intervals (0–24 h; Fig. 6A). No ladder formation could be detected in B-CMV1A2 cells treated with AFB1. Even when B-CMV1A2 cells were subjected to a 30-min exposure at a range of AFB1 concentrations (0.015–15 μM; 6-h exposure), no DNA ladder formation was detected (Fig. 6B). There was no clear indication of ladder formation detected in any AFB1- or staurosporine-treated B-CMV1A2 cells under any experimental protocol using three different methods for ladder detection (data not shown).
Activation of apoptotic executioner protease caspase-3 was then examined in was then examined in B-CMV1A2 cell cultures dosed with AFB1. B-CMV1A2 cells were exposed to 1 μM AFB1, 1 μM staurosporine, or DMSO for either 4 or 9 h. Proteolysed (activated) caspase-3 was detected in B-CMV1A2 cells exposed to either AFB1 or staurosporine for either 4 or 9 h, but was absent from control (DMSO-treated) cells for 9 h (Fig. 7A). To determine if BCMV1A2 cells possess a functional p53, we examined whether caffeine would cause caspase-3 cleavage. In BCMV-1A2 cells, the 24-h IC50 for caffeine was 281 μM as determined by the MTT assay (data not shown). Caspase-3 cleavage was not detected in BCMV-1A2 cells dosed with caffeine (150–450 μM) for 24 h (Fig. 7B).
DISCUSSION
In occupations where pulmonary exposures to AFB1-laden grain dusts are common, workers may be at an increased risk of developing lung cancer (Hayes et al., 1984). We previously showed that these CYP-transfected cells activate AFB1 to intermediate(s) that are cytotoxic and form AFB1–DNA adducts. When taking into consideration the expression of CYP mRNA, B-CMV1A2 cells were more efficient at activating AFB1 at low concentrations (<3 μM), while B3A4 cells were more efficient at higher concentrations (>3 μM) (Van Vleet et al., 2002b). Because of the reported relevance of p53 mutations and altered p53 expression in AFB1-induced hepatocarcinogenesis (Lee et al., 2000), we sought to determine if AFB1 exposure has an effect on p53 expression in these human bronchial epithelial cells.
This study indicates that, in contrast to normal cells, the immortalized cells used in this study—BEAS-2B, B3A4, and B-CMV1A2—express p53 constitutively. Since BEAS-2B cells are the progenitor cells of B3A4 and B-CMV1A2, constitutive expression in the latter cell lines was not the result of transfections inducing the expression of CYPs 1A2 and 3A4, but is likely due to SV-40 immortalization. Others have reported similar constitutive p53 expression in BEAS-2B cells (Gerwin et al., 1992). Importantly, our results also indicate that p53 function is impaired by exposure to AFB1. Our data demonstrate that AFB1 exposure inhibits p53 expression in B-CMV1A2 and B3A4 cells, an event associated with a reflective increase in MDM2 expression. It has been previously demonstrated that SV-40 affects p53 function. For example, SV-40 large T antigen causes continuous p53 inactivation and leads to immortalization of primary mouse embryonic fibroblasts (Carnero et al., 2000). Other SV-40 immortalized cell lines also constitutively express p53 (Miyazawa et al., 1998; Stein et al., 1991). It was also previously shown that BEAS cells, the progenitor cell line for the B-CMV1A2 and B3A4 cell lines, possess a p53 protein incapable of inducing the expression of downstream proteins under DNA-alkylating conditions (Technau et al., 2001). Researchers from that study noted that p53 expression was frequently decreased in cells exposed to mitomycin C, which is also in agreement with our data showing decreased p53 expression after AFB1 exposure. An increase in MDM2 and reduction in p53 was also observed with TCDD in HepG2 cells (Paajarvi et al., 2005). Interestingly, in another study, neuroblastoma cells where shown to possess a p53 that was also unable to induce the expression of p21 (and MDM2), after exposure to mitomycin C, even though p53 protein was able to bind DNA (Wolff et al., 2001). These discoveries support speculation that SV-40 immortalization not only inactivates p53 from its role in cell cycle control, but also inactivates its ability to protect the integrity of the genome, as seen in some cancer cells. Although these results provide insight into the function of p53 and the effects of SV-40 immortalization on p53 function, they also suggest that BEAS-2B cell physiology may present some limitations to studying the toxicological responses in certain molecular targets.
MDM2 induction, concomitant to the decline in p53 expression, indicates that the decline in p53 expression was not due to cell death from AFB1 treatment under these conditions. If the decrease were due to a general lack of protein synthesis from cell death, MDM2 would not be induced under these conditions. Other studies have shown that the induction of some MDM2 isoforms can cause a decrease in p53 expression (Carnero et al., 2000; Freedman et al., 1999), and that MDM2 induction can be independent of functional p53 (Hsing et al., 2000). Interestingly, the induction of MDM2 we observed was of the 76-kDa fragment, which typically attenuates the ability of the full-length p90MDM2 to decrease the level of p53 thereby increasing p53 (Perry et al., 2000).
Further evidence of the lack of p53 function can be seen in the inability of these (BCMV-1A2) SV-40 immortalized lung cells to undergo DNA laddering after treatment with AFB1 or staurosporine. A lack of functional p53 has been shown to perturb cellular growth arrest and apoptosis (May and May, 1995), while other studies have shown p53-independent induction of apoptosis in some SV-40 immortalized cell types (Gartenhaus et al., 1996; Levresse et al., 1998). Caffeine has been shown to induce apoptosis in a p53-dependent manner in p53+/+ mouse epidermal JB6 C141 cells, resulting in caspase-3 cleavage but not in p53–/– JB6 C141 cells (He et al., 2003). We were unable to observe caspase-3 activation in B-CMV1A2 cells by the p53-dependent apoptosis-inducer caffeine, further suggesting that p53 was nonfunctional in these cells. That both AFB1 and staurosporine were able to activate caspase-3 despite attenuated p53 indicates that apoptosis induced by these two compounds was p53 independent. Apoptosis has been previously reported in BEAS-2B cells (Agopyan et al., 2003; Nichols et al., 2003), which is consistent with our detection of caspase-3 activation. The absence of DNA ladder in the presence of caspase-3 activation is consistent with previous reports that caspase inhibitors are ineffective at preventing DNA fragmentation during apoptosis in multiple cell types from different species (Villa et al., 1998).
The AFB1-induced inhibition of p53 expression was greater in CYP1A2-expressing than in CYP3A4-expressing cells, except only at the highest AFB1 (15 μM) concentrations. Earlier studies from our laboratory demonstrated that B-CMV1A2 cells were substantially more efficient at AFB1 bioactivation to cytotoxic and DNA-alkylating intermediates than were B3A4 cells at low, environmentally relevant concentrations of AFB1 (Van Vleet et al., 2002a,b). In conclusion, the p53-mediated response to AFB1 treatment may indicate that these cells are at an increased risk of developing mutations. However, in the absence of a typical p53 response, these cells were able to undergo apoptosis, as evidenced by caspase-3 cleavage. The implications of our findings on the usefulness of these cells in in vitro studies of the effects of environmental carcinogens such as AFB1 are unclear. It is possible that the inhalation of AFB1-contaminated grain dusts may lead to modulation of p53 and cellular death under conditions where appropriate CYPs are expressed in the lung, resulting in adverse health effects.
NOTES
Portions of this report were presented at the 44th annual meeting of the Society of Toxicology, New Orleans, LA, March 2005 (Abstract # 1795).
1 Current address: Department of Toxicology, Bristol-Myers Squibb Company, Mt. Vernon, IN 47721.
2 Current address: Department of Surgery, Indiana University School of Medicine, Indianapolis, IN 46202.
ACKNOWLEDGMENTS
The authors wish to acknowledge Dr. Katherine Mace for the gift of B-CMV1A2 and B3A4 cells. This research was supported in part by the Marriner S. Eccles Foundation, by a competitive grant from USDA-NRI (2002–35204–12294), and by the Utah Agricultural Experiment Station, where it is paper no. 7738.
REFERENCES
Agopyan, N., Bhatti, T., Yu, S., and Simon, S. A. (2003). Vanilloid receptor activation by 2- and 10-μm particles induces responses leading to apoptosis in human airway epithelial cells. Toxicol. Appl. Pharmacol. 192, 21–35.
Aguilar, F., Hussain, S. P., and Cerutti, P. (1993). Aflatoxin B1 induces the transversion of G–>T in codon 249 of the p53 tumor suppressor gene in human hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 90, 8586–8590.
Autrup, H., Essigmann, J. M., Croy, R. G., Trump, B. F., Wogan, G. N., and Harris, C. C. (1979). Metabolism of aflatoxin B1 and identification of the major aflatoxin B1- DNA adducts formed in cultured human bronchus and colon. Cancer Res. 39, 694–698.
Ball, R. W., and Coulombe, R. A., Jr. (1991). Comparative biotransformation of aflatoxin B1 in mammalian airway epithelium. Carcinogenesis 12, 305–310.
Ball, R. W., Huie, J. M., and Coulombe, R. A., Jr. (1995). Comparative activation of aflatoxin B1 by mammalian pulmonary tissues. Toxicol. Lett. 75, 119–125.
Barton, C. M., Staddon, S. L., Hughes, C. M., Hall, P. A., O'Sullivan, C., Kloppel, G., Theis, B., Russell, R. C., Neoptolemos, J., Williamson, R. C., et al. (1991). Abnormalities of the p53 tumour suppressor gene in human pancreatic cancer [published erratum appears in Br. J. Cancer (1992) 65(3), 485]. Br. J. Cancer 64, 1076–1082.
Bondy, G. S., and Pestka, J. J. (2000). Immunomodulation by fungal toxins. J. Toxicol. Environ. Health B Crit. Rev. 3, 109–43.
Carnero, A., Hudson, J. D., Hannon, G. J., and Beach, D. H. (2000). Loss-of-function genetics in mammalian cells: the p53 tumor suppressor model. Nucleic Acids Res. 28, 2234–2241.
Cerutti, P., Hussain, P., Pourzand, C., and Aguilar, F. (1994). Mutagenesis of the H-ras protooncogene and the p53 tumor suppressor gene. Cancer Res. 54, 1934s–1938s.
Chang, F., Syrjanen, S., Tervahauta, A., and Syrjanen, K. (1993). Tumourigenesis associated with the p53 tumour suppressor gene. Br. J. Cancer 68, 653–661.
Chen, L., Marechal, V., Moreau, J., Levine, A. J., and Chen, J. (1997). Proteolytic cleavage of the mdm2 oncoprotein during apoptosis. J. Biol. Chem. 272, 22966–22973.
Coulombe, R. A., Huie, J. M., Ball, R. W., Sharma, R. P., and Wilson, D. W. (1991). Pharmacokinetics of intratracheally administered aflatoxin B1. Toxicol. Appl. Pharmacol. 109, 196–206.
Daniels, J. M., Liu, L., Stewart, R. K., and Massey, T. E. (1990). Biotransformation of aflatoxin B1 in rabbit lung and liver microsomes. Carcinogenesis 11, 823–827.
Daniels, J. M., and Massey, T. E. (1992). Modulation of aflatoxin B1 biotransformation in rabbit pulmonary and hepatic microsomes. Toxicology 74, 19–32.
Donnelly, P. J., Stewart, R. K., Ali, S. L., Conlan, A. A., Reid, K. R., Petsikas, D., and Massey, T. E. (1996). Biotransformation of aflatoxin B1 in human lung. Carcinogenesis 17, 2487–2494.
Erhardt, J. A., Ohlstein, E. H., Toomey, J. R., Gabriel, M. A., Willette, R. N., Yue, T.-L., Barone, F. C., and Parsons, A. A. (2001). Activation of caspase-3/caspase-3-like activity in rat cardiomyocytes by an RGD peptide, but not the GPIIb/IIIa antagonist lotrafiban. Thromb. Res. 103, 143–148.
Freedman, D. A., Wu, L., and Levine, A. J. (1999). Functions of the MDM2 oncoprotein. Cell. Mol. Life Sci. 55, 96–107.
Gartenhaus, R. B., Wang, P., and Hoffmann, P. (1996). Induction of the WAF1/CIP1 protein and apoptosis in human T-cell leukemia virus type I-transformed lymphocytes after treatment with adriamycin by using a p53-independent pathway. Proc. Natl. Acad. Sci. U.S.A. 93, 265–268.
Gerwin, B. I., Spillare, E., Forrester, K., Lehman, T. A., Kispert, J., Welsh, J. A., Pfeifer, A. M., Lechner, J. F., Baker, S. J., Vogelstein, B., et al. (1992). Mutant p53 can induce tumorigenic conversion of human bronchial epithelial cells and reduce their responsiveness to a negative growth factor, transforming growth factor beta 1. Proc. Natl. Acad. Sci. U.S.A. 89, 2759–2763.
Hainaut, P., and Vahakangas, K. (1997). p53 as a sensor of carcinogenic exposures: mechanisms of p53 protein induction and lessons from p53 gene mutations. Pathol. Biol. (Paris) 45, 833–844.
Hayes, R. B., van Nieuwenhuize, J. P., Raatgever, J. W., and Ten Kate, F. J. (1984). Aflatoxin exposures in the industrial setting: An epidemiological study of mortality. Food Chem. Toxicol. 22, 39–43.
He, Z., Ma, W. Y., Hashimoto, T., Bode, A. M., Yang, C. S., and Dong, Z. (2003). Induction of apoptosis by caffeine is mediated by the p53, Bax, and caspase 3 pathways. Cancer Res. 63, 4396–4401.
Hesketh, R. (1997). The Oncogene and Tumour Suppressor Gene Facts Book. Academic Press, San Diego. CA.
Hollstein, M. C., Wild, C. P., Bleicher, F., Chutimataewin, S., Harris, C. C., Srivatanakul, P., and Montesano, R. (1993). p53 mutations and aflatoxin B1 exposure in hepatocellular carcinoma patients from Thailand. Int. J. Cancer 53, 51–55.
Hsieh, J. K., Kletsas, D., Clunn, G., Hughes, A. D., Schachter, M., and Demoliou-Mason, C. (2000). p53, p21(WAF1/CIP1), and MDM2 involvement in the proliferation and apoptosis in an in vitro model of conditionally immortalized human vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 20, 973–981.
Hsing, A., Faller, D. V., and Vaziri, C. (2000). DNA-damaging aryl hydrocarbons induce Mdm2 expression via p53- independent post-transcriptional mechanisms. J. Biol. Chem. 275, 26024–26031.
Imaoka, S., Ikemoto, S., Shimada, T., and Funae, Y. (1992). Mutagenic activation of aflatoxin B1 by pulmonary, renal, and hepatic cytochrome P450s from rats. Mutat. Res. 269, 231–236.
Kastan, M. B., Zhan, Q., el-Deiry, W. S., Carrier, F., Jacks, T., Walsh, W. V., Plunkett, B. S., Vogelstein, B., and Fornace, A. J., Jr. (1992). A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 71, 587–597.
Kato, T., Nakasa, H., Ohmori, S., Kamataki, T., Itahashi, K., Shimada, T., Rikihisa, T., and Kitada, M. (1994). Possible occurrence of P450 related to P450 HFLb in extrahepatic tissues of human fetuses and its contribution to metabolic activation of promutagens. Mutat. Res. 310, 73–77.
Kelly, J. D., Eaton, D. L., Guengerich, F. P., and Coulombe, R. A., Jr. (1997). Aflatoxin B1 activation in human lung. Toxicol. Appl. Pharmacol. 144, 88–95.
Kew, M. C. (1992). Tumours of the liver. Scand. J. Gastroenterol. Suppl. 192, 39–42.
Klein, P. J., Buckner, R., Kelly, J., and Coulombe, R. A., Jr. (2000). Biochemical basis for the extreme sensitivity of turkeys to aflatoxin B1. Toxicol. Appl. Pharmacol. 165, 45–52.
Lane, D. P. (1992). Cancer p53, guardian of the genome [news; comment] [see comments]. Nature 358, 15–16.
Lasky, T., and Magder, L. (1997). Hepatocellular carcinoma p53 G > T transversions at codon 249: the fingerprint of aflatoxin exposure Environ. Health Perspect. 105, 392–397.
Lee, Y. I., Lee, S., Das, G. C., Park, U. S., and Park, S. M. (2000). Activation of the insulin-like growth factor II transcription by aflatoxin B1 induced p53 mutant 249 is caused by activation of transcription complexes; implications for a gain-of-function during the formation of hepatocellular carcinoma. Oncogene 19, 3717–3726.
Levresse, V., Moritz, S., Renier, A., Kheuang, L., Galateau-Salle, F., Mege, J. P., Piedbois, P., Salmons, B., Guenzburg, W., and Jaurand, M. C. (1998). Effect of simian virus large T antigen expression on cell cycle control and apoptosis in rat pleural mesothelial cells exposed to DNA damaging agents. Oncogene 16, 1041–1053.
Liu, L., Daniels, J. M., Stewart, R. K., and Massey, T. E. (1990). In vitro prostaglandin H synthase- and monooxygenase-mediated binding of aflatoxin B1 to DNA in guinea-pig tissue microsomes. Carcinogenesis 11, 1915–1919.
Liu, L., and Massey, T. E. (1992). Bioactivation of aflatoxin B1 by lipoxygenases, prostaglandin H synthase and cytochrome P450 monooxygenase in guinea-pig tissues. Carcinogenesis 13, 533–539.
Liu, L., Nakatsu, K., and Massey, T. E. (1993). In vitro cytochrome P450 monooxygenase and prostaglandin H-synthase mediated aflatoxin B1 biotransformation in guinea pig tissues: effects of beta-naphthoflavone treatment. Arch. Toxicol. 67, 379–385.
Mace, K., Aguilar, F., Wang, J. S., Vautravers, P., Gomez-Lechon, M., Gonzalez, F. J., Groopman, J., Harris, C. C., and Pfeifer, A. M. (1997). Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450- expressing human liver cell lines. Carcinogenesis 18, 1291–1297.
Mace, K., Bowman, E. D., Vautravers, P., Shields, P. G., Harris, C. C., and Pfeifer, A. M. (1998). Characterization of xenobiotic-metabolizing enzyme expression in human bronchial mucosa and peripheral lung tissues. Eur. J. Cancer 34, 914–920.
Mace, K., Gonzalez, F. J., McConnell, I. R., Garner, R. C., Avanti, O., Harris, C. C., and Pfeifer, A. M. (1994). Activation of promutagens in a human bronchial epithelial cell line stably expressing human cytochrome P450 1A2. Mol. Carcinog. 11, 65–73.
Maruyama, W., Youdim, M. B., and Naoi, M. (2001). Antiapoptotic properties of rasagiline, N-propargylamine-1(R)-aminoindan, and its optical (S)-isomer, TV1022. Ann. N.Y. Acad. Sci. 939, 320–329.
Matlashewski, G., Banks, L., Pim, D., and Crawford, L. (1986). Analysis of human p53 proteins and mRNA levels in normal and transformed cells. Eur. J. Biochem. 154, 665–672.
Maxwell, S. A. (1994). Selective compartmentalization of different mdm2 proteins within the nucleus. Anticancer Res. 14, 2541–2547.
May, P., and May, E. (1995). P53 and cancers. Pathol. Biol. (Paris) 43, 165–173.
Mendrysa, S. M., McElwee, M. K., and Perry, M. E. (2001). Characterization of the 5' and 3' untranslated regions in murine mdm2 mRNAs. Gene 264, 139–146.
Miyazawa, K., Mori, A., and Okudaira, H. (1998). Establishment and characterization of a novel human rheumatoid fibroblast-like synoviocyte line, MH7A, immortalized with SV40 T antigen. J. Biochem. (Tokyo) 124, 1153–1162.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63.
Nichols, W. K., Mehta, R., Skordos, K., Mace, K., Pfeifer, A. M., Carr, B. A., Minko, T., Burchiel, S. W., and Yost, G. S. (2003). 3-Methylindole-induced toxicity to human bronchial epithelial cell lines. Toxicol. Sci. 71, 229–236.
Nigro, J. M., Baker, S. J., Preisinger, A. C., Jessup, J. M., Hostetter, R., Cleary, K., Bigner, S. H., Davidson, N., Baylin, S., Devilee, P., et al. (1989). Mutations in the p53 gene occur in diverse human tumour types. Nature 342, 705–708.
Paajarvi, G., Viluksela, M., Pohjanvirta, R., Stenius, U., and Hogberg, J. (2005). TCDD activates Mdm2 and attenuates the p53 response to DNA damaging agents. Carcinogenesis 26, 201–208.
Perry, M. E., Mendrysa, S. M., Saucedo, L. J., Tannous, P., and Holubar, M. (2000). p76(MDM2) inhibits the ability of p90(MDM2) to destabilize p53. J. Biol. Chem. 275, 5733–5738.
Peterson, S. R., Gadbois, D. M., Bradbury, E. M., and Kraemer, P. M. (1995). Immortalization of human fibroblasts by SV40 large T antigen results in the reduction of cyclin D1 expression and subunit association with proliferating cell nuclear antigen and Waf1. Cancer Res. 55, 4651–4657.
Pochampally, R., Fodera, B., Chen, L., Lu, W., and Chen, J. (1999). Activation of an MDM2-specific caspase by p53 in the absence of apoptosis. J. Biol. Chem. 274, 15271–15277.
Pochampally, R., Fodera, B., Chen, L., Shao, W., Levine, E. A., and Chen, J. (1998). A 60 kd MDM2 isoform is produced by caspase cleavage in non-apoptotic tumor cells. Oncogene 17, 2629–2636.
Porras, A., Gaillard, S., and Rundell, K. (1999). The simian virus 40 small-t and large-T antigens jointly regulate cell cycle reentry in human fibroblasts. J. Virol. 73, 3102–3107.
Ralhan, R., Sandhya, A., Meera, M., Bohdan, W., and Nootan, S. K. (2000). Induction of MDM2-P2 transcripts correlates with stabilized wild-type p53 in betel- and tobacco-related human oral cancer. Am. J. Pathol. 157, 587–596.
Ramsdell, H. S., Parkinson, A., Eddy, A. C., and Eaton, D. L. (1991). Bioactivation of aflatoxin B1 by human liver microsomes: role of cytochrome P450 IIIA enzymes. Toxicol. Appl. Pharmacol. 108, 436–447.
Reddel, R. R., Ke, Y., Gerwin, B. I., McMenamin, M. G., Lechner, J. F., Su, R. T., Brash, D. E., Park, J. B., Rhim, J. S., and Harris, C. C. (1988). Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res. 48, 1904–1909.
Saucedo, L. J., Myers, C. D., and Perry, M. E. (1999). Multiple murine double minute gene 2 (MDM2) proteins are induced by ultraviolet light. J. Biol. Chem. 274, 8161–8168.
Shimada, T., and Guengerich, F. P. (1989). Evidence for cytochrome P-450NF, the nifedipine oxidase, being the principal enzyme involved in the bioactivation of aflatoxins in human liver. Proc. Natl. Acad. Sci. U.S.A. 86, 462–465.
Soini, Y., Chia, S. C., Bennett, W. P., Groopman, J. D., Wang, J. S., DeBenedetti, V. M., Cawley, H., Welsh, J. A., Hansen, C., Bergasa, N. V., et al. (1996). An aflatoxin-associated mutational hotspot at codon 249 in the p53 tumor suppressor gene occurs in hepatocellular carcinomas from Mexico. Carcinogenesis 17, 1007–1012.
Stein, L. S., Stoica, G., Tilley, R., and Burghardt, R. C. (1991). Rat ovarian granulosa cell culture: A model system for the study of cell–cell communication during multistep transformation. Cancer Res. 51, 696–706.
Technau, A., Wolff, A., Sauder, C., Birkner, N., and Brandner, G. (2001). p53 in SV40-transformed DNA-damaged human cells binds to its cognate sequence but fails to transactivate target genes. Int. J. Oncol. 18, 281–286.
Thorburn, J., Frankel, A. E., and Thorburn, A. (2003). Apoptosis by leukemia cell-targeted diphtheria toxin occurs via receptor-independent activation of Fas-associated death domain protein. Clin Cancer Res. 9, 861–865.
Van Vleet, T. R., Klein, P. J., and Coulombe, R. A., Jr. (2001). Metabolism of aflatoxin B1 by normal human bronchial epithelial cells. J. Toxicol. Environ. Health A 63, 525–540.
Van Vleet, T. R., Klein, P. J., and Coulombe, R. A., Jr. (2002a). Metabolism and cytotoxicity of aflatoxin B1 in cytochrome P450-expressing human lung cells. J. Toxicol. Environ. Health A 65, 853–867.
Van Vleet, T. R., Mace, K., and Coulombe, R. A., Jr. (2002b). Comparative aflatoxin B1 activation and cytotoxicity in human bronchial cells expressing cytochromes P450 1A2 and 3A4. Cancer Res. 62, 105–112.
Villa, P. G., Henzel, W. J., Sensenbrenner, M., Henderson, C. E., and Pettmann, B. (1998). Calpain inhibitors, but not caspase inhibitors, prevent actin proteolysis and DNA fragmentation during apoptosis. J. Cell Sci. 111, 713–722.
Wei, C., Caccavale, R. J., Kehoe, J. J., Thomas, P. E., and Iba, M. M. (2001). CYP1A2 is expressed along with CYP1A1 in the human lung. Cancer Lett. 171, 113–120.
Wolff, A., Technau, A., Ihling, C., Technau-Ihling, K., Erber, R., Bosch, F. X., and Brandner, G. (2001). Evidence that wild-type p53 in neuroblastoma cells is in a conformation refractory to integration into the transcriptional complex. Oncogene 20, 1307–1317.(Terry R. Van Vleet, Todd L. Watterson, P)
Department of Veterinary Sciences, Utah State University, Logan, UT 84322–4620
3 To whom correspondence should be addressed at Graduate Program in Toxicology, Utah State University, 4620 Old Main Hill, Logan, UT 84322. Fax: (435) 797-1598. E-mail: rogerc@cc.usu.edu.
ABSTRACT
Aflatoxin B1 (AFB1) is a potent dietary hepatocarcinogen in animals and probably in humans. Mutations (and altered expression) of the tumor suppresser gene p53 have been observed in liver tumors from patients exposed to high dietary AFB1. Inhalation of AFB1-laden grain dusts has been associated with an increased incidence of lung cancer in humans as well. We examined the effects of low concentrations of AFB1 on the expression of p53 and MDM2 in human bronchial epithelial cells (BEAS-2B) transfected with cDNA for either cytochrome P450 (CYP) 1A2 (B-CMV1A2) or CYP 3A4 (B3A4), two isozymes that are responsible for AFB1 activation in human liver and possibly the lung. Untreated B-CMV1A2 and B3A4 cells constitutively expressed p53. Exposure to a range (0.015–15 μM for 30 min) of AFB1 concentrations caused a concentration-dependent decline in p53 expression in B-CMV1A2 cells, and to a lesser extent, in B3A4 cells. The AFB1-mediated decrease in p53 continued for at least 12 h after 30-min exposures to 1.5 μM AFB1. Mirroring the decrease in p53 expression was a concentration-dependent increase in the expression of the 76-kDa MDM2 isoform in B-CMV1A2 and B-3A4 cells. Interestingly, AFB1 did not induce DNA laddering, an indicator of apoptotic cell death, but proteolytic activation of caspase-3 was detected in AFB1-treated B-CVM1A2 cells. In total, these data show that low, environmentally-relevant concentrations of AFB1 alter the expression of p53 and MDM2 in these human lung cells, and that cells that stably express CYP 1A2 were more susceptible to this effect than nontransfected, or 3A4-expressing cells.
INTRODUCTION
Aflatoxin B1 (AFB1) is a potent immunotoxicant and hepatocarcinogen in animals and probably in humans (Bondy and Pestka, 2000; Klein et al., 2000). The liver is the primary target organ, because AFB1 requires metabolic activation to form the reputed carcinogenic species AFB1-8,9-epoxide (AFBO) (Mace et al., 1994, 1997), but other organs are also affected by AFB1 exposures (Ball and Coulombe, 1991; Ball et al., 1995; Coulombe et al., 1991; Imaoka et al., 1992; Kato et al., 1994; Kelly et al., 1997; Liu et al., 1990, 1993; Liu and Massey, 1992). In human liver, CYPs 1A2 and 3A4 have been shown to be the principle enzymes responsible for AFB1 activation (Ramsdell et al., 1991; Shimada and Guengerich, 1989) and have also been detected in human lung tissues and cultured lung cells (Mace et al., 1998; Van Vleet et al., 2001; Wei et al., 2001).
Inhalation of respirable AFB1-contaminated grain dusts may pose a cancer hazard to susceptible individuals in certain agricultural occupations (Hayes et al., 1984). AFB1 is activated to AFBO in animal (Ball et al., 1995; Daniels et al., 1990; Daniels and Massey, 1992; Imaoka et al., 1992; Liu et al., 1990; Liu and Massey, 1992) and in human pulmonary tissues (Autrup et al., 1979; Donnelly et al., 1996; Kelly et al., 1997).
The tumor suppressor gene p53 is the most commonly mutated gene in human cancers, with mutations in approximately 50% of all human cancers (Chang et al., 1993; Kew, 1992; May and May, 1995; Nigro et al., 1989). Dietary AFB1 exposure has been linked to GT transversions at codon 249 of p53 in human primary hepatocellular carcinomas (PHC) (Cerutti et al., 1994; Hainaut and Vahakangas, 1997; Soini et al., 1996) and in cultured human hepatocytes exposed to AFB1 (Aguilar et al., 1993; Mace et al., 1997). Therefore, this mutation may inactivate p53, leading to AFB1-induced liver cancers (Hollstein et al., 1993; Lasky and Magder, 1997). It seems reasonable that a similar mutation may be produced by AFB1 in the lung.
After cellular DNA damage, p53 protein is phosphorylated by DNA-dependent protein kinase, which arrests the cell cycle in the G1 phase, thus preventing mitosis and allowing the repair of damaged sequences (Kastan et al., 1992). Thus, by preventing the proliferation of damaged cells, p53 acts to protect the integrity of the genome (Lane, 1992). Induction of p53 leads to increased MDM2 expression, which eventually inhibits p53 expression via a negative-feedback mechanism (Pochampally et al., 1998). To induce MDM2 expression, p53 acts as a transcriptional element on the MDM2-P2 promoter (Hesketh, 1997; Ralhan et al., 2000), but MDM2 induction is at least partly also due to mRNA stabilization (Hsing et al., 2000). To date, several MDM2 proteins have been identified (90, 76, 60, 46, and 35 kDa) (Maxwell, 1994; Mendrysa et al., 2001; Ralhan et al., 2000), which are produced by differential splicing of the mRNA transcript and caspase-3-mediated cleavage of the 90-kDa isoform (Chen et al., 1997; Maxwell, 1994; Mendrysa et al., 2001) and by internal initiation at codon 50 of the mdm2 mRNA (Saucedo et al., 1999).
The proteolytic activation of caspase-3 and DNA ladder formation are key steps in the apoptotic cascade (Maruyama et al., 2001) with caspase-3 activation regarded as a primary mechanism of apoptosis (He et al., 2003). Caspase-3-like activity has been implicated in the processing of MDM2 to a form that stabilizes p53 (Pochampally et al., 1999). Caspase-3 activation can be detected using Western immunoblotting to demonstrate proteolytic cleavage of the procaspase-3 protein (35 kDa) to the largest (17 kDa) proteolytic fragment (Erhardt et al., 2001).
The BEAS-2B cell line, a simian virus 40 (SV-40) large T antigen immortalized version of normal human bronchial epithelial (NHBE) cells, is an in vitro model for the study of the human lung toxicity (Reddel et al., 1988). Infection of normal cells with SV-40 interferes with p53 function, leading to immortalization (Carnero et al., 2000; Hsieh et al., 2000; Peterson et al., 1995; Porras et al., 1999). The SV-40 viral genome codes for the large T antigen, which binds p53, inhibiting its normal growth arrest and cell cycling functions (Levresse et al., 1998; Peterson et al., 1995; Porras et al., 1999).
We recently demonstrated that BEAS-2B cells transfected to stably express CYP 1A2 (BCMV-1A2) and 3A4 (B3A4) activate AFB1 to cytotoxic and DNA-alkylating species (Van Vleet et al., 2002a,b). Because AFB1 affects p53 expression in human liver cancer (Barton et al., 1991), we wished to determine if AFB1 would affect this tumor suppressor system in these immortalized lung cells. Our data indicate that AFB1 perturbs the expression of p53 and related proteins in these cells when critical CYPs are expressed. At the lowest concentrations studied, CYP 1A2-expressing cells were affected to a greater extent than those expressing 3A4, in support of previous results showing that expression of the former isoform may be more relevant to AFB1 toxicity. Despite compromised p53 activity, exposure to AFB1 activates caspase-3 in BCMV1A2 cells.
MATERIALS AND METHODS
Chemicals and reagents.
LHC-8, LHC-9, LHC Basal, epinephrine, retinoic acid, and bovine serum albumin (BSA) stock were obtained from BioWhittaker (Rockville, MD). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). Bovine fibronectin, 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), glycerol, AFB1, trypsin inhibitor, aminosalicylate, -mercaptoethanol, pyronin-Y, staurosporine, caffeine, and Coomassie blue were purchased from Sigma (St. Louis, MO). Collagen was a product of Collagen Corp. (Fremont, CA). CHAPS Cell Extraction buffer, primary polyclonal rabbit anti-human caspase-3 antibody, primary polyclonal rabbit p53 antibody used with the BEAS-2B cells, and secondary antirabbit IgG were purchased from Cell Signaling, (Beverly, MA). Primary monoclonal mouse p53 and MDM2 used with the BCMV-1A2, B3A4, and NHBE cells were from Calbiochem (San Diego, CA). Secondary (goat-anti-mouse) antibody was from Bio-Rad (Hercules, CA). ECL chemiluminescent reagent was from Amersham (Piscataway, NJ). Supersignal West Femto chemiluminescent substrate was from Fisher Scientific, (Pittsburg, PA). BEAS-2B, B-CMV1A2, and B3A4 cells were a generous gift from Dr. Katherine Mace (Nestle Research Centre; Lausanne, Switzerland). Normal human bronchial epithelial (NHBE) cells were purchased from BioWhittaker (San Diego, CA).
Cell culture.
BEAS-2B, B-CMV1A2, and B3A4 cells were cultured as previously described (Van Vleet et al., 2002a) for all experiments excluding caspase-3 activation and basal p53 expression in BEAS-2B wherein flasks were not coated with a plate coat consisting of 5 mg bovine fibronectin, 5 ml collagen, 50 ml BSA stock, and 500 ml LHC Basal. NHBE cells were cultured as previously described (Van Vleet et al., 2001).
Preparation of cell lysates.
Cells were seeded at a density of 9.5 x 105 cells/T75 flask and cultured for 48 h. For time-course studies, cultures were then exposed to 1.5 μM AFB1 for 30 min, after which flasks were washed with PBS, and then fresh media was added to cultures. Cells were harvested via trypsinization, at various time intervals thereafter (1, 2, 4, 6, 9, and 12 h). To determine the effects of a range of AFB1 concentrations on p53 expression, cells were exposed to AFB1 (0.015–15 μM) for 30 min, and flasks were then washed with PBS. The cells were cultured for 6 h in fresh media before they were harvested. Next, cells from each T75 flask were resuspended in 1 ml of LHC-9. The cell density was determined (Counter Model FN; Beckman-Coulter Fullerton, CA), and 0.5 ml of the cell suspension was centrifuged to collect cells. After removing the supernatant, cell lysing buffer (2% sodium dodecyl sulfate (SDS), 12% aminosalicylate, 2% NaCl, and 12% 2-butanol) was added to the pellet at a concentration of 100,000 cells/20 μl. Samples were stored at –80°C until separated by SDS–PAGE. Control groups were also run at each time point (time-course study), or at 6 h after exposure to AFB1 (concentration range study) for 30 min.
Measurement of p53 and MDM2 expression.
Cell lysates (20 μl) were heated in sample buffer (10% SDS, 0.5M Tris-HCl, 20% glycerol, 10% -mercaptoethanol, 0.1% pyronin-Y; 30 μl; total vol = 50 μl) to 70°C for 5 min and loaded into 10–15% SDS-PAGE gels (14 x 11 x 0.1 cm), with duplicate lanes, then electrophoresed for 8 h at 125 V. One-half of each gel was transferred to a Nitrobind nitrocellulose transfer membrane (Micron Separations, Inc.) using a semi-dry blotter (Buchler, Kansas City, MO). The other half of the gel was stained with Coomassie blue for molecular weight analysis. Molecular weight markers served as negative controls for nonspecific binding of antibodies to protein in the immunostained gel portions, and for molecular weight approximations of the Coomassie-stained gel halves. Nitrocellulose membranes were immunostained using the primary antibody (1:5000) in High Salt Tween (HST) blocking buffer (10 mM Tris, 1 M NaCl 0.5% Tween 20, pH 7.4). Membranes were washed with HST, Tris-buffered saline (TBS) (10 mM Tris and 140 mM NaCl, pH 7.4), and TBS-Tween (TBS with 0.1% Tween 20) as described previously (Klein et al., 2000). Secondary antibodies were also diluted in HST (1:2000). Proteins were detected by chemiluminescence generated by horseradish peroxidase-conjugated secondary antibody, using ECL reagent as a substrate, and quantified using a Nucleovision 920 chemiluminescence imaging workstation (Nucleotech Corp., Hayward, CA).
The following method was employed exclusively for examining p53 expression in BEAS-2B cells. Cells were grown to approximately 80% confluence and harvested via trypsinization. Cells were pelleted and resuspended in 100 μl of CHAPS (50 mM PIPES/NaOH (pH 6.5), 2 mM EDTA, 0.1% Chaps, 5 mM DTT, 20 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, and 1 mM PMSF) cell extract buffer and subjected to five freeze–thaw cycles. The cell lysate was then centrifuged at 13,000 rpm (16,060 x g) for 5 min, and the supernatant was retained and frozen at –80°C. Sample protein was measured using the Quick Start Bradford Protein assay kit (Bio-Rad, Hercules CA). Aliquots of the supernatant were diluted 1:1 with SDS sample buffer (2% SDS, 50 mM dithiothreitol (DTT), 0.01% bromophenyl blue, 10% glycerol), boiled for 5 min, and loaded onto 4–15% SDS/Tris–HCl mini acrylamide gradient gels (Bio-Rad Labs, Hercules, CA) at 15 μg protein per well. P53 standard (Oncogene, Boston, MA) was loaded at 10 μl per well. Samples were electrophoresed for 45 min at 200 V on a Bio-Rad Mini-Protean 3 Cell electrophoresis unit (Bio-Rad Labs, Hercules, CA). Gels were transferred at 100 V for 1 h to nitrocellulose transfer membranes (GE Osmonics, Minnetonka, MN) using the Bio-Rad Mini-Protean 3 Cell electrophoresis unit. Nitrocellulose membranes were washed in TBS for 5 min and then incubated in blocking buffer (TBS with 0.1% Tween 20 and 5% nonfat dry milk). Membranes were again washed in TBS-Tween (TBS with 0.1% Tween 20) three times for 5 min each, and immuno-stained overnight (at 4°C) with the primary antibody (1:1000) in 5% BSA, 1x TBS, 0.1% Tween-20 at 4°C with gentle shaking, overnight. Membranes were then washed with TBS-Tween three times. Secondary antibodies were diluted in blocking buffer (1:2000 and 1:1000) and incubated with the membrane at room temperature for 1 h. Proteins were detected by chemiluminescence generated by horseradish peroxidase-conjugated secondary antibody, using Supersignal West Femto chemiluminescent substrate, and captured using a Nucleovision 920 chemiluminescence imaging workstation (Nucleotech Corp., Hayward, CA).
Ladder assay for apoptosis.
B-CMV1A2 cells were seeded at a density of 9.5 x 105 cells/T75 flask, and cultured for 48 h. Actively dividing cells (approx. 50% confluency) were then dosed with AFB1 at various concentrations (0–15 μM) for 30 min and harvested after 6 h of culture in fresh media via trypsinization. To study the time course of apoptosis, actively dividing cells were dosed with 1.5 μM AFB1 for 30 min, washed with PBS, and harvested at various time intervals (1, 2, 4, 8, 12, 16, 20, 24 h) after PBS was replaced with fresh LHC-9. Cells were then harvested, and pellets were resuspended at a concentration of 2 x 106 cells/200 μl in PBS for use in assay. DNA samples (6 μg) were added to 10x loading buffer (1% SDS, 2.5 mg/ml bromophenyl blue, 30% glycerol) and loaded into 7 x 7.5 x 1 cm 1% agarose gels in TBE (0.04 M Tris, 0.04 M boric acid, 0.01 M EDTA). Samples were electrophoresed at 4°C for 35 min at 200 V. Laddering was also examined using the Qiagen DNeasy tissue Kit (Qiagen, Valencia, CA) and the proteinase K method described by Thorburn et al. (2003).
Detection of caspase-3 activation.
B-CMV1A2 and B3A4 cells were seeded at a density of 6.4 x 104 cells/T-75 flask and cultured for 48 h. Actively dividing cells (approx. 70% confluency) were then dosed with either AFB1 (1 μM), Staurosporine (1 μM; positive control), or DMSO (20 μl; negative control) for 4 and 9 h, and harvested in ice cold PBS, via scraping. Caffeine-dosed cells were exposed to 150, 250, 350, and 450 μM concentrations for 24 h. Cells were pelleted and resuspended in 100 μl of CHAPS (50 mM PIPES/NaOH (pH 6.5), 2 mM EDTA, 0.1% Chaps, 5 mM DTT, 20 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, and 1 mM PMSF) cell extract buffer and subjected to five freeze–thaw cycles. The cell lysate was then centrifuged at 16,000 rpm for 5 min, and the supernatant was retained and frozen at –80°C. Sample protein was measured using the Quick Start Bradford Protein Plate reader assay kit (Bio-Rad, Hercules CA) on a Labsystems Multiskan MCC/340 (Fisher Scientific, Pittsburg, PA). Aliquots of the supernatant (25 μl) were diluted 1:1 with SDS sample buffer (2% SDS, 50 mM DTT, 0.01% bromophenyl blue, 10% glycerol), boiled for 5 min, and loaded at 0.5 μg protein per well for AFB1 and 24 μg per well caffeine onto 15% SDS/Tris–HCl mini acrylamide gels (Bio-Rad Labs, Hercules, CA). Samples were electrophoresed for 45 min at 200 V on a Bio-Rad Mini-Protean 3 Cell electrophoresis unit (Bio-Rad Labs, Hercules, CA). Gels were transferred at 100 V for 1 h to nitrocellulose transfer membranes (GE Osmonics, Minnetonka, MN). Nitrocellulose membranes were washed in TBS for 5 min and then incubated in blocking buffer (TBS with 0.1% Tween 20 and 5% nonfat dry milk). Membranes were again washed in TBS-Tween (TBS with 0.1% Tween 20) three times for 5 min each, and immuno-stained overnight (at 4°C) with the primary antibody (1:1000) in blocking buffer. Membranes were then washed with TBS-Tween three times for 5 min. Secondary antibodies were also diluted in blocking buffer (1:2000 anti biotin and 1:1000 anti rabbit) and incubated with the membrane at room temperature for 1 h. Proteins were detected by chemiluminescence generated by horseradish peroxidase-conjugated secondary antibody, using Supersignal West Femto chemiluminescent substrate, and images were captured and archived.
Determination of cell viability.
The IC50 value for caffeine toxicity in BCMV-1A2 cells was determined via MTT in a 96-well format using a Labsystems Multiskan MCC/340 (Fisher scientific, Pittsburgh PA) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma Aldrich, St. Louis, MO) as previously described (Mosmann, 1983).
Statistical analysis and curve fitting.
Groups were compared for differences using one-way ANOVA (Sigma Stat Software). A p < 0.05 was judged significant. Curves generated from digital densitometry analysis were fit using Sigma Plot logistics curve fitting program (SPSS, Chicago, IL).
RESULTS
All SV-40 transformed cells, BEAS-2B, B-CMV1A2, and B3A4 expressed p53 constitutively (Figs. 1A and 1B). The doublets match those found by other researchers (Matlashewski et al., 1986) and those shown on the manufacturer's instructions. This is in contrast to normal human bronchial epithelial (NHBE) cells, where no constitutive p53 expression was observed (Fig. 1A).
When B-CMV1A2 and B3A4 cells were exposed to 1.5 μM AFB1 for 30 min, p53 expression decreased over time compared to their respective unexposed controls (Figs. 2A and 2B). This decrease in expression continued at least 12 h, with the effect being significantly greater in B-CMV1A2 cells than in B3A4 cells at all time points (Figs. 2A, 2B, and 2C). We then determined whether the inhibitory effect on p53 expression was dependent on AFB1 concentration. When exposed to a range of AFB1 concentrations (0–15 μM) for 30 min, a similar decrease in p53 expression was observed in both B-CMV1A2 and B3A4 cell types in a concentration-dependent manner 6 h post-exposure (Figs. 3A and 3B). Thus, the inhibitory effect of AFB1 on p53 expression was dependent on AFB1 concentration and duration of exposure. In cultures exposed to the highest AFB1 concentration (i.e., 15 μM), the decrease in p53 expression was greater in B3A4 than the B-CMV1A2 cells (Fig. 3C).
Because p53 expression was altered to a greater extent in B-CMV1A2 cells, we then examined the effect of AFB1 on the expression of MDM2 in these cells. As can be seen in Figure 4, AFB1 elicited an increase in MDM2 (76 kDa) expression in B-CMV1A2 cells in both a concentration- (0.15–15 μM; 6 h after 30-min exposures) and time-dependent (0–12 h after 30-min exposures to 1.5 μM AFB1) fashion (Figs. 4A and 4B). When data from Figures 3 and 4 are plotted together, the combined effect of AFB1 on both p53 and MDM2 can clearly be seen in Figure 5. The AFB1 concentration and time-dependent decrease in p53 expression were mirrored by a concomitant increase in MDM2 (76 kDa) expression (Figs. 5A and 5B) at each point. However, only expression of the 76-kDa isoform was consistently affected by AFB1. Other MDM2 proteins—90, 60, and 35 kDa—were detected, but their levels were not affected by AFB1 treatment (data not shown).
DNA ladder formation, an indicator of the onset of an irreversible stage in apoptosis, was examined in B-CMV1A2 cells. B-CMV1A2 cells were exposed to 1.5 μM at a range of post-AFB1 exposure intervals (0–24 h; Fig. 6A). No ladder formation could be detected in B-CMV1A2 cells treated with AFB1. Even when B-CMV1A2 cells were subjected to a 30-min exposure at a range of AFB1 concentrations (0.015–15 μM; 6-h exposure), no DNA ladder formation was detected (Fig. 6B). There was no clear indication of ladder formation detected in any AFB1- or staurosporine-treated B-CMV1A2 cells under any experimental protocol using three different methods for ladder detection (data not shown).
Activation of apoptotic executioner protease caspase-3 was then examined in was then examined in B-CMV1A2 cell cultures dosed with AFB1. B-CMV1A2 cells were exposed to 1 μM AFB1, 1 μM staurosporine, or DMSO for either 4 or 9 h. Proteolysed (activated) caspase-3 was detected in B-CMV1A2 cells exposed to either AFB1 or staurosporine for either 4 or 9 h, but was absent from control (DMSO-treated) cells for 9 h (Fig. 7A). To determine if BCMV1A2 cells possess a functional p53, we examined whether caffeine would cause caspase-3 cleavage. In BCMV-1A2 cells, the 24-h IC50 for caffeine was 281 μM as determined by the MTT assay (data not shown). Caspase-3 cleavage was not detected in BCMV-1A2 cells dosed with caffeine (150–450 μM) for 24 h (Fig. 7B).
DISCUSSION
In occupations where pulmonary exposures to AFB1-laden grain dusts are common, workers may be at an increased risk of developing lung cancer (Hayes et al., 1984). We previously showed that these CYP-transfected cells activate AFB1 to intermediate(s) that are cytotoxic and form AFB1–DNA adducts. When taking into consideration the expression of CYP mRNA, B-CMV1A2 cells were more efficient at activating AFB1 at low concentrations (<3 μM), while B3A4 cells were more efficient at higher concentrations (>3 μM) (Van Vleet et al., 2002b). Because of the reported relevance of p53 mutations and altered p53 expression in AFB1-induced hepatocarcinogenesis (Lee et al., 2000), we sought to determine if AFB1 exposure has an effect on p53 expression in these human bronchial epithelial cells.
This study indicates that, in contrast to normal cells, the immortalized cells used in this study—BEAS-2B, B3A4, and B-CMV1A2—express p53 constitutively. Since BEAS-2B cells are the progenitor cells of B3A4 and B-CMV1A2, constitutive expression in the latter cell lines was not the result of transfections inducing the expression of CYPs 1A2 and 3A4, but is likely due to SV-40 immortalization. Others have reported similar constitutive p53 expression in BEAS-2B cells (Gerwin et al., 1992). Importantly, our results also indicate that p53 function is impaired by exposure to AFB1. Our data demonstrate that AFB1 exposure inhibits p53 expression in B-CMV1A2 and B3A4 cells, an event associated with a reflective increase in MDM2 expression. It has been previously demonstrated that SV-40 affects p53 function. For example, SV-40 large T antigen causes continuous p53 inactivation and leads to immortalization of primary mouse embryonic fibroblasts (Carnero et al., 2000). Other SV-40 immortalized cell lines also constitutively express p53 (Miyazawa et al., 1998; Stein et al., 1991). It was also previously shown that BEAS cells, the progenitor cell line for the B-CMV1A2 and B3A4 cell lines, possess a p53 protein incapable of inducing the expression of downstream proteins under DNA-alkylating conditions (Technau et al., 2001). Researchers from that study noted that p53 expression was frequently decreased in cells exposed to mitomycin C, which is also in agreement with our data showing decreased p53 expression after AFB1 exposure. An increase in MDM2 and reduction in p53 was also observed with TCDD in HepG2 cells (Paajarvi et al., 2005). Interestingly, in another study, neuroblastoma cells where shown to possess a p53 that was also unable to induce the expression of p21 (and MDM2), after exposure to mitomycin C, even though p53 protein was able to bind DNA (Wolff et al., 2001). These discoveries support speculation that SV-40 immortalization not only inactivates p53 from its role in cell cycle control, but also inactivates its ability to protect the integrity of the genome, as seen in some cancer cells. Although these results provide insight into the function of p53 and the effects of SV-40 immortalization on p53 function, they also suggest that BEAS-2B cell physiology may present some limitations to studying the toxicological responses in certain molecular targets.
MDM2 induction, concomitant to the decline in p53 expression, indicates that the decline in p53 expression was not due to cell death from AFB1 treatment under these conditions. If the decrease were due to a general lack of protein synthesis from cell death, MDM2 would not be induced under these conditions. Other studies have shown that the induction of some MDM2 isoforms can cause a decrease in p53 expression (Carnero et al., 2000; Freedman et al., 1999), and that MDM2 induction can be independent of functional p53 (Hsing et al., 2000). Interestingly, the induction of MDM2 we observed was of the 76-kDa fragment, which typically attenuates the ability of the full-length p90MDM2 to decrease the level of p53 thereby increasing p53 (Perry et al., 2000).
Further evidence of the lack of p53 function can be seen in the inability of these (BCMV-1A2) SV-40 immortalized lung cells to undergo DNA laddering after treatment with AFB1 or staurosporine. A lack of functional p53 has been shown to perturb cellular growth arrest and apoptosis (May and May, 1995), while other studies have shown p53-independent induction of apoptosis in some SV-40 immortalized cell types (Gartenhaus et al., 1996; Levresse et al., 1998). Caffeine has been shown to induce apoptosis in a p53-dependent manner in p53+/+ mouse epidermal JB6 C141 cells, resulting in caspase-3 cleavage but not in p53–/– JB6 C141 cells (He et al., 2003). We were unable to observe caspase-3 activation in B-CMV1A2 cells by the p53-dependent apoptosis-inducer caffeine, further suggesting that p53 was nonfunctional in these cells. That both AFB1 and staurosporine were able to activate caspase-3 despite attenuated p53 indicates that apoptosis induced by these two compounds was p53 independent. Apoptosis has been previously reported in BEAS-2B cells (Agopyan et al., 2003; Nichols et al., 2003), which is consistent with our detection of caspase-3 activation. The absence of DNA ladder in the presence of caspase-3 activation is consistent with previous reports that caspase inhibitors are ineffective at preventing DNA fragmentation during apoptosis in multiple cell types from different species (Villa et al., 1998).
The AFB1-induced inhibition of p53 expression was greater in CYP1A2-expressing than in CYP3A4-expressing cells, except only at the highest AFB1 (15 μM) concentrations. Earlier studies from our laboratory demonstrated that B-CMV1A2 cells were substantially more efficient at AFB1 bioactivation to cytotoxic and DNA-alkylating intermediates than were B3A4 cells at low, environmentally relevant concentrations of AFB1 (Van Vleet et al., 2002a,b). In conclusion, the p53-mediated response to AFB1 treatment may indicate that these cells are at an increased risk of developing mutations. However, in the absence of a typical p53 response, these cells were able to undergo apoptosis, as evidenced by caspase-3 cleavage. The implications of our findings on the usefulness of these cells in in vitro studies of the effects of environmental carcinogens such as AFB1 are unclear. It is possible that the inhalation of AFB1-contaminated grain dusts may lead to modulation of p53 and cellular death under conditions where appropriate CYPs are expressed in the lung, resulting in adverse health effects.
NOTES
Portions of this report were presented at the 44th annual meeting of the Society of Toxicology, New Orleans, LA, March 2005 (Abstract # 1795).
1 Current address: Department of Toxicology, Bristol-Myers Squibb Company, Mt. Vernon, IN 47721.
2 Current address: Department of Surgery, Indiana University School of Medicine, Indianapolis, IN 46202.
ACKNOWLEDGMENTS
The authors wish to acknowledge Dr. Katherine Mace for the gift of B-CMV1A2 and B3A4 cells. This research was supported in part by the Marriner S. Eccles Foundation, by a competitive grant from USDA-NRI (2002–35204–12294), and by the Utah Agricultural Experiment Station, where it is paper no. 7738.
REFERENCES
Agopyan, N., Bhatti, T., Yu, S., and Simon, S. A. (2003). Vanilloid receptor activation by 2- and 10-μm particles induces responses leading to apoptosis in human airway epithelial cells. Toxicol. Appl. Pharmacol. 192, 21–35.
Aguilar, F., Hussain, S. P., and Cerutti, P. (1993). Aflatoxin B1 induces the transversion of G–>T in codon 249 of the p53 tumor suppressor gene in human hepatocytes. Proc. Natl. Acad. Sci. U.S.A. 90, 8586–8590.
Autrup, H., Essigmann, J. M., Croy, R. G., Trump, B. F., Wogan, G. N., and Harris, C. C. (1979). Metabolism of aflatoxin B1 and identification of the major aflatoxin B1- DNA adducts formed in cultured human bronchus and colon. Cancer Res. 39, 694–698.
Ball, R. W., and Coulombe, R. A., Jr. (1991). Comparative biotransformation of aflatoxin B1 in mammalian airway epithelium. Carcinogenesis 12, 305–310.
Ball, R. W., Huie, J. M., and Coulombe, R. A., Jr. (1995). Comparative activation of aflatoxin B1 by mammalian pulmonary tissues. Toxicol. Lett. 75, 119–125.
Barton, C. M., Staddon, S. L., Hughes, C. M., Hall, P. A., O'Sullivan, C., Kloppel, G., Theis, B., Russell, R. C., Neoptolemos, J., Williamson, R. C., et al. (1991). Abnormalities of the p53 tumour suppressor gene in human pancreatic cancer [published erratum appears in Br. J. Cancer (1992) 65(3), 485]. Br. J. Cancer 64, 1076–1082.
Bondy, G. S., and Pestka, J. J. (2000). Immunomodulation by fungal toxins. J. Toxicol. Environ. Health B Crit. Rev. 3, 109–43.
Carnero, A., Hudson, J. D., Hannon, G. J., and Beach, D. H. (2000). Loss-of-function genetics in mammalian cells: the p53 tumor suppressor model. Nucleic Acids Res. 28, 2234–2241.
Cerutti, P., Hussain, P., Pourzand, C., and Aguilar, F. (1994). Mutagenesis of the H-ras protooncogene and the p53 tumor suppressor gene. Cancer Res. 54, 1934s–1938s.
Chang, F., Syrjanen, S., Tervahauta, A., and Syrjanen, K. (1993). Tumourigenesis associated with the p53 tumour suppressor gene. Br. J. Cancer 68, 653–661.
Chen, L., Marechal, V., Moreau, J., Levine, A. J., and Chen, J. (1997). Proteolytic cleavage of the mdm2 oncoprotein during apoptosis. J. Biol. Chem. 272, 22966–22973.
Coulombe, R. A., Huie, J. M., Ball, R. W., Sharma, R. P., and Wilson, D. W. (1991). Pharmacokinetics of intratracheally administered aflatoxin B1. Toxicol. Appl. Pharmacol. 109, 196–206.
Daniels, J. M., Liu, L., Stewart, R. K., and Massey, T. E. (1990). Biotransformation of aflatoxin B1 in rabbit lung and liver microsomes. Carcinogenesis 11, 823–827.
Daniels, J. M., and Massey, T. E. (1992). Modulation of aflatoxin B1 biotransformation in rabbit pulmonary and hepatic microsomes. Toxicology 74, 19–32.
Donnelly, P. J., Stewart, R. K., Ali, S. L., Conlan, A. A., Reid, K. R., Petsikas, D., and Massey, T. E. (1996). Biotransformation of aflatoxin B1 in human lung. Carcinogenesis 17, 2487–2494.
Erhardt, J. A., Ohlstein, E. H., Toomey, J. R., Gabriel, M. A., Willette, R. N., Yue, T.-L., Barone, F. C., and Parsons, A. A. (2001). Activation of caspase-3/caspase-3-like activity in rat cardiomyocytes by an RGD peptide, but not the GPIIb/IIIa antagonist lotrafiban. Thromb. Res. 103, 143–148.
Freedman, D. A., Wu, L., and Levine, A. J. (1999). Functions of the MDM2 oncoprotein. Cell. Mol. Life Sci. 55, 96–107.
Gartenhaus, R. B., Wang, P., and Hoffmann, P. (1996). Induction of the WAF1/CIP1 protein and apoptosis in human T-cell leukemia virus type I-transformed lymphocytes after treatment with adriamycin by using a p53-independent pathway. Proc. Natl. Acad. Sci. U.S.A. 93, 265–268.
Gerwin, B. I., Spillare, E., Forrester, K., Lehman, T. A., Kispert, J., Welsh, J. A., Pfeifer, A. M., Lechner, J. F., Baker, S. J., Vogelstein, B., et al. (1992). Mutant p53 can induce tumorigenic conversion of human bronchial epithelial cells and reduce their responsiveness to a negative growth factor, transforming growth factor beta 1. Proc. Natl. Acad. Sci. U.S.A. 89, 2759–2763.
Hainaut, P., and Vahakangas, K. (1997). p53 as a sensor of carcinogenic exposures: mechanisms of p53 protein induction and lessons from p53 gene mutations. Pathol. Biol. (Paris) 45, 833–844.
Hayes, R. B., van Nieuwenhuize, J. P., Raatgever, J. W., and Ten Kate, F. J. (1984). Aflatoxin exposures in the industrial setting: An epidemiological study of mortality. Food Chem. Toxicol. 22, 39–43.
He, Z., Ma, W. Y., Hashimoto, T., Bode, A. M., Yang, C. S., and Dong, Z. (2003). Induction of apoptosis by caffeine is mediated by the p53, Bax, and caspase 3 pathways. Cancer Res. 63, 4396–4401.
Hesketh, R. (1997). The Oncogene and Tumour Suppressor Gene Facts Book. Academic Press, San Diego. CA.
Hollstein, M. C., Wild, C. P., Bleicher, F., Chutimataewin, S., Harris, C. C., Srivatanakul, P., and Montesano, R. (1993). p53 mutations and aflatoxin B1 exposure in hepatocellular carcinoma patients from Thailand. Int. J. Cancer 53, 51–55.
Hsieh, J. K., Kletsas, D., Clunn, G., Hughes, A. D., Schachter, M., and Demoliou-Mason, C. (2000). p53, p21(WAF1/CIP1), and MDM2 involvement in the proliferation and apoptosis in an in vitro model of conditionally immortalized human vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 20, 973–981.
Hsing, A., Faller, D. V., and Vaziri, C. (2000). DNA-damaging aryl hydrocarbons induce Mdm2 expression via p53- independent post-transcriptional mechanisms. J. Biol. Chem. 275, 26024–26031.
Imaoka, S., Ikemoto, S., Shimada, T., and Funae, Y. (1992). Mutagenic activation of aflatoxin B1 by pulmonary, renal, and hepatic cytochrome P450s from rats. Mutat. Res. 269, 231–236.
Kastan, M. B., Zhan, Q., el-Deiry, W. S., Carrier, F., Jacks, T., Walsh, W. V., Plunkett, B. S., Vogelstein, B., and Fornace, A. J., Jr. (1992). A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell 71, 587–597.
Kato, T., Nakasa, H., Ohmori, S., Kamataki, T., Itahashi, K., Shimada, T., Rikihisa, T., and Kitada, M. (1994). Possible occurrence of P450 related to P450 HFLb in extrahepatic tissues of human fetuses and its contribution to metabolic activation of promutagens. Mutat. Res. 310, 73–77.
Kelly, J. D., Eaton, D. L., Guengerich, F. P., and Coulombe, R. A., Jr. (1997). Aflatoxin B1 activation in human lung. Toxicol. Appl. Pharmacol. 144, 88–95.
Kew, M. C. (1992). Tumours of the liver. Scand. J. Gastroenterol. Suppl. 192, 39–42.
Klein, P. J., Buckner, R., Kelly, J., and Coulombe, R. A., Jr. (2000). Biochemical basis for the extreme sensitivity of turkeys to aflatoxin B1. Toxicol. Appl. Pharmacol. 165, 45–52.
Lane, D. P. (1992). Cancer p53, guardian of the genome [news; comment] [see comments]. Nature 358, 15–16.
Lasky, T., and Magder, L. (1997). Hepatocellular carcinoma p53 G > T transversions at codon 249: the fingerprint of aflatoxin exposure Environ. Health Perspect. 105, 392–397.
Lee, Y. I., Lee, S., Das, G. C., Park, U. S., and Park, S. M. (2000). Activation of the insulin-like growth factor II transcription by aflatoxin B1 induced p53 mutant 249 is caused by activation of transcription complexes; implications for a gain-of-function during the formation of hepatocellular carcinoma. Oncogene 19, 3717–3726.
Levresse, V., Moritz, S., Renier, A., Kheuang, L., Galateau-Salle, F., Mege, J. P., Piedbois, P., Salmons, B., Guenzburg, W., and Jaurand, M. C. (1998). Effect of simian virus large T antigen expression on cell cycle control and apoptosis in rat pleural mesothelial cells exposed to DNA damaging agents. Oncogene 16, 1041–1053.
Liu, L., Daniels, J. M., Stewart, R. K., and Massey, T. E. (1990). In vitro prostaglandin H synthase- and monooxygenase-mediated binding of aflatoxin B1 to DNA in guinea-pig tissue microsomes. Carcinogenesis 11, 1915–1919.
Liu, L., and Massey, T. E. (1992). Bioactivation of aflatoxin B1 by lipoxygenases, prostaglandin H synthase and cytochrome P450 monooxygenase in guinea-pig tissues. Carcinogenesis 13, 533–539.
Liu, L., Nakatsu, K., and Massey, T. E. (1993). In vitro cytochrome P450 monooxygenase and prostaglandin H-synthase mediated aflatoxin B1 biotransformation in guinea pig tissues: effects of beta-naphthoflavone treatment. Arch. Toxicol. 67, 379–385.
Mace, K., Aguilar, F., Wang, J. S., Vautravers, P., Gomez-Lechon, M., Gonzalez, F. J., Groopman, J., Harris, C. C., and Pfeifer, A. M. (1997). Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450- expressing human liver cell lines. Carcinogenesis 18, 1291–1297.
Mace, K., Bowman, E. D., Vautravers, P., Shields, P. G., Harris, C. C., and Pfeifer, A. M. (1998). Characterization of xenobiotic-metabolizing enzyme expression in human bronchial mucosa and peripheral lung tissues. Eur. J. Cancer 34, 914–920.
Mace, K., Gonzalez, F. J., McConnell, I. R., Garner, R. C., Avanti, O., Harris, C. C., and Pfeifer, A. M. (1994). Activation of promutagens in a human bronchial epithelial cell line stably expressing human cytochrome P450 1A2. Mol. Carcinog. 11, 65–73.
Maruyama, W., Youdim, M. B., and Naoi, M. (2001). Antiapoptotic properties of rasagiline, N-propargylamine-1(R)-aminoindan, and its optical (S)-isomer, TV1022. Ann. N.Y. Acad. Sci. 939, 320–329.
Matlashewski, G., Banks, L., Pim, D., and Crawford, L. (1986). Analysis of human p53 proteins and mRNA levels in normal and transformed cells. Eur. J. Biochem. 154, 665–672.
Maxwell, S. A. (1994). Selective compartmentalization of different mdm2 proteins within the nucleus. Anticancer Res. 14, 2541–2547.
May, P., and May, E. (1995). P53 and cancers. Pathol. Biol. (Paris) 43, 165–173.
Mendrysa, S. M., McElwee, M. K., and Perry, M. E. (2001). Characterization of the 5' and 3' untranslated regions in murine mdm2 mRNAs. Gene 264, 139–146.
Miyazawa, K., Mori, A., and Okudaira, H. (1998). Establishment and characterization of a novel human rheumatoid fibroblast-like synoviocyte line, MH7A, immortalized with SV40 T antigen. J. Biochem. (Tokyo) 124, 1153–1162.
Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63.
Nichols, W. K., Mehta, R., Skordos, K., Mace, K., Pfeifer, A. M., Carr, B. A., Minko, T., Burchiel, S. W., and Yost, G. S. (2003). 3-Methylindole-induced toxicity to human bronchial epithelial cell lines. Toxicol. Sci. 71, 229–236.
Nigro, J. M., Baker, S. J., Preisinger, A. C., Jessup, J. M., Hostetter, R., Cleary, K., Bigner, S. H., Davidson, N., Baylin, S., Devilee, P., et al. (1989). Mutations in the p53 gene occur in diverse human tumour types. Nature 342, 705–708.
Paajarvi, G., Viluksela, M., Pohjanvirta, R., Stenius, U., and Hogberg, J. (2005). TCDD activates Mdm2 and attenuates the p53 response to DNA damaging agents. Carcinogenesis 26, 201–208.
Perry, M. E., Mendrysa, S. M., Saucedo, L. J., Tannous, P., and Holubar, M. (2000). p76(MDM2) inhibits the ability of p90(MDM2) to destabilize p53. J. Biol. Chem. 275, 5733–5738.
Peterson, S. R., Gadbois, D. M., Bradbury, E. M., and Kraemer, P. M. (1995). Immortalization of human fibroblasts by SV40 large T antigen results in the reduction of cyclin D1 expression and subunit association with proliferating cell nuclear antigen and Waf1. Cancer Res. 55, 4651–4657.
Pochampally, R., Fodera, B., Chen, L., Lu, W., and Chen, J. (1999). Activation of an MDM2-specific caspase by p53 in the absence of apoptosis. J. Biol. Chem. 274, 15271–15277.
Pochampally, R., Fodera, B., Chen, L., Shao, W., Levine, E. A., and Chen, J. (1998). A 60 kd MDM2 isoform is produced by caspase cleavage in non-apoptotic tumor cells. Oncogene 17, 2629–2636.
Porras, A., Gaillard, S., and Rundell, K. (1999). The simian virus 40 small-t and large-T antigens jointly regulate cell cycle reentry in human fibroblasts. J. Virol. 73, 3102–3107.
Ralhan, R., Sandhya, A., Meera, M., Bohdan, W., and Nootan, S. K. (2000). Induction of MDM2-P2 transcripts correlates with stabilized wild-type p53 in betel- and tobacco-related human oral cancer. Am. J. Pathol. 157, 587–596.
Ramsdell, H. S., Parkinson, A., Eddy, A. C., and Eaton, D. L. (1991). Bioactivation of aflatoxin B1 by human liver microsomes: role of cytochrome P450 IIIA enzymes. Toxicol. Appl. Pharmacol. 108, 436–447.
Reddel, R. R., Ke, Y., Gerwin, B. I., McMenamin, M. G., Lechner, J. F., Su, R. T., Brash, D. E., Park, J. B., Rhim, J. S., and Harris, C. C. (1988). Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res. 48, 1904–1909.
Saucedo, L. J., Myers, C. D., and Perry, M. E. (1999). Multiple murine double minute gene 2 (MDM2) proteins are induced by ultraviolet light. J. Biol. Chem. 274, 8161–8168.
Shimada, T., and Guengerich, F. P. (1989). Evidence for cytochrome P-450NF, the nifedipine oxidase, being the principal enzyme involved in the bioactivation of aflatoxins in human liver. Proc. Natl. Acad. Sci. U.S.A. 86, 462–465.
Soini, Y., Chia, S. C., Bennett, W. P., Groopman, J. D., Wang, J. S., DeBenedetti, V. M., Cawley, H., Welsh, J. A., Hansen, C., Bergasa, N. V., et al. (1996). An aflatoxin-associated mutational hotspot at codon 249 in the p53 tumor suppressor gene occurs in hepatocellular carcinomas from Mexico. Carcinogenesis 17, 1007–1012.
Stein, L. S., Stoica, G., Tilley, R., and Burghardt, R. C. (1991). Rat ovarian granulosa cell culture: A model system for the study of cell–cell communication during multistep transformation. Cancer Res. 51, 696–706.
Technau, A., Wolff, A., Sauder, C., Birkner, N., and Brandner, G. (2001). p53 in SV40-transformed DNA-damaged human cells binds to its cognate sequence but fails to transactivate target genes. Int. J. Oncol. 18, 281–286.
Thorburn, J., Frankel, A. E., and Thorburn, A. (2003). Apoptosis by leukemia cell-targeted diphtheria toxin occurs via receptor-independent activation of Fas-associated death domain protein. Clin Cancer Res. 9, 861–865.
Van Vleet, T. R., Klein, P. J., and Coulombe, R. A., Jr. (2001). Metabolism of aflatoxin B1 by normal human bronchial epithelial cells. J. Toxicol. Environ. Health A 63, 525–540.
Van Vleet, T. R., Klein, P. J., and Coulombe, R. A., Jr. (2002a). Metabolism and cytotoxicity of aflatoxin B1 in cytochrome P450-expressing human lung cells. J. Toxicol. Environ. Health A 65, 853–867.
Van Vleet, T. R., Mace, K., and Coulombe, R. A., Jr. (2002b). Comparative aflatoxin B1 activation and cytotoxicity in human bronchial cells expressing cytochromes P450 1A2 and 3A4. Cancer Res. 62, 105–112.
Villa, P. G., Henzel, W. J., Sensenbrenner, M., Henderson, C. E., and Pettmann, B. (1998). Calpain inhibitors, but not caspase inhibitors, prevent actin proteolysis and DNA fragmentation during apoptosis. J. Cell Sci. 111, 713–722.
Wei, C., Caccavale, R. J., Kehoe, J. J., Thomas, P. E., and Iba, M. M. (2001). CYP1A2 is expressed along with CYP1A1 in the human lung. Cancer Lett. 171, 113–120.
Wolff, A., Technau, A., Ihling, C., Technau-Ihling, K., Erber, R., Bosch, F. X., and Brandner, G. (2001). Evidence that wild-type p53 in neuroblastoma cells is in a conformation refractory to integration into the transcriptional complex. Oncogene 20, 1307–1317.(Terry R. Van Vleet, Todd L. Watterson, P)