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Materials and Methods
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Several cytochrome P450 (CYP) enzymes are expressed in the human lung, where they participate in metabolic inactivation and activation of numerous exogenous and endogenous compounds. In this study, the expression pattern of all known xenobiotic-metabolizing CYP genes was characterized in the human alveolar type II cell-derived A549 adenocarcinoma cell line using qualitative reverse transcriptase/polymerase chain reaction (RT-PCR). In addition, the mechanisms of induction by chemicals of members in the CYP1 and CYP3A subfamilies were assessed by quantitative RT-PCR. The expression of messenger RNAs (mRNAs) of CYPs 1A1, 1B1, 2B6, 2C, 2E1, 3A5, and 3A7 was detected in the A549 cells. The amounts of mRNAs of CYPs 1A2, 2A6, 2A7, 2A13, 2F1, 3A4, and 4B1 were below the limit of detection. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced CYP1A1 and CYP1B1 mRNAs 56-fold and 2.5-fold, respectively. CYP3A5 was induced 8-fold by dexamethasone and 11-fold by phenobarbital. CYP3A4 was not induced by any of the typical CYP3A4 inducers used. The tyrosine kinase inhibitor genistein and the protein kinase C inhibitor staurosporine blocked TCDD-elicited induction of CYP1A1, but they did not affect CYP1B1 induction. Protein phosphatase inhibitors okadaic acid and calyculin A enhanced TCDD-induction of CYP1B1 slightly, but had negligible effects on CYP1A1 induction. These results suggest that CYP1A1 and CYP1B1 are differentially regulated in human pulmonary epithelial cells and give the first indication of the induction of CYP3A5 by glucocorticoids in human lung cells. These results establish that having retained several characteristics of human lung epithelial cell CYP expression, the A549 lung cell line is a valuable model for mechanistic studies on induction of the pulmonary CYP system.
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The lung constitutes a primary site of exposure for many inhaled chemical toxicants and carcinogens. As a consequence, the lung represents a major target for chemically induced diseases such as lung cancer, as well as parenchymal and obstructive lung diseases. Many of the hazardous chemicals, including constituents of cigarette smoke, are not active as such but require an enzymatic biotransformation to reactive forms (1). Attention has been primarily focused on cytochrome P450 (CYP)-dependent phase I activation of procarcinogens (2). Variations in the level of expression of this enzyme system have been implicated as being partly responsible for the interindividual differences in susceptibility to a multitude of cancers, including lung cancer (3, 4).
Lung tissues are known to activate several procarcinogens, such as the polycyclic aromatic hydrocarbon benzo- (a)pyrene (B[a]P), and several N-nitrosamines to form DNA adducts (5). It was shown recently (6) that benzo(a)pyrene diol epoxide, the ultimate carcinogenic metabolite of B(a)P, causes lung cancer-specific mutations in the p53 tumor suppressor gene in human bronchial epithelial cells, thus providing a direct link between lung cancer and B(a)P exposure. CYP enzymes taking part in the activation of B(a)P include several individual forms in families CYP1 and CYP3A (1). These CYP forms are expressed in various human pulmonary cell types. The main target of lung cancer caused by tobacco, the bronchial epithelial cells, expresses CYP1A1 (7) and CYP1B1 (10, 11), and these CYP forms are inducible by cigarette smoking. In addition, airway epithelial cells contain CYP3A5 messenger RNA (mRNA) and protein (12, 14, 15). Thus, the human lung tissue has the capacity to activate carcinogens and other toxins, and it is likely that factors affecting the activity of pulmonary CYPs may influence the risk of acquiring lung cancer and other chemically caused pulmonary diseases.
The induction of CYP enzymes in different human tissues is well characterized (16, 17). Induction of CYP1A1 and CYP3A4 by a variety of chemicals has been most intensively studied. Enzyme induction in the human lung has been less thoroughly studied, but we know that CYP1A1 and CYP1B1 induction occurs in response to cigarette smoking (7, 11). CYP3A enzymes are apparently not induced in human lung by cigarette smoke (15), and CYP3A5, the major pulmonary CYP3A form, is thought to be noninducible in the liver (18, 19).
The study on in vivo CYP induction in the human lung is technically demanding and requires the collection of lung biopsies or airway epithelial cell samples. Alternatively, surrogate cells such as alveolar macrophages can be used, but it is obvious that such surrogate cells do not accurately reflect the behavior of the actual target cells. To obtain insight into the mechanisms of human lung CYP induction, ex vivo models are needed. Primary cultures of bronchial epithelial cells and immortalized epithelial cells have been used, but these systems have a finite life span and show phenotypic alterations during subculturing. Transformed lung epithelial cell lines have been widely used, and one such continuous cell line, the alveolar epithelial type II cell-derived lung adenocarcinoma cell line A549 (20), has been a popular model. The A549 cells have retained some metabolic capacities of the normal type II alveolar cells, such as expressing CYP1A1, CYP1B1, and CYP2B6 (21, 22) and having the capability of forming DNA adducts after B(a)P treatment (23). This cell line thus provides a promising model for study of the regulation of human pulmonary epithelial cell CYP enzymes.
In this study, we have characterized in the A549 cell line the expression pattern of all known xenobiotic-metabolizing CYP genes. In addition, the induction profile caused by chemicals of members in the CYP1 and CYP3 families was determined. The results show that the A549 cell line is especially well suited for studying the control of expression of xenobiotic-metabolizing CYPs in the human lung because this cell line expresses most of the major constitutive and inducible CYP forms found in lung epithelial cells.
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Reagents
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was obtained
from the National Cancer Institute Chemical Carcinogen
Repository (Bethesda, MD). Rifampicin, dexamethasone,
phenobarbital, pregnenolone 16
-carbonitrile, and clotrimazole were purchased from Sigma (St. Louis, MO). Okadaic acid, calyculin A, staurosporine, and genistein were
purchased from Calbiochem (La Jolla, CA).
Cell Culture
The human lung adenocarcinoma cell line A549, originally
from the American Type Culture Collection (Rockville,
MD), was cultured in Ham's F12 medium with L-glutamine
(GIBCO BRL, Grand Island, NY) supplemented with
10% fetal bovine serum (GIBCO BRL) and 10 µg/ml gentamicin (GIBCO BRL). The cells were cultured at 37°C, 5% CO2, and saturated humidity. Nearly confluent cells
were incubated for 24 h in the presence of inducers (10 µM rifampicin, 10 µM dexamethasone, 1.5 mM phenobarbital, 10 nM TCDD, 10 µM PCN, or 10 µM clotrimazole).
Modulators of TCDD induction (10 nM okadaic acid, 3 nM
calyculin A, 50 µM genistein, or 50 nM staurosporine) were added together with TCDD. Control cultures received
the same concentration of vehicle (0.5% dimethyl sulfoxide). After the incubation, cells were washed with phosphate-buffered saline (pH 7.4), suspended in the same
buffer, and centrifuged. The cell pellet was suspended in
cell lysis buffer for mRNA extraction. The inducer and
modulator concentrations used were not cytotoxic judged
from cell morphology and 
-actin mRNA contents of the
cells. In pilot experiments, a range of inducer and modulator concentrations were tested. The concentrations chosen
for the actual studies were the ones causing maximal induction or inhibition without any signs of cytotoxicity.
mRNA and Complementary DNA
The mRNA of A549 cells was extracted with QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia Biotech, Uppsala, Sweden). The extraction of human liver and alveolar macrophage mRNAs used as positive controls have been described previously (24). To remove contaminating genomic DNA, mRNA preparations were treated with RNase-free DNase I and the DNase was removed by phenol/chloroform/isoamyl alcohol extraction. Complementary DNA (cDNA) was synthesized with the First-Strand Synthesis Kit (Amersham Pharmacia Biotech). All reagents were from Amersham Pharmacia Biotech. Human liver and human alveolar macrophage cDNAs used as positive controls were prepared the same way as mentioned previously for A549 cells.
RNA Blot Analysis
The amount and the integrity of mRNA in every sample
were verified with conventional RNA (Northern) blot analysis. An aliquot of mRNA was electrophoresed in an agarose
gel and transferred to Hybond-N+ nylon filter (Amersham,
Buckinghamshire, UK) followed by hybridization with [32P]-
labeled -actin cDNA probe. Filters were washed three times for 20 min in 0.1 × saline sodium citrate, 0.1% sodium dodecyl sulfate at 55°C. Radioactivity was measured with PhosphorImager SI equipment (Molecular Dynamics, Sunnyvale,
CA) and band intensities calculated with ImageQuaNT software from Molecular Dynamics (Sunnyvale, CA).
Qualitative Reverse Transcriptase/Polymerase Chain Reaction (RT-PCR)
The PCRs contained 1 µl of cDNA (of 15 µl of total cDNA), 2.0 U of DynaZyme DNA polymerase (Finnzymes, Helsinki, Finland), 5 µl of 10× DynaZyme reaction buffer, deoxynucleotide triphosphate reaction mix (Finnzymes) at a final concentration of 200 µM, 50 pmol of each primer and water to a final volume of 50 µl. A total of 35 PCR cycles of 1 min at 94°C, 1 min at 55-62°C and 2 min at 72°C was performed. In every series of PCRs was a negative control containing an aliquot of cDNA synthesis reaction performed with heat-inactivated reverse transcriptase enzyme. All PCRs were repeated at least twice. The primers were designed to be gene specific, except CYP2C primers, which detected all known human CYP2C forms (i.e., 2C8, 2C9, 2C18, and 2C19). To exclude chances of cross-hybridization with other sequences, each primer was compared with the European Molecular Biology Laboratory human gene bank using the FASTA program (Genetics Computer Group, Madison, WI). The primers were also designed to amplify regions containing at least one intron in the gene to exclude contamination of cDNA with genomic DNA. Because the genomic structure of CYP1B1 was not available at the time of the design of the primers, we were unable to target the CYP1B1 primers to separate exons. The primers, their locations, and the sizes of the PCR products were described previously (25). After the PCR, an 8-µl aliquot of each reaction mixture was electrophoresed in an agarose gel and stained with ethidium bromide. A visible band of the correct size was considered a sign of the specific mRNA being present in the sample.
Quantitative RT-PCR
Competitive PCR controls for CYP1A1, CYP1B1, CYP3A4, and CYP3A5 were prepared according to Jin and coworkers (29). The basis of this method is PCR amplification of a part of the target sequence with the same 3' primer and a recombinant 5' primer to produce a shortened template that can be amplified by the original primer pair. The obtained PCR products, containing a small 50-70 bp deletion but otherwise being identical in sequence to the target templates, were then cloned into the pCRII vector (Invitrogen BV, Leek, The Netherlands).
Quantitation was performed as follows. A master mix
of the PCR reagents (2.0 U Dynazyme DNA polymerase,
5 µl 10× DynaZyme reaction buffer, dNTP reaction mix
at a final concentration of 200 µM, 50 pmol each primer
and water to a final volume of 48 µl) was aliquoted into
sample tubes, and a constant amount of the studied cDNA
together with a serial dilution of control plasmid was
added to the reactions. Thirty-five PCR cycles were performed. An aliquot of reaction mixture was electrophoresed in agarose gel and stained with ethidium bromide. A
typical example is shown in Figure 1. The bands were measured directly from agarose gels with the ImageMaster
VDS densitometer and calculated with the ImageMaster
1D software from Amersham Pharmacia Biotech. The
amount of studied mRNA was quantitated by determining
graphically the point at which the amount of control molecules was equal to the target template. The results were
correlated with the amount of -actin mRNA in each sample quantitated by Northern blot. The numbers of experiments done are presented in Figures 2-5.
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Expression of CYP mRNAs in A549 Cells
Qualitative RT-PCR with gene-specific primers was performed to detect the general expression pattern of CYP mRNAs. As illustrated in Figure 6, correct-size amplification products, matching those derived from human liver cDNA, were seen for CYPs 1A1, 1B1, 2B6, 2C8-19, 2E1, 3A5, and 3A7 in the A549 cells. A summary of the RT-PCR results is provided in Table 1. CYP3A5 was the most abundantly expressed member of the CYP3A family, whereas CYP3A7 was observed in much lower quantities and CYP3A4 mRNA was missing altogether. CYP1A2, CYP2A6, CYP2A7, CYP2A13, CYP2F1, and CYP4B1 mRNAs were not detected at this level of sensitivity, which yielded clearly visible amplification products for each CYP in human liver or human alveolar macrophage (for CYP2F1 and CYP4B1) cDNA (data not shown). CYP2D6 amplification yielded multiple bands of different sizes, possibly reflecting the expression of pseudogenes and aberrantly spliced mRNAs (2, 30).
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Induction of CYP1 and CYP3A mRNAs in A549 Cells
To determine whether individual CYP forms in the families CYP1 and CYP3A are inducible by xenobiotics, the concentrations of CYP1A1, CYP1B1, CYP3A4, and CYP3A5 mRNAs were measured by quantitative RT-PCR after exposing the A549 cells to well-defined chemical inducers. A 24-h exposure to TCDD (10 nM) caused a 56-fold increase in the amount of CYP1A1 (Figure 2), whereas a similar treatment produced only a 2.5-fold induction of CYP1B1 mRNA (Figure 3). CYP1A1 and CYP1B1 mRNAs were not affected by treatment with either 10 µM rifampicin, 10 µM dexamethasone, or 1.5 mM phenobarbital. CYP3A4 mRNA was not present constitutively and its amount did not increase to detectable levels after exposure to any of the inducing compounds studied (PCN, clotrimazole, rifampicin, phenobarbital, dexamethasone, and TCDD). Interestingly, the amount of CYP3A5 mRNA was induced 8-fold and 11-fold by dexamethasone and phenobarbital, respectively, while PCN, clotrimazole, rifampicin, and TCDD did not cause any effect (Figure 4).
Modulation of TCDD-Induced CYP1A1 and CYP1B1
The role of intracellular protein phosphorylation in CYP1A1 and CYP1B1 induction was assessed by culturing the A549 cells in the presence of TCDD and protein kinase or protein phosphatase inhibitors. An amount of 50 nM staurosporine (a protein kinase C inhibitor) and 50 µM genistein (a tyrosine kinase inhibitor) blocked 79% and 65% of TCDD-elicited induction of CYP1A1, respectively (Figure 5). The protein phosphatase inhibitors calyculin A (3 nM) and okadaic acid (10 nM) had minor effects on CYP1A1 induction, but slightly enhanced the induction of CYP1B1 mRNA. Genistein and staurosporine had negligible effects on CYP1B1 induction by TCDD (Figure 5).
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The human adenocarcinoma cell line A549 is derived from
alveolar epithelial type II cells, a cell type that expresses
several CYP forms and also possesses metabolic activities
toward xenobiotics. CYP proteins in type II cells include
inducible CYP1A1 (9, 13, 31), CYP2B6 (31), CYP2E1 (31),
and CYP3A (14, 15, 32) (Table 1). Functional epoxide hydrolase and ethoxyresorufin O-deethylase (EROD) activities have also been detected in these cells (33). A549 cells
have retained many of the morphologic and metabolic features of normal type II cells (20). In earlier studies, A549
cells were shown to express at least CYP1A1, CYP1B1, and CYP2B6 enzymes (21, 22, 34, 35). These cells are also able to form DNA adducts after B(a)P treatment (23) followed by an increase in p53 protein expression (36). Both
adduct formation and the p53 response are inhibited by
the CYP1A1/CYP1B1 inhibitor -naphthoflavone (36). Döhr
and coworkers (21, 37) and Vogel and colleagues (38) have
studied extensively the transforming growth factor-1-mediated downregulation of TCDD-induced CYP1A1, CYP1B1,
and EROD activity in A549 cells and they have also detected the expression of aryl hydrocarbon receptor (AHR)
and AHR nuclear translocator (ARNT) in this cell line
(21, 37).
The objectives of this study were to determine comprehensively the expression pattern and induction of xenobiotic-metabolizing CYP mRNAs in the A549 cell line and to establish whether these characteristics resemble the ones found in alveolar type II cells. Qualitative and quantitative RT-PCR techniques were used because they are superior to conventional methods of mRNA detection owing to the great sensitivity and specificity of RT-PCR. Qualitative RT-PCR analysis showed that a number of major CYP forms are expressed in the A549 cells, and several individual CYPs are absent (Table 1, Figure 6). These data correlate well with earlier studies on in vivo pulmonary CYP expression. CYP1A1 has been shown to be inducible by cigarette smoking and it is mainly located in the peripheral lung (7, 13). Tobacco smoke-induced CYP1B1 mRNA has been found in bronchial cells (11) and alveolar macrophages (R. Piipari, unpublished data). CYP2B6 mRNA is consistently detected in the human lung (11, 24, 39), and there is evidence for the presence of the corresponding protein as well (12, 31). There are also indications for the presence of CYP2C isoforms in the human lung (12, 40). Despite numerous studies, there is still controversy about CYP2D6 expression in the pulmonary tissue (12, 24, 30, 40). It is likely that CYP2D6 mRNA is present in the lung as multiple splice variants (42), as corroborated by this and our previous study (24). CYP2E1 expression has been established in several studies (12, 43- 45). We did not detect in the A549 cells CYP2F1 and CYP4B1 mRNAs that have been found in whole lung tissue (10, 24, 39, 46, 47). In contrast to abundant hepatic expression of CYP3A4, this isoform seems to be of minor significance in the lung where it is expressed in only about 20% of the cases (15). It appears that in analogy with many other extrahepatic tissues, CYP3A4 is replaced by CYP3A5 in the human lung (12, 14, 15, 24).
The inducibility by chemicals of the major B(a)P activating CYP forms was assessed by quantitative RT-PCR. TCDD, a potent polycyclic aromatic hydrocarbon type inducer, clearly elevated the concentration of CYP1A1 mRNA (56-fold), whereas the increase in the amount of CYP1B1 mRNA was much more modest (2.5-fold), corroborating earlier findings in the same cell line (21, 37, 38). CYP3A4 was not affected by any of the typical inducing compounds studied. A major finding of this study is the approximately 10-fold induction of CYP3A5 elicited by dexamethasone and phenobarbital. CYP3A5, the minor hepatic CYP3A isoform, is not induced in human liver (18, 19), but there is evidence for its induction in human colon carcinoma cell lines (48). The promoter region of the CYP3A5 gene contains functional glucocorticoid-responsive element half-sites that have been shown to mediate induction by dexamethasone (49). The CYP3A4 gene is induced by binding of pregnane X receptor-retinoid X receptor (PXR-RXR) heterodimer to an everted repeat element (ER6) in the promoter area of the gene. However, the same heterodimer cannot activate the imperfect ER6 element found in the CYP3A5 gene (50). This is supported by the present finding that PXR-activating compounds like rifampicin and clotrimazole do not induce CYP3A5 in A549 cells. It is therefore likely that the glucocorticoid receptor conveys CYP3A5 induction as postulated by Schuetz and coworkers (49). These lines of evidence suggest that CYP3A5 induction is regulated in a tissue-specific manner in extrahepatic organs such as the lung and colon where this enzyme is abundantly expressed.
The role of intracellular protein phosphorylation on TCDD-induced CYP1A1 and CYP1B1 was determined using specific protein kinase and phosphatase inhibitors. The tyrosine kinase inhibitor genistein blocked CYP1A1 induction by TCDD but did not affect the inducibility of CYP1B1. A similar inhibition of CYP1A1 induction by genistein has previously been shown by Gradin and colleagues (51) in human primary keratinocytes and by Kikuchi and associates (52) in the human hepatoma HepG2 cell line. In contrast, genistein does not inhibit CYP1A1 induction by TCDD in rat (53) and mouse (54) hepatoma cells. Kikuchi and coworkers (52) postulated that the action of genistein is due to a dual action of inhibition of tyrosine kinase and topoisomerase I. Also topoisomerase I inhibitor camptothecin blocks CYP1A1 induction by TCDD (52, 55). According to Gradin and coworkers (51), the target for genistein action is the region of AHR harboring its ligand-binding domain. It was postulated that genistein inhibited ligand-induced release of hsp90 from the AHR (51). The lack of effect of genistein on CYP1B1 induction observed here indicates that its induction by TCDD is less dependent on AHR. This is supported by the fact that the CYP1B1 gene promoter is structurally and functionally similar to the promoters of constitutively expressed genes (56).
The protein kinase C (PKC) inhibitor staurosporine inhibited CYP1A1 induction but did not affect CYP1B1 induction. This is in agreement with findings that PKC inhibitors block the AHR-mediated induction of CYP1A1 (54, 57). The mechanism of PKC action on TCDD-elicited induction is controversial with conflicting reports in the literature. It seems that PKC inhibitors exert their action either by obstructing the DNA binding activity of AHR (54, 57) or by blocking the formation of a functional transcriptional complex capable of transactivating the CYP1A1 promoter without affecting DNA binding (59, 61, 62). The lack of effect of PKC inhibitors on CYP1B1 induction in the A549 cells provides further evidence of greater independence of CYP1B1 from the AHR. The serine/threonine protein phosphatase inhibitors calyculin A and okadaic acid enhanced CYP1B1 induction slightly but did not alter CYP1A1 induction. Previously these inhibitors (3 nM calyculin A, 50 nM okadaic acid) were shown to enhance the AHR-mediated induction in mouse Hepa-1 cells by affecting a step subsequent to xenobiotic response element (XRE) binding (63). There is also a report where 20 nM okadaic acid had no effect on polycyclic aromatic hydrocarbon induction of CYP1A1 mRNA in rat primary hepatocytes (60). Phosphatase treatments of AHR/ARNT have been shown to abolish the ability of the receptor complex to bind at XRE (54, 64, 65).
In conclusion, these results show that the A549 lung adenocarcinoma cell line is a valuable model for studies on human pulmonary xenobiotic metabolism. This cell line expresses many of the CYPs involved in activation of pulmonary toxicants and it has also retained the inducibility of CYP1 isoforms. The observed induction of CYP3A5 by phenobarbital and dexamethasone in this cell line is intriguing because this isoform is the major CYP3A form in the human lung. Current studies in our laboratory are aimed at assessing whether CYP3A5 is induced by other glucocorticoids, including the ones extensively used for the treatment of obstructive airway diseases, and ascertaining the mechanisms of this induction in the A549 cells.
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Address correspondence to: Hannu Raunio, Dept. of Pharmacology and Toxicology, University of Oulu, P.O. Box 5000, FIN-90401, Finland. E-mail: hannu.raunio{at}oulu.fi
(Received in original form July 1, 1999 and in revised form September 16, 1999).
Abbreviations: aryl hydrocarbon receptor, AHR; benzo(a)pyrene, B(a)P; complementary DNA, cDNA; cytochrome P450, CYP; messenger RNA, mRNA; protein kinase C, PKC; reverse transcriptase/polymerase chain reaction, RT-PCR; 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD.Acknowledgments: This work was funded in part by grants to J.H. from the Paulo Foundation, the Finnish Medical Society Duodecim, the Aino Soilis Foundation, and the Finnish Medical Foundation, and to O.P. from the European Commission (Biomed 2, BMH4-CT96-0254, "EUROCYP") and TEKES (Technology Development Center, Finland).
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1. Guengerich, F. P.. 1993. Bioactivation and detoxication of toxic and carcinogenic chemicals. Drug Metab. Dispos. 21: 1-6 [Medline].
2. Nelson, D. R., L. Koymans, T. Kamataki, J. J. Stegeman, R. Feyereisen, D. J. Waxman, M. R. Waterman, O. Gotoh, M. J. Coon, R. W. Estabrook, I. C. Gunsalus, and D. W. Nebert. 1996. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6: 1-42 [Medline].
3. Raunio, H., K. Husgafvel-Pursiainen, S. Anttila, E. Hietanen, A. Hirvonen, and O. Pelkonen. 1995. Diagnosis of polymorphisms in carcinogen-activating and inactivating enzymes and cancer susceptibility [review]. Gene 159: 113-121 [Medline].
4. Nebert, D. W., R. A. McKinnon, and A. Puga. 1996. Human drug-metabolizing enzyme polymorphisms: effects on risk of toxicity and cancer. DNA Cell Biol. 15: 273-280 [Medline].
5.
Autrup, H..
1990.
Carcinogen metabolism in cultured human tissues and
cells.
Carcinogenesis
11:
707-712
6.
Denissenko, M. F.,
A. Pao,
M. Tang, and
G. P. Pfeifer.
1996.
Preferential
formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in
P53.
Science
274:
430-432
7.
Omiecinski, C. J.,
C. A. Redlich, and
P. Costa.
1990.
Induction and developmental expression of cytochrome P450IA1 messenger RNA in rat and human tissues.
Cancer Res.
50:
4315-4321
8. McLemore, T. L., S. Adelberg, M. C. Liu, N. A. McMahon, S. J. Yu, W. C. Hubbard, M. Czerwinski, T. G. Wood, R. Storeng, R. A. Lubet, J. C. Eggleston, M. R. Boyd, and R. N. Hines. 1990. Expression of CYP1A1 gene in patients with lung cancer: evidence for cigarette smoke-induced gene expression in normal lung tissue and for altered gene regulation in primary pulmonary carcinomas. J. Natl. Cancer Inst. 83: 1333-1339 .
9. Anttila, S., E. Hietanen, H. Vainio, A.-M. Camus, H. V. Gelboin, S. S. Park, L. Heikkilä, A. Karjalainen, and H. Bartsch. 1991. Smoking and peripheral type of cancer are related to high levels of pulmonary cytochrome P450IA in lung cancer patients. Int. J. Cancer 47: 681-685 [Medline].
10. Willey, J. C., E. Coy, C. Brolly, M. J. Utell, M. W. Frampton, J. Hammersley, W. G. Thilly, D. Olson, and K. Cairns. 1996. Xenobiotic metabolism gene expression in human bronchial epithelial and alveolar macrophage cells. Am. J. Respir. Cell Mol. Biol. 14: 262-271 [Abstract].
11.
Willey, J. C.,
E. L. Coy,
M. W. Frampton,
A. Torres,
M. J. Apostolakos,
G. Hoehn,
W. H. Schuermann,
W. G. Thilly,
D. E. Olson,
J. R. Hammersley,
C. L. Crespi, and
M. J. Utell.
1997.
Quantitative RT-PCR measurement of
cytochromes p450 1A1, 1B1, and 2B7, microsomal epoxide hydrolase, and
NADPH oxidoreductase expression in lung cells of smokers and nonsmokers.
Am. J. Respir. Cell Mol. Biol.
17:
114-124
12. Macé, K., E. D. Bowman, P. Vautravers, P. G. Shields, C. C. Harris, and A. M. A. Pfeifer. 1998. Characterization of xenobiotic-metabolizing enzyme expression in human bronchial mucosa and peripheral lung tissue. Eur. J. Cancer 34: 914-920 .
13. Saarikoski, S. T., K. Husgafvel-Pursiainen, A. Hirvonen, H. Vainio, F. J. Gonzalez, and S. Anttila. 1998. Localization of CYP1A1 mRNA in human lung by in situ hybridization: comparison with immunohistochemical findings. Int. J. Cancer 77: 33-39 [Medline].
14. Kivistö, K. T., E.-U. Griese, P. Fritz, A. Linder, J. Hakkola, H. Raunio, P. Beaune, and H. K. Kroemer. 1996. Expression of cytochrome P4503A enzymes in human lung: a combined RT-PCR and immunohistochemical analysis of normal tissue and lung tumors. Naunyn-Schmiedeberg Arch. Pharmacol. 353: 207-212 [Medline].
15. Anttila, S., J. Hukkanen, J. Hakkola, T. Stjernvall, P. Beaune, R. J. Edwards, A. R. Boobis, O. Pelkonen, and H. Raunio. 1997. Expression and localization of CYP3A4 and CYP3A5 in human lung. Am. J. Respir. Cell Mol. Biol. 16: 242-249 [Abstract].
16. Pelkonen, O., J. Mäenpää, P. Taavitsainen, A. Rautio, and H. Raunio. 1998. Inhibition and induction of human cytochrome P450 (CYP) enzymes. Xenobiotica 28: 1203-1253 [Medline].
17. Raunio, H., M. Pasanen, J. Mäenpää, J. Hakkola, and O. Pelkonen. 1995. Expression of extrahepatic cytochrome P450 in humans. In Advances in Drug Metabolism in Man. G. M. Pacifici and G. N. Fracchia, editors. European Commission, Office for Official Publications of the European Communities, Luxembourg. 234-287.
18. Wrighton, S. A., B. J. Ring, P. B. Watkins, and M. VandenBranden. 1989. Identification of a polymorphically expressed member of the human cytochrome P-450III family. Mol. Pharmacol. 86: 97-105 .
19. Schuetz, E. G., J. D. Schuetz, S. C. Strom, M. T. Thompson, R. A. Fisher, D. T. Molowa, D. Li, and P. S. Guzelian. 1993. Regulation of human liver cytochromes P-450 in family 3A in primary and continuous culture of human hepatocytes. Hepatology 18: 1254-1262 [Medline].
20. Lieber, M., B. Smith, A. Szakal, W. Nelson-Rees, and G. Todaro. 1976. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int. J. Cancer 17: 62-70 [Medline].
21.
Döhr, O.,
R. Sinning,
C. Vogel,
P. Munzel, and
J. Abel.
1997.
Effect of
transforming growth factor-1 on expression of aryl hydrocarbon receptor
and genes of Ah gene battery: clues for independent down-regulation in
A549 cells.
Mol. Pharmacol.
51:
703-710
22. Foster, K. A., C. G. Oster, M. M. Mayer, M. L. Avery, and K. L. Audus. 1998. Characterization of the A549 cell line as a type II pulmonary epithelial cell model for drug metabolism. Exp. Cell. Res. 243: 359-366 [Medline].
23. Feldman, G., J. Remsen, K. Shinohara, and P. Cerutti. 1978. Excisability and persistence of benzo(a)pyrene DNA adducts in epithelioid human lung cells. Nature 274: 796-798 [Medline].
24. Hukkanen, J., J. Hakkola, S. Anttila, R. Piipari, A. Karjalainen, O. Pelkonen, and H. Raunio. 1997. Detection of mRNA encoding xenobiotic-metabolizing cytochrome P450s in human broncho-alveolar macrophages and peripheral blood lymphocytes. Mol. Carcinog. 20: 224-230 [Medline].
25.
Hakkola, J.,
M. Pasanen,
O. Pelkonen,
J. Hukkanen,
S. Evisalmi,
S. Anttila,
A. Rane,
M. Mäntylä,
R. Purkunen,
S. Saarikoski,
M. Tooming, and
H. Raunio.
1997.
Expression of CYP1B1 in human adult and fetal tissues and
differential inducibility of CYP1B1 and CYP1A1 by Ah receptor ligands
in human placenta and cultured cells.
Carcinogenesis
18:
391-397
26. Hakkola, J., M. Pasanen, J. Hukkanen, O. Pelkonen, J. Mäenpää, R. J. Edwards, A. R. Boobis, and H. Raunio. 1996. Expression of xenobiotic- metabolizing cytochrome P450 forms in human full-term placenta. Biochem. Pharmacol. 51: 403-411 [Medline].
27. Hakkola, J., H. Raunio, R. Purkunen, O. Pelkonen, S. Saarikoski, T. Cresteil, and M. Pasanen. 1996. Detection of cytochrome P450 gene expression in human placenta in first trimester of pregnancy. Biochem. Pharmacol. 52: 379-383 [Medline].
28. Koskela, S., J. Hakkola, J. Hukkanen, O. Pelkonen, M. Sorri, A. Saranen, S. Anttila, P. Fernandez-Salguero, F. J. Gonzalez, and H. Raunio. 1999. Expression of CYP2A genes in human liver and extrahepatic tissues. Biochem. Pharmacol. 57: 1407-1413 [Medline].
29. Jin, C.-F., M. Mata, and D. J. Fink. 1994. Rapid construction of deleted DNA fragments for use as internal standards in competitive PCR. PCR Methods Applic. 3: 252-255 .
30. Kivistö, K. T., E.-U. Griese, T. Stuven, P. Fritz, G. Freidel, H. K. Kroemer, and U. M. Zanger. 1997. Analysis of CYP2D6 expression in human lung: implications for the association between CYP2D6 activity and susceptibility to lung cancer. Pharmacogenetics 7: 295-302 [Medline].
31. Mori, M., F. Tezuka, R. Chiba, Y. Funae, M. Watanabe, T. Nukiwa, and T. Takahashi. 1996. Atypical adenomatous hyperplasia and adenocarcinoma of the human lung: their heterology in form and analogy in immunohistochemical characteristics. Cancer 77: 665-674 [Medline].
32. Kivistö, K. T., P. Fritz, A. Linder, G. Friedel, P. Beaune, and H. K. Kroemer. 1995. Immunohistochemical localization of cytochrome P450 3A in human pulmonary carcinomas and normal bronchial tissue. Histochemistry 103: 25-29 [Medline].
33.
Devereux, T. R.,
T. E. Massey,
M. R. Van Scott,
J. Yankaskas, and
J. R. Fouts.
1986.
Xenobiotic metabolism in human alveolar type II cells isolated by centrifugal elutriation and density gradient centrifugation.
Cancer
Res.
46:
5438-5443
34.
McLemore, T. L.,
S. Adelberg,
M. Czerwinski,
W. C. Hubbard,
S. J. Yu,
R. Storeng,
T. G. Wood,
R. N. Hines, and
M. R. Boyd.
1989.
Altered regulation
of the cytochrome P4501A1 gene: novel inducer-independent gene expression in pulmonary carcinoma cell lines.
J. Natl. Cancer Inst.
81:
1787-1794
35. Urani, C., M. Doldi, S. Crippa, and M. Camatini. 1998. Human-derived cell lines to study xenobiotic metabolism. Chemosphere 37: 2785-2795 [Medline].
36.
Rämet, M.,
K. Castrén,
K. Järvinen,
K. Pekkala,
T. Turpeenniemi-Hujanen,
Y. Soini,
P. Pääkkö, and
K. Vähäkangas.
1995.
p53 Protein expression is
correlated with benzo(a)pyrene-DNA adducts in carcinoma cell lines.
Carcinogenesis
16:
2117-2124
37.
Döhr, O., and
J. Abel.
1997.
Transforming growth factor-1 coregulates
mRNA expression of aryl hydrocarbon receptor and cell-cycle-regulating
genes in human cancer cell lines.
Biochem. Biophys. Res. Commun.
241:
86-91
[Medline].
38.
Vogel, C.,
O. Döhr, and
J. Abel.
1994.
Transforming growth factor-1 inhibits TCDD-induced cytochrome P450IA1 expression in human lung cancer
A549 cells.
Arch. Toxicol.
68:
303-307
[Medline].
39.
Czerwinski, M.,
T. L. McLemore,
H. V. Gelboin, and
F. J. Gonzalez.
1994.
Quantitation of CYP2B7, CYP4B1, and CYPOR messenger RNAs in normal human lung and lung tumors.
Cancer Res.
54:
1085-1091
40. Shimada, T., H. Yamazaki, M. Mimura, N. Wakamiya, Y.-F. Ueng, F. P. Guengerich, and Y. Inui. 1996. Characterization of microsomal cytochrome P450 enzymes involved in the oxidation of xenobiotic chemicals in human fetal livers and adult lungs. Drug Metab. Dispos. 24: 515-522 [Abstract].
41. Guidice, J.-M. L., D. Marez, N. Sabbagh, M. Legrand-Andreoletti, C. Spire, E. Alcaide, J.-J. Lafitte, and F. Broly. 1997. Evidence for CYP2D6 expression in human lung. Biochem. Biophys. Res. Commun. 241: 79-85 [Medline].
42.
Huang, Z.,
M. J. Fasco,
S. Spivack, and
L. S. Kaminsky.
1997.
Comparisons
of CYP2D messenger RNA splice variant profiles in human lung tumors
and normal tissues.
Cancer Res.
57:
2589-2592
43. Wheeler, C. W., S. A. Wrighton, and T. M. Guenthner. 1992. Detection of human lung cytochrome P450 that are immunochemically related to cytochrome P450IIE1 and cytochrome P450IIIA. Biochem. Pharmacol. 44: 911-917 .
44. Botto, F., E. Seree, S. E. Khyari, G. de Sousa, A. Massacrier, M. Placidi, P. Cau, W. Pellet, R. Rahmani, and Y. Barra. 1994. Tissue-specific expression and methylation of the human CYP2E1 gene. Biochem. Pharmacol. 48: 1095-1103 [Medline].
45. Kivistö, K. T., A. Linder, G. Friedel, P. Beaune, C. Belloc, H. K. Kroemer, and P. Fritz. 1995. Immunohistochemical localization of cytochrome P450 2E1 in human pulmonary carcinoma and normal bronchial tissue. Virchows Arch. 426: 243-247 [Medline].
46. Nhamburo, P. T., S. Kimura, O. W. McBride, C. A. Kozak, H. V. Gelboin, and F. J. Gonzalez. 1990. The human CYP2F gene family: identification of a cDNA encoding a new cytochrome P450, cDNA-directed expression, and chromosome mapping. Biochemistry 29: 5491-5499 [Medline].
47. Nhamburo, P. T., F. J. Gonzalez, O. W. McBride, H. V. Gelboin, and S. Kimura. 1989. Identification of a new P450 expressed in human lung: complete cDNA sequence, cDNA-directed expression, and chromosome mapping. Biochemistry 28: 8060-8066 [Medline].
48. Schuetz, E. G., W. T. Beck, and J. D. Schuetz. 1996. Modulators and substrates of P-glycoprotein and cytochrome P4503A coordinately up-regulate these proteins in human colon carcinoma cells. Mol. Pharmacol. 49: 311-318 [Abstract].
49. Schuetz, J. D., E. G. Schuetz, J. V. Thottassery, P. S. Guzelian, S. Strom, and D. Sun. 1996. Identification of a novel dexamethasone responsive enhancer in the human CYP3A5 gene and its activation in human and rat liver cells. Mol. Pharmacol. 49: 63-72 [Abstract].
50.
Blumberg, B.,
W. Sabbagh Jr.,
H. Juguilon,
J. Bolado Jr.,
C. M. van Meter,
E. S. Ong, and
R. M. Evans.
1998.
SXR, a novel steroid and xenobiotic-sensing nuclear receptor.
Genes Dev.
12:
3195-3205
51.
Gradin, K.,
M. L. Whitelaw,
R. Toftgård,
L. Poellinger, and
A. Berghard.
1994.
A tyrosine kinase-dependent pathway regulates ligand-dependent
activation of the dioxin receptor in human keratinocytes.
J. Biol. Chem.
269:
23800-23807
52.
Kikuchi, H.,
A. Hossain,
H. Yoshida, and
S. Kobayashi.
1998.
Induction of
cytochrome P-450 1A1 by omeprazole in human HepG2 cells is protein tyrosine kinase-dependent and is not inhibited by -naphthoflavone.
Arch.
Biochem. Biophys.
358:
351-358
[Medline].
53.
Backlund, M.,
I. Johansson,
S. Mkrtchian, and
M. Ingelman-Sundberg.
1997.
Signal transduction-mediated activation of the aryl hydrocarbon receptor in rat hepatoma H4IIE cells.
J. Biol. Chem.
272:
31755-31763
54.
Carrier, F.,
R. A. Owens,
D. W. Nebert, and
A. Puga.
1992.
Dioxin-dependent activation of murine Cyp1a-1 gene transcription requires protein kinase C-dependent phosphorylation.
Mol. Cell. Biol.
12:
1856-1863
55. Gradin, K., R. Toftgård, and A. Berghard. 1995. Differential effects of a topoisomerase I inhibitor on dioxin inducibility and high-level expression of the cytochrome P450IA1 gene. Mol. Pharmacol. 48: 610-615 [Abstract].
56.
Wo, Y.-Y. P.,
J. Stewart, and
W. F. Greenlee.
1997.
Functional analysis of the
promoter for the human CYP1B1 gene.
J. Biol. Chem.
272:
26702-26707
57.
Berghard, A.,
K. Gradin,
I. Pongratz,
M. Whitelaw, and
L. Poellinger.
1993.
Cross-coupling of signal transduction pathways: the dioxin receptor mediates induction of cytochrome P-450IA1 expression via a protein kinase
C-dependent mechanism.
Mol. Cell. Biol.
13:
677-689
58. Reiners, J. J., A. Schöller, P. Bischer, A. R. Cantu, and A. Pavone. 1993. Suppression of cytochrome P450 Cyp 1a-1 induction in murine hepatoma 1c1c7 cells by 12-O-tetradecanoylphorbol-13-acetate and inhibitors of protein kinase C. Arch. Biochem. Biophys. 301: 449-454 [Medline].
59.
Chen, Y.-H., and
R. H. Tukey.
1996.
Protein kinase C modulates regulation
of the CYP1A1 gene by the aryl hydrocarbon receptor.
J. Biol. Chem.
271:
26261-26266
60.
Xiao, G.-H.,
C. Falkner,
Y. Xie,
R. G. Lindahl, and
R. A. Prough.
1997.
cAMP-dependent negative regulation of rat aldehyde dehydrogenase class
3 gene expression.
J. Biol. Chem.
272:
3238-3245
61.
Long, W. P.,
M. Pray-Grant,
J. C. Tsai, and
G. H. Perdew.
1998.
Protein kinase C activity is required for aryl hydrocarbon receptor pathway-mediated signal transduction.
Mol. Pharmacol.
53:
691-700
62. Schafer, M. W., B. V. Madhukar, H. I. Swanson, K. Tullis, and M. S. Denison. 1993. Protein kinase C is not involved in Ah receptor transformation and DNA binding. Arch. Biochem. Biophys. 307: 267-271 [Medline].
63. Li, S.-Y., and J. J. Dougherty. 1997. Inhibitors of serine/threonine-specific protein phosphatases stimulate transcription by the Ah receptor/Arnt dimer by affecting a step subsequent to XRE binding. Arch. Biochem. Biophys. 340: 73-82 [Medline].
64.
Pongratz, I.,
P. E. Stromstedt,
G. G. Mason, and
L. Poellinger.
1991.
Inhibition of the specific DNA binding activity of the dioxin receptor by phosphatase treatment.
J. Biol. Chem.
266:
16813-16817
65. Mahon, M. J., and T. A. Gasiewicz. 1995. Ah receptor phosphorylation: localization of phosphorylation sites to the C-terminal half of the protein. Arch. Biochem. Biophys. 318: 166-174 [Medline].
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