* National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; and
Aichi Cancer Center Research Institute, Chikusa-Ku, Japan 464-8681
Received March 22, 2002; accepted June 18, 2002
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ABSTRACT |
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Key Words: toxicogenomics; microarray analysis; dioxin; TCDD; AhR; lung cells; A549; HPL1A; real time RT-PCR.
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INTRODUCTION |
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Previous studies in our laboratory (Tritscher et al., 1999, 2000
) as well as rodent bioassays (NTP, 1982
), confirm that the lung is a target organ for TCDD. In rats, TCDD disrupts the normal cellular proliferation/differentiation milieu resulting in alveolar-bronchiolar metaplasia and hyperplasia of the bronchiolar epithelium (Tritscher et al., 1999
), adenomatous hyperplasia (NTP, 1982
), and keratinizing squamous cell carcinoma of the lung (Kociba et al., 1978
). There is evidence that TCDD causes tumor promotion by interfering with intracellular signal transduction pathways related to growth factors and cytokines such as transforming growth factor and interleukin-1ß (IL1ß; Abbott and Birnbaum, 1990
; Sutter et al., 1991
). Investigations aimed at examining signaling pathways in lung are needed since the mechanisms whereby dioxins induce pulmonary diseases and/or cancer is largely unknown.
Multiple studies have identified a number of genes whose expression is altered by TCDD (Frueh et al., 2001; Kurachi et al., 2002
; Lai et al., 1996
; Puga et al., 2000b
; Sutter and Greenlee, 1992
). TCDD alters many of these genes by a direct receptor-mediated mechanism, involving a basic helix-loop-helix (bHLH) protein known as the aryl hydrocarbon receptor (AhR; Denison and Heath-Pagliuso, 1998
; Okey et al., 1994
; Rowlands and Gustafsson, 1997
). The AhR is maintained in a ligand-binding state by association with cytosolic proteins that include heat shock protein 90 (HSP90) dimer, c-SRC (Enan and Matsumura, 1996
), AIP1 (also known as ARA9 or XAP2; Ma and Whitlock, 1997
), and p23 (Kazlauskas et al., 1999
). Upon ligand binding, the AhR dissociates from the HSP90, is transferred into the nucleus where it forms a heterodimer with another bHLH protein, the aryl hydrocarbon nuclear translocator (ARNT). The AhR/ARNT heterodimer binds to the DNA xenobiotic responsive element (XRE, also known as the AhRE or DRE) in the promoter region of Ah responsive genes. Coactivators and general transcription factors are recruited with the result being transactivation of specific AhR-responsive genes. While this is a common mechanism, not all genes altered by exposure to dioxin necessarily have XRE in their DNA. It is becoming increasingly clear that the AhR is involved in a number of complex proteinprotein interactions. For example, the AhR can sequester ARNT preventing other known ARNT-dependent intracellular events to occur (Chan et al., 1999
). Alternately, it can bind directly to other proteins that interact with other transcription factors (Ge and Elferink, 1998
; Puga et al., 2000a
). Regardless of the downstream mechanism responsible for gene expression changes, the initial event is TCDD binding to the AhR.
Given the central role of the AhR in mediating the effects of TCDD, the identity of genes altered by activation of the AhR is crucial to understanding the pathological effects of TCDD. Early studies using differential hybridization screening of a human keratinocyte cell line identified novel target genes of TCDD, including cytochrome P450 1B1 (CYP1B1), plasminogen activator inhibitor-2 (PAI2), and IL1ß (Sutter et al., 1991). More recently, the complexity of the transcriptional response of human liver cells (HepG2) to TCDD have been reported (Frueh et al., 2001
; Puga et al., 2000b
). This study and others showed using cDNA microarray analysis that multiple genes are altered transcriptionally by TCDD, both directly and indirectly (Kurachi et al., 2002
; Thomas et al., 2001
). In mice, Park et al.(2001) also used cDNA microarray analysis, to identify in vivo novel TCDD targets with putative roles involved in toxicity of TCDD in the thymus. Tissue and cell specificity is a crucial factor in the biochemical and toxic effects of TCDD. With this in mind, the lung as a target organ for TCDD toxicity has widely been ignored although some reports of genes altered in the lung are available (DeVito and Birnbaum, 1995
; Santostefano et al., 1996
; Vogel et al., 1994
, 1998
). The lung is a known target organ for TCDD and other polyhalogenated aromatic hydrocarbons (PHAHs) in both rodent and humans. The fundamental mechanism for altered gene transcription is via activation of the AhR, hence there clearly is a need to identify the signaling pathway networks that are altered by TCDD and other PHAH compounds.
The majority of pulmonary adenocarcinomas are bronchogenic in origin, and some are derived from the periphery of the lung, that possesses type II pneumocyte characteristics. We were interested in the sensitivity of type II pneumocytes to TCDD since hyperplasia of these cells is linked to chronic lung disease, and this cell type is a possible precursor of pulmonary adenocarcinoma (Mori et al., 1998). Type II pneumocytes represent 35% alveoli surface area, are critical in maintaining normal alveolar function, and believed to play a role in the development of chronic lung diseases. These cells are a major source of surfactant that reduces surface tension in the lung providing protective functions for the peripheral lung. They undergo proliferation and transform into type I pneumocytes upon cellular injury and subsequent repair. To assess the effects of TCDD on type II pneumocytes, two cell lines were investigated; the HPL1A and the A549 cell lines. We chose the A549 cell line because it is a well-established and characterized human lung adenocarcinoma (type II pneumocytes) that expresses a number of cytochromes P450 including CYP1A1, a well-characterized TCDD-inducible gene (Hukkanen et al., 2000
). The A549 cells have proven valuable in elucidating some aspects of TCDD-regulation of cytochrome P450 responses in lung cells (Vogel et al., 1994
). However, as a malignant cell line it may not be the most appropriate model for assessing effects of TCDD on gene expression in "nonmalignant" human lung epithelium. The HPL1A cells were used to represent a "normal" cell line. They are a recently established immortalized human peripheral lung (HPL) epithelial cell line (Masuda et al., 1997
) that retains morphological and biochemical characteristics of peripheral epithelial cells (type II pneumocytes and Clara cells). These characteristics include cytokeratin staining, multivesicular bodies, incomplete multilamellar body-like structures, and expression of Clara cell specific genes (Masuda et al., 1997
).
The objective of this present study was to identify and compare concentration-dependent effects of TCDD on gene expression in a malignant tumorigenic and a nonmalignant lung cell line. To accomplish this, gene expression profiling by microarray dual fluorescence hybridizations was performed in cells treated with increasing concentrations of TCDD for 24 h. We identified novel genes altered by TCDD that are involved with differentiation and immune regulation. These genes represent integrated networks of signaling pathways that may be associated with pulmonary disease, particularly that of lung cancer.
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MATERIALS AND METHODS |
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RNA extraction.
Total RNA was prepared using Qiagen RNeasy midiprep columns (Qiagen, Valencia, CA) according to the manufacturer recommendations, with the following minor modifications: twice as many columns as recommended were used and RNA was eluted using maximal amount of H2O. Total RNA was concentrated using Microcon 30 spin columns (Millipore Corp., Bedford, MA), quantitated by UV spectroscopy at 260 nM, and stored in RNase-free H2O at 70°C. Samples were separated by electrophoresis on formaldehyde denaturing agarose gels stained with Sybergreen dye to confirm the integrity of the 18S and 28S ribosomal RNAs.
cDNA microarray labeling.
A detailed protocol for the synthesis of labeled cDNA used can be found at the following website: http://dir.niehs.nih.gov/microarray/methods.htm. Briefly, total RNA (35 µg for Cy3 label, 75 µg for Cy5 label) was annealed to the oligo d (T)1218 primer at 70°C for 10 min using: oligo d(T)1218, 500 µg/ml (Amersham-Pharmacia BioTech, Piscataway, NJ), and RNase Inhibitor, 10 U/µl (Life Technologies, Rockville, MD). The RNA was then reverse transcribed at 42°C for 3 h in a 40 µl total volume of first strand buffer (1X); Stanford dNTP mix, (0.75 mM dATP, 0.75 mM dGTP, 0.75 mM dCTP, 0.66 mM dTTP; Amersham-Pharmacia, Piscataway, NJ); FluoroLink Cy3-dUTP, 2.5 nM FluoroLink Cy5-dUTP, 2.5 nM (Amersham-Pharmacia BioTech, Piscataway, NJ); 0.1 M DTT (Life Technologies, Rockville, MD); SuperScript II RNase H- Reverse Transcriptase (18 U/µl; Life Technologies, Rockville, MD). RNA was then degraded using 0.1 M NaOH for 30 min at 70°C and neutralized with 0.1 M HCl. 1X TE buffer, (pH 7.5) and the fluorescent DNA probe pairs were combined and washed 3 times using a Microcon-30 filter (Millipore Corp., Bedford, MA) using 1X TE. During the last wash the following blockers were added to the TE buffer: 20 µg Human COT-1 DNA (Life Technologies, Rockville, MD), 20 µg yeast tRNA (Sigma, St. Louis, MO), 20 µg Poly dA (Amersham-Pharmacia BioTech, Piscataway, NJ). The probes were concentrated to 28 µl and 20X SSC (3 M NaCl, 0.3 M NaCitrate; 3X final conc.), 25X Denhardts (2X final conc.) and 10% SDS (0.3% final) were added as the hybridization solution. The mixture was heated at 100°C for 2 min, maintained at room temp (RT) for 10 min, then filtered through a Millipore Ultrafree-MC 0.45 µm filter unit (Millipore Corp., Bedford, MA).
cDNA microarray hybridization.
Labeled cDNAs were hybridized to the NIEHS Human ToxChip v1.0 (http://dir.niehs.nih.gov/microarray/chips.htm). DNA clones used for the Human ToxChip were obtained from Research Genetics (Research Genetics, Huntsville, AL), amplified by polymerase chain reaction and then spotted onto Poly L lysine-treated glass slides using a microarray printer (Beecher Instruments, Silver Spring, MD). All hybridizations were carried out in triplicate, control RNA was labeled in two hybridizations with Cy5 and in the third hybridization, a reciprocal fluorescent labeling was used where the control RNA was labeled with Cy3. Hybridizations were carried out for 1624 h at 65°C in a sealed, humidified chamber (National Health Genomic Research Institute). Following hybridization, slides were washed at room temperature (4 washes, 16 min each) using 0.5 x SSC, 0.01% SDS, and air-dried.
Microarray data acquisition and analysis.
Fluorescence signals were obtained using a Genepix array scanner (Axon Instruments, Inc., Union City, CA). Cy3 and Cy5 fluorescence were individually scanned using a 532 and 635 nM laser. The photomultiplier tubes were auto-adjusted to obtain equal signal strength of the two channels. Raw scanned images were processed initially using IPLab (Scanalytics, Inc.) with the ArraySuite extensions (originally developed at the National Human Genome Research Institute). Scatterplots of the channel intensities were individually examined and the intensity cut-off levels adjusted to limit dye-specific labeling artifacts.
Cy3 and Cy5 grayscale images were overlaid, to obtain a pseudo-color image, and the analysis software (Arraysuite v1.3; Scanalytics, Arlington VA) defined the spots in the ratio image by accessing the gene in plate order (GIPO) file. The GIPO file is a database that describes which gene is at that location on the microarray. The software shows spots having a ratio < 1 as green, those having a ratio > 1 as red, and with ratios = 1 as yellow (equal amounts of red and green). The distribution of log2-normalized spot ratio values was normalized to 84 "housekeeping" genes that were included on the chip. All spots whose ratio were outside of the 99% confidence interval of the population distribution for all genes on the chip were classified as statistically significant gene changes in this specific hybridization. The methods of the image analysis and quantitation are previously described (Chen et al., 1997). The statistical information generated for each spot was uploaded to the file server for further analysis at other workstations.
Expression profiles from the differentially expressed genes were managed in the MicroArray Project System (MAPS) relational database (Bushel et al., 2001): http://www.niehs.nih.gov/Connections/2000/feb/maps.htm. A binomial distribution (Casella and Berger, 1990
) was computed with the gene expression profiles obtained from the compound treatment to determine the probability of randomly detecting genes altered by treatment. That is for k = 0, 1, 2,...., n
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Real Time RT-PCR
Reverse transcription.
Total RNA (100 ng) was reverse transcribed using 1X RT, MgCl2 (2.2 mM), dNTP (2.0 mM), RNAsin (0.2 U/µl), random hexamer primers (0.5 mM), and MMLV reverse transcriptase (0.3 U/µl) in 10 µl reactions using a 3-step cycle: 25°C, 10 min; 48°C, 30 min; and 95°C, 5 min. Reverse transcription reagents were purchased from PE Applied Biosystems (Foster City, CA). The resulting cDNA was used in subsequent real time PCR reactions.
Sybergreen detection.
Real time fluorescence detection was carried out using an ABI Prism 7700 Sequence Detection System. Reactions were carried out in microAmp 96 well reaction plates, PCR buffer 1X (containing Sybergreen), MgCl2 (5 mM), dATP, dCTP, dGTP, dUTP (0.2 mM each), Taq Polymerase (0.25 units/µl; PE Applied Biosystems, Foster City, CA), forward and reverse primers (0.2 µM each, Research Genetics, Huntsville, AL) and cDNA (10 µl) in a final PCR reaction volume of 50 µl. Amplification parameters were: denaturation at 94°C 10 min, followed by 40 cycles of 95°C, 15 s; 60°C, 60 s. All primers and probes were designed using PrimerExpress Software (PE Applied Biosystems, Foster City, CA) and can be found in Table 1. Samples were analyzed in triplicate, and actin was used as an endogenous control. Fold induction was calculated using the formula 2-
CT, where
CT = target gene CT actin CT, and
CT is based on the mean
CT of respective control (non-TCDD treated). The CT value is determined as the cycle at where the fluorescence signal emitted is significantly above background levels and is inversely proportional to the initial template copy number. Amplification products using Sybergreen detection were initially checked by electrophoresis on ethidium bromide stained agarose gels. The estimated size of the amplified products matched the calculated size for transcript by visual inspection.
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RESULTS AND DISCUSSION |
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CYP1A1 was significantly induced by TCDD using specific and absolute quantitation by 5' nuclease (Taqman) probe-based real time RT-PCR. The effect was dependent on the concentration in both cell lines. In the HPL1A cell line, induction was 2-fold at 0.1 nM, 21-fold at 1 nM, and 29-fold at 10 nM (Fig. 1). In the A549 cell line, CYP1A1 was induced from 1.1-fold at 0.1 nM, 5-fold at 1 nM, and 13-fold at 10 nM. The fold induction from TCDD exposure for CYP1A1 in the A549 cell line was comparable to that found in the HepG2 cell line that is 1216-fold (Frueh et al., 2001
; Puga et al., 2000b
). CYP1B1 was concentration-dependently induced to similar degrees in both cell lines. In the HPL1A cell line, CYP1B1 was induced 3.1-fold at 0.1 nM, 4.9-fold at 1 nM, and 5.5-fold at 10 nM. In the A549 cell line, CYP1B1 was induced 2-fold at 0.1 nM, 3.7-fold at 1 nM, and 3.6-fold at 10 nM. In contrast, expression of actin remained essentially unchanged across the concentration-range (data not shown). These data confirm a preliminary report using intercalating dye-based (Sybergreen) real time RT-PCR detection for the responsiveness of this cell line (Martinez et al., 2000
).
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Based on the above criteria, 121 cDNA clones representing 117 independent genes were identified from the microarray analysis as being significantly altered by TCDD at any concentration and in either cell line. Of these, there were 15 genes altered in both cell lines, in at least one of the three treated (0.1, 1, or 10 nM TCDD) groups. These genes include AC083883, ALDH3, ALDH1A3, CDH1, CTNNB1, CYP1A1, CYP1B1, DKFZp434A1014, DUSP1, EGR1, F2R, HSPA4, KIAA1389, KIAA1007, and STK4. In both cell lines there were 68 genes significantly altered from control at any concentration, and 53 were cell line specific. A Venn diagram of the cell/concentration distribution of the genes that were identified as significantly altered by TCDD is shown in Figure 2. Genes are identified by UniGene symbol/accession number (see http://www.ncbi.nlm.nih.gov/UniGene/Hs.Home.html; where the complete name can be found upon typing in the accession number or the abbreviation). The Venn diagram represents genes that are common (or overlapping) with one concentration, two concentrations, or all three concentrations for each cell line. We found cell line to cell line differences as well as common gene responses to TCDD. This analysis focused only on those genes identified at 99% CL in replicate experiments. The majority (approximately 80%) of gene changes by TCDD in this analysis were cell-line specific. This indicates that caution should be used when extrapolating the specificity of mechanism of action of toxicants between tissue/cell lines and across species.
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The AhR battery of genes altered by TCDD in this study were primarily metabolic enzymes. Due to the overlap of genes involved in many different categories we attempted to categorize based on a biological functional. For example, a transcription factor or receptor that is involved in differentiation was so classified. The 5 major categories discussed are (1) signal transduction, (2) extracellular matrix and basement membrane components, growth factors, and chemokines, (3) genes involved with differentiation, (4) genes involved with cell growth or cell cycle, and (5) others that include stress, glucose regulation and NFB signaling. Other TCDD toxicogenomic studies have primarily examined TCDD effects in HepG2 cells (Frueh et al., 2001
; Puga et al., 2000b
) or mouse liver cells (Kurachi et al., 2002
; Thomas et al., 2001
). Thus tissue specificity may be responsible for the difficulty in making direct gene-to-gene comparisons. Other factors that may contribute to this difficulty are the inherent problems with nomenclature and differences in platforms used. Keeping this in mind, it is not surprising that there were only a few genes found in common with other array studies. Exact matches included CDN1A, CYP1A1, ODC1, THBD, and VEGF. Genes that matched others within the same family included ACY1, GSTM1, HSP70, PRKCA, STK4, S100P, and TCEB3 (Frueh et al., 2001
; Kurachi et al., 2002
; Puga et al., 2000b
; Thomas et al., 2001
). Hence, our study confirms approximately a dozen genes altered by TCDD in different systems. It extends these studies by identifying genes that are not necessarily involved in the toxic stress mediated by TCDD by using a dose continuum.
Concentration Responses
Examination of the concentration response curves for genes detected by microarray analysis using relative fold changes induced by TCDD is based on the 99% CL. Genes were both induced and repressed by TCDD treatment and a number of different concentration-response patterns were observed (Figs. 35). In genes that were repressed, the central tendencies of expression for concentration response were either:
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This study demonstrates that a number of genes altered by TCDD require concentrations that are 10100-fold lower than those widely used to analyze gene expression in other systems. These data suggest that a physiological response versus a toxic response may be initiated. We show that when one examines global TCDD concentration-response patterns the pattern of trends will vary substantially. Genes that were equally induced or repressed at all concentrations of TCDD may have unusually steep dose-response curves or alternatively, the threshold for effective concentration is lower than that measured in this study.
Signal Transduction Molecules
Many genes altered by TCDD are involved in signaling and lead to changes in cell morphology, motility, cell-cycle progression and the induction of malignant transformation. An important cell surface glycoprotein adhesion molecule is E-cadherin (CDH1). CDH1 was repressed in both cell lines (Table 2), and is implicated in carcinogenesis since it is often lost in human epithelial cancers and appears to be a rate-limiting step in the progression from adenoma to carcinoma. Catenin ß 1 (CTNNB1), an intracellular partner of CDH1, on the other hand was significantly induced in both cell lines (Table 2
). LRP6, IQGAP, and CDC42 (a Rho family kinase) are other genes involved in Wnt transduction (Fukata et al., 1999
, 2001
; Tamai et al., 2000
) that were altered (99% CL) only in the A549 cells suggesting a cell line specific pathway.
Another similarity between the two different cell lines was the induction of KIAA1389 (also known as SPA1). KIAA1389 belongs to a family of highly diverse, multifunctional signaling proteins that share a conserved 120 amino acid domain (RGS domain) known as regulators of G-protein signaling. KIAA1389 is known to inhibit activation of RAP1-GTP, which is required to maintain cell adhesion (Tsukamoto et al., 1999). KIAA1389 was altered in the HPL1A cell line at all concentrations of TCDD tested, and in the A549 cell line at 1 and 10 nM in a concentration-dependent manner. Signaling through the Ras cascade is implied by changes in gene levels of downstream mediators through pathways such as Rac, Rap, and Rho. Other evidence for p38 kinase is indicated by repression of DUSP1 in both cell lines and induction for MAPKAP2 in the HPL1A cells. It is unclear what membrane receptor may be activating Ras, but potential candidates indicated by microarray analysis include ROR2, IGF1R, and F2R (thrombin receptor). F2R, a proteolytically activated cell surface receptor that is involved in cytoskeletal signaling, was found altered in both cell lines. By microarray analysis F2R was detected as different from control (99% CL) at the low concentration for HPL1A and the lower two concentrations of A549; however, a concentration-dependent continuum was found for both cell lines. Thus, these data also show that there are several regulators of G protein signaling (1RAP1GA1, IQGAP, and a SPA-1 homologue KIAA1389, and LOC54453), as well as multiple signaling networks mediated by TCDD known to be involved with cell adhesion.
Extracellular Matrix, Basement Membrane Components, Growth Factors, and Chemokines
Effector genes involved in metastasis, cellular transformation, angiogenesis, cell growth, and invasion through the ECM and capillary basement membrane. Theses can be classified as (1) cytokines/chemokines, (2) angiogenic factors, or (3) extracellular matrix components/enzymes. Chemokines themselves regulate angiogenesis, promote cellular transformation, tumor growth, migration, homing, and metastasis (Strieter, 2001). In Figure 3
, the concentration-dependent trends for downstream effectors that were detected by microarray analysis are shown. Real time RT-PCR found similar fold induction for IL8 (also known as CXCL8; Table 2
). IL8 was induced in A549 cells and is known to be stimulated with tumor necrosis factor and IL1B (Standiford et al., 1990
). Chemokines also play a role in the inflammation of airways, and TCDD-induced chemokines expressed detected in this study include the following: IL8, SCYA2, LIF (Fig. 3
), and CXCR4 (Table 2
).
By facilitating angiogenesis during lung injury or repair, the highly vascular nature of the lung is critical to its function. Proangiogenic factors detected include VEGF, FGF, and ephrin A1 (EFNA1). Our data show that these angiogenic factors are differentially induced by TCDD, where induction of VEGF and EFNA1 was found in the "nonmalignant" HPL1A, while FGF2 was induced in the malignant A549 cell line. Increased vasculature and VEGF have been detected in asthma patients suggesting a role for VEGF in pulmonary disease, contributing to angiogenesis or chronic inflammation (Hoshino et al., 2001).
Induction of extracellular matrix components such as the hyaluronidase, matrix metalloproteinase 1 and 9 (MMP1 and MMP9), laminins (LAMC1 and LAMA3), and filamin (FLNB; Fig. 3) may contribute to tumor invasion and metastasis. The complex interplay of these compounds is observed by induction of MMP9, COL4A4, and TIMP3. MMP9 is gelatinase known to digest basement membrane collagen IV (COL4A4), and TIMP3 is an inhibitor of both MMP1 and MMP9 (Stamenkovic, 2000
).
Genes Involved with Cell Growth or Cell Cycle Control
Growth regulation is critical in carcinogenicity, in that uncontrolled growth of aberrant cells leads to tumors. TCDD is a known tumor promoter that modifies the normal cellular proliferation-differentiation process, a process that is linked to altered regulation of gene expression mediated by Ras and Rho GTPases. Many TCDD effects are attributed to its interference with growth regulation, yet the precise mechanism remains unclear. Transcripts detected in this study include previously reported as well as novel TCDD-modified genes. For example, the CDKN1A protein was recently detected by serial analysis of gene expression (SAGE) from the liver of TCDD-treated mice (Kurachi et al., 2002), and TCDD regulates MAD2L2 by an AhR independent manner (Oikawa et al., 2001
). EGR1, an immediate early gene that is required to activate transcription of genes involved in negative growth regulation was discovered to be induced by TCDD in HepG2 cells (Puga et al., 2000b
). Finally, TFDP2, an E2F dimerization partner that plays a role in oncogenesis was increased in a concentration-dependent manner. The E2F complex is an important indirect mediator of TCDD-induced alterations in cell cycle since the AhR is known to interact with the retinoblastoma protein and leads to repression of E2F-dependent transcription and G1 cell cycle progression (Puga et al., 2000a
).
There were a number of genes found altered by TCDD that are involved in cellular proliferation. Of these, only EGR1 was detected in both cell lines. In the HPL1A cell line, cell cycle genes induced by TCDD include CDKNIA (also known as p21, Cip1), MDM4 (also known as MDMX), and MAD2L2 (Fig. 2). Cell cycle genes altered by TCDD in the A549 cells included CKD6, CDC25A, MCM5, and TFDP2. Based on this array of genes, we conclude that TCDD is most likely affecting the G1 phase of the cell cycle, albeit through different effectors. CDKN1A, MDM4, CDK6, and CDC25 are all involved in the G1 phase of the cell cycle and TCDD is known to affect other cells at the G0/G1 progression (Ma and Whitlock, 1996
; Weber et al., 1997
). Other genes detected by microarray analysis that are not involved directly with cell cycle include negative growth regulators KNSL4 and KLF4 in HPL1A cells, ACVR2B and THBD for the A549 cells.
Genes Involved with Differentiation
A principal component of cancerous growth often depends on deranged patterns and control of differentiation. Thus changes in gene expression that alter the balance of growth and differentiation transcription and growth factors play important roles in pathophysiological changes like hyperplasia, fibrosis, and neoplasia. Cytodifferentiation is the cellular and molecular process that transforms precursor cells into phenotypically mature cells. The failure of a cell to normally differentiate can be dependent on the neoplastic phenotype (Harris et al., 1985; Nettesheim et al., 1985
). In this study, we report distinct differences in genes involved with differentiation altered by TCDD between the A549 and HPL1A cell lines. A hypothetical pathway of how TCDD may be altering differentiation is depicted in Figure 4
. Retinoic acid receptor ß (RARB) is activated by retinoic acid (RA) and causes suppression of tumor cell growth and/or inhibition of keratinization/differentiation (Sun et al., 2000
; Zou et al., 1999
). RARB is another important potential mediator of TCDD action and it was induced in the HPL1A in a concentration-dependent manner (1040%; Table 2
). These data suggest that the HPL1A cell line is retinoic acid responsive, since several RA responsive genes were detected by microarray analysis and include the following: RARB, NCOA2, CRABP1, ZNF42, and ELF3 (Fig. 4
). In contrast, alterations in RA-responsive genes were not detected in the A549 cells, which is consistent with prior observations that report the A549s as a retinoic acid resistant cell line (van der Leede et al., 1993
).
A role for TCDD in vitamin A homeostasis is evident by the depleted hepatic stores of vitamin A in chronically TCDD-treated rats (Kelley et al., 2000). Altered mobilization of vitamin A is evidenced by an increase in serum and kidney retinoic acid levels from TCDD treated rats (Nilsson et al., 2000
). Cross talk between signal transduction pathways for AhR and the RARs is suggested (Delescluse et al., 2000
; Gonzalez and Fernandez-Salguero, 1998
). We propose that induction of CYP1A1, as well as ALDH1A3 (also known as ALDH6) contribute to TCDD alterations of vitamin A levels. ALDH1A3 is a retinaldehyde dehydrogenase that can cause the irreversible synthesis of retinal to retinoic acid (Grun et al., 2000
; Vasiliou et al., 2000
). CYP1A1 has been linked to retinoic acid metabolism (Lampen et al., 2000
). Thus, an increase in the basic metabolic turnover of vitamin A without concurrent replacement may lead to loss of storage and altered homeostasis.
Immune Mediated Responses
It is known that alveolar cells play a role in host innate resistance to pulmonary infections by producing proinflammatory cytokines. Suppresion of an immune response will not only leave an organ or tissue susceptible to infections but also to neoplasms as shown by the association of immune defiencies with Kaposi sarcoma, non-Hodgkins lymphoma and anogenital carcinoma (Ioachim, 1997). In animal models, the immunotoxicity of TCDD is unequivocal (Holsapple et al., 1991
), and an extremely sensitive toxic parameter. In both humans and rodents, there are a number of immunotoxic endpoints altered by TCDD, yet the mechanism of immunosuppression is still not clear (Kerkvliet, 2002
; Vos and VanLoveren, 1995
). Both in vitro and in vivo data show that immune effects of TCDD may be direct (Sulentic et al., 1998
, 2000
) or indirect (Shepherd et al., 2000
). However, a clear role for the AhR is evidenced by the fact that AhR-deficient mice are resistant to TCDD immunotoxicity (Fernandez-Salguero et al., 1996
; Shepherd et al., 2001
).
Microarray data from both A549 and HPL1A cell lines suggest that TCDD may signal through the interferon pathway since the interferon gamma receptor 1 (IFNGR1) was upregulated in both cell lines (Fig. 2, Table 1
). While IFNGR1 was induced only in the A549- 0.1 nM TCDD group, we found a concentration-dependent induction based on hybridization values for IFNGR1 in the HPL1A cell line that was confirmed by real time RT-PCR (Table 2
). The mechanism for IFN signaling is through the IFNGR1 association with Jak1, upon activation by INF
. Transphosphorylation of janus kinases (Jaks) occurs, which phosphorylates IFNGR1, then initiates a cascade of events that leads to altered gene expression (Stark et al., 1998
). The data suggest that modulation of the Jak/STAT pathway was mediated through different pathways by the two cell lines since leukemia inhibitory factor (LIF), and interleukin 6 signal transducer (IL6ST, also known as gp130) were altered in the HPL1A cell line and not in the A549 cell line (Fig. 3
). LIF-activated IL6ST/LIFR signaling is mediated through the Jak/Stat pathway, where LIF binds to and activates a heterodimer composed of IL6ST and leukemia inhibitory factor receptor (LIFR; Gearing et al., 1992
). A number of genes that are downstream products of Stat activation (Heinrich et al., 1998
) were found altered in either the A549 cell line or the HPL1A cell line and include fos, interstitial collagenase (MMP1), vasoactive intestinal peptide (VIP), and IL6ST (gp130).
One of the most striking and novel immunomodulatory effects of TCDD that was only detected in the HPL1A cell line was the repression of several IFN-stimulated genes. These include interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), interferon-stimulated protein 15 (ISG15), EST, highly similar to IFT2-human interferon-induced protein with tetratricopeptide repeats 2 (IFT2), and myxovirus resistance 1 (homolog of murine interferon-inducible protein p78; MX1). These genes appear to be sensitive and robust targets of TCDD since IFIT1, IFT2, and ISG15 were repressed at all doses tested, and MX1 was repressed at the two higher doses. The sensitivity for repression for these genes is of interest since immunotoxicity of TCDD occurs at low doses. A potential target activated by TCDD is the interferon regulatory factor 4 (IRF4), which may lead to the repression of these genes (Fig. 5). The mechanism of IRF4 repression of ISG15 results from formation of a complex with interferon consensus sequence binding protein (ICSBP, also known as IRF8; Rosenbauer et al., 1999
). Alternatively, IRF4 binding to the immunophilin FKBP52 prevents association of IRF4 with its partner PU.1 (Sharma et al., 2000
) as well as transactivation (Mamane et al., 2000
). These data strongly suggest indirect immune-modulation by TCDD in lung epithelial cells.
Change in expression of surface antigens is a characteristic often seen in cancerous cells. There were cell line specific alterations of a number of surface antigens induced by TCDD (Fig. 2). These include epididymis-specific, putative ovarian carcinoma marker (HE4); EST, highly similar to tumor associated antigen (L6); transmembrane 4 superfamily member 1 (TM4SF1); and tumor-associated calcium signal transducer 2 (TACSTD2). Both L6 in the A549 cell line and TACSTD2 in the HPL1A cell line were induced at all doses of TCDD, suggesting that they may be potential biomarkers of TCDD exposure.
Other Pathways
Signaling pathways affected also include those involved in glucose homeostasis, stress, and NFB; a transcription factor for a multitude of genes. Direct interactions of the AhR and NF
B are reported by Tian et al.(1999), who propose that the immunosuppressive effects of TCDD are mediated by suppression of NF
B induced cytokines. NF
B interacts with the transcription factor AP-1 and upregulates the IL-8 gene (Shi et al., 1999
). Our data suggest a role of NF
B in lung epithelial cells as seen by modulation of IL8, REL, and BCL3. It is interesting to note that v-Rel causes induction of IRF-4 expression and is linked to transformation of fibroblasts (Hrdlickova et al., 2001
). BCL3 is a member of the I kappa B family and binds to the NF
B p50 subunit, thereby allowing activation as a positive regulator of NF
B activity (Franzoso et al., 1997
). Evidence of involvement of the AP-1 complex was seen by differential regulation by TCDD in the HPL1A and A549 cells. FOS was altered in the HPL1A cells and FOSL-1 was altered in the A549 cells (Figs. 2 and 5
). In studies using mammary epithelial cell line MCF-10A, TCDD increases the tyrosine phosphorylation of IGF1R (Tannheimer et al., 1998
). We have shown in addition to induction of IGF1R in both lung cell lines, other genes altered by TCDD that are involved with insulin signaling include INSIG1, PDK4, and PTPN1 (Table 2
, Fig. 2
).
Implications for Evaluating Human Lung Response to TCDD
The classification of TCDD as a known carcinogen and its association with human lung cancer and chronic obstructive pulmonary disease (Bertazzi et al., 2001; Pesatori et al., 1998
; Steenland et al., 1999
) warrants investigations on its effects in human lung cells. In the lung, there are over 40 different cell types, but tumors are derived mainly from bronchial epithelial cells, Clara cells, and type II pneumocytes. The focus of this study was to investigate TCDD effects using an in vitro model of type II pneumocytes comparing a malignant and a nonmalignant cell line. Consistent with the literature, many TCDD-altered gene expression profiles obtained from microarray analysis, included cytochrome P450s, quinone reductase, aldehyde dehydrogenases, fos, ras, protein kinase c, and vascular endothelial growth factor (Lai et al., 1996
; Sutter and Greenlee, 1992
).
It is becoming increasingly clear that TCDD is a multi-mechanistic xenobiotic that integrates a variety of signaling networks. This study and others using toxicogenomic approaches reflect the reliability that small changes in gene expression can be detected (Frueh et al., 2001; Kurachi et al., 2002
). A majority of the gene expression changes were less than 2-fold and may reflect modulation of signaling pathways, however only a dozen or so genes that had greater than 2-fold alterations are necessary to classify different toxicants (Thomas et al., 2001
). These data provide important potential insights into the mechanism of effects of TCDD in the lung; however, it is important to note that these studies have not been extended as yet to a large number of cell lines or in vivo conditions. Consequently, the cell line differences observed may simply reflect differences in cell type, growth conditions, or original source. Further validation of these genes affected by TCDD in vivo and transpecies comparisons will help to clarify how concordant these responses are with TCDD-induced lung toxicities.
Specific alterations in categories of genes altered from TCDD exposure, seem to depend on whether a cell is malignant or not, thus providing a valuable tool in assessing mechanism of action via toxicogenomic analysis. Gene expression profiles were different between the cell lines, as indicated by the nonmalignant HPL1A cells with altered genes that are immunosuppressive or involved in differentiation. The potential for TCDD-related decrease in immunocompetence or an imbalance in cell growth and differentiation is critical since it could lead to increases in a number of diseases including cancer. Whereas similar genes altered by TCDD in the two cell lines were those that are involved with growth control, cell-cell contact, cell motility, angiogenic factors, and metabolic enzymes.
The implication of these data is that some of these TCDD targets are known important contributors to pulmonary diseases and were only discovered by using nonmalignant cells. TCDD is a known human carcinogen that causes cancer promotion and enhances initiated cellular transformation (Tanaka et al., 1989). The hypothetical pathways presented in this study using genes altered by TCDD provide a thorough overview of the integration of signaling networks as well as added insights into the basic nature of its chemical carcinogenicity.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed at National Institute of Environmental Health Sciences, Rall Building 101, Room D452, 111 Alexander Drive, P.O. Box 12233, MD D4-01, Research Triangle Park, NC 27709. Fax: (919) 558-7053. E-mail: walker3{at}niehs.nih.gov.
Portions of this research were presented at the 40th annual meeting of the Society of Toxicology, March 2001, San Francisco, CA.
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REFERENCES |
---|
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---|
Bertazzi, P. A., Consonni, D., Bachetti, S., Rubagotti, M., Baccarelli, A., Zocchetti, C., and Pesatori, A. C. (2001). Health effects of dioxin exposure: A 20-year mortality study. Am. J. Epidemiol. 153, 10311044.
Boylan, J. F., and Gudas, L. J. (1992). The level of CRABP-I expression influences the amounts and types of all-trans-retinoic acid metabolites in F9 teratocarcinoma stem cells. J. Biol. Chem. 267, 2148621491.
Bushel, P. R., Hamadeh, H., Bennett, L., Sieber, S., Martin, K., Nuwaysir, E. F., Johnson, K., Reynolds, K., Paules, R. S., and Afshari, C. A. (2001). MAPS: A microarray project system for gene expression experiment information and data validation. Bioinformatics 17, 564565.
Casella, G., and Berger, R. L. (1990). Statistical Inference. Duxbury Press, Belmont, CA.
Chan, W. K., Yao, G., Gu, Y. Z., and Bradfield, C. A. (1999). Cross-talk between the aryl hydrocarbon receptor and hypoxia inducible factor signaling pathways. Demonstration of competition and compensation. J. Biol. Chem. 274, 1211512123.
Chen, Y., Dougherty, E. R., and Bittner, M. L. (1997). Ratio-based decisions and the quantitative analysis of cDNA microarray images. J. Biomed. Opt. 2, 364374.
Cheung, B., Hocker, J. E., Smith, S. A., Norris, M. D., Haber, M., and Marshall, G. M. (1998). Favorable prognostic significance of high-level retinoic acid receptor ß expression in neuroblastoma mediated by effects on cell cycle regulation. Oncogene 17, 751759.[ISI][Medline]
Delescluse, C., Lemaire, G., de Sousa, G., and Rahmani, R. (2000). Is CYP1A1 induction always related to AHR signaling pathway? Toxicology 153, 7382.[ISI][Medline]
Della Porta, G., Dragani, T. A., and Sozzi, G. (1987). Carcinogenic effects of infantile and long-term 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment in the mouse. Tumori 73, 99107.[ISI][Medline]
Denison, M. S., and Heath-Pagliuso, S. (1998). The Ah receptor: A regulator of the biochemical and toxicological actions of structurally diverse chemicals. Bull. Environ. Contam. Toxicol. 61, 557568.[ISI][Medline]
DeVito, M. J., and Birnbaum, L. S. (1995). The importance of pharmacokinetics in determining the relative potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran. Fundam. Appl. Toxicol. 24, 145148.[ISI][Medline]
Enan, E., and Matsumura, F. (1996). Identification of c-Src as the integral component of the cytosolic Ah receptor complex, transducing the signal of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through the protein phosphorylation pathway. Biochem. Pharmacol. 52, 15991612.[ISI][Medline]
Fernandez-Salguero, P. M., Hilbert, D. M., Rudikoff, S., Ward, J. M., and Gonzalez, F. J. (1996). Aryl-hydrocarbon receptor-deficient mice are resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxicity. Toxicol. Appl. Pharmacol. 140, 173179.[ISI][Medline]
Foster, K. A., Oster, C. G., Mayer, M. M., Avery, M. L., and Audus, K. L. (1998). Characterization of the A549 cell line as a type II pulmonary epithelial cell model for drug metabolism. Exp. Cell Res. 243, 359366.[ISI][Medline]
Franzoso, G., Carlson, L., Scharton-Kersten, T., Shores, E. W., Epstein, S., Grinberg, A., Tran, T., Shacter, E., Leonardi, A., Anver, M., Love, P., Sher, A., and Siebenlist, U. (1997). Critical roles for the Bcl-3 oncoprotein in T cell-mediated immunity, splenic microarchitecture, and germinal center reactions. Immunity 6, 479490.[ISI][Medline]
Frueh, F. W., Hayashibara, K. C., Brown, P. O., and Whitlock, J. P., Jr. (2001). Use of cDNA microarrays to analyze dioxin-induced changes in human liver gene expression. Toxicol Lett. 122, 189203.[ISI][Medline]
Fukata, M., Kuroda, S., Nakagawa, M., Kawajiri, A., Itoh, N., Shoji, I., Matsuura, Y., Yonehara, S., Fujisawa, H., Kikuchi, A., and Kaibuchi, K. (1999). Cdc42 and Rac1 regulate the interaction of IQGAP1 with ß-catenin. J. Biol. Chem. 274, 2604426050.
Fukata, M., Nakagawa, M., Itoh, N., Kawajiri, A., Yamaga, M., Kuroda, S., and Kaibuchi, K. (2001). Involvement of IQGAP1, an effector of Rac1 and Cdc42 GTPases, in cellcell dissociation during cell scattering. Mol. Cell Biol. 21, 21652183.
Ge, N. L., and Elferink, C. J. (1998). A direct interaction between the aryl hydrocarbon receptor and retinoblastoma protein. Linking dioxin signaling to the cell cycle. J. Biol. Chem. 273, 2270822713.
Gearing, D. P., Comeau, M. R., Friend, D. J., Gimpel, S. D., Thut, C. J., McGourty, J., Brasher, K. K., King, J. A., Gillis, S., Mosley, B., et al. (1992). The IL-6 signal transducer, gp130: An oncostatin M receptor and affinity converter for the LIF receptor. Science 255, 14341437.[ISI][Medline]
Gonzalez, F. J., and Fernandez-Salguero, P. (1998). The aryl hydrocarbon receptor: Studies using the AHR-null mice. Drug Metab. Dispos. 26, 11941198.
Grun, F., Hirose, Y., Kawauchi, S., Ogura, T., and Umesono, K. (2000). Aldehyde dehydrogenase 6, a cytosolic retinaldehyde dehydrogenase prominently expressed in sensory neuroepithelia during development. J. Biol. Chem.275, 4121041218.
Guigal, N., Seree, E., Nguyen, Q. B., Charvet, B., Desobry, A., and Barra, Y. (2001). Serum induces a transcriptional activation of CYP1A1 gene in HepG2 independently of the AhR pathway. Life Sci. 68, 21412150.[ISI][Medline]
Harris, C. C., Willey, J. C., Saladino, A. J., and Grafstrom, R. C. (1985). Effects of tumor promoters, aldehydes, peroxides, and tobacco smoke condensate on growth and differentiation of cultured normal and transformed human bronchial cells. Carcinog. Compr. Surv. 8, 159171.[Medline]
Heinrich, P. C., Behrmann, I., Muller-Newen, G., Schaper, F., and Graeve, L. (1998). Interleukin-6-type cytokine signalling through the gp130/Jak/STAT pathway. Biochem. J. 334, 297314.[ISI][Medline]
Holsapple, M. P., Morris, D. L., Wood, S. C., and Snyder, N. K. (1991).2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced changes in immunocompetence: Possible mechanisms. Annu. Rev. Pharmacol. Toxicol. 31, 73100.[ISI][Medline]
Hong, H., Kohli, K., Garabedian, M. J., and Stallcup, M. R. (1997). GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors. Mol. Cell Biol. 17, 27352744.[Abstract]
Hoshino, M., Nakamura, Y., and Hamid, Q. A. (2001). Gene expression of vascular endothelial growth factor and its receptors and angiogenesis in bronchial asthma. J. Allergy Clin. Immunol. 107, 10341038.[ISI][Medline]
Hrdlickova, R., Nehyba, J., and Bose, H. R., Jr. (2001). Interferon regulatory factor 4 contributes to transformation of v-Rel-expressing fibroblasts. Mol. Cell Biol. 21, 63696386.
Hromas, R., Collins, S. J., Hickstein, D., Raskind, W., Deaven, L. L., OHara, P., Hagen, F. S., and Kaushansky, K. (1991). A retinoic acid-responsive human zinc finger gene, MZF-1, preferentially expressed in myeloid cells. J. Biol. Chem. 266, 1418314187.
Hukkanen, J., Lassila, A., Paivarinta, K., Valanne, S., Sarpo, S., Hakkola, J., Pelkonen, O., and Raunio, H. (2000). Induction and regulation of xenobiotic-metabolizing cytochrome P450s in the human A549 lung adenocarcinoma cell line. Am. J. Respir. Cell Mol. Biol. 22, 360366.
Ioachim, H. L. (1997). Immune defiency: Opportunistic tumors. In Encyclopedia of Cancer, Vol. II. (J. R. Bertino, Ed.), pp. 901915. Academic Press, San Diego, CA.
Issemann, I., Prince, R. A., Tugwood, J. D., and Green, S. (1993). The peroxisome proliferator-activated receptor: Retinoidxreceptor heterodimer is activated by fatty acids and fibrate hypolipidaemic drugs. J. Mol. Endocrinol. 11, 3747.[Abstract]
Kazlauskas, A., Poellinger, L., and Pongratz, I. (1999). Evidence that the co-chaperone p23 regulates ligand responsiveness of the dioxin (Aryl hydrocarbon) receptor. J. Biol. Chem. 274, 1351913524.
Kelley, S. K., Nilsson, C. B., Green, M. H., Green, J. B., and Hakansson, H. (2000). Mobilization of vitamin A stores in rats after administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin: A kinetic analysis. Toxicol. Sci. 55, 478484.
Kerkvliet, N. I. (2002). Recent advances in understanding the mechanisms of TCDD immunotoxicity. Int. Immunopharmacol. 2, 277291.[ISI][Medline]
Kociba, R. J., Keyes, D. G., Beyer, J. E., Carreon, R. M., Wade, C. E., Dittenber, D. A., Kalnins, R. P., Frauson, L. E., Park, C. N., Barnard, S. D., Hummel, R. A., and Humiston, C. G. (1978). Results of a two-year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Toxicol. Appl. Pharmacol. 46, 279303.[ISI][Medline]
Kogevinas, M. (2000). Studies of cancer in humans. Food Addit. Contam. 17, 317324.[ISI][Medline]
Kogevinas, M., Becher, H., Benn, T., Bertazzi, P. A., Boffetta, P., Bueno-de-Mesquita, H. B., Coggon, D., Colin, D., Flesch-Janys, D., Fingerhut, M., Green, L., Kauppinen, T., Littorin, M., Lynge, E., Mathews, J. D., Neuberger, M., Pearce, N., and Saracci, R. (1997). Cancer mortality in workers exposed to phenoxy herbicides, chlorophenols, and dioxins. An expanded and updated international cohort study. Am. J. Epidemiol. 145, 10611075.[Abstract]
Kurachi, M., Hashimoto, S., Obata, A., Nagai, S., Nagahata, T., Inadera, H., Sone, H., Tohyama, C., Kaneko, S., Kobayashi, K., and Matsushima, K. (2002). Identification of 2,3,7,8-tetrachlorodibenzo-p-dioxin-responsive genes in mouse liver by serial analysis of gene expression. Biochem. Biophys. Res. Commun. 292, 368377.[ISI][Medline]
Lai, Z. W., Pineau, T., and Esser, C. (1996). Identification of dioxin-responsive elements (DREs) in the 5' regions of putative dioxin-inducible genes. Chem. Biol. Interact. 100, 97112.[ISI][Medline]
Lampen, A., Meyer, S., Arnhold, T., and Nau, H. (2000). Metabolism of vitamin A and its active metabolite all-trans-retinoic acid in small intestinal enterocytes. J. Pharmacol. Exp. Ther. 295, 979985.
Liu, M., Iavarone, A., and Freedman, L. P. (1996). Transcriptional activation of the human p21(WAF1/CIP1) gene by retinoic acid receptor. Correlation with retinoid induction of U937 cell differentiation. J. Biol. Chem. 271, 3172331728.
Ma, Q., and Whitlock, J. P., Jr. (1996). The aromatic hydrocarbon receptor modulates the Hepa 1c1c7 cell cycle and differentiated state independently of dioxin. Mol. Cell Biol.16, 21442150.[Abstract]
Ma, Q., and Whitlock, J. P., Jr. (1997). A novel cytoplasmic protein that interacts with the Ah receptor, contains tetratricopeptide repeat motifs, and augments the transcriptional response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Biol. Chem. 272, 88788884.
Mamane, Y., Sharma, S., Petropoulos, L., Lin, R., and Hiscott, J. (2000). Posttranslational regulation of IRF-4 activity by the immunophilin FKBP52. Immunity 12, 129140.[ISI][Medline]
Martinez, J. M., Masuda, A., Takahashi, T., and Walker, N. (2000). TCDD responsiveness of a nonmalignant human lung peripheral epithelial cell line. Organohalogen Compd. 49, 155158.
Masuda, A., Kondo, M., Saito, T., Yatabe, Y., Kobayashi, T., Okamoto, M., Suyama, M., Takahashi, T., and Takahashi, T. (1997). Establishment of human peripheral lung epithelial cell lines (HPL1) retaining differentiated characteristics and responsiveness to epidermal growth factor, hepatocyte growth factor, and transforming growth factor ß1. Cancer Res. 57, 48984904.[Abstract]
Means, A. L., Thompson, J. R., and Gudas, L. J. (2000). Transcriptional regulation of the cellular retinoic acid binding protein I gene in F9 teratocarcinoma cells. Cell Growth Differ. 11, 7182.
Mori, M., Kaji, M., Tezuka, F., and Takahashi, T. (1998). Comparative ultrastructural study of atypical adenomatous hyperplasia and adenocarcinoma of the human lung. Ultrastruct. Pathol. 22, 459466.[ISI][Medline]
Morris, D. L., Jeong, H. G., Stevens, W. D., Chun, Y. J., Karras, J. G., and Holsapple, M. P. (1994). Serum modulation of the effects of TCDD on the in vitro antibody response and on enzyme induction in primary hepatocytes. Immunopharmacology 27, 93105.[ISI][Medline]
NTP (1982). Report No 209, NIH Publication No. 821765. National Toxicology Program, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health.
Nettesheim, P., Gray, T., and Barrett, J. C. (1985). The toxic response of preneoplastic rat tracheal epithelial cells to 12-O-tetradecanoylphorbol-13-acetate. Carcinogenesis 6, 14271434.[Abstract]
Nilsson, C. B., Hoegberg, P., Trossvik, C., Azais-Braesco, V., Blaner, W. S., Fex, G., Harrison, E. H., Nau, H., Schmidt, C. K., van Bennekum, A. M., and Hakansson, H. (2000). 2,3,7,8-Tetrachlorodibenzo-p-dioxin increases serum and kidney retinoic acid levels and kidney retinol esterification in the rat. Toxicol. Appl. Pharmacol. 169, 121131.[ISI][Medline]
Nuwaysir, E. F., Bittner, M., Trent, J., Barrett, J. C., and Afshari, C. A. (1999). Microarrays and toxicology: The advent of toxicogenomics. Mol. Carcinog. 24, 153159.[ISI][Medline]
Oikawa, K., Ohbayashi, T., Mimura, J., Iwata, R., Kameta, A., Evine, K., Iwaya, K., Fujii-Kuriyama, Y., Kuroda, M., and Mukai, K. (2001). Dioxin suppresses the checkpoint protein, MAD2, by an aryl hydrocarbon receptor-independent pathway. Cancer Res. 61, 57075709.
Okey, A. B., Riddick, D. S., and Harper, P. A. (1994). The Ah receptor: Mediator of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Toxicol. Lett. 70, 122.[ISI][Medline]
Ong, D. E. (1987). Cellular retinoid-binding proteins. Arch. Dermatol. 123, 16931695a.[Abstract]
Park, J. H., Lee, S. W., Kim, I. T., Shin, B. S., Cheong, S. W., Cho, U. H., Huh, M. J., and Oh, G. S. (2001). TCDD up-regulation of IGFBP-6 and IL-5R subunit genes in vivo and in vitro. Mol. Cells 12, 372379.[ISI][Medline]
Pesatori, A. C., Zocchetti, C., Guercilena, S., Consonni, D., Turrini, D., and Bertazzi, P. A. (1998). Dioxin exposure and non-malignant health effects: A mortality study. Occup. Environ. Med. 55, 126131.[Abstract]
Pitt, J. A., Feng, L., Abbott, B. D., Schmid, J., Batt, R. E., Costich, T. G., Koury, S. T., and Bofinger, D. P. (2001). Expression of AhR and ARNT mRNA in cultured human endometrial explants exposed to TCDD. Toxicol. Sci. 62, 289298.
Puga, A., Barnes, S. J., Dalton, T. P., Chang, C., Knudsen, E. S., and Maier, M. A. (2000a). Aromatic hydrocarbon receptor interaction with the retinoblastoma protein potentiates repression of E2F-dependent transcription and cell cycle arrest. J. Biol. Chem. 275, 29432950.
Puga, A., Maier, A., and Medvedovic, M. (2000b). The transcriptional signature of dioxin in human hepatoma HepG2 cells. Biochem. Pharmacol. 60, 11291142.[ISI][Medline]
Rao, M. S., Subbarao, V., Prasad, J. D., and Scarpelli, D. G. (1988). Carcinogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the Syrian golden hamster. Carcinogenesis 9, 16771679.[Abstract]
Rosenbauer, F., Waring, J. F., Foerster, J., Wietstruk, M., Philipp, D., and Horak, I. (1999). Interferon consensus sequence binding protein and interferon regulatory factor-4/Pip form a complex that represses the expression of the interferon-stimulated gene-15 in macrophages. Blood 94, 42744281.
Rowlands, J. C., and Gustafsson, J. A. (1997). Aryl hydrocarbon receptor-mediated signal transduction. Crit. Rev. Toxicol. 27, 109134.[ISI][Medline]
Santostefano, M. J., Johnson, K. L., Whisnant, N. A., Richardson, V. M., DeVito, M. J., Diliberto, J. J., and Birnbaum, L. S. (1996). Subcellular localization of TCDD differs between the liver, lungs, and kidneys after acute and subchronic exposure: Species/dose comparisons and possible mechanism. Fundam. Appl. Toxicol. 34, 265275.[ISI][Medline]
Schmidt, S., Baniahmad, A., Eggert, M., Schneider, S., and Renkawitz, R. (1998). Multiple receptor interaction domains of GRIP1 function in synergy. Nucleic Acids Res. 26, 11911197.
Sharma, S., Mamane, Y., Grandvaux, N., Bartlett, J., Petropoulos, L., Lin, R., and Hiscott, J. (2000). Activation and regulation of interferon regulatory factor 4 in HTLV type 1-infected T lymphocytes. AIDS Res. Hum. Retroviruses 16, 16131622.[ISI][Medline]
Shepherd, D. M., Dearstyne, E. A., and Kerkvliet, N. I. (2000). The effects of TCDD on the activation of ovalbumin (OVA)-specific DO11. 10 transgenic CD4(+) T cells in adoptively transferred mice. Toxicol. Sci. 56, 340350.
Shepherd, D. M., Steppan, L. B., Hedstrom, O. R., and Kerkvliet, N. I. (2001). Anti-CD40 Treatment of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-exposed C57Bl/6 mice induces activation of antigen presenting cells yet fails to overcome TCDD-induced suppression of allograft immunity. Toxicol. Appl. Pharmacol. 170, 1022.[ISI][Medline]
Shi, Q., Le, X., Abbruzzese, J. L., Wang, B., Mujaida, N., Matsushima, K., Huang, S., Xiong, Q., and Xie, K. (1999). Cooperation between transcription factor AP-1 and NF-kappaB in the induction of interleukin-8 in human pancreatic adenocarcinoma cells by hypoxia. J. Interferon Cytokine Res. 19, 13631371.[ISI][Medline]
Spencer, D. L., Masten, S. A., Lanier, K. M., Yang, X., Grassman, J. A., Miller, C. R., Sutter, T. R., Lucier, G. W., and Walker, N. J. (1999). Quantitative analysis of constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1B1 expression in human lymphocytes. Cancer Epidemiol. Biomarkers Prev. 8, 139146.
Stamenkovic, I. (2000). Matrix metalloproteinases in tumor invasion and metastasis. Semin. Cancer Biol. 10, 415433.[ISI][Medline]
Standiford, T. J., Kunkel, S. L., Basha, M. A., Chensue, S. W., Lynch, J. P., 3rd, Toews, G. B., Westwick, J., and Strieter, R. M. (1990). Interleukin-8 gene expression by a pulmonary epithelial cell line. A model for cytokine networks in the lung. J. Clin. Invest. 86, 19451953.[ISI][Medline]
Stark, G. R., Kerr, I. M., Williams, B. R., Silverman, R. H. and Schreiber, R. D.(1998) How cells respond to interferons. Annu. Rev. Biochem. 67, 227264.[ISI][Medline]
Steenland, K., Piacitelli, L., Deddens, J., Fingerhut, M., and Chang, L. I. (1999). Cancer, heart disease, and diabetes in workers exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Natl. Cancer Inst. 91, 779786.
Strieter, R. M. (2001). Chemokines: Not just leukocyte chemoattractants in the promotion of cancer. Nat. Immunol. 2, 285286.[ISI][Medline]
Sulentic, C. E., Holsapple, M. P., and Kaminski, N. E. (1998). Aryl hydrocarbon receptor-dependent suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin of IgM secretion in activated B cells. Mol. Pharmacol. 53, 623629.
Sulentic, C. E., Holsapple, M. P., and Kaminski, N. E. (2000). Putative link between transcriptional regulation of IgM expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin and the aryl hydrocarbon receptor/dioxin-responsive enhancer signaling pathway. J. Pharmacol. Exp. Ther. 295, 705716.
Sun, S. Y., Wan, H. S., Yue, P., Hong, W. K., and Lotan, R. (2000). Evidence that retinoic acid receptor ß induction by retinoids is important for tumor cell growth inhibition. J. Biol. Chem. 275, 1714917153.
Sutter, T. R., and Greenlee, W. F. (1992). Classification of members of the Ah gene battery. Chemosphere 25, 223226.[ISI]
Sutter, T. R., Guzman, K., Dold, K. M., and Greenlee, W. F. (1991). Targets for dioxin: Genes for plasminogen activator inhibitor-2 and interleukin-1 ß. Science 254, 415418.[ISI][Medline]
Tamai, K., Semenov, M., Kato, Y., Spokony, R., Liu, C., Katsuyama, Y., Hess, F., Saint-Jeannet, J. P., and He, X. (2000). LDL-receptor-related proteins in Wnt signal transduction. Nature 407, 530535.[ISI][Medline]
Tanaka, N., Nettesheim, P., Gray, T., Nelson, K., and Barrett, J. C. (1989). 2,3,7,8-Tetrachlorodibenzo-p-dioxin enhancement of N-methyl-N'-nitro-N-nitrosoguanidineinduced transformation of rat tracheal epithelial cells in culture. Cancer Res. 49, 27032708.[Abstract]
Tannheimer, S. L., Ethier, S. P., Caldwell, K. K., and Burchiel, S. W. (1998). Benzo[a]pyrene- and TCDD-induced alterations in tyrosine phosphorylation and insulin-like growth factor signaling pathways in the MCF-10A human mammary epithelial cell line. Carcinogenesis 19, 12911297.[Abstract]
Thomas, R. S., Rank, D. R., Penn, S. G., Zastrow, G. M., Hayes, K. R., Pande, K., Glover, E., Silander, T., Craven, M. W., Reddy, J. K., Jovanovich, S. B., and Bradfield, C. A. (2001). Identification of toxicologically predictive gene sets using cDNA microarrays. Mol. Pharmacol. 60, 11891194.
Tian, Y., Ke, S., Denison, M. S., Rabson, A. B. and Gallo, M. A. (1999). Ah receptor and NF-B interactions, a potential mechanism for dioxin toxicity. J. Biol. Chem. 274, 510515.
Tritscher, A. M., Mahler, J., Portier, C. J., Lucier, G. L., and Walker, N. J. (1999). TCDD-induced lesions in rat lung after chronic oral exposure. Organohalogen Compd. 42, 285288.
Tritscher, A. M., Mahler, J., Portier, C. J., Lucier, G. W., and Walker, N. J. (2000). Induction of lung lesions in female rats following chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Pathol. 28, 761769.[ISI][Medline]
Tsukamoto, N., Hattori, M., Yang, H., Bos, J. L., and Minato, N. (1999). Rap1 GTPase-activating protein SPA-1 negatively regulates cell adhesion. J. Biol. Chem. 274, 1846318469.
Tymms, M. J., Ng, A. Y., Thomas, R. S., Schutte, B. C., Zhou, J., Eyre, H. J., Sutherland, G. R., Seth, A., Rosenberg, M., Papas, T., Debouck, C., and Kola, I. (1997). A novel epithelial-expressed ETS gene, ELF3: human and murine cDNA sequences, murine genomic organization, human mapping to 1q32.2 and expression in tissues and cancer. Oncogene 15, 24492462.[ISI][Medline]
van der Leede, B. M., van den Brink, C. E., and van der Saag, P. T. (1993). Retinoic acid receptor and retinoidxreceptor expression in retinoic acid-resistant human tumor cell lines. Mol. Carcinog. 8, 112122.[ISI][Medline]
Varanasi, U., Chu, R., Chu, S., Espinosa, R., LeBeau, M. M., and Reddy, J. K. (1994). Isolation of the human peroxisomal acyl-CoA oxidase gene: Organization, promoter analysis, and chromosomal localization. Proc. Natl. Acad. Sci. U.S.A. 91, 31073111.[Abstract]
Vasiliou, V., Pappa, A., and Petersen, D. R. (2000). Role of aldehyde dehydrogenases in endogenous and xenobiotic metabolism. Chem. Biol. Interact. 129, 119.[ISI][Medline]
Vogel, C., Dohr, O., and Abel, J. (1994). Transforming growth factor-ß 1 inhibits TCDD-induced cytochrome P450IA1 expression in human lung cancer A549 cells. Arch. Toxicol. 68, 303307.[ISI][Medline]
Vogel, C., Schuhmacher, U. S., Degen, G. H., Bolt, H. M., Pineau, T., and Abel, J. (1998). Modulation of prostaglandin H synthase-2 mRNA expression by 2,3,7,8- tetrachlorodibenzo-p-dioxin in mice. Arch. Biochem. Biophys. 351, 265271.[ISI][Medline]
Vos, J. G., and VanLoveren, H. (1995). Markers for immunotoxic effects in rodents and man. Toxicol. Let. 82 3, 385394.
Weber, T. J., Fan, Y. Y., Chapkin, R. S., and Ramos, K. S. (1997). Growth-related signaling in vascular smooth muscle cells is deregulated by TCDD during the G0/G1 transition. J. Toxicol. Environ. Health 51, 369386.[ISI][Medline]
Zou, C. P., Hong, W. K., and Lotan, R. (1999). Expression of retinoic acid receptor ß is associated with inhibition of keratinization in human head and neck squamous carcinoma cells. Differentiation 64, 123132.[ISI][Medline]