* Integrated Toxicology Program, Duke University Medical Center, Durham, North Carolina 27710; and
Product Evaluation Division, R. J. Reynolds Tobacco Co., Winston-Salem, North Carolina 27102
Received April 4, 2001; accepted June 11, 2001
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ABSTRACT |
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Key Words: c-myc; proto-oncogene; cycloheximide; MNNG; bronchial epithelial cells; mRNA; gene expression.
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INTRODUCTION |
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Chemical exposure can elicit changes in cellular morphology and cell survival (Vikhanskaya et al.1993). These changes result from coordinated gene expression modifications in many signaling pathways. For example, an imbalance among cell proliferation, DNA repair, and apoptosis is a primary cause of genetic instability and can lead to preneoplastic alterations (Yin et al.1999
). Maintenance of this balance requires critical control of the cell cycle. Many proto-oncogenes and tumor suppressor genes function to control the cell cycle and maintain cellular homeostasis. A notable proto-oncogene, the c-myc gene, encodes a transactivating factor involved in the control of cell proliferation, differentiation, and apoptosis (Dang, 1999
; Wagner et al.1994
; Yeilding et al., 1998
). Recent studies have shown that a pulse of c-myc expression can elicit genetic instability and tumorigenesis (Felsher and Bishop, 1999
). Proliferative rate can influence genetic stability, which in turn may contribute to the occurrence of mutations and other genetic alterations. Hence, appropriate expression of c-myc may be pivotal for maintaining a nontransformed phenotype.
Although lung tumors often exhibit altered expression of oncogenes and/or tumor suppressor genes, the role of specific chemicals in causing these alterations is not well defined. Quantitative gene expression methods (e.g., RT/PCR, Northern and Western blots) have been most often applied to the characterization of diseased tissue compared to normal tissue and preneoplastic tissue. Currently, there are few publications that have used the TaqManTM RT/PCR technology to ascertain the response of normal cells to chemical exposure. The objective of this study was to quantify changes in the levels of c-myc mRNA in NHBE cells and A549 cells following chemical treatment to test the hypothesis that the TaqManTM RT/PCR assay can be used to quantitatively assess potential biomarkers of chemical carcinogenesis as well as characterize the response of model cell systems from different donors and tissue states. Herein we describe the effect of two model toxins, cycloheximide (CHX) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), on c-myc gene expression and toxicity.
CHX, a protein synthesis inhibitor, was selected as a positive control for detecting changes in c-myc expression in this model system, as previous studies have demonstrated that CHX can increase c-myc mRNA levels (Borrelli et al.1992). It has been postulated that CHX increases levels of c-myc expression by stabilizing and/or elevating the cytoplasmic levels of c-myc mRNA. This occurs through the synthesis inhibition of a protein(s) that posttranscriptionally regulates c-myc mRNA. N-nitroso compounds are found in many airborne environmental pollutants, including tobacco smoke and air pollution (Tricker et al.1989
, 1991
). MNNG, a model N-nitroso compound, causes O6-methylguanine adducts in DNA (Marcelino et al.1998
). GC-AT transition mutations are frequently associated with MNNG-induced DNA damage (Chary et al.1991
; Furihata et al.1994
) and are consistent with the mispairing of O6-methylguanine adducted bases with thymine.
Specific quantitation and characterization of proto-oncogene and/or tumor suppressor gene expression in bronchial epithelial cells may serve as a useful model for determining the role of individual chemicals as well as complex mixtures during lung carcinogenesis. The resulting data may be used to characterize gene-specific responses to environmentally relevant doses of chemicals and thereby evaluate the roles of these chemicals in carcinogenesis. This study demonstrated a dose-dependent increase in c-myc expression following CHX exposure in NHBE and A549 cells, yet only A549 cells exhibited a response to MNNG exposure. In the latter case, a dose-dependent decrease was observed. These results support the utility of in vitro cell models for investigating the relationship of gene expression and chemically induced neoplasms.
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MATERIALS AND METHODS |
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Cell culture.
Adenocarcinoma (A549) cell line was obtained from the American Type Culture Collection (Rockville, MD). The NHBE donor cells (10 M: 10-year-old male, lot # 17378; 20 M: 20-year-old male, lot # 17684) were obtained from BioWhittaker, Inc. (Walkersville, MD). Cells were maintained in humidified incubators at 37°C and 5% CO2.The supplier's suggested media were used to maintain optimal growth conditions for each cell type. A549 cells were cultured using Kaighn's modified F12 medium (JRH Biosciences; Lenexa, KS) supplemented with 10% fetal bovine serum (FBS), 50 µg/ml gentamycin, and 2 mM glutamine. NHBE cells were cultured using a modified LHC-9 medium (BioWhittaker, Inc.; Walkersville, MD) supplemented with 52 µg/ml bovine pituitary extract, 0.5 µg/ml hydrocortisone, 0.5 ng/ml human epidermal growth factor, 0.5 µg/ml epinephrine, 10 µg/ml transferrin, 5 µg/ml insulin, 0.1 ng/ml retinoic acid, 6.5 ng/ml triiodothyronine, 50 µg/ml gentamycin, and 50 ng/ml amphotericin-B. Media was replenished every 23 days for all cell types, and the cells were harvested and passaged for exposure to the test agents while in logarithmic growth phase.
Cell treatment.
A549 cells were seeded at 500,000 cells per 60-mm dish, yielding 3040% confluence at seeding. Cells in log-phase growth were exposed to either CHX (0, 1, 10, or 100 µM) or MNNG (0, 1, 2.5, 5, 10, or 25 µM) in normal growth media for 1 h. The exposure medium was then removed, and one set of plates was harvested into 1 ml TRIzolTM reagent. In general, 23 plates were used per dose for each time point. The remaining plates (i.e., exposed cells) were maintained in normal growth media for the remainder of the experiment (i.e., 2, 5, or 24 h). NHBE cells were seeded at 100,000 or 500,000 cells per 60-mm dish, or at 30,000 cells per well for a 6-well dish and grown to 5070% confluence. The cells were then exposed to CHX or MNNG (as described above) using normal growth media. Solvent controls for both cell types (denoted as 0 µM) were 0.025% ethanol for the CHX exposures and 0.07% DMSO for the MNNG exposures.
Cytotoxicity assays.
Cytotoxicity of CHX and MNNG was evaluated by the neutral red (NR) assay. Cytoxicity was determined by quantifying the ability of cells to incorporate NR into the lysosomes of the cells. A decrease in NR dye uptake is indicative of injured or growth-inhibited cells (Borenfreund and Puerner, 1985). Cells were seeded at 10,000 cells per well using 96-well plates. On the following day, cells were treated with CHX or MNNG at concentrations of 0, 1, 10, 25, 50, or 100 µM for 1 h. Sixteen wells were treated per dose. The exposure medium was removed, and the cells were then rinsed with Hanks balanced salt solution (HBSS). The cells were subsequently maintained in normal growth media for 24 h posttreatment. For continuous exposure analysis, cells were treated as described and maintained for 24 h in the treatment media. At each time point, cells were rinsed with HBSS and screened as described below. Cells were incubated with 50 µg/ml NR in growth media for 4 h. The NR media was removed, cells were rinsed with HBSS, and elution buffer (50% EtOH, 1% acetic acid) was added to release the dye. Subsequently, absorbance readings were collected at 540 nm using a Multiskan plate reader. Cell survival was expressed as percent of solvent-exposed control survival, and IC50 (concentration that inhibits 50% of cell survival) values were calculated.
RNA isolation.
Isolation of total RNA using TRIzolTM reagent was conducted according to the manufacturer's recommendations (Gibco BRL; Gaithersburg, MD) as described previously (Fields et al.1999). The resulting RNA pellet was air-dried, resuspended in nuclease-free water (Promega Corp; Madison, WI), and treated with amplification-grade DNase I (Gibco, BRL; Gaithersburg, MD). RNA was quantified using a Beckman spectrophotometer; A260/280 readings between 1.8 and 2.0 were used to ensure purity. Additionally, formaldehyde gel electrophoresis was performed to verify intact 18S and 28S ribosomal RNA.
Reverse transcriptase/polymerase chain reaction.
c-myc RT/PCR was performed with PCR access reagents (Promega; Madison, WI). The reaction mix consisted of 1x AMVTfl buffer, 1 mM MgSO4, 200 µM each dNTPs, 0.1 U/µl AMV reverse transcriptase, 0.1 U/µl Tfl polymerase, 200 nM each c-myc forward and reverse primer (Gibco BRL; Grand Island, NY), and 50 nM c-myc fluorogenic probe (Perkin Elmer; Foster City, CA). The reverse transcription was performed at 48°C for 45 min, and inactivation of the RT enzyme was performed at 94°C for 2 min. The PCR cycling conditions were as follows: denaturation, 94°C for 30 sec; annealing, 60°C for 60 sec; extension, 68°C for 2 min. The number of cycles used depended upon the nature of the experiment; in general, 30 cycles were performed. ß-actin (control) RT/PCR was performed as above with the following reagent differences: 300 nM each ß-actin forward and reverse primers (Perkin Elmer; Foster City, CA), and 200 nM ß-actin fluorogenic probe (Perkin Elmer; Foster City, CA).
Gene-specific mRNA quantitation.
Quantitative comparison of c-myc expression was performed using a fluorescence-based RT/PCR method and the TaqManTM-LS50B luminescence spectrometer detection system (Perkin Elmer; Foster City, CA). Briefly, the TaqManTM-LS50B system utilized a fluorescent-tagged probe and spectrofluorometry for gene-specific quantitation as previously described (Fields et al.1999; Holland et al.1991
). The c-myc
RQ value was divided by the ß-actin
RQ value to yield normalized fluorescence for c-myc RNA accumulation. The respective
RQ values varied upon interassay comparison. However, the ratios determined by dividing the normalized c-myc fluorescence values of the control with the values of the treated samples were consistent as observed in intra- and interassay comparisons of samples. Statistical analysis of the data was performed by two-way ANOVA on the base-10 log of the
RQ values from at least three separate experiments. The level of statistical significance was set at p < 0.05.
c-myc and ß-actin primer and probe sequences.
c-myc forward primer: 5'-TAC CCT CTC AAC GAC AGC AG-3'; c-myc reverse primer: 5'-TCT TGA CAT TCT CCT CGG TG-3'; c-myc TaqManTM probe (Perkin Elmer; Foster City, CA): 5'-(FAM) CAA GAC TCC AGC GCC TTC TCT CCGp-X(TAMRA)-3'. ß-actin forward primer: 5'-TCA CCC ACA CTG TGC CCA TCT ACG A-3'; ß-actin reverse primer: 5'CAG CGG AAC CGC TCA TTG CCA ATG G-3'; ß-actin TaqManTM probe: 5'-(FAM) ATG CCC-X(TAMRA)-CCC CCA TGC CAT CCT GCG Tp-3' (Perkin Elmer; Foster City, CA).
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RESULTS |
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DISCUSSION |
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The expression of the proto-oncogene c-myc is involved in regulating cellular proliferation, differentiation, and apoptosis (Amati et al.1993; Gibson et al.1995
; Shichiri et al.1993
). Following mitogenic stimulation, c-myc expression can be induced in proliferating and resting cells. This increase has been associated with enhanced S-phase entry in keratinocytes via positive effects on cdk-2 and negative effects on cdk-4 (Alexandrow and Moses, 1998
). Elevated c-myc expression has been recently reported to induce genetic instability (Felsher and Bishop, 1999
; Yin et al.1999
). Additionally, changes in the expression of genes and proteins that regulate genetic stability are often observed in preneoplastic tissue.
Treatment of bronchial epithelial cells with compounds that may cause phenotypic and genotypic changes as well as promote a premalignant state could help determine the specific causative agents involved in the development of lung cancer. Although RT/PCR has historical presence in the literature, the methodology of quantitative PCR has advanced significantly in recent years. Hence, the potential for applying TaqManTM RT/PCR to detection of biomarkers of exposure may offer extreme benefits in the characterization of early steps of carcinogenesis. To this end, the authors believe that the initial publication from our laboratory in this area would warrant a survey of known and well-characterized test agents. In this regard, analysis of a control gene and cytotoxicity were also employed as described below to provide a concise and deliberate characterization of the cellular response to chemical treatment. Because gene expression data affected by cellular perturbation such as chemical exposure is routinely normalized to an endogenous standard (control gene; Vanden Heuval, 1998), the expression level of the housekeeping gene ß-actin was determined and used as the normalizing factor in this study. A constant level of ß-actin expression during modulation of c-myc gene expression was one means employed to demonstrate biologically relevant change in response to chemical exposure. Additionally, the NR assay was employed to ensure that any cytotoxicity that may have resulted from chemical exposure did not adversely impact the intent of the c-myc expression analyses.
In this study, we analyzed two chemicals that represent classes of compounds noted for inhibition of protein synthesis and for DNA methylation. The protein synthesis inhibitor cycloheximide elicited 1.4- and 2-fold increases in c-myc mRNA levels within A549 cells at 2 and 5 h, respectively, following chemical exposure. NHBE cells from two donors were affected similarly by cycloheximide exposure. The c-myc mRNA levels for the 10-M and 20-M donors were increased by 1.6- and 1.8-fold, respectively, at 5 h postexposure. The proposed mechanism by which CHX may be eliciting this effect involves CHX-induced suppression of the synthesis of a protein, which specifically degrades RNA by binding to the 3' untranslated region of the c-myc transcript (Alessenko et al.1997; Wisdom and Lee, 1991
). Therefore, the enhanced levels of c-myc mRNA are the result of increased RNA stability. The observed increase in c-myc expression occurs at noncytotoxic concentrations in both cell types, and the expression of ß-actin is not altered. Although the significance of the increase in c-myc mRNA is beyond the scope of this work, the mRNA modulation may be indicative of a subsequent physiological change. For example, enhanced cell proliferation and genomic instability has been associated with an increase in c-myc expression, whereas a decrease in c-myc expression has been associated with differentiation and a reduction in cell proliferation (Conzen et al.2000
; Schmidt, 1999
; Yeilding et al.1998
). Interestingly, an elevation in c-myc expression has also been observed in cells destined for apoptosis (Alessenko et al.1997
; Gibson et al.1995
). In the apoptotic response, c-myc is hypothesized to affect subsequent pathways/genes that liberate the cascade of events for programmed cell death (Dang, 1999
; Nesbit et al.2000
).
Analysis of N-nitroso compounds using NHBE cells is particularly advantageous because of the presence of these types of chemicals in cigarette smoke, air pollution, and certain industrial environments, and because NHBE cells represent the target tissue for these environmental exposures. A model type of N-nitroso compounds is N-methyl-N'-nitro-N-nitrosoguanidine. MNNG has been shown to upregulate c-myc expression in rat stomach mucosa (Marcelino et al.1998). Additionally, MNNG induced gene-specific damage to the c-neu and c-myc proto-oncogenes in rat lung epithelial cells and normal human bronchial epithelial cells (Tricker et al.1989
); a dose-dependent yet transient decrease in O6-methylguanine-DNA methyltransferase activity has also been observed (Krokan et al.1985
).
Chemical perturbations may induce permanent or transient yet dose-dependent effects on gene expression. With regard to MNNG treatment, short-term 0-, 2-, and 5-h postexposure samples were analyzed and did not exhibit a change in gene expression compared to control samples. Therefore, 24-h continuous and postexposure treatment regimens were characterized for cytotoxicity to determine whether the prolonged exposure would effect gene expression without undue toxicity. As indicated in the results, the toxicity was deemed excessive upon continuous exposure. Therefore, the continuous treatment protocol was not used. However, 24 h following the short-term (1-h) exposure, MNNG caused a dose-dependent decrease in c-myc expression in A549 cells at concentrations that did not alter the expression of the control gene ß-actin; however, MNNG did not significantly affect c-myc expression in NHBE cells. The observed differences in the effect on c-myc expression in rat stomach mucosa versus the results in this in vitro study may be due to the variation of the acidic environment in the stomach versus the neutral pH in the lung, as the reactive moiety of MNNG is more effectively liberated in an acidic environment. The distinct response of the human lung cancer cells as opposed to the rat stomach mucosa cells may also be due to inherent genetic differences between species or between cancerous and normal cells. For example, variations in activity levels of the DNA repair enzyme O6-methyl transferase may affect the removal of methylation sites in the target gene (Belanich et al.1996). Our data in NHBE cells suggest differences between normal and cancer cells. A comparison of the O6-methylguanine-DNA methyltransferase (O6-MGMT) activity demonstrated a wide range of DNA repair levels in normal and tumor tissue from brain, lung, and ovary (Citron et al.1992
). In particular, a 2.6-fold difference in O6-MGMT activity was observed in normal and lung adenocarcinoma tissue. In that analysis, the normal tissue was determined to have the higher activity (Citron et al.1991
). Studies by Okada et al. have demonstrated that dose-dependent methylation of the HpaII site of exon I of the c-myc gene occurs following MNNG exposure. The authors suggested that MNNG-induced methylation is associated with decreased repair in c-myc (Okada et al.1996
). Alterations in gene expression can be caused by a reduction in DNA repair; hence, the observance of a decrease in c-myc mRNA at 24 h posttreatment with MNNG may be indicative of specific damage to the c-myc gene.
The scope of this work was to investigate the quantitative capability of the TaqManTM RT/PCR assay for ascertaining gene expression response following chemical exposure. The results indicate that the TaqManTM gene expression detection system is capable of detecting expression changes following chemical treatment. The changes in c-myc expression due to CHX and MNNG treatment occurred without effect on the internal reference, ß-actin. The combined PCR and NR results indicate that gene expression profiling and cytotoxicity analysis of chemicals may help discern the threshold chemical concentrations that may cause cellular responses.
As demonstrated herein, in vitro cell systems may serve as useful models for the evaluation of gene expression following chemical exposure. The observed differential response to MNNG in the adenocarcinoma cell line compared to the normal lung cells may demonstrate the variation between cancerous and normal cell status. Furthermore, as NHBE cells are the progenitor cells for most types of lung cancer, quantitative analysis of mRNA from these cells by methods such as the TaqManTM gene expression detection system may generate data that can identify specific chemicals which may cause the genetic alterations that lead to lung cancer. Indeed, the cause of cancer involves a vast network of genes and gene products beyond c-myc. Hence, we are currently investigating how genes involved in cell cycle regulation, DNA damage response, and metabolism respond to chemicals (Fields et al., 2000, 2001
) more relevant to lung carcinogenesis.
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ACKNOWLEDGMENTS |
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NOTES |
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